Antibody or antigen-binding fragment thereof and use thereof

Abstract

Provided are an antibody or an antigen-binding fragment thereof and a use thereof. The antibody or the antigen-binding fragment thereof specifically binds to IL13Rα2 and does not cross-bind to IL13Rα1, exhibits a significant killing effect on tumor cells expressing IL13Rα2, and can promote the release of cytokines (particularly IFN-γ and IL-2).

Description

Antibody or antigen-binding fragment thereof and use thereof Technical Field

[0001] The present invention relates to the technical field of immunity and molecular biology, and in particular to an antibody or an antigen-binding fragment thereof and applications thereof. Background Art

[0002] IL13Rα2 is a glycosylated protein with a molecular weight of approximately 56 kDa. Human IL13Rα2 consists of a 26-amino acid signal sequence and a 17-amino acid intracellular region. A shorter intracellular protein contains an unknown signal sequence. In vitro, IL13Rα2 binds to IL-13 with high affinity. Human IL13Rα2 has an amino-terminal fibronectin-like domain, four conserved cysteine ​​residues, and a consistent WSXWS membrane structure in the extracellular domain. N-linked glycosylation is required for intracellular binding of IL-13.

[0003] Studies have shown that IL13Rα2 is associated with a variety of diseases, such as:

[0004] Non-patent literature: Study on the functional mechanism of IL-13Rα1 and IL-13Rα2 in atopic dermatitis (Xiao Song, Henan University, 2021) disclosed that the expression level of IL13Rα2 in healthy human skin is not high, while the expression level of IL13Rα2 in the skin of atopic dermatitis patients is significantly upregulated, and the upregulated expression of IL13Rα2 was further verified in the skin of atopic dermatitis model mice.

[0005] Non-patent literature: Progress in IL-13Rα2 research on tumors (He Xiaoyan, Xiong Lixia, Journal of Nanchang University (Medical Edition), 2013, 53(01):87-89) disclosed that IL13Rα2 is expressed in a variety of 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.

[0006] Non-patent literature: Research on the anti-human IL-13Rα2 single-domain antibody of the striped bamboo shark (Qin Lanyi, Zhejiang Sci-Tech University, 2021) disclosed that IL13Rα2 is closely related to the occurrence and development of gliomas, especially the specific high expression in brain gliomas. In addition, it was also disclosed that the expression level of IL13Rα2 in the lung tissue of patients with idiopathic pulmonary fibrosis was significantly higher than that in normal lung tissue; the lack of IL13Rα2 can moderately protect dextran sodium sulfate (DSS)-induced colitis; IL13Rα2 was also confirmed to be highly expressed on the surface of colorectal cancer cells; IL13Rα2 was also found to be an important marker for breast cancer, and its upregulation is the cause of breast cancer invasion; IL13Rα2 is also highly expressed on the surface of renal cell carcinoma and renal clear cell carcinoma, and in addition to enhancing the invasion and migration ability of cancer cells, the high expression of IL13Rα2 on the surface of renal clear cell carcinoma has been shown to be associated with the resistance of cancer cells to standard treatment drugs such as sunitinib.

[0007] Patent document (CN116194481A) discloses that IL13Rα2 is overexpressed in pancreatic cancer, ovarian cancer, melanoma, and malignant glioma.

[0008] Patent document (CN114014941A) discloses that IL13Rα2 is a receptor polypeptide of IL-13, which is almost exclusively expressed in cancer cells and not in normal tissue cells except testis.

[0009] The first IL13Rα2-specific CAR-T clinical trial was completed in 2015, enrolling three patients with recurrent glioblastoma (GBM). Overall, treatment was well-tolerated in these patients, with only transient encephalitis. In one patient, IL13Rα2 expression in the tumor decreased with CAR-T treatment, suggesting that the treatment promoted tumor regression.

[0010] Summary of the Invention

[0011] The present invention uses phage display technology to prepare and screen antibodies or antigen-binding fragments thereof. The screened antibodies or antigen-binding fragments thereof specifically bind to IL13Rα2 without cross-binding to IL13Rα1, and can effectively kill tumor cells expressing IL13Rα2 and increase the secretion level of cytokines. Specifically:

[0012] 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 thereof comprises CDR1, CDR2, and CDR3, wherein the amino acid sequence of CDR1 comprises the amino acid sequence shown in GX1X2FSX3AW (SEQ ID NO: 1) or GTIFSDSF (SEQ ID NO: 2), or comprises an amino acid sequence having more than 90% homology to the amino acid sequence shown in GX1X2FSX3AW (SEQ ID NO: 1) or GTIFSDSF (SEQ ID NO: 2);

[0013] The amino acid sequence of the CDR2 comprises the amino acid sequence shown in ITSX4GX5TV (SEQ ID NO: 3) or ITSGDITN (SEQ ID NO: 4), or comprises an amino acid sequence having more than 90% homology to the amino acid sequence shown in ITSX4GX5TV (SEQ ID NO: 3) or ITSGDITN (SEQ ID NO: 4);

[0014] The amino acid sequence of the CDR3 comprises the amino acid sequence shown as NARPGAVNSY (SEQ ID NO: 5), NSRIYTRSY (SEQ ID NO: 6) or NARPRGLSFSSY (SEQ ID NO: 7), or comprises an amino acid sequence having more than 90% homology with the amino acid sequence shown as NARPGAVNSY (SEQ ID NO: 5), NSRIYTRSY (SEQ ID NO: 6) or NARPRGLSFSSY (SEQ ID NO: 7).

[0015] Preferably, the amino acid sequence of the CDR1 is shown as GX1X2FSX3AW (SEQ ID NO: 1) or GTIFSDSF (SEQ ID NO: 2).

