KR20220030248A - Chimeric Antigen Receptor With 4-1BB Costimulatory Domain - Google Patents

Abstract

(i) an extracellular domain comprising an antigen-binding domain; (ii) a transmembrane domain; and (iii) an intracellular domain comprising a costimulatory endodomain, wherein the costimulatory endodomain is in a CAR comprising an intracellular domain comprising an intracellular signaling domain from 4-1BB/CD137 and five additional amino acids. Directed CAR-T compositions are provided. The disclosure also provides vectors, compositions, and methods of treatment using engineered immune cells comprising CARs and antigen binding molecules. The CAR compositions provided herein may be used in the treatment of certain cancers.

Description

Chimeric Antigen Receptor With 4-1BB Costimulatory Domain

CROSS-REFERENCE TO RELATED APPLICATIONS

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

Cancer remains one of the leading causes of death in the world. Recent statistics report that 13% of the world's population die from cancer. According to estimates from the International Agency for Research on Cancer (IARC), there were 14.1 million new cancer cases and 8.2 million cancer deaths worldwide in 2012. By 2030, the global burden is projected to increase to 21.7 million new cancer cases and 13 million deaths from cancer due to population growth and exposure to risk factors such as an aging population, smoking, unhealthy diet and lack of physical activity do. Moreover, the pain and medical costs of cancer treatment reduce the quality of life for both cancer patients and their families.

T cells engineered with chimeric antigen receptors (CAR-Ts) have great therapeutic potential to treat diseases such as cancer. CAR-T treatment confers strong target affinity and signaling function to T cells. However, the impressive efficacy of CAR-T therapy is often accompanied by serious side effects such as cytokine release syndrome (CRS). Therefore, there remains an unmet need to develop CAR-T treatments and strategies with reduced side effects.

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

In some embodiments, the chimeric antigen receptor is a single polypeptide. In some embodiments, the chimeric antigen receptor consists of two polypeptides.

In some embodiments, the costimulatory endodomain comprises an intracellular signaling domain from 4-1BB/CD137 and 5 additional amino acids, wherein the 5 additional amino acids are encoded by SEQ ID NO:1. In some embodiments, the costimulatory endodomain comprises SEQ ID NO:2.

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

In some embodiments, the transmembrane domain is 4-1BB/CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BALER, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18 , CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (tactile), CDlla, CDllb, CDllc, CDlld, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR , HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrins, ITGA4, ITGA6, ITGAD , ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand that specifically binds to CD83, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA) -1), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG/Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE/RANKL, VLA1 and a transmembrane domain selected from a protein selected from the group consisting of VLA-6. In some embodiments, the transmembrane domain is a transmembrane domain from CD8α. 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 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 an scFv.

In some embodiments, the immune cell is a human immune cell. In some embodiments, the human immune cell is an autologous human immune cell. In some embodiments, the human immune cell is an allogeneic human immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cells are NK cells.

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 costimulatory endodomain, wherein the costimulatory endodomain comprises an intracellular signaling domain from 4-1BB/CD137 and five additional amino acids.

In some embodiments, the chimeric antigen receptor is a single polypeptide. In some embodiments, the chimeric antigen receptor consists of two polypeptides.

In some embodiments, the costimulatory endodomain comprises an intracellular signaling domain from 4-1BB/CD137 and 5 additional amino acids, wherein the 5 additional amino acids are encoded by the nucleotide sequence of SEQ ID NO:1. In some embodiments, the costimulatory endodomain comprises SEQ ID NO:2.

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

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

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 leader sequence. In some embodiments, the chimeric antigen receptor further comprises a hinge region. In some embodiments, the hinge region is a CD8α hinge.

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

Provided herein is a method of producing an engineered immune cell, said method comprising introducing into an immune cell any one of the nucleic acids described herein or any one of the vectors described herein, thereby generating the engineered immune cell includes producing In some embodiments, the method further comprises culturing the engineered immune cell after the introducing step. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cells are NK cells.

In some embodiments, the method further comprises obtaining immune cells from the subject prior to the introducing step. In some embodiments, the method further comprises administering the engineered immune cells to the subject. In some embodiments, the subject has been diagnosed or confirmed to have cancer.

Provided herein are engineered immune cells produced by any one of the methods described herein.

Provided herein is a pharmaceutical composition comprising any one of the engineered immune cells described herein and a pharmaceutically acceptable carrier.

Provided herein is a method of treating cancer in a subject, comprising administering to the subject any one of the engineered immune cells described herein or any one of the pharmaceutical compositions described herein. In some embodiments, the cancer is an anti-glypican-3-associated cancer, an anti-CD19-associated cancer, or an anti-MVR-associated cancer. In some embodiments, the cancer is carcinoma, lymphoma (eg, Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, leukemia, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell of the lung 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 carcinoma, salivary carcinoma, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, other lymphoproliferative diseases, and various types of head and neck cancer. In some embodiments, the subject has previously received one or more additional anti-cancer therapies selected from the group consisting of ionizing radiation, a chemotherapeutic agent, a therapeutic antibody, and a checkpoint inhibitor. In some embodiments, the subject has been identified or diagnosed with cancer.

1 depicts a schematic diagram of an exemplary MVR CAR construct.
Figure 2 shows exemplary enzyme mapping results after MVRL2H2-4-1BB cloning.
3 shows the results of restriction enzyme digestion of huGC33(VH-VL)-euBBz, where the expected sizes are displayed on the DNA ladder, and the results are shown in gel electrophoresis images.
4 shows the results of restriction enzyme digestion of huGC33(VH-VL)-BBz, where the expected sizes are displayed on the DNA ladder, and the results are shown in gel electrophoresis images.
5A is a graph showing the total fold expansion of CAR-T cells over an 11 day period in vitro .
5B is a graph comparing fold expansion of CAR-T cells in vitro .
5C is a graph showing the cell viability of CAR-T cells in vitro .
6 depicts analysis of T cells transduced with huGC33(VH-VL)-euBBz and huGC33(VH-VL)-BBz for CAR expression.
7A is a graph showing an LDH-based cytotoxicity assay using target cells from the Huh-7 cell line.
7B is a graph showing an LDH-based cytotoxicity assay using target cells from PLC/PRF/5 cell lines.
8 is a set of graphs comparing the in vivo efficacy of huGC33(VH-VL)-euBBz and huGC33(VH-VL)-BBz CAR-T cells.
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.
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.
Figure 10 shows the analysis of CAR-T cells in mouse blood, bone marrow, spleen and liver 5 weeks after injection of huGC33(VH-VL)-euBBz and huGC33(VH-VL)-BBz CAR-T cells using FACS staining. do.
11A depicts CAR expression in T cells transduced with CD19-BBz and CD19-euBBz.
11B is a graph from a luciferase-based cytotoxicity assay showing killing activity in T cells transduced with CD19-BBz and CD19-euBBz.
12 shows the results of IVIS imaging of the effects of CD19-BBz CAR-T and CD19-euBBz CAR-T cells using an animal model.
13 is a graph showing the photon values of cancer cells in an animal model after injection of CD19-BBz CAR-T and CD19-euBBz CAR-T cells.
14A is a set of graphs showing the percentage of total CD19 CAR-T cells present in blood using FACS after orbital blood collection in mice at 3-4 day intervals.
14B is a set of graphs showing the percentage of CD4/CD8 CAR-T cells present in the blood using FACS after orbital blood collection in mice at 3-4 day intervals.
14C is a graph showing the total number of CD 19 CAR-T present in blood using FACS after orbital blood collection in mice at 3-4 day intervals.
15A depicts a schematic of an exemplary GPC3 CAR construct.
15B depicts a schematic of an exemplary GPC3 CAR construct.

The present disclosure describes a chimeric antigen receptor (CAR) comprising a 4-1BB costimulatory endodomain, as well as methods of making and using the same.

Justice

About: When used herein in reference to a value, the term “about” refers to a similar value in the context of the referenced value. In general, those skilled in the art familiar with the context will understand the appropriate degree of variation encompassed by "about" in that context. For example, in some embodiments, the term “about” refers to 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11% of the stated value. , 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less.

Administration: As used herein, the term “administration” refers to administration of a composition to a subject or system to effect delivery of an agent, typically of a composition or contained in a composition. One of ordinary skill in the art will be aware of the various routes that may be used for administration to a subject, eg, a human, in appropriate circumstances. For example, in some embodiments, administration can be ocular, oral, parenteral, topical, and the like. In some specific embodiments, administration is bronchial (eg, by bronchial instillation), buccal, dermal (which may be or include one or more topical, eg, dermal, intradermal, subcutaneous, transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intratracheal (eg, intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual , topical, tracheal (eg, by intratracheal instillation), vaginal, vitreous, and the like. In some embodiments, administration may comprise a single dose, multiple doses, or a fixed number of doses. In some embodiments, administration may include dosing that is intermittent (eg, multiple doses separated in time) and/or periodic (eg, individual doses separated by a common period of time) dosing. In some embodiments, administration can include continuous dosing (eg, perfusion) for at least a selected period of time.

Affinity: As is known in the art, “affinity” is a measure of the strength with which a particular ligand binds to its partner. Affinity can be measured in a variety of ways. In some embodiments, affinity is measured by a quantitative assay. In some such embodiments, the binding partner concentration can be fixed above the ligand concentration to mimic physiological conditions. Alternatively or additionally, in some embodiments, the binding partner concentration and/or ligand concentration may vary. In some such embodiments, affinity can be compared to a reference under comparable conditions (eg, concentration).

Antibody Agent: As used herein, the term “antibody agent” may refer to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex comprising immunoglobulin structural elements sufficient to confer specific binding. Exemplary antibody preparations include, but are not limited to, monoclonal antibodies, polyclonal antibodies, and fragments thereof. In some embodiments, the antibody preparation may comprise one or more sequence elements that are humanized, primatized, chimeric, etc., as is known in the art. In many embodiments, the term “antibody formulation” is used to refer to one or more of the constructs or formats known or developed in the art for exploiting antibody structural and functional characteristics in alternative presentations. For example, in some embodiments, antibody preparations used in accordance with the present invention include, but are not limited to, intact IgA, IgG, IgE, or IgM antibodies; bi- or multi-specific antibodies (eg, Zybodies®, etc.); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusion; single domain antibodies (eg, shark single domain antibodies such as IgNAR or fragments thereof); carmeloid antibodies; shielding antibodies (eg, Probodies®); small modular immunopharmaceuticals (“SMIPs™”); single chain or tandem diabodies (TandAb®); VHH; Anticalins®; Nanobodies® minibodies; BiTE®; ankyrin repeat proteins or DARPINs®; Avimers®; DART; TCR-like antibody; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimmerX®; microprotein; Fynomers®, Centyrins®; and KAFBITOR®. In some embodiments, antibody preparations may lack covalent modifications (eg, attachment of glycans) that they would have if they occur naturally. In some embodiments, the antibody agent contains covalent modifications (e.g., attachment of glycans, payloads [e.g., detectable moieties, therapeutic moieties, catalytic moieties, etc.], or other pendant groups [e.g., , poly-ethylene glycol, etc.] In many embodiments, the antibody preparation is or comprises a polypeptide whose amino acid sequence comprises one or more structural elements recognized by those skilled in the art as complementarity determining regions (CDRs). In an embodiment the antibody preparation is or comprises a polypeptide comprising at least one CDR (e.g., at least one heavy chain CDR and/or at least one light chain CDR) whose amino acid sequence is substantially identical to that found in the reference antibody. In some embodiments, the included CDR is substantially identical to the reference CDR in that the sequence is identical or contains between 1-5 amino acid substitutions compared to the reference CDR.In some embodiments, the included CDR is a reference CDR and at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100 Substantially identical to the reference CDR in that it exhibits % sequence identity.In some embodiments, the included CDR has at least 96%, 96%, 97%, 98%, 99% or 100% sequence identity with the reference CDR. It is substantially identical to the reference CDR in that it shows.In some embodiments, the included CDR is deleted, added or substituted in comparison with the reference CDR, at least one amino acid in the included CDR, but the included CDR is otherwise of the reference CDR. It is substantially identical to the reference CDR in that it has the same amino acid sequence as that of the reference CDR.In some embodiments, the included CDR is deleted, added, or substituted in comparison with the reference CDR, but 1-5 amino acids in the included CDR are included in the included CDR. is substantially identical to a reference CDR in that it otherwise has the same amino acid sequence as the reference CDR. In an embodiment, an included CDR is substantially identical to a reference CDR in that at least one amino acid in the included CDR is substituted as compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to that of the reference CDR. In some embodiments, an included CDR is linked to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared to the reference CDR, but the included CDR otherwise has the same amino acid sequence as the reference CDR. Practically the same. In some embodiments, the antibody preparation is or comprises a polypeptide whose amino acid sequence comprises a structural element recognized by one of skill in the art as an immunoglobulin variable domain. In some embodiments, the antibody preparation is a polypeptide protein having a binding domain that is homologous or generally homologous to an immunoglobulin-binding domain. In some embodiments, the antibody formulation is at least part of or comprises a chimeric antigen receptor (CAR).

Antigen: As used herein, the term “antigen” may refer to an agent that binds to an antibody agent. In some embodiments, the antigen may or may not bind to the antibody agent and induce a specific physiological response in the organism. In general, antigens include, for example, small molecules, nucleic acids, polypeptides, carbohydrates, lipids, polymers (biological polymers [eg, nucleic acid and/or amino acid polymers] and polymers other than biological polymers [eg, other than nucleic acid or amino acid polymers]. of polymers]), etc., or include them. In some embodiments, the antigen is or comprises a polypeptide. In some embodiments, the antigen is or comprises a glycan. One of ordinary skill in the art will generally know that the antigen may be provided in isolated or pure form, or alternatively in crude form (e.g., combined with other substances, e.g., cell extracts of antigen-containing sources or other relatively crude preparations) the same extract). In some specific embodiments, the antigen is present in a cellular context (eg, the antigen is expressed on the surface of the cell or is expressed in the cell). In some embodiments, the antigen is a recombinant antigen.

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 targeting moiety or entity can be of any chemical class, including, for example, carbohydrates, lipids, nucleic acids, metals, polypeptides, or small molecules. In some embodiments, the antigen binding domain may be or comprise a polypeptide (or 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).

Associated: Two events or entities are "associated" with each other as the term is used herein when the existence, level and/or form of one correlates with the other. For example, a particular entity (e.g., a polypeptide, genetic feature, metabolite, microorganism, etc.) is a disease, disorder or condition (e.g., throughout a related population) whose presence, level, and/or form It is considered to be associated with a particular disease, disorder or condition if it correlates with the incidence and/or susceptibility to it. In some embodiments, two or more entities are physically “associated with” each other when directly or indirectly interacting to remain in and/or physically proximate to each other. In some embodiments, two or more entities that are physically related to each other are covalently linked to each other. In some embodiments, two or more entities physically associated with each other are not covalently linked to each other but are non-covalently linked, for example, by means of hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof. are sharedly related.

