Fibronectin extra domain b (EDB)-specific car-t for cancer

Abstract

To provide a chimeric antigen receptor (CAR) specific for the extra domain B (EDB) of fibronectin.SOLUTION: A chimeric antigen receptor comprises: an extracellular antigen-binding domain having specificity for EDB; a transmembrane region; a hinge region between the extracellular antigen-binding domain and the transmembrane region; an intracellular signaling domain; and one or more costimulatory domains. Therein: the hinge region, the transmembrane region, and the intracellular signaling domain are derived from a CD3ε, CD3γ, CD3δ, or CD3ζ protein; the hinge region and the transmembrane region of the chimeric antigen receptor allow the chimeric antigen receptor to be incorporated into a T cell receptor complex; and when the chimeric antigen receptor is expressed on a surface of a T cell, the chimeric antigen receptor is able to activate the T cell upon binding to (a) soluble EDB, (b) membrane-bound EDB, and / or (c) EDB in an extracellular matrix.SELECTED DRAWING: None

Description

[Background technology]

[0001] Chimeric antigen receptors (CARs) mediate antigen binding and immune cell (e.g., T cell) activation. An engineered receptor in which the functions are fused into a single receptor, and a method for producing such an engineered receptor The drug gives immune cells new abilities to target specific proteins.

[0002] CARs have already been used in recent years in cancer treatment therapies, where modified T cells are used to target newly acquired cancer cells. The ability to recognize cancer antigens on cells allows for more effective targeting of them Typically, autologous T cells are collected from patients who need CAR-T therapy, and then the cells are treated with CAR-T therapy in vitro. By introducing R into T cells and then reinjecting the resulting CAR-T cells into the patient's body, It attacks tumors that have the antigen that CAR recognizes.

[0003] CAR-T cells can be derived from T cells in the patient's own blood (autologous) or from T cells from another healthy donor. For safety reasons, CAR-T cells may be derived from the patient's donor T cells (allogeneic). modified to have specificity for an antigen expressed in tumors but not in healthy cells Once CAR-T cells are introduced into the patient's body, they fight against cancer cells. It becomes a "living drug" that binds to cancer antigens, activates them, and grows to become cells that fight against cancer cells. It exerts toxicity.

[0004] The modification of CAR-T cells has the potential to enhance the proliferation ability of T cells after stimulation and to increase their ability to react to other living cells. Enhancement of cytotoxic effects of cytokines, interleukins, and growth factors It has been shown that the killing effect of T cells against tumor cells can be enhanced in many ways, including by increasing the secretion of cytokines. This can be done.

[0005] In recent years, CAR-T cell immunotherapy has shown highly effective results in the treatment of malignant hematological tumors. CAR-T therapy has made great strides in treating hematological malignancies, but its use in solid tumors remains limited. specificity, durability, safety, and immunosuppressive microenvironment encountered in the treatment of solid tumors in However, it faces many challenges, including the problem of Therefore, to overcome the limitations of current CAR-T cells in solid tumors, more reliable There is a need for high-throughput, safe, and effective CAR-T therapies that can be used to treat a broader range of tumors, including solid tumors. The treatment of Summary of the Invention [Means for solving the problem]

[0006] The present invention provides a chimeric antigen receptor (CAR), comprising: (1) fibronectin extra (2) an antigen-binding domain specific for domain B (EDB) and CD3, CD4, and CD 8. A transmembrane (TM) domain selected from CD28, OX40, or CD137 membrane proteins and (3) CD3ζ intracellular ITAMs (immunoreceptor receptor agonists) with or without costimulatory domains. and a CAR (carbohydrate) tyrosine-based activation motif) domain, which when expressed on the surface of a T cell (a) soluble EDB, (b) membrane-bound EDB, and / or (c) EDB in the extracellular matrix. When bound to DB (e.g., a component of the fibronectin network, The present invention provides a method for the activation of T cells using a T cell-binding protein (TCR), which acts as an adhesion support.

[0007] In certain embodiments, the antigen-binding domain is a single chain antibody (scFv), a nanobody antibody, (e.g. derivatives of VHH (camelid Ig)), single domain antibodies (dAbs, VH or derivatives of VL domains), bispecific T cell-inducing antibodies (BiTEs, bispecific antibodies ) and Dual Affinity ReTargeting ( DART, bispecific antibodies); anticalins (derivatives of lipoproteins); adnectins (10th FN3 (fibronectin)); designed ankyrin repeat protein (DARP in); or avimer.

[0008] In one embodiment, the antigen binding domain is a human scFv or a humanized scFv. .

[0009] In one embodiment, the CAR further comprises a nucleotide sequence between the antigen binding domain and the TM domain. It contains a hinge / spacer domain.

[0010] In one embodiment, the hinge / spacer domain and the TM domain are of the same tandem structure. It is derived from proteins.

[0011] In one embodiment, the similar protein is CD8α and the hinge / spacer domain. The main component is the extracellular domain of CD8α.

[0012] In one embodiment, (3) comprises a costimulatory domain.

[0013] In one embodiment, the costimulatory domain is from CD28.

[0014] In one embodiment, (3) comprises two costimulatory domains.

[0015] In one embodiment, the two costimulatory domains are a costimulatory domain from CD28; and / or a costimulatory domain from CD27, 4-1BB or OX-40.

[0016] In one embodiment, the CAR is an scFv of residues 21-236 of SEQ ID NO:1, CD8α It contains extracellular and transmembrane domains, the 4-1BB intracellular domain, and the CD3ζ intracellular domain. nothing.

[0017] In one embodiment, the CAR further comprises an N-terminal signal peptide sequence (e.g., The hIL-2 signal peptide sequence, or residues 1-20 of SEQ ID NO:1, is included.

[0018] In one embodiment, the CAR comprises the polypeptide of SEQ ID NO:1.

[0019] In another aspect of the invention, there is provided a polynucleotide encoding a CAR of the invention. For example, the polynucleotide can be SEQ ID NO:2.

[0020] In certain embodiments, the polynucleotide is codon-selected for expression in a human cell. has been optimized.

[0021] In another aspect of the invention, there is provided a vector comprising a nucleotide of the invention.

[0022] In one embodiment, the vector is administered to T cells, macrophages and / or NK cells, For example, infecting and administering the CAR to primary human T cells, macrophages, or NK cells. The present invention is directed to a viral vector capable of inducing and / or expressing a gene that is capable of inhibit ...

[0023] In certain embodiments, the vector is a human peripheral monocyte, a monocyte-derived dendritic cell, a hematopoietic stem cell, or and / or infecting and / or inducing PSCs (pluripotent stem cells) with said CAR. is a viral vector capable of expressing

[0024] In certain embodiments, the vector is a lentiviral vector.

[0025] In one embodiment, the lentiviral vector is a self-inactivating lentiviral vector. -It is.

[0026] In another aspect of the invention, a method for the preparation of a CAR of the invention is provided comprising administering to a subject a subject a nucleotide or A cell comprising the vector of the invention is provided.

[0027] In certain embodiments, the cell is an immune cell.

[0028] In one embodiment, the cell is a T cell.

[0029] In one embodiment, the cell is a NK cell.

[0030] In certain embodiments, the cell is a monocyte or a macrophage.

[0031] In certain embodiments, the cells are primary cells isolated from a patient.

[0032] In certain embodiments, the cells are from an established cell line, e.g., the cells are It is derived from a cell line allogeneic to the patient to whom it is administered.

[0033] In certain embodiments, the cells express a cytokine.

[0034] In one embodiment, the cytokine is IL-2, IL-7, IL-12, IL-15 or IL-21.

[0035] In one embodiment, the expression of a cytokine is a promoter that is activated upon activation of an immune cell. The controller is controlled by the

[0036] In certain embodiments, the cells further comprise a safety signaling pathway for downregulating the activity of immune cells. Including Itch.

[0037] In one embodiment, the safety switch is iCaspase9 (inducible caspase- 9) It contains a coding sequence for a monomer, e.g., FKBP dimers that are activated by the FKBP dimer and thus act as an immune regulator. Cell apoptosis is triggered.

[0038] In another aspect of the invention, a subject suffering from a disease or condition treatable by angiogenesis inhibitors is 20. A method of inhibiting angiogenesis in a subject comprising administering to said subject a therapeutically effective amount of a chimeric antibody. The method comprises administering immune cells expressing the chimeric antigen receptor (CAR), CAR) is an antibody specific for (1) fibronectin extra domain B (EDB). (2) a CD3, CD4, CD8, CD28, OX40, or CD13 binding domain; (7) A transmembrane (TM) domain selected from a membrane protein and (3) a costimulatory domain CD3ζ intracellular ITAM (immunoreceptor tyrosine-based activation motif) domain with or without The present invention provides a method comprising:

[0039] In one embodiment, the CAR is any of the CARs described herein.

[0040] In certain embodiments, the disease or condition is a solid tumor or a chronic inflammatory condition.

[0041] In one embodiment, cancer cells derived from solid tumors do not express EDB on the cell surface.

[0042] In one embodiment, the disease or condition is a solid tumor and the method further comprises: Epidemiological checkpoint inhibitors, such as PD-1 inhibitors (e.g., pembrolizumab ( pembrolizumab, nivolumab and cemiplimab emiplimab), PD-L1 inhibitors (e.g., atezolizumab izumab, avelumab and durvalumab ab), CTLA-4 targeted drugs (e.g., ipilimumab) or or immunomodulatory agents (e.g., thalidomide and lenalidomide) The method includes applying

[0043] In certain embodiments, the chronic inflammatory condition is psoriasis, rheumatoid arthritis, arthritis, ulcerative colitis, osteoarthritis, asthma, pulmonary fibrosis, IBD), inflammation-induced Lymphangiogenesis, obesity, diabetes, retinal neovascularization (RNV), diabetic retinopathy, choroidal neovascularization CNV, Age-related macular degeneration (AMD), Metabolic syndrome-related diseases, Long-term intermittent peritoneal dialysis, juvenile arthritis or atherosclerosis.

[0044] In some embodiments, the method further comprises administering to the patient a second therapeutic agent that effectively inhibits angiogenesis. The present invention includes applying

[0045] In certain embodiments, the second therapeutic agent is axitinib, bevacizumab, Bevacizumab, cabozantinib, Verolimus, lenalidomied, Zopanib (pazopanib), ramucirumab, regoraf Regorafenib, sorafenib, sunitinib (sunitinib), thalidomide, vandetanib andetanib and / or ziv-aflibercept cept).

[0046] In one embodiment, the vector of the present invention is administered ex vivo to primary immune cells isolated from a subject. and optionally culturing the vector-transduced primary immune cells ex vivo and / or By amplifying it, immune cells are generated.

[0047] In one embodiment, the method further comprises inhibiting cytokine release syndrome (CRS). Reagents for inhibiting IL-6, such as anti-IL-6 monoclonal antibodies (e.g., tocilizumab cilizumab) and / or globulin therapy. nothing.

[0048] Of course, any embodiment of the invention may be described only by way of example or claim. Any embodiment not expressly disclaimed or otherwise inappropriate. Unless otherwise specified, any one or more of the other embodiments of the present invention may be combined. It is possible. [Brief description of the drawings]

