Anti-ZP4 antibodies and chimeric antigen receptors and methods of using them

Abstract

Provided herein are ZP4 antibodies and ZP4-specific chimeric antigen receptors (CARs). Also provided are immune cells expressing ZP4-specific CARs and methods for treating cancer by administering such CAR immune cells.

Description

[Technical Field]

[0001] This invention was made with support from the National Institutes of Health under grant CA186784. The government has certain rights in this invention.

[0002] [Priority Claim] This application claims the benefit of U.S. Provisional Application No. 63 / 382,221, filed November 3, 2022, which is incorporated by reference herein in its entirety.

[0003] The present disclosure relates generally to the fields of immunology, cell biology, molecular biology, and medicine, and more particularly to Zona Pellucida 4 (ZP4) antibodies and related compositions, at least chimeric antigen receptors, and methods of their use. Summary of the Invention [Problem to be solved by the invention]

[0004] CAR T cells have the potential to cure patients with advanced cancer, as demonstrated by their remarkable success in hematological malignancies. This has led to six CAR T therapies being approved by the FDA since 2017. However, expanding their application to solid tumors such as breast cancer has proven challenging. One of the main obstacles is finding targets that are highly expressed in cancer cells but not in important normal tissues. Therefore, there is an urgent need to develop CAR T cell therapies directed against novel targets to further improve outcomes in these patients. [Means for solving the problem]

[0005] The present disclosure relates to methods and compositions related to certain antibodies. These antibodies can be used in any type of immunotherapy or any medical application in which targeting ZP4 has therapeutic significance. In some embodiments, the present disclosure provides isolated monoclonal antibodies that specifically bind to ZP4.

[0006] Embodiments of the present disclosure include isolated monoclonal antibodies that specifically bind to ZP4, including: (I): (a) a first VH CDR comprising SEQ ID NO: 3; (b) a second VH CDR comprising SEQ ID NO:4; (c) a third VH CDR comprising SEQ ID NO:5; (d) the first VL CDR comprising SEQ ID NO:8; (e) a second VL CDR comprising SEQ ID NO: 9; (f) a third VL CDR comprising SEQ ID NO: 10; (II): (a) a first VH CDR comprising SEQ ID NO: 13; (b) a second VH CDR comprising SEQ ID NO: 14; (c) a third VH CDR comprising SEQ ID NO: 15; (d) the first VL CDR comprising SEQ ID NO: 18; (e) a second VL CDR comprising SEQ ID NO: 19; (f) a third VL CDR comprising SEQ ID NO: 20; (III): (a) a first VH CDR comprising SEQ ID NO: 23; (b) a second VH CDR comprising SEQ ID NO: 24; (c) a third VH CDR comprising SEQ ID NO: 25; (d) a first VL CDR comprising SEQ ID NO: 28; (e) a second VL CDR comprising SEQ ID NO: 29; (f) a third VL CDR comprising SEQ ID NO: 30; (IV): (a) a first VH CDR comprising SEQ ID NO: 33; (b) a second VH CDR comprising SEQ ID NO: 34; (c) a third VH CDR comprising SEQ ID NO: 35; (d) the first VL CDR comprising SEQ ID NO: 38; (e) a second VL CDR comprising SEQ ID NO: 39; (f) a third VL CDR comprising SEQ ID NO: 40; or (V): (a) a first VH CDR comprising SEQ ID NO: 43; (b) a second VH CDR comprising SEQ ID NO: 44; (c) a third VH CDR comprising SEQ ID NO: 45; (d) the first VL CDR comprising SEQ ID NO: 48; (e) a second VL CDR comprising SEQ ID NO: 49; (f) the third VL CDR comprising SEQ ID NO: 50.

[0007] In certain embodiments, the antibody comprises: (a) a first VH CDR comprising SEQ ID NO: 3; (b) a second VH CDR comprising SEQ ID NO:4; (c) a third VH CDR comprising SEQ ID NO:5; (d) the first VL CDR comprising SEQ ID NO:8; (e) a second VL CDR comprising SEQ ID NO: 9; (f) the third VL CDR comprising SEQ ID NO: 10. In certain embodiments, the antibody comprises a VH domain that is at least about 80% identical to the VH domain of SEQ ID NO:2 and a VL domain that is at least about 80% identical to the VL domain of SEQ ID NO:7. In a specific embodiment, the antibody comprises a VH domain identical to the VH domain of SEQ ID NO:2 and a VL domain identical to the VL domain of SEQ ID NO:7.

[0008] In certain embodiments, the antibody comprises: (a) a first VH CDR comprising SEQ ID NO: 13; (b) a second VH CDR comprising SEQ ID NO: 14; (c) a third VH CDR comprising SEQ ID NO: 15; (d) the first VL CDR comprising SEQ ID NO: 18; (e) a second VL CDR comprising SEQ ID NO: 19; (f) the third VL CDR comprising SEQ ID NO: 20. In a specific embodiment, the antibody comprises a VH domain that is at least about 80% identical to the VH domain of SEQ ID NO:12 and a VL domain that is at least about 80% identical to the VL domain of SEQ ID NO:17. In a specific embodiment, the antibody comprises a VH domain identical to the VH domain of SEQ ID NO:12 and a VL domain identical to the VL domain of SEQ ID NO:17.

[0009] In certain embodiments, the antibody comprises: (a) a first VH CDR comprising SEQ ID NO: 23; (b) a second VH CDR comprising SEQ ID NO: 24; (c) a third VH CDR comprising SEQ ID NO: 25; (d) a first VL CDR comprising SEQ ID NO: 28; (e) a second VL CDR comprising SEQ ID NO: 29; (f) the third VL CDR comprising SEQ ID NO: 30. In a specific embodiment, the antibody comprises a VH domain that is at least about 80% identical to the VH domain of SEQ ID NO:22 and a VL domain that is at least about 80% identical to the VL domain of SEQ ID NO:27. In a specific embodiment, the antibody comprises a VH domain identical to the VH domain of SEQ ID NO:22 and a VL domain identical to the VL domain of SEQ ID NO:27.

[0010] In certain embodiments, the antibody comprises: (a) a first VH CDR comprising SEQ ID NO: 33; (b) a second VH CDR comprising SEQ ID NO: 34; (c) a third VH CDR comprising SEQ ID NO: 35; (d) the first VL CDR comprising SEQ ID NO: 38; (e) a second VL CDR comprising SEQ ID NO: 39; (f) the third VL CDR comprising SEQ ID NO: 40. In a specific embodiment, the antibody comprises a VH domain that is at least about 80% identical to the VH domain of SEQ ID NO:32 and a VL domain that is at least about 80% identical to the VL domain of SEQ ID NO:37. In a specific embodiment, the antibody comprises a VH domain identical to the VH domain of SEQ ID NO:32 and a VL domain identical to the VL domain of SEQ ID NO:37.

[0011] In certain embodiments, the antibody comprises: (a) a first VH CDR comprising SEQ ID NO: 43; (b) a second VH CDR comprising SEQ ID NO: 44; (c) a third VH CDR comprising SEQ ID NO: 45; (d) the first VL CDR comprising SEQ ID NO: 48; (e) a second VL CDR comprising SEQ ID NO: 49; (f) the third VL CDR comprising SEQ ID NO: 50. In a specific embodiment, the antibody comprises a VH domain that is at least about 80% identical to the VH domain of SEQ ID NO:42 and a VL domain that is at least about 80% identical to the VL domain of SEQ ID NO:47. In a specific embodiment, the antibody comprises a VH domain identical to the VH domain of SEQ ID NO:42 and a VL domain identical to the VL domain of SEQ ID NO:47.

[0012] In various embodiments, any of the antibodies encompassed herein may be recombinant. In certain embodiments, the antibody is IgG, IgM, IgA, or an antigen-binding fragment thereof. The antibody may be a Fab', F(ab')2, F(ab')3, monovalent scFv, bivalent scFv, or single-domain antibody. The antibody may be a human antibody, a humanized antibody, or a deimmunized antibody. In certain embodiments, the antibody is conjugated to an imaging agent, a chemotherapeutic agent, a toxin, or a radionuclide. The antibody may be part of a fusion protein or a chimeric protein.

[0013] Embodiments of the present disclosure include compositions comprising any of the antibodies encompassed herein in a pharmaceutically acceptable carrier. In certain embodiments, there is an isolated polynucleotide molecule comprising a nucleic acid sequence encoding any of the antibodies encompassed herein.

[0014] Embodiments of the present disclosure include recombinant polypeptides comprising an antibody VH domain comprising CDRs 1-3 of the VH domain of clone 46 (SEQ ID NOs: 3, 4, and 5) and CDRs 1-3 of the VL domain of clone 46 (SEQ ID NOs: 8, 9, and 10). Embodiments of the present disclosure include recombinant polypeptides comprising an antibody VH domain comprising CDRs 1-3 of the VH domain of clone 108 (SEQ ID NOs: 13, 14, and 15) and CDRs 1-3 of the VL domain of clone 108 (SEQ ID NOs: 18, 19, and 20). Embodiments of the present disclosure include recombinant polypeptides comprising an antibody VH domain comprising CDRs 1-3 of the VH domain of clone 2 (SEQ ID NOs: 23, 24, and 25) and CDRs 1-3 of the VL domain of clone 2 (SEQ ID NOs: 28, 29, and 30). Embodiments of the present disclosure include recombinant polypeptides comprising an antibody VH domain comprising CDRs 1-3 of the VH domain of clone 128 (SEQ ID NOS: 33, 34, and 35) and CDRs 1-3 of the VL domain of clone 128 (SEQ ID NOS: 38, 39, and 40). Embodiments of the present disclosure include recombinant polypeptides comprising an antibody VH domain comprising CDRs 1-3 of the VH domain of clone 164 (SEQ ID NOS: 43, 44, and 45) and CDRs 1-3 of the VL domain of clone 164 (SEQ ID NOS: 48, 49, and 50). In certain embodiments, there is an isolated polynucleotide molecule comprising a nucleic acid sequence encoding any of the polypeptides encompassed herein. Embodiments of the present disclosure include host cells containing one or more polynucleotide molecules encoding any of the antibodies and / or recombinant polypeptides encompassed herein. In certain embodiments, the host cell is an immune cell, a mammalian cell, a yeast cell, a bacterial cell, a ciliate cell, or an insect cell.

[0015] In various embodiments, there are methods for treating a subject with cancer, or preventing or delaying the onset or progression of cancer in a subject, comprising administering to the subject an effective amount of any antibody, any recombinant polypeptide, and / or any host cell encompassed herein. In certain embodiments, the cancer is a solid tumor, such as triple-negative breast cancer. In certain embodiments, the antibody is contained in a pharmaceutically acceptable composition. The antibody may be administered systemically, intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or locally. In certain embodiments, the method further comprises administering at least a second anti-cancer therapy to the subject. The second anti-cancer therapy may be surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, and / or cytokine therapy. The second anti-cancer therapy may include adoptive T-cell therapy.

[0016] In certain embodiments, there is a modified Zona Pellucida 4 (ZP4) CAR or TCR comprising an antigen binding domain, wherein the antigen binding domain comprises: (I): (a) a first VH CDR comprising SEQ ID NO: 3; (b) a second VH CDR comprising SEQ ID NO:4; (c) a third VH CDR comprising SEQ ID NO:5; (d) the first VL CDR comprising SEQ ID NO:8; (e) a second VL CDR comprising SEQ ID NO: 9; (f) a third VL CDR comprising SEQ ID NO: 10; (II): (a) a first VH CDR comprising SEQ ID NO: 13; (b) a second VH CDR comprising SEQ ID NO: 14; (c) a third VH CDR comprising SEQ ID NO: 15; (d) the first VL CDR comprising SEQ ID NO: 18; (e) a second VL CDR comprising SEQ ID NO: 19; (f) a third VL CDR comprising SEQ ID NO: 20; (III): (a) a first VH CDR comprising SEQ ID NO: 23; (b) a second VH CDR comprising SEQ ID NO: 24; (c) a third VH CDR comprising SEQ ID NO: 25; (d) a first VL CDR comprising SEQ ID NO: 28; (e) a second VL CDR comprising SEQ ID NO: 29; (f) a third VL CDR comprising SEQ ID NO: 30; (IV): (a) a first VH CDR comprising SEQ ID NO: 33; (b) a second VH CDR comprising SEQ ID NO: 34; (c) a third VH CDR comprising SEQ ID NO: 35; (d) the first VL CDR comprising SEQ ID NO: 38; (e) a second VL CDR comprising SEQ ID NO: 39; (f) a third VL CDR comprising SEQ ID NO: 40; or (V): (a) a first VH CDR comprising SEQ ID NO: 43; (b) a second VH CDR comprising SEQ ID NO: 44; (c) a third VH CDR comprising SEQ ID NO: 45; (d) the first VL CDR comprising SEQ ID NO: 48; (e) a second VL CDR comprising SEQ ID NO: 49; (f) the third VL CDR comprising SEQ ID NO: 50.

[0017] In certain embodiments for a CAR or TCR, the antigen binding domain comprises: (a) a first VH CDR comprising SEQ ID NO: 3; (b) a second VH CDR comprising SEQ ID NO:4; (c) a third VH CDR comprising SEQ ID NO:5; (d) the first VL CDR comprising SEQ ID NO:8; (e) a second VL CDR comprising SEQ ID NO: 9; (f) the third VL CDR comprising SEQ ID NO: 10. The antigen-binding domain may comprise a VH domain that is at least about 80% identical to the VH domain of SEQ ID NO:2 and a VL domain that is at least about 80% identical to the VL domain of SEQ ID NO:7. The antigen-binding domain may comprise a VH domain identical to the VH domain of SEQ ID NO:2 and a VL domain that is at least about 80% identical to the VL domain of SEQ ID NO:7. In certain embodiments for a CAR or TCR, the antigen binding domain comprises: (a) a first VH CDR comprising SEQ ID NO: 13; (b) a second VH CDR comprising SEQ ID NO: 14; (c) a third VH CDR comprising SEQ ID NO: 15; (d) the first VL CDR comprising SEQ ID NO: 18; (e) a second VL CDR comprising SEQ ID NO: 19; (f) the third VL CDR comprising SEQ ID NO: 20. In certain embodiments related to a CAR or TCR, the antigen binding domain may comprise a VH domain that is at least about 80% identical to the VH domain of SEQ ID NO: 12 and a VL domain that is at least about 80% identical to the VL domain of SEQ ID NO: 17. In certain embodiments for a CAR or TCR, the antigen binding domain comprises a VH domain identical to the VH domain of SEQ ID NO: 12 and a VL domain that is at least about 80% identical to the VL domain of SEQ ID NO: 17. In certain embodiments, the antigen binding domain comprises: (a) a first VH CDR comprising SEQ ID NO: 23; (b) a second VH CDR comprising SEQ ID NO: 24; (c) a third VH CDR comprising SEQ ID NO: 25; (d) a first VL CDR comprising SEQ ID NO: 28; (e) a second VL CDR comprising SEQ ID NO: 29; (f) the third VL CDR comprising SEQ ID NO: 30. In certain embodiments, the antigen-binding domain may comprise a VH domain that is at least about 80% identical to the VH domain of SEQ ID NO:22 and a VL domain that is at least about 80% identical to the VL domain of SEQ ID NO:27. In certain embodiments, the antigen-binding domain may comprise a VH domain identical to the VH domain of SEQ ID NO:22 and a VL domain that is at least about 80% identical to the VL domain of SEQ ID NO:27. In certain embodiments relating to a CAR or TCR, the antigen binding domain comprises: (a) a first VH CDR comprising SEQ ID NO: 33; (b) a second VH CDR comprising SEQ ID NO: 34; (c) a third VH CDR comprising SEQ ID NO: 35; (d) the first VL CDR comprising SEQ ID NO: 38; (e) a second VL CDR comprising SEQ ID NO: 39; (f) the third VL CDR comprising SEQ ID NO: 40. In certain embodiments, the antigen-binding domain may comprise a VH domain that is at least about 80% identical to the VH domain of SEQ ID NO:32 and a VL domain that is at least about 80% identical to the VL domain of SEQ ID NO:37. In certain embodiments, the antigen-binding domain may comprise a VH domain identical to the VH domain of SEQ ID NO:32 and a VL domain that is at least about 80% identical to the VL domain of SEQ ID NO:37. In certain embodiments relating to a CAR or TCR, the antigen binding domain comprises: (a) a first VH CDR comprising SEQ ID NO: 43; (b) a second VH CDR comprising SEQ ID NO: 44; (c) a third VH CDR comprising SEQ ID NO: 45; (d) the first VL CDR comprising SEQ ID NO: 48; (e) a second VL CDR comprising SEQ ID NO: 49; (f) the third VL CDR comprising SEQ ID NO: 50. In certain embodiments, the antigen-binding domain may comprise a VH domain that is at least about 80% identical to the VH domain of SEQ ID NO:42 and a VL domain that is at least about 80% identical to the VL domain of SEQ ID NO:47. In certain embodiments, the antigen-binding domain may comprise a VH domain identical to the VH domain of SEQ ID NO:42 and a VL domain that is at least about 80% identical to the VL domain of SEQ ID NO:47.

