Chimeric antigen receptors with 4-1BB co-stimulatory domains

Abstract

There is provided a CAR-T composition relating to a CAR, the CAR comprising: (i) an extracellular domain, the extracellular domain comprising an antigen binding domain; (ii) a transmembrane domain; and (iii) an intracellular domain comprising a co-stimulatory endodomain, where the co-stimulatory endodomain comprises an intracellular signaling domain from 4-1BB / CD137 and five further amino acids. The present disclosure also provides vectors, compositions, and methods of treatment using antigen binding molecules and engineered immune cells including 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 priority and benefits of the following: U.S. application 62 / 867,503 filed on June 27, 2019; International application PCT / KR2019 / 010244 filed on August 12, 2019; U.S. application 16 / 715,462 filed on December 16, 2019; U.S. application 62 / 991,493 filed on March 18, 2020; U.S. application 63 / 004,827 filed on April 3, 2020; U.S. application 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 died of 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, the global burden is expected to grow to 21.7 million new cancer cases and 13 million cancer deaths due to population growth and aging and exposure to risk factors such as smoking, unhealthy diet and lack of physical activity. Further, 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 gives T cells strong target affinity and signaling functions. However, the impressive efficacy of CAR-T therapy is often accompanied by serious side effects, such as cytokine release syndrome (CRS). Therefore, there is still an unmet need for the development of CAR-T treatments and strategies with reduced side effects. Summary of the invention

[0005] Provided herein is an immune cell comprising a chimeric antigen receptor (CAR), wherein the (CAR) comprises: (a) an extracellular domain comprising an antigen binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising a co-stimulatory endodomain, wherein the co-stimulatory endodomain 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 costimulatory endodomain comprises the 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 costimulatory endodomain 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 to an antigen associated with a disease. In some embodiments, the antigen binding domain specifically binds to a tumor antigen. In some embodiments, the antigen binding domain specifically binds to an antigen selected from the group consisting of: phosphatidylinositol glycan-3 (GPC3), malignant tumor variant receptor (MVR) and CD19.

[0009] In some embodiments, the transmembrane domain is a transmembrane domain selected 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, 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 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecules (SLAM proteins), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1 and VLA-6. In some embodiments, the transmembrane domain is a transmembrane domain from CD8α. In some embodiments, the intracellular domain further includes an 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 cells are human immune cells. In some embodiments, the human immune cells are autologous human immune cells. In some embodiments, the human immune cells are allogeneic human immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are NK cells.

[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 endodomain, wherein the co-stimulatory endodomain 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 endodomain 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 endodomain 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 an antigen associated with a disease. In some embodiments, the antigen binding domain specifically binds to a tumor antigen. In some embodiments, the antigen binding domain specifically binds to an antigen selected from the group consisting of glycan-3 (GPC3), malignant variant receptor (MVR) and CD19.

[0016] In some embodiments, the transmembrane domain is a transmembrane domain selected 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, 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 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecules (SLAM proteins), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1 and VLA-6. In some embodiments, the transmembrane domain is a transmembrane domain from CD8α.

[0017] In some embodiments, the intracellular domain further comprises an 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 a CD8α hinge.

[0018] Provided herein are vectors comprising any 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] Provided herein is a method for producing engineered immune cells, the method comprising: introducing any of the nucleic acids described herein or any of the vectors described herein into an immune cell, thereby producing the engineered immune cell. In some embodiments, the method further comprises, after the introduction step, culturing the engineered immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a NK cell.

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

[0021] Provided herein are engineered immune cells produced by any of the methods described herein.

[0022] Provided herein are pharmaceutical compositions comprising any of the engineered immune cells described herein and a pharmaceutically acceptable carrier.

[0023] Provided herein is a method for treating cancer in a subject, the method comprising administering to the subject any of the engineered immune cells described herein or any of the pharmaceutical compositions described herein. In some embodiments, the cancer is anti-Glypican-3 related cancer, anti-CD 19 related cancer, or 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, renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, other lymphoproliferative disorders, and various types of head and neck cancer. In some embodiments, the subject has previously 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 A schematic diagram of an exemplary MVR CAR construct is shown.

[0025] Figure 2 Exemplary enzyme mapping results after cloning of MVRL2H2-4-1BB are shown.

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

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

[0028] Figure 5B This is a chart comparing the in vitro expansion folds of CAR-T cells.

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

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

[0031] Fig. 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] Fig.9A is a graph showing CAR-T cell counts from a mouse model 5 weeks after injection of huGC33(VH-VL)-euBBz and huGC33(VH-VL)-BBz CAR-T cells.

[0035] Fig. 9B is a set of graphs comparing CAR-T cell counts from mouse models 5 weeks after injection of huGC33(VH-VL)-euBBz and huGC33(VH-VL)-BBz CAR-T cells.

[0036] Fig.10 Shown is 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)-BBzCAR-T cells using FACS staining.

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

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

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

[0040] Fig.13is 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] Fig.14 A is a set of graphs showing the percentage of total CD19 CAR-T cells present in the blood using FACS after orbital blood collection was performed in mice at 3 to 4 day intervals.

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

[0043] Fig.14 C is a graph showing the number of total CD19 CAR-Ts present in the blood using FACS after orbital blood collection was performed in mice at 3 to 4 day intervals.

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

[0045] Fig.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 of making and using the CARs.

[0047] definition

[0048] About: When used herein to refer to a value, the term "about" refers to a value that is similar to the referenced value in the context. Generally, one skilled in the art who is familiar with the context will understand the relevant degree of difference encompassed by "about" in the context. For example, in some embodiments, the term "about" can encompass 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 "administer" generally refers to a subject or systemic administration of a composition to achieve delivery of a medicament as a composition or contained in a composition. One of ordinary skill in the art will appreciate that various routes may be used for administration to, for example, a subject of a person, under appropriate circumstances. For example, in some embodiments, administration may be ocular administration, oral administration, parenteral administration, topical administration, etc. In some specific embodiments, administration may be bronchial administration (e.g., by bronchial instillation), buccal administration, skin administration (which may be or include, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral administration, intraarterial administration, intradermal administration, intragastric administration, intramedullary administration, intramuscular administration, intranasal administration, intraperitoneal administration, intrathecal administration, intravenous administration, intraventricular administration, administration in a specific organ (e.g., intrahepatic), mucosal administration, nasal administration, oral administration, rectal administration, subcutaneous administration, sublingual administration, topical administration, tracheal administration (e.g., by intratracheal instillation), vaginal administration, vitreous administration, etc. In some embodiments, administration may involve a single dose, multiple doses, or a fixed number of doses. In some embodiments, administration may involve administration that is intermittent administration (e.g., multiple doses separated in time) and / or periodic administration (e.g., separate doses separated by a common time period). In some embodiments, administration may involve continuous administration (e.g., perfusion) for at least a selected time period.

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

[0051] Antibody agent: As used herein, the term "antibody agent" may refer to an agent that specifically binds to a specific antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex comprising an immunoglobulin structural element 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 comprise one or more sequence elements of humanization, primatization, chimeric, etc., known in the art. In many embodiments, the term "antibody agent" is used to refer to one or more of the constructs or forms known or developed in the art for utilizing antibody structural and functional characteristics in alternative presentations. For example, in some embodiments, the antibody agent utilized in accordance with the present invention is in a form selected from, but not limited to, a complete IgA antibody, an IgG antibody, an IgE antibody, or an IgM antibody; a bispecific antibody or a multispecific antibody (e.g., , etc.); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments and isolated CDRs or collections thereof; single-chain Fv; polypeptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., ); Small modular immunopharmaceuticals (SMIPs) TM ), single-chain diabodies or tandem diabodies VHH; microantibodies; Ankyrin repeat protein or DART;TCR-like antibody; Trace protein; as well as In some embodiments, the antibody agent may lack covalent modifications (e.g., attachment of glycans) that it would have when produced naturally. In some embodiments, the 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, the antibody agent is or includes a polypeptide whose amino acid sequence includes one or more structural elements recognized as complementary determining regions (CDRs) by those skilled in the art. In some embodiments, the antibody agent is or includes a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) that is substantially identical to the CDR found in a reference antibody. In some embodiments, the included CDR is substantially identical to the reference CDR because the included CDR is identical in sequence to the reference CDR or contains between 1 and 5 amino acid substitutions. In some embodiments, the included CDR is substantially identical to a reference CDR in that the included CDR shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reference CDR. In some embodiments, the included CDR is substantially identical to a reference CDR in that the included CDR shows at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reference CDR. In some embodiments, the included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is deleted, added, or substituted compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to the amino acid sequence of the reference CDR. In some embodiments, the included CDR is substantially identical to 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 identical to the amino acid sequence of the reference CDR. In some embodiments, the included CDR is substantially identical to 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 identical to the amino acid sequence of the reference CDR. In some embodiments, the included CDR is substantially identical to 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 identical to the amino acid sequence of the reference CDR. In some embodiments, the antibody agent is or includes a polypeptide whose amino acid sequence includes a structural element that is recognized by those skilled in the art as an immunoglobulin variable domain.In some embodiments, the antibody agent is a polypeptide protein having a binding domain homologous or mostly homologous to an immunoglobulin binding domain. In some embodiments, the antibody agent is or includes at least a portion of a chimeric antigen receptor (CAR).

