Antibodies or their antigen-binding fragments, and their use

Abstract

The present invention relates to the field of immunology and molecular biology, and more specifically to antibodies or antigen-binding fragments thereof, and their use. The antibodies or antigen-binding fragments described in the present invention specifically bind to IL13Rα2, do not cross-bind to IL13Rα1, have a clear killing effect on tumor cells expressing IL13Rα2, and can promote the release of cytokines (particularly IFN-γ and IL-2).

Description

TECHNICAL FIELD

[0001] The present invention relates to the technical fields of immunology and molecular biology, and specifically to an antibody or an antigen-binding fragment thereof, and use thereof. BACKGROUND ART

[0002] IL13Rα2 is a glycosylated protein with a molecular weight of about 56 kDa. Human IL13Rα2 comprises a 26-amino acid signal sequence and a 17-amino acid intracellular domain, and the short intracellular protein contains an uncharacterized signal sequence. In vitro, IL13Rα2 and IL-13 bind with high affinity. Human IL-13Rα2 has a similar amino-terminal fibronectin region, four conserved cysteine residues, and a WSXWS motif whose extracellular domain is consistent. N-linked glycosylated protein is required for binding to IL-13 in cells.

[0003] Studies have shown that IL13Rα2 is associated with various diseases as follows, for example, Non-patent literature: Study on the Functional Mechanism of IL-13Rα1 and IL-13Rα2 in Atopic Dermatitis (Xiao Song, Henan University, 2021) discloses that the expression level of IL13Rα2 in the skin of healthy subjects is not high, while the expression level of IL13Rα2 in the skin of patients with atopic dermatitis is significantly increased, and the increased expression of IL13Rα2 has further been verified in the skin of atopic dermatitis model mice, Non-patent literature: Progress of IL-13Rα2 in Tumor Research (He Xiaoyan, Xiong Lixia, Medical Journal of Nanchang University, 2013, 53(01): 87-89) discloses that IL-13Rα2 is expressed in various malignant tumors, including glioblastoma, brain tumor, ovarian cancer, liver cancer, malignant mesothelioma, renal cell carcinoma, thyroid cancer, epidermoid carcinoma, AIDS-related Kaposi's sarcoma, prostate cancer, pancreatic cancer, etc., Non-patent literature: A study on a single-domain antibody against human IL-13Rα2 from the stag shark (Qin Lanyi, Zhejiang University of Science and Technology, 2021) revealed that IL13Rα2 is closely related to the development and progression of gliomas, and is specifically highly expressed in cerebral gliomas. Furthermore, it has been disclosed that the expression level of IL13Rα2 in the lung tissue of patients with idiopathic pulmonary fibrosis is significantly higher than in normal lung tissue, that IL13Rα2 deficiency can moderately protect against dextran sulfate sodium (DSS)-induced colitis, and that high expression of IL13Rα2 has also been confirmed on the surface of colon and rectal cancer cells. It has also been revealed that IL13Rα2 is an important marker for breast cancer, and that its elevated levels are a cause of breast cancer invasion. Additionally, IL13Rα2 is highly expressed on the surface of renal cell carcinoma and clear cell carcinoma, and high expression of IL13Rα2 on the surface of clear cell carcinoma not only enhances the invasive and migratory ability of cancer cells, but studies have also found that it is associated with the development of resistance to sunitinib, a standard treatment for cancer cells.

[0004] Patent document (CN116194481A) discloses the overexpression of IL13Rα2 in pancreatic cancer, ovarian cancer, melanoma, and malignant glioma.

[0005] Patent document (CN114014941A) discloses that IL13Rα2 is an IL-13 receptor polypeptide, which is not expressed in normal tissue cells except for the testes, and is almost exclusively expressed in cancer cells.

[0006] The first IL13Rα2-specific CAR-T clinical trial was completed in 2015, recruiting three patients with relapsed glioblastoma (GBM). In short, treatment in these patients was well-tolerated, with only transient encephalitis occurring. In one patient, IL13Rα2 expression in the tumor decreased with CAR-T treatment, suggesting that the treatment promoted tumor clearance. [Overview of the project] [Problems that the invention aims to solve]

[0007] This invention involves preparing and screening antibodies or their antigen-binding fragments using phage display technology. The screened antibodies or their antigen-binding fragments specifically bind to IL13Rα2, do not cross-bind to IL13Rα1, and can effectively kill tumor cells expressing IL13Rα2 and improve cytokine secretion levels. Specifically, the following applies:

[0008] In a first aspect of the present invention, an antibody or an antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment comprises CDR1, CDR2, and CDR3, and the amino acid sequence of CDR1 comprises the amino acid sequence represented by GX1X2FSX3AW (SEQ ID NO:1) or GTIFSDSF (SEQ ID NO:2), or comprises an amino acid sequence having 90% or more identity with the amino acid sequence represented by GX1X2FSX3AW (SEQ ID NO:1) or GTIFSDSF (SEQ ID NO:2). The amino acid sequence of CDR2 includes the amino acid sequence represented by ITSX4GX5TV (SEQ ID NO:3) or ITSGDITN (SEQ ID NO:4), or includes an amino acid sequence having 90% or more identity with the amino acid sequence represented by ITSX4GX5TV (SEQ ID NO:3) or ITSGDITN (SEQ ID NO:4). The amino acid sequence of CDR3 includes the amino acid sequence represented by NARPGAVNSY(SEQ ID NO:5), NSRIYTRSY(SEQ ID NO:6), or NARPRGLSFSSY(SEQ ID NO:7), or includes an amino acid sequence that has 90% or more identity with the amino acid sequence represented by NARPGAVNSY(SEQ ID NO:5), NSRIYTRSY(SEQ ID NO:6), or NARPRGLSFSSY(SEQ ID NO:7).

[0009] Preferably, the amino acid sequence of CDR1 is represented by GX1X2FSX3AW (SEQ ID NO:1) or GTIFSDSF (SEQ ID NO:2).

[0010] Preferably, the amino acid sequence of CDR2 is represented by ITSX4GX5TV (SEQ ID NO:3) or ITSGDITN (SEQ ID NO:4).

[0011] Preferably, the amino acid sequence of CDR3 is represented by NARPGAVNSY (SEQ ID NO: 5), NSRIYTRSY (SEQ ID NO: 6), or NARPRGLSFSSY (SEQ ID NO: 7).

[0012] Here, X1, X2, X3, X4, and X5 are each independently selected from either natural or non-natural amino acids. The aforementioned X1 is selected from I, S, F, T, or V, but is not limited thereto; preferably, X1 is selected from F and I. The aforementioned X2 is selected from I, T, or A, but is not limited to these, and is preferably selected from T and I. The aforementioned X3 is selected from G, N, S, V, L, A, D, Y, or I, but is not limited to these, and is preferably selected from S or G. The aforementioned X4 is selected from G, A, S, D, or T, but is not limited thereto; preferably, X4 is selected from S or G. The aforementioned X5 is selected from S, D, I, T, V, or M, but is not limited to these, and is preferably selected from D and S.

[0013] In one specific embodiment of the present invention, the amino acid sequence of CDR1 includes the amino acid sequence represented by GFTFSSAW (SEQ ID NO: 15), GTIFSDSF (SEQ ID NO: 2), or GIIFSGAW (SEQ ID NO: 17), or includes an amino acid sequence having 90% or more identity with the amino acid sequence represented by GFTFSSAW (SEQ ID NO: 15), GTIFSDSF (SEQ ID NO: 2), or GIIFSGAW (SEQ ID NO: 17). The amino acid sequence of CDR2 includes the amino acid sequence represented by ITSSGDTV (SEQ ID NO: 18), ITSGGSTV (SEQ ID NO: 19), or ITSGDITN (SEQ ID NO: 4), or includes an amino acid sequence having 90% or more identity with the amino acid sequence represented by ITSSGDTV (SEQ ID NO: 18), ITSGGSTV (SEQ ID NO: 19), or ITSGDITN (SEQ ID NO: 4). The amino acid sequence of CDR3 includes the amino acid sequence represented by NARPGAVNSY(SEQ ID NO:5), NSRIYTRSY(SEQ ID NO:6), or NARPRGLSFSSY(SEQ ID NO:7), or includes an amino acid sequence that has 90% or more identity with the amino acid sequence represented by NARPGAVNSY(SEQ ID NO:5), NSRIYTRSY(SEQ ID NO:6), or NARPRGLSFSSY(SEQ ID NO:7).