[0018] In specific embodiments of any CAR or TCR encompassed herein, the CAR comprises one or more signaling domains selected from CD3ξ, CD28, OX40 / CD134, 4-1BB / CD137, ICOS, CD27, and combinations thereof. The CAR or TCR may be encoded by a viral vector, such as a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a retroviral vector. The CAR may comprise a hinge and / or transmembrane domain. The CAR or TCR, or cells expressing them, may further comprise a transduction marker and / or a safety switch. The transduction marker and / or safety switch may be linked to the CAR on the vector by a cleavable peptide, such as a 2A peptide or an IRES. The CAR may further comprise a second antigen-binding domain that targets a cancer antigen different from the cancer antigen targeted by the other antigen-binding domain. Expression vectors encoding the CAR or TCR are encompassed herein.

[0019] In certain embodiments, there are host cells modified to express ZP4 CAR and / or ZP4 TCR. The cells can be modified to express any CAR or TCR encompassed herein. The cells can be immune cells, including T cells, including primary human T cells or TILs, as well as CD4 + T cells or CD8 +The immune cells include T cells. Primary human T cells can be derived from healthy donors or not. The immune cells can be autologous or allogeneic to the recipient individual. In certain embodiments, the cells are modified using CRISPR or transposase system.

[0020] Embodiments of the present disclosure include pharmaceutical compositions comprising ZP4-targeting T cells and a pharmaceutical carrier, wherein the ZP4-targeting T cells have been modified to express a CAR or TCR described herein.Embodiments of the present disclosure include compositions containing an effective amount of ZP4-targeting T cells for treating cancer in a subject, wherein the ZP4-targeting T cells have been modified to express a CAR or TCR described herein.

[0021] Embodiments of the present disclosure include the use of a composition containing an effective amount of ZP4-targeted T cells for treating cancer in a subject, wherein the ZP4-targeted T cells have been modified to express a CAR or TCR as described herein.

[0022] An embodiment of a method for treating cancer in a subject comprises administering to the subject an effective amount of ZP4-targeted T cells, wherein the ZP4-targeted T cells have been modified to express the CAR or TCR described herein. The cancer may or may not be a solid tumor. The cancer may be triple-negative breast cancer. In certain embodiments, the ZP4-targeted T cells are administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or topically. In certain embodiments, any method may further comprise administering a second anti-cancer therapy to the subject. The second anti-cancer therapy may be surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, and / or cytokine therapy. In certain embodiments, the cancer is a ZP4-expressing cancer.

[0023] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples herein, while indicating preferred embodiments of the present invention, are given by way of example only, and that various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art. [Brief explanation of the drawings]

[0024] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0025] [Figure 1A-D] Identification of ZP4 as a target for TNBC. Figure 1A: ZP4 mRNA expression in normal human tissues in the GTEx study. Figure 1B: MS / MS spectrum supporting one of the seven ZP4-specific peptides identified in CPTAC samples. Figure 1C: ZP4 mRNA expression in breast cancer tumors in the METABRIC cohort. Figure 1D: ZP4 IHC staining in normal tissues shows positivity restricted to the ZP surrounding the oocyte. [Figure 2A-B] ZP4 expression in BCM PDX models. Figure 2A: ZP4 mRNA was expressed in 33 of 103 (32%) TNBC PDX models, and ZP4 protein was detected in 4 of 103 (4%). Figure 2B: IHC confirmed ZP4 expression in two PDX models, BCM-9161 and BCM-0046. [Figure 3A-D] Engineering the ZP4 CAR. Figure 3A: Schematic diagram of an example ZP4 CAR retroviral vector. Figure 3B: Supernatants from hybridoma clones were analyzed by flow cytometry to detect ZP4 expression in ZP4.RFP+ 293T cells. 293T cells transfected with an empty RFP vector served as a negative control. Figure 3C: Flow cytometry showing ZP4-transfected SUM159. Figure 3D: Assay showing the cytotoxic activity of ZP4 CAR against ZP4+ TNBC cell lines. [Figure 4] Staining of ovarian tissue with mAB clone 46 demonstrated detection of ZP4 on the zona pellucida surrounding the oocyte. [Figure 5] When normal organ tissues were stained with mAB clone 46, no ZP4 was detected. [Figure 6] When normal organ tissues were stained with mAB clone 46, no ZP4 was detected. [Figure 7] When normal organ tissues were stained with mAB clone 46, no ZP4 was detected. [Figure 8] Staining of normal organ tissues with mAB clone 46 revealed findings suggesting the detection of ZP4 in testicular tissue. [Figure 9A-E] Figure 9A: Structure of an example CAR construct. Figure 9B: Graph showing ZP4 CAR expression on the surface of T cells. Figure 9C: Shows that all three CAR cells are predominantly CD8+. Figure 9D: Shows that CAR cells predominantly have an effector memory (Tem) phenotype. Figure 9E: Calculation of the percentage of specific lysis in each clone (clones 108, 128, and 164). [Figure 10A-G] Figure 10A: An example cycle of T cell replating, stimulation, and harvesting is shown. Figure 10B: A collection of representative flow diagrams showing the results of 72-hour co-cultures at weeks 1, 3, and 4. Figure 10C: A graph showing T cell proliferation quantified by cell number per well. Figure 10D: A graph showing T cell activation measured via 41BB after 72-hour co-culture with target cells. Figure 10E: Percentage of T cells remaining in each co-culture after quantification. Figure 10F: Percentage of tumor cells remaining in each co-culture after quantification. Figure 10G: A collection of graphs showing the polyfunctionality of ZP4 CAR T cells measured using Isoplexis. [Figure 11A-B] Figure 11A: Photograph of CAR T cell persistence monitored using the Lumina In Vivo Imaging System (IVIS). Figure 11B: Graph of in vivo persistence quantified as mean radioactivity. [Figures 12A-E]Figure 12A: Collection of photographs of tumor burden monitored using IVIS. Figure 12B: Graph of tumor burden quantified as mean radioactive dose. Figure 12C: Graph of tumor volume in days from tumor injection. Figure 12D: Graph of survival probability. Figure 12E: Collection of photographs and graphs showing that clone 128 and clone 108 CAR T cells reduced overall metastatic burden, particularly in the lung and liver. DETAILED DESCRIPTION OF THE INVENTION

[0026] [II. Definition] As used herein, "essentially free" means that the specified component is not intentionally incorporated into the composition or is present only as a contaminant or trace component. Thus, the total amount of the specified component incorporated into the composition is less than 0.05%, preferably less than 0.01%. Most preferred is a composition in which the component is not detectable by conventional analytical techniques.

[0027] As used herein, "a" or "an" may mean one or more. When used in conjunction with the word "comprising" in the claims, "a" or "an" may mean one or more than one.

[0028] The use of "or" in the claims means "and / or" unless explicitly referring to only one of the alternatives or where they are mutually exclusive. As used herein, "another" may mean at least a second or more. The words "about," "substantially," and "approximately" generally refer to a range of ±5% of the stated value.

[0029] "Treating" or "treatment" refers to the administration of one or more medications with the goal of alleviating the signs and symptoms of a disease or condition. Desirable therapeutic effects include slowing the rate of disease progression, amelioration or alleviation of the condition, remission, or improved prognosis. Alleviation can occur either before or after the appearance of signs or symptoms of the disease or condition. Thus, "treating" can include "prevention." "Treatment" also does not require complete alleviation of symptoms or a cure, and explicitly includes treatments that have only a marginal benefit to the patient.

[0030] As used throughout this application, the term "therapeutic benefit" or "therapeutically effective" refers to any effect associated with the medical treatment of a condition that promotes or improves the well-being of a subject. This includes, but is not limited to, reducing the frequency or severity of signs or symptoms of a disease. For example, treating cancer can include reducing tumor size, reducing tumor invasiveness, reducing the rate of cancer growth, or preventing metastasis. Treating cancer can also mean extending the survival of a subject with cancer.

[0031] "Subject" and "patient" refer to a human or non-human (e.g., primate, mammal, vertebrate). In certain embodiments, the subject is a human.

[0032] The phrase "pharmaceutical or pharmacologically acceptable" refers to a molecular entity or composition that does not produce adverse, allergic, or other undesirable reactions when administered to an animal, such as a human. In view of the present disclosure, the preparation of pharmaceutical compositions containing an antibody or additional active ingredient will be well known to those of skill in the art. Furthermore, it is understood that for administration to an animal (e.g., a human), the preparation should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biologics Standards.

[0033] As used herein, the term "pharmaceutically acceptable carrier" includes any aqueous solvent (e.g., water, alcoholic / aqueous solutions, saline, sodium salts, injectable carriers such as Ringer's sugar solution), non-aqueous solvent (e.g., propylene glycol, polyethylene glycol, vegetable oils, injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, inert gases), isotonicity agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, coloring agents, fluid or nutrient replenishers, similar materials, and combinations thereof. The pH and exact concentration of each component in a pharmaceutical composition are adjusted according to known parameters.

[0034] [III. ZP4 antibody] In certain embodiments, an antibody or fragment thereof is contemplated that binds to at least a portion of ZP4 and inhibits ZP4 activity (at least any type of activity, including signal transduction). As used herein, the term "antibody" is intended to broadly refer to any immunological binding agent, including IgG, IgM, IgA, IgD, IgE, and genetically modified IgG, as well as polypeptides containing antibody CDR domains that retain antigen-binding activity. The antibody may be selected from the group consisting of a chimeric antibody, an affinity-matured antibody, a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, an antigen-binding antibody fragment, or a natural or synthetic ligand. In a specific example, the anti-ZP4 antibody is a monoclonal antibody or a humanized antibody.

[0035] Thus, by known means and those described herein, it is possible to produce polyclonal or monoclonal antibodies, antibody fragments, binding domains, and CDRs (including modified versions of any of the above) specific for ZP4, one or more of its epitopes, or conjugates thereof. These antigens or epitopes may be naturally occurring or may be synthetic derivatives or variants of the naturally occurring compounds.

[0036] Examples of antibody fragments suitable for this embodiment include, but are not limited to: (i) V L , V H , C L and C H1 (ii) Fab fragments consisting of V domains; H and C H1 (iii) a Fd fragment consisting of a single antibody V domain; L and V H (iv) an Fv fragment consisting of a V H (v) an isolated CDR region; (vi) a bivalent F(ab')2 fragment formed by linking two Fab fragments; (vii) a V H Domains and V L (viii) bispecific single-chain Fv dimers (see U.S. Pat. No. 5,091,513); (ix) diabodies, multivalent or multispecific fragments constructed by gene fusion (see U.S. Pat. App. Pub. No. 20050214860). Fv, scFv or diabody molecules may be composed of V H and V L It can be stabilized by introducing a disulfide bond linking the domains. Minibodies can also be made in which an scFv is linked to a CH3 domain.

[0037] Antibody-like binding peptidomimetics are also contemplated in embodiments. Liu et al. (2003) describe "antibody-like binding peptidomimetics" (ABiPs), which are peptides that act as simplified antibodies and have the advantages of long serum half-lives and ease of synthesis.

[0038] Antigens such as the ZP4 extracellular domain (ECD) protein can be used to vaccinate animals to produce antibodies specific to ZP4. Antigens are often bound or conjugated to other molecules to enhance immune responses. As used herein, "conjugate" refers to any peptide, polypeptide, protein, or non-proteinaceous substance attached to an antigen for the purpose of inducing an immune response in an animal. Antibodies produced in animals in response to antigen immunization include a population of non-identical molecules (polyclonal antibodies) derived from multiple antibody-producing B lymphocytes. Polyclonal antibodies are a mixed antibody population composed of multiple antibody species, each recognizing a different epitope on the same antigen. Under appropriate conditions for polyclonal antibody production in an animal, the majority of antibodies in the animal's serum will recognize a collective epitope on the immunized antigenic compound. This specificity can be further enhanced by affinity purification, which selects only antibodies that recognize the desired antigen or epitope.

[0039] A monoclonal antibody is a single species of antibody in which all antibody molecules recognize the same epitope because all antibody-producing cells are derived from a single B lymphocyte cell line. The process for producing monoclonal antibodies (MAbs) generally begins with steps similar to those used for producing polyclonal antibodies. In some embodiments, rodents such as mice or rats are used to produce monoclonal antibodies. In other embodiments, rabbit, sheep, or frog cells may be used. The use of rats is well-known and may offer certain advantages. Mice (e.g., BALB / c mice) are routinely used and are known for their high rate of stable cell fusion.

[0040] Hybridoma technology involves fusing a single B lymphocyte from a mouse previously immunized with the ZP4 antigen with an immortalized myeloma cell (usually a mouse). This technology provides a means to propagate a single antibody-producing cell for infinite generations, allowing the unlimited production of structurally identical antibodies (monoclonal antibodies) with the same antigen or epitope specificity.

[0041] In certain embodiments, ZP4 cells can be isolated. In a specific embodiment, human ZP4 is overexpressed on the surface of mouse L cells (an immortalized fibroblast cell line). These cells can be sorted to enrich for ZP4-high expressing populations and then used to immunize mice to generate antibody clones. This method ensures that the antibodies produced specifically bind only to extracellular epitopes. After immunization, mouse lymph nodes can be harvested and used to generate hybridomas. The supernatant of each hybridoma clone can be screened for specificity against ZP4. RNA can be obtained from hybridomas producing selected ZP4 mAb clones, and the variable region sequences of the monoclonal antibodies can be obtained and used to construct CARs.

[0042] Plasma cells (CD45) were isolated from freshly prepared peripheral blood mononuclear cells (PBMCs) from immunized rabbits. + CD5 - CD19 + ) can be isolated and further selected for cells that bind to ZP4. After enrichment of antibody-producing B cells, total RNA can be isolated and cDNA synthesized. The DNA sequences of both heavy and light chain antibody variable regions can be amplified, constructed into phage display Fab expression vectors, and introduced into Escherichia coli. ZP4-specific binding Fabs can be selected through multiple rounds of enrichment by panning and sequenced. Selected C ZP4 D79b-binding hits can be expressed as full-length IgG in rabbit or rabbit / human chimeric form using a mammalian expression vector system in Invitrogen's human embryonic kidney (HEK293) cells and purified using protein G resin and FPLC (high-performance liquid chromatography) separation equipment.

[0043] In one embodiment, the antibody is a chimeric antibody, e.g., one in which antigen-binding sequences of non-human origin are incorporated into heterologous non-human, human, or humanized sequences (e.g., framework and / or constant region sequences). Techniques have been developed to replace the light and heavy chain constant regions of monoclonal antibodies with homologous regions of human origin, while retaining the variable regions of the foreign antibody. Alternatively, "fully human" monoclonal antibodies have been produced from transgenic mice carrying human immunoglobulin genes. Furthermore, techniques have been developed to convert variable regions of rodents, such as mice, to more human forms using recombinant antibody variable regions containing human and mouse amino acid sequences. In "humanized" monoclonal antibodies, only the hypervariable CDRs are derived from mouse monoclonal antibodies, while the framework and constant regions are derived from human amino acid sequences (see U.S. Patent Nos. 5,091,513 and 6,881,557). Replacing rodent-specific amino acid sequences with amino acid sequences found in the corresponding positions in human antibodies is believed to reduce the likelihood of an immune response in therapeutic applications. Additionally, the hybridoma or other cells that produce the antibody may undergo genetic mutations or other changes that may or may not alter the binding specificity of the antibody.