[0049] [Figure 1] FIG. 1 shows EDB-CAR expression in lentivirally transduced human T cells in flow cytometry analysis. M1: mock transduced T cells. T: untransduced T cells. [Figure 2A] Figure 2A shows that in the presence of recombinant EDB protein, EDB-CAR T cells produced IFN-γ. [Figure 2B] Figure 2B shows the lysis of U87-MG cells after 2-24 h co-culture with EDB-CAR T cells at an effector:target (E:T) ratio of 5:1. Cell lysis was measured by LDH method. N=3, each data point reflects the mean SEM of triplicates. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; two-tailed Student's t test.) [Figure 3A] 3A-3B show the expression levels of EDB in multiple cell lines detected at the protein level by Western blotting (FIG. 3A) and at the mRNA level by qPCR (FIG. 3B). [Figure 3B] 3A-3B show the expression levels of EDB in multiple cell lines detected at the protein level by Western blotting (FIG. 3A) and at the mRNA level by qPCR (FIG. 3B). [Figure 4A] Figures 4A-4B show the cytotoxicity of EDB-CAR T cells against human or mouse cancer cells and HUVEC cells after 24 h of co-culture at each E:T ratio. Target cell destruction was measured by LDH assay. (N = 3; two-tailed Student's t-test.) [Figure 4B] Figures 4A-4B show the cytotoxicity of EDB-CAR T cells against human or mouse cancer cells and HUVEC cells after 24 h of co-culture at each E:T ratio. Target cell destruction was measured by LDH assay. (N = 3; two-tailed Student's t-test.) [Diagram 5] Figure 5 shows that EDB-CAR T cells produced IFN-γ in vitro in the presence of tumor cells. EDB CAR-T cells were co-cultured with cancer cells at different E:T ratios for 24 h, and supernatants were collected to detect IFN-γ. N = 3; two-tailed Student's t-test. [Figure 6] Figure 6 shows that EDB-CAR T cells produced TNF-α in the presence of tumor cells. EDB-CAR T cells were co-cultured with cancer cells at different E:T ratios for 24 h, and the supernatants were collected to detect TNF-α. (N = 3; two-tailed Student's t-test.) [Figure 7] FIG. 7 shows expression of EDB-CAR in NK-92 after transduction analyzed by flow cytometry. [Figure 8A] Figure 8A shows the cytotoxicity of EDB-CAR NK-92 cells, and Figure 8B shows that EDB-CAR NK-92 cells released IFN-γ into the supernatant after 24 hours of co-culture with U87-MG cells at each E:T ratio (N = 3; two-tailed Student's t-test). [Figure 9] Figure 9 shows the histopathological analysis of mouse organ tissues by hematoxylin and eosin staining, which shows that no pathological changes / toxicity occurred in normal mice injected with very high doses of EDB-specific CAR-T cells. Images were taken at 20x magnification by a Leica Aperio VERSA 8 section scanner. Each division indicates 100 μm. [Figure 10] Figures 10A-10B show the purity of CD14+ monocytes and the expression of EDB-CAR. Specifically, Figure 10A shows the purity of CD14+ monocytes / macrophages separated from PBMCs with CD14 MicroBeads (proven by flow cytometry). Figure 10B shows the expression of EDB-CAR in lentiviral-transduced human monocytes analyzed by flow cytometry. M1 represents the mock-transduced negative control. Also shown is the transduction efficiency. [Figure 11A]Figures 11A-11J show the characterization of EDB-targeted CAR monocytes. Specifically, EDB-CAR-monocytes / macrophages were incubated with each EDB-expressing cell line at different effector:target (E:T) ratios (Figures 11A-11F) or 5 μg / mL EDB protein (Figures 11G-11J) for 24 h. Culture supernatants were collected to measure the expression of TNF-α (Figures 11C, 11F, and 11J), IL-12 (Figures 11B, 11E, and 11H), and IFN-γ (Figures 11A, 11D, 11G). Data represent three independent experiments. Each data point reflects the mean SEM of triplicates. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; two-tailed Student's t test.) [Figure 11B] Figures 11A-11J show the characterization of EDB-targeted CAR monocytes. Specifically, EDB-CAR-monocytes / macrophages were incubated with each EDB-expressing cell line at different effector:target (E:T) ratios (Figures 11A-11F) or 5 μg / mL EDB protein (Figures 11G-11J) for 24 h. Culture supernatants were collected to measure the expression of TNF-α (Figures 11C, 11F, and 11J), IL-12 (Figures 11B, 11E, and 11H), and IFN-γ (Figures 11A, 11D, 11G). Data represent three independent experiments. Each data point reflects the mean SEM of triplicates. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; two-tailed Student's t test.) [Figure 11C]Figures 11A-11J show the characterization of EDB-targeted CAR monocytes. Specifically, EDB-CAR-monocytes / macrophages were incubated with each EDB-expressing cell line at different effector:target (E:T) ratios (Figures 11A-11F) or 5 μg / mL EDB protein (Figures 11G-11J) for 24 h. Culture supernatants were collected to measure the expression of TNF-α (Figures 11C, 11F, and 11J), IL-12 (Figures 11B, 11E, and 11H), and IFN-γ (Figures 11A, 11D, 11G). Data represent three independent experiments. Each data point reflects the mean SEM of triplicates. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; two-tailed Student's t test.) [Figure 11D] Figures 11A-11J show the characterization of EDB-targeted CAR monocytes. Specifically, EDB-CAR-monocytes / macrophages were incubated with each EDB-expressing cell line at different effector:target (E:T) ratios (Figures 11A-11F) or 5 μg / mL EDB protein (Figures 11G-11J) for 24 h. Culture supernatants were collected to measure the expression of TNF-α (Figures 11C, 11F, and 11J), IL-12 (Figures 11B, 11E, and 11H), and IFN-γ (Figures 11A, 11D, 11G). Data represent three independent experiments. Each data point reflects the mean SEM of triplicates. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; two-tailed Student's t test.) [Figure 11E]Figures 11A-11J show the characterization of EDB-targeted CAR monocytes. Specifically, EDB-CAR-monocytes / macrophages were incubated with each EDB-expressing cell line at different effector:target (E:T) ratios (Figures 11A-11F) or 5 μg / mL EDB protein (Figures 11G-11J) for 24 h. Culture supernatants were collected to measure the expression of TNF-α (Figures 11C, 11F, and 11J), IL-12 (Figures 11B, 11E, and 11H), and IFN-γ (Figures 11A, 11D, 11G). Data represent three independent experiments. Each data point reflects the mean SEM of triplicates. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; two-tailed Student's t test.) [Figure 11F] Figures 11A-11J show the characterization of EDB-targeted CAR monocytes. Specifically, EDB-CAR-monocytes / macrophages were incubated with each EDB-expressing cell line at different effector:target (E:T) ratios (Figures 11A-11F) or 5 μg / mL EDB protein (Figures 11G-11J) for 24 h. Culture supernatants were collected to measure the expression of TNF-α (Figures 11C, 11F, and 11J), IL-12 (Figures 11B, 11E, and 11H), and IFN-γ (Figures 11A, 11D, 11G). Data represent three independent experiments. Each data point reflects the mean SEM of triplicates. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; two-tailed Student's t test.) [Figure 11G]Figures 11A-11J show the characterization of EDB-targeted CAR monocytes. Specifically, EDB-CAR-monocytes / macrophages were incubated with each EDB-expressing cell line at different effector:target (E:T) ratios (Figures 11A-11F) or 5 μg / mL EDB protein (Figures 11G-11J) for 24 h. Culture supernatants were collected to measure the expression of TNF-α (Figures 11C, 11F, and 11J), IL-12 (Figures 11B, 11E, and 11H), and IFN-γ (Figures 11A, 11D, 11G). Data represent three independent experiments. Each data point reflects the mean SEM of triplicates. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; two-tailed Student's t test.) [Figure 11H] Figures 11A-11J show the characterization of EDB-targeted CAR monocytes. Specifically, EDB-CAR-monocytes / macrophages were incubated with each EDB-expressing cell line at different effector:target (E:T) ratios (Figures 11A-11F) or 5 μg / mL EDB protein (Figures 11G-11J) for 24 h. Culture supernatants were collected to measure the expression of TNF-α (Figures 11C, 11F, and 11J), IL-12 (Figures 11B, 11E, and 11H), and IFN-γ (Figures 11A, 11D, 11G). Data represent three independent experiments. Each data point reflects the mean SEM of triplicates. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; two-tailed Student's t test.) [Figure 11J]Figures 11A-11J show the characterization of EDB-targeted CAR monocytes. Specifically, EDB-CAR-monocytes / macrophages were incubated with each EDB-expressing cell line at different effector:target (E:T) ratios (Figures 11A-11F) or 5 μg / mL EDB protein (Figures 11G-11J) for 24 h. Culture supernatants were collected to measure the expression of TNF-α (Figures 11C, 11F, and 11J), IL-12 (Figures 11B, 11E, and 11H), and IFN-γ (Figures 11A, 11D, 11G). Data represent three independent experiments. Each data point reflects the mean SEM of triplicates. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; two-tailed Student's t test.) [Figure 12] Figure 12 shows expression of EDB-CAR (SEQ ID NO: 1-17) in primary human T cells in flow cytometry analysis. M1: mock transduced T cells. T: untransduced T cells. [Figure 13] Figure 13 shows that repeated stimulation of primary human T cells transduced with EDB-CAR (SEQ ID NO: 1, 3-7) can activate proliferation of the transduced cells. Proliferation was confirmed by cell counting. M1: mock transduced T cells. T: untransduced T cells. [Figure 14] Figure 14 shows that primary human T cells transduced with EDB-CAR (SEQ ID NO: 1, 3-7) are cytotoxic to U87MG cancer cells. M1: mock-transduced T cells. T: untransduced T cells. [Figure 15] Figure 15 shows that repeated stimulation of primary human T cells transduced with EDB-CAR (SEQ ID NO: 1, 3, 18) can activate proliferation of the transduced cells. The proliferation was confirmed by flow cytometry analysis after labeling of T cells with Celltrace dye. M1: mock transduced T cells. T: untransduced T cells. [Figure 16]Figure 16 shows that primary human T cells transduced with EDB-CAR are cytotoxic to U87MG cancer cells. EDB BB: SEQ ID NO: 1; EDB137pro: SEQ ID NO: 10; EDB-αCD3: SEQ ID NO: 18; EDB-αCD3 / EDB BB: co-transduced with SEQ ID NO: 18 and SEQ ID NO: 1; EDB-αCD3 / 137pro: co-transduced with SEQ ID NO: 18 and SEQ ID NO: 10. M1: mock transduced T cells. T: untransduced T cells. [Figure 17] Figure 17 shows that repeated stimulation of primary human T cells transduced with EDB-CAR can activate the proliferation of transduced cells. EDB BB: SEQ ID NO: 1; EDB28: SEQ ID NO: 3; CD3z: SEQ ID NO: 12; CD3z / 28pro: co-transduced with SEQ ID NO: 12 and SEQ ID NO: 9; CD3z / 137pro: co-transduced with SEQ ID NO: 12 and SEQ ID NO: 10; CD3z / CD4CD28: co-transduced with SEQ ID NO: 12 and SEQ ID NO: 8. Proliferation was confirmed by flow cytometry analysis after labeling of T cells with Celltrace dye. M1: mock transduced T cells. T: untransduced T cells. [Figure 18] Figure 18 shows that primary human T cells transduced with EDB-CAR are cytotoxic to U87MG cancer cells. EDB BB: SEQ ID NO: 1; EDB28: SEQ ID NO: 3; CD3z: SEQ ID NO: 12; CD3z / 28pro: co-transduced with SEQ ID NO: 12 and SEQ ID NO: 9; CD3z / 137pro: co-transduced with SEQ ID NO: 12 and SEQ ID NO: 10; CD3z / CD4CD28: co-transduced with SEQ ID NO: 12 and SEQ ID NO: 8. M1: mock transduced T cells. T: untransduced T cells. [Figure 19] Figure 19 shows that repeated stimulation of primary human T cells transduced with EDB-CAR can activate proliferation of transduced cells. EDB BB: SEQ ID NO: 1; EDB28: SEQ ID NO: 3; CD3zFL: SEQ ID NO: 11; CD3eFL: SEQ ID NO: 15. Proliferation was confirmed by flow cytometry analysis after labeling T cells with Celltrace dye. M1: mock transduced T cells. T: untransduced T cells. [Figure 20] Figure 20 shows that primary human T cells transduced with EDB-CAR are cytotoxic to U87MG cancer cells. EDB BB: SEQ ID NO: 1; EDB28: SEQ ID NO: 3; CD3zFL: SEQ ID NO: 11; CD3zFL / 28pro: co-transduced with SEQ ID NO: 11 and SEQ ID NO: 9; CD3zFL / 137pro: co-transduced with SEQ ID NO: 11 and SEQ ID NO: 10; CD3zFL / CD4CD28: co-transduced with SEQ ID NO: 11 and SEQ ID NO: 8; CD3eFL: SEQ ID NO: 15; CD3eFL / 28pro: co-transduced with SEQ ID NO: 15 and SEQ ID NO: 9; CD3eFL / 137pro: co-transduced with SEQ ID NO: 15 and SEQ ID NO: 10; CD3eFL / CD4CD28: co-transduced with SEQ ID NO: 15 and SEQ ID NO: 8. M1: mock transduced T cells. T: untransduced T cells. [Figure 21] Figure 21 shows that repeated stimulation of primary human T cells transduced with EDB-CAR can activate proliferation of transduced cells. EDB BB: SEQ ID NO: 1; EDB28: SEQ ID NO: 3; CD3eFLCD28: SEQ ID NO: 16; CD3eFLCD137: SEQ ID NO: 17. Labeling of T cells with Celltrace dye was followed by flow cytometry analysis and imaging showing proliferative T cell population status. M1: mock transduced T cells. T: untransduced T cells. [Figure 22] Figure 22 shows that primary human T cells transduced with EDB-CAR are cytotoxic to U87MG cancer cells. EDB BB: SEQ ID NO: 1; EDB28: SEQ ID NO: 3; CD3eFL / 28pro: co-transduced with SEQ ID NO: 15 and SEQ ID NO: 9; CD3eFL / 137pro: co-transduced with SEQ ID NO: 15 and SEQ ID NO: 10; CD3eFLCD28: SEQ ID NO: 16; CD3eFLCD137: SEQ ID NO: 17. M1: mock transduced T cells. T: untransduced T cells. [Diagram 23]Figure 23 shows that the "Hijack Plus" EDB-CAR based on SEQ ID NOs: 16 and 17 was incorporated into the TCR complex. Human primary T cells were transduced with the "Hijack Plus" EDB-CAR and immunoprecipitated with a biotin-labeled polyclonal goat anti-human IgG F(ab')2 fragment antibody. The CD3ζ subunit of the TCR complex was detected with an anti-CD3ζ antibody (Figure 23A). Jurkat cells were transduced with the "Hijack Plus" EDB-CAR and immunoprecipitated with a biotin-labeled polyclonal goat anti-human IgG F(ab')2 fragment antibody. The CD3ζ subunit of the TCR complex was detected with an anti-CD3ζ antibody (Figure 23B). [Figure 24] Figure 24 shows the in vivo efficacy of each EDB CAR T cell in treating tumors formed by U87MG cells using NCG mice. The second generation EDB CAR (Figure 24A), "trans" EDB CAR (Figure 24B), bispecific EDB-αCD3 (Figure 24C), "Hijack Plus" EDB-CAR (Figure 24D) or "Hijack Plus" EDB-CAR (Figure 24E) showed different levels of tumor growth inhibition or tumor regression.

[0050] Specific embodiments 1. Overview Part of the invention described herein is an antibody specific for the fibronectin EDB domain. or its antigen-binding fragment is useful for constructing CAR (chimeric antigen receptor) structures and is a membrane-bound ED B, or EDB in the extracellular matrix (deposited in tumor tissue) as well as in solution. This is based on the finding that the soluble form of EDB can also be recognized in the presence of β-

[0051] Another part of the invention described herein is the discovery of a curious finding: the EDB-specific CA R-bearing immune cells (e.g., CAR T cells) are transduced in vitro with normal human umbilical vein endothelial cells ( Although it has cytotoxicity for HUVECs, it is difficult to detect large amounts of immune cells carrying such CARs. For example, the injection of T cells into mice does not result in expected toxicity. As is known in the art, toxicity is a major obstacle to the widespread use of CAR T cell therapy, primarily Cytokine Release Syndrome (CRS) and neurotoxicity. For example, Toxicities associated with Her2 and carbonic anhydrase IX have led to the development of CARs to target Her2 and carbonic anhydrase IX for the treatment of solid tumors. Early attempts to do so were unsuccessful and led to uncontrolled inflammation, tissue damage, and ultimately death. Symptoms of CRS include fever, hypotension, hypoxia, end-organ dysfunction, cytopenia, and blood coagulation. Neurotoxicity is variable and includes encephalopathy, cognitive impairment, speech disorders, However, when using the CAR construct of the present invention, There seem to be no symptoms.

[0052] As such, the CAR constructs of the present invention are useful in CAR-based immunotherapy, e.g., CAR T Alternatively, the present invention may be useful for treating diseases in which angiogenesis by CAR NK cells is a pathological condition. These diseases include cancer and inflammatory diseases.

[0053] Thus, in one aspect, the present invention provides a chimeric antigen receptor (CAR), comprising: (1) an antigen-binding domain specific for fibronectin extra domain B (EDB); (2) from CD3, CD4, CD8, CD28, OX40 or CD137 membrane proteins (3) CD3 with or without a costimulatory domain; and ζ contains an intracellular ITAM (immunoreceptor tyrosine-based activation motif) domain and is expressed on T cells. When expressed on the surface of the CAR, it produces (a) soluble EDB, (b) membrane-bound EDB and or (c) upon binding to EDB in the extracellular matrix (e.g., fibronectin). (a component that acts as a cell attachment support), and can activate T cells. A representative CAR of the present invention is SEQ ID NO:1.

[0054] Other exemplary CARs of the present invention are SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 11, 12, Shown here are 13, 14, 15, 16, and 17.

[0055] Another aspect of the invention provides a polynucleotide encoding a CAR of the invention, e.g. For example, SEQ ID NO:2 is provided.

[0056] In another aspect of the invention, a vector comprising a polynucleotide of the invention, e.g. A lentiviral vector comprising SEQ ID NO:2 is provided.

[0057] In another aspect of the invention, the CAR of the invention, the polypeptide of the invention and / or A cell, e.g., an immune cell, is provided that comprises a vector of the invention. The cell may be a T cell or NK cells may also be used.

[0058] In another aspect of the invention, a subject suffering from a disease or condition treatable by angiogenesis inhibitors is 20. A method of inhibiting angiogenesis in a subject comprising administering to said subject a therapeutically effective amount of a chimeric antibody. The method comprises administering immune cells expressing the chimeric antigen receptor (CAR), CAR) is an antibody specific for (1) fibronectin extra domain B (EDB). (2) a CD3, CD4, CD8, CD28, OX40, or CD13 binding domain; (7) A transmembrane (TM) domain selected from a membrane protein and (3) a costimulatory domain CD3ζ intracellular ITAM (immunoreceptor tyrosine-based activation motif) domain with or without The present invention provides a method comprising:

[0059] The disease or condition may be a solid tumor or a chronic inflammatory condition.

[0060] The above are general aspects of the invention described herein, and the following are further details of different aspects of the invention. Provide details.

[0061] 2. EDB of Fibronectin (FN) Fibronectin is a high molecular weight glycoprotein in the extracellular matrix (ECM) and acts as a transmembrane The receptor proteins integrins and ECM components (e.g., collagen, fibrin Fibronectin binds to heparan sulfate proteoglycans. It exists as a dimer consisting of two nearly identical monomers linked via a pair of disulfide bonds. Fibronectin is encoded by a single gene, but alternative versions of its precursor mRNA are Splicing produces at least 20 different isoforms in humans. (White and Muro's general discussion of FN function, "Fibronect in splice variants: understanding their multiple roles in health and disease usi ng engineered mouse models.” IUBMB Life. 63(7):538-546, 2011, incorporated herein by reference. ).

[0062] Fn monomers are approximately 250 kda in size and share a disulfide bond near the C-terminus. FN is a member of three different homologies: type I, type II, and type III. These independent sequences consist of overlapping units of approximately 40, 60 and 90 amino acids, respectively. Many of the domains that fold in a specific manner are also present in various ECM proteins. Among them, type III module is the most abundant module in FN molecules and is Type I modules have been found in many proteins from many species, whereas type I modules have only been found in vertebrates. It has not been done.

[0063] In humans, FN protein diversity consists of two alternatively spliced ​​ends. Extra Domains A and B (EIIIA and EIII A) and two other type III repeats. This is due to the connecting fragment - type III connecting segment (IIICS). DA and EDB splicing is similar (or completely included) in all species splicing of the IIICS region is species specific ( Humans have five variants, rodents have three, and birds have two.

[0064] FN can be a soluble dimer in plasma or it can be secreted intact from hepatocytes into the circulation (plasma FN or pFN) or deposited in the tissue ECM as insoluble fibers (cellular F These two FN isoforms are characterized by the presence of EDA and EDB domains. (a) pFN lacks the alternatively spliced ​​EDA and EDB sequences. and (b) cFNs contain different ratios of these domains.

[0065] As used herein, the terms "EDB," "EIIIB," and "EDB domain" Or "ED-B domain" refers to the extra domain B of (human) fibronectin. In humans, EDB is a type III homology domain with approximately 91 residues. EDB is nearly undetectable in healthy adult tissues, but is abundant in the vasculature of many aggressive solid tumors. Its abundant presence makes EDB a suitable target for anti-cancer and / or anti-inflammatory treatments of the present invention.

[0066] In one embodiment, the antigen recognized by the CAR of the present invention is a fibronectin splice. ising isoforms, for example, the ED-B domain of FN.

[0067] 3. Antibodies and antigen-binding fragments of EDB In one embodiment, a CAR that binds to the EDB domain of fibronectin has high affinity. sum force, e.g., nanomolar or subnanomolar K D It has value in the field. Any method that can measure the concentration of a substance, such as biolayer interferometry (BLI), surface plasmon resonance (S Affinity can be measured by PCR or BIACORE, or other methods. do.

[0068] In one embodiment, the antigen-binding portion of the CAR is an EDB-specific antibody or its antigen-binding Fragment-based, e.g., those described in WO99 / 058570 (both of which are incorporated herein by reference). (incorporated herein).

[0069] In one embodiment, the EDB-specific antibody or antigen-binding fragment thereof is CAA06864. .2 (incorporated herein by reference).

[0070] In one embodiment, the EDB-specific antibody or antigen-binding fragment thereof is at least one of the L19 antibody. It is based on at least one CDR sequence.

[0071] In one embodiment, the EDB-specific antibody or antigen-binding fragment thereof is based on huBC1. The huBC1 antibody is a humanized antibody that targets the extracellular matrix of many invasive tumors. EDB-FN is involved in carcinoembryonic antigens and angiogenesis. Related.

[0072] In one embodiment, the antigen-binding portion of a CAR is an amino acid sequence of a CAR provided herein. Any antigen-binding portion of the nucleic acid sequence may be bound to at least 80%, 81%, 82%, 83%, 84%, or 85% of the nucleic acid sequence. 5%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 9 In one embodiment, the nucleic acid has 5%, 96%, 97%, 98% or 99% identity. The antigen-binding portion of a CAR is one or more CDR regions of an antibody exemplified herein. and a diversity of 5 or less (e.g., 4, 3, 2, or 1) amino acid residues in and bind to the same epitope on EDB with approximately the same affinity (e.g., K D In one embodiment, the amino acid residue diversity is conservative amino acid residues. As used herein, the term "conservative amino acid substitution" refers to a substitution of a is an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution occurs. This is about exchange.

[0073] As used herein, an "antibody" or "globulin (Ig)" is generally a compound that is It contains polypeptide chains of two heavy chains (HC) and two light chains (LC), and the like, for example, nanobodies from camelids (e.g., alpacas). Antibodies (containing only heavy chains), single domain antibodies (dAbs) (derived from heavy or light chains) ), and full length or functional specific mutants, variants or derivatives thereof (including nucleotides, These include, but are not limited to, human, chimeric, humanized and fully human antibodies. However, these antibodies retain the basic epitope binding characteristics of Ig molecules and are bispecific. Antibodies or globulins include any of the following: isomeric, multispecific, and dual variable region globulins. Types, e.g., IgG, IgE, IgM, IgD, IgA and IgY (globulins) It is the main antibody in the blood of birds, reptiles, and lungfish, and in the egg yolk. concentration), or subtype, e.g., IgG1, IgG2, IgG3, IgG 4, IgA1 and IgA2 and may be allotypes.

[0074] The amino acid sequences of naturally occurring non-human antibodies or fragments thereof, such as VHH sequences (particularly In addition, one or more amino acid residues in the frame sequence are replaced with a normal four-chain antibody of human origin. by replacing one or more amino acid residues that appear at the corresponding positions in the VH domain of the target polypeptide. This results in a "humanized" antibody or antigen-binding fragment thereof. Methods for humanization are well known and include Humanized antibodies or domains thereof may be more distinct from the corresponding naturally occurring non-human antibodies or domains thereof. Such antigen-binding fragments have several advantages, including reduced immunogenicity.