[0014] Preferably, the antibody or its antigen-binding fragment comprises one of the following groups. A) CDR1 contains GFTFSSAW (SEQ ID NO: 15), or contains an amino acid sequence having 90% or more identity with the amino acid sequence represented by GFTFSSAW (SEQ ID NO: 15); CDR2 contains ITSSGDTV (SEQ ID NO: 18), or contains an amino acid sequence having 90% or more identity with the amino acid sequence represented by ITSSGDTV (SEQ ID NO: 18); CDR3 contains NARPGAVNSY (SEQ ID NO: 5), or contains an amino acid sequence having 90% or more identity with the amino acid sequence represented by NARPGAVNSY (SEQ ID NO: 5); B) CDR1 contains GTIFSDSF (SEQ ID NO: 2), or contains an amino acid sequence having 90% or more identity with the amino acid sequence represented by GTIFSDSF (SEQ ID NO: 2); CDR2 contains ITSGGSTV (SEQ ID NO: 19), or contains an amino acid sequence having 90% or more identity with the amino acid sequence represented by ITSGGSTV (SEQ ID NO: 19); CDR3 contains NSRIYTRSY (SEQ ID NO: 6), or contains an amino acid sequence having 90% or more identity with the amino acid sequence represented by NSRIYTRSY (SEQ ID NO: 6); or C) CDR1 includes GIIFSGAW (SEQ ID NO: 17) or an amino acid sequence having 90% or more identity with the amino acid sequence represented by GIIFSGAW (SEQ ID NO: 17), CDR2 includes ITSGDITN (SEQ ID NO: 4) or an amino acid sequence having 90% or more identity with the amino acid sequence represented by ITSGDITN (SEQ ID NO: 4), and CDR3 includes NARPRGLSFSSY (SEQ ID NO: 7) or an amino acid sequence having 90% or more identity with the amino acid sequence represented by NARPRGLSFSSY (SEQ ID NO: 7).

[0015] In one specific embodiment of the present invention, the antibody or its antigen-binding fragment comprises CDR1, CDR2, and CDR3, wherein CDR1 is represented by GFTFSSAW (SEQ ID NO: 15), CDR2 is represented by ITSSGDTV (SEQ ID NO: 18), and CDR3 is represented by NARPGAVNSY (SEQ ID NO: 5).

[0016] In one specific embodiment of the present invention, the antibody or its antigen-binding fragment comprises CDR1, CDR2, and CDR3, wherein CDR1 is represented by GTIFSDSF (SEQ ID NO: 2), CDR2 is represented by ITSGGSTV (SEQ ID NO: 19), and CDR3 is represented by NSRIYTRSY (SEQ ID NO: 6).

[0017] In one specific embodiment of the present invention, said antibody or antigen-binding fragment thereof comprises CDR1, CDR2 and CDR3, wherein said CDR1 is represented by GIIFSGAW (SEQ ID NO: 17), said CDR2 is represented by ITSGDITN (SEQ ID NO: 4), and said CDR3 is represented by NARPRGLSFSSY (SEQ ID NO: 7).

[0018] Preferably, the variable regions of said antibody are in the order of CDR1, CDR2, CDR3. A framework region may further be comprised between said CDR1 and CDR2, and / or a framework region may further be comprised between said CDR2 and CDR3.

[0019] Preferably, said antibody or antigen-binding fragment thereof includes, but is not limited to, Fab, Fd, Fab', Fab'-SH, Fv, scFv, F(ab')2, single domain antibodies, diabodies (dAb), full-length antibodies, or linear antibodies.

[0020] In one specific embodiment of the present invention, said antibody or antigen-binding fragment thereof is a single domain antibody.

[0021] Said single domain antibody comprises one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions.

[0022] Preferably, the amino acid sequence of said antibody or antigen-binding fragment thereof comprises the amino acid sequence represented by any one of SEQ ID NO: 8 to 9 or 16, or comprises an amino acid sequence having at least 90% identity with the amino acid sequence represented by any one of SEQ ID NO: 8 to 9 or 16.

[0023] More preferably, the amino acid sequence of said antibody or antigen-binding fragment thereof is represented by any one of SEQ ID NO: 8 to 9 or 16.

[0024] Preferably, the antibody or its antigen-binding fragment binds to the IL13Rα2 protein.

[0025] Preferably, the IL13Rα2 protein includes human or non-human animal IL13Rα2 protein.

[0026] The non-human animal is preferably a rodent. More preferably, the rodent includes, but is not limited to, rats or mice.

[0027] A second aspect of the present invention provides nucleic acids encoding CDR1, CDR2, and CDR3 of the present invention.

[0028] A third aspect of the present invention provides a nucleic acid which encodes an antibody or an antigen-binding fragment thereof as described in any one of the first aspects described above.

[0029] Preferably, the nucleotide sequence of the nucleic acid includes a nucleotide sequence represented by any one of SEQ ID NO: 10 to 12, or includes a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by any one of SEQ ID NO: 10 to 12.

[0030] In one specific embodiment of the present invention, the nucleotide sequence of the nucleic acid is represented by one of SEQ ID NO: 10 to 12.

[0031] A fourth aspect of the present invention provides an expression vector comprising the nucleic acid described in any one of the above descriptions.

[0032] Preferably, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

[0033] More preferably, the prokaryotic expression vector is selected from the Escherichia coli expression system series vector, the Bacillus subtilis expression system series vector, and the streptomycin expression system series vector.

[0034] More preferably, the eukaryotic expression vector is selected from yeast expression system series vectors, fungal expression system series vectors, insect cell expression system series vectors, or mammalian cell expression system series vectors.

[0035] In one specific embodiment of the present invention, the expression vector is a eukaryotic expression vector.

[0036] A fifth aspect of the present invention provides a host cell.

[0037] Preferably, the host cell comprises the nucleic acid described in any one of the above clauses, and / or the expression vector described in any one of the above clauses.

[0038] Preferably, the host cell may be a eukaryotic cell or a prokaryotic cell.

[0039] A sixth aspect of the present invention provides a chimeric antigen receptor comprising an extracellular antigen-binding domain, wherein the extracellular antigen-binding domain comprises the antibody or antigen-binding fragment described in the first aspect above.

[0040] Preferably, the chimeric antigen receptor further comprises an intracellular domain, a transmembrane domain, and / or a hinge region.

[0041] Preferably, the hinge region connects the extracellular antigen-binding domain and the transmembrane domain.

[0042] Preferably, the transmembrane domain is one or more combinations selected from CD3ζ polypeptide, CD4 polypeptide, CD8 polypeptide, CD28 polypeptide, CD28-41BB polypeptide, OX40 polypeptide, ICOS polypeptide, CTLA-4 polypeptide, PD-1 polypeptide, LAG-3 polypeptide, 2B4 polypeptide, or BTLA polypeptide.

[0043] Preferably, the intracellular domain includes a signal transduction domain, preferably CD3ζ.

[0044] More preferably, the intracellular domain further comprises a co-stimulatory domain.

[0045] Preferably, the intracellular domain is one or more combinations selected from CD28, ICOS, 4-1BB, OX-40, CD27, or CD3ζ. More preferably, it is 4-1BB-CD3ζ.

[0046] Preferably, the hinge region is selected from the extracellular hinge regions of CD8, CD8α, CD28, or IgG, and is preferably CD8.

[0047] In one specific embodiment of the present invention, the structure of the chimeric antigen receptor is the above-mentioned antibody or its antigen-binding fragment -CD8HT-4-1BB-CD3ζ.

[0048] A seventh aspect of the present invention provides a method for constructing a chimeric antigen receptor as described in the sixth aspect above, the construction method being: Step A) obtaining a nucleic acid encoding the antibody or antigen-binding fragment described in the first embodiment above, Step B) involves transforming host cells with the nucleic acid obtained in Step A) to induce its expression.

[0049] Preferably, the nucleic acid is the nucleic acid described in the third aspect of the present invention.

[0050] An eighth aspect of the present invention provides immune cells that express the antibody or antigen-binding fragment described in the first aspect and / or the chimeric antigen receptor described in the sixth aspect.

[0051] Preferably, the immune cells are obtained by transfecting them with the nucleic acid described in any one of the above items.