[0044] Methods for producing polyclonal antibodies in various animal species, as well as various types of monoclonal antibodies, including humanized, chimeric, and fully human, are widely known and highly predictable to those skilled in the art. For example, the following U.S. patents and patent applications provide enabling descriptions of such methods: U.S. Patent Application Publication Nos. 2004 / 0126828 and 2002 / 0172677; and U.S. Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,196,265, 4,275,149, 4,275,149, 4,375,160, 4,475,160, 4,575,160, 4,675,160, 4,775,160, 4,817,160, 4,850,160, 4,939,350, 4,996,345, 4,196,265, 4,275,149, 4,575,160, 4,775,160, 4,817,160, 4,939,160, 4,939,160, 4,939,160, 4,196,265, 4,275,149, 4,575,160, 4,775,160, 4,817,160, 4,817,160, 4,939 ... No. 277,437, No. 4,366,241, No. 4,469,797, No. 4,472,509, No. 4,606,855, No. 4,703,003, No. 4,742,159, No. 4, No. 767,720, No. 4,816,567, No. 4,867,973, No. 4,938,948, No. 4,946,778, No. 5,021,236, No. 5,164,296, No. 5, No. 196,066, No. 5,223,409, No. 5,403,484, No. 5,420,253, No. 5,565,332, No. 5,571,698, No. 5,627,052, No. 5, No. 656,434, No. 5,770,376, No. 5,789,208, No. 5,821,337, No. 5,844,091, No. 5,858,657, No. 5,861,155, No. 5, Nos. 871,907, 5,969,108, 6,054,297, 6,165,464, 6,365,157, 6,406,867, 6,709,659, 6,709,873, 6,753,407, 6,814,965, 6,849,259, 6,861,572, 6,875,434, and 6,891,024. All patents, patent application publications, and other publications cited herein are hereby incorporated by reference.

[0045] Antibodies can be produced from any animal origin, including birds and mammals. Preferably, the antibodies are from sheep, murines such as mice and rats, rabbits, goats, guinea pigs, camels, horses, or chickens. Furthermore, new technologies have made it possible to develop and screen human antibodies from human recombinant antibody libraries. For example, bacteriophage antibody expression technology allows the production of specific antibodies without animal immunization (as described in U.S. Pat. No. 6,946,546, which is incorporated herein by reference).

[0046] Antibodies against ZP4 are fully expected to be capable of neutralizing or antagonizing the effects of ZP4, regardless of the animal species, monoclonal cell line, or other origin of the antibody. Certain animal species may be less favorable for generating therapeutic antibodies because they are more likely to cause allergic reactions due to activation of the complement system via the antibody's "Fc" region. However, intact antibodies can be enzymatically digested to separate "Fc" (complement-binding) fragments and antibody fragments containing binding domains and CDRs. Removal of the Fc region reduces the likelihood that antigen-antibody fragments will induce unwanted immune responses, so Fc-free antibodies may be preferred for prophylactic or therapeutic treatments. As mentioned above, antibodies can be constructed as chimeric, partially human, or fully human to reduce or avoid immunological side effects that can occur when antibodies from other species or containing other sequences are administered to animals.

[0047] Substitutional variants involve the substitution of one amino acid for another at one or more positions in the protein and can be designed to alter one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions can be conservative, i.e., substitutions of amino acids with similar shape and charge. Conservative substitutions are well known to those skilled in the art and include, for example, the following changes: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine or leucine. In contrast, non-conservative substitutions affect the function or activity of a polypeptide. Non-conservative changes include substitutions of chemically distinct residues, such as between polar or charged amino acids and non-polar or uncharged amino acids.

[0048] The protein may be a recombinant protein or may be synthesized in vitro. Alternatively, the non-recombinant or recombinant protein may be isolated from bacteria. It is also contemplated that bacteria containing such mutants may be used in the compositions and methods. Thus, the protein need not be isolated.

[0049] It is contemplated that the composition contains about 0.001 mg to about 10 mg of total polypeptide, peptide, and / or protein per mL. Thus, the concentration of protein in the composition can be about, at least about, or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / mL, or any value within these ranges. Of these, about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% may be antibodies that bind to ZP4.

[0050] Antibodies, or preferably immunological portions of antibodies, can be chemically conjugated to other proteins or expressed as fusion proteins, and for purposes of this specification and the appended claims, all such fusion proteins are included in the definition of an antibody or immunological portion of an antibody.

[0051] This embodiment provides antibodies and antibody-like molecules against ZP4, as well as polypeptides and peptides that are conjugated with at least one agent to form antibody conjugates or payloads. To enhance the effectiveness of antibody molecules as diagnostic or therapeutic agents, it is common to conjugate, covalently bind, or complex at least one molecule or moiety of interest. Such molecules or moieties may be, but are not limited to, at least one effector molecule or reporter molecule. Effector molecules include molecules with desired activity (e.g., cytotoxic activity). Non-limiting examples of effector molecules conjugated to antibodies include toxins, therapeutic enzymes, antibiotics, radiolabeled nucleotides, etc. Meanwhile, a reporter molecule is defined as any moiety that can be detected by an assay. Non-limiting examples of reporter molecules conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles, or ligands (e.g., biotin).

[0052] Numerous methods for binding or conjugating antibodies to their conjugate moieties are known to those skilled in the art. Some conjugation methods use metal chelate complexes, such as the attachment of organic chelators such as diethylenetriaminepentaacetic anhydride (DTPA), ethylenetriaminetetraacetic acid, N-chloro-p-toluenesulfonamide, and / or tetrachloro-3,6-diphenylglycouril-3 to antibodies. Monoclonal antibodies can also be reacted with enzymes in the presence of binding agents such as glutaraldehyde or periodic acid. Conjugates with fluorescein markers are prepared in the presence of these binding agents or by reaction with isothiocyanates.

[0053] [IV. Cell therapy] Certain embodiments of the present disclosure relate to obtaining and administering cells to a subject as immunotherapy targeting cancer cells. These cells may deliver antibody compositions encompassed herein, but may or may not themselves be immune cells. In certain embodiments, the cells are immune cells. Examples of cells include T cells (including αβT cells or γδT cells), natural killer (NK) cells, immutable NKT (iNKT) cells, B cells, macrophages, stem cells of any type (including mesenchymal stem cells (MSCs) or induced pluripotent stem cells (iPS cells)), or dendritic cells.

[0054] Several basic approaches for the induction, activation, and expansion of functional effector T cells against tumors have been reported over the past 20 years. These include: autologous cells such as tumor-infiltrating lymphocytes (TILs); T cells activated ex vivo using autologous dendritic cells (DCs), lymphocytes, artificial antigen-presenting cells (APCs), or beads coated with T cell ligands and activating antibodies; cells isolated by capturing target cell membranes; allogeneic cells that naturally express T cell receptors (TCRs) against the host tumor; and non-tumor-specific autologous or allogeneic cells that have been genetically reprogrammed or "redirected" to express tumor-reactive TCRs or chimeric TCR molecules with antibody-like tumor recognition capabilities (so-called "T-bodies"). These approaches have given rise to a number of protocols for the preparation and immunization of T cells that can be used in the methods of the present disclosure.

[0055] A. Preparation of T Cells In some embodiments, the T cells are obtained from blood, bone marrow, lymph, or lymphoid organs. In some embodiments, the cells are human cells. These cells are typically primary cells, e.g., isolated directly from a subject or frozen after isolation from a subject. In some embodiments, the cells are selected from the overall T cell population, CD4 + cells, CD8 +The cells may include one or more of T cells or other cell types, including cells and subsets thereof (defined by function, activation state, maturity, differentiation potential, proliferation potential, recirculation potential, localization and / or persistence, antigen specificity, type of antigen receptor, presence in specific organs or compartments, marker or cytokine secretion profile, and / or differentiation stage). With respect to the subject to be treated, these cells may be allogeneic or autologous. In some embodiments, for use in off-the-shelf technologies, the cells are pluripotent and / or multipotent, e.g., stem cells, e.g., induced pluripotent stem cells (iPSCs). In certain embodiments, the method involves isolating the cells from a subject, preparing, processing, culturing, and / or genetically modifying them, and reintroducing them into the same patient, before or after cryopreservation.

[0056] T cells (e.g., CD4 + and / or CD8 + T cell) subtypes and subpopulations include naive T cells (T N cells), effector T cells (T EFF ), memory T cells and their subtypes (stem cell-like memory T cells (TSCs) M ), central memory T cells (T CM ), effector memory T cells (T EM ), terminally differentiated effector memory T cells (T TEMRA )), tumor-infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T cells (MAITs), innate and adaptive regulatory T cells (Tregs), TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, α / β T cells, and δ / γ T cells.

[0057] In certain embodiments, one or more T cell populations are enriched for cells that are positive for a particular marker (positive selection) or depleted for cells that are negative for a particular marker (negative selection). In some cases, the marker is one that is absent or expressed at low levels in some T cell populations (e.g., non-memory cells) but present or expressed at high levels in other T cell populations (e.g., memory cells). In certain embodiments, cells (e.g., CD8 + cells or CD3 + The cells) are enriched for cells positive for or with high surface expression of CD45RO, CCR7, CD28, CD27, CD44, CD127, and / or CD62L, and / or depleted for cells positive for or with high surface expression of CD45RA. In certain embodiments, the cells are enriched or depleted for cells positive for or with high surface expression of CD122, CD95, CD25, CD27, and / or IL7-Rα (CD127). In some examples, CD8 + T cells are enriched for CD45RO positive (or CD45RA negative) and CD62L positive cells.

[0058] In one embodiment, T cells are isolated from a PBMC sample by negative selection based on markers expressed on non-T cells (e.g., B cells, monocytes, other leukocytes, such as CD14). + or CD8 + Using a selection step, CD4 + Helper T cells and CD8 + Isolate cytotoxic T cells, such as CD4 + and CD8 + The population can be further sorted into subpopulations by positive or negative selection based on markers that are expressed at high levels in one or more of the naive, memory and / or effector T cell subpopulations.

[0059] In one embodiment, CD8 +The cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells by positive or negative selection based on surface antigens associated with the subpopulation. In one embodiment, central memory T (T CM Enrichment for T ) cells is performed to improve efficacy, such as improving long-term survival, proliferation, and / or engraftment after transplantation, which is particularly pronounced in certain aspects for that subpopulation. In one embodiment, CM enriched CD8 + T cells and CD4 + Combining T cells further improves efficacy.

[0060] In one embodiment, the T cells are autologous T cells. In this method, a tumor specimen is obtained from a patient and a single cell suspension is prepared. The single cell suspension can be obtained by any suitable method, for example, by mechanical means (e.g., tumor dissociation using a gentleMACS® Dissociator from Miltenyi Biotec, Auburn, Calif.) or enzymatic means (e.g., collagenase or DNase). The single cell suspension of the tumor enzymatic digest is cultured in the presence of interleukin-2 (IL-2). Cells are cultured until confluent (e.g., approximately 2 x 10 6 The cells are cultured for about 5 to about 21 days, preferably about 10 to about 14 days. For example, the cells can be cultured from 5, 5.5, or 5.8 days to 21, 21.5, or 21.8 days, or from 10, 10.5, or 10.8 days to 14, 14.5, or 14.8 days.

[0061] The cultured T cells can be pooled and rapidly expanded. Rapid expansion increases the number of antigen-specific T cells by at least about 50-fold (e.g., 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more) in about 10 to about 14 days, preferably about 14 days. More preferably, rapid expansion increases the number of antigen-specific T cells by at least about 200-fold (e.g., 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, or more) in about 10 to about 14 days, preferably about 14 days.

[0062] Expansion can be achieved by any of a number of methods known to those skilled in the art. For example, T cells can be rapidly expanded using nonspecific T cell receptor stimulation in the presence of feeder lymphocytes and interleukin 2 (IL-2) or interleukin 15 (IL-15), with IL-2 being preferred. Nonspecific T cell receptor stimulation includes OKT3 (a murine monoclonal anti-CD3 antibody available from Ortho-McNeil®, Raritan, NJ) at approximately 30 ng / mL. Alternatively, T cells can be rapidly expanded by in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with one or more cancer antigens (including antigenic portions such as epitopes and cells), which may be expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2)-binding peptide, in the presence of a T cell growth factor (e.g., 300 IU / mL IL-2 or IL-15, preferably IL-2). In vitro induced T cells can be rapidly expanded by restimulating HLA-A2-expressing antigen-presenting cells with the same cancer antigen. Alternatively, T cells can be stimulated with irradiated autologous lymphocytes or HLA-A2 + They can be restimulated with allogeneic lymphocytes and IL-2.

[0063] Autologous T cells can be modified to express T cell growth factors that promote the proliferation and activation of autologous T cells. Suitable T cell growth factors include, for example, interleukin (IL)-2, IL-7, IL-15, and IL-12. Suitable modification methods are known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY, 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. In certain embodiments, the modified autologous T cells express high levels of T cell growth factors. The coding sequences of T cell growth factors such as IL-12 are known to those skilled in the art, as are the operative links of promoters that promote high-level expression.

[0064] (B. Genetically Engineered Antigen Receptors) The cells can be genetically engineered to express a modified antigen receptor, such as a modified TCR or chimeric antigen receptor (CAR). For example, autologous T cells can be engineered to express a T cell receptor (TCR) with antigen specificity for a cancer antigen (e.g., ZP4). Suitable TCRs include those with antigen specificity for melanoma antigens, i.e., gp100 or MART-1. Suitable engineering methods are known to those skilled in the art. See, e.g., Sambrook and Ausubel, supra. For example, T cells can be transduced to express a TCR with antigen specificity for a cancer antigen using the transduction techniques described in Heemskerk et al., Hum Gene Ther. 19:496-510 (2008) and Johnson et al., Blood 114:535-546 (2009).

[0065] In some embodiments, the T cells contain one or more genetically engineered nucleic acids, including one or more nucleic acids encoding one or more antigen receptors and genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are exogenous, i.e., not normally present in the cell or a sample obtained from the cell, e.g., obtained from another organism or cell, and not normally found in the modified cell and / or its derived organism. In some embodiments, the nucleic acids are non-natural, e.g., non-naturally occurring chimeric nucleic acids.

[0066] In some embodiments, CAR comprises an extracellular antigen recognition domain that specifically binds to ZP4.In some embodiments, the antigen is a protein that is expressed on the cell surface.In some embodiments, CAR is a TCR-like CAR, and the antigen is a processed peptide antigen, i.e., a peptide antigen derived from intracellular protein, which is recognized on the cell surface in the context of major histocompatibility complex (MHC) molecules, similar to TCR.

[0067] Representative antigen receptors, including CARs and recombinant TCRs, and methods for introducing and modifying these receptors into cells are described, for example, in International Published Patent Application Publication Nos. WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publication Nos. US2002131960, US2013287748 ... 130149337, U.S. Patent Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European Patent Application No. EP2537416, or those described in Sadelain et al., 2013, Davila et al., 2013, Turtle et al., 2012, Wu et al., 2012. In some embodiments, the engineered antigen receptors include CARs described in U.S. Patent No. 7,446,190, as well as those described in International Published Patent Application Publication No. WO / 2014 / 055668 A1.

[0068] <1. Chimeric Antigen Receptor> In one embodiment, the CAR comprises: (a) an intracellular signaling domain, (b) a transmembrane domain, and (c) an extracellular domain comprising an antigen-binding domain.

[0069] In certain embodiments, modified antigen receptors include CARs, including activating or stimulatory CARs, costimulatory CARs (see WO2014 / 055668), and / or inhibitory CARs (iCARs, see Fedorov et al., 2013). CARs generally comprise an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components, in certain embodiments linked via a linker and / or transmembrane domain. Such molecules typically mimic or approximate signaling via a natural antigen receptor, the combination of that receptor and a costimulatory receptor, or a costimulatory receptor alone.

[0070] Certain embodiments of the present disclosure relate to the use of nucleic acids, including nucleic acids encoding antigen-specific CAR polypeptides (including humanized CARs (hCARs) to reduce immunogenicity), which comprise an intracellular signaling domain, a transmembrane domain, and an extracellular domain containing one or more signal motifs. In certain embodiments, the CAR can recognize an epitope comprising a shared space between one or more antigens. In certain embodiments, the binding site can comprise a complementarity-determining region of a monoclonal antibody, a variable region of a monoclonal antibody, and / or an antigen-binding fragment thereof. In another embodiment, the specificity is derived from a peptide (e.g., a cytokine) that binds to a receptor.