[0075] Nucleotide sequences encoding naturally occurring antibodies are provided, and further novel nucleotide sequences are One or more modifications in the nucleotide sequence may be made so that the code sequence encodes the "humanized" version thereof. "Humanization" can be achieved by modifying multiple codons. Expression of the humanized antibody or fragment can provide a humanized antibody or fragment. Based on the amino acid sequence of the non-human sequence, a humanized form is designed, and then the peptide is synthesized by peptide synthesis technology. Alternatively, the skilled artisan can synthesize one or more naturally occurring saccharides in an appropriate form. The sequences present (e.g., one or more FR sequences or CDR sequences) and / or or combining one or more portions of one or more synthetic or semi-synthetic sequences. and providing a nucleotide sequence or nucleic acid encoding the humanized antibody or fragment thereof. Optionally, the humanized sequence can be codon optimized and then expressed in host immune cells, e.g., human T cells. It may also be expressed in cells, NK cells, monocytes or macrophages.

[0076] As used herein, an "antibody derivative or antigen-binding fragment" refers to an antibody that is (i) a Fab fragment, optionally including a molecule comprising a polypeptide chain derived from a non-full-length antibody of Variable light chain (VL), variable heavy chain (VH), constant light chain (CL) and constant heavy chain 1 (CH1) (ii) a F(ab')2 fragment, which is linked via a disulfide bond in the hinge region (iii) a bivalent fragment containing two linked Fab fragments; (iv) a variable region (Fv) fragment, which consists of the VH and CH1 domains of an antibody; (v) single-arm VL and VH domains; (v) single-domain antibodies (dAbs ) fragments, including single variable domains; (vi) isolated complementarity determining regions (CDRs); (vii) single chain Fv fragments (scFv); (viii) bivalent antibodies, which contain VH and VL domains The enzymes are expressed on a single polypeptide chain, but the linker used is too short to be integrated into the same chain. Since pairing between the two domains in the chain is not possible, these domains are not part of another chain. The complementary domains of the two antibodies are paired together to form a bivalent, bispecific antibody, resulting in two antigen-binding sites. (ix) a linear antibody, which comprises a pair of linearly linked Fv segments (VH-CH1-VH- CH1) which forms a pair of antigen-binding regions with a complementary light chain polypeptide; (x) other non-full-length portions of the heavy and / or light chains of globulins, or mutations thereof This includes, but is not limited to, any form, variant or derivative, either alone or in any combination. Not determined.

[0077] In certain embodiments, the antigen-binding domain is a single chain antibody (scFv), a nanobody antibody, (e.g. derivatives of VHH (camelid Ig)), single domain antibodies (dAbs, VH or derivatives of VL domains), bispecific T cell-inducing antibodies (BiTEs, bispecific antibodies ) and Dual Affinity ReTargeting ( DART, bispecific antibodies); anticalins (derivatives of lipoproteins); adnectins (10th FN3 (fibronectin)); designed ankyrin repeat protein (DARP in); or avimer.

[0078] In one embodiment, the antigen binding domain is a human scFv or a humanized scFv. .

[0079] In either case, the derivative or fragment retains or nearly retains the target binding properties of the full-length antibody. Maintain (for example, K D 5%, 10%, 20%, 30%, 40%, 50%, 80% at least 2, 3, 5, 7, 8 or 10 times higher than the full-length antibody.

[0080] In certain embodiments, the antigen-binding fragments of the present invention further comprise an "antibody-based binding protein." Proteins include, but are not limited to, other non-globulin or non-antibody inducers, as used herein. If the derivative is a nucleotide sequence, it is preferable to use at least one VH (heavy chain variable region), VL (light chain variable region) derived from Such antibody-based proteins contain a heavy chain constant region (CH), or a heavy chain constant region (CH). (i) Fc fusion proteins of binding proteins, which contain all or part of the globulin CH domain (ii) those that contain the main receptor or receptor component, the VH and / or VL domains; (iii) a binding protein coupled to a replaceable molecular scaffold; and / or VL and / or CH domains not normally found in naturally occurring antibodies or antibody fragments. These include, but are not limited to, molecules that are combined and / or assembled in a convenient manner. I can't.

[0081] In certain embodiments, the antigen-binding fragments of the present invention include "modified antibody forms," As used herein, antibody-drug conjugates, polyalkylene oxide-modified s cFv, Monobody, Diabody, Camelidae Animal (e.g., alpaca) antibodies, domain antibodies, bispecific or trispecific antibodies, I Two IgG structures linked via gA or J chain and secretory component, shark antibodies, New World spirits Protozoan framework + non-New World primate CDRs, IgG4 with hinge region removed Antibody, IgG engineered with two extra binding sites in the CH3 domain, for Fcγ receptors An antibody with an altered Fc region to enhance affinity for Includes the body.

[0082] In certain embodiments, the antigen-binding fragments of the present invention further include "antibody mimetics." However, as used herein, proteins that do not belong to the globulin family include Some are not proteins but, for example, aptamers or synthetic polymers. "Antibody mimetics" or "alternative scaffolds" have a β-sheet structure similar to that of antibodies. Potential advantages over glycerol include better solubility, greater tissue penetration, and improved resistance to heat and enzymes. Higher stability and relatively low production costs. A number of antibody mimetics are provided, providing candidates that specifically bind to each possible target. As with antibodies, high-throughput screening (HTS) techniques and Display technologies (e.g., phage display, bacterial display, yeast or Target-specific antibody mimetics can be developed by mammalian display. Currently, the developed antibody mimetics are based on designed ankyrin repeat proteins (DARPins). (also called C-type lectins, A-domain proteins of Staphylococcus aureus, transferrin , lipoproteins, fibronectin type III domain 10, Kunitz domain Protease inhibitors, ubiquitin-derived binders (called affilins), Listeria-derived binding agents, cystine knots or knot proteins, thioredoxins SynA-based binders, SH-3 domains, stradobodies ), disulfide bonds and Ca 2+ The "A domain" of the membrane receptor, CTLA4, stabilized by Compounds based on Fyn SH3 and aptamers (peptides that bind to specific target molecules) Contains the tide molecule.

[0083] The antigen-binding portion of the CAR, specifically CAA068, which specifically recognizes EDB fibronectin The scFv based on 64.2 may utilize the various antibody formats described herein. For example, other than scFv, the CDR sequences based on CAA06864.2 include Fab, (F ab')2, diabody, minibody or nanobody antibodies In one embodiment, the antigen-binding fragment is in the form of an scFv. In one embodiment, the heavy and light chains are linked via a peptide linker.

[0084] In one embodiment, the CAR is selected from SEQ ID NOs: 1, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16 or 17 sequences.

[0085] 4. CAR specific to EDB In one aspect of the present invention, a method for the preparation of a fibronectin-derived antigen-binding fragment having specificity for the EDB of fibronectin is provided. When expressed on the surface of a T cell, the CAR is a) soluble EDB, (b) membrane-bound EDB, and / or (c) EDB in the extracellular matrix B (e.g., a component of the fibronectin network, which mediates cell adhesion) The present invention provides a method for the activation of T cells by using a cellular marker that acts as a support.

[0086] In one embodiment, the chimeric antigen receptor comprises an extracellular antigen-binding domain, a transmembrane (TM) domain, domain, one or more costimulatory domains and an intracellular signaling domain. In embodiments, the CAR further comprises a hinge region between the antigen binding domain and the TM domain. The hinge and TM domains are derived from the same protein. The proteins may be the same or may be derived from different proteins.

[0087] For example, in one embodiment, the CAR comprises: (1) a fibronectin extradomain (2) an antigen-binding domain specific for, for example, CD3, CD4 , CD8, CD28, OX40 or CD137 membrane protein. ) domain, (3) intracellular ITAM capable of CD3ζ with or without a costimulatory domain (immunoreceptor tyrosine-based activation motif) domain.

[0088] In one embodiment, the extracellular antigen binding region is an sc-Fv, Fab, scFab or or an scIgG fragment.

[0089] In one embodiment, the transmembrane domain is a domain selected from the group consisting of CD3ζ, CD4, CD8, CD28, OX40 or comprising the transmembrane domain of CD137.

[0090] In one embodiment, the transmembrane region comprises the transmembrane region of the CD8 transmembrane domain.

[0091] In one embodiment, the transmembrane region is a CD8 transmembrane domain, e.g., a CD8α transmembrane domain. The CD8α hinge region of the CD8α signaling domain (e.g., the CD8α hinge region of SEQ ID NO:1) .

[0092] In one embodiment, the CAR further comprises an extracellular antigen binding domain and a transmembrane domain. In one embodiment, the hinge region comprises a CD8 hinge region, For example, it is derived from the CD8α hinge region contained in SEQ ID NO:1.

[0093] In one embodiment, the hinge region and the TM region are from the same protein, e.g. For example, both may be derived from the CD8 protein.

[0094] In one embodiment, the hinge and TM regions are from different proteins, e.g. For example, the hinge region is from the CD8α protein, and the TM region is from CD3 or CD28. It may be derived from the TM region.

[0095] In one embodiment, the hinge region is selected from the group consisting of CD3γ, CD3δ, CD3ε, CD3ζ, CD1 37 or CD28 protein, and the TM region is CD3γ, CD3δ, or CD3ε. , CD3ζ, CD137 or CD28.

[0096] In one embodiment, the hinge and / or transmembrane region of the chimeric receptor is a chimeric protein. This allows proteins to mix with the TCR complex.

[0097] In one embodiment, the chimeric receptor that is mixed into the TCR complex provides an extra costimulatory signal. It may also have the following.

[0098] In one embodiment, one polypeptide comprises a primary T cell activation signal, e.g. , CD3γ, CD3δ, CD3ε, and CD3ζ-derived, while another type The peptide is induced by costimulatory signals, such as those from CD28, CD137, and OX40. Things like that can be seen.

[0099] In one embodiment, primary T cell activation signals are generated with or without T cell costimulation. The effect of immunization is mediated by a bispecific polypeptide that binds tumor antigens and T cell receptors. .

[0100] In one embodiment, the primary T cell activation signal binds to a tumor antigen and a T cell receptor. The binding is mediated by a bispecific polypeptide which binds to the polypeptide and which is active. It may be secreted by activated T cells or added exogenously.

[0101] In one embodiment, the length of the hinge region in the CAR is SEQ ID NO: 1, 3, 4, 5, Hinge at 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 For example, the hinge region is a region of SEQ ID NO: 1, 3, 4, 5, 6, Hinge region at 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residue longer or shorter than good.

[0102] In one embodiment, the CAR activates immune cells expressing one or more CARs. It contains a signaling domain that activates the

[0103] In one embodiment, the CAR stimulates one or more (e.g., two) T cells. In one embodiment, one or more The signaling domains are TCRζ, FcRγ, FcRβ, FcRε, CD3γ, and CD3 δ, CD3ε, CD3ζ, and CD5, CD22, CD79a, CD79b and CD66 In one embodiment, the signaling domain of C AR is a CD3ζ signaling domain, e.g., the CD3ζ signaling domain in SEQ ID NO:1. Includes a transduction domain.

[0104] In one embodiment, the two costimulatory domains are a costimulatory domain from CD28; and / or a costimulatory domain from CD27, 4-1BB or OX-40.

[0105] In one embodiment, the CAR further comprises one or more of the following: CD2, CD3, CD4, CD5, CD7 , CD27, CD28, CD30, CD40, CD83, CD86, CD127, CD1 34, CD137 / 4 -1BB, 4-1BBL, OX-40, PD-1, LFA-1, Lck, DAP10, LIGHT, NKG2C, B7-H3, CD3ζ or ICOS In one embodiment, the antibody comprises one or more costimulatory domains from one or more of: One or more of the costimulatory domains are CD3ζ, FcεRIγ, PKCθ, or ZAP70 In one embodiment, the CAR comprises an intracellular signaling region derived from CD28 costimulatory In one embodiment, the CAR comprises a co-stimulatory signaling domain of the CAR. 4-1BB (CD137) as a marker for enhanced antigen activation and increased potency. In another embodiment, the CAR comprises an ITAM derived from the costimulatory domain of CD28. It also contains ITAM-derived CAR-mediated T cell activation.

[0106] In one embodiment, the CAR further comprises a primary stimulatory signal, e.g., CD3ζ, A single polypeptide encoding CD3ε, CD3γ, and CD3δ. 5, CD7, CD27, CD28, CD30, CD40, CD83, CD86, CD12 7, CD134, CD137 / 4-1BB, 4-1BBL, OX-40, PD-1, LFA1, Lck, DAP10, LIGHT, NKG2C, B7-H3 or ICOS In some embodiments, the co-stimulatory signal is encoded by a polypeptide that encodes a co-stimulatory signal. In the present invention, the one or more costimulatory domains are selected from the group consisting of CD3ζ, FcεRIγ, PKCθ, and It contains the intracellular signaling domain derived from ZAP70. In this conformation, or the membrane proximity of the costimulatory signaling domain resembles that of the native configuration.

[0107] In one embodiment, a leader sequence is added to the N-terminus of the CAR to facilitate expression of the CAR. In one embodiment, the GM-CSF receptor is fused to a sequence or signal peptide. In one embodiment, the leader sequence is human IL-2 This is the leader sequence of

[0108] In one embodiment, the CAR further comprises a polypeptide that displays or tracks CAR expression. The reporter molecule includes a reporter molecule such as GFP.

[0109] In one embodiment, the CAR is a CD8α extracellular and transmembrane domain, a 4-1BB cell Based on CAA06864.2, fused to the CD3 ζ intracellular domain and the CD3ζ intracellular domain In one embodiment, the CAR comprises an scFv selected from SEQ ID NOs: 1, 3, 4, 5, 6, 7, It comprises a sequence of 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acids. In one embodiment, the entire EDB CAR is provided with a signal peptide for guidance to the plasma membrane. The gene is expressed with a signal peptide (eg, the signal peptide of human interleukin-2).

[0110] In another embodiment, the CAR comprises the CD8α extracellular and transmembrane domains, CD28 CAA06864.2-based antibody fused to the CD3 ζ intracellular domain and the CD3ζ intracellular domain. In one embodiment, the CAR comprises an scFv based on the amino acid sequence of SEQ ID NO:3. nothing.

[0111] In another embodiment, the CAR comprises the CD8α extracellular and transmembrane domains, 4-1B fused to the B intracellular domain, the CD4 intracellular domain and the CD3 ζ intracellular domain, In one embodiment, the CAR comprises an scFv based on CAA06864.2. Contains the amino acid sequence numbered 4.

[0112] In another embodiment, the CAR comprises the CD8α extracellular and transmembrane domains, 4-1B fused to a B intracellular domain, a CD8 intracellular domain, and a CD3 ζ intracellular domain, In one embodiment, the CAR comprises an scFv based on CAA06864.2. Contains the amino acid sequence numbered 5.

[0113] In yet another embodiment, the CAR comprises a CD8α extracellular and transmembrane domain, a CD fused to 4 intracellular domain, 4-1BB intracellular domain and CD3ζ intracellular domain In one embodiment, the CAR comprises an scFv based on CAA06864.2. It comprises the amino acid sequence of SEQ ID NO:6.

[0114] In yet another embodiment, the CAR comprises a CD8α extracellular and transmembrane domain, a CD fused to the CD8 intracellular domain, the CD3ζ intracellular domain, and the CD3ζ intracellular domain In one embodiment, the CAR comprises an scFv based on CAA06864.2. It comprises the amino acid sequence of SEQ ID NO:7.

[0115] In yet another embodiment, the CAR comprises a CD8α extracellular and transmembrane domain, a CD 4 intracellular domain and fused to the CD28 intracellular domain, CAA06864.2 In one embodiment, the CAR comprises an scFv based on the amino acid sequence of SEQ ID NO:8. include.

[0116] In yet another embodiment, the CAR comprises a CD28 extracellular and transmembrane domain and CAA068, fused to the CD28 intracellular domain (CD28 aa138-220) In one embodiment, the CAR comprises an scFv based on the amino acid sequence of SEQ ID NO: 9. It contains the amino acid sequence.

[0117] In yet another embodiment, the CAR comprises the 4-1BB extracellular and transmembrane domains and and CAA fused to the 4-1BB intracellular domain (CD137 aa160-255). In one embodiment, the CAR comprises an scFv based on SEQ ID NO: 1. It contains the amino acid sequence of 0.

[0118] In yet another embodiment, the CAR comprises a CD8 hinge domain and a CD3 ζ domain. CAA0686, fused to the extracellular and transmembrane domains and the CD3ζ intracellular domain In one embodiment, the CAR comprises an scFv based on the amino acid sequence of SEQ ID NO: 10. It contains the amino acid sequence.

[0119] In yet another embodiment, the CAR comprises a CD8α extracellular and transmembrane domain, a CD It contains an scFv based on CAA06864.2 fused to the 3ζ intracellular domain. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO:12.

[0120] In yet another embodiment, the CAR is subsequently synthesized by transfection with the CD3ε extracellular and transmembrane domains, and CAA fused to a short linker (GRASG) followed by the intracellular domain. In one embodiment, the CAR comprises an scFv based on SEQ ID NO: 1. It contains the amino acid sequence of 3.

[0121] In yet another embodiment, the CAR is subsequently synthesized by transfection with the CD3ε extracellular and transmembrane domains, and the intracellular domain, fused to a 10 amino acid linker (2XG4S). In one embodiment, the CAR comprises an scFv based on CAA06864.2. It comprises the amino acid sequence of SEQ ID NO:14.

[0122] In yet another embodiment, the CAR is subsequently synthesized by transfection with the CD3ε extracellular and transmembrane domains, and the intracellular domain, fused to a 15 amino acid linker (3XG4S). In one embodiment, the CAR comprises an scFv based on CAA06864.2. It comprises the amino acid sequence of SEQ ID NO:15.

[0123] In yet another embodiment, the CAR is subsequently synthesized by transfection with the CD3ε extracellular and transmembrane domains, and the intracellular domain, followed by the CD28 intracellular domain, and Contains an scFv based on CAA06864.2 fused to a linker (3XG4S) In one embodiment, the CAR comprises the amino acid sequence of SEQ ID NO:16.

[0124] In yet another embodiment, the CAR is subsequently synthesized by transfection with the CD3ε extracellular and transmembrane domains, and the intracellular domain, followed by the 4-1BB intracellular domain, The scFv was based on CAA06864.2 and fused to the linker (3XG4S). In one embodiment, the CAR comprises the amino acid sequence of SEQ ID NO:17.

[0125] In one embodiment, the CAR is composed of two single polypeptide chains, The tide chain comprises the amino acid sequence of SEQ ID NO:11 and the amino acid sequence of SEQ ID NO:8.

[0126] In one embodiment, the CAR is composed of two single polypeptide chains, The tide chain comprises the amino acid sequence of SEQ ID NO:12 and the amino acid sequence of SEQ ID NO:8.

[0127] In one embodiment, the CAR is composed of two single polypeptide chains, The tide chain comprises the amino acid sequence of SEQ ID NO:13 and the amino acid sequence of SEQ ID NO:8.