[0052] Preferably, the immune cells include, but are not limited to, one or more of the following: lymphocytes (including T cells, B cells, and NK cells), dendritic cells, monocytes / macrophages, granulocytes, or mast cells.

[0053] In one specific embodiment of the present invention, the immune cells are CAR-T cells.

[0054] A ninth aspect of the present invention provides an immunoconjugate.

[0055] The immunoconjugate comprises an antibody or its antigen-binding fragment according to the first embodiment, or a chimeric antigen receptor according to the sixth embodiment, which is conjugated to a therapeutic agent or diagnostic reagent.

[0056] A tenth aspect of the present invention provides the use of the antibody or its antigen-binding fragment, the nucleic acid, the expression vector, the host cell, the chimeric antigen receptor, and the immune cell of the present invention in the preparation of an antibody-drug conjugate or a multifunctional antibody.

[0057] An eleventh aspect of the present invention provides a drug or pharmaceutical composition comprising one or more of the following: the antibody or its antigen-binding fragment, the nucleic acid, the expression vector, the host cell, the chimeric antigen receptor, or the immune cell.

[0058] The drug or pharmaceutical composition specifically targets IL13Rα2 cells.

[0059] Preferably, the drug or pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant.

[0060] Preferably, the pharmaceutically acceptable auxiliary material is one or more combinations selected from carriers, excipients, diluents, lubricants, wetting agents, emulsifiers, preservatives, antioxidants, buffers, antimicrobial agents, solutes that make the formulation isotonic with the recipient's blood, suspending agents, suspension aids, solubilizers, thickeners, stabilizers, sweeteners, and flavorings.

[0061] Preferably, the formulation of the drug or pharmaceutical composition may be in the form of a syrup, elixir, suspension, powder, granule, tablet, capsule, lozenge, aqueous solution, cream, ointment, lotion, gel, or emulsion. Various dosage forms of the drug can be prepared according to common production methods in the pharmaceutical field.

[0062] Preferably, the pharmaceutical preparation is a unit dose preparation.

[0063] The amount of the pharmacoactive ingredient in a unit dose formulation can be varied or adjusted from 0.001 mg to 1000 mg depending on the specific use and efficacy of the pharmacoactive ingredient.

[0064] If necessary, the pharmaceutical composition may further contain other suitable therapeutic agents.

[0065] Formulations suitable for non-gastrointestinal administration, such as intravenous, intramuscular, intradermal, and subcutaneous routes, include aqueous and non-aqueous isotonic sterile injectable preparations that may contain, for example, antioxidants, buffers, bactericides, and solutes that make the preparation isotonic with the recipient's blood, as well as aqueous and non-aqueous sterile suspensions that may contain suspension aids, solubilizers, thickeners, stabilizers, or preservatives. In the embodiment of the present invention, the drug or pharmaceutical composition can be administered, for example, by intravenous infusion, oral, topical, intraperitoneal, intravesical, and intrathecal administration. The preparations may be filled into sealed containers for single or multiple doses, such as ampoules or vials. Injectable solutions and suspensions can be prepared from the sterile powders, granules, and tablets of the types described above.

[0066] Preferably, the drug or pharmaceutical composition may be used alone or in combination with other therapeutic agents.

[0067] A twelfth aspect of the present invention provides a diagnostic kit comprising one or more of the following: the antibody or its antigen-binding fragment, the nucleic acid, the expression vector, the host cell, the chimeric antigen receptor, or the immune cell.

[0068] The aforementioned diagnostic kit diagnoses diseases associated with IL13Rα2 expression.

[0069] Preferably, the diseases associated with IL13Rα2 expression include diseases in which inhibition of IL13Rα2 expression is beneficial for treatment.

[0070] Preferably, the diseases associated with IL13Rα2 expression include diseases in which IL13Rα2 is expressed, highly expressed, or overexpressed.

[0071] Preferably, the diseases associated with IL13Rα2 expression include, but are not limited to, dermatitis, lung disease, intestinal disease, and tumors.

[0072] More preferably, the dermatitis includes, but is not limited to, atopic dermatitis.

[0073] More preferably, the lung disease includes, but is not limited to, pulmonary fibrosis (preferably idiopathic pulmonary fibrosis) or a lung tumor.

[0074] More preferably, the intestinal disease includes colitis (preferably dextran sulfate sodium-induced colitis) or a tumor of the intestine.

[0075] More preferably, the tumor includes, but is not limited to, lymphoma, lung cancer, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, liver cancer, brain tumor, rectal cancer, stomach cancer, bladder cancer, glioma (e.g., glioma), lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma, and the leukemia is one or more selected from acute lymphoblastic leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, or chronic myeloid leukemia.

[0076] More preferably, the lymphoma includes Hodgkin lymphoma and non-Hodgkin lymphoma, and includes one or more of the following: B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, or Waldenström macroglobulinemia.

[0077] More preferably, the sarcoma includes one or more of the following: osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, or chondrosarcoma.

[0078] In one specific embodiment of the present invention, the tumor is one or more selected from glioma, brain tumor, ovarian cancer, liver cancer, malignant mesothelioma, kidney cancer, thyroid cancer, epidermal carcinoma, Kaposi's sarcoma, prostate cancer, pancreatic cancer, breast cancer, or melanoma, but is not limited to these.

[0079] The aforementioned gliomas include, but are not limited to, glioblastomas and gliomas.

[0080] The aforementioned kidney cancer includes, but is not limited to, renal cell carcinoma or clear cell renal carcinoma.

[0081] The Kaposi's sarcoma, prostate cancer, and pancreatic cancer mentioned above are preferably those associated with AIDS.

[0082] In one specific embodiment of the present invention, the tumor includes melanoma and glioma.

[0083] A thirteenth aspect of the present invention provides the use of the antibody or its antigen-binding fragment, the nucleic acid, the expression vector, the host cell, the chimeric antigen receptor, or the immune cell.

[0084] Preferably, the use is in the preparation of products for diagnosing and / or treating diseases associated with IL13Rα2 expression.

[0085] More preferably, the diseases associated with IL13Rα2 expression include diseases in which inhibition of IL13Rα2 expression is advantageous for treatment.

[0086] More preferably, the diseases associated with IL13Rα2 expression include diseases in which IL13Rα2 is expressed, highly expressed, or overexpressed.

[0087] Preferably, the product includes, but is not limited to, a diagnostic kit, a drug, or a pharmaceutical composition.

[0088] Preferably, the diseases associated with IL13Rα2 expression include, but are not limited to, dermatitis, lung disease, intestinal disease, and tumors.

[0089] More preferably, the dermatitis includes, but is not limited to, atopic dermatitis.

[0090] More preferably, the lung disease includes, but is not limited to, pulmonary fibrosis (preferably idiopathic pulmonary fibrosis) or a lung tumor.

[0091] More preferably, the intestinal disease includes, but is not limited to, colitis (preferably dextran sulfate sodium-induced colitis) or intestinal tumors.

[0092] More preferably, the tumor is selected from lymphoma, lung cancer, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, liver cancer, brain tumor, rectal cancer, stomach cancer, bladder cancer, glioma, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma, and the leukemia includes, but is not limited to, one or more of acute lymphoblastic leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, or chronic myeloid leukemia.

[0093] More preferably, the lymphoma includes Hodgkin lymphoma and non-Hodgkin lymphoma, and includes one or more of the following: B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, or Waldenström macroglobulinemia.

[0094] More preferably, the sarcoma includes one or more of the following: osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, or chondrosarcoma.

[0095] In one specific embodiment of the present invention, the tumor is one or more selected from glioma, brain tumor, ovarian cancer, liver cancer, malignant mesothelioma, kidney cancer, thyroid cancer, epidermal carcinoma, Kaposi's sarcoma, prostate cancer, pancreatic cancer, breast cancer, or melanoma, but is not limited to these.

[0096] The aforementioned gliomas include, but are not limited to, glioblastomas and gliomas.

[0097] The aforementioned kidney cancer includes renal cell carcinoma or clear cell renal carcinoma.

[0098] The Kaposi's sarcoma, prostate cancer, and pancreatic cancer mentioned above are preferably those associated with AIDS.

[0099] In one specific embodiment of the present invention, the tumor includes melanoma and glioma.

[0100] A fourteenth aspect of the present invention provides a method for treating diseases related to IL13Rα2 expression.

[0101] Preferably, the method comprises administering the antibody or its antigen-binding fragment, the chimeric antigen receptor, or the immune cells of the present invention to a subject in need.