[0071] In the present disclosure, the human CAR nucleic acid is assumed to be a human gene used to enhance cellular immunotherapy for human patients. In certain embodiments, the present disclosure includes a full-length CAR cDNA or coding region. The antigen-binding domain or domains can include fragments of the VH and VL chains of a single-chain variable fragment (scFv) derived from a specific human monoclonal antibody. For example, those described in U.S. Patent No. 7,109,304 (incorporated herein by reference). The fragments can be any of the antigen-binding domains of a human antigen-specific antibody. In a more specific embodiment, the fragments are antigen-specific scFvs encoded by a sequence optimized for human codon usage for expression in human cells.

[0072] The construct may be in a multimeric form, such as a diabody or multimer. Multimers are most likely formed as diabodies by cross-pairing of the light and heavy chain variable regions. Several options exist for the hinge region of the construct, ranging from complete deletion, maintaining the first cysteine ​​residue, substituting proline for serine, or truncating up to the first cysteine. The Fc portion may be deleted. Any protein that is stable and / or dimerizes may be used for this purpose. For example, one of the Fc domains (either the CH2 or CH3 domain from a human immunoglobulin) may be used, or the hinge, CH2, and CH3 regions of a human immunoglobulin modified to improve dimerization may be used. Only the hinge portion of an immunoglobulin may be used, or a portion of CD8α may be used.

[0073] In one embodiment, the CAR nucleic acid comprises a sequence encoding another costimulatory receptor, such as a transmembrane domain and a modified CD28 intracellular signaling domain, including, but not limited to, one or more of CD28, CD27, OX-40 (CD134), and 4-1BB (CD137).

[0074] In some embodiments, CARs are constructed with specificity for a particular antigen (or marker, ligand), such as an antigen expressed on a particular cell type to be targeted by adoptive immunotherapy (e.g., a cancer marker) and / or an antigen expressed on a normal or non-diseased cell type intended to induce a suppressive response. To this end, CARs generally comprise one or more antigen-binding molecules (e.g., one or more antigen-binding fragments, domains, portions, or one or more antibody variable domains and / or antibody molecules) in their extracellular portion. In some embodiments, CARs comprise the antigen-binding domain or a portion thereof of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy chain (VH) and variable light chain (VL) of a monoclonal antibody (mAb).

[0075] The sequence of the open reading frame encoding the chimeric receptor can be obtained from genomic DNA sources, cDNA sources, or synthesized (e.g., via PCR), or a combination thereof. Depending on the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof, as introns have been shown to stabilize mRNA. It may also be advantageous to use endogenous or exogenous non-coding regions to enhance mRNA stability.

[0076] It is believed that the chimeric construct can be introduced into immune cells by nude DNA or suitable vector.Methods for stably transfecting cells by electroporation using nude DNA are known to those skilled in the art (see, for example, U.S. Patent No. 6,410,319).Nude DNA generally refers to the DNA encoding the chimeric receptor contained in a plasmid expression vector configured in the appropriate direction for expression.In some embodiments, RNA can be transfected via lipid nanoparticles.

[0077] Alternatively, chimeric constructs can be introduced into immune cells using viral vectors (for example, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, or lentiviral vectors).Suitable vectors used according to the method of the present disclosure are replication-incompetent in immune cells.Many virus-based vectors are known, which keep the virus copy number in cells low enough to maintain cell viability, for example, include HIV, SV40, EBV, HSV, or BPV-based vectors.

[0078] In some embodiments, the antigen-specific binding or recognition component is linked to one or more transmembrane and intracellular signaling domains.In some embodiments, CAR comprises a structure in which a transmembrane domain is fused to the extracellular domain of the CAR.In some embodiments, the transmembrane domain that is naturally linked to one of the domains contained in the CAR is used.In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding with the transmembrane domain of the same or different cell membrane protein and minimize interaction with other components of the receptor complex.

[0079] In one embodiment, the transmembrane domain is derived from either natural or synthetic sources. If the source is natural, the domain can be obtained from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e., including at least) the transmembrane regions of the α, β, or ζ chains of the T cell receptor, CD28, CD3ζ, CD3ε, CD3γ, and CD3δ. In another embodiment, the transmembrane domain is synthetic. In one embodiment, a synthetic transmembrane domain is composed primarily of hydrophobic residues such as leucine and valine. In one embodiment, a synthetic transmembrane domain has a triadic motif of phenylalanine, tryptophan, and valine at each end.

[0080] 2. T cell receptor (TCR) In one embodiment, the genetically engineered antigen receptor comprises a recombinant TCR and / or a TCR cloned from a naturally occurring T cell. "T cell receptor" or "TCR" refers to a molecule that contains variable α and β chains (also known as TCRα and TCRβ, respectively) or variable γ and δ chains (also known as TCRγ and TCRδ, respectively), and is capable of specifically binding to an antigenic peptide bound to an MHC receptor. In one embodiment, the TCR is an αβ type.

[0081] While αβ and γδ TCRs are generally structurally similar, the T cells that express them may have differences in anatomical location or function. TCRs can exist on the cell surface or in soluble forms. Generally, TCRs are present on the surface of T cells (or T lymphocytes) and are responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. In certain embodiments, TCRs can contain a constant domain, a transmembrane domain, and / or a short cytoplasmic tail (see, e.g., Janeway et al., 1997). For example, in certain aspects, each chain of a TCR can have an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a C-terminal short cytoplasmic tail. In certain embodiments, TCRs are associated with invariant proteins of the CD3 complex, which are involved in mediating signal transduction. Unless otherwise specified, the term "TCR" should be understood to encompass functional fragments thereof. The term also encompasses complete or full-length TCRs, including αβ and γδ TCRs.

[0082] Thus, reference herein to a TCR is intended to include a TCR or functional fragment thereof (e.g., antigen-binding domain) that binds to an MHC molecule bound to a specific antigen peptide, i.e., an MHC-peptide complex. The terms "antigen-binding domain" or "antigen-binding fragment" can be used interchangeably and refer to a molecule that includes a portion of the structural domain of a TCR but is capable of binding to the antigen (e.g., an MHC-peptide complex) bound by the intact TCR. In some cases, the antigen-binding domain includes the variable domain of a TCR, e.g., the variable α chain and variable β chain of the TCR, each chain containing three complementarity-determining regions sufficient to form a binding site for binding to a specific MHC-peptide complex.

[0083] In some embodiments, the variable domains of TCR chains associate to form immunoglobulin-like loops, i.e., complementarity-determining regions (CDRs), which confer antigen recognition ability and define the binding site of the TCR molecule, thereby determining peptide specificity. Typically, as in immunoglobulins, CDRs are separated by framework regions (FRs) (see, e.g., Jores et al., 1990; Chothia et al., 1988; Lefranc et al., 2003). In some embodiments, CDR3 is the CDR primarily responsible for recognizing processed antigens, while CDR1 of the α chain has been shown to interact with the N-terminal portion of antigenic peptides, and CDR1 of the β chain interacts with the C-terminal portion of peptides. CDR2 is thought to recognize MHC molecules. In some embodiments, the variable region of the β chain may further comprise a hypervariable region (HV4).

[0084] In some embodiments, a TCR chain comprises a constant domain. For example, similar to an immunoglobulin, the extracellular portion of a TCR chain (e.g., an α chain, a β chain) can comprise two immunoglobulin domains: an N-terminal variable domain (e.g., Va or Vp, typically amino acids 1-116 according to Kabat numbering; Kabat et al., "Sequences of Proteins of Immunological Interest," U.S. Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.) and a constant domain adjacent to the cell membrane (e.g., an α chain constant domain, or Ca, amino acids 117-259 according to Kabat; a β chain constant domain, or Cp, amino acids 117-295). For example, in some cases, the extracellular portion of a TCR formed by two chains comprises two membrane-proximal constant domains and two membrane-distal variable domains containing CDRs. The constant domain of a TCR comprises a short connective sequence containing cysteine ​​residues that form disulfide bonds linking the two chains. In one embodiment, the TCR has an additional cysteine ​​residue in each of the α and β chains and contains two disulfide bonds in the constant domain.

[0085] In some embodiments, the TCR chain may comprise a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain comprises a cytoplasmic tail. In some cases, this structure allows the TCR to associate with other molecules, such as CD3. For example, a TCR comprising a constant domain with a transmembrane region may anchor the protein to the cell membrane and associate with the invariant subunit of the CD3 signaling apparatus or complex.

[0086] Generally, CD3 is a multiprotein complex containing three distinct chains (γ, δ, ε) and a ζ chain. For example, in mammals, it can contain a homodimer of CD3γ, CD3δ, two CD3ε chains, and a CD3ζ chain. The CD3γ, CD3δ, and CD3ε chains are cell surface proteins belonging to the immunoglobulin superfamily, each containing a single immunoglobulin domain, and are highly similar to each other. The transmembrane regions of the CD3γ, CD3δ, and CD3ε chains are negatively charged, a feature that allows these chains to associate with positively charged T cell receptor chains. The cytoplasmic tails of the CD3γ, CD3δ, and CD3ε chains each contain one conserved motif known as an immunoreceptor tyrosine-based activation motif (ITAM), and the CD3ζ chain each contains three ITAMs. Generally, ITAMs are involved in the signaling function of the TCR complex. These accessory molecules have negatively charged transmembrane regions and contribute to the propagation of signals from the TCR into the cell. The CD3 and ζ chains, together with the TCR, form a structure called the T cell receptor complex.

[0087] In some embodiments, the TCR may be a heterodimer of two chains, α and β (or optionally γ and δ), or may be a single-chain TCR construct. In some embodiments, the TCR is a heterodimer comprising two independent chains (α and β, or γ and δ), linked, for example, by a disulfide bond. In some embodiments, a TCR against a target antigen (e.g., a cancer antigen) is identified and introduced into a cell. In some embodiments, nucleic acids encoding the TCR may be obtained from various sources, for example, by polymerase chain reaction (PCR) amplification of a known TCR DNA sequence. In some embodiments, the TCR may be obtained from a biological source, for example, a T cell (e.g., a cytotoxic T cell), a T cell hybridoma, or cells from other publicly available sources. In some embodiments, the T cell may be obtained from cells isolated in vivo. In some embodiments, a high-affinity T cell clone is isolated from a patient and its TCR is isolated. In some embodiments, the T cell may be a cultured T cell hybridoma or clone. In some embodiments, TCR clones against target antigens are generated in transgenic mice that have been introduced with human immune system genes (e.g., human leukocyte antigen system, HLA). In some embodiments, phage display is used to isolate TCRs against target antigens. In some embodiments, TCRs or their antigen-binding domains can be synthetically generated from TCR sequence information.

[0088] (C. Installation method) Those skilled in the art will be fully able to construct vectors for expression of any antigen receptor of the present disclosure using standard recombinant techniques (see, e.g., Sambrook et al., 2001 and Ausubel et al., 1996, both of which are incorporated herein by reference). Vectors include, but are not limited to, plasmids, cosmids, viruses (bacteriophages, animal viruses, plant viruses), and artificial chromosomes (e.g., YACs). Examples include retroviral vectors (e.g., those derived from Moloney murine leukemia virus (MoMLV), MSCV, SFFV, MPSV, SNV, etc.), lentiviral vectors (e.g., those derived from HIV-1, HIV-2, SIV, BIV, FIV, etc.), adenoviral (Ad) vectors (including replication-competent, replication-defective, and genome-deleted Ad vectors), adeno-associated virus (AAV) vectors, simian polyoma virus 40 (SV-40) vectors, bovine papillomavirus vectors, Epstein-Barr virus vectors, herpesvirus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, mouse mammary tumor virus vectors, Ruth sarcoma virus vectors, parvovirus vectors, poliovirus vectors, vesiculostamatis virus vectors, Maraba virus vectors, and group B adenovirus-derived enadenotucirev vectors.

[0089] 1. Viral Vectors In some embodiments, the present disclosure may provide a viral vector encoding an antigen receptor. In creating a recombinant viral vector, non-essential genes are typically replaced with genes or coding sequences for foreign (or non-native) proteins. A viral vector is a type of expression construct that uses viral sequences to introduce nucleic acids and, optionally, proteins into cells. The ability of certain viruses to infect or enter cells via receptor-mediated endocytosis and integrate into the host cell genome, resulting in stable and efficient expression of viral genes, has made them promising candidates for transferring foreign nucleic acids into cells (e.g., mammalian cells). Non-limiting examples of viral vectors that can be used to deliver nucleic acids in certain embodiments of the present disclosure are provided below.

[0090] Lentiviruses are complex retroviruses that contain the common retroviral genes gag, pol, and env, as well as other genes with regulatory or structural functions. Lentiviral vectors are well known to those skilled in the art (see, e.g., U.S. Patent Nos. 6,013,516 and 5,994,136).

[0091] Recombinant lentiviral vectors can infect non-dividing cells and can be used for gene transfer and expression of nucleic acid sequences both in vivo and ex vivo. For example, recombinant lentiviruses capable of infecting non-dividing cells, in which suitable host cells for the lentivirus are transfected with two or more vectors carrying rev and tat in addition to packaging functions (i.e., gag, pol, and env), are described in U.S. Patent No. 5,994,136, which is incorporated herein by reference.

[0092] 2. Preparation sequence The expression cassette contained in a vector useful in the present disclosure contains, inter alia, a eukaryotic transcriptional promoter operably linked to a protein-coding sequence, splice signals containing intronic sequences, and transcription termination / polyadenylation sequences, in a 5' to 3' direction. Promoters and enhancers that control the transcription of protein-coding genes in eukaryotic cells are composed of multiple genetic elements. The intracellular machinery can collect and integrate the regulatory information conveyed by each element, allowing different genes to acquire often complex transcriptional regulation patterns over time. Promoters used in the context of the present disclosure include constitutive promoters, inducible promoters, and tissue-specific promoters.

[0093] [a. Promoter / Enhancer] The expression constructs provided herein include a promoter for driving the expression of an antigen receptor. Promoters generally contain sequences that function to position the start point of RNA synthesis. The most well-known example is the TATA box, but some promoters lacking a TATA box, such as the promoters of the mammalian terminal deoxynucleotidyl transferase gene and the SV40 late gene promoter, have distinct elements that overlap the start point itself to help fix the start position. Additional promoter elements control the frequency of transcription initiation. These are usually located upstream of the start point (within the range of 30–110 bp), although some promoters have been shown to contain functional elements downstream of the start point. To place a coding sequence "under the control" of a promoter, the 5' end of the transcription start point of the transcriptional reading frame must be positioned "downstream" (i.e., 3') of the selected promoter. An "upstream" promoter promotes DNA transcription and expression of the encoded RNA.

[0094] Spacing between promoter elements is often flexible, and promoter function can be maintained even when elements are inverted or shifted relative to one another. For example, in the tk promoter, spacing between promoter elements can be increased by up to 50 bp before activity begins to decline. Depending on the promoter, individual elements appear to function cooperatively or independently to activate transcription. Promoters may or may not be used in conjunction with "enhancers." The term "enhancer" refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[0095] A promoter can be one that is naturally associated with a nucleic acid sequence (e.g., obtained by isolating 5' non-coding sequences located upstream of the coding segment and / or exons), and such a promoter is sometimes referred to as "endogenous." Similarly, an enhancer can be one that is naturally associated with a nucleic acid sequence and can be located upstream or downstream of that sequence. Alternatively, certain advantages may be gained by placing a coding nucleic acid segment under the control of a recombinant or exogenous promoter. A "recombinant" or "exogenous" promoter refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A "recombinant" or "exogenous" enhancer similarly refers to an enhancer that is not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers include promoters or enhancers from other genes, from viruses, or from either prokaryotic or eukaryotic cells, as well as promoters or enhancers that are "non-naturally occurring," i.e., those that contain different elements of different transcriptional regulatory regions and / or contain mutations that alter expression. For example, among the most commonly used promoters in recombinant DNA construction are the β-lactamase (penicillinase), lactose, and tryptophan (trp) promoter systems. Promoter and enhancer nucleic acid sequences can be produced synthetically as well as using recombinant cloning and / or nucleic acid amplification techniques (including, in the context of the compositions of the present disclosure, PCR™). Furthermore, it is contemplated that control sequences that direct transcription and / or expression of sequences in non-nuclear organelles, such as mitochondria and chloroplasts, may also be used.