[0128] In one embodiment, the CAR is composed of two single polypeptide chains, The tide chain comprises the amino acid sequence of SEQ ID NO:14 and the amino acid sequence of SEQ ID NO:8.

[0129] In one embodiment, the CAR is composed of two single polypeptide chains, The tide chain comprises the amino acid sequence of SEQ ID NO:15 and the amino acid sequence of SEQ ID NO:8.

[0130] In one embodiment, the CAR is composed of two single polypeptide chains, The tide chain comprises the amino acid sequence of SEQ ID NO:11 and the amino acid sequence of SEQ ID NO:9.

[0131] In one embodiment, the CAR is composed of two single polypeptide chains, The tide chain comprises the amino acid sequence of SEQ ID NO:12 and the amino acid sequence of SEQ ID NO:9.

[0132] In one embodiment, the CAR is composed of two single polypeptide chains, The tide chain comprises the amino acid sequence of SEQ ID NO:13 and the amino acid sequence of SEQ ID NO:9.

[0133] In one embodiment, the CAR is composed of two single polypeptide chains, The tide chain comprises the amino acid sequence of SEQ ID NO:14 and the amino acid sequence of SEQ ID NO:9.

[0134] In one embodiment, the CAR is composed of two single polypeptide chains, The tide chain comprises the amino acid sequence of SEQ ID NO:15 and the amino acid sequence of SEQ ID NO:9.

[0135] In one embodiment, the CAR is composed of two single polypeptide chains, The tide chain comprises the amino acid sequence of SEQ ID NO:11 and the amino acid sequence of SEQ ID NO:10.

[0136] In one embodiment, the CAR is composed of two single polypeptide chains, The tide chain comprises the amino acid sequence of SEQ ID NO:12 and the amino acid sequence of SEQ ID NO:10.

[0137] In one embodiment, the CAR is composed of two single polypeptide chains, The tide chain comprises the amino acid sequence of SEQ ID NO:13 and the amino acid sequence of SEQ ID NO:10.

[0138] In one embodiment, the CAR is composed of two single polypeptide chains, The tide chain comprises the amino acid sequence of SEQ ID NO:14 and the amino acid sequence of SEQ ID NO:10.

[0139] In one embodiment, the CAR is composed of two single polypeptide chains, The tide chain comprises the amino acid sequence of SEQ ID NO:15 and the amino acid sequence of SEQ ID NO:10.

[0140] In one embodiment, the bispecific molecule comprises an scFv based on CAA06864.2 and and scFv based on anti-CD3ε antibody. Thus, such polypeptides capable of binding to the EDB antigen and to the T cell receptor have been described.

[0141] Table 1 lists all of the sequences disclosed in this invention, including their sequence ID numbers, synopsis, and nomenclature. Chimeric antigen receptors are summarized.

[0142] TIFF2025066806000001.tif1221225.Polynucleotide In another aspect of the invention, a polynucleotide encoding a CAR of the invention as described herein is In one embodiment, the polynucleotide comprises SEQ ID NO:2.

[0143] In some embodiments, the nucleic acid is a synthetic nucleic acid. In some embodiments, the nucleic acid is a DNA In one embodiment, the nucleic acid is an RNA molecule (e.g., encoding a CAR). In one embodiment, the mRNA is a capped, polyadenotic, cytidine substitution, 5-methylcytidine substitution, pseudouridine substitution, or a combination thereof. It is being done.

[0144] In one embodiment, the nucleic acid (e.g., DNA) is In one embodiment, the promoter is linked to a regulatory element (e.g., a promoter). In some embodiments, the promoter is an inducible promoter. In one embodiment, the promoter is a cell-specific promoter. In some embodiments, the promoter is an organism-specific promoter.

[0145] The most widely used promoters are the pol I promoter and the pol II promoter. promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter Motor, retrovirus Rous sarcoma virus LTR promoter, cytomegalovirus CMV promoter, SV40 promoter, dihydrofolate reductase promoter It contains the β-actin promoter and the β-actin promoter.

[0146] In one aspect, the nucleic acid sequences provided by the present invention encode a CAR as described herein. The nucleic acid sequence to be loaded is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% %, 99% or 100% identity.

[0147] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, To align sequences for a purpose (e.g., a first and a second amino acid or nucleic acid sequence Optimal alignment is achieved by inserting gaps into one or two of the (For purposes of comparison, non-homologous sequences may be ignored). is at least 80% of the length of the reference sequence, and in one embodiment, the length of the reference sequence 90%, 95% or 100% of the length of the corresponding amino acid position or The amino acid residues or nucleotides at the nucleotide positions are compared. the same amino acid residue or nucleotide at the corresponding position in the second sequence If the position is occupied by a 1, the molecules are similar at that position. The fraction is a function of the number of similar positions shared by the two sequences and is the optimal alignment of the two sequences. Considering this, the number of gaps to be inserted and the length of each gap are also necessary. For this purpose, the comparison of two sequences and the determination of the percentage identity between the two sequences are carried out by the method It can be completed with the ossum 62 scoring matrix, with gap penalties The penalty is 12, the gap extension penalty is 4, and the frameshift gap penalty is 5. I did.

[0148] In one embodiment, a nucleic acid encoding a CAR protein and derivatives or functional fragments thereof is provided. The nucleic acid molecule is expressed in a host cell or organism after undergoing codon optimization. The cells may include established cell lines (e.g., T / NK cells) or isolated primary cells. The nucleic acids may be codon-optimized for any target organism, particularly human immune cells. Don usage tables are easily available, for example at www.kazusa.orjp / codon / These tables can be modified in a number of ways. (Nakamura et al., Nucl. Acids Res. 28:2 92, 2000). In addition, specific sequences may be used for expression in specific host cells. A sequence-optimizing codon algorithm, e.g., Gene Forge (Aptage n; Jacobus, Pa.) is provided.

[0149] Examples of codon-optimized sequences in such cases are those expressed in eukaryotic organisms, e.g., humans. (i.e., optimized for expression in humans), or another eukaryotic, animal or mammalian CAR coding sequence contemplated herein. This is preferred, but of course other examples are possible, and host species other than human are also possible. Codon optimization for a gene or for a particular organ is known. Codon optimization refers to the process of optimizing the codon sequence of a gene in a host cell while maintaining the natural amino acid sequence. At least one of the most frequently used codons (e.g., about 1 2, 3, 4, 5, 10, 15, 20, 25, 50 or more) of the codons of the native sequence This method enhances the expression of a modified nucleic acid sequence in a host cell by replacing the Some species show a particular bias for certain codons for certain amino acids. The difference in codon usage between the two genes is usually related to the efficiency of translation of messenger ribonucleic acid (mRNA) and Messenger RNA is specifically determined by the nature of the codon being translated and the availability of a particular transfer RNA (tRNA) molecule. The predominance of available tRNAs in a cell is usually determined by the availability of tRNAs for peptide synthesis. Therefore, the optimal gene sequence for a given organism is determined by the codons most commonly used in the organism. Genes can be customized based on codon optimization to achieve expression Codon usage tables are readily available, for example at www.kazusa.orjp / codon These tables are found in the "codon usage database" of (Nakamura et al., Nucl. Acids Res. 28 :292, 2000). Algorithms that optimize the sequence of codons, such as Gene Forge (Apta gen; Jacobus, Pa. In one embodiment, CAR is One or more codons in the coding sequence (e.g., 1, 2, 3, 4, 5, 10 , 15, 20, 25, 50 or more, or all codons for a particular amino acid Corresponding to the most used codons.

[0150] In one embodiment, one or more polynucleotides or nucleic acids of the invention are The vector may be present in a host (e.g., a viral vector).

[0151] As used herein, the term "vector" refers to a nucleic acid that is typically linked to another nucleic acid. A vector is a nucleic acid molecule capable of transporting a nucleic acid. A double-stranded nucleic acid molecule, which may contain one or more free ends or no free ends (e.g., a circular Nucleic acid molecules, including DNA, RNA or both, as well as other nucleic acid molecules known in the art. Polynucleotide variants of the present invention include, but are not limited to,

[0152] In certain embodiments, the vector may be a cloning vector or an expression vector. The vector may be a plasmid, a phagemid, a cosmid, etc. The target cell (e.g., a mammalian cell, e.g., a human immune cell, e.g., a T / N The vector may contain one or more regulatory elements that allow the vector to grow in a specific host cell (K cell).

[0153] In one embodiment, the vector is a "plasmid", which is a circular double stranded DNA loop, by inserting another DNA segment into it, for example by standard molecular cloning techniques It refers to something that can be done.

[0154] In one embodiment, the vector is a viral vector, wherein the virally derived DNA A or RNA sequence is present in the vector, and the vector is a virus (e.g., retrovirus, lentivirus, Virus, replication-deficient retrovirus, adenovirus, replication-deficient adenovirus, H It is packaged in SV and adeno-associated virus (AAV). The vector further comprises a polynucleotide for transfecting a host cell carrying the virus. .

[0155] In some embodiments, the vector is a lentiviral vector. In this case, the lentiviral vector is a self-inactivating lentiviral vector. , Zufferey et al., “Self-Inactivating Lentiviru s Vector for Safe and Efficient In vivo Gene Delivery.” J Virol. 72(12): 9873‐98 80, 1998, incorporated herein by reference.

[0156] In one embodiment, the vector is "Sleeping Beauty" ng Beauty, SB) based on the transposon, To introduce genes into the genome of vertebrates as non-viral vectors and to The SB system is composed only of DNA, so it is used to treat viruses. Compared with vectors, production and transportation costs are significantly lower. SB transposons have already been used in human clinical trials. It has been used to genetically modify T cells in clinical trials.

[0157] In certain embodiments, vectors are autonomously replicating in a host cell into which they are introduced. In certain embodiments, after introduction into a host cell, the vector (e.g., The vector integrates into the genome of the host cell and thereby In one embodiment, the vector is a vector as used herein. These vectors are called "expression vectors" and can direct the expression of genes to which they are operatively linked. A vector that is expressed in eukaryotes is a "eukaryotic expression vector."

[0158] In one embodiment, the vector is a recombinant expression vector and is capable of being expressed in a host cell. The recombinant expression vector comprises a nucleic acid of the invention in a form suitable for expression in a mammalian cell. The regulatory elements may be selected depending on the host cell used for expression. In this context, "operably linked" may be a nucleic acid sequence that is expressed by a nucleic acid sequence that is expressed by a nucleic acid sequence that is not specifically linked to ... The term "link" refers to a method for adjusting a target nucleotide sequence so as to express the nucleotide sequence. The first step is to link the gene to a section element (e.g., in an in vitro transcription / translation system or in a vector). (inside the host cell when the marker is introduced into the host cell).

[0159] The term "regulatory element" includes promoters, enhancers, internal ribosome entry sites (IRSs), and RES) and other expression control elements (e.g., transcription termination signals, e.g., Re-adenylation signal and poly U sequence). See, e.g., Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS in ENZYMO LOGY 185, Academic Press, San Diego, cal. Such regulatory elements are described in Ifornia (1990). The vector directs the constitutive expression of nucleotide sequences in many types of host cells. and those that direct the expression of a nucleotide sequence in a given host cell (e.g., tissue-specific Tissue-specific promoters contain a specific regulatory sequence (e.g., heterologous regulatory sequence) that is specific to the target tissue in which they are primarily required, e.g., muscle. , neurons, skeleton, skin, blood, specific organs (e.g., liver, pancreatic gland) or specific Guiding expression in cell types (e.g., lymphocytes, e.g., T cells or NK cells) Regulatory elements may also be involved in chronologically dependent manners, e.g., cell cycle dependent or Expression can be directed in a developmental stage-dependent manner, which may vary depending on the tissue or cell type. It may or may not be genus-specific.

[0160] In one embodiment, the vector comprises one or more pol III promoters (e.g., For example, one, two, three, four, five or more pol III promoters), one or Multiple pol II promoters (e.g., 1, 2, 3, 4, 5 or more pol II promoter), one or more pol I promoters (e.g., 1, 2, 3, 4, 5 or more pol I promoters) or combinations thereof Examples of pol III promoters include the U6 and H1 promoters. Non-limiting examples of pol II promoters include those derived from the retrovirus Rous Sarcoma Virus ( RSV) LTR promoter (optionally with RSV enhancer), Cytomegalovirus (CMV) promoter (optionally with a CMV enhancer) [e.g., Boshart et al., Cell, 41:521-530 (1985)], SV40 promoter promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglucose These include the lysine kinase (PGK) promoter and the EF1a promoter. However, the present invention is not limited to the above.

[0161] The term "regulatory element" further includes enhancer elements, such as the WPRE, CMV enhancer, R-U5' fragment in the HTLV-1 LTR [Mol. Cel l Biol., 8(1), p.466-472, 1988], SV40 enhancer , and the intron sequence between exons 2 and 3 of rabbit b-globin [Proc. Na tl. Acad. Sci. USA., Vol.78(3), p.1527-3 1, 1981].

[0162] Those skilled in the art will appreciate that the design of an expression vector requires careful consideration of, for example, the choice of the host cell to be transformed, the desired expression vector, and the desired It can be seen that the level of expression is determined by factors such as the level of expression of the vector. Thus, to produce the transcripts, proteins or peptides encoded by the nucleic acids described herein The polypeptide may be a fusion protein or peptide.

[0163] In one embodiment, the vector is a lentiviral or AAV vector, and is specific to the species. The antibody can be selected to target a variety of cells (e.g., tissues and / or cells). species-specific).

[0164] Many art-recognized methods, such as transfection, lipid vectors, infection, electolysis, poration, microinjection, parenteral injection, aerosol, gene gun, etc. The vectors of the present invention are delivered to target cells, for example primary T / NK cells or Alternatively, it can be introduced into "existing" allogeneic T / NK cells.

[0165] In certain embodiments, transfection can be achieved using, for example, calcium phosphate, lipid or protein. Chemical transfection involves the introduction of vectors into the protein complex. Dextran, liposomes and lipoplexes (for oral gene delivery) interfaces The activator and perfluorochemical liquid are for aerosol delivery of genes.

[0166] In one embodiment, the combination of plasmid DNA and a lipid solution forms liposomes. By combining these two proteins, lipid vectors can be generated that fuse with the cell membrane of many cell types, allowing vector The DNA is introduced into the cytoplasm and nucleus of the cell where it is expressed. Therefore, by linking folic acid to DNA or DNA-lipid complexes, vectors can be efficiently expressed and The targeting moiety is introduced into cells that express the folate receptor at low levels. The vector can be delivered to a particular cell type targeted by the targeting moiety.

[0167] In one embodiment, the vector DNA is internalized by receptor-mediated uptake. It will be turned into.

[0168] In one embodiment, the vector is a lentivirus, and the packaging of the vector By substituting the genes for surface glycoproteins with genes from another viral genome in a human cell line (PCL), This broadens the vector's spectrum of infection with target cells.

[0169] 6.Immune cells The CAR of the present invention can be introduced into various immune cells for CAR-mediated therapy. Immune cells that can be transduced with CAR include T cells, NK cells, and mononuclear cells (including peripheral mononuclear cells). , monocyte-derived dendritic cells, macrophages, hematopoietic stem cells and / or induced pluripotent stem cells (PSC) and others.

[0170] Therefore, in one aspect, the present invention also provides a method for the preparation of a CAR, CAR protein, or a combination thereof. A vector of the invention comprising a polynucleotide encoding a gene or a polynucleotide of the invention. The present invention provides a cell comprising the

[0171] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is an immune cell. T cells, e.g., CD4 + or CD8 + In one embodiment, the The cells are NK cells. In some embodiments, the cells are monocytes. In one embodiment, the cells are isolated from the patient. The primary cells are transfected with a CAR expression vector to express the CAR before the cells are returned to the patient. In one embodiment, the cells are derived from a healthy donor, and the cells Before transferring the cells back to a healthy donor and another patient, the cells were transfected with a CAR expression vector to express the CAR. is introduced into the cells. Optionally, the HLA type of a healthy donor is matched with the HLA type of the patient.

[0172] In one embodiment, T cells and / or NK cells and / or monocytes and / or Alternatively, macrophages can be isolated from peripheral blood mononuclear cells (PBMCs), bone marrow cells, or other tissues by a variety of non-limiting methods. Many types of tissue, including but not limited to lymph node tissue, umbilical cord blood, thymus tissue, ascites, pleural effusion, spleen tissue, and tumors, are also used. It is obtained from a variety of sources.

[0173] In one embodiment, the immune cells are administered to a patient in need of CAR therapy (e.g., a patient suffering from cancer or inflammation). In this example, T cells were isolated from the body of a patient diagnosed with a genital tract infection. / NK cells / monocytes / macrophages are autologous cells.

[0174] As used herein, "autologous" refers to cells derived from the subject of cell therapy. It refers to a system or group of cells.

[0175] In one embodiment, the immune cells are isolated from a healthy donor who is not in need of treatment. In this embodiment, the immune cells are derived from an allogeneic host, preferably human leukocytes. It is derived from a host with matching human leukocyte antigen (HLA) receptors.

[0176] In one embodiment, the T cells are CD4 + In one embodiment, the T The cells are CD8 + Includes T cells.

[0177] The CAR T cells of the present invention can be produced by any method known in the art. For example, an expression vector, such as a CAR of the present invention, comprising a polynucleotide thereof, and Viral-based vectors capable of expressing (1) is a method for obtaining positive CAR-T, CAR-NK, and other cells by isolating immune cells. Those skilled in the art can readily use expression constructs, e.g., Suitable viral vectors for the expression of can be constructed.

[0178] In certain embodiments, the cells (e.g., immune cells) may further be administered cytokines, e.g. For example, IL-2, IL-7, IL-12, IL-15 or IL-21, or any of these. In one embodiment, when the CAR binds to its target antigen, the CAR expresses a combination of In one embodiment, the expression of one or more cytokines is activated. Expression of the kine is controlled by a promoter that is activated upon immune cell activation.

[0179] In certain embodiments, the cells further comprise a safety signaling pathway for downregulating the activity of immune cells. Including Itch.

[0180] In one embodiment, the safety switch is iCaspase9 (inducible caspase- 9) It contains a coding sequence for a monomer, e.g., FKBP dimerization activates the immune system. It can trigger apoptosis in cells.