[0102] In one specific embodiment of the present invention, the immune cells are CAR-T cells.

[0103] Preferably, for the treatment of diseases related to IL13Rα2 expression, CAR-T cells in concentrations of 1.0E+05 cells / mL to 1.0E+08 cells / mL are used, for example, 1.0E+05 cells / mL, 3.0E+05 cells / mL, 5.0E+05 cells / mL, 6.0E+05 cells / mL, 7.0E+05 cells / mL, 8.0E+05 cells / mL, 9.0E+05 cells / mL, 1.0E+06 cells / mL, 3.0E+06 cells / mL, 4.0E+06 cells / mL, 5 This includes the use of 0.0E+06 particles / mL, 6.0E+06 particles / mL, 7.0E+06 particles / mL, 8.0E+06 particles / mL, 9.0E+06 particles / mL, 1.0E+07 particles / mL, 2.0E+07 particles / mL, 3.0E+07 particles / mL, 4.0E+07 particles / mL, 5.0E+07 particles / mL, 6.0E+07 particles / mL, 7.0E+07 particles / mL, 8.0E+07 particles / mL, 9.0E+07 particles / mL, and 1.0E+08 particles / mL.

[0104] Preferably, the treatment of the disease associated with IL13Rα2 expression includes the use of CAR-T cells, more preferably CAR-T cells, where the target cells are 1 / 10, 1 / 5, 1 / 3, 1 / 2, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0105] A fifteenth aspect of the present invention provides a method for detecting IL13Rα2, the method comprising contacting the above-mentioned antibody or its antigen-binding fragment, the above-mentioned chimeric antigen receptor, or the above-mentioned immune cells with a sample to be detected.

[0106] The sample to be detected includes, but is not limited to, body fluids, cells, tissues, or organs. Preferably, it is blood, serum, plasma, or tumor cells.

[0107] Preferably, the method further comprises adding a labeling substance, more preferably, the labeling substance including, but not limited to, a radioactive marker, a fluorescent marker, or a reporter cell.

[0108] In one specific embodiment of the present invention, the method includes contacting a sample to be detected with a detectable amount of the above-mentioned antibody or its antigen-binding fragment, the above-mentioned chimeric antigen receptor, or the above-mentioned immune cells, and adding a labeling substance.

[0109] Preferably, the detectable amount is at least 0.1 μg / mL, for example, 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, 0.5 μg / mL, 0.6 μg / mL, 0.7 μg / mL, 0.8 μg / mL, 0.9 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL or more.

[0110] The terms "contains" or "composes" as used in this invention are open expressions and include certain components or steps described, as well as other certain components or steps that do not have a substantial effect. When used to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may consist of the sequence, and the protein or nucleic acid may have additional amino acids or nucleotides at one or both ends, but will have the same or similar activity as the original sequence.

[0111] In this invention, the term "and / or" encompasses all combinations of items linked by that term, and each combination should be considered as individually listed herein. For example, "A and / or B" includes "A", "A and B", and "B". Also, for example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C", and "A, B and C".

[0112] The “treatment” of the present invention refers to alleviating, interrupting, preventing, controlling, stopping, reducing, or reversing signs, symptoms, impairments, conditions, or the progression or severity of a disease after it has begun to progress, but does not necessarily mean completely eliminating all signs, symptoms, conditions, or impairments associated with the disease.

[0113] In this invention, the term "diagnosis" refers to determining whether a patient has had a disease or condition in the past, at the time of diagnosis, or in the future, or determining the progression or potential for future progression of a disease.

[0114] In this invention, "pharmaceutically acceptable" means that it does not significantly irritate the organism or inhibit the biological activity and properties of the active substance in the administered product.

[0115] The "antigen-binding fragment" of the present invention refers to a portion of an antibody that retains the specific binding activity of the antibody, that is, any portion of the antibody can specifically bind to an epitope on the antibody's target molecule. This includes, for example, Fab, Fv, Fd, Fab', Fab'-SH, F(ab')2, and variants of these fragments. For example, the heavy and / or light chain of an antibody, the variable region of the heavy and / or light chain of an antibody, or one or more CDRs derived from the heavy or light chain of an antibody. Of these, Fab is a monovalent fragment consisting of VL, VH, CL, and CH1 domains. F(ab')2 is a bivalent fragment containing two Fab fragments linked by a disulfide bond in the hinge region. Fd is an Fd fragment consisting of a VH domain and a CH1 domain. Fv is an Fv fragment consisting of a VL domain and a VH domain of a single arm of an antibody. Fab' is a Fab fragment having one or more cysteine ​​residues at the C-terminus of the CH1 domain. Fab'-SH is a Fab' that has at least one free thiol at a cysteine ​​residue in its constant domain. Single-domain antibodies are antibodies that lack an antibody light chain and only have a heavy chain, and are also called nanobodies because of their small molecular weight. Here, VH represents the variable region of the heavy chain, VL represents the variable region of the light chain, CL represents the light chain, and CH1 represents the CH1 that constitutes the constant region of the heavy chain.

[0116] In this invention, the "non-natural amino acid" refers to a compound containing an amino group and a carboxyl group that do not naturally exist in proteins. Preferably, the non-natural amino acid is any non-natural amino acid known in the art. More preferably, the non-natural amino acids include, but are not limited to, N-ethyl asparagine, hydroxylysine, 3-hydroxyproline, 2-aminobutyric acid, β-alanine, β-aminopropionic acid, 2-aminoadipic acid, 3-aminoadipic acid, 4-aminobutyric acid, 6-aminohexanoic acid, 2-aminoheptanoic acid, allo-isoleucine, heterochain lysine, 4-hydroxyproline, allo-hydroxylysine, 2-aminoisobutyric acid, N-methylglycine, N-methylisoleucine, 3-aminoisobutyric acid, 6-N-methyllysine, 2,4-diaminobutyric acid, N-methylvaline, ornithine, norleucine, n-valine, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, or 2-aminopimelic acid. Of course, the "non-natural amino acids" in this application may also be modified derivatives of natural amino acids.

[0117] In this invention, the term "CDR" refers to the complementarity-determining region within the antibody variable sequence. Each variable region has three CDRs, one in the heavy chain and / or light chain, which are referred to as CDR1, CDR2, and CDR3. The precise boundaries of these CDRs are defined differently depending on the system. These systems include the Kabat, Chothia, IMGT, AbM, and Contact systems, and the CDRs described herein can be defined based on any of these systems. [Brief explanation of the drawing]

[0118] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. [Figure 1] This is the detection result for the purified antibody. [Figure 2] This represents the average fluorescence intensity of six groups: 1-E9, 1-E10, 2-G01, 1-C10, 10-C4, and 7-G8. [Figure 3]This represents the average fluorescence intensity of the three groups: 4-C2, 4-F10, and 7-F2. [Figure 4] The invention describes the toxic effects of the single-domain antibodies screened in this invention on 293T, 293T-IL13Rα2, A375, and U251 cells, where T represents T cells, G01 represents CAR-T cells prepared with the 2-G01 single-domain antibody, C2 represents CAR-T cells prepared with the 4-C2 single-domain antibody, F10 represents CAR-T cells prepared with the 4-F10 single-domain antibody, and EQ represents the positive control. [Figure 5] The effects of the single-domain antibodies screened in this invention on IFN-γ cytokine release concentrations in 293T, A375, and U251 cells, where T represents T cells, G01 represents CAR-T cells prepared with 2-G01 single-domain antibody, C2 represents CAR-T cells prepared with 4-C2 single-domain antibody, F10 represents CAR-T cells prepared with 4-F10 single-domain antibody, EQ represents positive control, and Alone represents spontaneous release of effector cells. The bar graphs for each group are arranged from left to right in the order of T, EQ, G01, C2, and F10. [Figure 6] The effects of the single-domain antibodies screened in this invention on IL-2 cytokine release concentrations in 293T, A375, and U251 cells, where T represents T cells, G01 represents CAR-T cells prepared with 2-G01 single-domain antibody, C2 represents CAR-T cells prepared with 4-C2 single-domain antibody, F10 represents CAR-T cells prepared with 4-F10 single-domain antibody, EQ represents positive control, and Aron represents spontaneous release of effector cells. The bar graphs for each group are arranged from left to right in the order of T, EQ, G01, C2, and F10. [Figure 7] The effect of the single-domain antibody screened in this invention on tumor volume, where G01 represents CAR-T cells prepared with the 2-G01 single-domain antibody. [Figure 8]The mean tumor volume of mice subcutaneously injected with A375 in each dose treatment group, where G01 represents CAR-T cells prepared with the 2-G01 single-domain antibody, and **** represents p<0.0001. [Figure 9] This represents the body weight of tumor-bearing mice in each treatment group, and G01 represents CAR-T cells prepared with the 2-G01 single-domain antibody. [Figure 10] This involves optimizing the concentration of the 2-G01 antibody for screening. [Figure 11] This involves optimizing the concentration of the 4-F10 antibody for screening. [Figure 12] This is the screening result of the 2-G01 antibody using a membrane protein screening array. [Figure 13] This is the screening result of the 4-F10 antibody using a membrane protein screening array. [Figure 14] The binding indices of 2-G01 antibodies at different concentrations are shown as the binding indices for the top 20 targets. [Figure 15] The binding indices of 4-F10 antibodies at different concentrations are shown as the binding indices for the top 20 targets. [Modes for carrying out the invention]