[0052] Antigen: As used herein, the term "antigen" may refer to an agent in conjunction with an antibody agent. In certain embodiments, an antigen in conjunction with an antibody agent may or may not induce a specific physiological response in an organism. Typically, an antigen may be or include any chemical entity, such as a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, a polymer (including a biopolymer [e.g., a nucleic acid and / or an amino acid polymer] and a polymer other than a biopolymer [e.g., a polymer other than a nucleic acid or an amino acid polymer]), etc. In certain embodiments, an antigen is or includes a polypeptide. In certain embodiments, an antigen is or includes a polysaccharide. It will be appreciated by those skilled in the art that, typically, an antigen may be provided or used in an isolated or pure form, or may alternatively be provided in a crude form (e.g., together with other materials, such as in an extract of other relatively crude preparations such as a cell extract or a source containing an antigen).

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

[0054] Antigen binding domain: As used herein, the term "antigen binding domain" may refer to an antibody agent or portion thereof that specifically binds to a target moiety or entity. Typically, the interaction between an antigen binding domain and its target is non-covalent. In some embodiments, the target moiety or entity may belong to any chemical class, including, for example, carbohydrates, lipids, nucleic acids, metals, polypeptides, or small molecules. In some embodiments, the antigen binding domain may be or 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] Related to: Two events or entities are "associated with" one another, as that term is used herein, if the presence, level, and / or form of one event or entity is related to the presence, level, and / or form of another event or entity. For example, a particular entity (e.g., a polypeptide, a genetic signature, a metabolite, a microorganism, etc.) is considered to be associated with a particular disease, disorder, or condition if the presence, level, and / or form of the entity is associated with the incidence and / or susceptibility of the disease, disorder, or condition (e.g., across a related population). In some embodiments, two or more entities are physically "associated" with one another if they interact, directly or indirectly, so that they are physically close to one another and / or remain in close proximity to one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another, but are non-covalently associated, for example, by hydrogen bonding, 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 a non-covalent association between or among two or more entities. "Direct" binding involves physical contact between the entities or moieties. Indirect binding involves physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can generally be assessed in any of a variety of situations, including studying the interacting entities or moieties 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 precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic cells. The present disclosure specifically identifies certain cancers whose teachings may be particularly relevant. In some embodiments, a relevant cancer may be characterized by a solid tumor. In some embodiments, a relevant cancer may be characterized by a hematological tumor.

[0058] In general, examples of different types of cancer known in the art include, for example, cancers of the hematopoietic system, including leukemias, lymphomas (Hodgkin and non-Hodgkin), myelomas and myeloproliferative disorders; sarcomas, melanomas, adenomas, solid tissue cancers, squamous cell carcinomas of the oral cavity, larynx, larynx and lung, liver cancer, genitourinary system cancers such as prostate cancer, cervical cancer, bladder cancer, uterine cancer and endometrial cancer and renal cell carcinoma, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancers of the endocrine system, thyroid cancer, parathyroid cancer, head and neck cancer, breast cancer, gastrointestinal cancer and cancers of the nervous system, benign lesions such as papilloma, and the like.

[0059] CDR: As used herein, "CDR" may refer to a complementarity determining region within the variable region of an antibody agent. There are three CDRs in each variable region of the heavy and light chains, designated CDR1, CDR2, and CDR3 for each variable region. A "set of CDRs" or "CDR set" refers to a set of three or six CDRs that appear in a single variable region that is capable of binding an antigen or CDRs of homologous heavy and light chain variable regions that are capable of binding an antigen. Certain systems for defining CDR boundaries have been established in the art (e.g., Rabat, Chothia, etc.); those skilled in the art understand the differences between these systems and can understand CDR boundaries to the extent necessary to understand and practice 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 pro-apoptotic agents, cell growth inhibitors and / or cytotoxic agents, for example, specifically including agents used and / or recommended for treating one or more diseases, disorders or conditions associated with undesirable cell proliferation. In many embodiments, chemotherapeutic agents 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 cytoskeletal disruptors (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 of the following substances (i.e., sharing relevant antiproliferative activity).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, racil), 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] In some embodiments, chemotherapeutic agents may be used in the context of antibody-drug conjugates. 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, 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 manipulated artificially. For example, when a polypeptide sequence is manipulated artificially, the polypeptide is considered to be "engineered". For example, in some embodiments of the present invention, an engineered polypeptide includes a sequence that includes one or more amino acid mutations, deletions and / or insertions that have been artificially introduced into a reference polypeptide sequence. In some embodiments, an engineered polypeptide includes a polypeptide that has been artificially fused (i.e., covalently linked) to one or more other polypeptides to form a fusion polypeptide that does not occur naturally in vivo. Similarly, if a cell or organism has been manipulated so that its genetic information changes (e.g., new genetic material that did not exist previously has been introduced, such as by transformation, matching, somatic cell hybridization, transfection, transduction or other mechanisms, or, for example, by substitution or deletion mutations or by matching schemes to change or remove previously existing genetic material), the cell or organism is considered to be "engineered". As commonly practiced and understood by those skilled in the art, even if the actual manipulation is performed on an existing entity, the derivatives and / or offspring of an engineered polypeptide or cell are generally still referred to as "engineered".

[0063] Host cell: As used herein, the term "host cell" may refer to a cell of an organism that is selected, modified, transformed, grown, used or manipulated in any manner to produce material by the cell, such as expression of a gene, DNA or RNA sequence, protein or enzyme in the cell. Host cells may 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 (eg, in a test tube or reaction vessel, in cell culture, etc.) rather than in a multicellular organism.

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

[0066] Isolated: As used herein, the term "isolated" may refer to a substance and / or entity that has been (1) separated from at least some of the components with which it was originally associated when it was produced (whether in nature and / or in an experimental setting), and / or (2) designed, produced, prepared and / or manufactured by the hand of man. An isolated substance and / or entity may be separated from the other components with which it was originally 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 those skilled in the art, a substance may still be considered "isolated" or even "pure" after being combined with certain other components, such as one or more carriers or excipients (e.g., buffers, solvents, water, etc.); in such embodiments, the separation or purity percentage of the substance is calculated without the inclusion of such carriers or excipients. As just one example, in some embodiments, a biopolymer such as a polypeptide or polynucleotide that occurs in nature is considered "isolated" when: a) by virtue of its origin or source of derivation, it is not associated with some or all of the components that accompany it in its natural state; b) the biopolymer is substantially free of other polypeptides or nucleic acids from the same species in which it is produced 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 in which the biopolymer is produced in nature. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cellular system other than that in which the polypeptide is produced 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 may be considered an "isolated" polypeptide to the extent that the polypeptide has been separated from: a) other components with which it is associated in nature; and / or b) other components with which it was originally associated when produced.

[0067] Operably linked: As used herein, the term "operably linked" may refer to a juxtaposition in which the components described are in a relationship that allows the components to function in their intended manner. A control element that is "operably linked" to a functional element is associated in a manner that achieves expression and / or activity of the functional element under conditions compatible with the control element. In some embodiments, an "operably linked" control element is contiguous (e.g., covalently linked) with the coding element of interest. In some embodiments, the control element functions in trans or otherwise with 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 a unit dose suitable for administration in a treatment regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.

[0069] Polypeptide: As used herein, the term "polypeptide" generally refers to its art-recognized polymer of at least three amino acids. It will be understood by those of ordinary skill in the art that the term "polypeptide" is intended to be general enough to cover polypeptides having the complete sequences listed herein, while covering polypeptides representing functional fragments of such complete polypeptides (e.g., fragments retaining at least one activity). In addition, it will be understood by those of ordinary skill in the art that protein sequences generally tolerate some substitutions without destroying activity. Therefore, the activity is retained and shares at least about 30%-40% overall sequence identity with another polypeptide of the same class, usually greater than about 50%, 60%, 70% or 80%, and is usually further included in one or more highly conserved regions that usually cover at least 3-4 and often up to 20 or more amino acids. The polypeptide of at least one region is included in the relevant term "polypeptide" as used herein. The polypeptide may contain L-amino acids, D-amino acids, or both, and may 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, the protein may include natural amino acids, non-natural 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, the protein is an antibody agent, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0070] Prevent / Prevention: As used herein, the terms "prevent" or "prevention" when used in conjunction with the occurrence of a disease, disorder, and / or condition may 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 assessed on a population basis such that if a statistically significant reduction 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, then the agent is considered to "prevent" the particular disease, disorder, or condition.

[0071] Recombinant: As used herein, the term "recombinant" may refer to a polypeptide 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 manipulated to express one or more genes or gene components that encode and / or direct the expression of a polypeptide or one or more components, parts, elements or domains thereof; and / or a polypeptide prepared, expressed, produced or isolated by any other means, the other means involving splicing or connecting selected nucleic acid sequence elements to each other, chemically synthesizing selected sequence elements and / or otherwise generating nucleic acids that encode and / or direct the expression of a polypeptide or one or more components, parts, elements or domains thereof. In some embodiments, one or more of such selected sequence elements are found in nature. In some embodiments, one or more of such selected sequence elements are designed on a computer. In some embodiments, one or more such selected sequence elements are generated by mutagenesis (e.g., in vivo or in vitro) of known sequence elements, e.g., from a natural or synthetic source, 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" may refer to the ability to distinguish between possible binding partners in an environment where binding may occur. A binding agent that interacts with a particular target in the presence of other potential targets is said to "specifically bind" to the target with which it interacts. In some embodiments, specific binding is assessed by detecting or determining the degree of association between a binding agent and its partner. In some embodiments, specific binding is assessed by detecting or determining the degree of dissociation of a binding agent-partner complex; in some embodiments, specific binding is assessed by detecting or determining the ability of a binding agent to counteract alternative interactions between its partner and another entity. In some embodiments, specific binding is assessed by performing such detection or determination over a range of concentrations.