Binding: As used herein, the term “binding” will typically be understood to refer to a non-covalent association between or among two or more entities. A “direct” association includes physical contact between entities or moieties. Indirect bonding includes physical interaction by way of physical contact with one or more intervening entities. Binding between two or more entities is typically an entity or moiety that interacts in any of a variety of circumstances - (eg, while covalently or otherwise associated with a carrier entity and/or in a biological system or cell). can be evaluated in situations, including those being studied in isolation or in the context of more complex systems.

Cancer: The terms “cancer”, “malignant”, “neoplastic”, “tumor” and “carcinoma” herein refer to relatively abnormal, uncontrolled and/or autonomous growth resulting in significant loss of control of cell proliferation. It is used to refer to cells exhibiting an abnormal growth phenotype characterized by In some embodiments, a tumor may be or comprise cells that are precancerous (eg, benign), malignant, pre-metastatic, metastatic and/or non-metastatic. The present disclosure specifically identifies certain cancers to which its teachings may be particularly relevant. In some embodiments, the relevant cancer may be characterized as a solid tumor. In some embodiments, the relevant cancer may be characterized by a hematological tumor. In general, examples of the various types of cancer known in the art include, for example, hematopoietic cancer, including leukemia, lymphoma (Hodgkin's and non-Hodgkin's), myeloma, and myeloproliferative disorders; sarcoma, melanoma, adenoma, carcinoma of solid tissue, squamous cell carcinoma of the oral cavity, throat, larynx, and lung, liver cancer, genitourinary cancer such as prostate cancer, cervical cancer, bladder cancer, uterine and endometrial and renal cell carcinoma, bone cancer , pancreatic cancer, skin cancer, skin or intraocular melanoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancer, breast cancer, gastrointestinal cancer and nervous system cancer, benign lesions such as papillomas, and the like.

CDR: As used herein, “CDR” may refer to the complementarity determining region within the variable region of an antibody preparation. There are three CDRs in each of the variable regions of the heavy and light chains, designated CDR1, CDR2 and CDR3 for each variable region. "Set of CDRs" or "set of CDRs" refers to a single variable region capable of binding antigen or a group of three or six CDRs that occur in the CDRs of cognate heavy and light chain variable regions capable of binding antigen. Certain systems for defining CDR boundaries are established in the art (eg, Kabat, Chothia, etc.); Those skilled in the art can recognize the differences between or between these systems and understand CDR boundaries to the extent necessary to understand and practice the claimed invention.

chemotherapeutic agents; As used herein, the term “chemotherapeutic agent” refers to one or more pro-apoptotic, cytostatic and/or cytotoxic agents, for example, one or more diseases, disorders or conditions associated with undesirable cell proliferation. It has an art-understood meaning specifically including agents used and/or recommended to treat In many embodiments, the chemotherapeutic agent is useful for the treatment of cancer. In some embodiments, the chemotherapeutic agent is one or more alkylating agents, one or more anthracyclines, one or more cytoskeletal disruptors (eg, microtubule targeting agents such as taxanes, maytansine and analogs thereof), one or more epothilones, or one or more histone deacetylase inhibitor HDAC), one or more topoisomerase inhibitors (eg inhibitors of topoisomerase I and/or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogues or nucleotides 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 (i.e., sharing related anti-proliferative activity) one or more of the following: can In some specific embodiments, the chemotherapeutic agent is actinomycin, all-trans retinoic acid, auristatin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chloram Bucil, cyclophosphamide, curcumin, cytarabine, daunorubicin, docetaxel, doxyfluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, maytansine and/or analogs thereof (eg DM1) mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, maytansinoids, oxaliplatin, paclitaxel, pemetrexed, teniposide, thio It may include or include one or more of guanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, and combinations thereof. In some embodiments, chemotherapeutic agents may be used in the context of antibody-drug conjugates. In some embodiments, the chemotherapeutic agent is hLL1-doxorubicin, hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLLl-SN-38 , hRS7-Pro-2-P-Dox, hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20-Pro-2-P-Dox, hPAM4-Pro-2-P -Dox, hLLl-Pro-2-P-Dox, P4/D 10-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glembatuzon 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, an antibody-drug conjugate selected from the group consisting of borsetuzumab mapodotin and robotuzumab mertansine.

Engineered: In general, the term “engineered” may refer to an aspect that has been manipulated by a human hand. For example, a polypeptide is considered "engineered" when the polypeptide sequence has been manipulated by human hands. For example, in some embodiments of the invention, the engineered polypeptide comprises a sequence comprising one or more amino acid mutations, deletions and/or insertions introduced into a reference polypeptide sequence by human hand. In some embodiments, the engineered polypeptide comprises a polypeptide that has been fused (ie, covalently linked) to one or more additional polypeptides by human hand to form a fusion polypeptide that does not naturally occur in vivo . Similarly, a cell or organism can be engineered to alter its genetic information ( e.g., new genetic material not previously present in It is considered "engineered" when genetic material introduced by, or previously present, has been altered or removed, for example, by substitution or deletion mutations or a crossover protocol). As is common practice and is understood by one of ordinary skill in the art, an engineered polypeptide or derivative and/or progeny of a cell is typically still referred to as "engineered" even though the actual manipulation has been performed on the previous entity.

Host cell: As used herein, the term “host cell” refers to a cell, e.g., a cell, DNA or RNA sequence of a gene, selected, modified, transformed, grown, It may refer to a cell of an organism that is used or manipulated in any way. Host cells can include immune cells including, but not limited to, lymphocytes (eg, T cells, B cells, and NK cells), neutrophils, and monocytes/macrophages.

In vitro: As used herein, the term “in vitro ” refers to an event that occurs not within a multi-cell organism, but within an artificial environment, eg, in a test tube or reaction vessel, in cell culture, and the like.

In vivo: As used herein, the term “in vivo ” refers to events that occur within multi-cellular organisms such as humans and non-human animals. In the context of cell-based systems, the term may be used to refer to events that occur within living cells (eg, as opposed to in vitro systems).

Isolated: As used herein, the term “isolated” means (1) separated from at least some of its associated components when it is first produced (whether in nature and/or in an experimental setting) and/or (2) a human may refer to a substrate and/or entity designed, produced, prepared, and/or fabricated by the hands of Isolated substrates and/or entities are 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 greater 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 greater than 98%, about 99%, or about 99% pure. As used herein, a substrate is " pure " when it is substantially free of other components. In some embodiments, as will be understood by one of ordinary skill in the art, the substrate is still " isolated " or after combination with certain other components, such as, for example, one or more carriers or excipients (eg, buffers, solvents, water, etc.) It can even be considered " pure "; In such embodiments, the percent isolation or purity of the substrate is calculated without including such carriers or excipients. To give one example, in some embodiments, a biological polymer, such as a naturally occurring polypeptide or polynucleotide, comprises: a) some or all of the components that it accompanies in its natural state in nature by the origin or origin of its derivation; not related; b) is substantially free of other polypeptides or nucleic acids of the same species from the species that produce it in nature; c) is considered " isolated " when it is expressed by or otherwise associated with a cell or other component of an expression system that is not the species that produces it in nature. Thus, for example, in some embodiments, a polypeptide that is synthesized chemically or synthesized in a cellular system different from that in which it occurs in nature is considered to be an “ isolated ” polypeptide. Alternatively or additionally, in some embodiments, a polypeptide that has undergone one or more purification techniques is: a) to which it is associated in nature; and/or b) a polypeptide that is “ isolated ” to the extent that it has been separated from other components with which it is first produced.

operatively connected; As used herein, the term “operably linked” may refer to a juxtaposition in a relationship that permits the described components to function in their intended manner. A control element “ operably linked ” to a functional element is associated with it in such a way that expression and/or activity of the functional element is achieved under conditions compatible with the control element. In some embodiments, an “ operably linked ” control element is contiguous (eg, covalently linked) with a coding element of interest. In some embodiments, the control element operates on or otherwise from the functional element of interest.

Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to a composition in which an active agent is formulated together 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 amount suitable for administration in a treatment regimen that, when administered to a relevant population, exhibits a statistically significant probability of achieving a predetermined therapeutic effect.

Polypeptide: As used herein, the term “polypeptide” generally refers to the art-recognized meaning of a polymer of at least three amino acids. Those of skill in the art recognize that the term "polypeptide" is intended to be sufficiently general to encompass the polypeptides having the complete sequence recited herein, as well as polypeptides that represent functional fragments of such complete polypeptides (ie, fragments that retain at least one activity). something to do. Moreover, those skilled in the art understand that protein sequences generally allow for some substitutions without destroying activity. thus retains activity and comprises at least one region of at least about 30-40%, often greater than about 50%, 60%, 70%, or 80%, and generally even higher, overall sequence identity, often more than 90% or even 95%, 96%, 97%, 98% or 99%, usually at least 3-4 and often up to 20 or more amino acids in one or more highly conserved regions, so that other members of the same class Any polypeptide shared with the polypeptide is encompassed by the related term “polypeptide,” as used herein. Polypeptides may contain L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, and the like. In some embodiments, a 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 a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, the protein is an antibody preparation, an antibody fragment, a biologically active portion thereof, and/or a characteristic portion thereof.

Prevent or prevent: When used in reference to the occurrence of a disease, disorder and/or condition, the term “prevent” or “prevention,” as used herein, refers to reducing the risk of developing a disease, disorder and/or condition. and/or delaying the onset and/or severity of one or more features or symptoms of a disease, disorder or condition. In some embodiments, prophylaxis is when an agent is administered to a particular disease, disorder, or condition when a statistically significant decrease in the development, frequency, and/or intensity of one or more symptoms of the disease, disorder, or condition is observed in a population susceptible to the disease, disorder, or condition. Evaluated on a population basis to be considered "preventing" the condition.

Recombinant: As used herein, the term “recombinant” refers to a polypeptide designed, engineered, manufactured, expressed, produced, constructed and/or isolated by recombinant means, such as a polypeptide expressed using a recombinant expression vector transfected into a host; polypeptides isolated from recombinant, combinatorial human polypeptide libraries; has been transgenic to express a gene or genes or genetic component that encodes and/or directs expression of a polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof; polypeptides isolated from otherwise engineered animals (eg, mice, rabbits, sheep, fish, etc.); and/or a polypeptide or one or more component(s), portion(s), element(s) or domains thereof that splices or ligates selected nucleic acid sequence elements to each other and chemically synthesizes and/or otherwise selects sequence elements ( ) can refer to a polypeptide prepared, expressed, produced or isolated by any other means, including generating a nucleic acid encoding and/or directing the expression of In some embodiments, one or more of these selected sequence elements are found in nature. In some embodiments, one or more of these selected sequence elements are designed in silico . In some embodiments, one or more of these selected sequence elements is a known sequence from a natural or synthetic source, e.g., such as in the germline of the source organism of interest (e.g., human, mouse, etc.) from mutagenesis (eg, in vivo or in vitro ) of the element.

Specific binding: As used herein, the term “specific binding” may refer to the ability to discriminate between possible binding partners in an environment in which binding may occur. A binding agent that interacts with one particular target when other potential targets are present is said to " specifically bind " to the target with which it interacts. In some embodiments, specific binding is assessed by detecting or determining the degree of association between the binding agent and its partner. In some embodiments, specific binding is assessed by detecting or determining the degree of dissociation of the binder-partner complex; In some embodiments, specific binding is assessed by detecting or determining the ability of the binding agent to compete for alternative interactions between its partner and another entity. In some embodiments, specific binding is assessed by performing such detection or determination over a range of concentrations.

Subject : As used herein, the term “subject” refers to an organism, typically a mammal (eg, a human, including prenatal human forms in some embodiments). In some embodiments, the subject suffers from a related disease, disorder or condition. In some embodiments, the subject is susceptible to a disease, disorder or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of the disease, disorder or condition. In some embodiments, the subject is a person with one or more characteristics characteristic of susceptibility or risk for a disease, disorder or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom a diagnosis and/or therapy has been administered and/or administered.

Therapeutic agent: As used herein, the phrase “therapeutic agent” generally refers to any agent that, when administered to an organism, induces a desired pharmaceutical effect. In some embodiments, an agent is considered a therapeutic agent if it exhibits a statistically significant effect across an appropriate population. In some embodiments, a suitable population may be a population of model organisms. In some embodiments, an appropriate population may be defined by various criteria, such as a particular age group, gender, genetic background, pre-existing clinical condition, and the like. In some embodiments, a therapeutic agent is a substrate that can be used for alleviating, ameliorating, alleviating, suppressing, preventing, delaying the onset, reducing the severity, and/or reducing the incidence of one or more symptoms or characteristics of a disease, disorder and/or condition. In some embodiments, a "therapeutic agent" is an agent that has been approved or needs to be approved by a government agency before it can be marketed for administration to humans. In some embodiments, a “therapeutic agent” is an agent that requires a medical prescription for administration to a human.