[0096] Naturally, it is important to use a promoter and / or enhancer that effectively directs the expression of the DNA segment in the desired organelle, cell type, tissue, organ, or organism. Those skilled in the art of molecular biology are generally familiar with the combination of promoters, enhancers, and cell types for protein expression (see, e.g., Sambrook et al., 1989, incorporated herein by reference). The promoter used may be constitutive, tissue-specific, inducible, or usefully used under appropriate conditions to direct high-level expression, which is advantageous in large-scale production of recombinant proteins and / or peptides. The promoter may be exogenous or endogenous.

[0097] Additionally, any promoter / enhancer combination (e.g., as described in the Eukaryotic Promoter Data Base EPDB (available on the World Wide Web at epd.isb-sib.ch / )) can be used to drive expression. Use of T3, T7, or SP6 cytoplasmic expression systems is another embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided as part of a delivery complex or as an additional gene expression construct.

[0098] Non-limiting examples of promoters include viral early or late promoters, such as the SV40 early or late promoter, the cytomegalovirus (CMV) immediate early promoter, and the Ruth sarcoma virus (RSV) early promoter; eukaryotic promoters, such as the β-actin promoter, the GADPH promoter, and the metallothionein promoter; and promoters linked to response elements, such as the cyclic AMP response element promoter (cre), the serum response element promoter (sre), the phorbol ester promoter (TPA), and the minimal TATA box proximal response element promoter (tre). Human growth hormone promoter sequences (e.g., the human growth hormone minimal promoter described in GenBank accession number X05244, nucleotides 283-341) and mouse mammary tumor promoters (available from the American College of Cancer (ATCC), catalog number ATCC 45007) can also be used. In certain embodiments, the promoter is a CMV IE, Dectin-1, Dectin-2, human CD11c, F4 / 80, SM22, RSV, SV40, Ad MLP, β-actin, MHC class I or MHC class II promoter, although any other promoter useful for driving expression of therapeutic genes is applicable to the present disclosure.

[0099] In certain embodiments, the methods of the present disclosure also relate to enhancer sequences, i.e., nucleic acid sequences that enhance promoter activity, are cis-acting, orientation-independent, and have the potential to function over relatively long distances (several kilobases from the target promoter), although enhancer function is not necessarily limited to such long distances and may also function in the proximal region of a particular promoter.

[0100] b. Initiation signal and linked expression Specific initiation signals may also be used in the expression constructs provided herein for efficient translation of the coding sequence. These signals include the ATG start codon or its adjacent sequences. Exogenous translational control signals (including the ATG start codon) may need to be provided. This is within the skill of the art and would be readily apparent to one skilled in the art. It is well known that the initiation codon must be located "in frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert. Exogenous translational control signals and initiation codons may be natural or synthetic. The efficiency of expression can be enhanced by including appropriate transcriptional enhancer elements.

[0101] In certain embodiments, internal ribosome entry site (IRES) elements may be used to create multigene or polycistronic messages. IRES elements can bypass the ribosome scanning model of 5' methylated cap-dependent translation and initiate translation at internal sites. IRES elements from two members of the picornaviridae family (poliovirus and encephalomyocarditis virus) and from mammalian messages have been described. IRES elements can be linked to exogenous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, to form polycistronic messages. IRES elements ensure that each open reading frame is available for efficient translation by the ribosome. Multiple genes can be efficiently expressed as a single message using a single promoter / enhancer.

[0102] Additionally, specific 2A sequence elements can be used in the constructs provided herein to create linked or co-expression of genes. For example, cleavage sequences can be used to achieve co-expression of genes by linking open reading frames to form a single cistron. Exemplary cleavage sequences include F2A (foot-and-mouth disease virus 2A) or "2A-like" sequences (e.g., Thosea asigna virus 2A; T2A).

[0103] <3. Replication origin> To allow a vector to replicate in a host cell, it may contain one or more origin of replication sites (often referred to as "ori"). For example, it may contain a nucleic acid sequence corresponding to the oriP derived from Epstein-Barr virus (EBV) described above, or a genetically engineered oriP designed to have similar or superior functions. This is a specific nucleic acid sequence from which replication begins. Alternatively, an origin of replication derived from another extrachromosomally replicating virus or an autonomously replicating sequence (ARS) can also be used.

[0104] <4. Selectable and Screenable Markers> In certain embodiments, cells containing the constructs of the present disclosure can be identified in vitro or in vivo by including a marker in the expression vector. Such a marker confers a distinguishable change to the cell, allowing for easy identification of cells containing the expression vector. Generally, a selection marker is a marker that confers a selectable property. A positive selection marker is one in which the presence of the marker allows selection, while a negative selection marker is one in which the presence of the marker inhibits selection. An example of a positive selection marker is a drug resistance marker.

[0105] Typically, the inclusion of a drug selection marker facilitates cloning and identification of transformants. For example, genes conferring resistance to neomycin, puromycin, hydomycin, dihydrofolate reductase (DHFR), guanylate phosphoribosyltransferase (GPT), zeocin, and histidinol are useful selection markers. In addition to markers that confer phenotypes that allow for the identification of transformants based on the implementation of conditions, other types of markers are contemplated, such as colorimetric screenable markers like GFP. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) and chloramphenicol acetyltransferase (CAT) can be used as negative selection markers. Those skilled in the art will also understand how to use immunological markers in conjunction with FACS analysis. The marker used is not considered critical, so long as it is capable of being expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selection and screenable markers are widely known to those skilled in the art.

[0106] <5. Other nucleic acid transfer methods> In addition to viral delivery of nucleic acids encoding antigen receptors, the following methods for recombinant gene transfer into a given host cell also exist and are therefore contemplated in this disclosure.

[0107] Nucleic acids (such as DNA or RNA) can be introduced into the immune cells of the present disclosure using any method suitable for nucleic acid delivery for cell transformation, including those described herein or known to those skilled in the art. Such methods include, but are not limited to, direct delivery of DNA by ex vivo transfection, injection (including microinjection), electroporation, calcium phosphate precipitation, the use of DEAE-dextran followed by polyethylene glycol, direct sonication, liposome-mediated transfection and receptor-mediated transfection, microprojectile bombardment, silicon carbide fiber agitation, Agrobacterium-mediated transformation, desiccation / inhibition-mediated DNA uptake, and any combination of these methods. By applying these techniques, organelles, cells, tissues, or organisms can be stably or transiently transformed.

[0108] [V. Treatment method] Certain aspects of this embodiment can be used to prevent or treat diseases or disorders related to ZP4 signal transduction, including those in which killing ZP4-positive cells improves at least one symptom of the disease or disorder.ZP4 signal transduction can be reduced by any suitable composition to suppress the proliferation of cancer cells.In a specific example, the substance is anti-ZP4 antibody or anti-ZP4 CAR-expressing cell.

[0109] In certain embodiments, the present disclosure provides a method of immunotherapy comprising administering an effective amount of an antibody of the present disclosure, a composition comprising at least CAR T cells. In certain embodiments, a medical disease or disorder is treated by administering a cell population expressing a CAR to induce an immune response. Furthermore, in certain embodiments of the present disclosure, cancer is treated by administering a CAR immune cell population to induce an immune response. Provided herein is a method of treating or delaying the progression of cancer, comprising administering to an individual an effective amount of antigen-specific cell therapy. The method can be applied to the treatment of immune disorders, solid cancers, and hematological cancers. Specifically, the cancer can be a B-cell malignancy, including, for example, B-cell acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, Burkitt's lymphoma, and chronic lymphocytic leukemia.

[0110] Tumors for which the present therapeutic methods are useful include any malignant cell type found in solid tumors or hematologic tumors. Exemplary solid tumors include, but are not limited to, tumors in organs selected from the group consisting of the pancreas, colon, cecum, stomach, brain, head, neck, ovaries, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. Exemplary hematologic tumors include tumors of the bone marrow, T- or B-cell malignancies, leukemia, lymphoma, blastoma, myeloma, and the like. Further examples of cancers treatable by the methods provided herein include, but are not limited to, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, stomach or gastric cancer (including gastrointestinal cancer and gastrointestinal stromal tumors), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, ductal carcinoma, vulvar cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0111] The cancer may be of the following histological types, but is not limited to: malignant neoplasm; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; mixed hepatocellular and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; adenocarcinoma in familial polyposis coli; parenchymal carcinoma; malignant carcinoid tumor; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma ;Esinophilic carcinoma;Esinophilic pigmented adenocarcinoma;Basophilic carcinoma;Clear cell adenocarcinoma;Granular cell carcinoma;Follicle adenocarcinoma;Papillary and follicular adenocarcinoma;Non-encapsulating sclerosing carcinoma;Adrenal cortical carcinoma;Endometrioid carcinoma;Cutaneous adenocarcinoma;Apocrine gland carcinoma;Sebaceous gland adenocarcinoma;Ear gland adenocarcinoma;Mucoepidermoid carcinoma;Cystadenocarcinoma;Papillary cystadenocarcinoma;Papillary serous cystadenocarcinoma;Mucinous cystadenocarcinoma;Mucinous adenocarcinoma;Signet ring cell carcinoma;Invasive ductal carcinoma;Medullary carcinoma;Lobular carcinoma;Inflammatory carcinoma;Paget's disease (breast);Acinic cell carcinoma;Adenosquamous carcinoma;Adenocarcinoma with squamous metaplasia;Malignant thymoma;Malignant ovarian stromal tumor;Malignant secoma;Malignant granulosa cell tumor;Malignant androblastoma;Certo Leydig cell carcinoma; Malignant Leydig cell tumor; Malignant lipid cell tumor; Malignant paraganglioma; Extramammary malignant paraganglioma; Pheochromocytoma; Glomangiosarcoma; Malignant melanoma; Non-pigmented melanoma; Superficial spreading melanoma; Lentigo malignant melanoma; Acral lentiginous melanoma; Nodular melanoma; Malignant melanoma within giant pigmented nevus; Epithelioid cell melanoma; Malignant blue nevus; Sarcoma; Fibrosarcoma; Malignant fibrous histiocytoma; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonic rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Malignant mixed tumor; Müllerian mixed tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma; Malignant mesenchymal tumor; Malignant Brenner tumor; Malignant philoid tumor; Synovial sarcoma; Malignant mesothelioma; Atypical germinoma (dysgerminoma); Germ cell carcinoma; Malignant teratoma; Malignant struma ovari; Choriocarcinoma; Malignant mesonephroma; Angiosarcoma; Malignant hemangioendothelioma; Kaposi's sarcoma; Malignant hemangiopericytoma; Malignant lymphangiosarcoma; Osteosarcoma; Subcortical osteosarcoma; Chondrosarcoma; Malignant chondroblastoma; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Malignant odontogenic tumor; Ameloblastic odontogenic sarcoma; Malignant ameloblastoma; Ameloblastic fibrosarcoma; Malignant pinealoma; Chordoma; Malignant glioma; Ependymoma; Astrocytoma; Protoplasmic astrocytoma;Fibrous astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal tumor; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory nerve tumor; malignant meningioma; neurofibrosarcoma; malignant neurilemmoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's type; paragranulomatous type; small lymphocytic lymphoma; large cell diffuse lymphoma; follicular lymphoma; mycosis fungoides; non-Hodgkin's lymphoma otherwise specified; B-cell lymphoma; low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL ;High-grade lymphoblastic NHL;High-grade small anaplastic cell NHL;Bulky mass NHL;Mantle cell lymphoma;AIDS-related lymphoma;Waldenstrom's macroglobulinemia;Malignant histiocytosis;Multiple myeloma;Mast cell sarcoma;Immunoproliferative small intestinal disease;Leukemia;Lymphocytic leukemia;Plasma cell leukemia;Erythroleukemia;Lymphosarcomatous cell leukemia;Myeloid leukemia;Basophilic leukemia;Eosinophilic leukemia;Monocytic leukemia;Mast cell leukemia;Megakaryoblastic leukemia;Myeloid sarcoma;Hairy cell leukemia;Chronic lymphocytic leukemia (CLL);Acute lymphoblastic leukemia (ALL);Acute myeloid leukemia (AML);Chronic myeloid leukemia.

[0112] Certain embodiments relate to methods for treating leukemia. Leukemia is a cancer of the blood or bone marrow, usually characterized by abnormal proliferation (production by growth) of leukocytes (white blood cells). It is part of a group of diseases called hematological malignancies. Leukemia is a broad term that encompasses a wide range of diseases. Leukemia is clinically and pathologically classified into acute and chronic forms.

[0113] In some embodiments of the disclosed methods, activated CD4 and / or CD8 T cells in an individual are characterized as γ-IFN-producing CD4 and / or CD8 T cells and / or by having enhanced cytotoxic activity compared to before administration. γ-IFN can be measured by any means known to those skilled in the art, such as intracellular cytokine staining (ICS), which involves cell fixation, permeabilization, and staining with an antibody against γ-IFN. Cytotoxic activity can also be measured by any means known to those skilled in the art, such as a cell killing assay using mixed effector and target cells.

[0114] In some embodiments, a subject may be administered non-myeloablative lymphodepleting chemotherapy prior to T cell therapy. The non-myeloablative lymphodepleting chemotherapy may be any suitable therapy and may be administered by any suitable route. Non-myeloablative lymphodepleting chemotherapy may include, for example, administration of cyclophosphamide and fludarabine, which is particularly suitable when the cancer is melanoma, which may be metastatic. An example of a route of administration for cyclophosphamide and fludarabine is intravenous. Similarly, any suitable dose of cyclophosphamide and fludarabine may be administered. In certain aspects, approximately 60 mg / kg of cyclophosphamide is administered for two days, followed by approximately 25 mg / m 2 of fludarabine is administered for 5 days.

[0115] In certain embodiments, a T cell growth factor that promotes the proliferation and activation of autologous T cells is administered to a subject simultaneously with or after the administration of autologous T cells. The T cell growth factor can be any suitable growth factor that promotes the proliferation and activation of autologous T cells. Examples of suitable T cell growth factors include interleukin (IL)-2, IL-7, IL-15, and / or IL-12, which can be used alone or in various combinations, such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL-2. IL-12 is a particularly important T cell growth factor.

[0116] Therapeutically effective amounts of immune cells can be administered by multiple routes of administration, including, for example, intravenous, intraperitoneal, intramuscular, intrasternal or intraarticular injection, or parenteral administration such as infusion.

[0117] Intratumoral injection, or injection into the tumor vasculature, is particularly contemplated for well-defined, solid, and accessible tumors. Local, regional, or systemic administration may also be appropriate. For tumors larger than 4 cm, a dose volume of approximately 4 to 10 ml (especially 10 ml) is appropriate, while for tumors smaller than 4 cm, a dose volume of approximately 1 to 3 ml (especially 3 ml) is used. Multiple injections administered as a single dose may contain a volume of about 0.1 to about 0.5 ml.

[0118] The T cell population can be administered in a treatment regimen depending on the nature of the disease. For example, it can be administered once or several times over several days to improve the disease state, or it can be administered periodically over a long period of time to suppress the progression of the disease and prevent recurrence. The exact dosage used will depend on the route of administration and the severity of the disease or disorder, and should be determined according to the judgment of the practitioner and each patient's circumstances. The therapeutically effective amount of T cells will depend on the subject being treated, the type and severity of the disease, and the method of administration. In some embodiments, the dosage used for treatment in human subjects is at least 3.8 x 10 4 , at least 3.8 × 10 5 , at least 3.8 × 10 6 , at least 3.8 × 10 7 , at least 3.8 × 10 8 , at least 3.8 × 10 9 , or at least 3.8 × 10 10 T cells / m 2 In certain embodiments, the dosage used to treat a human subject may range from about 3.8×10 9 to approximately 3.8 x 10 10 T cells / m 2 In yet another embodiment, the therapeutically effective amount of T cells is in the range of about 5 x 10 per kg of body weight. 6 Approximately 7.5 × 10 cells 8 range of cells, e.g., about 2 x 10 7 Approximately 5 × 10 cells 8 cells / kg body weight, or approximately 5 x 10 7 Approximately 2 x 10 cells 8 The range is 100 cells / kg body weight. The exact amount of T cells can be easily determined by one skilled in the art based on the age, weight, sex and physiological condition of the subject. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0119] In certain embodiments of the present disclosure, an effective amount of immune cells expressing ZP4 CAR is delivered to an individual in need, for example, an individual with cancer.These cells strengthen the individual's immune system to attack cancer cells.In some cases, the individual is provided with immune cells one or more times.When the individual is provided with immune cells more than once, the interval between administrations should be long enough to allow the cells to grow in the body, and in certain embodiments, the interval between administrations is 1, 2, 3, 4, 5, 6, 7 days or more.