[0181] 7. Drug Compositions and Their Applications In another aspect of the invention, a method for treating a disease or condition, such as cancer or an inflammatory disease, is provided. A pharmaceutical composition for administering the modified T / NK cells / monocytes / macrophages of the present invention to The present invention also provides a composition comprising the compound and a pharma- ceutical acceptable carrier. Use of the modified T / NK cells / monocytes / macrophages of the present invention in the manufacture of a medicament for treating Protection of use is claimed.

[0182] As used herein, a "pharmaceutical acceptable carrier" refers to any physiologically compatible Solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents In certain embodiments, the carrier is administered intravenously, intramuscularly, subcutaneously, parenterally, spinally or via a suitable route. Suitable for epidermal application (eg by injection or infusion).

[0183] In one embodiment, the present invention provides a CAR-based immune cell expressing a CAR of the present invention. The present invention relates to a method for treating solid tumors or inflammatory diseases by administering human T cells or NK cells. In another embodiment, the present invention provides a method for treating a patient suffering from a disease comprising: Immune cells are activated by administering CAR-T or CAR-NK cells expressing CARs. In one embodiment, the method includes infusion of lymphocytes to a solid tumor in a patient. CAR-T / CAR-NK cells can be administered by autologous administration in the treatment. A patient in need of treatment by the methods described herein and known in the art is infused with lymphocytes. Autologous PBMCs were collected from patients, and T / NK cells were activated and expanded, and then used to treat patients. It is injected into the subject's body.

[0184] 8.How to use In another aspect of the invention, a subject suffering from a disease or condition treatable by angiogenesis inhibitors is The method of the present invention includes administering to the subject a therapeutically effective amount of a chimeric antigen receptor. administering immune cells expressing a receptor (CAR) or a pharmaceutical composition comprising the immune cells; The chimeric antigen receptor (CAR) comprises: (1) fibronectin extracellular matrix (FIM) (2) an antigen-binding domain specific for domain B (EDB) and CD3, CD4, and CD 8. A transmembrane (TM) domain selected from CD28, OX40, or CD137 membrane proteins and (3) CD3ζ intracellular ITAMs (immunoreceptor receptor agonists) with or without costimulatory domains. and a tyrosine-based activation motif (Tyrosine-Based Inhibitor Motif) domain.

[0185] As used herein, a "therapeutically effective amount" or a "therapeutically effective dose" or an "effective "Amount" refers to a sufficient amount of a substance, compound, material, or cell to be administered to achieve the desired treatment. The objective of the present invention is to provide an effective treatment for at least one symptom of a disease or condition. Prevent, cure or ameliorate a disease or condition, or completely or partially inhibit the progression / aggravation of the disease or condition The application rate is also an amount that is below the threshold level of toxicity and is sufficient to prevent said threshold toxicity. Once the sexually significant level is exceeded, the subject may terminate or discontinue treatment.

[0186] For example, when administered to a subject in an effective amount, the immune cells of the invention and the immune cells of the invention The pharmaceutical composition comprising the compound reduces / delays / eliminates the symptoms of one or more diseases and reduces the frequency of attacks thereof. Reduce the intensity and / or duration of, or anticipate pain resulting from injury or disorder due to disease For example, in the treatment of tumors, the In comparison, the immune cells and pharmaceutical compositions containing the immune cells of the present invention inhibit the growth of cancer cells at least About 10%, at least about 20%, at least about 30%, at least about 40%, at least Suppression by about 50%, at least about 60%, at least about 70%, or at least about 80% The immune cells and drugs comprising the immune cells of the present invention can be used in suitable animal model systems. The ability of the compound to inhibit tumor growth is evaluated to predict therapeutic efficacy against human tumors. Alternatively or alternatively, a model system that is reasonably relevant to the disease or condition may be provided. The ability of the stem to inhibit tumor cell growth in vitro can be measured.

[0187] The amount and dosage level of the immune cells in the pharmaceutical composition of the present invention may vary depending on the specific needs of the patient. the type and / or extent of the subject's cancer; the desired therapeutic response; The toxicity of the drug to the patient and other factors considered relevant by the attending physician: The dosage level selected will depend on the particular composition used, the route of administration, the age of the patient, concurrent use, Other drug compositions, duration of administration, excretion or clearance rate, sex, weight, condition The patient's condition, overall health and medical history, and similar characteristics of the patient as are known in the medical field. The amount of pharmacokinetic activity that can be determined by the dosage form is determined by many pharmacokinetic factors, including the dosage form itself. Those of skill in the art will appreciate without undue experimentation. Effective amounts of the present invention can be determined empirically. Taken together, various activated immune cells and weighting factors, such as potency, relative bioavailability, By selecting the appropriate dosage form based on the patient's weight, the severity of adverse side effects, and the appropriate administration method, A suitable prophylactic or therapeutic plan can be developed that does not cause substantial toxicity. , is completely effective in treating a particular subject.

[0188] Toxicity and efficacy of the present invention are evaluated using standard pharmaceutical protocols in cell cultures or experimental animals. For example, LD50 (the dose that produces 50% of the amount of a substance) and ED50 (the dose that produces 50% of the amount of a substance) The dose ratio between toxic and therapeutic effects is called the therapeutic index. The therapeutic index can be expressed as the ratio LD50 / ED50. Prophylactic and / or therapeutic agents that have toxic or side effects are preferred. However, this should be done so that potential damage to uninfected cells is minimized. By carefully designing a delivery system that targets the appropriate drug to the infected tissue site, This should reduce side effects.

[0189] In certain embodiments, data obtained from cell culture assays, animal studies, and clinical studies can be used to determine dosage ranges of prophylactic and / or therapeutic agents for humans. The dosage of such agents is preferably within the range of circulating concentrations and has little or no toxicity. Depending on the dosage form and route of administration used, the dose may vary from this Any of the agents used in the methods of the present invention may have preliminary results from cell culture assays. The therapeutically effective dose can be estimated based on the results of animal models and cell culture. The IC50 (i.e., the concentration of the test compound at which half-maximal inhibition of symptoms is achieved) was included. Such information can be used to determine the dosage to achieve a circulating plasma concentration range that satisfies the therapeutic objectives. can be used to more accurately determine useful doses in humans. Levels in plasma may be measured by liquid chromatography.

[0190] In one embodiment, the CAR in the immune cell is any of the CARs of the invention described herein. It's AR.

[0191] In one embodiment, the immune cells are autologous or allogeneic T cells, NK cells, monocytes or It is a macrophage.

[0192] In one embodiment, the disease or condition is a solid tumor, a chronic inflammatory condition, an atheroma. arteriosclerosis, myocardial infarction, fibrosis or wound.

[0193] Examples of cancers or solid tumors are lung cancer, ovarian cancer, colon cancer, colorectal cancer, melanoma, and renal cancer. , bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancies, head and neck cancer, glioma, gastric cancer, nasopharyngeal cancer Includes head cancer, laryngeal cancer, cervical cancer, uterine cancer and osteosarcoma.

[0194] Other examples of cancers that can be treated with the methods or pharmaceutical compositions of the present invention include bone cancer, pancreatic cancer, skin cancer, Prostate cancer, skin or intraocular malignant melanoma, uterine cancer, anal cancer, testicular cancer, uterine cancer, intrauterine Membrane cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, Thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia (Includes acute myelocytic leukemia, myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia) (including pediatric solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cancer, central nervous system Central nervous system cancer (CNS), primary central nervous system lymphoma, spinal column tumor, brain stem glioma, pituitary adenoma , Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, and asbestos-induced cancer. The present invention includes cancers caused by various cancers, including cancers caused by cancers caused by cancerous tissues, cancers caused by ... various cancers, and combinations of said cancers.

[0195] In some embodiments, the cancer is a solid tumor / cancer. In some embodiments, the cancer is lung cancer, For example, lung squamous cell carcinoma. In some embodiments, the cancer is ovarian cancer. In the present invention, the cancer is colon cancer.

[0196] In one embodiment, cancer cells derived from solid tumors do not express EDB on the cell surface.

[0197] In some embodiments, the method further comprises administering to a patient an immune checkpoint inhibitor, such as , PD-1 inhibitors (e.g., pembrolizumab, nivolumab, and cemiplimab), PD- L1 inhibitors (e.g., atezolizumab, avelumab, and durvalumab), CTLA-4 Targeted drugs (e.g., ipilimumab) or immunomodulatory agents (e.g., thalidomide and lenalidomide) This includes applying

[0198] In one embodiment, the method further comprises administering to the subject radiation therapy and / or chemotherapy. Therapy and / or surgery and / or other tumor-targeting drugs (e.g., other The method involves administering a monoclonal antibody (such as a mAb) that targets an antigen or a small molecule compound.

[0199] In one embodiment, chemotherapy includes all-trans retinoic acid, actinomycin D , Adriamycin, Anastrozole, Azacitidine, Azathioprine, Alkeran, Ara-C, arsenic trioxide, BiCNU bleomycin, busulfan, CCNU, cal Boplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cyta Rabin, DTIC, Daunorubicin, Docetaxel, Doxifluridine, Adriamycin , 5-fluorouracil, epirubicin, epothilone, etoposide, exemestane, Erlotinib, fludarabine, fluorouracil, hydroxyurea, idarubicin, ibuprofen Matinib, letrozole, lapatinib, leustatin, 6-MP, mithramycin, mycobacterium tomycin, mitoxantrone, mechlorethamine, megestrol, mercaptopurine, Methotrexate, mitoxantrone, navelbine, nitrogen mustard, oxalate Liplatin, Paclitaxel, Disodium Pamidronate, Pemetrexed, Rituxan , 6-TG, taxol, topotecan, tamoxifen, taxotere, teniposide, Oguanine, toremifene, trimetrexate, trastuzumab, valrubicin, bin Blastine, Vincristine, Vindesine, Vinorelbine, Velban, VP-16 and / or contains Xeloda.

[0200] In certain embodiments, the chronic inflammatory disease is psoriasis, rheumatoid arthritis, Arthritis, ulcerative colitis, osteoarthritis, asthma, pulmonary fibrosis, IBD, inflammation-induced Lymphangiogenesis, Obesity, Diabetes, Retinal neovascularization (RNV), Diabetic retinopathy, Choroidal neovascularization Vascular (CNV), Age-related macular degeneration (AMD), Metabolic syndrome-related diseases, Long-term Peritoneal dialysis, juvenile arthritis or atherosclerosis.

[0201] In some embodiments, the method further comprises administering to the patient a second therapeutic agent that effectively inhibits angiogenesis. The present invention includes applying

[0202] In certain embodiments, the second therapeutic agent is axitinib, bevacizumab, cabozantinib, , everolimus, lenalidomide, pazopanib, ramucirumab, regorafenib, sorafenib sunitinib, thalidomide, vandetanib and / or diabetofibercept Includes

[0203] In one embodiment, the vector of the present invention is administered ex vivo to primary immune cells isolated from a subject. and optionally culturing the vector-transduced primary immune cells ex vivo and / or By amplifying it, immune cells are generated.

[0204] In one embodiment, the method further comprises inhibiting cytokine release syndrome (CRS). A reagent that inhibits IL-6 expression, such as an anti-IL-6 monoclonal antibody (e.g., tocilizumab), is administered. This includes administering anti-inflammatory drugs and / or globulin therapy.

[0205] In certain embodiments, the subject is a human, a non-human primate, a cow, a horse, a pig, a sheep, In one embodiment, the subject is a human. In aspects of the invention relating to predictive therapy in cancer, the subject is a person having a suspected / narrowed diagnosis of cancer. A human being who is at high risk or has been diagnosed with cancer. Methods for identifying subjects who are suspected of having cancer This can be done through physical examination, the subject's family history, the subject's medical history, biopsy or a number of imaging techniques, For example, ultrasound, computed tomography, magnetic resonance imaging, magnetic resonance spectroscopy and positive The diagnostic methods for cancer and the clinical description of cancer diagnosis are widely known to medical engineers. It is something that should be known.

[0206] 9. Kit Another aspect of the present invention is a kit for the method of producing the immune cells of the present invention. Reagents for isolating immune cells from patients, culturing and amplifying isolated immune cells, a medium for infecting an immune cell expressing an isolated CAR of the present invention; Reagents including vectors, reagents for activating immune cells (e.g., T cells), A reagent for detecting / verifying the expression of the CAR of the present invention in a subject's diseased tissue, Reagents to confirm the presence or absence of B (e.g., immunohistochemical or immunofluorescent reagents or Other imaging modalities, such as non-invasive in vivo Immuno-PET / CT, The kit includes one or more of the following:

[0207] The kit further includes immune cells bearing a CAR by carrying out the method of the present invention and their use. Includes instructions for use.

[0208] The kit may comprise any one or more of the components described herein in one or more containers. For example, in one embodiment, the kit may comprise one of the above kits or Mixing of multiple components and / or separation and mixing of samples and application to subjects The kit may include containers housing the reagents described herein. The reagents can be prepared sterile, packaged in syringes, and shipped refrigerated. Alternatively, it can be stored in a vial or other container. The container may contain other reagents prepared sterilely. Alternatively, the kit may be premixed and ready for injection. The composition may include an active agent delivered in a vessel, vial, test tube, or other container. EXAMPLES

[0209] Example 1: Generation of EDB-CAR-T cells EDB-CAR (SEQ ID NO:1) chimeric antigen receptor is a newly designed molecule whose code The sequence (SEQ ID NO:2) of EDB-CAR was synthesized by Genewiz. The variable region (scFv) was designed based on CAA06864.2 and specifically binds the EDB antigen. Specifically, the construction of EDB CAR is the fusion of gene fragments, among which EDB-specific The scFv binds the CD8α extracellular and transmembrane domains, the 4-1BB intracellular domain, and the CD3 By using the human IL-2 signal peptide fused to the ζ intracellular domain Guides the entire EDB-CAR receptor to the plasma membrane. The otide sequence (SEQ ID NO:2) was synthesized from scratch by Genewiz.

[0210] To express EDB CAR, the fusion gene DNA fragment was transfected into a lentiviral vector. After cloning into M1, pseudotyped lentiviruses were transduced into activated T cells. .

[0211] Protocol provided by the manufacturer (Miltenyi, 130-096-535) Isolate T cells from peripheral blood mononuclear cells (PBMCs) by magnetic bead negative selection according to the protocol. Complete RPMI (10% heat-inactivated fetal bovine serum and 100 U / mL penicillin) was used. RPMI supplemented with cyclosporine / streptomycin, 500 U / mL rhIL-2 ( SinoBiological, GMP-11848-HNAE), 10 ng / mL IL-7 (SinoiBologic, GMP-11840-HNAE) and 10 n g / mL IL-15 (SinoBiologic, GMP- 11846-HNAE) ) Magnetic beads were incubated with anti-human CD3 and CD28 antibodies (Thermo Fisheye r Scientific, 11131D) and incubated with T cells for 24 hours. After that, it was mixed with FuSure (Boston 3T Biotechnologies). The cells were then spin-fected with the combined lentiviral vectors. It was amplified and used in in vitro assays.

[0212] Fab fragments that recognize human variable framework sequences were used for flow cytometry. Therefore, we detected the expression of EDB-CAR on the surface of T cells. 6 Individual shape Transfected cells were incubated with 8 μg / mL of reconstituted biotin-labeled goat anti-human Ig GF(ab')2 fragment polyclonal antibody (Jackson Immunoresea rch, Cat#109-066-097) and FACS buffer (PBS + 0.4% FB The cells were then incubated at 4°C for 25 min in FACS buffer. 5.5 μl phycoerythrin (R-phycoerythrin streptavidin, Ja ckson Immunoresearch, 016-110-084) and FACS buffer The cells were incubated in ice for 20 minutes in a dark place. The cells were washed three times and analyzed by ACEA Novocyte flow cytometer. The Fab fragment recognized only T cells transduced with the EDB chimeric receptor (Figure 1). .

[0213] In addition, T cells transduced with EDB-CAR recognize soluble EDB antigen and express IFN- It was found that EDB-CAR in this design produced soluble antigen γ. Notably, EDB-specific antibodies were partially activated by IFN- γ induction was suppressed (Figure 2A). G cells bearing the EDB antigen on their surface were able to be killed (Fig. 2B).

[0214] Therefore, both soluble and membrane-bound antigens stimulate the receptors to activate T cells. The current design of EDB-CAR is unique because it can

[0215] Example 2: EDB CAR-T cells are cytotoxic to cells expressing the EDB protein have To confirm the presence of the EDB domain of fibronectin in target cells, anti-E Standard Western blot analysis with DB monoclonal antibody BC-1 (Abcam, ab154210) Blotting analysis was performed, followed by detection with a secondary antibody. EDB was detected in human colon cancer cells. cell line Caco-2, human breast cancer cell line MCF-7, HS578T, MDA-MB-468, The expression was observed in several representative human cancer cell lines, including the human glioblastoma cell line U87-MG. In addition, the IL-11 expression was observed in mouse colorectal cancer cell line CT26 and human umbilical vein endothelial cell line HU. EDB protein expression was also observed in VEC cells (Fig. 3A ).

[0216] To verify the Western blotting analysis, we used a plasmid with specificity for the EDB domain. Using a marker, the mRNA level of EDB protein in each cell was analyzed by qPCR. The expression of the target cells was confirmed. Total RNA was extracted from the target cells (19221, YEASEN). The total RNA was then reverse transcribed using the template (11121ES60, YEASEN) to obtain cDNA. Using the obtained cDNA as a template, qPCR was performed with the following primers: GADPH F -Primer 5'-ACCCAGAAGACTGTGGATGG-3' and R-Primer Inducer 5'-TCTAGACGGCAGGTCAGGTC-3' and EDB F-probe Primer 5'-AAC TCA CTG ACC TAA GCT TT-3' and R-primer 5'-CGT TTG TGT CAG TGT AG-3' and S YBR Green dye (11199ES03, YEASEN). From the data, EDB characteristics Although heterologous mRNAs were present at different levels in many cancer cell lines and HUVEC cells, MC It was shown that it was not detected in F-7 and MDA-MB-468 (Figure 3B).

[0217] Cytotoxicity of EDB CAR-T cells in a set of cells exhibiting different levels of EDB expression The versatility of the 10 4 Transducing target cells The T cells were mixed with effector-target ratios of 1:1, 5:1, and 10:1. After 4 hours of incubation, the LDH was detected by a LDH detection kit (Yeasen, 40209ES76). Consistent with the expression analysis (Figures 3A-3B), we detected degradation of target cells. MG, Hs578, A549, and F9 are involved in cell lysis induced by EDB-CAR T However, MCF-7 and MDA-MB468 were not affected (Figure 4A-4 B) In the Caco-2 cell line, low levels of cell killing were associated with a tendency for cells to aggregate in vitro. This may be due to (data not shown).