Therapeutically effective amount: As used herein, the term “therapeutically effective amount” refers to a population suffering from or susceptible to a disease, disorder and/or condition depending on a therapeutic dosing regimen to treat the disease, disorder and/or condition. It means a sufficient amount when administered to In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and/or severity of, stabilizes one or more characteristics, and/or delays the onset of one or more symptoms of a disease, disorder and/or condition. Those skilled in the art will recognize that the term “therapeutically effective amount” does not actually require successful treatment to be achieved in a particular subject. Rather, a therapeutically effective amount may be an amount that, when administered to a patient in need of such treatment, provides a particular desired pharmacological response in a significant number of subjects. For example, in some embodiments, the term “therapeutically effective amount” means in the context of a therapy of the present invention to block, stabilize, attenuate or otherwise prevent cancer-supporting processes occurring in an individual in need thereof when administered to said individual. Refers to an amount that will reverse or enhance or increase the cancer-suppressing process in the subject. 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 the further development of cancer in the individual. A particularly preferred “therapeutically effective amount” of the compositions described herein helps to reverse the pathogenesis of a malignancy, such as pancreatic carcinoma, or to achieve or prolong remission of a malignancy (in therapeutic treatment). A therapeutically effective amount administered to an individual to treat cancer in that individual may be the same or different from a therapeutically effective amount administered to promote remission or inhibit metastasis. As with most cancer therapies, the methods of treatment described herein should not be construed, limited, or otherwise limited to "cure" for cancer, rather, the method of treatment is intended to "treat" cancer, i.e., cancer. to the use of the disclosed compositions to produce a desirable or beneficial change in the health of an individual suffering from These benefits are recognized by health care providers skilled in the field of oncology and include stabilization of the patient's condition, reduction of tumor size (tumor regression), improvement of vital functions (eg, improvement of function of cancerous tissues or organs), and reduction of further metastases. or suppression, reduction of opportunistic infections, increase survival rate, reduction of pain, improvement of motor function, improvement of cognitive function, improvement of feeling of energy (reduction of vitality, malaise), improvement of mood of well-being, restoration of normal appetite, restoration of healthy weight gain, and its combinations, including but not limited to. In addition, regression of a particular tumor in an individual (eg, as a result of a treatment described herein) also results in (eg, over the course of treatment) taking a sample of cancer cells from a site of a tumor, such as a pancreatic adenocarcinoma, and It can be assessed by testing cancer cells for levels of metabolic and signaling markers to monitor the status of cancer cells to determine whether the cancer cells regress to a less malignant phenotype at the molecular level. For example, tumor regression induced using the methods of the present invention may result in a decrease in any of the anti-angiogenic markers discussed above, an increase in the anti-angiogenic markers described herein, or abnormal in an individual diagnosed with cancer. It will be marked by finding normalization of metabolic pathways, intercellular signaling pathways, or intracellular signaling pathways that exhibit activity (ie, a change to a state found in a normal individual not suffering from cancer). One of ordinary skill in the art will recognize 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.

Transfection: As used herein, the term “transfection” may refer to the introduction of a foreign nucleic acid into a cell using recombinant DNA technology. As used herein, the term “transformation” may refer to the introduction of a foreign gene, DNA or RNA sequence into a host cell such that the host cell expresses the introduced gene or sequence to produce an encoded protein or enzyme.

Transduction: As used herein, the term “transduction” may refer to the introduction of a foreign nucleic acid into a cell using a viral vector.

Variant: The term "variant" as used herein in the context of a molecule, e.g., a nucleic acid, protein or small molecule, exhibits significant structural identity to a reference molecule, but contains, for example, one or more chemical moieties compared to a reference entity. It may refer to a molecule that is structurally different from a reference molecule in the presence or absence or level of t. In some embodiments, the variant is also functionally different from its reference molecule. In general, whether a molecule is properly considered to be a "variant" of a reference molecule is based on the degree of structural identity with 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 distinct molecule that shares one or more of these characteristic structural elements but differs in at least one aspect from the reference molecule. To name a few, polypeptides may have specific structural motifs and/or characteristic sequence elements composed of a plurality of amino acids that contribute to specific structural motifs and/or biological functions and/or have designated positions with respect to each other in linear or three-dimensional space; Nucleic acids may have characteristic sequence elements consisting of a plurality of nucleotide residues having positions directed relative to one another in linear or three-dimensional space. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence. In some embodiments, the variant polypeptide or nucleic acid is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 99% overall sequence identity to a reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, the variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid.

Vector: As used herein, the term “vector” may refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors can encompass both non-viral and viral carriers for introducing nucleic acids into cells in vitro , ex vivo or in vivo .

The vector may be a replicon with another DNA fragment attached to amplify the attached fragment. The term “replicon” refers to any genetic element (eg, plasmid, phage, cosmid, chromosome, or virus) capable of acting as an autonomous unit of DNA replication in vivo . Many vectors known in the art can be used to manipulate nucleic acids, integrate response elements and promoters into genes, and the like. Preferred vectors include, but are not limited to, plasmids (eg, PBR322 or pUC plasmid derivatives), modified viruses (eg, adenoviruses, retroviruses, adeno-associated viruses or herpes viruses), or bluescript vectors. . For example, DNA fragments corresponding to response elements and promoters can be inserted into an appropriate vector by combining the appropriate DNA fragment with a selected vector having complementary aggregation ends. In some embodiments, the terminus of the DNA molecule may be enzymatically modified, or an arbitrary site may be created by joining the nucleotide sequence with the DNA terminus via a linker. In some embodiments, the vector can be engineered to contain a selectable marker gene for screening cells that have integrated the marker into the cell genome. Such markers make it possible to identify and/or screen host cells expressing the protein encoded by the marker.

One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can integrate into the genome of a host cell upon introduction into the host cell, thereby being replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of operably linked genes. Such vectors are referred to herein as "expression vectors". Non-limiting examples of expression vectors and packaging constructs that can be used to deliver the chimeric antigen receptors described herein include retroviral vectors (eg, 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, pGALV).

In some embodiments, the vector provides the necessary regulatory sequences (eg, transcriptional and translational elements) to regulate expression of the fusion protein in an appropriate host cell. Regulatory sequences may include promoter regions, enhancer regions, transcription termination sites, ribosome binding sites, initiation codons, splice signals, introns, polyadenylation signals, Shine/Dalgarno translation sequences and Kozak consensus sequences. Regulatory sequences are selected in view of the host cell in which the fusion protein will be produced. In some embodiments, suitable bacterial promoters include, but are not limited to, bacteriophage λpL or pR, T6, T7, T7/lacO, lac, recA, gal, trp, ara, hut, and trp-lac. In some embodiments, suitable eukaryotic promoters are PRBI, GAPDH, metallothionein, thymidine kinase, viral LTR, cytomegalovirus, SV40, or tissue-specific or tumor-specific promoters such as α-fetal protein, amylase , cathepsin E, M1 muscarinic receptor or γ-glutamyl transferase.

In some embodiments, additional vectors include lipoplexes (cationic liposome-DNA complexes), polyplexes (cationic polymer-DNA complexes), and protein-DNA complexes. In addition to nucleic acids, vectors may also include one or more regulatory regions and/or selectable markers useful for selecting, measuring, and monitoring the outcome of nucleic acid delivery (eg, delivery to a particular tissue, or duration of expression). there is.

Vectors can be obtained by 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)), salt method of liposome-mediated texture (Wong, TK et al., Gene, 10:87 (1980), Nicolau and Sene, Biochim. Biophys. Acta, 721: 185-190) (1982), Nicolau et al., Methods Enzymol., 149: 157-176 (1987)), DEAE-dextran treatment (Gopal, Mol. Cell Biol., 5: 1188-1190 (1985)), genetic species or Gene shock using DNA vector transporters (Yang et al, Proc. Natl. Acad. Sci., 87: 9568-9572 (1990)) (Wu et al, J. Biol. Chem. 267: 963 (1992), It can be introduced into a desired host cell by methods known in the art, such as Wu et al., J. Biol. Chem. 263: 14621 (1988), Canadian Patent Application No. 2,012,311 to Hartmut et al.).

In some embodiments, viral vectors have been used for a wide range of gene transfer applications in cells as well as live animal subjects. Viral vectors that can be used include adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus, herpes simplex virus, lentivirus, baculovirus, Sendai virus, measles virus, simian virus 40 and Epstein-Barr virus vectors. including but not limited to. Non-viral vectors include plasmids, lipoplexes (cationic liposome-DNA complexes), polyplexes (cationic polymer-DNA complexes) and protein-DNA complexes. In addition to nucleic acids, vectors may also include one or more regulatory regions and/or selectable markers useful for screening, measuring, and monitoring the outcome of nucleic acid delivery (eg, delivery to a particular tissue, or persistence of expression).

In some embodiments, polynucleotides can be introduced in vivo by lipofection. The use of liposomes for encapsulating and transfecting nucleic acids in vitro has increased. In some embodiments, liposomes for in vivo transfection of genes can be prepared using synthetic cationic lipids designed to limit the difficulties and risks encountered by liposome-mediated transfection (Feigner et al, Proc. Natl). Acad. Sci. USA 84: 7413 (1987), Mackey et al., Proc. Natl. Acad. Sci. USA 85: 8027 (1988), Ulmer et al., Science 259: 1745 (1993)). In some embodiments, the use of cationic lipids may promote encapsulation of negatively charged nucleic acids and may also promote fusion with negatively charged cell membranes (Feigner et al., Science 337: 387 (1989)). ). Lipid compounds and compositions particularly useful for the delivery of nucleic acids are described in WO95/18863, W096/17823, and U.S. Pat. 5,459,127, which are incorporated herein by reference in their entirety. In some embodiments, direct transfection of a particular cell type would obviously be particularly desirable for tissues with cellular heterogeneity, such as pancreas, liver, kidney and brain. In some embodiments, lipids are capable of chemically binding other molecules for targeting (Mackey et al, 1988). In some embodiments, targeted peptides, such as hormones or neurotransmitters, and proteins, such as antibodies, or non-peptide molecules can be chemically bound to the liposome.

Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (eg, electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally 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, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 2 ^nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), which is incorporated herein by reference for any purpose. Included.

engineered immune cells

As used herein, “immune cell” refers to cells of the immune system that can be classified into lymphocytes (eg, 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 cells are NK cells. 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 expresses a non-naturally occurring protein (eg, a chimeric antigen receptor) or has been genetically modified to include an exogenous nucleic acid.

Immune cells (eg, T cells) may be modified in one or more ways. Immune cells (eg, T cells) may express at least one non-native molecule that is a receptor for an antigen present on the surface of one or more types of cells. In some embodiments, the immune cells comprise immune cells not found in nature (eg, T cells) because they are engineered to contain or express at least one synthetic molecule not found in nature. In certain embodiments, the immune cells (eg, T cells) express certain tumor antigens, such as glypican-3 (GPC3), malignant variant receptor (MVR), HLA-DR (human leukocyte antigen-D related), or CD19. engineered to express at least one chimeric antigen receptor (CAR) comprising a CAR of interest. In certain embodiments, the immune cells are T cells, e.g., CD4 ⁺ T cells, CD8 ⁺ T cells, Treg cells, Th1 T cells, Th2 T cells, Th17 T cells, non-specific T cells, or any of the foregoing. It may be a population of T cells comprising a combination of those. Immune cells (eg, T cells) (CAR T cells) engineered with chimeric antigen receptors have great therapeutic potential for the treatment of cancer. Using CARs, receptors can be programmed to recognize antigens, which when bound activate immune cells to kill cells expressing the antigen. Thus, immune cells expressing CAR(s) for antigens expressed on tumor cells are able to target and kill the tumor cells. For example, a recent clinical trial of CD19-targeted CAR-transduced T cells (CD19-CAR T cells) against hematologic tumours showed the potent effect of CAR T technology. (Kochenderfer, JN et al. (2010) Blood 116: 4099-4102; Porter, D. L, 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 due, at least in part, to the fusion structure of the CAR created 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).

A CAR comprises an extracellular antigen-binding domain, a transmembrane domain and an intracellular signaling domain. In some embodiments, the extracellular antigen-binding domain comprises a single chain variable fragment (scFv) capable of recognizing a tumor-associated antigen, and the transmembrane domain uses transmembrane domains from molecules such as CD8 and CD28 and , intracellular signaling domains use immunoreceptor tyrosine-based activation motifs (eg, CD3ζ) and intracellular signaling domains of co-stimulatory signaling molecules (eg, CD28 and CD137(4-1BB)).

"Single chain variable fragment, scFv," as used herein, 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) joined by a linker, wherein the two domains are Associated together to form an antigen-binding site.

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

In some embodiments, the intracellular signaling domain is 4-1BB/CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3delta, CD3epsilon, CD3gamma, CD3zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7 , CD84, CD8, CD8alpha, CD8beta, CD96 (tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRTAM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR , HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2Rbeta, IL-2R gamma, IL-7R alpha, inducible T cell co-stimulator (ICOS), integrins, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, ligand that specifically binds to CD83, LIGHT, LTBR, Ly9 (CD229), Lyl08, lymphocyte function-associated antigen-1 (LFA- 1), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG/Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling Lymphocyte activating molecule (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE/RANKL, VLA1, and and an intracellular signaling domain from a protein selected from VLA-6, or any combination thereof. .

In some embodiments, the chimeric antigen receptor further comprises an additional antigen-binding domain. In some embodiments, the chimeric antigen receptor is a dual-specific CAR (ie, targets two antigen binding domains). In some embodiments, the chimeric antigen receptor is multivalent (ie, targets multiple antigen binding domains). In some embodiments, the additional antigen-binding domain is an scFv.

Immune cells (eg, T cells) can be from any source known in the art. For example, immune (eg, T) cells can be differentiated from a hematopoietic stem cell population in vitro or immune (eg, T) cells can be obtained from a subject. T cells can be obtained from peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural fluid, spleen tissue, or tumor. Additionally, immune (eg, T) cells may be derived from one or more immune cell lines available in the art. In some embodiments, T cells can be obtained from blood collected from a subject using any number of techniques known to those of skill in the art, such as FICOLL™ isolation and/or plasmapheresis. Additional methods of isolating T cells for T cell therapy are disclosed in US 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. included in this specification.

In some embodiments, the immune cell is an autologous T cell. In some embodiments, the immune cells are obtained from a subject other than the patient. In some embodiments, the T cells for use in the method of treatment are syngeneic (donor and recipient are different but identical twins). In some embodiments, the T cells for use in a method of treatment are allogeneic (same species but from a different donor) as the recipient subject. In some embodiments, the T cell is an autologous stem cell (for autologous stem cell therapy or ASCT). In some embodiments, the immune cell is a non-autologous T-cell. In some embodiments, the immune cells are obtained from a healthy donor. In some embodiments, the immune cells are obtained from a patient suffering from cancer or a tumor.

T cells can be engineered to express, for example, a chimeric antigen receptor (CAR). In some embodiments, the CAR-T cell can be engineered to express an extracellular single chain variable fragment (scFv). In some embodiments, the CAR is engineered such that the costimulatory domains are expressed as separate polypeptide chains. Exemplary CAR-T cell therapies and constructs are described in US Patent Publication Nos. 2013/0287748, 2014/0227237, 2014/0099309, and 2014/0050708, which are incorporated herein by reference in their entirety.

CAR construct

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 is capable of providing immune effector cells with specificity for a particular antigen. In some embodiments, a CAR refers to a receptor used to transfer the specificity of a monoclonal antibody preparation to a T cell. In general, a CAR comprises an extracellular binding domain (ectodomain), a transmembrane domain and an intracellular signaling domain (endodomain).

In some embodiments, to achieve potent immune (eg, CAR-T) cell expansion, function, persistence and anti-tumor activity, the costimulatory signal is from an immune (eg, T cell) cell costimulatory molecule. may be provided by incorporation of an intracellular signaling domain into the CAR construct. In some embodiments, the selection and localization of the co-stimulatory domain within the CAR construct can affect immune (eg, CAR-T) cell function and fate and can affect immune (eg, CAR-T) cell function. It may have differential effects on kinetics, cytotoxic functions, and potential safety profiles. Non-limiting examples of costimulatory molecules include CD28, ICOS, CD27, 4-1BB/CD137, 0X40, and CD40L.