[0016] Preferably, the amino acid sequence of the CDR2 is shown as ITSX4GX5TV (SEQ ID NO: 3) or ITSGDITN (SEQ ID NO: 4).

[0017] Preferably, the amino acid sequence of the CDR3 is shown as NARPGAVNSY (SEQ ID NO: 5), NSRIYTRSY (SEQ ID NO: 6) or NARPRGLSFSSY (SEQ ID NO: 7).

[0018] Wherein, X1, X2, X3, X4, and X5 are independently selected from natural amino acids or non-natural amino acids.

[0019] Said X1 is selected from but not limited to I, S, F, T or V. Preferably, X1 is selected from F and I.

[0020] The X2 is selected from but not limited to I, T, or A. Preferably, X2 is selected from T, I.

[0021] The X3 is selected from but not limited to G, N, S, V, L, A, D, Y or I. Preferably, X3 is selected from S or G.

[0022] The X4 is selected from but not limited to G, A, S, D or T. Preferably, X4 is selected from S or G.

[0023] The X5 is selected from but not limited to S, D, I, T, V or M. Preferably, X5 is selected from D and S.

[0024] In a specific embodiment of the present invention, the amino acid sequence of the CDR1 comprises the amino acid sequence shown in GFTFSSAW (SEQ ID NO: 15), GTIFSDSF (SEQ ID NO: 2) or GIIFSGAW (SEQ ID NO: 17), or comprises an amino acid sequence having more than 90% homology with the amino acid sequence shown in GFTFSSAW (SEQ ID NO: 15), GTIFSDSF (SEQ ID NO: 2) or GIIFSGAW (SEQ ID NO: 17).

[0025] The amino acid sequence of the CDR2 comprises the amino acid sequence shown in ITSSGDTV (SEQ ID NO: 18), ITSGGSTV (SEQ ID NO: 19) or ITSGDITN (SEQ ID NO: 4), or comprises an amino acid sequence having more than 90% homology with the amino acid sequence shown in ITSSGDTV (SEQ ID NO: 18), ITSGGSTV (SEQ ID NO: 19) or ITSGDITN (SEQ ID NO: 4).

[0026] The amino acid sequence of the CDR3 comprises the amino acid sequence shown as NARPGAVNSY (SEQ ID NO: 5), NSRIYTRSY (SEQ ID NO: 6) or NARPRGLSFSSY (SEQ ID NO: 7), or comprises an amino acid sequence having more than 90% homology with the amino acid sequence shown as NARPGAVNSY (SEQ ID NO: 5), NSRIYTRSY (SEQ ID NO: 6) or NARPRGLSFSSY (SEQ ID NO: 7).

[0027] Preferably, the antibody or antigen-binding fragment thereof includes any one of the following groups:

[0028] A) the CDR1 comprises GFTFSSAW (SEQ ID NO: 15), or comprises an amino acid sequence having 90% or greater homology to the amino acid sequence of GFTFSSAW (SEQ ID NO: 15); the CDR2 comprises ITSSGDTV (SEQ ID NO: 18), or comprises an amino acid sequence having 90% or greater homology to the amino acid sequence of ITSSGDTV (SEQ ID NO: 18); and the CDR3 comprises NARPGAVNSY (SEQ ID NO: 5), or comprises an amino acid sequence having 90% or greater homology to the amino acid sequence of NARPGAVNSY (SEQ ID NO: 5);

[0029] B) the CDR1 comprises GTIFSDSF (SEQ ID NO: 2), or comprises an amino acid sequence having 90% or more homology to the amino acid sequence of GTIFSDSF (SEQ ID NO: 2); the CDR2 comprises ITSGGSTV (SEQ ID NO: 19), or comprises an amino acid sequence having 90% or more homology to the amino acid sequence of ITSGGSTV (SEQ ID NO: 19); the CDR3 comprises NSRIYTRSY (SEQ ID NO: 6), or comprises an amino acid sequence having 90% or more homology to the amino acid sequence of NSRIYTRSY (SEQ ID NO: 6); or,

[0030] C) the CDR1 comprises GIIFSGAW (SEQ ID NO: 17), or comprises an amino acid sequence having 90% or more identity to the amino acid sequence of GIIFSGAW (SEQ ID NO: 17); the CDR2 comprises ITSGDITN (SEQ ID NO: 4), or comprises an amino acid sequence having 90% or more identity to the amino acid sequence of ITSGDITN (SEQ ID NO: 4); and the CDR3 comprises NARPRGLSFSSY (SEQ ID NO: 7), or comprises an amino acid sequence having 90% or more identity to the amino acid sequence of NARPRGLSFSSY (SEQ ID NO: 7).

[0031] In a specific embodiment of the present invention, the antibody or antigen-binding fragment thereof comprises CDR1, CDR2, and CDR3, wherein the CDR1 is shown as GFTFSSAW (SEQ ID NO: 15), the CDR2 is shown as ITSSGDTV (SEQ ID NO: 18), and the CDR3 is shown as NARPGAVNSY (SEQ ID NO: 5).

[0032] In a specific embodiment of the present invention, the antibody or antigen-binding fragment thereof comprises CDR1, CDR2, and CDR3, wherein the CDR1 is represented by GTIFSDSF (SEQ ID NO: 2), the CDR2 is represented by ITSGGSTV (SEQ ID NO: 19), and the CDR3 is represented by NSRIYTRSY (SEQ ID NO: 6).

[0033] In a specific embodiment of the present invention, the antibody or antigen-binding fragment thereof comprises CDR1, CDR2, and CDR3, wherein the CDR1 is represented by GIIFSGAW (SEQ ID NO: 17), the CDR2 is represented by ITSGDITN (SEQ ID NO: 4), and the CDR3 is represented by NARPRGLSFSSY (SEQ ID NO: 7).