[0218] Interestingly, there was no clear correlation between high EDB expression and the level of cell destruction. Without wishing to be bound by any particular theory, the EDB The accessibility or specific conformation of a domain can be altered, affecting the susceptibility of cells to degradation. It may resonate.

[0219] Overall, these data support the use of EDB-CAR T cells for cancer therapy. do.

[0220] Example 3: EDB-CAR T cells have cytotoxicity against HUVEC endothelial cells Angiogenesis is a prerequisite for tumor growth and metastasis. Successful treatment (Keating 2014, Syed 2015) may result in tumor vasculature The feasibility of targeting angiogenesis as a treatment has been demonstrated.

[0221] The present example shows that EDB-CAR T cells can cause cytotoxicity against HUVEC cells. These results indicate that the CAR T cells of the present invention are useful for therapeutic targeting of angiogenesis. Cytoplasmic cells are endothelial cells that can form tube-like structures. The levels increased with increasing effector-to-target ratio (Fig. A, lower panel, middle).

[0222] This indicates that EDB-CAR T cells can be effective angiogenesis inhibitors. EDB in the process of vascular structure formation + Because fibronectin is involved, EDB-C AR T cells may be used alone or in combination with currently available antiangiogenic agents (e.g., bevacizumab). It may be used in combination with rivaroxaban or aflibercept.

[0223] Example 4: EDB-positive cancer cells can activate EDB CAR-T cells IFN-γ is a marker of T cell activation. EDB-CAR T cells based on SEQ ID NO:1 To evaluate the activation of EDB during the cytotoxic reaction, -IFN-γ expression in CAR T cells was detected. Indeed, in the presence of target cells, IFN-γ was detected in the supernatant (Fig. 5). No IFN-γ induction was observed in these cells, which may be due to low or undetectable EDB in these cells. These findings support the finding that EDB-CAR T cells It is clear that EDB-induced cell destruction is due to binding to fibronectin. It was clearly shown.

[0224] Interestingly, although the cytotoxicity was low, high levels of IFN-γ were observed in Caco-2 cells. This finding was unexpected and is consistent with the commonly observed effects of cytotoxicity (Figure 2). This is inconsistent with the observation that the IFN-γ induction pathway is involved in the differentiation of EDB-CAR T cells. This indicates independence from toxicity pathways.

[0225] Indeed, preliminary data suggest that after cytotoxic reactions, the EDB-CAR T of the present invention This is quite surprising, since the data show that This indicates that treatment with the EDB-CAR T of the present invention is safer.

[0226] In addition, CAR T cells can produce TNF-α after cytotoxic killing. was observed (Jiang et al. 2018). Consistent with IFN-γ expression, TNF-α induction was tested after infusion of Caco-2 and Incubation of HS578T cells with IL-1 induces the production of TNF-α and results in high efficacy. The expression of β-lactam α-lactam was enhanced by the β-lactam α-lactam target ratio, but was not enhanced by incubation with MCF-7 and MDA-MB-468 cells. When the cells were incubated with 100 mM NaCl, they did not produce significant amounts of TNF-α (Figure 6).

[0227] Example 5: EDB-CAR NK cells also have cytotoxicity against cancer cells The experiment showed that natural The potential application of treatment regimes based on killer (NK) cells has been demonstrated.

[0228] The NK-92 cell line is a patient-derived immortalized cell line that is already in clinical studies. To demonstrate the applicability of B-CAR in NK-based therapy, we have launched EDB-CAR. The NK-92 cell line was transduced with a lentiviral vector expressing CAR Similar to the method for generating T cells, anti-Fab fragment antibodies were used to generate flow cytometry T cells, as described above. Expression of EDB-CAR was analyzed by cytometry. More than 55% of NK-92 cells We demonstrated the expression of EDB-CAR on the cell surface (Figure 7).

[0229] Glioblastoma cell line by incubation with EDB-CAR NK-92 cells The cytolysis of U87-MG was induced (Figure 8A). This result was consistent with the cytotoxicity measurements. Upon target cell activation, EDB-CAR NK-92 cells produced IFN-γ.

[0230] These results suggest that EDB-CAR-transduced natural killer cells play a key role in cancer The potential therapeutic effects of

[0231] Example 6: Intracellular injection of EDB-CAR T cells CAR-T cells have been shown to have cytotoxicity against HUVEC cells in vitro. Such EDB-specific CAR-T cells may cause unacceptable toxicity to normal blood vessels in patients in vivo. The experiment showed that the compound has in vitro cytotoxicity against HUVEC cells. However, the test CAR-T cells were shown to be safe for use in the body.

[0232] In this experiment, mice were 7 T cells, 1 x 10 7 EDB CAR-T thin cells or 2 × 10 7 of EDB CAR-T cells were injected. 21 days after T cell infusion Mice were sacrificed. Different tissues were collected, fixed in formalin, embedded in paraffin, and The tissue was then stained with H&E. Representative micrographs are shown in Figure 9. The photographs were taken with a Leica Ap The images were taken at 20x magnification using an Erio VERSA 8 section scanner. Each scale bar represents 100 μm.

[0233] The results showed that there were no obvious pathological changes in any of the tissues examined, and there was no significant difference between the groups. It can be seen that there was no difference. See FIG.

[0234] In the study, mice weighing about 20 g were given a high dose of EDB-CAR-T cells. The amount of CAR-T cells injected was 20 million per animal. This amount of CAR-T cells is equivalent to approximately 1000 cells per animal weighing 60 kg. This is equivalent to injecting 60 billion cells, a very high dose that is not achievable in practice. be.

[0235] Example 7: Intracellular injection of EDB-CAR T cells In recent years, CAR-mediated immunotherapy has gained broader therapeutic applications. The exact mechanism by which immunotherapy kills cancer cells is still incompletely understood. Long-term killing capacity of T cells is mediated by CAR in CAR T cells It has been reported that the quality of IS positively correlates with the efficacy of CAR-modified immune cells. Data from xenotransplantation models have demonstrated that the quality of in vitro IS predicts the quality of in vivo CA. It was also demonstrated that the performance of R-modified immune cells correlated positively with that of R-modified immune cells. Xiong et al., Molecular See ar Therapy 26(4):963-975, 2018.

[0236] However, this example demonstrated that macrophages after EDB-CAR transduction produce TNFα. This indicates that EDB-CAR-mediated cancer cell killing is in part due to its anti-cancer activity. This is due to the stimulation of cytokine secretion and is less dependent on immune synapse mechanisms. Indicates whether it is present or not.

[0237] In this experiment, the protocol provided by the manufacturer (130-050 -201, Miltenyi), and isolated mononuclear cells from PBMCs by magnetic bead positive selection. I let go. CD14 was chosen. + Mononuclear cells were cultured in 10% FBS and 10 ng / ml human recombinant The uncultured cells were incubated in RPMI containing GM-CSF (300-03-20, PeproTech). After seeding in treated cell culture flasks for 8 days, FuSure (Bosto n 3T Biotechnologies) On day 7, monocytes / macrophages were transfected with lentiviral vectors expressing The cells were harvested and tested for expression of EDB-CAR (SEQ ID NO: 1). See:

[0238] Detecting cytokines released by monocytes / macrophages after EDB-CAR transduction To achieve this, target cells were transfected with EDB protein at effector-target ratios of 10, 20, and 40. The mixture was mixed in a 96-well U-bottom plate at the ratio of 0.01 to 0.01. After 24 hours of incubation, ELISA ( The expression of IFN-γ, TNF-α and IL-12 was measured by DAKEWE. See 11A-11J.

[0239] Below, an overview of the experimental procedure is provided.

[0240] TIFF2025066806000002.tif201411 Day: Mononuclear cells were isolated from peripheral blood and identified as CD14+ by flow cytometry. The cell ratio was detected and 10 ng / ml GM-CSF was added and continued for 5 days.

[0241] Day 5: The attached cells were digested and stained with trypan blue to calculate cell vitality.

[0242] Day 6: Virus was transduced.

[0243] Day 7: Cellular activity was observed.

[0244] Day 8: CAR positive rate was detected by flow cytometry.

[0245] Day 9: Incubated with target cells or EDB antigen.

[0246] Day 10: Cytokine expression was detected by ELISA.

[0247] Early observations of the cytotoxicity of EDB-CAR T cells demonstrated that their killing activity was suppressed in target cells. The expression levels of EDB-fibronectin in the cells did not appear to be proportional to the expression levels of EDB-fibronectin in the cells. In addition, when cultured with target cells, EDB-CAR T cells inhibited TNFα was secreted.

[0248] TNFα has a cytotoxic function. It has been shown that nucleocytes / macrophages also secrete TNFα. See Figures 11C and 11J. Both interferon-γ and TNF-α are strong stimulators of immune cells and inhibitors of cancer cells. Therefore, immune cells modified with the EDB-CAR of the present invention By delivering cytokines, e.g., IFN-γ and / or TNFα, to cancer tissues. This confirmed its usefulness in achieving EDB-CAR-mediated killing.

[0249] Example 8: Another EDB-CAR expressed in T cells CD4 and CD8 intracellular domains are involved in lymphocyte-specific T cell signaling It is known to interact with protein tyrosine kinase (Lck). and the intracellular domain of CD8ic into the second generation EDB CAR (SEQ ID NO: 1, 3). By this, EDB CARs (SEQ ID NOs: 4, 5, 6, 7, 8) were generated. CD4ic and CD8ic EDB C were generated as described in Example 1. The expression of AR was tested (see FIG. 12). The sequence IDs for the corresponding antibodies can be found in Table 1. Negative controls were untransduced T cells (T) or mock-transduced T cells (MCT). In this example, the T cells were transduced with BBCD4CD3 Expression of EDB CARs, named z and BBCD8CD3z (sequence IDs 4, 5). was hardly detected (Fig. 12). However, BBCD4CD3z and BBCD8 CD3z CAR T cells showed no significant proliferative activity or cytotoxicity against cancer cells after antigen induction. Other second-generation EDB CAR T cells (see Examples 9 and 10) This suggests that BBCD4CD3z and BBCD8CD3z CAR T cells have potent biological activity. Indicates that it has.

[0250] Example 9: EDB-CAR T cells activated by cancer cells Unlike targets of transmembrane proteins, EDB CAR T cells target the soluble EDB protein or EDB CA to target EDB-containing fibronectin in the extracellular matrix. Whether antigen stimulation of RT cells is sufficient to drive proliferation and cytotoxic activity remains to be determined. It is unclear. The second generation EDB CAR (a single CAR containing primary and costimulatory signals) (defined as a single chimeric antigen receptor), and EDB containing CD4ic- and CD8ic- The proliferation activity of CAR after antigen stimulation was measured. On the first and seventh days of the measurement, U87MG Stimulate the transduced T cells with 10% heat-inactivated FBS-containing RPM. In I 1640, the effector to target ratio was 5:1. As shown in Figure 13 In addition, the transduced T cells rapidly grew upon stimulation with U87MG and expressed low levels of BBCD. Includes 4CD3z and BBCD8CD3z (sequence IDs 4, 5) CAR T cells. What should be noted in the present invention is that the transduced EDB CAR was used in proliferation and cytotoxicity assays. The T cells were adjusted to about 20% of the total.

[0251] Example 10: In vitro cytotoxicity of various EDB-CAR T cells against cancer cells Induced and transduced with EDB-CAR with primary and costimulatory signals The cytotoxic activity of T cells was measured. Primary human T cells were transduced with the indicated lentiviral vectors. and incubated with U87MG cells at various E:T ratios for 24 hours. Cell lysis was measured by H assay. Data are representative of three independent experiments (Figure 14). Second generation EDB CAR (defined as including primary and costimulatory signals) and Both CD4ic- and CD8ic-containing EDB CARs expressed in U87MG cancer cells. EDB28 CAR T cells show significant cytotoxic activity against U87MG cancer cells. Similar to the observation in Example 9, the in vitro cytotoxicity was highest in the low expression BBCD4 CD3z and BBCD8CD3z (sequence ID 4, 5) CAR T cells highly expressed EDB They showed similar levels of cytotoxicity as CAR T cells.

[0252] Example 11: Bispecific molecule / EDB-CAR T cells activated by cancer cells In one aspect of the invention, there is provided an anti-EDB ScFv fused to an anti-CD3ε ScFv. However, it can be a bispecific molecule and can be used alone, with CD28, OX40 or CD1 Chimeric antigen receptor consisting of anti-EDB fibronectin ScFv fused to 37 protein Also, a bispecific anti-E fused to an anti-CD3ε ScFv may be used. DB ScFv is secreted by modified T cells. The bispecific anti-EDB ScFv fused to D3ε ScFv was generated and used alone. The bispecific conjugate, EDB antigen, and CD3ε are expressed on T cells. Interaction with these proteins activates T cell proliferation and cytotoxicity.

[0253] In this example, T cells were targeted with the bispecific conjugate EDB-αCD3 (SEQ ID NO: 18). Transduced with CellTrace (Invitrogen, C34564). The T cells were labeled and stimulated with U87MG cells at an effector-target ratio of 1:5. All cells were cultured in RPMI 1640 containing 10% heat-inactivated FBS. At different time points, cells were analyzed by flow cytometry with a 630 nm excitation source. The trailing fluorescence (area in brackets) relative to the main peak indicates the proliferation of T cells. The numbers in parentheses represent the percentage of the total area and represent the proliferative area of ​​T cells. By way of example, bispecific EDB-αCD3 alone can stimulate T cell proliferation. It has been proven that.

[0254] Example 12: Cytotoxicity of bispecific molecules / EDB-CAR T cells against cancer cells Bispecific ED transduced alone or together with EDB-targeting CAR in U87MG cells The in vitro cytotoxic activity of the B-αCD3 (SEQ ID NO: 18) molecule was measured (FIG. 16). Primary human T cells transduced with EGFR-1 were co-incubated with U87MG cells at various E:T ratios for 24 h. Cell lysis was measured by LDH assay. Data are from three independent experiments. The bispecific EDB-αCD3 (SEQ ID NO: 18) and 137Pro (SEQ ID NO: 10) The combination appears to have synergistic cytotoxicity. The lentiviral vector used to transduce the cells was reduced by half, but the cytotoxic activity was doubled. It is equivalent to the 3rd generation EDB CAR.

[0255] Example 13: "Trans" EDB-CAR T cells activated by cancer cells In the native TCR complex, the ITAM-containing CD3ζ intracellular domain is associated with the plasma membrane. Many so-called second-generation CARs combine the CD3 intracellular domain with CD28, 4-1B Fusion of costimulatory signals from B or OX40 localizes ITAMs to the distal membrane end. Phosphorylation of CD3ζ is influenced by its membrane proximity, and the distal end of the ITIM is located at the phosphorylation site. Therefore, the EDB-specific ScFv and the CD3ζ and CD28 extracellular domains Similarly, the EDB-specific ScFv was generated to express CD4 CD3ε, CD3γ, CD3δ, CD4, CD8, OX40, CD28 or CD137 The extracellular domain, transmembrane (TM) domain and intracellular domain of a membrane protein selected from the group consisting of A linker region (e.g., a sequence The extracellular domain may or may not be present. The amino acid sequence and transmembrane domains can be found in other proteins, e.g., CD8 (e.g., SEQ ID NO:1). 2) may be derived.

[0256] Anti-EDB fibronectin single chain antibody and full-length CD3ε, CD3γ, CD3δ or CD When fused to the 3ζ protein, the anti-EDB ScFv is targeted to the T cell receptor (TCR) complex of the cell. and administering to the body a partial or full-length CD3ε, CD3γ, CD3δ or CD3ζ protein. When the anti-EDB fibronectin ScFv fused to the protein binds to the EDB cancer antigen, the TCR The complex assembles. In addition, costimulatory signals are CD4, CD8, CD28, OX40, or Provided by anti-EDB ScFv, a single polypeptide fused to the CD137 protein Since the costimulatory signal region is provided by a single polypeptide, such We define these fusion molecules as “trans” versions of CARs. In contrast to the “cis” CAR structure, in this structure, the costimulatory domain is a single polypeptide. Located in the peptide chain.

[0257] Two polypeptides are used to generate "trans" EDB-CAR, but one ScFv is fused to CD3ε or CD3ζ and is used as the primary T cell activation signal, and the other Fusion with CD137 or CD28 costimulatory domains. Fusion with CD3ζ or costimulatory domains. Since the ITAM domain is close to the plasma membrane, its morphology is predicted to be most similar to the native structure. Other molecules involved in TCR signaling (e.g., LCK) are anchored to the plasma membrane. To induce CAR, the “trans” form of CAR is required for the formation of a signaling complex involving LCK. More ideally, the primary stimulatory signal (e.g., SEQ ID NOs: 11, 12, 13, 14 or Any EDB-CAR having a costimulatory signal (e.g., SEQ ID NO: 8, 9, 10) to generate a “trans” EDB-CAR. In the present example, the T cells are immunized with anti-E DB ScFv and transduced with either alone or a costimulatory signal (SEQ ID NOs: 9 and 10, respectively). or 8) is co-transduced with an EDB CAR molecule.

[0258] The expression test of the "trans" EDB CAR was carried out as described in Example 1 (Figure 1 2). Transduction was performed using CellTrace (Invitrogen, C34564). Transfected T cells were labeled and stimulated with U87MG cells at an effector-target ratio of 1:5. All cells were cultured in RPMI 1640 containing 10% heat-inactivated FBS. At different time points, cells were analyzed by flow cytometry with a 630 nm excitation source. The trailing fluorescence (area in brackets) relative to the main peak was analyzed (Figure 17). The numbers above the brackets represent the percentage of the total area and represent the proliferative portion of T cells.

[0259] Example 14: Cytotoxicity of "trans" EDB-CAR T cells against cancer cells "Trans" EDB-CAR is a primary signaling site that occurs in two independent polypeptide chains. Generating “trans” EDB CAR T cells by combining priming and costimulatory signals To achieve this, we performed stimulation alone or at the same time with anti-EDB ScFv fused to CD3ζ (SEQ ID NO: 12). EDB CAR molecules having the signal (SEQ ID NO: 9, 10, or 8, respectively) Transduce cells. Incubate target U87MG cells with various E:T ratios for 24 hours. The induction of cytotoxic activity of transduced T cells was tested by incubating the cells with 100 μg / mL of ... Cell lysis was measured by the method. Data are representative of three independent experiments (Figure 18). In the transduction mode, the lentiviral vector used to transduce T cells was reduced by half. However, the cytotoxic activity of the “trans” EDB CAR T cells was higher than that of the second-generation EDB CAR. or the first generation CD3ζ CAR.