[0073] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, including prenatal human forms in some embodiments). In some embodiments, the subject suffers from a disease, disorder, or condition of interest. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, a subject refers to a person having one or more characteristics that are susceptible to or at risk for a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom 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 induces a desired pharmacological effect when administered to an organism. In some embodiments, an agent is considered a therapeutic agent if it shows a statistically significant effect across an appropriate population. In some embodiments, an appropriate population may be a population of a model organism. In some embodiments, an appropriate population may 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, alleviate, 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 before it can be sold to humans for administration. In some embodiments, a "therapeutic agent" is an agent that requires a medical prescription to be administered 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 suffering from or susceptible to a 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 specific desired pharmacological response in a large number of subjects when administered to a patient in need of such treatment. For example, in some embodiments, the term "therapeutically effective amount" refers to an amount that will block, stabilize, attenuate, or reverse a cancer support process occurring in the individual, or will enhance or increase a cancer suppression process in the individual, when administered to an individual in need in the context of the therapy of the present invention. 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 therapeutic treatment) the development of malignancies such as pancreatic cancer, or helps achieve or prolong remission of a malignancy. The therapeutically effective amount administered to an individual to treat the individual's cancer may be the same or different from the therapeutically effective amount administered to promote remission or inhibit metastasis. As with most cancer therapies, the methods of treatment described herein should not be interpreted as, limited to, or otherwise limited to "curing" cancer; rather, the methods of treatment involve the use of the described compositions for "treating" cancer, i.e., producing a desired or beneficial change in the health of an individual suffering from cancer. Such benefits are recognized by healthcare providers skilled in the field of oncology and include, but are not limited to, stabilization of patient condition, reduction in tumor size (tumor regression), improvement in vital function (e.g., improvement in function of cancerous tissue or organ), reduction or inhibition of further metastasis, reduction in opportunistic infections, increase in survivability, pain relief, improvement in motor function, improvement in cognitive function, improvement in energy perception (vitality, reduced discomfort), improvement in well-being, restoration of normal appetite, restoration of healthy weight gain, and combinations thereof. In addition, regression of a particular tumor in an individual (e.g., as a result of treatment as described herein) can also be assessed by collecting cancer cell samples from a tumor site such as pancreatic cancer (e.g., during treatment) and testing the levels of metabolic and signaling markers of the cancer cells to monitor the status of the cancer cells in order to verify regression of the cancer cells to a less malignant phenotype at the molecular level.For example, tumor regression induced by employing 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 metabolic pathways, intercellular signaling pathways, or intracellular signaling pathways that exhibit abnormal activity in individuals diagnosed with cancer (i.e., a change to a state found in normal individuals without cancer). One of ordinary skill in the art will appreciate that in some embodiments, a therapeutically effective amount may be formulated and / or administered in a single dose. In some embodiments, a therapeutically effective amount may be formulated and / or administered in multiple doses, for example, as part of a dosing regimen.

[0076] Transfection: As used herein, the term "transfection" may refer to the introduction of exogenous nucleic acids into cells using recombinant DNA technology. As used herein, the term "transformation" may refer to the introduction of exogenous genes, DNA or RNA sequences into host cells so that the host cells will express the introduced genes or sequences to produce encoded proteins or enzymes.

[0077] Transduction: As used herein, the term "transduction" may refer to the introduction of exogenous nucleic acid into cells 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" may refer to a molecule that shows significant structural identity with a reference molecule compared to a reference entity but is structurally different from the reference molecule, for example, 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 appropriately considered 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 appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. By definition, a variant is a unique molecule that shares one or more such characteristic structural elements but is different from a reference molecule in at least one aspect. To name just a few examples, a polypeptide may 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 specific structural motif and / or biological function; a nucleic acid may 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 may be different from a reference polypeptide or nucleic acid due to one or more differences in an amino acid or nucleotide sequence. In some embodiments, the variant polypeptide or nucleic acid shows an overall sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 99% with a reference polypeptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid does not share at least one characteristic sequence element with the reference polypeptide or nucleic acid. In some embodiments, the reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, the variant polypeptide or nucleic acid shares one or more biological activities of the reference polypeptide or nucleic acid.

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

[0080] A vector may be a replicon to which another DNA segment is attached to amplify the attached segment.The term "replicon" refers to any genetic element (eg, a plasmid, bacteriophage, cosmid, chromosome, or virus) capable of acting as an autonomous unit of DNA replication in vivo.

[0081] Many vectors known in the art can be used for nucleic acid engineering, responsive elements and promoters are incorporated 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, by combining suitable DNA fragments with selected vectors with complementary sticky ends, the DNA fragments corresponding to the response element and promoter can be inserted into suitable vectors. In certain embodiments, the ends of the DNA molecules can be enzymatically modified, or any site can be produced by combining the nucleotide sequence with the DNA ends via a connexon. In certain embodiments, the vector can be manipulated to contain a selection marker gene to screen for cells that have incorporated markers into the cell genome. Such markers make it possible to identify and / or screen host cells expressing proteins encoded by the markers.

[0082] One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which other DNA segments can be connected. Another type of vector is a viral vector, in which other DNA segments can be connected to the viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, and thus replicated with the host genome. In addition, some vectors can instruct the expression of the gene operatively connected thereto. Such vectors are referred to as "expression vectors" in this article. 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 necessary regulatory sequences (e.g., transcription elements and translation elements) to regulate the expression of the fusion protein in a suitable host cell. Regulatory sequences may include promoter regions, enhancer regions, transcription termination sites, ribosome binding sites, start codons, splicing signals, introns, polyadenylation signals, Shine / Dalgarno translation sequences, and Kozak consensus sequences. In view of the host cell in which the fusion protein will be produced, regulatory sequences are selected. In some embodiments, suitable bacterial promoters include, but are not limited to, bacteriophage λpL or pR, T6, T7, T7 / lacO, lac, recA, gal, trp, ara, hut, and trp-lac. In some embodiments, suitable eukaryotic promoters include, but are not limited to, PRBI, GAPDH, metallothionein, thymidine kinase, viral LTR, cytomegalovirus, SV40, or tissue-specific or tumor-specific promoters, such as α-fetoprotein, amylase, cathepsin E, M1 muscarinic receptor, or γ-glutamyl transferase.

[0084] In some embodiments, additional vectors include lipid complexes (cationic liposome-DNA complexes), polycomplexes (cationic polymer-DNA complexes), and protein-DNA complexes. In addition to nucleic acids, vectors may 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] The vector can be introduced into the desired host cell by methods known in the art, such as injection, transfection, electroporation, microinjection, transduction, cell fusion, lipofection, calcium phosphate precipitation (Graham, FL et al., Virology, 52:456 (1973), Chen and Okayama, Mol. Cell. Biol. 7:2745-2752 (1987)), liposome-mediated structured salt method (Wong, TK et al., Gene, 10:87 (1980), Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190 (1982), Nicolau et al., Methods of Enzymology, 730:194-197 (1983)), and the like. Enzymol., 149:157-176 (1987)), DEAE-dextran treatment (Gopal, Mol. Cell. Biol., 5:1188-1190 (1985)), gene bombardment (Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572 (1990)) 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).

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

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

[0088] In certain embodiments, polynucleotide can be introduced in vivo by lipofection.Liposomes are used for increasing the use of in vitro encapsulation and transfection nucleic acid.In certain embodiments, synthetic cationic lipids designed to limit the difficulties and risks encountered by liposome-mediated transfection can be used to prepare liposomes for in vivo transfection of genes (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 certain embodiments, the use of cationic lipids can promote the encapsulation of negatively charged nucleic acids, and can also promote 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 US5,459,127, which are incorporated herein by reference in their entirety. In certain embodiments, for tissues with cellular heterogeneity, such as pancreas, liver, kidney and brain, direct transfection of specific cell types is obviously particularly desirable. In certain embodiments, lipids can be chemically combined with other molecules for targeting (Mackey et al., 1988). In certain embodiments, targeting peptides such as hormones or neurotransmitters and proteins such as antibodies or non-peptide molecules can be chemically combined with liposomes.

[0089] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis and tissue culture and transformation (e.g., electroporation, liposome transfection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions or as generally achieved in the art or as described herein. The aforementioned 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 edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989), which is incorporated herein by reference for any purpose.

[0090] Engineered immune cells

[0091] As used herein, "immune cell" refers to a cell 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 a 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., a chimeric antigen receptor) or to include exogenous nucleic acids.