As used herein, 4-1BB/CD137 is an activation-induced T cell costimulatory molecule that is expressed on a subset of resting CD8+ T cells and is upregulated on both CD4+ and CD8+ T cells after activation. In some embodiments, the T cell expressing a CAR incorporating the 4-1BB/CD137 domain is capable of expressing granzyme B, IFN-γ, TNF-α, GM-CSF and the anti-apoptotic protein Bcl-XL, and (Zhong et al., Mol. Ther. 2010; 18: 413-420), where CARs incorporating the 4-1BB/CD137 costimulatory domain may exhibit longer CAR-T cell persistence (Zhao et al., Cancer Cell 2015; 28: 415-428). In some embodiments, the intracellular domain of a chimeric receptor described herein comprises a 4-1BB signaling domain followed by a 5 amino acid sequence, which is any other desired extracellular, transmembrane and/or useful in the context of a chimeric receptor. It may be further combined with the intracellular domain(s).

In some embodiments, the CAR comprises (a) an extracellular domain comprising an antigen-binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising a costimulatory endodomain, wherein the costimulatory endodomain comprises an intracellular signaling domain from 4-1BB/CD137 and five additional amino acids. As contemplated herein, a CAR construct may comprise an extracellular domain directed against any desired antigen-binding domain. In some embodiments, the costimulatory endodomain comprises an intracellular signaling domain from 4-1BB/CD137 and 5 additional amino acids, wherein the 5 additional amino acids are encoded by SEQ ID NO:1. In some embodiments, the costimulatory endodomain comprises SEQ ID NO:2. In some embodiments, the co-stimulatory endodomain is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 in SEQ ID NO: 2 or 4. %, 99% or 100% identical sequences.

SEQ ID NO: 1 - 5 additional amino acids (DNA sequence)

CGTTTCTCTGTTTGTT

SEQ ID NO: 4-1BB costimulatory domain with 2 - 5 additional amino acids (DNA sequence)

CGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG

SEQ ID NO: 3 - 5 additional amino acids (amino acid sequence)

RFSVV

SEQ ID NO: 4-1BB costimulatory domain with 4-5 additional amino acids (amino acid sequence)

RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

In some embodiments, the extracellular binding domain of the CAR comprises an antigen-binding domain. In some embodiments, the antigen-binding domain specifically binds an antigen associated with a disease. In some embodiments, the antigen-binding domain specifically binds a tumor antigen. In some embodiments, the antigen-binding domain specifically binds any number of targets, including surface antigens, cytoplasmic or nuclear antigens. For example, the antigen binding domain may be BCMA, CD2, CD3, CD4, CD8, CD10, CD19, CD20, CD22, CD23, CD33, CD38, CD44, CD52, CD70, CD99, CD138, CD123, CD274, TIM-3, Members of the epidermal growth factor receptor family (erbl, erb2, erb3, erb4 and mutants thereof), members of the ephrin receptor family (EphA1-10, EphBl-6), prostate specific antigens (eg, prostate stem cell antigens) PSCA, prostate specific membrane antigen PSMA), embryonic antigen (eg, carcinoembryonic antigen CEA, fetal acetylcholine receptor), member of the vascular endothelial growth factor family (VEGFR 1-3), epithelial cell junction molecule EpCAM, alpha Fetoprotein AFP, member of the mucin protein family (eg MUC1, MUC16), follicle stimulating hormone receptor (FSHR), human high molecular weight-melanoma-associated antigen (HMW-MAA), folate binding protein FBP, a-folic acid receptors, ligands of the NKG2D receptor, members of the epithelial glycoprotein family (eg EGP-2, EGP-4), diasiarogangliosides (eg GD2, GD3), members of the carbonic anhydrase family (eg, EGP-2, EGP-4) CAIX), and members of the carbohydrate antigen family (eg, Ley), including named proteins and mutations of the protein family. In some embodiments, the antigen-binding domain is capable of binding an antibody or fragment thereof that binds to a cytoplasmic or nuclear antigen, such as a La/SSB antigen, a member of the Rho family of GTPases, a member of the high-mobility family of proteins, and the like. Similarly, the antigen binding domain is capable of binding to the alpha and beta or gamma and delta chains of a T cell receptor (TCR) or fragment thereof. In some embodiments, the antigen binding domain is capable of binding peptides presented by human leukocyte antigen class (HLA) I and II protein complexes. Examples are the EGFR family, survivin, the srylike high motility group box (SOX) protein family, melanoma-associated antigens (eg, autoimmune cancer/testis antigen NY-ESO-1, the melanoma antigen family). A member of MAGEA, antigens preferentially expressed in melanoma PRAME, gp100, MART-1), and leukemia-associated antigens (eg, AML1-ETO, DEK-CAN, PML-RARalpha, Flt3-ITD, NPM1) , AurA, Bcl-2, Bl-1, BMI1, BRAP, CML28, CML66, Cyclin A, Cyclin Bl, 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, sirbibim, Wilms oncogene 1 (WT1)) TCR specific for peptides derived from proteins such as, but not limited to. The antigen binding domain may bind to a cytokine receptor (eg, IL-13 receptor, IL-22 receptor), NKG2D receptor (eg, ULBP1, ULBP2), an EGFR family member, or a self-reactive TCR. In some embodiments, the antigen-binding domain specifically binds a tumor antigen. Examples are glypican-3 (GPC), malignant variant receptor (MVR), HLA-DR (human leukocyte antigen-D related), AFP, CEA, CA-125, MUC-1, ETA, tyrosinase, MAGE, immature laminin receptor , TAG-72, HPV E6, HPV E7, BING-4, calcium-activated chloride channel 2, cyclin-Bl, 9D7, Ep-CAM, EphA3, Her2/neu, telomerase, mesothelin, SAP-1, cer Vibin, NY-ESO-1/LAGE-1, PRAME, SSX-2, BRCA1/2, CDK4, CML66 or CD19. In some embodiments, the antigen-binding domain is or comprises an antibody agent. In some embodiments, the antigen-binding domain is or comprises an antibody agent that specifically binds to GPC3, MVR, HLA-DR or CD19.

In some embodiments, the transmembrane domain is 4-1BB/CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18 , CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8alpha, CD8beta, CD96 (tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR , HVEM (LIGHTR), IA4, ICAM-1,Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell co-stimulator (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand that specifically binds to CD83, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 ( LFA-1), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG/Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162) , signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE/RANKL, VLA1 and a transmembrane domain from a protein selected from VLA-6. In some embodiments, the transmembrane domain is a transmembrane domain from CD8α. In some embodiments, the transmembrane domain comprises SEQ ID NO:5. In some embodiments, the transmembrane domain is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of SEQ ID NO:5. or 100% identical sequences.

SEQ ID NO: 5 - CD8/hinge/transmembrane

ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCTAGCCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTTGCTGGGGTCCTTCTCCTGTCACTGGTTATC

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 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of SEQ ID NO:6. or 100% identical sequences.

SEQ ID NO: 6 - CD3ζ

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA

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

SEQ ID NO: 7 - CD8α leader sequence

GGATCCATGGCCTTACCAGTGACCGCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCG

SEQ ID NO: 8 - Flag-tag sequence

GACTACAAGGACGACGATGACAAG

GPC3 CAR

Glypican-3 (GPC3) is a cell surface protein encoded by the GPC3 gene in humans and a high frequency oncofetal antigen re-expressed in neoplastic hepatocytes (Vidali, et al, 2008, J hepatol 48:399-406) ). GPC3 is highly expressed in fetal liver and not in normal adult liver tissue, but its expression is reactivated in hepatocellular carcinoma, and the detection rate of GPC3 expression during the early stages of liver cancer is closely related to the development of hepatocarcinoma. increases with deployment. Moreover, GPC3 is also expressed in tumors such as melanoma, ovarian clear cell carcinoma, yolk sac tumor, neuroblastoma and other tumors. Given its particularly high expression in hepatocellular carcinoma, melanoma and other tumors, GPC3 is 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).

GPC3 is a member of the proteoglycan family that functions as a receptor for extracellular matrix or cell growth factors in cell adhesion in organogenesis. The protein core of GPC3 comprises two subunits, an N-terminal subunit and a C-terminal subunit. A glycosyl phosphatidylinositol (GPI) anchor is added to the serine at position 560 located on the carboxyl (C)-terminal side of GPC3. GPI anchors serve to localize GPC3 on the cell surface through covalent binding to cell membrane lipids. In addition, serine at position 495 and serine at position 509 of GPC3 are transformed into heparan sulfate chains (HS chains), wherein the HS chains have multiple growth signal traits such as Wnt signaling, FGF signaling, and BMP signal transduction pathways. It is known to regulate the entry pathway. It is known that the growth signal transduction pathways involved are different depending on the type of cancer. For example, in hepatocellular carcinoma (HCC), cells grow by stimulation of the Wnt signaling pathway.

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

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

In some embodiments, the CAR polypeptide comprises i) at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 in SEQ ID NO: 15. a light chain variable domain comprising a sequence that is % or 100% identical to SEQ ID NO: 16 and at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 an extracellular antigen-binding domain comprising a heavy chain variable domain comprising a sequence that is %, 99%, or 100% identical; ii) a transmembrane domain; and iii) an intracellular signaling domain that induces T cell activation when the antigen binds to the antibody agent. In some embodiments, the CAR polypeptide comprises i) at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 of SEQ ID NO: 17. a light chain variable domain comprising a sequence that is % or 100% identical to SEQ ID NO: 18 and at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 an extracellular antigen-binding domain comprising a heavy chain variable domain comprising a sequence that is %, 99%, or 100% identical; ii) a transmembrane domain; and iii) an intracellular signaling domain that induces T cell activation when the antigen binds to the antibody agent.

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 induces T cell activation when the antigen binds to the antibody agent.

SEQ ID NO: 15 - human GC33 light chain variable region (amino acid sequence)

DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSNGNTYLHWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGSGTKLEIK

SEQ ID NO: 16 - human GC33 heavy chain variable region (amino acid sequence)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWIGALDPKTGDTAYSQKFKGRATLTADKSTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSS

SEQ ID NO: 17 - human GC33 light chain variable region (DNA sequence)

GACGTCGTTATGACACAGAGTCCCCTCTCCTTGCCGGTGACCCTGGGTCAGCCTGCGTCCATCTCTTGCAGATCCTCCCAGTCTCTGGTACACTCCAACGGCAACACATACTTGCACTGGTACCAACAAAGACCTGGTCAGTCACCGCGACTTCTCATATATAAAGTTTCCAATAGGTTCAGTGGAGTGCCAGACAGGTTCAGTGGTTCAGGATCAGGCACTGATTTCACGCTTAAAATCAGTCGGGTTGAGGCGGAGGACGTAGGAGTTTACTATTGCAGCCAGAATACGCACGTGCCGCCTACTTTTGGCTCTGGAACCAAGTTGGAAATAAAG

SEQ ID NO: 18 - human GC33 heavy chain variable region (DNA sequence)

CAAGTGCAACTCGTACAATCAGGTGCTGAAGTCAAAAAGCCGGGAGCCTCTGTTAAAGTGTCCTGTAAAGCCAGCGGCTACACCTTTACCGATTATGAGATGCACTGGGTTCGGCAGGCTCCGGGCCAAGGTCTGGAGTGGATCGGGGCTCTTGACCCAAAGACGGGCGACACGGCTTATTCACAAAAATTCAAAGGTAGGGCTACTCTGACTGCCGATAAGTCCACCAGCACCGCGTATATGGAGCTCTCTAGCTTGCGAAGCGAGGACACGGCGGTGTACTATTGCACACGCTTCTATAGTTACACATATTGGGGTCAAGGCACGCTTGTGACCGTGTCTAGC

MYR CAR

Human leukocyte antigen-DR (HLA-DR) is a classical major histocompatibility complex II molecule (Shackelford, DA et al., 1982 Immunol. Rev. 66: 133-187). A peptide of more than 9 amino acids in length, HLA-DR and its ligands constitute ligands for the T cell receptor (TCR). HLA-DR molecules are upregulated in response to signaling. In case of infection, peptides (such as staphylococcal enterotoxin I peptides) are bound into DR molecules and presented to T-cell receptors found on T-helper cells. These cells then bind antigens on the surface of the B-cells that stimulate B-cell proliferation.

The main function of HLA-DR is to present to the immune system peptide antigens of potentially foreign origin for the purpose of eliciting or inhibiting T-(helper)-cell responses that ultimately lead to the production of antibodies to the same peptide antigen. HLA-DR is an αβ heterodimer, a cell surface receptor, each subunit containing two extracellular domains, a membrane-spanning domain and a cytoplasmic tail. Both α and β chains are anchored to the membrane. The N-terminal domain of the mature protein forms the alpha-helix constituting the exposed portion of the binding groove, and the C-terminal cytoplasmic region interacts with other chains to form a beta-sheet below the binding groove spanning the cell membrane. . Most of the peptide contact sites are in the first 80 residues of each chain.

HLA-DR has limited expression on antigen presenting cells such as dendritic cells, macrophages, monocytes and B cells. HLA-DR 'antigens' of increased abundance on the cell surface are often in response to stimulation, and thus HLA-DR is also a marker for immune stimulation. Due to the high expression level of HLA-DR in B-cell malignancies and the limited expression spectrum in normal cells, antibodies to HLA-DR have been developed and tested against 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 discontinued due to limited efficacy (Lin, TS, et al. (2009) Leuk. Lymphoma 50: 1958-1963).