[0034] Preferably, the variable regions of the antibody are CDR1, CDR2, and CDR3 in order. A framework region may be included between the CDR1 and CDR2, and / or a framework region may be included between the CDR2 and CDR3.

[0035] Preferably, the antibody or antigen-binding fragment thereof includes but is not limited to Fab, Fd, Fab', Fab'-SH, Fv, scFv, F(ab')2, single domain antibody, diabody (dAb), full length antibody or linear antibody.

[0036] In a specific embodiment of the present invention, the antibody or antigen-binding fragment thereof is a single domain antibody.

[0037] The single-domain antibody comprises a heavy chain variable region (VHH) and two conventional CH2 and CH3 regions.

[0038] Preferably, the amino acid sequence of the antibody or antigen-binding fragment thereof comprises the amino acid sequence shown in any one of SEQ ID NOs: 8-9 or 16, or comprises an amino acid sequence having more than 90% homology with the amino acid sequence shown in any one of SEQ ID NOs: 8-9 or 16.

[0039] Further preferably, the amino acid sequence of the antibody or antigen-binding fragment thereof is shown in any one of SEQ ID NOs: 8-9 or 16.

[0040] Preferably, the antibody or antigen-binding fragment thereof binds to IL13Rα2 protein.

[0041] Preferably, the IL13Rα2 protein includes IL13Rα2 protein from human or non-human animals.

[0042] The non-human animal is preferably a rodent. More preferably, the rodent includes but is not limited to rats or mice.

[0043] In a second aspect, the present invention provides a nucleic acid encoding the CDR1, CDR2 and CDR3 of the present invention.

[0044] The third aspect of the present invention provides a nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of the first aspects.

[0045] Preferably, the nucleotide sequence of the nucleic acid comprises a nucleotide sequence shown in any one of SEQ ID NOs: 10-12 or a nucleotide sequence having more than 90% homology with a nucleotide sequence shown in any one of SEQ ID NOs: 10-12.

[0046] In a specific embodiment of the present invention, the nucleotide sequence of the nucleic acid is shown in any one of SEQ ID NOs: 10-12.

[0047] The fourth aspect of the present invention provides an expression vector comprising any of the above-mentioned nucleic acids.

[0048] Preferably, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

[0049] Further preferably, the prokaryotic expression vector is selected from the group consisting of an Escherichia coli expression system series vector, a Bacillus subtilis expression system series vector, and a Streptomycin expression system series vector.

[0050] Further preferably, the eukaryotic expression vector is selected from a yeast expression system series vector, a fungal expression system series vector, an insect cell expression system series vector or a mammalian cell expression system series vector.

[0051] In a specific embodiment of the present invention, the expression vector is a eukaryotic expression vector.

[0052] The fifth aspect of the present invention provides a host cell.

[0053] Preferably, the host cell contains any of the nucleic acids described above, and / or any of the expression vectors described above.

[0054] Preferably, the host cell can be a eukaryotic cell or a prokaryotic cell.

[0055] In a sixth aspect, the present invention provides a chimeric antigen receptor, wherein the chimeric antigen receptor comprises an extracellular antigen-binding domain, and the extracellular antigen-binding domain comprises the antibody or antigen-binding fragment thereof described in the first aspect.

[0056] Preferably, the chimeric antigen receptor further comprises an intracellular domain, a transmembrane domain and / or a hinge region.

[0057] Preferably, the hinge region connects the extracellular antigen binding domain and the transmembrane domain.

[0058] Preferably, the transmembrane domain is selected from one or a combination of two or more of 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.

[0059] Preferably, the intracellular domain includes a signal transduction domain, preferably CD3ζ.

[0060] Further preferably, the intracellular domain also includes a costimulatory domain.

[0061] Preferably, the intracellular domain is selected from one or a combination of two or more of CD28, ICOS, 4-1BB, OX-40, CD27, or CD3ζ, and more preferably 4-1BB-CD3ζ.

[0062] Preferably, the hinge region is selected from the extracellular hinge region of CD8, CD8α, CD28 or IgG, preferably CD8.

[0063] In a specific embodiment of the present invention, the chimeric antigen receptor structure is: the above-mentioned antibody or antigen-binding fragment thereof-CD8HT-4-1BB-CD3ζ.

[0064] A seventh aspect of the present invention provides a method for constructing the chimeric antigen receptor described in the sixth aspect, the method comprising:

[0065] A) obtaining a nucleic acid encoding the antibody or antigen-binding fragment thereof according to the first aspect;

[0066] B) transforming the nucleic acid obtained in step A) into a host cell and inducing its expression.

[0067] Preferably, the nucleic acid is the nucleic acid described in the third aspect of the present invention.

[0068] In an eighth aspect, the present invention provides an immune cell, wherein the immune cell expresses the antibody or antigen-binding fragment thereof according to the first aspect and / or the chimeric antigen receptor according to the sixth aspect.

[0069] Preferably, the immune cells are obtained by transfecting any of the above-mentioned nucleic acids into immune cells.

[0070] Preferably, the immune cells include, but are not limited to, one or more of lymphocytes (including T cells, B cells, NK cells), dendritic cells, monocytes / macrophages, granulocytes or mast cells.

[0071] In a specific embodiment of the present invention, the immune cells are CAR-T cells.

[0072] The ninth aspect of the present invention provides an immunoconjugate.

[0073] The immunoconjugate comprises the antibody or antigen-binding fragment thereof described in the first aspect or the chimeric antigen receptor described in the sixth aspect conjugated to a therapeutic agent or a diagnostic agent.

[0074] The tenth aspect of the present invention provides a use of the antibody or antigen-binding fragment thereof, 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.