[0092] Immune cells (e.g., T cells) can be modified in one or more than one way. Immune cells (e.g., T cells) can express at least one non-natural molecule that is a receptor for an antigen present on the surface of one or more types of cells. In some embodiments, the immune cells include immune cells (e.g., T cells) that are not found in nature because the immune cells are 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), malignant variant receptor (MVR), HLA-DR (human leukocyte antigen-D related), or CD19. In specific embodiments, the immune cells can be T cells, for example, CD4 + T cells, CD8 +T cell, Treg cell, Th1 T cell, Th2 T cell, Th17 T cell, non-specific T cell or T cell group including any combination of the above.Immune cells (e.g., T cells) (CAR T cells) engineered with chimeric antigen receptors have great therapeutic potential in treating cancer. Using CAR, receptors can be programmed to recognize antigens, which activate immune cells to kill cells expressing the antigen when combined. Therefore, immune cells expressing CARs for antigens expressed on tumor cells can target and kill the tumor cells. For example, recent clinical trials of CD19-targeted CAR transduction T cells (CD19-CAR T cells) for hematological malignancies show the powerful effect of CAR T technology. (Kochenderfer, JN et al., (2010) Blood 116:4099-4102; Porter, DL et al., (2011) N. Engl. J. Med. 365:725-733; Grupp, SA et al., (2013) N. Engl. J. Med. 368:1509-1518; Kochenderfer, JN et al., (2015) J. Clin. Oncol. 33:540-549; Brown, CE et al., (2016) N. Engl. J. Med. 375:2561-2569). The clinical success of CAR T is at least partly attributed to the fusion structure of CAR, which is formed by artificially combining a high-affinity antigen-binding domain with multiple signaling domains (Maus, MV et al., (2014) Blood 123:2625-2635; van der Stegen, SJ et al., (2015) Nat. Rev. Drug Discov. 14:499-509).

[0093] CAR includes an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain. In some embodiments, the extracellular antigen binding domain includes a single-chain variable fragment (scFv) capable of recognizing a tumor-associated antigen, the transmembrane domain uses a transmembrane domain from molecules such as CD8 and CD28, and the intracellular signaling domain uses an intracellular signaling domain of an immunoreceptor tyrosine-based activation motif (e.g., CD3ζ) and a costimulatory signaling molecule (e.g., CD28 and CD137 (4-1BB)).

[0094] As used herein, "single-chain variable fragment, scFv" refers to a fragment of an antibody defined as a recombinant protein comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) connected by a linker, which associates the two domains together so as to form an antigen binding site.

[0095] In some embodiments, the transmembrane domain is a transmembrane domain from a protein selected from the group consisting of 4-1BB / CD137, an activated NK cell receptor, an immunoglobulin, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, 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 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecules (SLAM proteins), 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 an intracellular signaling domain from a protein selected from the group consisting of 4-1BB / CD137, an activated NK cell receptor, an immunoglobulin, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, 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, ligand specifically binding to CD83, LIGHT, LTBR, ​​Ly9 (CD229), Lyl08, lymphocyte function-associated antigen-1 (LFA-1), MHC Class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecules (SLAM proteins), 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 includes 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 hematopoietic stem cell populations, or immune (e.g., T) cells can be obtained from a subject. T cells can be obtained from peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, or a tumor. In addition, immune (e.g., T) cells can be derived from one or more immune cell lines available in the art. In some embodiments, any number of techniques known to the skilled person (e.g., FICOLL TM T cells are obtained from blood collected from a subject by separation and / or apheresis. Additional methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748. Other non-limiting examples can be found in International 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, immune cells are autologous T cells. In some embodiments, immune cells are obtained from subjects who are not patients. In some embodiments, T cells used for treatment methods are isogenic (donors and recipients are different, but identical twins). In some embodiments, T cells used for treatment methods are allogeneic (from the same species but different donors) with recipient subjects. In some embodiments, T cells are autologous stem cells (for autologous stem cell therapy or ASCT). In some embodiments, immune cells are non-autologous T cells. In some embodiments, immune cells are obtained from healthy donors. In some embodiments, immune cells are obtained from patients with cancer or tumors.

[0100] T cells can be engineered to express, for example, chimeric antigen receptors (CAR). In some embodiments, CAR-T cells can be engineered to express extracellular single-chain variable fragments (scFv). In some embodiments, CAR is engineered so that the costimulatory domain is expressed as a separate polypeptide chain. Exemplary CAR-T cell therapies and constructs are described in U.S. Patent Publications No. 2013 / 0287748, No. 2014 / 0227237, No. 2014 / 0099309, and No. 2014 / 0050708, which are incorporated herein by reference in their entirety.

[0101] CAR constructs

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

[0103] In some embodiments, in order to achieve robust immune (e.g., CAR-T) cell expansion, function, persistence and anti-tumor activity, costimulatory signals can be provided by incorporating intracellular signaling domains from immune (e.g., T cells) cell costimulatory molecules into CAR constructs. In some embodiments, the selection and positioning of costimulatory domains in CAR constructs may affect immune (e.g., CAR-T) cell function and fate, and have different effects on immune (e.g., CAR-T) cell dynamics, cytotoxicity function and potential safety curves. Non-limiting examples of costimulatory molecules include CD28, ICOS, CD27, 4-1BB / CD137, OX40 and CD40L.

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

[0105] In some embodiments, 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. As contemplated herein, the CAR construct may comprise an extracellular domain involving any desired antigen binding domain. In some embodiments, the co-stimulatory endodomain 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 endodomain comprises SEQ ID NO: 2. In some embodiments, the co-stimulatory endodomain 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: 2 or 4.

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

[0107] CGTTTCTCTGTTGTT

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

[0109]

[0110] SEQ ID NO:3—five additional amino acids (amino acid sequence)

[0111] RFSVV

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

[0113] RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0114] In some embodiments, the extracellular binding domain of CAR includes an antigen binding domain. In some embodiments, the antigen binding domain specifically binds to an antigen associated with a disease. In some embodiments, the antigen binding domain specifically binds to a tumor antigen. In some embodiments, the antigen binding domain specifically binds to any number of targets, including surface antigens, cytoplasmic antigens, or nuclear antigens. For example, the antigen binding domain can bind to 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 mutants thereof), 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 (VEGFR 1-3), 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, ligand of NKG2D receptor, epithelial glycoprotein family members (e.g., EGP-2, EGP-4), diasialogangliosides (e.g., GD2, GD3), carbonic anhydrase family members (e.g., CAIX) and carbohydrate antigen family members (e.g., Ley), including mutants of named proteins and protein families. In some embodiments, the antigen binding domain can bind to an antibody or fragment thereof that binds to a cytoplasmic antigen or a nuclear antigen, such as a La / SSB antigen, a member of the Rho family of GTPases, a member of a high mobility group protein, etc. Similarly, the antigen binding domain can bind to the α and β or γ and δ chains of a T cell receptor (TCR) or a fragment thereof. In some embodiments, the antigen binding domain can bind to peptides presented by human leukocyte antigen class (HLA) I and II protein complexes.Examples are, but are not limited to, TCRs specific for peptides derived from proteins such as the EGFR family, survivin, the SRY-like high-mobility group box (SOX) protein family, melanoma-associated antigens (e.g., the autoimmune cancer / testis antigen NY-ESO-1, melanoma antigen family A Members of MAGEA, antigens 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 a cytokine receptor (e.g., IL-13 receptor, IL-22 receptor), a NKG2D receptor (e.g., ULBP1, ULBP2), an EGFR family member, or an autoreactive TCR. In some embodiments, the antigen binding domain specifically binds to a tumor antigen. Examples are, but are not limited to, glypican-3 (GPC), malignant tumor 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 includes an antibody agent. In some embodiments, the antigen binding domain is or includes 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 group consisting of 4-1BB / CD137, an activated NK cell receptor, an immunoglobulin, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, 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 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecules (SLAM proteins), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1 and VLA-6. In some embodiments, the transmembrane domain is a transmembrane domain from CD8α. In some embodiments, the transmembrane domain includes SEQ ID NO: 5. In some embodiments, the transmembrane domain comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:5.

[0116] SEQ ID NO:5—CD8 / hinge / transmembrane domain

[0117]

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

[0119] SEQ ID NO:6-CD3ζ

[0120]

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

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

[0123]

[0124] SEQ ID NO:8—Flag tag sequence

[0125] GACTACAAGGACGACGATGACAAG

[0126] GPC3CAR

[0127] Glypican-3 (GPC3) is a cell surface protein encoded by the GPC3 gene in humans and is a carcinoembryonic antigen re-expressed by neoplastic hepatocytes at high frequency (Vidali et al., 2008, J Hepatol 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, wherein the detection rate of GPC3 expression is relatively high in the early stages of liver cancer and increases with the development of liver cancer. Furthermore, GPC3 is also expressed in tumors such as melanoma, ovarian clear cell carcinoma, yolk sac tumor, neuroblastoma and other tumors. Given its high expression in hepatocellular carcinoma, melanoma, and other tumors, GPC3 has emerged as a useful immunohistochemical diagnostic test (Anatelli et al., 2008, Am J Clin Path 130:219-223) and potential biomarker (Aburatani, 2005, 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 protein core of GPC3 includes two subunits, an N-terminal subunit and a C-terminal subunit. A glycosylphosphatidylinositol (GPI) anchor is added to the serine at position 560 located on the carboxyl (C) terminal side of GPC3. The GPI anchor plays a role in positioning GPC3 on the cell surface by covalently 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), wherein the HS chains are known to regulate multiple growth signal transduction pathways, such as Wnt signaling, FGF signaling, and BMP signaling pathways. It is known that the growth signal transduction pathways involved vary depending on the type of cancer. For example, in hepatocellular carcinoma (HCC), cells grow by stimulation of the Wnt signaling pathway.