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

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

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

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

DIQMTQSPSSLSASVGDRVTITCKASDfflNNWLAWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGKDYTLTISSLQPEDFATYYCQQYWSTPFTFGQGTKVEIKGGGGSGGGGSGSGGGGSQVQLSKVTWGDTWGVQLSKVTWGDTAAGTSGSSALVTSLETGVPSRFSGSGSGKDYTLTISSLQPEDFATYYVETVTSGVTSGTS

SEQ ID NO: 20 - MVRL2H2 (DNA sequence)

GATATTCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCAAGGCCAGTGACCACATCAACAACTGGCTGGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAACTACTGATCAGCGGCGCCACCTCTCTGGAAACCGGAGTCCCTTCTCGCTTCTCTGGTTCCGGATCTGGGAAGGATTACACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGCAGTACTGGTCCACCCCCTTCACCTTCGGACAGGGTACCAAGGTGGAGATCAAAGGCGGAGGCGGATCTGGCGGCGGAGGAAGTGGCGGAGGGGGATCTCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTTTCTCCCTGAGTCGGTACTCTGTGCATTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGCTGGGGATGATCTGGGGAGGCGGCAGCACCGACTACAACAGCGCCCTGAAGTCCCGACTGACCATATCAAAGGACAACTCCAAGAACCAGGTGTCCTTGAAGCTGAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAAATGAGGGCGATACCACCGCCGGCACTTGGTTTGCCTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

CD19 CAR

CD19 is a biomarker for normal and neoplastic B cells as well as follicular dendritic cells. CD19 is critically involved in establishing an intrinsic B cell signaling threshold by regulating both B cell receptor-dependent and independent signaling. Moreover, CD19 functions in conjunction with the complement receptor CD21, and the tetraspanin membrane protein CD81 (TAPA-1), as well as CD225, as the dominant signaling component of the multimolecular complex on the surface of mature B cells, where CD19 also acts in bodily fluids. It plays an important role in maintaining the balance between sexual antigen-induced responses and resistance induction.

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

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

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

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

DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPWGSLVAPSKDTYYQSLSVTCTVSGVSLPDYGVSWLGKGMDYQDTYYQSLSVTCTVSGVSLPDYGVSWLGKMDY

SEQ ID NO: 22 - CD19 (DNA sequence)

GACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCA

nucleic acid

As used herein, "nucleic acid" is used to include any compound and/or substrate comprising a polynucleotide. Exemplary nucleic acids or polynucleotides may include, but are not limited to, ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA).

In some embodiments, the nucleic acid construct comprises a region encoding a CAR, wherein the CAR comprises (a) an extracellular domain comprising an antigen-binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising an intracellular signaling domain from 4-1BB/CD137 and five additional amino acids. In some embodiments, the nucleic acid construct may be inserted into an expression vector or viral vector by methods known in the art, and the nucleic acid molecule may be operably linked to an expression control sequence. Non-limiting examples of expression vectors include plasmid vectors, transposon vectors, cosmid vectors, and virus-derived vectors (eg, any adenovirus-derived vector (AV), cytomegalovirus-derived (CMV) vector, simian virus-derived vector). (SV40) vectors, adeno-associated virus (AAV) vectors, lentiviral vectors and retroviral vectors). In some embodiments, the expression vector is a viral vector. In some embodiments, the viral vector is a lentiviral vector. 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 NOs: 23, 24, 25, 26, 27, and/or 28. In some embodiments, the expression vector comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% of SEQ ID NOs: 23, 24, 25, 26, 27, and/or 28 , 96%, 97%, 98%, 99% or 100% identical sequences.

SEQ ID NO: 23 - EF1a-promoter

CGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACTGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGG TTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA

SEQ ID NO: 24 - U5 repeat

AGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAG

SEQ ID NO: 25- Gag/Pol

CGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTTGGAATCACACGACCTGGATGG AGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTAT

SEQ ID NO: 26 - cPPT

TAGACTGTAGCCCAGGAATATGGCAGCTAGATTGTACACATTTAGAAGGAAAAGTTATCTTGGTAGCAGTTCATGTAGCCAGTGGATATATAGAAGCAGAAGTAATTCCAGCAGAGACAGGGCAAGAAACAGCATACTTCCTCTTAAAATTAGCAGGAAGATGGCCAGTAAAAACAGTACATACAGACAATGGCAGCAATTTCACCAGTACTACAGTTAAGGCCGCCTGTTGGTGGGCGGGGATCAAGCAGGAATTTGGCATTCCCTACAATCCCCAAAGTCAAGGAGTAATAGAATCTATGAATAAAGAATTAAAGAAAATTATAGGACAGGTAAGAGATCAGGCTGAACATCTTAAGACAGCAGTACAAATGGCAGTATTCATCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTCGGGTTTATTACAGGGACAGCAGAGATCCAGTTTGGCT

SEQ ID NO: 27 - Woodchuck/PRE

ATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG

SEQ ID NO: 28 - R/region

GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCA

Lentiviral vectors are derived from lentiviruses. Lentiviral vectors are based on single-stranded RNA lentiviruses, a subclass of retroviruses. They can transduce dividing and non-dividing cells, including neurons, by combining the advantage of mid-range cloning ability with stable gene expression. Upon 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 examples of retroviral vectors used for gene transfer. Unlike other retroviruses, HIV-based vectors are known to integrate their passenger genes into non-dividing cells, and thus can be used to treat persistent forms of the disease.

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

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

Production of CAR-T cells

Provided herein are methods of producing immune cells comprising a CAR. In some embodiments, the immune cell into which the CAR has been introduced is a human immune cell. In some embodiments, the immune cell is an autologous human immune cell. In some embodiments, the immune cell is an allogeneic human immune cell. In some embodiments, the immune cells are CD4 ⁺ T cells (helper T cells, T _H cells), CD8 ⁺ T cells (cytotoxic T cells, CTLs), memory T cells, regulatory T cells (Treg cells), apoptotic T cells. cells, but are not limited thereto. In some embodiments, the immune cells are NK cells.

In some embodiments, viral infection of immune cells comprises transfecting host cells (eg, 293T cells, PBMCs, Plat-GP cells, or PA317) with a CAR expression vector and packaging plasmid to produce a recombinant virus and immune cells may include transfection with a recombinant virus. The viral infection method can be performed by any method known in the art. In some embodiments, delivery of the CAR expression vector to immune cells examines the expression of the CAR by flow cytometry, northern blotting, southern blotting, PCR (eg, RT-PCR), ELISA, or western blotting. or by examining the expression of the marker gene inserted into the vector.

In some embodiments, the disclosure provides into an immune cell comprising (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 costimulatory endodomain, wherein the costimulatory endodomain comprises an intracellular signaling domain from 4-1BB/CD137 and five additional amino acids, (ii) encoding a CAR A vector comprising a nucleic acid comprising: (a) an extracellular domain comprising an antigen-binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising a costimulatory endodomain, wherein the costimulatory endodomain comprises an intracellular signaling domain from 4-1BB/CD137 and 5 additional amino acids. Methods of producing engineered immune cells are provided.

In some embodiments, 5 amino acids are added to the 4-1BB cytoplasmic domain used to generate the CAR as a costimulatory signaling factor to increase immunological potency in the cytoplasmic signaling domain 4-1BB. In some embodiments, the finished construct comprises an antigen-binding domain that is an scFv; EF1-α promoter; the hinge region and transmembrane domain of human CD8; and an intracellular signaling domain. In particular, the intracellular signaling domain comprises a stimulatory domain and a costimulatory signaling domain. In some embodiments, the transmembrane domain is an alpha, beta or zeta chain of a T cell receptor, or CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154. In some embodiments, the transmembrane domain comprises CD8. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain within the CD3zeta major signaling domain selected from CD28, 0X40, CD27, ICAM-1, ICOS (CD278), and 4-1BB/CD137. In some embodiments, the costimulatory domain comprises 4-1BB with 5 consecutive amino acids added. In some embodiments, the costimulatory domain is linked to CD3zeta.

In some embodiments, the method of producing an engineered immune cell of the present disclosure comprises generating 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. , further comprising culturing for 11 or 12 days.

In some embodiments, the method of producing an engineered immune cell further comprises culturing the engineered immune cell after the introducing step. In some embodiments, the method of producing the engineered immune cells further comprises obtaining the immune cells from the subject prior to the introducing step.

Any method known in the art for expressing a CAR in an immune cell can be used in the context of the present disclosure. For example, there are various nucleic acid vectors for expression known in the art, such as, for example, linear polynucleotides, polynucleotides bound with ionic or amphiphilic compounds, plasmids or viral vectors, but the present invention is not limited thereto. In some embodiments, a vector for expression of a CAR in an immune cell may be or may comprise a self-replicating plasmid or virus or derivative thereof. Viral vectors may include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like. Lentiviral vectors that are retroviral vectors in some embodiments may be used. In some embodiments, vectors are non-plasmids and non-viral compounds, such as, for example, liposomes.

The present disclosure encompasses the recognition that CAR-T cells produced by the methods described herein may be therapeutically useful (eg, for the treatment of cancer).

therapeutic application

Provided herein are methods of treating a subject having cancer or other malignancy, wherein the method comprises administering to the subject a composition comprising or delivering immune cells comprising a CAR. In some embodiments, the cancer is an anti-glypican-3-associated cancer. In some embodiments, the cancer is an anti-CD19-associated cancer. In some embodiments, the cancer is an anti-MVR-related cancer.

Cancer can refer to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth leads to the formation of malignant tumors that invade surrounding tissues and can metastasize to distant parts of the body via the lymphatic system or bloodstream. The cancer or cancerous tissue may comprise a tumor.

An “anti-glypican-3-associated cancer” is a cancer characterized by cancer cells with glypican-3 present on their surface. GPC3, a membrane-bound heparan sulfate proteoglycan, is overexpressed in approximately 70-80% of hepatocellular carcinomas but is generally not expressed in healthy tissues. In addition, GPC3 overexpression is found in several tumors, such as, but not limited to, hepatocellular carcinoma, hepatoblastoma, germ cell tumors (eg, yolk tumor, choriocarcinoma), Wilms' tumor, gastric carcinoma, arsenic lung cancer and thyroid cancer.

An “anti-CD19-associated cancer” is a cancer characterized by CD19 expression, wherein CD19 exhibits an essential role in B cell development and maturation. CD19 expression is highly conserved in most B cell tumors. It is expressed in most acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL) and B-cell lymphomas.

An “anti-MVR-associated cancer” is characterized by cancer cells having increased expression of the HLA-DR antigen compared to non-cancer cells from a subject. In some embodiments, the cancer with higher expression of the HLA-DR antigen is 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, the disease associated with HLA-DR expression includes, but is not limited to, atypical and/or non-classical cancer, malignancy, precancerous condition, or proliferative disease expressing HLA-DR, or any combination thereof. does not

In some embodiments, cancers for treatment by the methods of the present disclosure may include, but are not limited to, carcinoma, lymphoma (eg, Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. . In some embodiments, the cancer is squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, stomach cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer , hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer can do.

In some embodiments, the cancer suitable for treatment by the methods of the present disclosure is a hematologic cancer. In some embodiments, the hematologic cancer is leukemia. In some embodiments, the cancer is one or more acute, including but not limited to, B-cell acute lymphoblastic leukemia (“BALL”), T-cell acute lymphoblastic leukemia (“TALL”), acute lymphoblastic leukemia (ALL). leukemia; one or more chronic leukemias including, but not limited to, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL); B-cell prolymphocytic leukemia, blast plasmacytoid dendritic cell neoplasia, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, blast leukemia, small- or large-cell-follicular lymphoma, malignant lymphoproliferative condition, MALT lymphoma , mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic and myelodysplastic syndromes, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia and myeloid blood with "proleukemia" is selected from the group consisting of a diverse set of hematologic conditions associated by inefficient production (or dysplasia) of cells.

In some embodiments, the cancer for treatment by the methods of the present disclosure is a B cell lymphoma (ie, a malignant lymphoma derived from B cells). B-cell lymphomas include Hodgkin's lymphoma and non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, mucosal-associated lymphoid tissue lymphoma (MALT), chronic lymphocytic leukemia, mantle cell lymphoma (MCL), Burkitt. Lymphoma, mediastinal giant B-cell lymphoma, Waldenstrom's macroglobulinemia, nodular marginal zone B-cell lymphoma (NMZL), splenic marginal zone lymphoma (SMZL), endovascular large B-cell lymphoma, primary exudative lymphoma, lymphoma granulomatosis, and AIDS-related lymphoma, but is not particularly limited as long as it is a lymphoma of B cell origin.

Immune cells (eg, CAR-T cells) can be administered to a patient in need thereof in a therapeutically effective amount. For example, a therapeutically effective amount of immune cells (eg, CAR-T cells) is at least about 10 ⁴ cells, at least about 10 ⁵ cells, at least about 10 ⁶ cells, at least about 10 ⁷ cells, at least about 10 ⁸ cells. cells, at least about 10 ⁹ , or at least about 10 ¹⁰ . In some embodiments, a therapeutically effective amount of T cells is about 10 ⁴ cells, about 10 ⁵ cells, about 10 ⁶ cells, about 10 ⁷ cells, or about 10 ⁸ cells. In some embodiments, the therapeutically effective amount of T cells is from about 0.4×10 ⁸ to about 2×10 ⁸ T cells. In some embodiments, a therapeutically effective amount of T cells is about 0.4×10 ⁸ , about 0.5×10 ⁸ , about 0.6×10 ⁸ , about 0.7×10 ⁸ , about 0.8×10 ⁸ , about 0.9×10 ⁸ , about 1.0×10 ⁸ , about 1.1×10 ⁸ , about 1.2×10 ⁸ , about 1.3×10 ⁸ , about 1.4×10 ⁸ , about 1.5×10 ⁸ , about 1.6×10 ⁸ , about 1.7×10 ⁸ , about 1.8× 10 ⁸ , about 1.9×10 ⁸ , or about 2.0×10 ⁸ T cells.

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 ⁷ cells/kg, about 8×10 ⁷ cells/kg, about 9×10 ⁷ cells/kg. In some embodiments, a therapeutically effective amount of immune cells (eg, CAR-T cells) is from about 1×10 ⁶ to about 2×10 ⁶ T cells/kg body weight, up to a maximum dose of about 1×10 ⁸ T cells. am. In some embodiments, a therapeutically effective amount of T cells is about 1×10 ⁶ or about 2×10 ⁶ T cells per kg body weight, up to a maximum dose of about 1×10 ⁸ T cells.

The number of cells will depend on the ultimate use for which the composition is intended as well as 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, the population of T cells comprising a CAR contains between 10% and 50%, between 15% and 45%, between 20% and 40%, between 25% and 35%, or between 20% and 30% of such T cells. something to do. In some embodiments, the population of T cells for administration is in a volume of no more than a liter. In some embodiments, the T cells for administration are in a volume of less than 500 ml, less than 250 ml, or 100 ml or less. In some embodiments, the desired density of T cells is typically greater than 10 ⁶ cells/ml and generally greater than 10 ⁷ cells/ml, typically greater than or equal to 10 ⁸ cells/ml. The number of clinically relevant immune cells may be divided into multiple injections that are cumulatively equal to or greater than 10 ⁷ cells, 10 ⁸ cells, 10 ⁹ cells, 10 ¹⁰ cells, 10 ¹¹ cells or 10 ¹² cells.