[0075] In the eleventh aspect of the present invention, a drug or pharmaceutical composition is provided, which comprises one or more of the antibody or antigen-binding fragment thereof, the nucleic acid, the expression vector, the host cell, the chimeric antigen receptor or the immune cell.

[0076] The drug or pharmaceutical composition specifically targets IL13Rα2 cells.

[0077] Preferably, the medicine or pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0078] Preferably, the pharmaceutically acceptable excipient is selected from one or a combination of two or more of a carrier, an excipient, a diluent, a lubricant, a wetting agent, an emulsifier, a preservative, an antioxidant, a buffer, an antibacterial agent, a solute that makes the preparation isotonic with the recipient's blood, a suspending agent, a suspending aid, a solubilizer, a thickener, a stabilizer, a sweetener, and a flavoring.

[0079] Preferably, the drug or pharmaceutical composition can 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 conventional production methods in the pharmaceutical field.

[0080] Preferably, the pharmaceutical preparation is a unit dose preparation.

[0081] The amount of the pharmaceutically active ingredient in a unit dose of preparation may be varied or adjusted from 0.001 mg to 1000 mg, depending on the specific application and potency of the pharmaceutically active ingredient.

[0082] According to specific needs, the pharmaceutical composition may also contain other suitable therapeutic agents.

[0083] The medicine or pharmaceutical composition is suitable for parenteral administration such as, for example, preparations for administration by intravenous, intramuscular, intradermal and subcutaneous routes include aqueous and non-aqueous isotonic sterile injections, which may contain antioxidants, buffers, antibacterial agents, and solutes that make the preparation isotonic with the recipient's blood, as well as aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers or preservatives. In the practice of the present invention, the medicine or pharmaceutical composition can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically and intrathecally. The preparation can be present in unit dose or multidose sealed containers such as ampoules and bottles. Injectable solutions and suspensions can be prepared from sterile powders, granules and tablets of the types previously described.

[0084] Preferably, the drug or pharmaceutical composition can be used alone or in combination with other therapeutic agents.

[0085] The twelfth aspect of the present invention provides a diagnostic kit, which comprises one or more of the antibody or antigen-binding fragment thereof, the nucleic acid, the expression vector, the host cell, the chimeric antigen receptor or the immune cell.

[0086] The diagnostic kit diagnoses diseases related to IL13Rα2 expression.

[0087] Preferably, the diseases associated with IL13Rα2 expression include diseases that require inhibition of IL13Rα2 expression for treatment.

[0088] Preferably, the diseases associated with IL13Rα2 expression include diseases in which IL13Rα2 is expressed, highly expressed, or overexpressed.

[0089] Preferably, the diseases associated with IL13Rα2 expression include but are not limited to dermatitis, lung diseases, intestinal diseases, tumors, etc.

[0090] Further preferably, the dermatitis includes but is not limited to atopic dermatitis.

[0091] Further preferably, the lung disease includes but is not limited to pulmonary fibrosis (preferably idiopathic pulmonary fibrosis) or lung tumor.

[0092] Further preferably, the intestinal disease includes colitis (preferably dextran sulfate sodium induced colitis) or intestinal tumors.

[0093] Further 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 cancer, rectal cancer, gastric cancer, bladder cancer, glioma (such as brain 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; wherein the leukemia is selected from one or more of acute lymphocytic leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia or chronic myeloid leukemia.

[0094] More preferably, the lymphoma includes Hodgkin's lymphoma and non-Hodgkin's lymphoma, including one or more of B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma or Waldenstrom's macroglobulinemia.

[0095] More preferably, the sarcoma includes one or more of osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma or chondrosarcoma.

[0096] In a specific embodiment of the present invention, the tumor is selected from, but not limited to, one or more of glioma, brain tumor, ovarian cancer, liver cancer, malignant mesothelioma, kidney cancer, thyroid cancer, epidermoid cancer, Kaposi's sarcoma, prostate cancer, pancreatic cancer, breast cancer or melanoma.

[0097] The glioma includes but is not limited to glioblastoma and brain glioma.

[0098] The renal cancer includes renal cell carcinoma or renal clear cell carcinoma.

[0099] The Kaposi's sarcoma, prostate cancer, and pancreatic cancer are preferably Kaposi's sarcoma, prostate cancer, and pancreatic cancer associated with AIDS.

[0100] In a specific embodiment of the present invention, the tumor includes melanoma and glioma.

[0101] The thirteenth aspect of the present invention provides a use of the antibody or antigen-binding fragment thereof, the nucleic acid, the expression vector, the host cell, the chimeric antigen receptor or the immune cell.

[0102] Preferably, the application includes application in the preparation of products for diagnosing and / or treating diseases associated with IL13Rα2 expression.

[0103] Further preferably, the diseases associated with IL13Rα2 expression include diseases that require inhibition of IL13Rα2 expression for treatment.

[0104] Further preferably, the diseases associated with IL13Rα2 expression include diseases in which IL13Rα2 is expressed, highly expressed, or overexpressed.

[0105] Preferably, the product includes but is not limited to a diagnostic kit, a drug or a pharmaceutical composition.

[0106] Preferably, the diseases associated with IL13Rα2 expression include but are not limited to dermatitis, lung diseases, intestinal diseases, tumors, etc.

[0107] Further preferably, the dermatitis includes but is not limited to atopic dermatitis.

[0108] Further preferably, the lung disease includes but is not limited to pulmonary fibrosis (preferably idiopathic pulmonary fibrosis) or lung tumor.

[0109] Further preferably, the intestinal disease includes colitis (preferably dextran sulfate sodium induced colitis) or intestinal tumors.