[0119] The following describes the technical solutions in embodiments of the present invention clearly and completely with reference to the drawings, and it is clear that the embodiments described are only a part of the present invention, not all of it. All other embodiments that can be obtained without creative work by those skilled in the art based on the embodiments of the present invention are all within the scope of the protection of the present invention.

[0120] Some of the methods used in embodiments of the present invention:

[0121] Method for preparing CAR-T cells:

[0122] (1) Cell preparation: PBMCs isolated from peripheral blood were activated with CD3 / CD28 magnetic beads and infected with lentivirus after 24 hours. (2) Lentivirus infection: According to the requirements of the experimental design, collect an appropriate amount of activated T cells, centrifuge at room temperature and 300 g for 5 minutes, discard the supernatant, resuspend the T cells in the culture medium, add an appropriate amount of lentivirus concentrate (viral amount determined according to viral titer and MOI), and gently pipette to obtain a final cell concentration of 2.0E+10 6 The solution was diluted to / mL and incubated overnight in a 5% CO2 incubator to induce infection. (3) After 24 hours of lentivirus infection, the culture medium was changed and the amplification culture of CAR T cells was continued. Cell count and cell infection efficiency were observed. (4) Cell toxicity detection was performed 10 days after lentivirus infection.

[0123] Example 1: IL13Rα2 single-domain antibody development and phage display library screening

[0124] 1. After immunizing alpacas, immunoassay titers were detected using ELISA. The results are shown in Table 1 below. Once a certain titer was reached, peripheral blood was collected from the alpacas, PBMC cells were isolated, and an antibody phage library was constructed.

[0125] 2. Panning of the phage display library Panning was performed using the IL13Rα2 recombinant protein, and then the phage display library was incubated with the recombinant protein. Recombinant phages bound to the target antigen were eluted with TEA and amplified. After 3-4 panning cycles, monoclones were selected and sequenced.

[0126] [Table 1]

[0127] Recombinant proteins of the target antigen were co-incubated with a single-domain antibody phage display library, and phages that specifically bind to the target antigen were enriched. This enrichment was performed four times. The input and output for each enrichment were statistically analyzed, and the enrichment factor for each enrichment was calculated. The results are shown in Table 2 below.

[0128] [Table 2]

[0129] 3. Expression and purification of candidate single-domain antibodies Bacterial suspension of the target clone was collected and amplified by PCR, and the PCR product was transiently transfected into CHO-K1 cells. The supernatant of the expressed antibody was detected by flow cytometry fluorescence sorting (FACS). Thirteen different clones that specifically bind to IL13Rα2—1-E9, 1-E10, 1-C10, 2-A01, 2-G01, 4-F10, 4-C2, 4-D6, 5-A4, 10-C4, 6-A10, 7-F2, and 7-G8—were selected, and eukaryotic expression vectors were constructed. The detection results of the purified antibodies are shown in Figure 1. Of these, 6-A10 was excluded because no band was observed. The remaining 12 clones were used as candidate single-domain antibodies.

[0130] 4. Serial dilution and detection of candidate single-domain antibodies by FACS Detection was performed using CHO-K1 / IL13Rα2 cells. After adding purified candidate single-domain antibodies and setting appropriate starting concentrations, the cells were diluted 11 times with a 3-fold gradient, and FACS detection was performed using secondary antibodies. Examples of average fluorescence intensity are shown in Figures 2-3, and examples of EC50 (μg / mL) for each antibody are shown in Table 3 below.

[0131] [Table 3]

[0132] Results Analysis: All of the candidate single-domain antibodies listed above bound to CHO-K1 / IL13Rα2 cells. All other candidate antibodies, except for the 10-C4 candidate antibody, bound strongly to CHO-K1 / IL13Rα2 cells.

[0133] 5. FACS detection of cells transiently transfected with candidate single-domain antibodies and IL13Rα1. The results showed that none of the candidate single-domain antibodies mentioned above cross-bound to 293F / IL13Rα1 cells. Furthermore, after analyzing and verifying the above sequences, we conducted further research on the three key sequence antibodies shown below: 2-G01, 4-F10, and 4-C2. Using the EQ antibody as a positive control, its sequence is as follows.

[0134] Nucleotide sequence encoding 2-G01: CAGGTACAGCTGAAAGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGTAGCCTCTGGATTCACATTCAGTAGCGCGTGGATGGGCTGGTACCGCCAGGCTGCAGGGAAGCAGCGCGAATTGTTCGCATCTATTACTAGTAGTGGTGACACAGTCTA TGCAGGCTCCGTGAAGGGCCGATTCTCCATCTCTAAGAACAACGCCGAGAACACTCTTTATCTGCAAATGGATGCGCTGAGGCCTGAAGATACAGCCGTCTATTACTGCAATGCCCGTCCGGGGGCGGTCAATAGTTACTGGGCCAGGGGGACCCAGGTCACCGTCTCCACA(SEQ ID NO:10); Nucleotide sequence encoding 4-C2: CAGTTGCGGCTCTTGGAGTCTGGGGGAGGCCTGGTGCAGGCTGGGGAGTCTCTGAGACTCTCCTGTGCTGTCTCTGGAACCATCTTTAGTGACAGTTTCCTGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGATCGCAAGTATTACTAGTGGTGGTAGCACAGTT TATGCAGGTCCGTAAAGGGCCGATTCACCATCTCCAGAGACAACGCCCAGGACAGGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTCTATTACTGTAATTCACGCATCTATACTCGATCCTACTGGGGCCAGGGGACCCAAGTCACCGTCTCTTCT(SEQ ID NO:11); 4-Nucleotide sequence encoding F10: CAGGTGCAGCTGGTAGAGTCTGGGGGCGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAATCATCTTCAGTGGCCGTGGATGGGCTGGTACCGCCAGGCTGCAGGCGAGCAGCGCGAGTTGGTCGCATCTATTACTAGTGGTGATATCACAAACTATGC AGACTCCGTGAAGGGCCGATTCACCATCTCTAGAGACAACGCCGAAAACAGGGCTATCTGCAAATGAATACGCTGAAGCCTGAGGATACAGCCGTCTTACTGTAAGCCCCGTCTAGGGGTTTAAGTTTCAGTTCTTACTGGGGCCAGGGGACCCTGGTCACCGTCTCCTCA(SEQ ID NO:12); Nucleic acid sequence that encodes EQ antibodies: GGCCCCGTGCCCCCTAGCACCGCCCTGCGCTACCTGATCGAGGAACTGGTGAACATCACCCAGAACCAGAAAGCCCCCCTGTGCAACGGCAGCATGGTGTGGAGCATCAACCTGACCGCCGGCATGTACTGTGCCGCCCTGGAAAGCCTGATCAACGTGAGCGGCTGCAG CGCCATCGAGAAAACCCAGCGGATGCTGTCCGGCTTCTGCCCCCACAAGGTGTCCGCCGGACAGTTCAGCAGCCTGCACGTGCGGGACACCAAGATCGAGGTGGCCCAGTTCGTGAAGGACCTGCTGCTGCACCTGAAGAAGCTGTTCCGGGAGGGCCGGTTCAAC(SEQ ID NO:13); Amino acid sequence of 2-G01: QVQLKESGGGLVQAGGSLRLSCVASGFTFSSAWMGWYRQAAGKQRELFASITSSGDTVYAGSVKGRFSISKNNAENTLYLQMDALRPEDTAVYYCNARPGAVNSYWGQGTQVTVST(SEQ ID NO:16); 4-C2 amino acid sequence: QLRLLESGGGLVQAGESLRLSCAVSGTIFSDSFLGWYRQAPGKQRELIASITSGGSTVYAGSVKGRFTISRDNAQDTVYLQMNSLKPEDTAVYYCNSRIYTRSYWGQGTQVTVSS(SEQ ID NO:8); Amino acid sequence of 4-F10: QVQLVESGGGLVQAGGSLLSCAASGIIFSGAWMGWYRQAAGEQRELVASITSGDITNYADSVKGRFTISRDNAENTGYLQMNTLKPEDTAVYYCNARPRGLSFSSYWGQGTLVTVSS(SEQ ID NO:9); Amino acid sequence of EQ antibody: GPVPPSTALRYLIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN(SEQ ID NO:14).