In some embodiments, the composition may be administered parenterally to a patient. In some embodiments, a composition comprising or delivering a T cell comprising a CAR may be administered parenterally to a patient in one or multiple doses. In some embodiments, the composition comprising or delivering T cells comprising a CAR is administered once daily, once every 2 to 7 days, once weekly, once every 2 weeks, once every month, once every 3 months, or It may be administered parenterally to the patient once every 6 months.

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 is (a) an extracellular domain comprising an antigen-binding domain; (b) a transmembrane domain; and (c) a costimulatory endodomain, wherein the costimulatory endodomain comprises an intracellular domain comprising an intracellular signaling domain from 4-1BB/CD137 and five additional amino acids.

In some embodiments, the subject has previously received one or more additional anti-cancer therapies selected from the group consisting of ionizing radiation, a chemotherapeutic agent, a therapeutic antibody, and a checkpoint inhibitor. In some embodiments, the subject has been identified or diagnosed with cancer.

pharmaceutical composition

In some embodiments, the present disclosure provides a pharmaceutical composition comprising a T 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 costimulatory endodomain, wherein the costimulatory endodomain comprises an intracellular signaling domain from 4-1BB/CD137 and five additional amino acids and a pharmaceutically acceptable possible carriers. In some embodiments, the T cell comprising the CAR is an autologous T cell. In some embodiments, the pharmaceutical composition may include a buffer, diluent, solubilizer, emulsifier, preservative, adjuvant, excipient, or any combination thereof. In some embodiments, if desired, the composition may also contain one or more additional therapeutically active substances.

In some embodiments, the T cells of the present disclosure are first harvested from their culture medium, and then the cells are washed and washed in a medium and container system suitable for administration in a therapeutically-effective amount (“pharmaceutically acceptable” carrier) and It is formulated by concentration. A suitable infusion medium may be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), although 5% dextrose in water or fluid may also be used. The infusion medium may be supplemented with human serum albumin.

In some embodiments, the composition is formulated for parenteral administration. For example, the pharmaceutical compositions provided herein may be provided in sterile injectable form (eg, 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 a powder (eg, lyophilized and/or sterilized) that can be reconstituted with an aqueous diluent (eg, water, buffer, salt solution, etc.) prior to infusion, optionally under vacuum. is provided as 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, and the like. In some embodiments, the powder should be mixed gently (eg, not shaken) with the aqueous diluent.

In some embodiments, a T cell comprising a CAR and/or a nucleic acid encoding a CAR of the present disclosure is formulated with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution and 1-10% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The vehicle or lyophilized powder may contain additives that maintain isotonicity (eg, sodium chloride, mannitol) and chemical stability (eg, buffers and preservatives). In some embodiments, the formulation is sterilized by known or suitable techniques. The pharmaceutical composition may further comprise a pharmaceutically acceptable excipient, which, as used herein, comprises any and all solvents, dispersion media, diluents, or other liquid vehicles suitable for the particular dosage form desired; dispersing or suspending aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like. Remington's The Science and Practice of Pharmacy, 21st Edition, AR Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006) discloses various excipients used in formulating pharmaceutical compositions and known techniques for their preparation. . Except where any conventional excipient medium is incompatible with the substrate or derivative thereof, such as interacting in a deleterious manner with any other ingredient(s) of the pharmaceutical composition that would otherwise produce an undesirable biological effect or otherwise; Their use is considered to be within the scope of this disclosure.

In some embodiments, a composition comprising a CAR and/or a population of T cells comprising a nucleic acid encoding a CAR of the present disclosure is stably formulated. In some embodiments, a stable formulation of a CAR and/or a population of T cells comprising a nucleic acid encoding a CAR of the present disclosure comprises saline or a phosphate buffer with a selected salt, as well as a preservative suitable for pharmaceutical or veterinary use. but also preservative solutions and formulations containing multi-use preservative formulations. Preserved formulations may contain at least one known preservative or optionally at least one of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, Magnesium chloride (eg, hexahydrate), alkylparabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dihydroacetate and thimerosal, or mixtures thereof are added to the aqueous diluent contains Any suitable concentration or mixture is 0.001-5%, or any range or value therein, such as, but not limited to, 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3 , 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8 , 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range or value therein. can be used as is known in Non-limiting examples include, without preservatives, 0.1-2% m-cresol (eg 0.2, 0.3. 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol (eg 0.5, 0.9) , 1.1, 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (eg 0.005, 0.01), 0.001-2.0% phenol (eg 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1.0% alkylparaben(s) (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.

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 is administered for a specified period of time after reconstitution (eg, 2 hours, 12 hours, 24 hours, 2 days, 5 days, 7 days, 10 days, 2 weeks, 1 month). , two months or more). In some embodiments, storage of a composition comprising an antibody preparation for a period of time greater than a designated time results in degradation of the antibody preparation. Liquid dosage forms and/or reconstituted solutions may contain particulate matter and/or discoloration prior to administration. In some embodiments, the solution should not be used if it becomes discolored or cloudy and/or particulate matter remains after filtration. General considerations in the formulation and/or manufacture of pharmaceutical agents can be found, for example, in Remington: Science and Practice of Pharmacy 21 ^st ed., Lippincott Williams & Wilkins, 2005.

In some embodiments, a pharmaceutical composition comprising a CAR and/or a T cell comprising a nucleic acid encoding a CAR of the present disclosure is a container for storage or administration, e.g., a vial, a syringe (e.g., an IV syringe) ), or in a bag (eg, an IV bag). A pharmaceutical composition according to the present disclosure may be prepared, packaged, and/or sold in bulk, in a single unit dose and/or in a plurality of 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 active ingredient is generally equivalent to the dosage of active ingredient to be administered to a subject and/or to a convenient fraction of such dosage, eg, one-half or one-third of such dosage.

kit

The present disclosure further provides a kit comprising one or more containers filled with at least one CAR and/or a nucleic acid encoding a CAR as described herein. The kit can be used in any applicable method including, for example, a method of treatment, a method of diagnosis, a method of cell proliferation and/or isolation, and the like. Optionally associated with such container(s) may be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of a pharmaceutical or biological product, the notice being (a) manufacturing for human administration , approval by the agency of use or sale, (b) instructions for use, or both.

In some embodiments, a kit may include one or more reagents for detection (eg, detection of a CAR and/or a nucleic acid encoding a CAR. In some embodiments, the kit comprises a CAR and/or a nucleic acid encoding the CAR. Nucleic acid can be included in detectable form (e.g., covalently associated with a detectable moiety or entity. In some embodiments, one or more CARs and/or CARs as provided herein are encoded. nucleic acid can be included in the kit used for the treatment of the subject, in some embodiments, the CAR as provided herein and/or the nucleic acid encoding the CAR is used to prepare an autologous T cell expressing the CAR. can be included in

In some embodiments, the kit may provide one, two, three, four or more antigen-specific antibody preparations, each suitable for cloning into a CAR construct. In some embodiments, the kit may provide antibody preparations to T cells identified or isolated from a subject and/or other reagents for assaying the binding affinity of the CAR and/or CAR T cells. In some embodiments, the kit may provide antibody preparations and/or other reagents for assaying the functional affinity of a CAR and/or CAR T cells to a subject's T cells.

Example

The present disclosure is further described in the following examples which do not limit the scope of the disclosure set forth in the claims.

Example 1 - GPC3 Lentiviral Transfer Plasmid

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

[Table 1].

huGC33 VH-VL-scFv was cloned into a lentiviral vector, pELPS4-MVRL2H2-euBBz, where pELPS4-MVRL2H2-euBBz is a lentiviral vector comprising a costimulatory domain 4-1BB with 5 additional amino acids. The lentiviral vector construct, pELPS4-huGC33 VH-VL, was digested with restriction enzymes, where the results of restriction enzyme digestion are shown in FIG. 3 .

To generate a CAR construct lacking 5 additional amino acids for the 4-1BB costimulatory domain, huGC33 VH-VL-scFv was cloned into a lentiviral vector, pELPS2-CD19-BBz, where pELPS2-CD19-BBz is 5 It is a lentiviral vector comprising the costimulatory domain 4-1BB without additional amino acids. The lentiviral vector construct, huGC33(VH-VL)-BBz, was digested with restriction enzymes, where the results of restriction enzyme digestion are shown in FIG. 4 .

Example 2 - Pharmaceutical composition of GPC3 CAR-T cells

PBMCs were thawed and activated by placing PBMC freezing vials (5×10 ⁷ cells/1 mL/vial) in a water bath for 2-3 minutes. 10 mL CAR-T cell culture medium and 1 mL PBMC were placed in a 50 mL conical tube and centrifuged at 1500 rpm for 5 minutes. The supernatant was removed and the CAR-T cells were counted after resuspending the cells in 5 mL of fresh medium. The cell density was adjusted to 1× ^{10 6} cells/mL by adding fresh cell medium. 10 μL of T cell activation beads were added for all 1× ^{10 6} cells and the medium was supplemented with IL-2. Cell culture medium was cultured in a T75 flask at 5% CO ₂ and 37° C. in an incubator.

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

Cultured CAR-T cells were counted every 2-3 days, and fresh culture medium and IL-2 were added after each counting ( FIG. 5 ). CAR-T cells cultured on day 12 were harvested and stored at -80°C in a frozen container.

CAR expression was analyzed on day 12 of culture, and the results indicate that the control group showed no expression whereas the CAR-T cell group showed 54-66% CAR expression ( FIG. 6 ).

Target cells (GPC3 positive cell line) were harvested and seeded in 96-well U bottom plates. Effector cells (CAR-T cells) were then added to the wells at an effector cell:target cell ratio 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 absorbance was measured at 490 nm to quantify cytotoxicity ( FIG. 7 ).

Example 3 - in vivo huGC33(VH-VL)-BBz CAR-T cells and huGC33(VH-VL)-euBBz CAR-T cells

To validate 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 (2x10 ⁶ cells/200 μL/head) were NSG mice (6-8 weeks old, male) were injected, and 35 days after injection, mice with a tumor size of about 200 mm 3 were separated into 5 groups, each group of 4 mice. The control group received injections of 5% HSA and the other group received injections with CAR-T cells. Tumor growth was observed by measuring the tumor size twice a week using TM900 ( FIG. 8 ).

After CAR-T cell administration, orbital blood was collected from mice once a week, and 100 μL of each blood sample was centrifuged at 12,000 rpm for 10 minutes to check the percentage of CAR-T cells and cell count. 100 μL of blood was placed in a FACS tube and live/dead cell staining was performed using the Zombie NIR™ Fixable Viability Kit. After reaching a concentration of 0.1 μL/100 μDPBS/tube, staining was performed at room temperature for 10 min. Add 25 µL of counting beads, 0.5 µL of CD45, 0.5 µL of CD8, 1.0 µL of CD45RO, 1.0 µL of CD62L, 1.0 µL of PD-1, 1.0 µL of Tim-3, 0.5 µL of CD4, 0.5 µL of CD69, and 0.0125 µL of flag to 100 µL of FACS buffer. and stained for 30 minutes at room temperature. After 30 minutes, 1X RBC elution buffer was added and reacted at room temperature for 5 minutes. After centrifugation at 2,000 rpm for 4 min, all supernatant was discarded. After adding 2 mL of FACS buffer to the tube, centrifugation was performed at 2,000 rpm for 4 minutes, and analysis was performed using FACSCelesta ( FIG. 9 ).

Six weeks after CAR-T injection, mouse spleen, liver and bone marrow were harvested to evaluate the ratio of huGC33(VH-VL)-BBz CAR-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 min and all supernatant was 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 ).

Example 4 - Construction of CD19-euBBz CAR

To increase the immunological efficacy in the cytoplasmic signaling domain 4-1BB, the CAR expression vector (CD19-euBBz CAR) was newly created by adding 5 amino acids as a co-stimulatory signaling factor to the 4-1BB cytoplasmic domain used in CAR-T. built. The finished construct contains the EF1 alpha promoter, an anti-CD19 scFv comprising a hinge region and a transmembrane domain of human CD8, and an intracellular signaling domain. In particular, the intracellular signaling domain consists of a stimulatory domain and a co-stimulatory signaling domain. The intracellular signaling domain is 4-1BB, a co-stimulatory signaling domain with 5 consecutive amino acids added, to which CD3 zeta is linked. Finally, the resulting CAR gene fragment was spliced into an ELPS lentiviral expression vector digested with BamHI and Sal I. In addition, cloning was performed using BarnH I/Nhe I restriction enzymes to replace only the scFv portion.

Example 5 - CD19-euBBz and CD19-BBz CAR-T cells

The 293T cell culture used for the production of recombinant lentivirus was prepared with 10% FBS (Millipore, TMS-013-BKR) and 1 × P/S (Gibco, 15140-122) in high glucose DMEM (Welgene, LM001-05). containing medium. 293T cells were incubated in DMEM medium containing 10% FBS for 24 hours prior to transduction in a 37° C. 5% CO2 incubator. The next day, for transfection, the transfection reagent and the lentiviral plasmid were mixed in an appropriate ratio and incubated for 48 hours. The supernatant containing the lentivirus was collected and centrifuged at 400 x g for 10 min. In addition, the supernatant was filtered through a 0.45 μm syringe filter using a 50 mL syringe. The obtained supernatant was mixed with a lentivirus enrichment kit (Clontech, 631231) at a ratio of 3:1, and reacted at 4°C for 24 to 48 hours. This was then centrifuged at 4° C. and 4,000 rpm for 2 hours to obtain a virus, which was resuspended in 0.5 mL of RPMI (Welgene, LMO1-O1) without FBS to produce a lentivirus.

To determine the transduction efficiency of mammalian cells, transformation units (TU/mL) were determined by analyzing the particle counts of actual transducible-capable lentiviruses using Jurkat cells. CAR expression can be assayed by FACS. On the first day, Jurkat cells were seeded in 96-well plates at 1×10 ⁵ cells/100 μL per well. On the second day, serial dilutions of lentivirus in 96-well plates were performed by 1/3 and lentiviral transduction was performed on already inoculated Jurkat cells. At this time, lentivirus transduction was further increased by introducing polybrene (Millipore) into RPMI medium (10% FBS and 1xP/S). After centrifugation at 1200×g and 32° C. for 2 hours, cells were incubated for 3 hours in a 37° C. 5% CO2 incubator and only 100 μL of RPMI was added per well. On day 5, flags of lentiviruses infected into cells were stained with anti-flag-DYKDDDDK (Biolegend, Cat No. 637310) to analyze the percentage of transduced cells by flow cytometry. Using this, titers were calculated as described in Follenzi and Naldini, 2002 (Follenzi and Naldini, 2002).