[0110] Further 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 cancer, rectal cancer, gastric 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; wherein the leukemia is selected from one or more of acute lymphocytic leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia or chronic myeloid leukemia.

[0111] More preferably, the lymphoma includes Hodgkin's lymphoma and non-Hodgkin's lymphoma, including one or more of B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma or Waldenstrom's macroglobulinemia.

[0112] More preferably, the sarcoma includes one or more of osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma or chondrosarcoma.

[0113] In a specific embodiment of the present invention, the tumor is selected from, but not limited to, one or more of glioma, brain tumor, ovarian cancer, liver cancer, malignant mesothelioma, kidney cancer, thyroid cancer, epidermoid cancer, Kaposi's sarcoma, prostate cancer, pancreatic cancer, breast cancer or melanoma.

[0114] The glioma includes but is not limited to glioblastoma and brain glioma.

[0115] The renal cancer includes renal cell carcinoma or renal clear cell carcinoma.

[0116] The Kaposi's sarcoma, prostate cancer, and pancreatic cancer are preferably Kaposi's sarcoma, prostate cancer, and pancreatic cancer associated with AIDS.

[0117] In a specific embodiment of the present invention, the tumor includes melanoma and glioma.

[0118] A fourteenth aspect of the present invention provides a method for treating diseases related to IL13Rα2 expression.

[0119] Preferably, the method comprises administering the antibody or antigen-binding fragment thereof of the present invention, the chimeric antigen receptor of the present invention, or the immune cell of the present invention to a subject in need thereof.

[0120] In a specific embodiment of the present invention, the immune cells are CAR-T cells.

[0121] Preferably, the treatment of diseases related to IL13Rα2 expression includes using 1.0E+05 / mL to 1.0E+08 / mL CAR-T cells, such as 1.0E+05 / mL, 3.0E+05 / mL, 5.0E+05 / mL, 6.0E+05 / mL, 7.0E+05 / mL, 8.0E+05 / mL, 9.0E+05 / mL, 1.0E+06 / mL, 3.0E+06 / mL, 4.0E+06 / mL, 5.0E+06 cells / mL, 6.0E+06 cells / mL, 7.0E+06 cells / mL, 8.0E+06 cells / mL, 9.0E+06 cells / mL, 1.0E+07 cells / mL, 2.0E+07 cells / mL, 3.0E+07 cells / mL, 4.0E+07 cells / mL, 5.0E+07 cells / mL, 6.0E+07 cells / mL, 7.0E+07 cells / mL, 8.0E+07 cells / mL, 9.0E+07 cells / mL, and 1.0E+08 cells / mL.

[0122] Preferably, the treatment of diseases related to IL13Rα2 expression includes the use of CAR-T cells. Further preferably, the CAR-T cells: target cells are greater than or equal to 1 / 10, 1 / 5, 1 / 3, 1 / 2, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.

[0123] In a fifteenth aspect, the present invention provides a method for detecting IL13Rα2, which comprises contacting the above-mentioned antibody or antigen-binding fragment thereof, the above-mentioned chimeric antigen receptor, or the above-mentioned immune cell with a sample to be detected.

[0124] The sample to be tested includes but is not limited to body fluids, cells, tissues or organs, preferably blood, serum, plasma or tumor cells.

[0125] Preferably, the method further comprises adding a labeling substance. Further preferably, the labeling substance includes but is not limited to a radioactive marker, a fluorescent marker or a reporter cell, etc.

[0126] In a specific embodiment of the present invention, the method comprises contacting the sample to be tested with a detectable amount of the above-mentioned antibody or antigen-binding fragment thereof or the above-mentioned chimeric antigen receptor or the above-mentioned immune cell, and adding a labeling substance.

[0127] 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.

[0128] The terms "comprising" or "including" as used herein are open-ended and encompass the specified components or steps described, as well as other specified components or steps that do not substantially affect them. When used to describe a protein or nucleic acid sequence, the protein or nucleic acid may consist of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, while still having the same or similar activity as the original sequence.

[0129] The term "and / or" in this invention includes all combinations of the items connected by the term, and each combination should be considered to have been listed separately herein. For example, "A and / or B" includes "A," "A and B," and "B." For another example, "A, B and / or C" includes "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0130] As used herein, "treating" means slowing, interrupting, preventing, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-associated signs, symptoms, conditions, or disorders.

[0131] The term "diagnosis" in the present invention refers to finding out whether a patient has had a disease or condition in the past, at the time of diagnosis, or in the future, or to finding out the progression or possible future progression of a disease.

[0132] The term "pharmaceutically acceptable" as used herein means that the pharmaceutical composition neither significantly stimulates the organism nor inhibits the biological activity and properties of the active substance of the administered product.

[0133] The "antigen-binding fragment" described in the present invention is a portion of an antibody that retains the specific binding activity of the antibody, that is, any part of the antibody that is capable of specifically binding to an epitope on the antibody's target molecule. It includes, for example, Fab, Fv, Fd, Fab', Fab'-SH, F(ab')2, and variants of these fragments. For example, the heavy and / or light chains of an antibody, the heavy and / or light chain variable regions of an antibody, or a single or two or more CDRs from the heavy or light chains of an antibody. Fab is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains. F(ab')2 is a bivalent fragment consisting of two Fab fragments connected by a disulfide bridge in the hinge region. Fd is an Fd fragment consisting of the VH and CH1 domains. Fv is an Fv fragment consisting of the VL and VH domains 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' in which the cysteine ​​residue in the constant domain carries at least one free thiol group. Single-domain antibodies (SDOs) are antibodies that lack light chains and only have heavy chains. Due to their small molecular weight, they are also called nanobodies. VH represents the heavy chain variable region, VL represents the light chain variable region, CL represents the light chain, and CH1 represents the CH1 component of the heavy chain constant region.