[0129] The present disclosure provides, at least in part, a GPC3 CAR polypeptide. In some embodiments, the extracellular binding domain of the GPC3 CAR includes an antigen binding domain. In some embodiments, the antigen binding domain is or includes an antibody agent. In some embodiments, the antigen binding domain is or includes an antibody agent that specifically binds to GPC3.

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

[0131] sequence SEQ ID NO: Light chain CDR1 RSSQSLVHSNGNTYLH 9 Light chain CDR2 KVSNRFS 10 Light chain CDR3 SQNTHVPPT 11 Heavy chain CDR1 DYEM 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 comprising 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, which, when the antigen binds to the antibody agent, causes T cell activation. In some embodiments, the CAR polypeptide comprises: i) an extracellular antigen binding domain, the extracellular antigen binding domain comprising 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 results in T cell activation when the antigen binds to the 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 results in T cell activation when the 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 classic major histocompatibility complex II molecule (Shackelford, DA et al., 1982 "Immunol. Rev." 66: 133-187). HLA-DR and its ligand (peptide with a length of 9 amino acids or longer) constitute the ligand of the T cell receptor (TCR). HLA-DR molecules are upregulated in response to signal transduction. In the case of infection, peptides (such as staphylococcal enterotoxin I peptides) are bound to DR molecules and presented to T cell receptors 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 trigger 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. 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, which constitutes the exposed part of the binding groove, and the C-terminal cytoplasmic region interacts with the other chain, thereby forming a β sheet across the cell membrane under the binding groove. Most of the peptide contact positions are located in the first 80 residues of each chain.

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

[0148] As used herein, malignant tumor variant receptor (MVR) antibody agents recognize the polymorphic region of HLA-DR (described in U.S. Patent Application Publication No. US2016-0257762, which is incorporated herein by reference in its entirety). The present disclosure provides, at least in part, MVR CAR polypeptides. Figure 1 A schematic diagram of an exemplary MVR CAR construct according to the present disclosure is shown in . In some embodiments, the extracellular domain of the CAR includes an antigen binding domain. In some embodiments, the antigen binding domain is or includes an antibody agent. In some embodiments, the antigen binding domain is or includes an antibody agent that specifically binds to HLA-DR.

[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 for normal and neoplastic B cells and follicular dendritic cells. CD19 plays a key role in establishing intrinsic B cell signaling thresholds by regulating both B cell receptor-dependent and -independent signaling. In addition, CD19 plays a role together with complement receptor CD21 and four-transmembrane protein CD81 (TAPA-1) and CD225 as the main signaling component of multimolecular complexes on the surface of mature B cells, wherein 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, a CD19 CAR polypeptide. In some embodiments, the extracellular domain of the CAR includes an antigen binding domain. In some embodiments, the antigen binding domain is or includes an antibody agent. In some embodiments, the antigen binding domain is or includes an antibody agent that specifically binds to 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 Acids

[0165] As used herein, "nucleic acid" is used to include any compound and / or substance including polynucleotides. Exemplary nucleic acids or polynucleotides may 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 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 connected 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 sequence

[0171] SEQ ID NO:25-Gag / Pol

[0172]

[0173] SEQ ID NO:26-cPPT

[0174]

[0175] SEQ ID NO:27 - Woodchuck / PRE

[0176]

[0177] SEQ ID NO:28-R / region

[0178]

[0179] Lentiviral vectors are derived from lentiviruses. Lentiviral vectors are based on single-stranded RNA lentiviruses, which are subclasses of retroviruses. Lentiviral vectors combine the advantages of moderate cloning ability with stable gene expression, wherein the lentiviral vectors are capable of transducing dividing cells 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. Unlike other retroviruses, HIV-based vectors are known to incorporate their passenger genes into non-dividing cells, and therefore can be used to treat persistent disease forms.

[0180] Additional sequences may 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, adapters and linkers is well known in the art.

[0181] In some embodiments, the nucleic acid molecule is inserted into a vector capable of expressing a CAR of the present disclosure when introduced into an engineered immune cell. In some embodiments, the engineered immune cell is a T cell.

[0182] Generation of CAR-T cells

[0183] Provided herein are methods for generating immune cells comprising CARs. In some embodiments, the immune cells into which the CARs are 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, CTL), memory T cells, regulatory T cells (Treg cells), apoptotic T cells, but not limited thereto. In some embodiments, the immune cells are NK cells.

[0184] In some embodiments, viral infection of immune cells may include transfecting host cells (e.g., 293T cells, PBMC, Plat-GP cells, or PA317) with a CAR expression vector and a packaging plasmid to prepare a recombinant virus and infect immune cells with the recombinant virus. The viral infection method may be performed by any method known in the art. In some embodiments, the transfer of CAR expression vectors to immune cells may be confirmed by checking the expression of CAR or checking the expression of marker genes inserted in the vector by flow cytometry, Northern blotting, Southern blotting, PCR (e.g., RT-PCR), ELISA, or Western blotting.

[0185] In some embodiments, the present disclosure provides a method for producing an engineered immune cell, the method comprising: introducing the following into an immune cell: (i) a nucleic acid encoding a CAR, wherein the CAR comprises: (a) an extracellular domain comprising an antigen binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising a 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.

[0186] In some embodiments, in order to increase the immune efficacy in the cytoplasmic signaling domain 4-1BB, 5 amino acids are added to the 4-1BB cytoplasmic domain used to produce CAR as a costimulatory signaling factor. In some embodiments, the complete construct includes: 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 includes a stimulatory domain and a costimulatory signaling domain. In some embodiments, the transmembrane domain may include 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 includes CD8. In some embodiments, the intracellular signaling domain comprises a co-stimulatory signaling domain in a CD3 zeta primary signaling domain, the co-stimulatory signaling domain being selected from CD28, OX40, 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 connected to CD3 zeta.

[0187] In some embodiments, a method of producing an engineered immune cell of the present disclosure further comprises culturing the engineered immune cell 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.

[0188] In some embodiments, the method of producing engineered immune cells further comprises culturing the engineered immune cells after the introducing step. In some embodiments, the method of producing engineered immune cells further comprises obtaining the immune cells from a subject before the introducing step.

[0189] 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 a variety of nucleic acid vectors known in the art for expression, such as linear polynucleotides, polynucleotides binding ions or amphipathic compounds, plasmids or viral vectors, even if the present disclosure is not limited thereto. In some embodiments, the vector for expressing CAR in immune cells may be or include an autonomously replicating plasmid or virus or a derivative thereof. The viral vector may include but is not limited to an adenoviral vector, an adeno-associated viral vector, a retroviral vector, and the like. In some embodiments, a lentiviral vector that is a retroviral vector may be used. In some embodiments, the vector is a non-plasmid and non-viral compound, such as a liposome.

[0190] The present disclosure encompasses the recognition that CAR-T cells produced by the methods described herein can be therapeutically useful (e.g., for treating cancer).

[0191] Therapeutic applications

[0192] Provided herein is a method for treating a subject with cancer or other malignancies, wherein the method comprises administering to the subject a composition comprising or delivering an immune cell comprising a CAR. In some embodiments, the cancer is an anti-Glypican-3 related cancer. In some embodiments, the cancer is an anti-CD19 related cancer. In some embodiments, the cancer is an anti-MVR related cancer.

[0193] Cancer may refer to a broad class 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 may also metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer or cancerous tissue may comprise a tumor.

[0194] "Anti-glypican-3-associated cancers" are cancers 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 usually expressed in healthy tissues. In addition, GPC3 overexpression is found in several 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.

[0195] "Anti-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 lymphocytic leukemias (ALL), chronic lymphocytic leukemias (CLL), and B cell lymphomas.

[0196] "Anti-MVR associated cancers" are characterized in that cancer cells have increased expression of HLA-DR antigens relative to non-cancerous cells from the subject. In some embodiments, cancers with higher expression of HLA-DR antigens may include, but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, blood cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer, and prostate cancer. In some embodiments, diseases associated with HLA-DR expression include, but are not limited to, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative diseases expressing HLA-DR, or any combination thereof.

[0197] In some embodiments, cancers treated by the methods of the present disclosure may include, but are not limited to, carcinomas, lymphomas (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastomas, sarcomas, and leukemias. In some embodiments, cancers may include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, leukemia and other lymphoproliferative disorders and various types of head and neck cancer.

[0198] 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 a 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 lymphoid leukemia ("BALL"), T-cell acute lymphoid leukemia ("TALL"), acute lymphoid leukemia (ALL); one or more chronic leukemias, including but not limited to chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL); additional blood cancers or blood conditions, including but not limited to B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasms, Burkitt's lymphoma (Burkitt's The term "leukemia" refers to a variety of hematologic conditions that are united by ineffective production (or dysplasia) of myeloid blood cells.