[0120] In certain embodiments, cells genetically modified to express ZP4 CAR are administered in a therapeutically effective amount (10 3 ~10 10 A therapeutically effective amount is provided to an individual in the range of 10 3 ~10 10 , 10 3 ~10 9 , 10 3 ~10 8 , 10 3 ~10 7 , 10 3 ~10 6 , 10 3 ~10 5 , 10 3 ~10 4 , 10 4 ~10 10 , 10 4 ~10 9 , 10 4 ~10 8 , 10 4 ~10 7 , 10 4 ~10 6 , 10 4 ~10 5 , 10 5 ~10 10 , 10 5 ~10 9 , 10 5 ~10 8 , 10 5 ~10 7 , 10 5 ~10 6 , 10 6 ~1010 , 10 6 ~10 9 , 10 6 ~10 8 , 10 6 ~10 7 , 10 7 ~10 10 , 10 7 ~10 9 , 10 7 ~10 8 , 10 8 ~10 10 , 10 8 ~10 9 , or 10 9 ~10 10 Thus, in certain embodiments, an individual with certain cancers is provided one or more times with a therapeutically effective amount of cells expressing a ZP4 CAR.

[0121] A. Pharmaceutical Compositions Also provided herein are pharmaceutical compositions and formulations comprising cells expressing a CAR and a pharmaceutically acceptable carrier.

[0122] The pharmaceutical compositions and formulations described herein can be prepared in the form of a lyophilized formulation or aqueous solution by mixing an active ingredient (e.g., an antibody or polypeptide) having the desired purity with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 22nd ed., 2012). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, m-cresol, etc.). steroids); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and non-ionic surfactants such as polyethylene glycol (PEG). Further exemplary pharmaceutically acceptable carriers herein include interstitial drug dispersing agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), including, for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). In one aspect, the sHASEGP is combined with one or more other glycosaminoglycanases, such as chondroitinases.

[0123] (B. Combination Therapy) In certain embodiments, the compositions and methods of the present invention include a T cell population in combination with at least one additional treatment. The additional treatment may be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination thereof. The additional treatment may be in the form of adjuvant or neoadjuvant therapy.

[0124] In some embodiments, the additional treatment is administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional treatment is administration of a side effect-reducing agent (e.g., an agent intended to reduce the occurrence and / or severity of side effects of treatment, such as an antiemetic). In some embodiments, the additional treatment is radiation therapy. In some embodiments, the additional treatment is surgery. In some embodiments, the additional treatment is a combination of radiation therapy and surgery. In some embodiments, the additional treatment is gamma irradiation. In some embodiments, the additional treatment is a therapy targeting the PBK / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. The additional treatment may be one or more chemotherapeutic agents known to those skilled in the art.

[0125] Immune cell therapy can be administered before, during, after, or in various combinations with an additional cancer therapy (e.g., immune checkpoint therapy). These administrations may occur simultaneously or within minutes, days, or weeks. In embodiments in which immune cell therapy is provided to a patient separately from an additional therapeutic agent, it is generally desirable not to allow a significant amount of time to pass between administrations so that the two compounds can still exert their beneficial synergistic effects on the patient. In such cases, it is contemplated that the antibody therapy and anti-cancer therapy will be administered to the patient within about 12-24 hours or 72 hours, more specifically within about 6-12 hours. In some circumstances, it may be desirable to extend the interval between administrations from a few days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks).

[0126] Various combinations may be used. In the following examples, immune cell therapy is "A" and anti-cancer therapy is "B": A / B / A, B / A / B, B / B / A, A / A / B, A / B / B, B / A / A, A / B / B / B, B / A / B / B, B / B / B / A, B / B / A / B, A / A / B / B, A / B / A / B, A / B / B / A, B / B / A / A, B / A / B / A, B / A / A / B, A / A / A / B, B / A / A / A, A / B / A / A, A / A / B / A

[0127] Administration of any compound or treatment of the present embodiments to a patient will be carried out according to general protocols for the administration of such compounds, taking into account any toxicity of the compound. Thus, in some embodiments, there will be a step of monitoring for toxicity resulting from the combination therapy.

[0128] <1. Chemotherapy> There is a wide variety of chemotherapeutic agents that may be used in embodiments of the present disclosure. Examples of chemotherapeutic agents include alkylating agents (e.g., thiotepa and cyclophosphamide), alkylsulfonates (e.g., busulfan, improsulfan, and piposulfan), aziridines (e.g., benzdopa, carboquone, meturedopa, and uredopa), ethyleneimines and methylameramines (including arretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine), acetogenins (especially bullatacin and bullatacin), and the like. non), camptothecins (including the synthetic analog topotecan), bryostatin, kallistatin, CC-1065 (including the synthetic analogs adozelesin, carzelesin, and bizelesin), cryptophycins (especially cryptophycin 1 and cryptophycin 8), dolastatins, duocarmycins (including the synthetic analogs KW-2189 and CBI-TMI), eleutherobin, pancratistatin, sarcodictin, spongistatin, nitrogen mustards (e.g., chlorambucil, chlornaphazine, colofosfamidis), methicillin-resistant Staphylococcus aureus (e.g., methicillin-resistant Staphylococcus aureus ... nates (e.g., clodronate), esperamicin, neocarzinostatin chromosomes and related chromoprotein endiine antibiotic chromosomes, aclacinomycin, actinomycin, auxaramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins (such as mitomycin C), mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, putricin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin), antimetabolites (such as methotrexate and 5-fluorouracil (5-FU)), folic acid analogs (such as denoptera purine analogues (e.g., fludarabine, 6-mercaptopurine, thiamiprine and thioguanine), pyrimidine analogues (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine and fluorouridine), androgens (e.g., calsterone, dromostanolone propionate, epithiostanol, mepitiostane and testolactone), anticorticosteroids (e.g., mitotane and trimethoprim), lostan), folic acid supplements (e.g., folinic acid), aceglatone, aldophosphamide glycosides, aminolevulinic acid, eniluracil, amsacrine, bestravcil, bisantrene, edatrexate, defolamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, epothilone, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidamine, maytansinoids (such as maytansine and ansamitocin), mitoguazone, mitoxantrone, mopidanmol, nitraserine, pentostatin, furuncle Enammet, pirarubicin, losoxantrone, podophyllinic acid, 2-ethylhydrazide, procarbazine, PSK polysaccharide complex, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2,2',2''-trichlorotriethylamine, trichoceten (especially T-2 toxin, veracrin A, roridin A, and anguidine), urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, cytosine arabinoside ("Ara-C"), cyclophosphamide,Taxanes (e.g., paclitaxel and docetaxel), gemcitabine, 6-thioguanine, mercaptopurine, platinum complexes (e.g., cisplatin, oxaliplatin, and carboplatin), vinblastine, platinum, etoposide (VP-16), ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronic acid, irinotecan (e.g., CPT-11), topoisomerase inhibitors RFS2000, difluoromethylornithine (DFMO), retinoids (e.g., retinoic acid), capecitabine, carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyltransferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above agents.

[0129] 2. Radiation Therapy Other commonly used agents that induce DNA damage include commonly known gamma rays, X-rays, and / or the directed administration of radioisotopes to tumor cells. Other forms of DNA damage have also been considered, such as microwaves, proton beam radiation, and ultraviolet (UV) radiation. All of these agents likely cause widespread damage to DNA itself, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray dosages range from 50–200 roentgens per day over prolonged periods (3–4 weeks) to 2000–6000 roentgens as a single dose. The range of dosages for radioisotopes is very wide and depends on the half-life of the isotope, the strength and type of radiation emitted, and the amount of uptake by tumor cells.

[0130] <3. Immunotherapy> Those skilled in the art will understand that immunotherapy can be used in conjunction with or in combination with the methods herein. In the context of cancer treatment, immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is one example. The immune effector may be, for example, an antibody specific to a marker on the surface of tumor cells. The antibody alone can provide a therapeutic effect, or it can induce other cells that actually cause cytotoxicity. Antibodies can also be conjugated to drugs or toxins (e.g., chemotherapeutic agents, radionuclides, ricin A chain, cholera toxin, pertussis toxin, etc.) to act as targeting agents. Alternatively, the effector may be a lymphocyte bearing a surface molecule that interacts directly or indirectly with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0131] Antibody-drug conjugates (ADCs) comprise monoclonal antibodies (MAbs) covalently linked to cytotoxic drugs and can be used in combination therapy. This approach combines the high specificity of MAbs for antigen targets with highly potent cytotoxic drugs, allowing the "armed" MAbs to deliver the drug (payload) to tumor cells. Targeted delivery of the drug minimizes exposure to normal tissues, reducing toxicity and improving the therapeutic index. Examples of ADCs include ADCETRIS® (brentuximab vedotin) and KADCYLA® (trastuzumab emtansine or T-DM1).

[0132] One aspect of immunotherapy is that tumor cells must have a marker suitable for targeting, i.e., a marker not present on the majority of other cells. Many tumor markers exist, and any of these may be suitable for targeting in the context of this specification. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, erb b2, and p155. Another aspect of immunotherapy is combining anti-cancer effects with immunostimulatory effects. Examples of immunostimulatory molecules include cytokines (e.g., IL-2, IL-4, IL-12, GM-CSF, interferon-γ (γ-IFN)), chemokines (e.g., MIP-1, MCP-1, IL-8), and growth factors (e.g., FLT3 ligand).

[0133] Examples of immunotherapies include immune adjuvants (e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, aromatic compounds), cytokine therapy (e.g., interferon alpha, beta, and gamma, IL-1, GM-CSF, TNF), gene therapy (e.g., TNF, IL-1, IL-2, and p53), and monoclonal antibodies (e.g., anti-CD20 antibodies, anti-ganglioside GM2 antibodies, and anti-p185 antibodies). It is contemplated that one or more anti-cancer therapies will be used in conjunction with the antibody therapies described in this disclosure.

[0134] In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints either enhance signals (e.g., costimulatory molecules) or attenuate signals. Immune checkpoints that can be inhibited include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B- and T-lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene-3 (LAG3), programmed cell death 1 (PD-1), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), and V-domain Ig suppressor of T-cell activation (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0135] Immune checkpoint inhibitors may be drugs such as small molecules, recombinant ligands or receptors, or antibodies (e.g., human antibodies). Known immune checkpoint protein inhibitors or their analogs may be used, particularly chimeric, humanized, or human antibodies. Those skilled in the art will recognize that different aliases or equivalent names are used for the antibodies described herein. Such aliases or equivalent names are interchangeable in the context of this specification. For example, lambrolizumab is also known by the aliases or equivalent names MK-3475 and pembrolizumab.

[0136] In one embodiment, a PD-1 binding antagonist is a molecule that inhibits PD-1 from binding to its ligand binding partner. In a specific embodiment, the ligand binding partner of PD-1 is PD-L1 and / or PD-L2. In another embodiment, a PD-L1 binding antagonist is a molecule that inhibits PD-L1 from binding to its binding partner. In a specific embodiment, the binding partner of PD-L1 is PD-1 and / or B7-1. In another embodiment, a PD-L2 binding antagonist is a molecule that inhibits PD-L2 from binding to its binding partner. In a specific embodiment, the binding partner of PD-L2 is PD-1. The antagonist may be an antibody, antigen-binding fragment, immunoadhesin, fusion protein, or oligopeptide.

[0137] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin in which the extracellular domain or PD-1 binding site of PDL1 or PDL2 is fused to a constant region of an immunoglobulin sequence (e.g., an Fc region)). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab (also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, OPDIVO®) is a usable anti-PD-1 antibody. Pembrolizumab (also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475) is an exemplary anti-PD-1 antibody. CT-011 (also known as hBAT or hBAT-1) is also an anti-PD-1 antibody. AMP-224 (also known as B7-DCIg) is a PDL2-Fc fusion soluble receptor.

[0138] Another immune checkpoint that can be targeted in the methods disclosed herein is cytotoxic T lymphocyte antigen 4 (CTLA-4) (also known as CD152). The complete cDNA sequence of human CTLA-4 is Genbank accession number L15006. CTLA-4 is present on the surface of T cells and acts as an "off" switch when it binds to CD80 or CD86 present on the surface of antigen-presenting cells. CTLA-4 is a member of the immunoglobulin superfamily, expressed on the surface of helper T cells, and transmits inhibitory signals to T cells. CTLA-4 is similar to the T cell costimulatory protein CD28; both bind to CD80 and CD86 (also known as B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA-4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA-4 is also present on regulatory T cells and may be important for the function of these cells. T cell activation via the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules.

[0139] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0140] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the methods of the present invention can be generated by methods well known to those of skill in the art. Alternatively, anti-CTLA-4 antibodies recognized by those of skill in the art can be used. An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof. In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains in the VH region and the CDR1, CDR2, and CDR3 domains in the VL region of ipilimumab. In another embodiment, the antibody binds to and / or competitively binds to the same epitope as the above-described antibodies. In another embodiment, the antibody has at least about 90% (eg, at least about 90%, 95%, or 99%) variable region amino acid sequence identity with the antibody (eg, ipilimumab).

[0141] <4. Surgery> Approximately 60% of cancer patients undergo some type of surgery, including preventative, diagnostic or staging, curative, or palliative surgery. Curative surgery includes resection, which physically removes, excises, and / or destroys all or part of cancerous tissue, and may be combined with other treatments, such as current embodiment treatments, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to the physical removal of at least part of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgical surgery (Mohs surgery).

[0142] When cancer cells, tissues, or tumors are partially or completely removed, a cavity may form within the body. Treatment can be achieved by perfusion, direct injection, or local administration of an additional anticancer therapy to the site. Such treatment can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments can also be administered at various dosages.

[0143] <5. Other drugs> The use of other agents in combination with certain aspects of embodiments of the present invention to improve the therapeutic efficacy of the treatment is contemplated. These additional agents include agents that enhance the expression of cell surface receptors and gap junctions, cytostatic and differentiation-inducing agents, inhibitors of cell adhesion, agents that sensitize hyperproliferative cells to apoptosis-inducing agents, or other biological agents. Enhancing intercellular signaling by increasing the number of gap junctions may enhance the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cytostatic or differentiation-inducing agents may be used in combination with certain aspects of embodiments of the present invention to improve the anti-hyperproliferative efficacy of the treatment. Cell adhesion inhibitors are contemplated to improve the efficacy of embodiments of the present invention. Examples of cell adhesion inhibitors include focal adhesion kinase (FAK) inhibitors and lovastatin.

[0144] VI. ARTICLE OF MANUFACTURE OR KITS Also provided herein are articles of manufacture or kits containing immune cells, antibodies, reagents, buffers, or combinations thereof. The articles of manufacture or kits can further include a package insert containing instructions for using the immune cells to treat or delay the progression of cancer in an individual or to enhance the immune function of an individual with cancer. Any of the antigen-specific immune cells described herein can be included in an article of manufacture or kit. Suitable containers include, for example, bottles, vials, bags, and syringes. Containers can be formed from a variety of materials, such as glass, plastic (e.g., polyvinyl chloride or polyolefin), or metal alloys (e.g., stainless steel or Hastelloy). In some embodiments, the container holds a formulation, and a label on or associated with the container may indicate directions for use. The articles of manufacture or kits may further include other materials desirable from a commercial and user standpoint, such as other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the articles of manufacture may further include one or more additional pharmaceutical agents (e.g., chemotherapeutic agents, anti-tumor agents). Suitable containers for one or more agents include, for example, bottles, vials, bags, and syringes.

[0145] VII. SEQUENCES USED IN SPECIFIC EMBODIMENTS All of the following are variable chain sequences. [Table 1] JPEG2025540600000003.jpg221170JPEG2025540600000004.jpg216170JPEG2025540600000005.jpg22217 0JPEG2025540600000006.jpg214170JPEG2025540600000007.jpg215170JPEG2025540600000008.jpg51170 [Example]

[0146] VIII. Working Examples The following examples are included to demonstrate specific embodiments of the invention. Those of skill in the art should appreciate that the techniques disclosed in the examples which follow are techniques discovered by the inventors to function well in the practice of the invention, and as such, can be considered to constitute preferred modes for practicing the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments disclosed and still obtain like or similar results without departing from the spirit and scope of the invention.