[0135] Example 2: Verification of antibody efficacy by in vitro cell killing experiment

[0136] 1. Target cells 293T, 293T-IL13Rα2, A375 (human malignant melanoma cells), and U251 (human glioma cells) were individually counted, collected, centrifuged at room temperature at 300g for 5 minutes, and the supernatant was discarded. 2. The cells were resuspended in the culture medium and centrifuged at room temperature at 300g for 5 minutes. 3. Discard the supernatant and repeat step 2. 4. The cells were resuspended in an appropriate amount of culture medium, and the cell density was statistically measured. 5. Preparation of CAR-T cells: The CAR structure is an antibody sequence (the antibody sequence is 2-G01, 4-C2, or 4-F10, which were screened in Example 1)-CD8HT-4-1BB-CD3ζ structure. CAR-T cells were prepared using the method described above. 6. CAR-T cells were collected, centrifuged at 300g for 5 minutes, and the supernatant was discarded. 7. The cells were resuspended in an appropriate amount of culture medium, samples were taken and counted, and the cells were centrifuged at room temperature at 300g for 5 minutes. 8. Discard the supernatant and repeat step 7. 9. CAR-T cells were resuspended in PBS and the cell density was statistically measured. Table 4 shows information on exemplary CAR-T cells prepared with the 2-G01 antibody.

[0137] [Table 4] 96-well cell culture plates (purchased from Corning, catalog number: 3599) were prepared, and CAR-T / NC cells and target cells were co-incubated. The effector / target ratios of target cells per well and effective CAR-T / NC cells were 5:1, 3:1, 1:1, and 1:2, with a total volume of less than 100 μL. Detection was performed by co-incubating at 10.37°C. 11. Cell killing efficiency was detected. 12. The experimental data was analyzed and plotted to express the experimental results and conclusions.

[0138] As a result, as shown in Figure 4, the three single-domain antibodies of the present invention did not have a lethal effect on 293T cells, but they did have a lethal effect on 293T cells, A375 cells, and U251 cells expressing IL13Rα2, with the lethal effect being strongest when the effector / target ratio (E:T) was 5:1.

[0139] Example 3: Cytokine Detection

[0140] 1. Target cells 293T, A375, and U251 were counted individually, collected, centrifuged at room temperature at 300g for 5 minutes, and the supernatant was discarded. 2. The cells were resuspended in the culture medium and centrifuged at room temperature at 300g for 5 minutes. 3. Discard the supernatant and repeat step 2. 4. The cells were resuspended in an appropriate amount of culture medium, and the cell density was statistically measured. 5. CAR-T cells were collected, centrifuged at 300g for 5 minutes, and the supernatant was discarded (the preparation of CAR-T cells was the same as in step 5 of Example 2). 6. The cells were resuspended in an appropriate amount of culture medium, samples were taken and counted, and the cells were centrifuged at room temperature at 300g for 5 minutes. 7. Discard the supernatant and repeat step 6. 8. CAR-T cells were resuspended in PBS and the cell density was statistically measured. A 96-well cell culture plate was prepared, and CAR-T / NC cells and target cells were co-incubated. The effector / target ratio of target cells per well to effective CAR-T / NC cells was 1:1, and the total volume was less than 200 μL. After co-incubation at 9.37°C, the cell supernatant was collected, detected using ELISA, and the data was analyzed to obtain the results. As a result, as shown in Figures 5-6, the use of the single-domain antibody of this application significantly improved the release concentrations of cytokines IFN-γ and IL-2.

[0141] Example 4: Antitumor activity of IL-13Rα2 VHH CAR T cells in an A375 homologous mouse melanoma model.

[0142] Cell Culture: A375 cells (CL-0014, Procell) were rapidly thawed in a 37°C water bath, resuspended in RPMI-1640 (purchased from Gibco, catalog number: C11875500BT) containing 10% fetal bovine serum (purchased from ExCell Bio, catalog number: FSP500) and 100 μg / mL penicillin-streptomycin solution (purchased from Procell, catalog number: PB180120, 100 μg / mL penicillin and 100 μg / mL streptomycin), and subcultured once every two days in a 37°C, 5% CO2 incubator (purchased from Thermo Scientific, catalog number: 371). A375 cells were seeded in 150 mm culture dishes (purchased from NEST, catalog number: 715001) with a cell count of 2 × 10⁶. 7 Cells were collected when the number reached one per plate.

[0143] Preparation of CAR-T cell reinfusion solution: CAR-T cells prepared using cryopreserved T cells and 2-G01 antibody (hereinafter referred to as G01 CAR-T cells) were removed from liquid nitrogen, thawed in a 37°C water bath, and transferred to a centrifuge tube containing PBS for centrifugation (centrifuge purchased from Eppendorf, catalog number: 5804R). The supernatant was discarded and the cells were resuspended in PBS. The number of cells was counted (cell counter purchased from Invitrogen, catalog number: Countess II). The T cell concentration was adjusted to 5.0E+07 cells / mL, and the effective cell concentrations of G01 CAR-T cells were adjusted to 1.0E+06 cells / mL, 3.0E+06 cells / mL, and 1.0E+07 cells / mL.

[0144] Construction of an A375 tumor mouse model: 2 × 10⁶ tumor cells per mouse. 6 The drug was subcutaneously inoculated into 30 NCG mice (purchased from Jiangsu Jiecui Yaokang Biotechnology Co., Ltd., animal production license number: SCXK(Su)2018-0008) at a dose of one cell. Four days after inoculation, the tumor volume (approximately 30 mm) was measured. 3 Mice with relatively uniform characteristics were randomly divided into five groups as shown in Table 5. Group A1 (n=6): 100 μL of PBS was reinjected into the tail vein of each mouse as a solvent control. Group A2 (n=6): 100 μL of T cells at 5.0E+07 cells / mL was reinjected into the tail vein of each mouse. Group A3 (n=6): 100 μL of effective G01 CAR-T cells at 1.0E+06 cells / mL was reinjected into the tail vein of each mouse. Group A4 (n=6): 100 μL of G01 CAR-T cells at 3.0E+06 cells / mL was reinjected into the tail vein of each mouse. Group A5 (n=6): 100 μL of G01 CAR-T cells at 1.0E+07 cells / mL was reinjected into the tail vein of each mouse. Tumor volume was measured twice a week using calipers. Tumor volume calculation formula: Tumor volume = (Tumor long axis length) × (Tumor short axis length) 2 The tumor inhibition rate was calculated using the formula: / 2. Tumor inhibition rate = (average tumor volume of the treatment group - average tumor volume of the control group) / average tumor volume of the control group × 100%. Statistical comparison of tumor growth was performed using two-way ANOVA. In addition, the body weight of mice in each group was measured twice a week.

[0145] [Table 5]

[0146] Data Analysis: Statistical analysis was performed using Graph Pad 10. Experimental data are presented as mean ± standard deviation (Mean ± SD), and the data were tested using the F-test and two-sample ANOVA. If p < 0.05, the calculation was performed using the two-sample heteroscedasticity hypothesis t-test. If p > 0.05, the calculation was performed using the equal variance hypothesis t-test (test criterion α = 0.05, two-tailed test).