[0199] In some embodiments, the cancer treated by the methods of the present disclosure is a B-cell lymphoma (i.e., a malignant lymphoma of B-cell origin). B-cell lymphomas include Hodgkin's lymphoma and non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, mucosa-associated lymphoid tissue lymphoma (MALT), chronic lymphocytic leukemia, mantle cell lymphoma (MCL), Burkitt's lymphoma, mediastinal large B-cell lymphoma, Waldenstrom's macroglobulinemia, nodal marginal zone B-cell lymphoma (NMZL), splenic marginal zone lymphoma (SMZL), intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, and AIDS-related lymphoma, but are not particularly limited thereto, as long as the B-cell lymphoma is a lymphoma of B-cell origin.

[0200] Immune cells (e.g., CAR-T cells) can be administered to a patient in need thereof in a therapeutically effective amount. For example, a therapeutically effective amount of an immune cell (e.g., CAR-T cell) 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 10In some embodiments, the therapeutically effective amount of T cells is about 10 4 cells, about 10 5 cells, about 10 6 cells, about 10 7 cells or about 10 8 In some embodiments, the therapeutically effective amount of T cells is between about 0.4×10 8 and about 2×10 8 In some embodiments, the therapeutically effective amount of T cells is about 0.4×10 8 pcs, about 0.5×10 8 pcs, about 0.6×10 8 pcs, about 0.7×10 8 pcs, about 0.8×10 8 pcs, about 0.9×10 8 pcs, about 1.0×10 8 pcs, about 1.1×10 8 pcs, about 1.2×10 8 pcs, about 1.3×10 8 pcs, about 1.4×10 8 pcs, about 1.5×10 8 pcs, about 1.6×10 8 pcs, about 1.7×10 8 pcs, about 1.8×10 8 pcs, about 1.9×10 8 or about 2.0×10 8 T cells.

[0201] In some embodiments, the 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 In some embodiments, the therapeutically effective amount of immune cells (e.g., CAR-T cells) is between about 1×10 cells / kg body weight. 6 and about 2×10 6 T cells to a maximum dose of about 1 × 10 8 In some embodiments, the therapeutically effective amount of T cells is about 1×10 6 or about 2×10 6 T cells to a maximum dose of about 1 × 10 8 T cells.

[0202] The number of cells will depend on the desired end use of the composition, as will the type of cells contained therein. 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 T cell population 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 density of the desired T cells is generally greater than 10 6 cells / ml and usually greater than 10 7 cells / ml, usually 10 8 Clinically relevant numbers of immune cells can be assigned to cells that accumulate to or exceed 10 7 cells, 10 8 cells, 10 9 cells, 10 10 cells, 10 11 cells or 10 12 Multiple infusions of cells.

[0203] In some embodiments, the composition can be administered parenterally to the patient. In some embodiments, the composition comprising or delivering a T cell comprising a CAR can be administered parenterally to the patient in one or more administrations. In some embodiments, the composition comprising or delivering a T cell comprising a CAR can be administered parenterally to the patient once a day, once every 2 to 7 days, once a week, once every two weeks, once a month, once every three months, or once every 6 months.

[0204] 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 an engineered immune cell 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 endodomain, wherein the co-stimulatory endodomain comprises an intracellular signaling domain from 4-1BB / CD137 and five additional amino acids.

[0205] 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.

[0206] Pharmaceutical composition

[0207] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a T cell including a CAR and a pharmaceutically acceptable carrier, wherein the CAR includes: (a) an extracellular domain including an antigen binding domain; (b) a transmembrane domain; and (c) an intracellular domain including a co-stimulatory endodomain, wherein the co-stimulatory endodomain includes an intracellular signaling domain from 4-1BB / CD137 and five additional amino acids. In some embodiments, the T cell including CAR is an autologous T cell. In some embodiments, the pharmaceutical composition may include a buffer, a diluent, a solubilizer, an emulsifier, a preservative, an adjuvant, an excipient, or any combination thereof. In some embodiments, if desired, the composition may also contain one or more additional therapeutically active substances.

[0208] In some embodiments, the T cells of the present disclosure are formulated by first collecting the T cells from the culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration in a therapeutically effective amount (a "pharmaceutically acceptable" carrier). A suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott), or plasma-lysate A (Baxter), but 5% dextrose in water or Ringer's lactate can also be used. The infusion medium can be supplemented with human serum albumin.

[0209] In some embodiments, the composition is formulated for parenteral administration. For example, the pharmaceutical composition 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 a powder form (e.g., lyophilized and / or sterilized), optionally under vacuum, and it can be reconstituted with an aqueous diluent (e.g., water, buffer, saline solution, etc.) before 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 an aqueous diluent (e.g., without oscillation).

[0210] In some embodiments, the T cells of the present disclosure including CAR and / or nucleic acid encoding CAR are formulated with pharmaceutically acceptable parenteral vehicles. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 1%-10% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used. The vehicle or lyophilized powder may 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 may additionally include a pharmaceutically acceptable excipient, as used herein, and the excipient includes any and all solvents, dispersion media, diluents or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid adhesives, lubricants, etc., suitable for the desired specific dosage form. Remington's The Science and Practice of Pharmacy, 21st edition, A.R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006) discloses various excipients used in formulating pharmaceutical compositions and known techniques for their preparation. Unless any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component or components of the pharmaceutical composition, it is contemplated that it will be used within the scope of the present disclosure.

[0211] In some embodiments, the compositions of the T cell groups of nucleic acids including CAR and / or CAR are stably prepared. In some embodiments, the stable formulations of T cell groups including CAR and / or CAR encoding nucleic acids of the present disclosure may include phosphate buffer containing saline or selected salts and preservative solutions and formulations containing preservatives and multi-purpose preservative formulations suitable for pharmaceutical or veterinary use. In aqueous diluents, the preservative formulations contain at least one known preservative or are optionally selected from the following groups: at least one phenol, meta-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 as known in the art can 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, 4.5, 4.6, 4.7, 4.8, 4.9, or any range or value therein. Non-limiting examples include no preservatives, 0.1-2% m-cresol (e.g., 0.2, 0.30.4, 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 the like.

[0212] In some embodiments, the pharmaceutical composition is provided in a form that can be refrigerated and / or frozen. In some embodiments, the pharmaceutical composition is provided in a form that cannot be refrigerated and / or frozen. In some embodiments, the reconstituted solution and / or liquid dosage form can be stored for a certain 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 reconstruction. In some embodiments, the storage of the composition comprising the antibody agent for more than a specified time will cause the antibody agent to degrade. Before administration, the liquid dosage form and / or the reconstituted solution may include particulate matter and / or decolorization. In some embodiments, if decolorization or turbidity and / or if particulate matter is retained after filtration, the solution should not be used. General considerations in the deployment and / or manufacture of medicaments can be found in, for example, Remington: Pharmaceutical Science and Practice, 21st edition, Lippincott Williams and Wilkins Publishing Company, 2005.

[0213] In some embodiments, a pharmaceutical composition comprising a T cell comprising a CAR of the present disclosure and / or a nucleic acid encoding a CAR may be contained in a container for storage or administration, for example, 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 may be prepared, packaged and / or sold in batches as a single unit dose and / or as multiple single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient to be administered to the subject and / or a convenient fraction of such a dosage, such as half or one-third of such a dosage.

[0214] Reagent test kit

[0215] The present disclosure further provides a kit, the kit includes one or more containers, the container is filled with at least one CAR as described herein and / or a nucleic acid encoding CAR. The kit can be used for any applicable method, including, for example, a method of treatment, a method of diagnosis, a method of cell proliferation and / or a method of separation, etc. Such containers may optionally be associated with a notice in a form specified by a government agency regulating the manufacture, use or sale of a drug or biological product, the notice reflecting (a) approval of manufacture, use or sale by the agency for human administration, (b) instructions for use or both.

[0216] In some embodiments, the kit may include one or more reagents for detecting (e.g., detecting CAR and / or nucleic acids encoding CAR). In some embodiments, the kit may include CAR in a detectable form (e.g., covalently associated with a detectable portion or entity) and / or nucleic acids encoding CAR. In some embodiments, one or more CARs and / or nucleic acids encoding CAR as provided herein may be included in a kit for treating a subject. In some embodiments, CARs and / or nucleic acids encoding CAR as provided herein may be included in a kit for preparing autologous T cells expressing CAR.

[0217] In some embodiments, the kit may 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 may provide other reagents for determining the binding affinity of antibody agents and / or CARs and / or CAR T cells to T cells identified or isolated from a subject. In some embodiments, the kit may provide other reagents for determining the functional affinity of antibody agents and / or CARs and / or CAR T cells to T cells of a subject.

[0218] Examples

[0219] The present disclosure is further described in the following examples, which do not limit the scope of the present disclosure described in the claims.

[0220] Example 1 - GPC3 Lentiviral Transfer Plasmid

[0221] A single-chain variable fragment (scFv) version of the humanized anti-GC33 antibody agent was generated by joining the VH and VL regions using standard DNA cloning techniques known in the art. Figure 2 Table 1 shows the lentiviral transfer plasmids used in this article.

[0222] Table 1.

[0223]

[0224] huGC33 VH-VL-scFv was cloned into the lentiviral vector pELPS4-MVRL2H2-euBBz, which 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, Figure 3 Restriction enzyme digestion results are shown in .