[0147] Example 1 - Chimeric Antigen Receptor (T-cell therapy targeting Zona Pellucida 4 for the treatment of solid tumors) CAR-T cells have the potential to cure patients with advanced cancer, as demonstrated by their remarkable success in hematological malignancies, resulting in five FDA-approved CAR-T therapies since 2017. However, expanding their application to solid tumors such as breast cancer has proven challenging. One major obstacle is finding targets that are highly expressed in cancer cells but not in critical normal tissues. The inventors attempted to find such targets for triple-negative breast cancer (TNBC) and identified zona pellucida 4 (ZP4) as a promising novel target for TNBC. ZP4 is a component of the human zona pellucida (ZP) matrix, an extracellular structure that surrounds oocytes and is involved in follicle formation, species-specific fertilization, and early development. ZP4 expression is restricted to the ovary, and its expression in other normal tissues has not been reported.

[0148] First, mRNA expression data from 54 human normal tissues in the Genotype-Tissue Expression (GTEx) project were analyzed. ZP4 showed no detectable mRNA expression in any of the GTEx tissues, except for some ovarian samples (Figure 1A). Furthermore, data analysis from the Breast Cancer International Consortium (METABRIC) showed significant ZP4 expression in a subset of breast cancer samples belonging to the basal / TNBC subgroup (Figure 1C). Similar findings were obtained in The Cancer Genome Atlas (TCGA) and Clinical Proteomic Tumor Analysis Consortium (CPTAC) (proteome) cohorts. Based on this analysis, ZP4-expressing tumors represented approximately 10% of basal / TNBC samples. Importantly, mass spectrometry-based proteome data from TCGA / CPTAC samples further demonstrated that ZP4 protein was detectable in some samples with ZP4 mRNA expression (Figure 1B). By immunohistochemistry (IHC), we confirmed a complete lack of ZP4 expression in all vital organs, with expression in the ovary restricted to the ZP matrix surrounding oocytes (Figure 1D). Therefore, redirecting T cells targeting ZP4 should not cause on-target or off-target toxicity, a drawback of other CAR-T cell targets currently being explored for solid tumors. Breast cancer patient-derived xenograft (PDX) mouse models more accurately recapitulate patient disease and the tumor microenvironment (TME). We confirmed variable levels of ZP4 expression in TNBC PDX models by mRNA sequencing, proteomic analysis, and IHC staining (Figure 2). The RNA sequencing and proteomic data obtained from these models provide a unique opportunity to examine the antitumor activity of ZP4 CAR-T cells against tumors with different ZP4 expression levels and further understand the suppressive mechanisms affecting CAR-T cells in the breast cancer TME.

[0149] To construct second-generation ZP4-specific CARs, we immunized mice with mouse L cells transduced and sorted to express ZP4, generating monoclonal antibodies against ZP4. A clone superset containing over 80 candidate monoclonal antibodies was obtained. We selected clones 108 and 46 with high specificity as candidates (Figure 3B). The cDNA sequences of the antibodies were obtained and used to design second-generation CARs containing the CD28 costimulatory signal domain and the TCR ζ chain signal domain (Figure 3A). To evaluate the activity of these CAR-T cells in vitro and in vivo, we overexpressed ZP4 in the triple-negative breast cancer cell line SUM159 and used it as a target in cytotoxicity assays. ZP4 CAR-T cells killed ZP4-positive SUM159 cells (Figure 3C-D).

[0150] The specificity of the antibody clone was also verified by IHC staining. Monoclonal antibody clone 46 was used to stain human ovarian tissue and detected ZP4 in the zona pellucida region of oocytes (Figure 4). Clone 46 was also used to stain various normal organ tissues and showed no cross-reactivity with any of the normal organ tissues tested (Figures 5-8).

[0151] [Example 2] We aimed to evaluate the effector function, migration, proliferation, and persistence of ZP4 CAR T cells in vitro and in vivo using ZP4-positive MDA-MB-231 and SUM-159 cell lines as targets. Three ZP4-specific CAR constructs were generated using the variable region sequences of monoclonal antibodies clone 108, clone 128, and clone 164. Each single-chain variable fragment (scFv) was cloned into a second-generation retroviral CAR construct, which contained a CD3 spacer, CD28 transmembrane and signaling domains, and a CD3ζ signaling domain (Figure 9A). A truncated CD19 (CD19) reporter separated by a T2A sequence was also included to enable enrichment of CAR T cells by magnetic bead selection. ZP4 CAR expression was confirmed by flow cytometry using a human Fcγ fragment-specific antibody. After transduction, stable ZP4 CAR expression was confirmed on the surface of activated T cells from healthy donors (Figure 9B). Analysis of T cell phenotype by flow cytometry revealed no significant differences in CD4 / CD8 distribution or differentiation status. All three CARs were predominantly CD8+ (Figure 9C) and predominantly effector memory (Tem) phenotype (Figure 9D). The short-term cytotoxicity of ZP4 CAR T cells was assessed by targeting ZP4-positive MDA-MB-231 cells at E:T ratios (effector:target) of 40:1, 20:1, 10:1, and 5:1 with standard 4-6 h chromium 51 ( 51 Effector cells (untransduced T cells or ZP4 CAR T cells) were used to measure the cytotoxicity of CAR T cells. 51 The cells were co-cultured with Cr-labeled target cells in triple wells. After incubation, the supernatants were collected and radioactivity was measured using a γ-counter. To calculate cytotoxic activity, the percentage of specific lysis (% specific lysis) was calculated. CAR T cells from clone 108, clone 128, and clone 164 each exhibited antigen-specific cytotoxicity against ZP4-positive target cells, killing at least 80% of the cells at an E:T ratio of 40:1 (Figure 9E).

[0152] The cytotoxicity and persistence of ZP4 CAR T cells were also assessed in long-term coculture assays. T cells were stimulated weekly with ZP4-positive irradiated MDA-MB-231 cells. At the end of each week, T cells were counted to assess proliferation, and 72-hour cocultures were performed to assess CAR T cell efficacy. The remaining T cells were then re-cocultured with fresh irradiated tumor cells for the next stimulation cycle (Figure 10A). Representative flow diagrams show the results of the 72-hour cocultures at weeks 1, 3, and 4. T cells are in the upper left quadrant (stained with anti-CD3 antibody), and residual tumor cells are in the lower right quadrant (tumor cells express RFP). While all three CARs effectively eliminated tumor cells at week 1, only z128 CAR T cells consistently eliminated tumor cells over four stimulations (Figure 10B). z128 CAR T cells demonstrated superior proliferation after multiple stimulations, as quantified by cell number per well (Figure 10C). T cell activation after 72 hours of coculture with target cells was measured using 41BB (Figure 10D), and the percentage of T cells and the percentage of residual tumor cells in each coculture were quantified (Figure 10E, F).

[0153] The polyfunctionality of ZP4 CAR T cells was measured using the Isoplexis method, which enables multiplex cytokine analysis at the single-cell level. This method calculates the "polyfunctional intensity index (PSI)," defined by the manufacturer, based on the percentage of cells producing multiple cytokines and the cytokine secretion intensity of each cell. Polyfunctionality is considered an important indicator of efficacy in CAR T-cell therapy. In both CD4 and CD8 subsets, z128 CAR T cells exhibited the highest PSI, indicating a potentially more potent product (Figure 10G). This result may explain why z128 CAR T cells outperform other CARs even after multiple stimulations, and further investigation is planned.

[0154] The persistence and effector function of ZP4 CAR T cells were further evaluated in vivo. All animal experiments were performed under protocols approved by the Baylor College of Medicine Institutional Animal Care and Use Committee. To assess CAR T cell migration and proliferation, 6- to 8-week-old female NSG mice were inoculated with 5 × 10 ZP4-positive MDA-MB-231 cells. 6 1.5 × 10 cells were injected into the mammary fat pad and allowed to form tumors. Animals were then randomized to receive either non-transduced T cells (NT) or ZP4 CAR T cells (1.5 × 10 cells) co-expressing a green fluorescent protein / firefly luciferase (GFP.ffLuc) fusion gene. 6 100 mg / kg / day were administered intravenously (iv). CAR T cell persistence was monitored using a Lumina In Vivo Imaging System (IVIS). Although statistical significance was not observed due to high variability, z128 CAR T cells demonstrated the highest persistence in vivo, visualized by IVIS (Figure 11A) and quantified as mean radiance (Figure 11B). To evaluate the antitumor efficacy of ZP4 CAR T cells, a similar protocol was used, except tumor cells were labeled with GFP.ffLuc, and T cells were injected into tumors approximately 100 mm in size. 3 At the time when the tumor reached 14-16 days after inoculation, 2 × 10 6 Two doses of each drug were administered. Tumor burden was monitored via IVIS, and tumors were measured weekly with calipers. Bioluminescence images taken 7 days after tumor inoculation showed that all tumors had fully implanted. From day 21 onward, the scale was adjusted to more accurately display tumor signal at later time points (Figure 12A). z128 CAR T cells effectively controlled tumor growth, as indicated by low tumor radiance (Figure 12B) and small tumor volume (Figure 12C). Compared with z164 CAR T cells, mice treated with z108 and z128 CAR T cells showed significantly extended survival (Figure 12D). Furthermore, z128 and z108 CAR T cells also reduced overall metastatic burden, particularly in the lungs and liver (Figure 12E).

[0155] All methods disclosed and claimed herein can be practiced and constructed without undue experimentation in light of the present disclosure. While the compositions and methods of the present invention have been described based on preferred embodiments, those skilled in the art will recognize that various modifications to the steps or sequence of the methods described herein can be made without departing from the concept, spirit, and scope of the present invention. More specifically, it will be apparent that certain chemically and physiologically similar agents may be substituted for the agents described herein while still achieving the same or similar results. Such similar substitutes and modifications will be apparent to those skilled in the art and are deemed to be within the spirit, scope, and concept of the present invention as defined by the appended claims.

[0156] [References] The following references, to the extent that they provide procedural or other details supplementary to those set forth herein, are expressly incorporated herein. Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Chothia et al., 1988 . Davila et al., 2013 . European patent application number EP2537416 Heemskerk et al. Hum Gene Ther. 19:496-510, 2008. International Patent Publication No. WO / 2014055668 Al. International Patent Publication No. WO200014257 International Patent Publication No. WO2012 / 129514 International Patent Publication No. WO2013 / 071154 International Patent Publication No. WO2013 / 123061 International Patent Publication No. WO2013 / 166321 International Patent Publication No. WO2013126726 International Patent Publication No. WO2014031687 Johnson et al. Blood 114:535-46, 2009. Jores et al., 1990. Kabat et al., "Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5 th ed. Lefranc et al., 2003. Liu et al., 2003. Remington's Pharmaceutical Sciences 22 nd edition, 2012. Sadelain et al., 2013. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001. Turtle et al., 2012. U.S. Patent No. 5,091,513 U.S. Patent No. 5,091,513 U.S. Patent No. 5,994,136 U.S. Patent No. 6,013,516 U.S. Patent No. 6,410,319 U.S. Patent No. 6,410,319 U.S. Patent No. 6,451,995 U.S. Patent No. 6,881,557 U.S. Patent No. 6,946,546 U.S. Patent No. 7,070,995 U.S. Patent No. 7,265,209 U.S. Patent No. 7,354,762 U.S. Patent No. 7,446,179 U.S. Patent No. 7,446,190 U.S. Patent No. 7,446,191 U.S. Patent No. 8,252,592 U.S. Patent No. 8,324,353 U.S. Patent No. 8,339,645 U.S. Patent No. 8,398,282 U.S. Patent No. 8,479,118 U.S. Patent No.: 7,446,190 U.S. Patent Publication No. US2002131960 U.S. Patent Publication No. US20050214860 U.S. Patent Publication No. US20130149337 U.S. Patent Publication No. US2013287748 Wu et al., 2012

Claims

1. 1. An isolated monoclonal antibody, wherein the antibody specifically binds to ZP4, comprising: (I)： (a) First V H The CDRs correspond to SEQ ID NO:3; (b) Second V H The CDRs correspond to SEQ ID NO: 4; (c) The Third V H The CDRs correspond to SEQ ID NO: 5; (d) First V L The CDRs correspond to SEQ ID NO:8; (e) Second V L The CDRs correspond to SEQ ID NO: 9; and (f) The third V L DR corresponds to SEQ ID NO: 10; (II): (a) First V H CDRs correspond to SEQ ID NO: 13; (b) Second V H The CDRs correspond to SEQ ID NO: 14; (c) The Third V H CDRs correspond to SEQ ID NO: 15; (d) First V L CDRs correspond to SEQ ID NO: 18; (e) Second V L The CDRs correspond to SEQ ID NO: 19; and (f) The third V L The CDRs correspond to SEQ ID NO: 20; (III): (a) First V H CDRs correspond to SEQ ID NO: 23; (b) Second V H The CDRs correspond to SEQ ID NO: 24; (c) The Third V H The CDRs correspond to SEQ ID NO: 25; (d) First V L CDRs correspond to SEQ ID NO: 28; (e) Second V L The CDRs correspond to SEQ ID NO: 29; and (f) The third V L CDRs correspond to SEQ ID NO: 30; (IV): (a) First V H CDRs correspond to SEQ ID NO: 33; (b) Second V H CDRs correspond to SEQ ID NO: 34; (c) The Third V H CDRs correspond to SEQ ID NO: 35; (d) First V L CDRs correspond to SEQ ID NO: 38; (e) Second V L The CDRs correspond to SEQ ID NO: 39; and (f) The third V L The CDRs correspond to SEQ ID NO: 40; or (V)： (a) First V H CDRs correspond to SEQ ID NO: 43; (b) Second V H CDRs correspond to SEQ ID NO: 44; (c) The Third V H CDRs correspond to SEQ ID NO: 45; (d) First V L CDRs correspond to SEQ ID NO: 48; (e) Second V L The CDRs correspond to SEQ ID NO: 49; and (f) The third V L The CDRs correspond to SEQ ID NO:

50.

2. The antibody of claim 1, comprising: (a) First V H The CDRs correspond to SEQ ID NO:3; (b) Second V H The CDRs correspond to SEQ ID NO: 4; (c) The Third V H The CDRs correspond to SEQ ID NO: 5; (d) First V L The CDRs correspond to SEQ ID NO:8; (e) Second V L The CDRs correspond to SEQ ID NO: 9; and (f) The third V L The CDRs correspond to SEQ ID NO:

10.

3. The antibody is V of SEQ ID NO:2 H V having at least about 80% identity with the domain H domain, as well as V of SEQ ID NO: 7 L V having at least about 80% identity with the domain L The antibody of claim 2, comprising a domain.

4. The antibody is V of SEQ ID NO:2 H Domain identical to V of SEQ ID NO: 7 L The antibody of claim 2, wherein the domain is identical to the

5. The antibody of claim 1, comprising: (a) First V H CDRs correspond to SEQ ID NO: 13; (b) Second V H The CDRs correspond to SEQ ID NO: 14; (c) The Third V H CDRs correspond to SEQ ID NO: 15; (d) First V L CDRs correspond to SEQ ID NO: 18; (e) Second V L The CDRs correspond to SEQ ID NO: 19; and (f) The third V L The CDRs correspond to SEQ ID NO: 20;

6. The antibody is V of SEQ ID NO: 12 H V having at least about 80% identity with the domain H domain, as well as V of SEQ ID NO: 17 L V having at least about 80% identity with the domain L The antibody of claim 5, comprising a domain.

7. The antibody is V of SEQ ID NO: 12 H domain and V of SEQ ID NO: 17 L The antibody of claim 5, wherein the domain is identical to the

8. The antibody of claim 1, comprising: (a) First V H CDRs correspond to SEQ ID NO: 23; (b) Second V H The CDRs correspond to SEQ ID NO: 24; (c) The Third V H The CDRs correspond to SEQ ID NO: 25; (d) First V L CDRs correspond to SEQ ID NO: 28; (e) Second V L The CDRs correspond to SEQ ID NO: 29; and (f) The third V L CDRs correspond to SEQ ID NO: 30;

9. The antibody is V of SEQ ID NO: 22 H V having at least about 80% identity with the domain H domain, as well as V of SEQ ID NO: 27 L V having at least about 80% identity with the domain L The antibody of claim 8, comprising a domain.