[0147] Experimental Results: Compared to the control group, reinjection of G01 CAR-T cells into groups A4 and A5 significantly inhibited tumor growth. Tumor inhibition rates were 14.80% in group A2, 8.36% in group A3, 67.59% in group A4, and 99.89% in group A5. In group A4, tumors in two mice almost completely disappeared 14 days after G01 CAR-T cell reinjection, but tumors in the remaining four mice grew slowly. Tumor growth was significantly inhibited compared to group A2. In group A5, tumors in six mice almost completely disappeared 14 days after G01 CAR-T cell reinjection. Tumor growth was significantly inhibited compared to group A2, with an inhibition rate of nearly 100% (see Figures 7-8).

[0148] Furthermore, when the body weight of the five groups of mice was detected, no significant difference in body weight was observed among the five groups (see Figure 9). This indicates that the use of CAR-T cells described in this application is safe.

[0149] As described above, IL-13Rα2 CAR-T cells showed a significant tumor-inhibiting effect in A735 melanoma model mice. A significant tumor-inhibiting effect was observed when the effective cell reinjection rate reached 3.0E+06 cells / mouse, and the tumor inhibition rate approached 100% when the effective cell reinjection rate reached 1.0E+07 cells / mouse. Furthermore, there was no effect on the body weight of the individual mice.

[0150] Example 5: Verification of antibody binding specificity to IL-13Rα2 using a membrane protein screening array.

[0151] The binding specificity of membrane proteins (receptors, transporters, enzymes, etc.) from 4032 humans to the antibodies screened in Example 1 was investigated using the AB5000 membrane protein screening array. The steps were as follows:

[0152] 1) Test setup: The optimal concentration of the test sample for screening was determined.

[0153] To determine the optimal sample concentration used in the targeted screening phase, target cells were transfected into 96-well plates with a sample-binding target gene (positive) vector and an empty vector (negative control). After transfection, the samples were diluted with a 3.16-fold gradient to the highest concentration of 20 μg / ml, establishing a total of nine detection concentrations. The samples were added to culture plates along with the same number of reporter cells as the target cells and co-incubated. Fluorescence signals were collected using a fluorescence microplate reader. The optimal screening concentration of the test samples was determined by binding index to achieve low background and high sensitivity. The optimal screening concentration was determined to be 0.1 μg / mL (Figures 10-11) and used in subsequent experiments.

[0154] 2) Membrane protein screening array: Test samples were screened using the membrane protein screening array AB5000 to determine the binding target.

[0155] To identify binding targets, 4032 human membrane proteins were expressed in HEK-293T cells placed in individual wells of a 96-well plate. After culturing, the test samples and reporter cells were added to transfected cells. The optimal screening concentration of the test samples was 0.1 μg / mL. Fluorescence signals were collected using a fluorescence microplate reader. The resulting binding values ​​were then normalized and converted to obtain a single numerical value (i.e., binding index) of the binding of the test samples to each target protein.

[0156] See Figures 12-13 for the results of membrane protein screening arrays using the 2-G01 antibody and the 4-F10 antibody.

[0157] Taking the top 20 targets by binding index as an example, the target information and binding index are as follows:

[0158] 2-G01 antibodies: IL13Rα2 (UniPort ID: Q14627, binding index: 38.49), CACNG3 (UniPort ID: O60359, binding index: 2.10), GOLM2 (UniPort ID: Q6P4E1, binding index: 2.10), CSF1R (UniPort ID: P07333, binding index: 2.08), RIGI (UniPort ID: O95786, binding index: 2.03), ICOS (UniPort ID: Q9Y6W8, binding index: 2.02), GDPD5 (UniPort ID: Q8WTR4, binding index: 2.02), IGSF5 (UniPort ID: Q9NSI5, binding index: 2.01), GFRA1 (UniPort ID: P56159, binding index: 2.00), MUSK (UniPort... (UniPort ID: O15146, binding index: 1.99), TNFSF9 (UniPort ID: P41273, binding index: 1.98), GPM6A (UniPort ID: P51674, binding index: 1.97), FGFR4 (UniPort ID: P22455, binding index: 1.96), ASGR1 (UniPort ID: P07306, binding index: 1.96), ILDR1 (UniPort ID: Q86SU0, binding index: 1.95), ROR1 (UniPort ID: Q01973, binding index: 1.95), CALCRL (UniPort ID: Q16602, binding index: 1.94), OR2B3 (UniPort ID: O76000, binding index: 1.92), SMO ... ID:Q99835, Binding Index: 1.92), RNF19A (UniPort ID:Q9NV58, Binding Index: 1.91). 4-F10 antibody: IL13Rα2 (UniPort ID: Q14627, binding index: 24.82), DDX58 (UniPort ID: O95786, binding index: 2.48), GPM6A (UniPort ID: P51674, binding index: 2.4), OR4A47 (UniPort ID:Q6IF82, Coupling index: 2.22), CLEC10A (UniPort ID: Q8IUN9, Coupling index: 2.15), C2CD2L (UniPort ID: O14523, Coupling index: 2.03), SELL (UniPort ID: P14151, Coupling index: 1.99), OR1D5 (UniPort ID:P58170, binding index: 1.95), GPR33 (UniPort ID:Q49SQ1, binding index: 1.93), CDHR5 (UniPort ID:Q9HBB8, Bonding index: 1.93), ANKK1(UniPort ID:Q8NFD2, Bonding index: 1.91), CPNE7(UniPort ID:Q9UBL6, Bonding index: 1.90), KCNH4(UniPort ID:Q9UQ05, Bonding index: 1.89), FAM189A2(UniPort ID:Q15884, bonding index: 1.89), DPY19L3(UniPort ID:Q6ZPD9, bonding index: 1.86), PRIMA1(UniPort ID:Q86XR5, bonding index: 1.83), CYYR1(UniPort ID:Q96J86, bonding index: 1.82), PTPRK(UniPort ID:Q15262, Coupling index: 1.82), DIRAS1(UniPort ID:O95057, Coupling index: 1.81), CHIC1(UniPort ID:Q5VXU3, bonding index: 1.76).

[0159] 3) Target validation: The newly identified targets were validated using reporter cells.

[0160] To determine the optimal sample concentration used in the targeted screening phase, target cells were transfected into 96-well plates with a sample-binding target gene (positive) vector and a blank vector (negative control). After transfection, the samples were diluted with a 3.16-fold gradient to the highest concentration of 20 μg / ml, establishing a total of nine detection concentrations. The samples were added to culture plates along with the same number of reporter cells as the target cells and co-incubated. Fluorescence signals were collected using a fluorescence microplate reader.

[0161] The results are shown in Figures 14-15.

[0162] As described above, the antibodies screened in this application specifically bind to IL13Rα2 and can be used for the detection of the IL13Rα2 protein.

[0163] Although preferred embodiments of the present invention have been described in detail above, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical idea of ​​the present invention, various simple modifications can be made to the technical solution means of the present invention, and all of these simple modifications fall within the scope of protection of the present invention.

[0164] Furthermore, each specific technical feature described in the above specific embodiments can be combined in any suitable manner, provided they do not contradict each other. To avoid unnecessary redundancy, the present invention will not separately describe various possible combinations.

Claims

1. An antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is GX １ X ２ FSX ３ It contains an amino acid sequence indicated by AW (SEQ ID NO: 1) or GTIFSDSF (SEQ ID NO: 2), or GX １ X ２ FSX ３ It contains an amino acid sequence that has 90% or more identity with the amino acid sequence indicated by AW (SEQ ID NO: 1) or GTIFSDSF (SEQ ID NO: 2), The amino acid sequence of said CDR2 is ITSX ４ GX ５ TV (SEQ ID NO: 3) or comprises the amino acid sequence represented by ITSGDITN (SEQ ID NO: 4), or ITSX ４ GX ５ TV (SEQ ID NO: 3) or comprises an amino acid sequence having at least 90% identity with the amino acid sequence represented by ITSGDITN (SEQ ID NO: 4), The amino acid sequence of CDR3 includes the amino acid sequence represented by NARPGAVNSY (SEQ ID NO: 5), NSRIYTRSY (SEQ ID NO: 6), or NARPRGLSFSSY (SEQ ID NO: 7), or includes an amino acid sequence having 90% or more identity with the amino acid sequence represented by NARPGAVNSY (SEQ ID NO: 5), NSRIYTRSY (SEQ ID NO: 6), or NARPRGLSFSSY (SEQ ID NO: 7). Here, X １ It is selected from I, S, F, T, or V. X ２ It is selected from I, T, or A. X ３ It is selected from G, N, S, V, L, A, D, Y, or I. X ４ It is selected from G, A, S, D, or T. X ５ An antibody or its antigen-binding fragment, characterized in that it is selected from S, D, I, T, V, or M.