FACS staining was performed to determine the production rate of the two types of CAR-T cells after 14 days of incubation. For each CAR-T cell type, 2×10 ⁵ cells were collected in FACS tubes (FALCON, Cat. No. 352052), then 2 mL of FACS buffer was added and centrifuged (Thermo, ST16) at 2,000 rpm. Centrifugation was performed for 5 minutes. After discarding the supernatant, 0.5 μL/tube anti-CD8 APC (SKI, Biolegend, Cat. No. 344722), 0.5 μL/tube anti-CD4 BV650 (RPA-T4, Biolegend, Cat. No. 300536) and 0.125 μL/tube Tube anti-flag PE (L5, Biolegend, Cat. No. 637310) was added and staining was performed at room temperature for 30 min. After adding 2 mL of FACS buffer and centrifuging at 2,000 rpm for 5 minutes, this process was repeated once more. For live/dead cell staining, 1 μL/tube 7-AAD (Biolegend, Cat. No. 420404) was added and the mixture was left at room temperature for 5 min and then analyzed using FACS (BD, FACSCelesta).

Confirmation of the proportion of generated CD19 CAR-T cells using FACS staining showed that for the improved-construct CD19-euBBZ CAR-T cells, the cell proportion was CD4+/CAR+ 29.4%, CD8+/CAR+ 50.8%, and total It was confirmed that the CAR-T was 80.2%. For non-construct-improved CD19-BBz CAR-T cells, it was confirmed that the cell ratio was 42.7% for CD4+/CAR+, 29.3% for CD8+/CAR+, and 72.0% for total CAR-T. Therefore, it was confirmed that CD19-euBBz CAR-T cells showed 8.2% higher CAR expression than CD19-BBz CAR-T cells, and CD8+/CAR+ cells were 21.5%, that is, about twice as much ( FIG. 11a ).

Example 6 - Confirmation of Cytotoxicity of Produced CD19 CAR-T Cells

To determine the cytotoxicity of two CAR-T cell types cultured for 14 days, the CAR-T(E):LCL(T) ratio was 30: 30 in 96-well white plates (Corning, Cat. No. 3917). 1, 10:1, 3:1 and 1:1. First, CAR-T cells were placed in wells at 6×10 ⁵ cells/50 μL, 2×10 ⁵ cells/50 μL, 9×10 ⁴ cells/50 μL, and 2×10 ⁴ cells/50 μL, respectively. Next, the target cell line, that is, the CBK LCL-Luc cell line was added to a 37° C. CO ₂ incubator (Mammert, INC0153med) at 2×10 ⁴ cells/50 μL and reacted for 4 hours. After 4 hours, 100 μL of Bright-Glo™ (Promega, Cat. No. E2620) was added to each well, and after 5 minutes, relative illumination units (RLU) values were measured using a Luminometer (Thermo, Floroscan, FL). did

There was no difference in cytotoxicity between CAR-T cells into which 4-1BB was introduced and CAR-T cells into which euBBz containing 5 amino acids added to the 4-1BB domain were introduced.

As a result, when two CAR-T cells and a CBK LCL-Luc cell line were incubated together at a ratio of 30:1, the cytotoxicity was about 80% after 4 hours, and when incubated at 10:1, the cytotoxicity was about shows that it was 50%. Cytotoxicity decreased approximately 3-fold with a 3-fold decrease in each number of CAR-T cells incubated with cancer cells; 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. 11b ).

Example 7 - Induction of Subcutaneous Animal Models and Validation of CAR-T via Automated Caliper and IVIS Imaging

In the case of experimental animals, NSG (NOD-scid IL2rypL1) mice (The Jackson Laboratory) were used under constant conditions in an animal kennel. At a 12 h light/dark cycle and 50±10% humidity, the temperature was 23±2° C.; Feed and drinks were provided ad libitum. In efficacy experiments using CD19-euBBz CAR-T with 5 amino acids added to the 4-1BB domain, the CBK LCL-Luc cell line was prepared with 2×10 ⁶ cells/100 μL DPBS/head and injected subcutaneously into 6-week-old female mice. was induced in a subcutaneous animal model. When the cancer size, measured using automatic calipers (Youngbio, TM900), reached between 50 and 100 mm 3 , CD19-euBBz CAR-T cells and CD19-BBz CAR-T cells were mixed with 2×10 ⁶ cells/100 μL DPBS/head ( Dose 1) and 6×10 ⁶ cells/100 μL DPBS/head (dose 2) were administered once via tail vein to confirm efficacy. Cancer size and viability were checked periodically in all animal experiments.

More specifically, arm size and photon values were measured using automatic calipers and IVIS imaging equipment (PerkinElmer, Luna III) at intervals of 3 and 4 days after CAR-T administration ( FIGS. 12 and 13 ). When using the TM900, the arm size was determined after the device was placed at the arm site. When imaging and photon values were confirmed using an IVIS imaging device, 150 mg/kg XenoLight™ D-luciferin (PerkinElmer, Cat. No. 122799) was first administered intraperitoneally to mice. After 15 minutes, inhalation anesthesia was induced using isoflurane and the presence of cancer cells was imaged using IVIS after 5 minutes. After imaging, normalization was performed and luciferase values (photon values) were then checked and graphed. 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. As shown in FIG . 12 , the effect could be confirmed within one week after administration of two types of CAR-T cells.

In the experimental group in which CD19-euBBz CAR-T cells were administered with 2×10 ⁶ cells/100 μL DPBS/head and 6×10 ⁶ cells/100 μL DPBS/head, cancer cells were observed on IVIS imaging one week after administration. In addition, when 6×10 ⁶ cells/100 μL DPBS/head was administered to the group treated with CD19-BBz CAR-T, almost no cancer cells were observed by imaging within one week of administration. However, in the experimental group administered with CD19-BBz CAR-T, cancer cells were confirmed after 1 week.

One week after CAR-T administration, the value of luciferase imaged in each subject after the imaging procedure was determined as shown in the images; Luciferase values were confirmed only in the group administered with CD19 CAR-T in 2×10 ⁶ cells/100 μL DPBS/head. When observed after 3 weeks or more, tumors continued to grow in mice not administered with CAR-T cells, and cancer cells were not identified in the three experimental groups in which the cancer cells initially disappeared. However, although luciferase levels decreased in the group receiving 2×10 ⁶ cells/100 μL DPBS/head for CD19 CAR-T after 10 days, the cancer cells did not completely disappear after 3 weeks. Experimental imaging of cancer cells when administered with CD19-euBBz CAR-T 2×10 ⁶ cells/100 μL DPBS/head compared to the group treated with CD19-BBz CAR-T at 6×10 ⁶ cells/100 μL DPBS/head and It was confirmed that the efficacy was similar. The results showed that there was no difference in the cytotoxicity of CD19-euBBz CAR-T and CD19-BBz CAR-T in vitro, but it was confirmed that there was a 5-fold better efficacy in CD19-euBBz CAR-T in animal models. .( Fig. 12 )

Example 8 - in vivo Identification of the ratio of CD19-euBBz CAR-T in animal models

To confirm the efficacy of the improved-construct CAR-T cells, the presence of CAR-T in mouse blood was confirmed after administration of CAR-T cells to a subcutaneous animal model. More specifically, orbital blood collections were performed from mice at intervals of 3 and 4 days after CAR-T administration. At each blood collection, 70 μL of blood was collected and 60 μL of blood was used to check the percentage and cell count of CAR-T cells. 60 μL blood was placed in 5 mL FACS tubes and live/dead cell staining was performed using a Zombie Aqua BV510 (Biolegend, Cat. No. 423101). After reaching a concentration of 0.1 μL/100 μL DPBS/tube, staining was performed at room temperature for 10 min. Counting beads (Molecularprobes, Cat. No. C36950) followed by anti-CD45 FITC (HI30, Biolegend, Cat. No. 304006), anti-CD8 BV786 (SK-1, Biolegend, Cat. No. 344740), anti-CD4 BV650 and anti-flag PE were added and stained for 30 min at room temperature. Each antibody was mixed with 0.5 μL/100 μL FACS buffer/tube, to which 25 μL of counting beads were added. After 30 minutes, 2 mL of 1X RBC elution buffer (Biolegend, Cat. No. 422401) was added and reacted at room temperature for 5 minutes. After centrifugation at 2,000 rpm for 5 minutes using a centrifuge, all of the supernatant was discarded. 2 mL of FACS washing buffer was added to this tube and centrifugation was performed at 2,000 rpm for 5 minutes. This process was repeated once more, then 50 μL of FACS buffer was added and analyzed using FACS.

One week after CAR-T cell administration, in the CD19-euBBz CAR-T treated group, approximately 20% CD19-euBBz in both the 2×10 ⁶ cells/100 μL DPBS/head and 6×10 ⁶ cells/100 μL DPBS/head groups. CAR-T cells were identified in blood; However, in the CD19-BBz CAR-T-administered group, only 5% CD19 CAR-T was identified when 6×10 ⁶ cells/100 μL DPBS/head was administered. After 3 days, the number and percentage of CAR-T cells in the mouse reached a maximum and decreased. 3 experimental groups in which CAR-T cells were identified within 1 week (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; At 6×10 ⁶ cells/100 μL DPBS/head), cancer cells were rapidly killed because they were able to contact relatively many CAR-T cells before they proliferated in the mouse body. However, in the experimental group administered with CD19-BBz CAR-T at 2×10 ⁶ cells/100 μL DPBS/head, the ratio and number of CAR-T cells reached a maximum at week 2, and CAR-T with relatively well proliferating cancer cells. The percentage was about 25%. CD19-euBBz CAR-T was quantitatively more stable than CD19-BBz CAR-T after the CAR-T ratio initially increased and then decreased. However, in the case of CD19-BBz CAR-T, the proportion of CAR-T cells increased and later decreased, as a result, it took a longer time for the tumor to disappear in the mouse body. As a result, as in the results of this experiment, the group administered with CD19-euBBz CAR-T at 2×10 ⁶ cells/100 μL DPBS/head produced CD19-BBz CAR-T at 6×10 ⁶ cells/100 μL DPBS/head. CAR-T levels and effects similar to those of the administered group were shown in mice, indicating that CD19-euBBz CAR-T had superior effects ( FIG. 14 ).