[0134] The "non-natural amino acids" of the present invention are compounds containing an amino group and a carboxyl group that are not naturally found in proteins. Preferably, the non-natural amino acids are any non-natural amino acids known in the art. Further preferably, the non-natural amino acids include, but are not limited to, N-ethylaspartyl, 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, isodesmosine, 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, norvaline, desmosine, 2,2'-diaminopimelane, 2,3-diaminopropionic acid, N-ethylglycine, or 2-aminopimelane, and the like. Of course, the "non-natural amino acids" mentioned in this application may also be derivatives of natural amino acids after modification.

[0135] As used herein, "CDR" refers to the complementarity determining regions within the variable sequences of antibodies. For each variable region, there are three CDRs in each variable region of the heavy and / or light chain, referred to as CDR1, CDR2, and CDR3. The exact boundaries of these CDRs are defined differently according to different systems. These systems include the Kabat, Chothia, IMGT, AbM, or Contact systems, among others, and CDRs herein may be defined according to any of these systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0136] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0137] Figure 1: Detection results of purified antibodies;

[0138] Figure 2: Average fluorescence intensity of six groups: 1-E9, 1-E10, 2-G01, 1-C10, 10-C4, and 7-G8;

[0139] Figure 3: Average fluorescence intensity of the three groups 4-C2, 4-F10, and 7-F2;

[0140] Figure 4: Cytotoxic effects of the single-domain antibodies screened by the present invention on 293T, 293T-IL13Rα2, A375, and U251 cells, wherein T represents T cells, G01 represents CAR-T cells prepared with 2-G01 single-domain antibodies, C2 represents CAR-T cells prepared with 4-C2 single-domain antibodies, F10 represents CAR-T cells prepared with 4-F10 single-domain antibodies, and EQ represents a positive control;

[0141] [Corrected 29.03.2024 according to Rule 91] Figure 5: Effects of the single-domain antibodies screened by the present invention on the release concentration of IFN-γ cytokines from 293T, A375, and U251 cells; wherein T represents T cells, G01 represents CAR-T cells prepared with 2-G01 single-domain antibodies, C2 represents CAR-T cells prepared with 4-C2 single-domain antibodies, F10 represents CAR-T cells prepared with 4-F10 single-domain antibodies, EQ represents positive control, and Alone represents spontaneous release from effector cells. The bar graphs in each group are T, EQ, G01, C2, and F10 from left to right;

[0142] [Corrected 29.03.2024 according to Rule 91] Figure 6: Effects of the single-domain antibodies screened by the present invention on the release concentration of IL-2 cytokines from 293T, A375, and U251 cells; wherein T represents T cells, G01 represents CAR-T cells prepared with 2-G01 single-domain antibodies, C2 represents CAR-T cells prepared with 4-C2 single-domain antibodies, F10 represents CAR-T cells prepared with 4-F10 single-domain antibodies, EQ represents positive control, and Alone represents spontaneous release from effector cells. The bar graphs in each group are T, EQ, G01, C2, and F10 from left to right;

[0143] Figure 7: Effects of the single-domain antibodies screened by the present invention on tumor volume, wherein G01 represents CAR-T cells prepared with 2-G01 single-domain antibody;

[0144] Figure 8: Average tumor volume of mice subcutaneously injected with A375 at different doses. G01 represents CAR-T cells prepared with 2-G01 single-domain antibody, **** indicates p < 0.0001;

[0145] Figure 9: Body weight of tumor-bearing mice in different treatment groups. G01 represents CAR-T cells prepared with 2-G01 single-domain antibody;

[0146] Figure 10: Optimization of the concentration of 2-G01 antibody for screening;

[0147] Figure 11: Optimization of 4-F10 antibody concentration for screening;

[0148] Figure 12: Membrane protein screening array screening results for the 2-G01 antibody;

[0149] Figure 13: Membrane protein screening array screening results for the 4-F10 antibody;

[0150] Figure 14: Binding indices of different concentrations of 2-G01 antibody to the top 20 targets;

[0151] Figure 15: Binding indices of 4-F10 antibody at different concentrations to the top 20 targets. DETAILED DESCRIPTION

[0152] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0153] Some of the methods used in the embodiments of the present invention:

[0154] Preparation method of CAR-T cells:

[0155] ① Cell preparation: PBMCs isolated from peripheral blood were activated with CD3 / CD28 magnetic beads and infected with lentivirus 24 hours later;

[0156] ② Lentivirus infection: According to the experimental design requirements, take an appropriate amount of activated T cells, centrifuge at 300g for 5 minutes at room temperature, discard the supernatant, resuspend the T cells in culture medium, and then add an appropriate amount of lentivirus concentrate (the amount of virus is determined according to the virus titer and MOI) and gently pipette to a final cell concentration of 2.0E+10 6 / mL, incubate the infection in a 5% CO2 incubator overnight.

[0157] ③ 24 hours after lentiviral infection, change the medium and continue to expand and culture the CAR T cells. Observe the cell number and cell infection efficiency.

[0158] ④. Cell killing assay was performed 10 days after cells were infected with lentivirus.

[0159] Example 1: Development of IL13Rα2 single domain antibody and screening of phage display library

[0160] 1. After the alpaca was immunized, the immune titer was tested using ELISA. The results are shown in Table 1 below. After a certain titer was reached, peripheral blood was drawn from the alpaca for PBMC cell separation and antibody phage library was constructed.

[0161] 2. Panning of phage display library:

[0162] IL13Rα2 recombinant protein is used for panning, and then the phage display library is incubated with the recombinant protein. The recombinant phage bound to the target antigen is eluted using TEA and amplified. After 3-4 rounds of panning, single clones are selected for sequencing.