[0225] To create a CAR construct without the five additional amino acids in the 4-1BB costimulatory domain, huGC33 VH-VL-scFv was cloned into the lentiviral vector pELPS2-CD19-BBz, which is a lentiviral vector containing the costimulatory domain 4-1BB without the five additional amino acids. The lentiviral vector construct huGC33 (VH-VL) -BBz was digested with restriction enzymes, wherein Figure 4 Restriction enzyme digestion results are shown in .

[0226] Example 2-Pharmaceutical Composition of GPC3 CAR-T Cells

[0227] PBMC cryovials (5 × 10 7 10 mL of CAR-T cell culture medium and 1 mL of PBMC were placed in a water bath for 2-3 minutes to thaw and activate PBMC. 10 mL of CAR-T cell culture medium and 1 mL of PBMC were placed in a 50 mL conical tube and centrifuged at 1500 rpm for 5 minutes. The supernatant was removed and the CAR-T cells were counted after 5 mL of cells were resuspended in fresh culture medium. Fresh cell culture medium was added to adjust the cell density to 1×10 6 cells / mL. 6 10 μL of T cell activation beads were added per 10 cells and the medium was supplemented with IL-2. The cells were cultured in T75 flasks in an incubator at 5% CO2 and 37°C.

[0228] CAR-T cells were then generated by spinoculating activated T cells with lentivirus encoding CAR. Activated PBMCs from cell culture were counted and cells were seeded in 24-well plates in the presence of 500 μL of cell culture medium and lentivirus. After spinoculation transduction, transduced cells from 1 well were cultured in cell culture medium supplemented with IL-2.

[0229] The cultured CAR-T cells were counted every 2-3 days, and fresh culture 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 freezing container at -80°C.

[0230] CAR expression was analyzed on day 12 of culture, and the results showed that the control group showed no expression, while the CAR-T cell group showed 54-66% CAR expression ( Figure 6 ).

[0231] Target cells (GPC3 positive cell line) were collected and seeded in 96-well U-shaped bottom plates. Effector cells (CAR-T cells) were then added to the wells at effector cell: target cell ratios of 10:1, 3:1, 1:1, 0.3:1, and incubated at 37°C for 24 hours. After incubation, CytoTox96 reagent was added to each well, and cytotoxicity was quantified by measuring the absorbance at 490nm (Figure 7).

[0232] Example 3 - In vivo huGC33(VH-VL)-BBz CAR-T cells and huGC33(VH-VL)-euBBz CAR-T cells

[0233] 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, the tumor size was about 200 mm 3 The mice were divided into 5 groups, each with 4 mice. The control group received 5% HSA injection, while the other groups received CAR-T cell injection. The growth of the tumor was observed by measuring the tumor size twice a week using TM900 ( Figure 8 ).

[0234] After CAR-T cell administration, mice were subjected to orbital blood collection once a week, where 100 μL of each blood sample was centrifuged at 12,000 rpm for 10 minutes to confirm the proportion and cell count of CAR-T cells. 100 μL of blood was placed in a FACS tube and analyzed using Zombie NIR TM Vitality kit can be fixed to carry out live / dead cell staining.After reaching the concentration of 0.1 μL / 100 μDPBS / tube, dyeing is performed at room temperature for 10 minutes. Counting beads 25 μL, CD45 0.5 μL, CD8 0.5 μL, CD45RO 1.0 μL, CD62L 1.0 μL, PD-1 1.0 μL, Tim-3 1.0 μL, CD4 0.5 μL, CD69 0.5 μL and Flag0.0125 μL are added to 100 μL FACS buffer, and dyeing is continued for 30 minutes at room temperature. After 30 minutes, IX RBC lysis buffer is added and reacted at room temperature for 5 minutes. After centrifugation at 2,000 rpm for 4 minutes, all supernatants are discarded. 2 mL FACS buffer is added to the tube and centrifuged at 2,000 rpm for 4 minutes, and FACSCelesta is used to analyze (Fig. 9).

[0235] Six weeks after CAR-T injection, mouse spleen, liver and bone marrow were collected to evaluate the ratio of huGC33 (VH-VL) -BBzCAR-T cells to huGC33 (VH-VL) -euBBz CAR-T cells. 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 IX ACK buffer was added and reacted for 10 minutes. Then, 10 mL DPBS was added and centrifuged at 2,000 rpm for 5 minutes. FACS staining was performed as described above ( Fig.10 ).

[0236] Example 4 - Construction of CD19-euBBz CAR

[0237] In order to increase the immune efficacy of the cytoplasmic signaling domain 4-1BB, 5 amino acids are added to the 4-1BB cytoplasmic domain used in CAR-T as costimulatory signal factors to newly construct a CAR expression vector (CD19-euBBz CAR). The completed construct includes anti-CD 19 of scFv, which includes a transmembrane domain of EF1α promoter, hinge region and human CD8 and an intracellular signaling domain. Specifically, the intracellular signaling domain consists of a stimulatory domain and a costimulatory signaling domain. The intracellular signaling domain is a costimulatory signaling domain 4-1BB, in which 5 consecutive amino acids are added, and CD3ζ is connected to the amino acids. The CAR gene fragment finally produced is conjugated with an ELPS lentiviral expression vector cut with BamH I and Sal I. In addition, cloning is performed using BamH I / Nhe I restriction enzymes to replace only the scFv portion.

[0238] Example 5 - CD19-euBBz and CD19-BBz CAR-T cells

[0239] The 293T cell culture for producing recombinant lentivirus contained medium including 10% FBS (Millipore, TMS-013-BKR) and high glucose DMEM (Welgene, LM001-05) containing 1×P / S (Gibco, 15140-122). 293T cells were incubated in DMEM medium including 10% FBS for 24 hours before transduction in a 37°C 5% CO2 incubator. During transfection on the second day, the transfection reagent and lentiviral plasmid were mixed in appropriate proportions, and the incubation lasted for 48 hours. The supernatant containing the lentivirus was then collected and centrifuged at 400×g for 10 minutes. In addition, the supernatant was filtered using a 50 mL syringe with a 0.45 μm syringe filter. 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. The virus was then obtained by centrifugation at 4,000 rpm for 2 hours at 4°C, and the virus was resuspended in 0.5 mL RPMI (Weijian, LM001-01) excluding FBS to produce lentivirus.

[0240] To determine the transduction efficiency of mammalian cells, transforming units (TU / mL) were measured by analyzing the particle counts of lentivirus that actually had transduction capacity using Jurkat cells. CAR expression can be measured by FACS. On the first day, Jurkat cells were plated at 1×10 5 Cells / 100 μL were inoculated into 96-well plates. The next day, the lentivirus was serially diluted 1 / 3 in a 96-well plate, and lentiviral transduction was performed on the Jurkat cells that had been inoculated. At this point, by introducing polybrene (Millipore) into RPMI culture medium (10% FBS and 1×P / S), the transduction of the lentivirus was further increased. After centrifugation for 2 hours at 1200xg and 32°C, the cells were incubated in a 37°C 5% CO2 incubator for 3 hours, and only 100 μL RPMI was added to each well. On the 5th day, the flag of the lentivirus infected into the cell was stained with anti-Flag-DYKDDDDK (Biolegend, catalog number 637310) to analyze the percentage of cells transduced with a flow cytometer. Using this, the titer was calculated as described in Follenzi and Naldini, 2002 (Follenzi and Naldini, 2002).

[0241] FACS staining was performed to confirm the production ratio of the two CAR-T cells after 14 days of incubation. For each CAR-T cell type, 2×105 Cells, and then add 2mL FACS buffer, and use centrifuge (Thermo, ST16) to centrifuge at 2,000rpm for 5 minutes. After discarding the supernatant, add 0.5μL / tube anti-CD8 APC (SKI, Bio Legend, catalog number 344722), 0.5μL / tube anti-CD4 BV650 (RPA-T4, Bio Legend, catalog number 300536) and 0.125μL / tube anti-flag PE (L5, Bio Legend, catalog number 637310), and dye at room temperature for 30 minutes. After adding 2mL FACS buffer and centrifuging at 2,000rpm for 5 minutes, repeat this process once more. In order to dye the survival / dead cells, add 1μL / tube 7-AAD (Bio Legend, catalog number 420404) and the mixture is placed at room temperature for 5 minutes, and then use FACS (BD, FACSCelesta) to analyze.

[0242] The ratio of the generated CD19 CAR-T cells was confirmed using FACS staining, and 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 the 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 about twice as much. ( Fig.11 A)

[0243] Example 6 - Confirmation of cytotoxicity of generated CD19 CAR-T cells

[0244] To determine the cytotoxicity of the two CAR-T cell types cultured for 14 days, CAR-T(E):LCL(T) was present in a 96-well white plate (Corning, catalog number 3917) at a ratio of 30:1, 10:1, 3:1, and 1:1. First, CAR-T cells were cultured at 6×10 5 cells / 50μL, 2×10 5 cells / 50μL, 9×10 4 cells / 50 μL and 2×10 4Next, the target cell line, i.e., the CBK LCL-Luc cell line, was added with 2×10 4 cells / 50 μL and reacted for 4 hours. After 4 hours, 100 μL Bright-Glo TM (Promega, catalog number E2620) was added to each well, and after 5 minutes, the relative light unit (RLU) value was measured using a luminometer (Thermo, Fluoroskan FL).