SEQUENCE LISTING <110> Euutilex Co., Ltd. <120> CHIMERIC ANTIGEN RECEPTOR WITH 4-1BB COSTIMULATORY DOMAIN <130> 47683-0044WO1 <150> 62/867,503 <151> 2019-06-27 <150> PCT/KR2019/010244 <151> 2019-08-12 <150 > 16/715,462 <151> 2019-12-16 <150> 62/991,493 <151> 2020-03-18 <150> 63/004,827 <151> 2020-04-03 <150> 63/043,237 <151> 2020 -06-24 <160> 28 <170> PatentIn version 3.5 <210> 1 <211> 15 <212> DNA <213> Artificial <220> <223> synthetic oligonucleotide <400> 1 cgtttctctg ttgtt 15 <210> 2 < 211> 141 <212> DNA <213> Artificial <220> <223> synthetic oligonucleotide <400> 2 cgtttctctg ttgttaaacg gggcagaaag aaactcctgt atatattcaa acaaccattt 60 atgagaccag tacaaactac tcaagaggaa gatagctg t 212> PRT <213> Artificial <220> <223> synthetic polypeptide <400> 3 Arg Phe Ser Val Val 1 5 <210> 4 <211> 47 <212> PRT <213> Artificial <220> <223> synthetic polyptpdie <400> 4 Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 1 5 10 15 Lys G ln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 20 25 30 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 45 <210> 5 <211> 207 <212> DNA <213 > Artificial <220> <223> CD8 / Hinge / Transmembrane <400> 5 accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgctag ccagcccctg 60 tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120 gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 180 ctgtcactgg ttatcaccct ttactgc 207 <210> 6 <211> 339 <212> DNA <213> Artificial <220> <223> synthetic oligonucleotide <400> 6 agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180 gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240 cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300 tacgacgccc ttcacatgca ggccctgccc cctcgctaa 339 <210> 7 <211> 69 <212> DNA <213> Artificial <220> <223> CD8 alpha leader sequence <400> 7 ggatccatgg ccttaccagt gaccgccttg ctcctgccgc tggccttgct gctccacgcc 60 gccaggccg 69 <210> 8 <211> 24 <212> DNA <213> Artificial <220> <223> Flag-tag sequence < 400> 8 gactacaagg acgacgatga caag 24 <210> 9 <211> 16 <212> PRT <213> Artificial <220> <223> Light chain CDR1 <400> 9 Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His 1 5 10 15 <210> 10 <211> 7 <212> PRT <213> Artificial <220> <223> Light chain CDR2 <400> 10 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 11 <211 > 9 <212> PRT <213> Artificial <220> <223> Light chain CDR3 <400> 11 Ser Gln Asn Thr His Val Pro Thr 1 5 <210> 12 <211> 5 <212> PRT <213> Artificial <220> <223> Heavy chain CDR1 <400> 12 Asp Tyr Glu Met His 1 5 <210> 13 <211> 17 <212> PRT <213> Artificial <220> <223> Heavy chain CDR2 <400> 13 Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys 1 5 10 15 Gly <210> 14 <211> 6 <212> PRT <213> Artificial <220> <223> Heavy chain CDR3 <400> 14 Phe Tyr Ser Tyr Thr Tyr 1 5 <210> 15 <211> 112 <212> PRT <213> Artificial <220> <223> human GC33 light chain variable region <400> 15 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 16 <211> 115 <212> PRT <213> Artificial <220> <223> human GC33 heavy chain variable region <400> 16 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 17 <211> 336 <212> DNA <213> Artificial <220> <223> human GC33 light chain variable region <400> 17 gacgtcgtta tgacacagag tcccctctcc ttgccggtga ccctgggtca gcctgcgtcc 60 atctcttgca gatggt cactccaacg gcaacacata cttgcactgg 120 taccaacaaa gacctggtca gtcaccgcga cttctcatat ataaagtttc caataggttc 180 agtggagtgc cagacaggtt cagtggttca ggatcaggca ctgatttcac gcttaaaatc 240 agtcgggttg aggcggagga cgtaggagtt tactattgca gccagaatac gcacgtgccg 300 cctacttttg gctctggaac caagttggaa ataaag 336 <210> 18 <211> 345 <212> DNA <213> Artificial <220> < 223> human GC33 heavy chain variable region <400> 18 caagtgcaac tcgtacaatc aggtgctgaa gtcaaaaagc cgggagcctc tgttaaagtg 60 tcctgtaaag ccagcggcta cacctttacc gattatgaga tgcactgggt tcggcaggct 120 ccgggccaag gtctggagtg gatcggggct cttgacccaa agacgggcga cacggcttat 180 tcacaaaaat tcaaaggtag ggctactctg actgccgata agtccaccag caccgcgtat 240 atggagctct ctagcttgcg aagcgaggac acggcggtgt actattgcac acgcttctat 300 agttacacat attggggtca aggcacgctt gtgaccgtgt ctagc 345 <210> 19 <211> 243 <212> PRT <213> Artificial <220> <223> synthetic polypeptide <400> 19 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Asp His Ile Asn Asn Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu 115 120 125 Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys 130 135 140 Thr Val Ser Gly Phe Ser Leu Ser Arg Tyr Ser Val His Trp Ile Arg 145 150 155 160 Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly Met Ile Trp Gly Gly 165 170 175 Gly Ser Thr Asp Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ser 180 185 190 Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr 195 200 205 Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asn Glu Gly Asp Thr 210 215 220 Thr Ala Gly Thr Trp Phe Ala Tyr Trp Gly Gin Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser <210> 20 <211> 729 <212> DNA <213> Artificial <220> <223> synthetic oligonucleotide <400> 20 gatattcaga tgacccagtc cccgagctcc ctgtccgcct ctgtgggcga tagggtcacc 60 atcacctgca agactc aggccagtga ccaagct cctgaact c acca acct tggaaaccgg agtcccttct 180 cgcttctctg gttccggatc tgggaaggat tacactctga ccatcagcag tctgcagccg 240 gaagacttcg caacttatta ctgtcagcag tactggtcca cccccttcac cttcggacag 300 ggtaccaagg tggagatcaa aggcggaggc ggatctggcg gcggaggaag tggcggaggg 360 ggatctcagg tgcagctgca ggagtcgggc ccaggactgg tgaagccttc ggagaccctg 420 tccctcacct gcactgtctc tggtttctcc ctgagtcggt actctgtgca ttggatccgg 480 cagcccccag ggaagggact ggagtggctg gggatgatct ggggaggcgg cagcaccgac 540 tacaacagcg ccctgaagtc ccgactgacc atatcaaagg acaactccaa gaaccaggtg 600 tccttgaagc tgagctctgt gaccgctgcg gacacggccg tgtattactg tgcgagaaat 660 gagggcgata ccaccgccgg cacttggttt gcctatt210ggg 21 gccagggaac cctggtcacc <212> Pctggtcacc 720 gtctcctca <213> Artificial <220> <223> synthetic polypeptide <400> 21 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu 115 120 125 Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys 130 135 140 Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg 145 150 155 160 Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser 165 170 175 Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile 180 185 190 Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln 195 200 205 Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly 210 215 220 Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gly Gly Thr Ser Val Thr Val 225 230 235 240 Ser Ser <210> 22 <211> 726 <212 > DNA <213> Artificial <220> <223> synthetic oligonucleotide <400> 22 gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60 atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120 gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180 aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240 gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300 gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360 ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 420 tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 480 cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 540 tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 600 ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 660 tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc 720 tcctca 726 < 210> 23 <211> 1184 <212> DNA <213> Artificial <220> <223> synthetic oligonucleotide <400> 23 cgtgaggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt 60 tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg 120 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa 180 gtgcagtagt cgccgtgaac gttcttttttc gcaacgggtt tgccgccaga acacaggtaa 240 gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt 300 gaattacttc gg cacctggctg cagtactggt ggagtgt 360 gatggt ggagtgt ag ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgagg 420 cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg 480 ctgctttcga taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt 540 tctggcaaga tagtcttgta aatgcgggcc aagatctgca cactggtatt tcggtttttg 600 gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc 660 tgcgagcgcg gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg 720 tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg 780 caccagttgc gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat 840 ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct 900 ttccgtcctc agccgtcgct tcatgtgact ccactgagta ccgggcgccg tccaggcacc 960 tcgattagtt ctcgagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg 1020 cgatggagtt tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga 1080 tgtaattctc cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc 1140 agacagtggt tcaaagtttt tttcttccat ttcaggtgtc gtga 1184 <210> 24 <211> 84 <212> DNA <213> Artific ial <220> <223> synthetic oligonucleotide <400> 24 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta 60 gtcagtgtgg aaaatctcta gcag 84 <210> 25 <211> 1377 <212> DNA <213> Artificial <220> <223> synthetic oligonucleotide > 25 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc 60 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt 120 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga 180 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat 240 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt 300 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag 360 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa 420 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa 480 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg 540 gacaattgga agaagtgaaccagga gga aagaga gacca accagga agaga agat aaaccagat gaatagga 660 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg 720 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg 780 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc 840 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg 900 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat 960 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac 1020 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat 1080 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca 1140 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga 1200 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg 1260 tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa 1320 ggtggagaga gagacagaga cagatccatt cgattagtga acggatctcg acggtat 1377 <210> 26 <211> 547 <212> DNA <213 > Artificial <220> <223> synthetic oligonucleotide <400> 26 tagactgtag cccaggaata tggcagctag attgtacaca tttaga agga aaagttatct 60 tggtagcagt tcatgtagcc agtggatata tagaagcaga agtaattcca gcagagacag 120 ggcaagaaac agcatacttc ctcttaaaat tagcaggaag atggccagta aaaacagtac 180 atacagacaa tggcagcaat ttcaccagta ctacagttaa ggccgcctgt tggtgggcgg 240 ggatcaagca ggaatttggc attccctaca atccccaaag tcaaggagta atagaatcta 300 tgaataaaga attaaagaaa attataggac aggtaagaga tcaggctgaa catcttaaga 360 cagcagtaca aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt 420 acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac 480 aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag 540 tttggct 547 <210> 27 <211> 591 <212> DNA <213> Artificial <220> <223> synthetic oligonucleotide <400> 27 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc 60 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta 120 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt 180 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgcctgca acctgtggcat tgctgctgca acctgtgg cgggac tttcgctttc cccctcccta 300 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt 360 tgggcactga caattccgtg gtgttgtcgg ggaagctgac gtcctttcca tggctgctcg 420 cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca 480 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc 540 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcct g 591 <210> 28 <211> 98 <212> DNA <213> Artificial <220> <223> synthetic oligonucleotide <400> 28 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca 60ctgcttaagc ctcaataaag cttgccttga gtgcttca 98

Claims (61)

An immune cell comprising a chimeric antigen receptor (CAR), said (CAR) comprising:
(a) an extracellular domain comprising an antigen-binding domain;
(b) a transmembrane domain; and
(c) an intracellular domain comprising a costimulatory endodomain, wherein the costimulatory endodomain comprises an intracellular domain comprising an intracellular signaling domain from 4-1BB/CD137 and 5 additional amino acids. The immune cell of claim 1 , wherein the chimeric antigen receptor is a single polypeptide. The immune cell of claim 1 , wherein the chimeric antigen receptor consists of two polypeptides. 4. The method according to any one of claims 1 to 3, wherein the costimulatory endodomain comprises an intracellular signaling domain from 4-1BB/CD137 and 5 additional amino acids, wherein the 5 additional amino acids are SEQ ID NO: 1 encoded by an immune cell. 5. The immune cell of claim 4, wherein the costimulatory endodomain comprises SEQ ID NO:2. 6. The immune cell according to any one of claims 1 to 5, wherein the antigen-binding domain is humanized. 7. The immune cell according to any one of claims 1 to 6, wherein the antigen-binding domain is human. 8. The immune cell according to any one of claims 1 to 7, wherein the antigen-binding domain is an scFv. 9. The immune cell of any one of claims 1-8, wherein the antigen-binding domain specifically binds to an antigen associated with a disease. 10. The immune cell of any one of claims 1-9, wherein the antigen-binding domain specifically binds a tumor antigen. 11. The immune system according to any one of claims 1 to 10, wherein the antigen-binding domain specifically binds to an antigen selected from the group consisting of glypican-3 (GPC3), malignant variant receptor (MVR) and CD19. cell. 12. The method of any one of claims 1 to 11, wherein the transmembrane domain is 4-1BB/CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D) ), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8alpha, CD8beta, CD96 (tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 ( CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator ( ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, a ligand that specifically binds to CD83, LIGHT, LTBR, Ly9 (CD229) , Lymphocyte function-associated antigen-1 (LFA-1), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG/Cbp, programmed death-1 (PD-) 1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAM1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, a membrane selected from a protein selected from the group consisting of Toll ligand receptor, TRANCE/RANKL, VLA1, and VLA-6. Tong domain, immune cell. 13. The immune cell according to any one of claims 1 to 12, wherein the transmembrane domain is a transmembrane domain from CD8α. 14. The immune cell of any one of claims 1-13, wherein the intracellular domain further comprises an intracellular domain from CD3ζ. 15. The immune cell of any one of claims 1-14, wherein the chimeric antigen receptor further comprises a signal peptide or leader sequence. 16. The immune cell of any one of claims 1-15, wherein the chimeric antigen receptor further comprises a hinge region. 17. The immune cell of claim 16, wherein the hinge region is a CD8α hinge. 18. The immune cell of any one of claims 1-17, wherein the chimeric antigen receptor further comprises an additional antigen-binding domain. The immune cell of claim 18 , wherein the additional antigen-binding domain is an scFv. 20. The immune cell according to any one of claims 1 to 19, wherein the immune cell is a human immune cell. 21. The immune cell of claim 20, wherein the human immune cell is an autologous human immune cell. 21. The immune cell of claim 20, wherein the human immune cell is an allogeneic human immune cell. 23. The immune cell of any one of claims 1-22, wherein the immune cell is a T cell. 24. The immune cell according to any one of claims 1 to 23, wherein the immune cell is an NK cell. A nucleic acid encoding a chimeric antigen receptor (CAR), said chimeric antigen receptor comprising:
(a) an extracellular domain comprising an antigen-binding domain;
(b) a transmembrane domain; and
(c) an intracellular domain comprising a costimulatory endodomain, wherein the costimulatory endodomain comprises an intracellular domain comprising an intracellular signaling domain from 4-1BB/CD137 and 5 additional amino acids. 26. The nucleic acid of claim 25, wherein the chimeric antigen receptor is a single polypeptide. 26. The nucleic acid of claim 25, wherein the chimeric antigen receptor consists of two polypeptides. 28. The method of any one of claims 25-27, wherein the costimulatory endodomain comprises an intracellular signaling domain from 4-1BB/CD137 and 5 additional amino acids, wherein the 5 additional amino acids are SEQ ID NO: 1 nucleic acid encoded by the nucleotide sequence of 29. The nucleic acid of claim 28, wherein the costimulatory endodomain comprises SEQ ID NO:2. 30. The nucleic acid of any one of claims 25-29, wherein the antigen-binding domain is humanized. 31. The nucleic acid of any one of claims 25-30, wherein the antigen-binding domain is human. 32. The nucleic acid of any one of claims 25-31, wherein the antigen-binding domain is an scFv. 33. The nucleic acid of any one of claims 25-32, wherein the antigen-binding domain specifically binds an antigen associated with a disease. 34. The nucleic acid of any one of claims 25-33, wherein the antigen-binding domain specifically binds a tumor antigen. 35. The nucleic acid of any one of claims 25-34, wherein the antigen-binding domain specifically binds an antigen selected from the group consisting of glycan-3 (GPC3), malignant variant receptor (MVR) and CD19. . 36. The method of any one of claims 25-35, wherein the transmembrane domain is 4-1BB/CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D). ), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (tactile), CD11a, CD11b , CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 ( CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator ( ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM,ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, CD83-specific ligand, LIGHT, LTBR, Ly9 (CD229) , Lymphocyte function-associated antigen-1 (LFA-1), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG/Cbp, programmed death-1 (PD-) 1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Select from a protein selected from the group consisting of Toll ligand receptor, TRANCE/RANKL, VLA1, and VLA-6 A transmembrane domain that has been described as a nucleic acid. 37. The nucleic acid of any one of claims 25-36, wherein the transmembrane domain is a transmembrane domain from CD8 alpha. 38. The nucleic acid of any one of claims 25-37, wherein the intracellular domain further comprises an intracellular domain from CD3ζ. 39. The nucleic acid of any one of claims 25-38, wherein the chimeric antigen receptor further comprises a signal peptide or leader sequence. 40. The nucleic acid of any one of claims 25-39, wherein the chimeric antigen receptor further comprises a hinge region. 41. The nucleic acid of claim 40, wherein the hinge region is a CD8α hinge. 42. A vector comprising the nucleic acid of any one of claims 25-41. 43. The vector of claim 42, further comprising a promoter operably linked to the nucleic acid. 44. The vector of claim 43, wherein the promoter is a constitutive promoter. 44. The vector of claim 43, wherein the promoter is an inducible promoter. 46. The vector according to any one of claims 42 to 45, wherein the vector is a viral vector. 47. The vector of claim 46, wherein the viral vector is a lentiviral vector. A method of producing an engineered immune cell, said method comprising:
A method comprising introducing the nucleic acid of any one of claims 25-41 or the vector of any one of claims 42-47 into an immune cell, thereby producing an engineered immune cell. 49. The method of claim 48, further comprising culturing the engineered immune cell after the introducing step. 50. The method of any one of claims 48-49, wherein the immune cell is a T cell. 51. The method of any one of claims 48-50, wherein the immune cells are NK cells. 52. The method of any one of claims 48-51, further comprising obtaining immune cells from the subject prior to the introducing step. 53. The method of claim 52, further comprising administering the engineered immune cells to the subject. 54. The method of claim 52 or 53, wherein the subject has been diagnosed or confirmed to have cancer. 55. An engineered immune cell produced by the method of any one of claims 48-54. A pharmaceutical composition comprising the engineered immune cell of claim 55 and a pharmaceutically acceptable carrier. 57. A method of treating cancer in a subject, the method comprising administering to the subject the engineered immune cell of claim 55 or the pharmaceutical composition of claim 56. 58. The method of claim 57, wherein the cancer is an anti-glypican-3-associated cancer, an anti-CD19-associated cancer, or an anti-MVR-associated cancer. 58. The method of claim 57, wherein the cancer is carcinoma, lymphoma (eg, Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, leukemia, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, of the lung. Squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, stomach cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary carcinoma, kidney cancer, prostate cancer, vulvar cancer cancer, thyroid cancer, liver carcinoma, other lymphoproliferative diseases and various types of head and neck cancer. 60. The method of any one of claims 57-59, wherein the subject has previously received one or more additional anti-cancer therapies selected from the group consisting of ionizing radiation, a chemotherapeutic agent, a therapeutic antibody, and a checkpoint inhibitor. 61. The method of any one of claims 57-60, wherein the subject has been identified or diagnosed with cancer.

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