[0163] Table 1

[0164] The target antigen recombinant protein was co-incubated with the single-domain antibody phage display library to enrich phages that specifically bind to the target antigen. After four rounds of enrichment, the input and output of each round were counted, and the enrichment factor for each round was calculated. The results are shown in Table 2 below.

[0165] Table 2

[0166] 3. Expression and purification of candidate single domain antibodies

[0167] The bacterial solution of the target clone was used for PCR amplification, the PCR product was transiently transfected into CHO-K1 cells, and the expressed antibody supernatant was used for flow cytometry fluorescence sorting (FACS) detection.

[0168] Thirteen differential clones, including 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, which specifically bind to IL13Rα2, were selected to construct eukaryotic expression vectors. The results of antibody detection after purification are shown in Figure 1. Among them, 6-A10 had no band and was therefore excluded. The remaining 12 clones were used as candidate single-domain antibodies.

[0169] 4. FACS gradient dilution test of candidate single domain antibodies

[0170] CHO-K1 / IL13Rα2 cells were used for detection. Purified candidate single domain antibodies were added, and after setting an appropriate starting concentration, 11 points of 3-fold serial dilution were performed. FACS detection was performed using a secondary antibody. Exemplary mean fluorescence intensities are shown in Figures 2-3. Exemplary EC50 (μg / mL) of each antibody are shown in Table 3 below.

[0171] Table 3

[0172] Result analysis: All the candidate single domain antibodies above bound to CHO-K1 / IL13Rα2 cells. Except for the 10-C4 candidate antibody, the other candidate antibodies bound strongly to CHO-K1 / IL13Rα2 cells.

[0173] 5. FACS detection of candidate single domain antibodies and IL13Rα1 transiently transfected cells

[0174] The results showed that none of the above candidate single domain antibodies cross-binded to 293F / IL13Rα1 cells.

[0175] Furthermore, after analysis and verification of the above sequences, the following three key sequence antibodies were further studied, namely 2-G01, 4-F10, and 4-C2, with the sequences shown below. EQ antibody was used as a positive control, with the sequence shown below.

[0176] Nucleotide sequence encoding 2-G01:

[0177] Nucleotide sequence encoding 4-C2:

[0178] Nucleotide sequence encoding 4-F10:

[0179] Nucleic acid sequence encoding EQ antibody:

[0180] Amino acid sequence of 2-G01:

[0181] Amino acid sequence of 4-C2:

[0182] Amino acid sequence of 4-F10:

[0183] Amino acid sequence of EQ antibody:

[0184] Example 2: In vitro cell killing experiment to verify the effectiveness of the antibody

[0185] 1. Count target cells 293T, 293T-IL13Rα2, A375 (human malignant melanoma cells), and U251 (human glioma cells), collect the cells, centrifuge at 300g for 5 minutes at room temperature, and discard the supernatant;

[0186] 2. Resuspend the cells in culture medium and centrifuge at 300g for 5 minutes at room temperature;

[0187] 3. Discard the supernatant and repeat step 2;

[0188] 4. Resuspend the cells with appropriate amount of culture medium and calculate the cell density;

[0189] 5. Preparation of CAR-T cells: The CAR structure is an antibody sequence (the antibody sequence is 2-G01, 4-C2, or 4-F10 screened in Example 1)-CD8HT-4-1BB-CD3ζ structure, and CAR-T cells are prepared using the above method;

[0190] 6. Take the CAR-T cells, centrifuge at 300g for 5 minutes, and discard the supernatant;

[0191] 7. Resuspend the cells with appropriate amount of culture medium, take samples for counting, and centrifuge at 300g for 5 minutes at room temperature;

[0192] 8. Discard the supernatant and repeat step 7;

[0193] 9. Resuspend CAR-T cells in PBS and count the cell density;

[0194] Exemplary information of CAR-T cells prepared with 2-G01 antibody is shown in Table 4:

[0195] Table 4

[0196] A 96-well cell culture plate (purchased from Corning, catalog number: 3599) was used for co-incubation of CAR-T / NC cells and target cells: the number of target cells per well and the effective CAR-T / NC cell effector-target ratio were 5:1, 3:1, 1:1, and 1:2, and the total volume did not exceed 100 μL.

[0197] 10. Incubate at 37°C for testing.

[0198] 11. Detect cell killing efficiency.

[0199] 12. Analyze experimental data and draw graphs to present experimental results and conclusions.

[0200] The results are shown in Figure 4. The three single-domain antibodies described in the present invention have no killing effect on 293T cells, but have a killing effect on 293T cells, A375 cells and U251 cells expressing IL13Rα2, especially when the effector-target ratio (E:T) is 5:1, the killing effect is the strongest.

[0201] Example 3: Cytokine Detection

[0202] 1. Count the target cells 293T, A375, and U251 respectively, collect the cells, centrifuge at 300g for 5 minutes at room temperature, and discard the supernatant;

[0203] 2. Resuspend the cells in culture medium and centrifuge at 300g for 5 minutes at room temperature;

[0204] 3. Discard the supernatant and repeat step 2;

[0205] 4. Resuspend the cells with an appropriate amount of culture medium and calculate the cell density.

[0206] 5. Take the CAR-T cells, centrifuge at 300g for 5 minutes, and discard the supernatant (the preparation of CAR-T cells is the same as step 5 in Example 2);

[0207] 6. Resuspend the cells with appropriate amount of culture medium, take samples for counting, and centrifuge at 300g for 5 minutes at room temperature;

[0208] 7. Discard the supernatant and repeat step 6;

[0209] 8. Resuspend CAR-T cells in PBS and count the cell density;

[0210] Take a 96-well cell culture plate and co-incubate CAR-T / NC cells and target cells: the target cell number and target-efficacy ratio of effective CAR-T / NC cells in each well is 1:1, and the total volume does not exceed 200 μL.