[0245] It was found that there was no difference in cytotoxicity between CAR-T cells introduced with conventional 4-1BB and CAR-T cells introduced with euBBz containing 5 amino acids added to the 4-1BB domain.

[0246] The results showed that when the two CAR-T cells and the CBK LCL-Luc cell line were incubated together at a ratio of 30: 1, it was found that the cytotoxicity was 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. ( Fig.11 B) Example 7 - Subcutaneous animal model induction and CAR-T validation by automated caliper and IVIS imaging

[0247] For experimental animals, NSG (NOD-scid IL2rγμL1) mice (The Jackson Laboratory) were used and managed under constant conditions in an animal nursery. The temperature was 23±2°C, with a 12-hour light / dark cycle and a humidity of 50±10%; feed and drinks were provided ad libitum. In an efficacy experiment using CD19-euBBz CAR-T and adding five amino acids to the 4-1BB domain, the CBK LCL-Luc cell line was grown at 2×10 6 10 cells / 100 μL DPBS / head were prepared and injected subcutaneously into 6-week-old female mice to induce a subcutaneous animal model. When the tumor size, as measured using an automatic caliper (Youngbio, TM900), reached between 50 and 100 mm 3 When the number of CD19-euBBz CAR-T cells and CD19-BBz CAR-T cells was 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 regularly confirmed.

[0248] More specifically, using an automated caliper and IVIS imaging equipment (PerkinElmer, Luna III), cancer size and photon counts ( Fig.12 , 13 ). In the case of TM900, the size of the cancer was determined after the device was placed at the cancer site. When imaging and photon values ​​were confirmed using an IVIS imaging device, mice were first intraperitoneally administered 150 mg / kg XenoLight TM D-luciferin (PerkinElmer, catalog number 122799). After 15 minutes, inhalation anesthesia was induced using isoflurane, and 5 minutes later, the presence of cancer cells was imaged using IVIS. After imaging, normalization was performed, and then the luciferase value (photon value) was confirmed and plotted. After constructing a subcutaneous animal model using the CBK LCL-Luc cell line, IVIS imaging was used to compare the effects of CD19-BBz CAR-T and CD19-euBBz CAR-T cells. Fig.12 As shown in , the effects can be confirmed within 1 week after administration of the two types of CAR-T cells.

[0249] In 2×10 6 cells / 100 μL DPBS / head and 6×10 6 In the experimental group treated with CD19-euBBz CAR-T cells, cancer cells were observed on IVIS imaging one week after administration. In addition, in the group treated with CD19-BBzCAR-T, when 6×10 6 When the number of cells / 100 μL DPBS / head was 100 μL, cancer cells were rarely observed by imaging within 1 week of administration. However, after 1 week, cancer cells were identified in the experimental group administered with CD19-BBz CAR-T.

[0250] One week after CAR-T administration, the value of luciferase imaged in each subject after the imaging process was determined as shown in the imaging; only 6 Luciferase values ​​were confirmed in the group that received CD19 CAR-T cells. When observed 3 weeks or more thereafter, tumors continued to grow in mice that were not administered CAR-T cells, and no cancer cells were identified in the three experimental groups where cancer cells initially disappeared. However, 10 days later, mice that received 2×10 6The luciferase level in the CD19 CAR-T group with 2×10 cells / 100μL DPBS / head decreased, but the cancer cells did not disappear completely after 3 weeks. 6 When the concentration of 6×10 cells / 100 μL DPBS / head was 10 μL, the effect was similar to that of 6×10 6 The results showed that there was no difference in the in vitro cytotoxicity between CD19-euBBzCAR-T and CD19-BBz CAR-T, but confirmed that the efficacy was 5 times that of CD19-euBBz CAR-T in the animal model. Fig.12 )

[0251] Example 8-Confirmation of the proportion of CD19-euBBz CAR-T in an in vivo animal model

[0252] After CAR-T cells were administered in a subcutaneous animal model to verify the efficacy of the improved construct CAR-T cells, the presence of CAR-T was confirmed from mouse blood. More specifically, after CAR-T was administered, orbital blood collection was performed on mice at intervals of 3 and 4 days. At each blood collection, 70 μL of blood was collected, and 60 μL of blood was used to confirm CAR-T cell ratios and cell counts. 60 μL of blood was placed in 5mL FACS tubes, and live / dead cell staining was performed using Zombie Aqua BV510 (Bio Legend, catalog number 423101). After reaching a concentration of 0.1 μL / 100 μL DPBS / tubes, staining was performed at room temperature for 10 minutes. Since beads (Molecularprobes, catalog number C36950) were counted, anti-CD45 FITC (HI30, Bio Legend, catalog number 304006), anti-CD8 BV786 (SK-1, Bio Legend, catalog number 344740), anti-CD4 BV650 and anti-flag PE were added and stained for 30 minutes at room temperature. Each antibody was mixed with 0.5 μL / 100 μL FACS buffer / tube, and 25 μL counting beads were added thereto. After 30 minutes, 2 mL 1X RBC lysis buffer (Bio Legend, catalog number 422401) was added and stained at room temperature.

[0253] The reaction was carried out at room temperature for 5 minutes. After centrifugation at 2,000 rpm for 5 minutes using a centrifuge, all 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 more, and then 50 μL of FACS buffer was added and analyzed using FACS.

[0254] One week after CAR-T cell administration, in the CD19-euBBz CAR-T treatment group, at 2×10 6 cells / 100 μL DPBS / head group and 6×10 6 About 20% CD19-euBBzCAR-T cells were confirmed in the blood of both groups; however, in the group administered with CD19-BBz CAR-T, when 6×10 6 When the number of cells / 100 μL DPBS / head was 5%, only 5% CD19 CAR-T was confirmed. After 3 days, the number and proportion of CAR-T cells in mice reached the maximum and then decreased. Within one week, in the three experimental groups where 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 killed quickly because the cancer cells may have been exposed to relatively more CAR-T cells before they proliferated in the mouse body. 6 In the experimental group where CD19-BBz CAR-T was administered with 2×10 cells / 100 μL DPBS / head, the ratio and number of CAR-T cells reached the maximum at 2 weeks, and the CAR-T ratio was about 25%, where cancer cell proliferation was relatively good. After the CAR-T ratio 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 ratio of CAR-T cells increased and decreased at a slightly later time, and therefore it took longer for the tumor to disappear in the mouse body. Therefore, as in the results of this experiment, at 2×10 6 The group administered with CD19-euBBz CAR-T cells / 100 μL DPBS / head showed the same results as the group administered with 6×10 6 The groups administered with CD19-BBz CAR-T cells / 100 μL DPBS / head showed similar CAR-T levels and effects, indicating that CD19-euBBz CAR-T has a superior effect ( Fig.14 ).

Claims

1. An immune cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises: (a) an extracellular domain, the extracellular domain comprising an antigen binding domain capable of specifically binding to glypican-3 (GPC3); (b) a transmembrane domain; and (c) an intracellular domain comprising a costimulatory endodomain, wherein the costimulatory endodomain comprises the intracellular signaling domain from 4-1BB / CD137 and five additional amino acids; wherein the co-stimulatory endodomain comprises SEQ ID NO:4, The antigen binding domain can specifically bind to Glypican-3 (GPC3), and comprises a light chain variable domain represented by SEQ ID NO:15 and a heavy chain variable domain represented by SEQ ID NO:

16.

2. The immune cell of claim 1, wherein the chimeric antigen receptor is a single polypeptide.

3. The immune cell of claim 1, wherein the chimeric antigen receptor comprises two polypeptides.

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

5. The immune cell of claim 1, wherein the antigen binding domain is human. The immune cell of claim 1 , wherein the antigen binding domain is a scFv.

7. The immune cell according to claim 1, wherein the antigen binding domain can specifically bind to a tumor antigen.

8. The immune cell of claim 1, wherein the transmembrane domain is a transmembrane domain selected from a protein selected from the group consisting of 4-1BB / CD137, activated NK cell receptor, immunoglobulin, CDS, cytokine receptor, signaling lymphocyte activation molecule (SLAM protein), TNF receptor protein, 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), CEACAM1, CRT AM, DAP-10, DNAM1 (CD226), GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, inducible T cell co-stimulator (ICOS), integrin, KIRDS2, LAT, LFA-1, ligand that specifically binds to CD83, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), MHC class 1 molecules, OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), SLP-76, Toll ligand receptor, VLA1 and VLA-6. 9 . The immune cell according to claim 8 , wherein the transmembrane domain is a transmembrane domain from CD8α.

10. The immune cell of claim 1, wherein the intracellular domain further comprises an intracellular domain from CD3ζ.

11. The immune cell of claim 1, wherein the chimeric antigen receptor further comprises a hinge region.

12. The immune cell of claim 11, wherein the hinge region is a CD8α hinge.

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

14. The immune cell of claim 13, wherein the additional antigen binding domain is a scFv.

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

16. The immune cell of claim 15, wherein the human immune cell is an autologous human immune cell.

17. The immune cell of claim 15, wherein the human immune cell is an allogeneic human immune cell.

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

19. The immune cell according to claim 1, wherein the immune cell is a NK cell.

20. A pharmaceutical composition comprising the immune cell according to claim 1 and a pharmaceutically acceptable carrier.

Images