10. The antibody is V of SEQ ID NO: 22 H domain and V of SEQ ID NO: 27 L The antibody of claim 8, wherein the domain is identical to the

11. The antibody of claim 1, comprising: (a) First V H CDRs correspond to SEQ ID NO: 33; (b) Second V H CDRs correspond to SEQ ID NO: 34; (c) The Third V H CDRs correspond to SEQ ID NO: 35; (d) First V L CDRs correspond to SEQ ID NO: 38; (e) Second V L The CDRs correspond to SEQ ID NO: 39; and (f) The third V L The CDRs correspond to SEQ ID NO: 40;

12. The antibody is V of SEQ ID NO: 32 H V having at least about 80% identity with the domain H domain, as well as V of SEQ ID NO: 37 L V having at least about 80% identity with the domain L The antibody of claim 11, comprising a domain.

13. The antibody is V of SEQ ID NO: 32 H domain and V of SEQ ID NO: 37 L The antibody of claim 11, wherein the domain is identical to the

14. The antibody of claim 1, comprising: (a) First V H CDRs correspond to SEQ ID NO: 43; (b) Second V H CDRs correspond to SEQ ID NO: 44; (c) The Third V H CDRs correspond to SEQ ID NO: 45; (d) First V L CDRs correspond to SEQ ID NO: 48; (e) Second V L The CDRs correspond to SEQ ID NO: 49; and (f) The third V L The CDRs correspond to SEQ ID NO:

50.

15. The antibody is V of SEQ ID NO: 42 H V having at least about 80% identity with the domain H domain, as well as V of SEQ ID NO: 47 L V having at least about 80% identity with the domain L 15. The antibody of claim 14, comprising a domain.

16. The antibody is V of SEQ ID NO: 42 H domain and V of SEQ ID NO: 47 L The antibody of claim 14, wherein the domain is identical to the

17. The antibody of any one of claims 1 to 16, wherein the antibody is a recombinant antibody.

18. The antibody of claim 1, wherein the antibody is an IgG, IgM, IgA, or an antigen-binding fragment thereof.

19. The antibody of any one of claims 1 to 18, wherein the antibody is a Fab', F(ab')2, F(ab')3, monovalent scFv, bivalent scFv, or single domain antibody.

20. The antibody of any one of claims 1 to 19, wherein the antibody is a human antibody, a humanized antibody, or a deimmunized antibody.

21. The antibody of any one of claims 1 to 20, wherein the antibody is conjugated to an imaging agent, a chemotherapeutic agent, a toxin, or a radioisotope.

22. The antibody of any one of claims 1 to 21, wherein the antibody is part of a fusion protein.

23. The antibody of any one of claims 1 to 22, wherein the antibody is part of a chimeric protein.

24. A composition comprising an antibody according to any one of claims 1 to 23 in a pharmaceutically acceptable carrier.

25. An isolated polynucleotide molecule comprising a nucleic acid sequence encoding the antibody of any one of claims 1 to 24.

26. Clone 46 V H CDR1-3 of the domain (SEQ ID NOs: 3, 4, 5) and V of clone 46 L Antibody V comprising CDRs 1-3 of the domain (SEQ ID NOs: 8, 9, 10) H A recombinant polypeptide comprising a domain.

27. Clone 108 V H CDR1-3 of the domain (SEQ ID NOs: 13, 14, 15) and V of clone 108 L Antibody V comprising CDR1-3 of domain (SEQ ID NOs: 18, 19, 20) H A recombinant polypeptide comprising a domain.

28. Clone 2 V H CDR1-3 of domain (SEQ ID NOs: 23, 24, 25) and V of clone 2 L Antibody V comprising CDR1-3 of domain (SEQ ID NOs: 28, 29, 30) H A recombinant polypeptide comprising a domain.

29. Clone 128 V H CDR1-3 of the domain (SEQ ID NOs: 33, 34, 35) and V of clone 128 L Antibody V comprising CDR1-3 of domain (SEQ ID NOs: 38, 39, 40) H A recombinant polypeptide comprising a domain.

30. Clone 164 V H CDR1-3 of the domain (SEQ ID NOs: 43, 44, 45) and V of clone 164 L Antibody V comprising CDR1-3 of domain (SEQ ID NOs: 48, 49, 50) H A recombinant polypeptide comprising a domain.

31. 31. An isolated polynucleotide molecule comprising a nucleic acid sequence encoding the polypeptide of any one of claims 26 to 30.

32. A host cell comprising one or more polynucleotide molecules encoding the antibody of any one of claims 1 to 23 or the recombinant polypeptide of any one of claims 26 to 30.

33. 33. The host cell of claim 32, wherein the host cell is a mammalian cell, a yeast cell, a bacterial cell, a ciliate cell or an insect cell.

34. 34. A method for treating a subject having cancer, or for preventing or delaying the onset or progression of cancer in said subject, said method comprising administering to said subject an effective amount of an antibody of any one of claims 1 to 23, a recombinant polypeptide of any one of claims 26 to 30, and / or a host cell of claim 32 or 33.

35. 35. The method of claim 34, wherein the cancer is a solid tumor.

36. 36. The method of claim 34 or 35, wherein the cancer is triple-negative breast cancer.

37. The method of any one of claims 34 to 36, wherein the antibody is in a pharmaceutically acceptable composition.

38. The method of any one of claims 34 to 37, wherein the antibody is administered systemically.

39. 39. The method of any one of claims 34 to 38, wherein the antibody is administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or topically.

40. 40. The method of any one of claims 34 to 39, further comprising administering to said subject at least a second anti-cancer treatment.

41. 41. The method of claim 40, wherein the second anti-cancer treatment is surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, and / or cytokine therapy.

42. 42. The method of claim 40 or 41, wherein the second anti-cancer treatment comprises adoptive T cell therapy.

43. A modified Zona Pellucida 4 (ZP4) CAR or TCR comprising the following antigen-binding domains: (I)： (a) First V H The CDRs correspond to SEQ ID NO:3; (b) Second V H The CDRs correspond to SEQ ID NO: 4; (c) The Third V H The CDRs correspond to SEQ ID NO: 5; (d) First V L The CDRs correspond to SEQ ID NO:8; (e) Second V L The CDRs correspond to SEQ ID NO: 9; and (f) The third V L The CDRs correspond to SEQ ID NO: 10; (II): (a) First V H CDRs correspond to SEQ ID NO: 13; (b) Second V H The CDRs correspond to SEQ ID NO: 14; (c) The Third V H CDRs correspond to SEQ ID NO: 15; (d) First V L CDRs correspond to SEQ ID NO: 18; (e) Second V L The CDRs correspond to SEQ ID NO: 19; and (f) The third V L The CDRs correspond to SEQ ID NO: 20; (III): (a) First V H CDRs correspond to SEQ ID NO: 23; (b) Second V H The CDRs correspond to SEQ ID NO: 24; (c) The Third V H The CDRs correspond to SEQ ID NO: 25; (d) First V L CDRs correspond to SEQ ID NO: 28; (e) Second V L The CDRs correspond to SEQ ID NO: 29; and (f) The third V L CDRs correspond to SEQ ID NO: 30; (IV): (a) First V H CDRs correspond to SEQ ID NO: 33; (b) Second V H CDRs correspond to SEQ ID NO: 34; (c) The Third V H CDRs correspond to SEQ ID NO: 35; (d) First V L CDRs correspond to SEQ ID NO: 38; (e) Second V L The CDRs correspond to SEQ ID NO: 39; and (f) The third V L The CDRs correspond to SEQ ID NO: 40; or (V)： (a) First V H CDRs correspond to SEQ ID NO: 43; (b) Second V H CDRs correspond to SEQ ID NO: 44; (c) The Third V H CDRs correspond to SEQ ID NO: 45; (d) First V L CDRs correspond to SEQ ID NO: 48; (e) Second V L The CDRs correspond to SEQ ID NO: 49; and (f) The third V L The CDRs correspond to SEQ ID NO:

50.

44. 44. The CAR or TCR of claim 43, comprising the following antigen binding domain: (a) First V H The CDRs correspond to SEQ ID NO:3; (b) Second V H The CDRs correspond to SEQ ID NO: 4; (c) The Third V H The CDRs correspond to SEQ ID NO: 5; (d) First V L The CDRs correspond to SEQ ID NO:8; (e) Second V L The CDRs correspond to SEQ ID NO: 9; and (f) The third V L The CDRs correspond to SEQ ID NO: 10;

45. the antigen-binding domain is V of SEQ ID NO: 2 H V having at least about 80% identity with the domain H domain, and V of SEQ ID NO: 7 L V having at least about 80% identity with the domain L 45. The CAR or TCR of claim 44, comprising a domain.

46. the antigen-binding domain is V of SEQ ID NO: 2 H Domain identical to V of SEQ ID NO: 7 L V having at least about 80% identity with the domain L 45. The CAR or TCR of claim 44, comprising a domain.

47. 44. The CAR or TCR of claim 43, comprising the following antigen binding domain: (a) First V H CDRs correspond to SEQ ID NO: 13; (b) Second V H The CDRs correspond to SEQ ID NO: 14; (c) The Third V H CDRs correspond to SEQ ID NO: 15; (d) First V L CDRs correspond to SEQ ID NO: 18; (e) Second V L The CDRs correspond to SEQ ID NO: 19; and (f) The third V L The CDRs correspond to SEQ ID NO: 20;

48. the antigen-binding domain is V of SEQ ID NO: 12 H V having at least about 80% identity with the domain H domain, and V of SEQ ID NO: 17 L V having at least about 80% identity with the domain L 48. The CAR or TCR of claim 47, comprising a domain.

49. the antigen-binding domain is V of SEQ ID NO: 12 H domain and V of SEQ ID NO: 17 L V having at least about 80% identity with the domain L 48. The CAR or TCR of claim 47, comprising a domain.

50. 44. The CAR or TCR of claim 43, comprising the following antigen binding domain: (a) First V H CDRs correspond to SEQ ID NO: 23; (b) Second V H The CDRs correspond to SEQ ID NO: 24; (c) The Third V H The CDRs correspond to SEQ ID NO: 25; (d) First V L CDRs correspond to SEQ ID NO: 28; (e) Second V L The CDRs correspond to SEQ ID NO: 29; and (f) The third V L CDRs correspond to SEQ ID NO: 30;

51. the antigen-binding domain is V of SEQ ID NO: 22 H V having at least about 80% identity with the domain H domain, and V of SEQ ID NO: 27 L V having at least about 80% identity with the domain L 51. The CAR or TCR of claim 50, comprising a domain.

52. the antigen-binding domain is V of SEQ ID NO: 22 H domain and V of SEQ ID NO: 27 L V having at least about 80% identity with the domain L 51. The CAR or TCR of claim 50, comprising a domain.

53. 44. The CAR or TCR of claim 43, comprising the following antigen binding domain: (a) First V H CDRs correspond to SEQ ID NO: 33; (b) Second V H CDRs correspond to SEQ ID NO: 34; (c) The Third V H CDRs correspond to SEQ ID NO: 35; (d) First V L CDRs correspond to SEQ ID NO: 38; (e) Second V L The CDRs correspond to SEQ ID NO: 39; and (f) The third V L The CDRs correspond to SEQ ID NO: 40;

54. the antigen-binding domain is V of SEQ ID NO: 32 H V having at least about 80% identity with the domain H domain, and V of SEQ ID NO: 37 L V having at least about 80% identity with the domain L 54. The CAR or TCR of claim 53, comprising a domain.

55. the antigen-binding domain is V of SEQ ID NO: 32 H domain and V of SEQ ID NO: 37 L V having at least about 80% identity with the domain L 54. The CAR or TCR of claim 53, comprising a domain.

56. 44. The CAR or TCR of claim 43, comprising the following antigen binding domain: (a) First V H CDRs correspond to SEQ ID NO: 43; (b) Second V H CDRs correspond to SEQ ID NO: 44; (c) The Third V H CDRs correspond to SEQ ID NO: 45; (d) First V L CDRs correspond to SEQ ID NO: 48; (e) Second V L The CDRs correspond to SEQ ID NO: 49; and (f) The third V L The CDRs correspond to SEQ ID NO:

50.

57. the antigen-binding domain is V of SEQ ID NO: 42 H V having at least about 80% identity with the domain H domain, and V of SEQ ID NO: 47 L V having at least about 80% identity with the domain L 57. The CAR or TCR of claim 56, comprising a domain.

58. the antigen-binding domain is V of SEQ ID NO: 42 H domain and V of SEQ ID NO: 47 L V having at least about 80% identity with the domain L 57. The CAR or TCR of claim 56, comprising a domain.

59. 59. The CAR or TCR of any one of claims 43 to 58, wherein the CAR comprises one or more signaling domains selected from CD3zeta, CD28, OX40 / CD134, 4-1BB / CD137, and combinations thereof.

60. 60. The CAR or TCR of any one of claims 43 to 59, wherein the CAR or TCR is encoded by a viral vector.

61. The CAR or TCR of claim 60, wherein the viral vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a retroviral vector.

62. 62. The CAR or TCR of any one of claims 43 to 61, wherein the CAR or TCR comprises a hinge region.

63. 63. The CAR or TCR of any one of claims 43 to 62, wherein the CAR comprises a transmembrane domain.

64. 64. The CAR or TCR of any one of claims 43 to 63, wherein the CAR or TCR further comprises a transduction marker and / or a safety switch.

65. 65. The CAR or TCR of claim 64, wherein the transduction marker and / or safety switch is linked to the CAR by a cleavage peptide or an IRES.

66. 66. The CAR or TCR of claim 65, wherein the cleavage peptide is a 2A peptide.

67. 67. The CAR or TCR of any one of claims 43 to 66, wherein the CAR further comprises a second antigen-binding domain.

68. 68. The CAR or TCR of claim 67, wherein the second antigen binding domain targets a cancer antigen that is different from the antigen targeted by the other antigen binding domain.

69. An expression vector encoding a CAR or TCR according to any one of claims 43 to 68.

70. A host cell engineered to express ZP4 CAR and / or ZP4 TCR.

71. 71. The cell of claim 70, wherein the cell is modified to express a CAR of any one of claims 43 to 68.

72. 72. The cell of claim 71, wherein the cell is an immune cell.

73. 73. The cell of claim 72, wherein the immune cell is a T cell.

74. 74. The cell of claim 73, wherein the T cell is a primary human T cell or a TIL.

75. 74. The cell of claim 73, wherein the T cell is a CD4+ cell or a CD8+ T cell.

76. 75. The cell of claim 74, wherein the primary human T cells are obtained from a healthy donor.

77. 77. The cell of any one of claims 73 to 76, wherein the T cell is autologous.

78. 77. The cell of any one of claims 73 to 76, wherein the T cell is allogeneic.

79. 79. The cell of any one of claims 70 to 78, wherein the cell is modified using a CRISPR system or a transposase system.

80. 69. A pharmaceutical composition comprising ZP4-targeted T cells modified to express a CAR or TCR of any one of claims 43 to 68 and a pharmaceutically acceptable carrier.

81. A composition for treating cancer comprising an effective amount of ZP4-targeting T cells modified to express a CAR or TCR according to any one of claims 43 to 68.

82. 69. Use of a composition comprising an effective amount of ZP4-targeted T cells modified to express a CAR or TCR according to any one of claims 43 to 68 for the treatment of cancer in a subject.

83. 69. A method of treating cancer in a subject, comprising administering to the subject an effective amount of ZP4-targeted T cells modified to express a CAR or TCR of any one of claims 43 to 68.

84. 84. The method of claim 83, wherein the cancer is a solid tumor.

85. 85. The method of claim 83 or 84, wherein the cancer is triple-negative breast cancer.

86. 86. The method of any one of claims 83 to 85, wherein the ZP4-targeted T cells are administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or topically.

87. 87. The method of any one of Claims 83-86, further comprising administering to said subject at least a second anti-cancer treatment.

88. 88. The method of claim 87, wherein the second anti-cancer treatment is surgery, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy, and / or cytokine therapy.

89. 89. The method of claim 88, wherein the cancer is a ZP4-expressing cancer.

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