2. X １ It is selected from F or I, X ２ It is selected from T or I, X ３ It is selected from S or G, X ４ It is selected from S or G, X ５ The antibody or antigen-binding fragment according to claim 1, characterized in that is selected from D or S.

3. The amino acid sequence of CDR1 includes the amino acid sequence shown as GTFFSSAW (SEQ ID NO: 15), GTIFSDSF (SEQ ID NO: 2), or GIIFSGAW (SEQ ID NO: 17), or includes an amino acid sequence having 90% or more identity with the amino acid sequence shown as GTFFSSAW (SEQ ID NO: 15), GTIFSDSF (SEQ ID NO: 2), or GIIFSGAW (SEQ ID NO: 17). The amino acid sequence of CDR2 includes the amino acid sequence represented by ITSSGDTV (SEQ ID NO: 18), ITSGGGSTV (SEQ ID NO: 19), or ITSGDITN (SEQ ID NO: 4), or includes an amino acid sequence having 90% or more identity with the amino acid sequence represented by ITSSGDTV (SEQ ID NO: 18), ITSGGGSTV (SEQ ID NO: 19), or ITSGDITN (SEQ ID NO: 4). The antibody or antigen-binding fragment according to claim 1 or 2, characterized in that the amino acid sequence of CDR3 includes the amino acid sequence represented by NARPGAVNSY (SEQ ID NO: 5), NSRIYTRSY (SEQ ID NO: 6), or NARPRGLSFSSY (SEQ ID NO: 7), or includes an amino acid sequence having 90% or more identity with the amino acid sequence represented by NARPGAVNSY (SEQ ID NO: 5), NSRIYTRSY (SEQ ID NO: 6), or NARPRGLSFSSY (SEQ ID NO: 7).

4. The antibody or its antigen-binding fragment comprises one of the following groups: A) CDR1 contains GTFFSSAW (SEQ ID NO: 15), or contains an amino acid sequence having 90% or more identity with the amino acid sequence represented by GTFFSSAW (SEQ ID NO: 15), CDR2 contains ITSSGDTV (SEQ ID NO: 18), or contains an amino acid sequence having 90% or more identity with the amino acid sequence represented by ITSSGDTV (SEQ ID NO: 18), CDR3 contains NARPGAVNSY (SEQ ID NO: 5), or contains an amino acid sequence having 90% or more identity with the amino acid sequence represented by NARPGAVNSY (SEQ ID NO: 5), B) CDR1 contains GTIFSDSF (SEQ ID NO: 2), or contains an amino acid sequence having 90% or more identity with the amino acid sequence represented by GTIFSDSF (SEQ ID NO: 2), CDR2 contains ITSGGGSTV (SEQ ID NO: 19), or contains an amino acid sequence having 90% or more identity with the amino acid sequence represented by ITSGGGSTV (SEQ ID NO: 19), CDR3 contains NSRIYTRSY (SEQ ID NO: 6), or contains an amino acid sequence having 90% or more identity with the amino acid sequence represented by NSRIYTRSY (SEQ ID NO: 6), or C) The antibody or antigen-binding fragment according to any one of claims 1 to 3, characterized in that CDR1 contains GIIFSGAW (SEQ ID NO: 17) or an amino acid sequence having 90% or more identity with the amino acid sequence represented by GIIFSGAW (SEQ ID NO: 17), CDR2 contains ITSGDITN (SEQ ID NO: 4) or an amino acid sequence having 90% or more identity with the amino acid sequence represented by ITSGDITN (SEQ ID NO: 4), and CDR3 contains NARPRGLSFSSY (SEQ ID NO: 7) or an amino acid sequence having 90% or more identity with the amino acid sequence represented by NARPRGLSFSSY (SEQ ID NO: 7).

5. The antibody or antigen-binding fragment according to any one of claims 1 to 4, characterized in that the antibody or antigen-binding fragment comprises Fab, Fd, Fab', Fab'-SH, Fv, scFv, F(ab')2, single-domain antibody, diabody (dAb), full-length antibody, or linear antibody.

6. The antibody or its antigen-binding fragment binds to the IL13Rα2 protein, Preferably, the antibody or antigen-binding fragment according to any one of claims 1 to 5, wherein the IL13Rα2 protein comprises a human or non-human animal IL13Rα2 protein.

7. The antibody or antigen-binding fragment according to any one of claims 1 to 6, characterized in that the amino acid sequence of the antibody or antigen-binding fragment includes an amino acid sequence represented by any one of SEQ ID NO: 8 to 9 or 16, or includes an amino acid sequence having 90% or more identity with the amino acid sequence represented by any one of SEQ ID NO: 8 to 9 or 16.

8. A nucleic acid, wherein the nucleic acid encodes an antibody or antigen-binding fragment according to any one of claims 1 to 7, Preferably, the nucleic acid is characterized in that it includes a nucleotide sequence represented by any one of SEQ ID NO: 10 to 12, or a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by any one of SEQ ID NO: 10 to 12.

9. An expression vector or host cell, wherein the expression vector or host cell comprises the nucleic acid described in claim 8.

10. A chimeric antigen receptor, wherein the chimeric antigen receptor comprises an extracellular antigen-binding domain, and the extracellular antigen-binding domain comprises an antibody or antigen-binding fragment according to any one of claims 1 to 7.

11. The chimeric antigen receptor further comprises an intracellular domain, a transmembrane domain and / or a hinge region, Preferably, the hinge region connects the extracellular antigen-binding domain and the transmembrane domain, characterized in that the chimeric antigen receptor is as described in claim 10.

12. The chimeric antigen receptor according to claim 11, characterized in that the transmembrane domain is one or more combinations selected from CD3ζ polypeptide, CD4 polypeptide, CD8 polypeptide, CD28 polypeptide, CD28-41BB polypeptide, OX40 polypeptide, ICOS polypeptide, CTLA-4 polypeptide, PD-1 polypeptide, LAG-3 polypeptide, 2B4 polypeptide, or BTLA polypeptide.

13. The chimeric antigen receptor according to claim 11 or 12, characterized in that the intracellular domain is one or more combinations selected from CD28, ICOS, 4-1BB, OX-40, CD27, or CD3ζ.

14. The chimeric antigen receptor according to any one of claims 11 to 13, characterized in that the hinge region is selected from the extracellular hinge region of CD8, CD28, or IgG.

15. An immune cell, characterized in that the immune cell expresses an antibody or antigen-binding fragment according to any one of claims 1 to 7, or a chimeric antigen receptor according to any one of claims 10 to 14.

16. The immune cells are one or more selected from lymphocytes (e.g., T cells, B cells, NK cells), dendritic cells, monocytes / macrophages, granulocytes, or mast cells. Preferably, the immune cell is a CAR-T cell, as described in claim 15.

17. A drug or diagnostic kit, wherein the drug or diagnostic kit comprises one or more of the following: an antibody or antigen-binding fragment according to any one of claims 1 to 7, a nucleic acid according to claim 8, an expression vector or host cell according to claim 9, a chimeric antigen receptor according to any one of claims 10 to 14, or an immune cell according to any one of claims 15 to 16.

18. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 7, a nucleic acid according to claim 8, an expression vector or host cell according to claim 9, a chimeric antigen receptor according to any one of claims 10 to 14, or an immune cell according to any one of claims 15 to 16, wherein such use includes use in the preparation of a product for diagnosing and / or treating a disease associated with IL13Rα2 expression. Preferably, the disease associated with IL13Rα2 expression includes diseases in which inhibition of IL13Rα2 expression is advantageous for treatment.

19. The aforementioned disease associated with IL13Rα2 expression is a tumor, Preferably, the tumor is selected from lymphoma, lung cancer, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, liver cancer, brain tumor, rectal cancer, stomach cancer, bladder cancer, glioma, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma, and the leukemia is one or more selected from acute lymphoblastic leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, or chronic myeloid leukemia, characterized in that the use according to claim 18.

20. A method for detecting IL13Rα2, the method comprising contacting an antibody or antigen-binding fragment according to any one of claims 1 to 7, a chimeric antigen receptor according to any one of claims 10 to 14, or an immune cell according to any one of claims 15 to 16 with a sample to be detected.

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