[0260] Example 15: "Hi jack" / EDB-CAR T cells activated by cancer cells cell In the present invention, a chimeric antigen receptor is generated using full-length CD3ε and CD3ζ proteins, With or without a linker region connecting the EDB fibronectin binding domain. The typical TCR complex consists of TCRα, TCRβ, CD3γ, CD3δ, CD3ζ and and CD3ε subunits. The protein is delivered to the T cell receptor (TCR) complex of the cell, allowing the chimeric EDB CAR to be delivered to the recipient. Chimeric TCR introduced into the recipient binds to the cancer antigen EDB in an MHC I complex-independent manner. Contrary to conventional CAR structures, TCR-complexed CAR is expected to suppress TCR signaling. The function of the hijack CAR is to obtain the antigen specificity and chirality of the antigen-specific ScFv. The activation of T cells is due to the TCR binding to tumor antigens. Any primary T cell bearing the antigen is activated by the presence of the cancer antigen independent of the MHC complex. In short, the conventional CAR structure is considered to be an independent molecule. "EDB CAR is a receptor engineered into the TCR complex. ijack" EDB CAR engagement triggers TCR activation by EDB antigen , leading to EDB CAR T cell proliferation and cytotoxicity.

[0261] "Hijack" EDB CAR can provide primary T cell stimulatory signals However, the costimulatory signal is provided by a single polypeptide, among which the EDB-specific Sc T cell activation with Fv of CD4, CD8, CD28, OX40 or CD137 protein Such an embodiment of the EDB CAR may also be referred to as "trans."

[0262] In this example, the anti-EDB ScFv is linked to a linker sequence (SEQ ID NOs: 11, 13, 14 , 15) or without the full-length CD3ε or CD3ζ protein. The CD3ε or CD3ζ polypeptide fragments used in the examples are designed to enter the TCR complex. In fact, one "hijacked" EDB CAR (SEQ ID NO: 11, 13, 14, 15) may also be used in the form of a "trans" EDB CAR. In the experiment, anti-EDB ScFv (SEQ ID NO: 11) or 15 amino acids fused to CD3ζ were used. Anti-EDB ScFv fused to CD3ε via a 5'-amino acid linker (SEQ ID NO: 15) was used to express T cells. EDB CAR molecules (SEQ ID NO: 9, 10 or 8) alone or with costimulatory signals were administered to cells. ) were transduced with the "hijack" EDB CAR The expression test was performed using CellTrace (Invitrogen) (see FIG. 12). n, C34564) and incubated at 1:5 with U87MG cells. All cells were stimulated at an effector-target ratio. The cells were grown in RPMI 1640 containing 100% ethanol. At different times, the cells were analyzed using a flow cytometer with a 630 nm excitation source. Cells were analyzed by cytometry (Figure 19). Trailing fluorescence to the main peak (area in brackets) represents the proliferation activity of T cells. The numbers above the brackets are the percentage of the total area. It represents the growing part of the cell. By combining the peptide with the CD28 costimulatory domain in trans, enhanced proliferation was observed. It was made.

[0263] Example 16: Cytotoxicity of "Hijack" EDB-CAR T cells against cancer cells The "Hijack" EDB CAR is fused to full-length CD3ζ or CD3ε protein The antibody contains an anti-EDB ScFv and has a costimulatory molecule produced as a single polypeptide chain. , or not. "hijack" EDB To generate CAR T cells, Anti-EDB ScFv fused to full-length CD3ε (SEQ ID NO: 11) alone or as a co-stimulatory EDB CAR molecules having the signal (SEQ ID NO: 9, 10, or 8, respectively) Transduce cells. Another version of "hijacks" EDB CAR T cells In this study, T cells were co-transfected with full-length CD3ε CAR alone or in combination with costimulatory EDB CAR molecules. Signaling with anti-EDB ScFv fused to (SEQ ID NO: 13, 14 or 15) (SEQ ID NO: 9, 10, or 8, respectively). The cytotoxic activity of the transduced T cells was tested by incubating for 4 h. Cell lysis was measured by LDH assay. Data are representative of three independent experiments (Figure 2). 0) In addition, when the “hijack” EDB CAR bound to the costimulatory EDB CAR molecule, This reduces the amount of lentiviral vector used to transduce T cells by half.

[0264] Example 17: "Hijack Plus" EDB-CAR activated by cancer cells T cells In the "trans" form of the "hijack" CAR, anti-EDB fibronectin Sc Fv is full-length CD3ε, CD3γ, CD3δ or CD3ζ (hijack CAR) , or fused to partial or full-length CD4, CD8, CD28, OX40, or CD137 and providing a costimulatory signaling domain in trans as a single polypeptide. The "Hijack" EDB CAR was engineered into the TCR complex. Although the receptors themselves lack the costimulatory domains necessary for enhanced proliferation, E For targets such as DB, it is possible to separate the primary and costimulatory signals onto separate polypeptide chains. It is still unclear what optimal activation of T cells occurs when In addition, the primary and costimulatory signals are generated in a "cis" manner, i.e., the costimulatory domain is hijack" EDB CAR (SEQ ID NO: 16, 17). The chimeric TCR recognizes the EDB antigen and provides the primary survival signal upon TCR activation. The co-stimulatory domain enhances T cell proliferation and cytotoxicity. .

[0265] To design a “hijack” EDB CAR with a costimulatory domain, we used EDB CD3εFL (SEQ ID NO: 15) contains the costimulatory domain of CD28 or CD137 at its C-terminus. The "hijack plus" EDB CAR construct (SEQ ID NOs: 16 and 17) was ligated to In fact, in the "hijack plus" CAR, anti-EDB fibronectin S The cFvs are fused to full-length CD3ε, CD3γ, CD3δ or CD3ζ, and are also available as partial or can be fused to full-length CD4, CD8, CD28, OX40, or CD137 to provide costimulatory It is a similar polypeptide chain that can provide a signaling domain. "Hijack plus" EDB CAR T cells that are activated by releasing To test, we used anti-hijack plus EDB CARs (sequence numbers 1 and 2 are respectively T cells were transduced with either hijack or 17 as described in Example 1. The expression test of "plus" EDB CAR was performed (see FIG. 12). T cells were labeled with CellTrace (Invitrogen, C34564) and The cells were stimulated with U87MG cells at an effector-target ratio of 1:5, and all cells were heat-inactivated with 10% The cells were grown in RPMI 1640 containing activated FBS and at different time points The excitation source of the cells was analyzed by flow cytometry at 630 nm (Figure 21, upper The trailing fluorescence (bracketed area) relative to the main peak represents the proliferation activity of T cells. The numbers above the brackets are the percentage of the total area, which represents the area of ​​T cell proliferation. Compared with controls, "hijack plus" EDB CAR T cells showed increased proliferation It has been proven that.

[0266] The lower panel of Figure 21 shows the “hijack plus” ED after exposure to U87MG cells. Morphology of B CAR T cells is depicted. EDB28 CAR T (SEQ ID NO: 3) Cells showed the highest aggregation, which was mainly due to surface adhesion proteins after T cell activation. After induction with U87MG cells, the “hijack plus” CD3eFLCD28 CAR T (SEQ ID NO: 16) cells proliferated and aggregated significantly.

[0267] Example 18: Antibody activity of "Hijack Plus" EDB-CAR T cells against cancer cells cell toxicity "Hijack plus" EDB-CAR is a full-length CD3ζ or CD3ε protein. It contains an anti-EDB ScFv fused to a protein and a costimulatory molecule on the same polypeptide chain. Therefore, the primary and costimulatory signals of the “hijack plus” EDB CAR The cis form of the molecule is still capable of activating the cytotoxicity of transduced T cells. In order to test whether the "hijack plus" EDB CAR T cells were generated and incubated with target U87MG cells at various E:T ratios for 24 hours. The induction of cytotoxic activity of transduced T cells was tested by injecting the T cells with LDH. Cytolysis was measured by the CD2 "hijack plus" EDB CAR with 8 costimulatory signals (SEQ ID NO: 16) has cytotoxic activity. jack plus” EDB CAR exhibits mild but significant cytotoxic activity.

[0268] Example 19: “hijack plus” EDB CAR cells are attached to the TCR complex Enter "hijack plus" EDB CAR is a CD3ζ or CD It contains the ε polypeptide fragment, which is incorporated into the TCR complex, but does not bind to CD28 or CD 137 Whether fusion with a costimulatory domain inhibits uptake remains unclear. In this example, the NP-40 detergent was used to remove the "hijack plus" EDB CAR The transduced T cells were lysed with IgG and then incubated with a biotin-labeled polyclonal goat anti-human IgG antibody. IgG F(ab')2 fragment antibody (Jackson Immunoresearch, Immunoprecipitation was performed using a CD3ζ specific antibody (Cat. No. 109-066-097). Eastern blotting (Figure 23A) or Western blotting using a CD3δ-specific antibody Immunoprecipitates were analyzed by blotting (Figure 23B). k plus" EDB CAR complexed band of approximately 17 kDa (SEQ ID NOs: 16 and 17) In CD3δ, approximately 19 A band of 100 kDa was detected. Alternatively, the "hijack" EDB CAR (SEQ ID NO: 1 5, 16, and 17) (Figure 23B). jack plus EDB CAR (SEQ ID NO: 15, 16 or 17) binds CD3δ and These EDB CARs were shown to associate favorably with the TCR complex. The co-stimulatory domain is fused to the C-terminus of CD3ζ or CD3ε, forming a “hijack” plus" does not interfere with the assembly of the EDB CAR.

[0269] Example 20: Tumor reduction and inhibition of tumor growth in vivo In the present invention, a series of EDB CARs were constructed, all of which were shown to be T The induction of antigen-specific activation and cytotoxic activity of cells was demonstrated. Can engineered EDB CAR T cells penetrate tumor tissue and kill tumor cells? It is still unclear. Published work (Wagner et al., 2021, DO I: 10.1158 / 2326-6066.CIR-20-0280), the second Using EDB CAR T cells, mild inhibition of tumor growth was observed and has already been achieved. It can be seen that the amount of tumor tissue was not reduced. The aim of the study was to analyze the effect of the drug on immune-deficient NCG mice in the treatment of U87MG tumors. To construct the model, we inject approximately 1 million mice into 6-week-old NCG mice to develop tumors on the dorsal side. U87MG cells were injected subcutaneously. Once tumors were palpable and measured, The size of the tumor is approximately 20-50 mm 3 Once the mice reached 4 days of age, they were divided into groups (5 / group). Mice were injected with 5 million of each transduced EDB CAR T cell via intravenous injection. The literature has reported that the second generation EDB CAR has little inhibitory effect on tumor growth. (Figure 24.A). By providing a costimulatory signal to the “trans” or bispecific EDB CAR, When combined, no significant inhibition of tumor growth was observed (Fig. 24, B and C). ijack" EDB CAR T cells showed mild inhibition of tumor growth (Figure 24. D) The most effective treatment with “hijack plus” EDB CAR T cells An inhibitory effect was observed, with a long-term delay in tumor growth (upper panel in Figure 24E). The main difference is that the tumors were reduced to almost undetectable levels in the early stages of treatment. (Figure 24E, lower panel) This shows that "hijack plus" EDB CAR T cells It is able to penetrate tumor tissue and decompose tumor cells, resulting in almost complete regression. Ten days after infusion of the “hijack plus” EDB CAR T cells, tumor tissue The fabric became inaccessible. Therefore, "hijack plus" EDB CAR T thin The cytotoxic activity of the cells is not affected by the immunosuppressive microenvironment, i.e., activated T cells are able to attack tumor tissue. This is because the immune system is not suppressed by the immune system and can degrade tumor cells, which is called "h ijack plus EDB CAR T cells are useful as a therapeutic agent for cancer treatment , even in tumors with a strongly immunosuppressive microenvironment.

[0270] The results of the in vivo study showed that only the "hijack plus" EDB CAR caused significant swelling. It has been shown that the drug can inhibit tumor growth and promote tumor regression, but this discovery is in line with previous findings. Consistent with the observations, i.e., the second generation E DB CAR is not good at suppressing tumor growth. The defect in the EDB antigen is still unknown, but may be due to the specificity of the EDB antigen itself. DB-containing fibronectin is a component of the extracellular matrix (ECM) and is involved in the ECM and interstitial It exists in many forms, including endothelial and perivascular structures. None of these proteins are completely membrane proteins, but rather have typical lateral structures similar to those of membrane proteins in vivo. Therefore, the CD3ζ and CD28 intracellular domains are not easily separated in vivo. The second generation EDB CAR forms cluster-like structures and promotes T cell activation and proliferation. may not be enough to enhance it.

[0271] Conversely, the cytotoxicity of “hijack plus” EDB CAR T cells in vitro and in vivo Both were confirmed. Our findings are at least as good as the "hijack plus" EDB C This shows that AR T cells are activated in vivo and have cytotoxicity. Activate the cytotoxic activity of T cells to eliminate large tumor burdens in the body. Although the mechanism of activation is still unclear, it is thought that TCR activation and cis-mediated costimulatory signaling pathways may be involved. The second generation CD3ζ-containing EDB CAR inhibits tumor growth in vivo. Combined with the insufficient reduction in curbing the length of the "hijack" EDB CA R further comprises elements in the TCR complex and CD3 ITAMs (e.g., CD3 It is also shown that the chimera " "hijack plus" TCR complex activation involves both primary and costimulatory signals This is because the proximity of the signalosome to primary and costimulatory signals in vivo is essential. We show that EDB is important for the activation of chimeric antigen receptors targeting EDB in mice.

[0272] Sequence listing in FASTA format: >EDB137ic MYRMQLLSCIALSLALVTNSEVQLLESGGGLVQPGGSLRL SCAASGFTFSSFSMSWVRQAPG KGLEWVSSISGSSGTTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKP FPYFD YWGQGTLVTVSSGDGSSSGGSGGASEIVLTQSPGT LSLSPGERATLSCRASQSVSSSFLAWYQ QKPGQAPRLLI YYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQTGRIPPTFG QGTKVEIKAKPTTTPAPRPPTPAPTIAS QPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAP LAGTC GVLLLSLVITLYKRGRKKLLYIFKQPFMRPVQTTQEEDGC SCRFPEEEEGGCELRVK FSRSADAPAYQQGQNQLYNELN LGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPR (SEQ ID NO: 1) >EDB137 ic nucleotide sequence ATGTACAGAATGCAGCTGCTGTCCTGCATCGCCCTGAGCC TGGCCCTGGTGACCAATAGCGA GGTGCAACTCCTGGAGT CCGGCGGAGGCCTGGTCCAACCTGGAGGAAGCCTGAGGCT GAGCT GTGCCGCCAGCGGCTTCACCTTTTCCAGCTTCTC CATGAGCTGGGTGAGACAGGCCCCCGGC AAAGGCCTGGA GTGGGTGTCCAGCATCTCCGGCAGCTCCGGCACCACCTAC TATGCTGATTC CGTGAAGGGCAGGTTCACCATCTCCAGG GACAACAGCAAGAACACACTGTACCTCCAAATGA ACTCC CTGAGGGCCGAAGACACCGCCGTGTACTACTGCGCCAAGC CCTTTCCCTATTTCGAC TATTGGGGCCAGGGCACACTGG TCACCGTGAGCTCCGGCGATGGAAGCAGCGGAGGAAGCGG AGGCGCTAGCGAAATCGTGCTGACCCAGAGCCCTGGCAC ACTGTCCCTGAGCCCTGGAGAAA GAGCCACCCTGAGCTG TAGGGCCTCCCAGAGCGTGAGCAGCAGCTTCCTGGCCTGG TACCAA CAGAAGCCCGGACAGGCCCCCAGGCTGCTGATC TACTATGCCTCCTCCAGGGCCACAGGCAT CCCCGACAGG TTCTCCGGCTCCGGTTCTGGCACCGATTTTACCCTGACCA TCTCCAGGCTGG AGCCCGAAGACTTCGCCGTGTATTACT GCCAGCAGACCGGACGTATTCCTCCTACCTTTGGC CAGG GCACCAAGGTGGAGATCAAAGCCAAGCCCACCACCACACC TGCCCCTAGACCCCCTAC ACCTGCCCCCACAATCGCTTC CCAGCCTCTGTCCCTGAGGCCTGAGGCTTGTAGGCCTGCC G CTGGAGGAGCTGTGCACACCAGAGGCCTCGACTTCGCC TGCGACATCTATATCTGGGCTCCT CTGGCCGGCACCTGT GGAGTCCTCCTGCTGAGCCTGGTGATCACACTGTACAAGA GAGGCAG GAAGAAGCTGCTGTACATCTTCAAGCAACCCT TCATGAGGCCTGTGCAGACCACCCAGGAAG AAGATGGCT GCAGCTGCAGGTTCCCTGAGGAAGAAGAGGGCGGATGCGA GCTGAGAGTGAAG TTCAGCAGGTCCGCCGATGCCCCTGC CTATCAGCAGGGCCAGAACCAGCTGTACAACGAACT CAA CCTGGGCAGGAGGGAGGAGTACGACGTCCTCGACAAGAGG AGAGGCAGGGACCCCGAGA TGGGAGGCAAGCCTCAGAGG AGGAAGAACCCTCAAGAGGGACTGTACAACGAGCTGCAGA AG GACAAGATGGCCGAGGCCTACTCCGAGATCGGCATGA AGGGCGAGAGAAGAAGAGGCAAGGG CCATGATGGCCTCT ACCAGGGCCTGAGCACCGCCACCAAGGACACATACGATGC CCTGCATA TGCAGGCCCTCCCCCCTAGGTGA(SEQ ID NO: 2) >EDB28ic MYRMQLLSCIALSLALVTNSEVQLLESGGGLVQPGGSLRL SCAASGFTFSSFSMSWVRQAPG KGLEWVSSISGSSGTTY YADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKP FPYFD YWGQGTLVTVSSGDGSSGGSGGASEIVLTQSPGT LSLSPGERATLSCRASQSVSSSFLAWYQ QKPGQAPRLLI YYASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQTGRIPPTFG QGTKVEIKAKPTTTPAPRPPTPAPTIAS QPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAP LAGTC GVLLLSLVITLYRSKRSRLLHSDYMNMTPRRPGPTRKHYQ PYAPPRDFAAYRSRVKF SRSADAPAYQQGQNQLYNELNL GRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR (SEQ ID NO:3) >EDB 41BB-CD4ic-CD3z MYRMQLLSCIALSLALVTN SEVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQ APG KGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNT LYLQMNSLRAEDTAVYYCAKPFPYFD YWGQGTLVTVSSG DGSSGGSGGASEIVLTQSPGTLSLSPGERATLSCRASQSV SSSFLAWYQ QKPGQAPRLLIYYASSRATGIPDRFSGSGS GTDFTLTISRLEPEDFAVYYCQQTGRIPPTFG QGTKVEI KAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH TRGLDFACDIYIWAP LAGTCGVLLLSLVITLYKRGRKKL LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELCVR C RHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTCSPIRVKF SRSADAPAYQQGQNQLYNELN LGRREEYDVLDKRRGRDP EMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR GKGH DGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 4) >EDB 41BB-CD8ic-CD3Z MYRMQLLSCIALSLALVTN SEVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQ APG KGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNT LYLQMNSLRAEDTAVYYCAKPFPYFD YWGQGTLVTVSSG DGSSGGSGGASEIVLTQSPGTLSLSPGERATLSCRASQSV SSSFLAWYQ QKPGQAPRLLIYYASSRATGIPDRFSGSGS GTDFTLTISRLEPEDFAVYYCQQTGRIPPTFG QGTKVEI KAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH TRGLDFACDIYIWAP LAGTCGVLLLSLVITLYKRGRKKL LYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELLYC N HRNRRRVCKCPRPVVKSGDKPSLSARYVRVKFSRSADAPA YQQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPQR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLS TATKDTYDALHMQALPPR (SEQ ID NO: 5) >EDB CD4ic-41BB-CD3Z MYRMQLLSCIALSLALVTN SEVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQ APG KGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNT LYLQMNSLRAEDTAVYYCAKPFPYFD YWGQGTLVTVSSG DGSSGGSGGASEIVLTQSPGTLSLSPGERATLSCRASQSV SSSFLAWYQ QKPGQAPRLLIYYASSRATGIPDRFSGSGS GTDFTLTISRLEPEDFAVYYCQQTGRIPPTFG QGTKVEI KAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH TRGLDFACDIYIWAP LAGTCGVLLLSLVITLYLYCNHRN RRRVCKCPRPVVKSGDKPSLSARYVKRGRKKLLYIFKQ P FMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPA YQQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPQR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLS TATKDTYDALHMQALPPR(SEQ ID NO: 6) >EDB CD8ic - BB - CD3z MYRMQLLSCIALSLALVTNSE VQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAP G KGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCAKPFPYFD YWGQGTLVTVSSGDG SSGGSGGASEIVLTQSPGTLSLSPGERATLSCRASQSVSS SFLAWYQ QKPGQAPRLLIYYASSRATGIPDRFSGSGSGT DFTLTISRLEPEDFAVYYCQQTGRIPPTFG QGTKVEIKA KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR GLDFACDIYIWAP LAGTCGVLLLSLVITLYLYCNHRNRRRVCKCPRPVVKSGD KPSLSARYVKRGRKKLLYIFKQ PFMRPVQTTQEEDGCSC RFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGR REEYD VLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLS TATKDTYDALH MQALPPR (SEQ ID NO: 7) >EDB CD4ic-CD28ic MYRMQLLSCIALSLALVTNSEV QLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPG KGLEWVSSISGSSGTTYYADSVKGRFTISRDNSKNTLYL QMNSLRAEDTAVYYCAKPFPYFD YWGQGTLVTVSSGDGS SGGSGGASEIVLTQSPGTLSLSPGERATLSCRASQSVSSS FLAWYQ QKPGQAPRLLIYYASSRATGIPDRFSGSGSGTD FTLTISRLEPEDFAVYYCQQTGRIPPTFG QGTKVEIKAK PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG LDFACDIYIWAP LAGTCGVLLLSLVITLYCVRCRHRRRQ AERMSQIKRLLSEKKTCQCPHRFQKTCSPIRSKRS RLLH SDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 8) >EDB CD28 aa138-220 MYRMQLLSCIALSLALVTNS EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQA PGKGLEWVSSI SGSSGTTYYADSVKGRFTISRDNSKNTL YLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSSGDG SSG GSGGASEIVLTQSPGTLSLSPGERATLSCRASQSVS SSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSG SGSG TDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIKK HLCPSPLFPGPSKPFWVLVVVGGVLAC YSLLVTVAFIIF WVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAA YRS (SEQ ID NO: 9) >EDB CD137 aa160 - 255 MYRMQLLSCIALSLALVTN SEVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQ APGKGLEWVSSI SGSSGTTYYADSVKGRFTISRDNSKNT LYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGD GSSG GSGGASEIVLTQSPGTLSLSPGERATLSCRASQSV SSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSG SGS GTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIK PSPADLSPGASSVTPPAPAREPGHSPQI ISFFLALTSTA LLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEE DGCSCRFPEEEEGGCEL (SEQ ID NO: 10) >EDB CD3zFL MYRMQLLSCIALSLALVTNSEVQLLESG GGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWV SSI SGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCAKPFPYFDYWGQGTLVTVSSGDGSSG GSGG ASEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQ QKPGQAPRLLIYYASSRATGIPDRFSG SGSGTDFTLTIS RLEPEDFAVYYCQQTGRIPPTFGQGTKVEIKAKPTTTPAP RPPTPAPTIASQPLSLRPE CARPAAGGAVHQSFGLLDPK LCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQ LYNELNLGRRE EYDVLDKRRGRDPEMGGKPQRRKNPQEG LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT KDT YDALHMQALPPR (SEQ ID NO: 11) >EDB CD3zic MYRMQLLSCIALSLALVTNSEVQLLESG GGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWV SSI SGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLR AEDTAVYYCAKPFPYFDYWGQGTLVTVSSGDGSSG GSGG ASEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQ QKPGQAPRLLIYYASSRATGIPDRFSG SGSGTDFTLTIS RLEPEDFAVYYCQQTGRIPPTFGQGTKVEIKAKPTTTPAP RPPTPAPTIASQPLSLRPE ACRPAAGGAVHTRGLDFACD IYIWAPLAGTCGVLLLSLVITLYRVKFSRSADAPAYQQGQ NQLYNELNLGR REEYDVLDKRRGRDPEMGGKPQRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST ATK DTYDALHMQALPPR (SEQ ID NO: 12) >EDB 5aaCD3eFL MYRMQLLSCIALSLALVTNSEVQLL ESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGL EWVSSI SGSSGTTYYADSVKGRFTISRDNSKNTLYLQMN SLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGDGSSG G SGGASEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLA WYQQKPGQAPRLLIYYASSRATGIPDRFSG SGSGTDFTL TISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIKGRASGD GNEEMGGITQTPYKVSISGTTV ILTCPQYPGSEILWQHN DKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRG SKPEDANFYLYLRA RVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRK AKAKPVTRGAGAGGRQRGQNKERPPPVPNPD YEPIRKGQ RDLYSGLNQRRI (SEQ ID NO: 13) >EDB 10aa linker CD3eFL MYRMQLLSCIALSLALVTN SEVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQ APGKGLEWVSSI SGSSGTTYYADSVKGRFTISRDNSKNT LYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGD GSSG GSGGASEIVLTQSPGTLSLSPGERATLSCRASQSV SSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSG SGS GTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIK GGGGSGGGGSDGNEEMGGITQTPYKVSI SGTTVILTCPQ YPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELE QSGYYVCYPRGSKPEDANFY LYLRARVCENCMEMDVMSV ATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGG RQRGQNKERPPP VPNPDYEPIRKGQRDLYSGLNQRRI(sequence number 14) 列番号14) >EDB 15aa linker CD3eFL MYRMQLLSCIALSLALVTN SEVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQ APGKGLEWVSSI SGSSGTTYYADSVKGRFTISRDNSKNT LYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGD GSSG GSGGASEIVLTQSPGTLSLSPGERATLSCRASQSV SSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSG SGS GTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIK GGGGSGGGGSGGGGSDGNEEMGGITQTP YKVSISGTTVI LTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKE FSELEQSGYYVCYPRGSKPE DANFYLYLRARVCENCMEM DVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRG AGAGGRQRGQNK ERPPPVPNPDYEPIRKGQRDLYSGLNQ RRI (SEQ ID NO: 15) >EDB 15aa linker CD3eFL CD28ic MYRMQLLSCIAL SLALVTNSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSF SMSWVRQAPGKGLEWVSSI SGSSGTTYYADSVKGRFTIS RDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTL VTVSSGDGSSG GSGGASEIVLTQSPGTLSLSPGERATLS CRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDR FSG SGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQ GTKVEIKGGGGSGGGGSGGGGSDGNEEMGGITQTP YKVS ISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDE DHLSLKEFSELEQSGYYVCYPRGSKPE DANFYLYLRARV CENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAK AKPVTRGAGAGGRQRGQNK ERPPPVPNPDYEPIRKGQRD LYSGLNQRRIRSKRSRLLHSDYMNMTPRRPGPTRKHYQPY APPRDFAAYRS (SEQ ID NO:16) >EDB 15aa linker CD3eFL CD137ic MYRMQLLSCIA LSLALVTNSEVQLLESGGGLVQPGGSLRLSCAASGFTFSS FSMSWVRQAPGKGLEWVSSI SGSSGTTYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGT LVTVSSGDGSSG GSGGASEIVLTQSPGTLSLSPGERATL SCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPD RFSG SGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFG QGTKVEIKGGGGSGGGGSGGGGSDGNEEMGGITQTP YKV SISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSD EDHLSLKEFSELEQSGYYVCYPRGSKPE DANFYLYLRAR VCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKA KAKPVTRGAGAGGRQRGQNK ERPPPVPNPDYEPIRKGQR DLYSGLNQRRIKRGRKKLLYIFKQPFMRPVQTTQEEDGCS CRFPEEEEGGCE L (SEQ ID NO: 17) >EDB-αCD3 MYRMQLLSCIALSLALVTNSEVQLLESGGG LVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSS I SGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAE DTAVYYCAKPFPYFDYWGQGTLVTVSSGDGSSG GSGGAS EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQK PGQAPRLLIYYASSRATGIPDRFSG SGSGTDFTLTISRL EPEDFAVYYCQQTGRIPPTFGQGTKVEIKGRASGDIKLQQ SGAELARPGASVKMSCK TSGYTFTRYTMHWVKQRPGQGL EWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSS LTSEDSAVY YCARYYDDHYCLDYWGQGTTLTVSSVEGGS GGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASS S VSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGS GTSYSLTISSMEAEDAATYYCQQWSSNPLTFGA GTKLEL KHHHHHHHH (SEQ ID NO: 18)

Claims

1. A chimeric antigen receptor (CAR), (1) Antigen binding specific to the extra domain B (EDB) of fibronectin A domain, (2) CD3ζ, CD3ε, CD3γ, CD3δ, CD4, CD8, CD28, OX4 0 or CD137 membrane protein and / or an extracellular domain and / or a transmembrane domain (T M) domain; (3) CD3ζ intracellular ITAM (immunoreceptor activity domain) with or without costimulatory domain and a tyrosine-linked phosphatase (tyrosine-linked phosphatase) domain, When the CAR is expressed on the surface of a T cell, the CAR is (a) soluble EDB, (b ) membrane-bound EDB, and / or (c) extracellular matrix (e.g., the fibril of a cell attachment support). When it binds to EDB in the extracellular matrix (ECM), which is a component of the endothelial cell network, it CAR, which can activate cells.

2. The antigen-binding domain can be an scFv, a single chain antibody, a nanobody antibody (e.g., a VHH ( Derivatives of camelid Ig), single domain antibodies (dAbs, VH or VL domains Derivatives), bispecific T cell-inducing antibodies (BiTEs, bispecific double-chain antibodies) and bispecific Affinity retargeting proteins (DARTs, bispecific double-chain antibodies); anticalins (lipoproteins); Protein derivatives); Adnectins (10th FN3 (fibronectin)); Design ankyrin repeat proteins (DARPins); or avimers The CAR according to claim 1.

3. The antigen-binding domain of claim 1 is a human scFv or a humanized scFv. C.A.R.

4. In addition, it contains a hinge / spacer domain between the antigen-binding domain and the TM domain. The CAR according to any one of claims 1 to 3.

5. The hinge / spacer domain and the TM domain are derived from the same protein. The CAR according to claim 4.

6. The similar protein is CD8α, and the hinge / spacer domain is C The CAR of claim 5, which is the extracellular domain of D8α.

7. The CAR according to any one of claims 1 to 6, wherein (3) comprises the costimulatory domain.

8. The CAR of claim 7, wherein the costimulatory domain is derived from CD28.

9. The CAR according to any one of claims 1 to 8, wherein (3) comprises two costimulatory domains.

10. The two costimulatory domains are one costimulatory domain from CD28, and / or one costimulatory domain from C 10. The method according to claim 9, comprising one costimulatory domain from D27, 4-1BB or OX-40. The CAR described herein.

11. scFv of residues 21-236 of SEQ ID NO:1, CD8α extracellular domain and transmembrane domain 1, comprising the C1-, C2-, C3-, C4-1BB intracellular domain and the CD3ζ intracellular domain. A.R.

12. Additionally, an N-terminal signal peptide sequence (e.g., the hIL-2 signal peptide sequence, or residues 1-20 of SEQ ID NO:

1.

13. The CAR of claim 12, comprising the polypeptide of SEQ ID NO:

1.

14. A polynucleotide encoding the CAR according to any one of claims 1 to 13, for example , a polynucleotide of SEQ ID NO:

2.

15. The polypeptide of claim 14, which is codon-optimized for expression in human cells. Ligands.

16. A vector comprising the polynucleotide of claim 14 or 15.

17. The vector is a viral vector, and can be used to infect T cells, macrophages and / or NK cells. For example, infecting the CAR in primary human T cells, macrophages or NK cells. and / or expressible according to claim 16. Vector.

18. The vector of claim 17 which is a lentiviral vector.

19. 1 , wherein the lentiviral vector is a self-inactivating lentiviral vector.

8. The vector described in 8.

20. A cell expressing the CAR according to any one of claims 1 to 13, 15 or the vector according to any one of claims 16 to 19. -containing cells.

21. The cell of claim 20 , wherein the cell is an immune cell.

22. The cell of claim 20 , wherein the cell is a T cell.

23. The cell of claim 20 , wherein the cell is a NK cell.

24. The cell of claim 20 , wherein the cell is a macrophage.

25. The method according to any one of claims 20 to 24, wherein the cells are primary cells isolated from a patient. cells.

26. The cells are derived from an established cell line, e.g., in a patient to whom the cells are administered. A cell according to any one of claims 20 to 24, which is derived from an allogeneic cell line to 。

27. The cell of any one of claims 20-26, wherein the cell expresses a cytokine.

28. The cytokine is IL-2, IL-7, IL-12, IL-15 or IL-21. The cell of claim 27 .

29. The expression of the cytokine is controlled by a promoter that is activated upon activation of immune cells. The cell of claim 27 or 28.

30. Claims 20-30 further comprising a safety switch for downregulating the activity of immune cells. A cell according to any one of claims 1 to 5.

31. The safety switch is a code for an iCaspase9 (inducible caspase-9) monomer. The iCaspase9 (inducible caspase-9) monomer comprises, for example, Activation of FKBP by dimerization triggers apoptosis in immune cells.

31. The cell according to item 30.

32. Inhibition of angiogenesis in subjects suffering from a disease or condition treatable by anti-angiogenic effects The method of the present invention comprises administering to a subject a therapeutically effective amount of a chimeric antigen receptor (CAR). administering immune cells to the patient, the chimeric antigen receptor (CAR) comprising: (1) An antigen-binding domain specific for fibronectin extra domain B (EDB) In and (2) CD3ζ, CD3ε, CD3γ, CD3δ, CD4, CD8, CD28, OX4 a transmembrane (TM) domain selected from CD137 or CD138 membrane protein; (3) CD3ζ intracellular ITAM (immunoreceptor activity domain) with or without costimulatory domain and a polypeptide (polysaccharide tyrosine motif) domain.

33. The method according to claim 32, wherein the CAR is any one of claims 1 to 13. Law.

34. 34. The method of claim 32 or 33, wherein the disease or condition is a solid tumor or a chronic inflammatory condition. The method described.

35. 35. The method of claim 34, wherein the cancer cells derived from a solid tumor do not express EDB on the cell surface. 。

36. The disease or condition is a solid tumor, and the method further comprises administering to the subject an immune checkpoint inhibitor. inhibitors, e.g., PD-1 inhibitors (e.g., pembrolizumab, nivolumab, and serotoninib); miprimimab), PD-L1 inhibitors (e.g., atezolizumab, avelumab, and durval mab), CTLA-4 targeted drugs (e.g., ipilimumab) or immunomodulatory agents (e.g., The method according to claim 34 or 35, comprising administering a medicament (such as thalidomide or lenalidomide) to the patient. Law.

37. The chronic inflammatory conditions include psoriasis, rheumatoid arthritis, psoriatic arthritis, ulcerative colitis, enteritis, osteoarthritis, asthma, pulmonary fibrosis, IBD), inflammation-induced lymphangiogenesis, hypertrophy diabetes, retinal neovascularization (RNV), diabetic retinopathy, choroidal neovascularization (CNV), Age-related macular degeneration (AMD), metabolic syndrome-related diseases, long-term peritoneal dialysis, young onset 35. The method of claim 34, wherein the condition is arthritis or atherosclerosis.

38. The method of claim 32 further comprises administering a second therapeutic agent that effectively inhibits angiogenesis.

37. A method according to any one of claims 37 to 37.

39. The second therapeutic agent is axitinib, bevacizumab, cabozantinib, everolimus, Lenalidomide, pazopanib, ramucirumab, regorafenib, sorafenib, sunitinib , thalidomide, vandetanib and / or dib-aflibercept.

8. The method according to claim 8.

40. A primary culture of a subject isolated from the vector according to any one of claims 16 to 19 in vitro. Transduction into immune cells, and optionally culturing the primary immune cells transduced with the vector in vitro. The method of any of claims 32-39, wherein the immune cells are generated by inducing and / or amplifying the immune cells.

2. The method according to claim 1.

41. Additionally, agents that inhibit cytokine release syndrome (CRS), such as anti-IL-6 antibodies, administering monoclonal antibodies (e.g., tocilizumab) and / or globulins; 41. The method of any one of claims 32-40, comprising administering a therapeutic dose of

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