[0211] 9. After incubation at 37°C, collect the cell supernatant and detect it using ELISA, and analyze the data to obtain the results.

[0212] The results are shown in Figures 5-6. The use of the single-domain antibody described in the present application can significantly increase the release concentrations of cytokines IFN-γ and IL-2.

[0213] Example 4: Anti-tumor effect of IL-13Rα2 VHH CAR T cells in the A375 syngeneic mouse melanoma tumor model

[0214] Cell culture: A375 cells (CL-0014, Procell) were quickly thawed in a 37°C water bath and resuspended in RPMI-1640 (Gibco, C11875500BT) containing 10% fetal bovine serum (ExCell Bio, FSP500) and 100 μg / mL penicillin-streptomycin solution (Procell, PB180120, 100 μg / mL penicillin and 100 μg / mL streptomycin). The cells were then incubated at 37°C in a 5% CO2 incubator (Thermo Scientific, 371). The cells were passaged every 2 days. A375 cells were seeded in 150 mm culture dishes (NEST, 715001) and plated when the cell count reached 2 × 10 7 Cells were collected at 100 cells / dish.

[0215] Preparation of CAR-T cell infusion: Frozen T cells and CAR-T cells prepared based on 2-G01 antibody (hereinafter referred to as G01CAR-T cells) were removed from liquid nitrogen and thawed in a 37°C water bath. The cells were transferred to a centrifuge tube containing PBS and centrifuged (centrifuge purchased from Eppendorf, catalog number: 5804R). The supernatant was removed, and the cells were resuspended in PBS. The cells were counted (using a cell counter purchased from Invitrogen, catalog number: Countess II). The concentration of T cells was adjusted to 5.0E+07 cells / mL, and the effective cell concentration of G01CAR-T cells was adjusted to 1.0E+06 cells / mL, 3.0E+06 cells / mL, and 1.0E+07 cells / mL.

[0216] Establishment of A375 tumor mouse model: A375 tumor cells were inoculated at a rate of 2×10 per mouse. 630 NCG mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., Animal Production License No.: SCXK (Su) 2018-0008) were subcutaneously inoculated with 10 cells. Four days after inoculation, the tumor volume (about 30 mm 3 Mice with relatively uniform serotypes were randomly divided into five groups, as shown in Table 5. Group A1 (n = 6) received a tail vein infusion of 100 μL PBS per mouse, serving as the solvent control group; group A2 (n = 6) received a tail vein infusion of 100 μL 5.0E+07 T cells per mouse; group A3 (n = 6) received a tail vein infusion of 100 μL 1.0E+06 effective G01CAR-T cells per mouse; group A4 (n = 6) received a tail vein infusion of 100 μL 3.0E+06 G01CAR-T cells per mouse; and group A5 (n = 6) received a tail vein infusion of 100 μL 1.0E+07 G01CAR-T cells per mouse. Tumor volume was measured twice weekly with a vernier caliper. Tumor volume calculation formula: Tumor volume = (tumor long axis length) × (tumor short axis length) 2 / 2, and the tumor inhibition rate was calculated, tumor inhibition rate = (mean tumor volume of the treatment group - mean tumor volume of the control group) / mean tumor volume of the control group × 100%. Statistical comparison of tumor growth was analyzed by two-way ANOVA. In addition, the body weight of mice in each group was measured twice a week.

[0217] Table 5

[0218] Data analysis: Statistical analysis was performed using GraphPad 10. Data are expressed as mean ± SD and analyzed using F-test and two-sample analysis of variance. When p < 0.05, a two-sample t-test assuming heteroscedasticity was used. When p > 0.05, a t-test assuming equal variance was used (test criteria: α = 0.05, two-sided).

[0219] Experimental Results: Compared with the control group, G01CAR-T cell infusion significantly inhibited tumor growth in groups A4 and A5. The inhibition rates were 14.80% in group A2, 8.36% in group A3, 67.59% in group A4, and 99.89% in group A5. Fourteen days after G01CAR-T cell infusion, tumors in two mice in group A4 were nearly eliminated, while tumors in the remaining four mice grew slowly, demonstrating significant tumor growth inhibition compared to group A2. Fourteen days after G01CAR-T cell infusion, tumors in six mice in group A5 were nearly completely eliminated, demonstrating significant tumor growth inhibition, approaching 100%, compared to group A2 (see Figures 7-8).

[0220] In addition, the body weights of the five groups of mice were tested, and it was found that there was no significant difference in the body weights of the five groups of mice (see Figure 9), indicating that the use of the CAR-T cells described in this application is safe.

[0221] In summary, IL-13Rα2 CAR-T cells demonstrated a significant tumor-suppressing effect in A735 melanoma model mice. A significant tumor-suppressing effect was observed when the effective cell infusion rate reached 3.0E+06 cells per mouse, and the tumor rate approached 100% when the effective cell infusion rate reached 1.0E+07 cells per mouse. Furthermore, the treatment had no effect on the mice's body weight.

[0222] Example 5: Membrane protein screening array to verify the specificity of antibody binding to IL-13Rα2

[0223] The membrane protein screening array AB5000 was used to examine the binding specificity of 4032 human membrane proteins (receptors, transporters, enzymes, etc.) to the antibodies screened in Example 1. The steps are as follows:

[0224] 1) Experimental setup: Determine the optimal test sample concentration for screening.

[0225] In order to determine the optimal concentration of samples used in the target screening stage, target cells were transfected with vectors encoding sample-binding target genes (positive) and blank vectors (negative control) in 96-well plates. After transfection, the samples were diluted 3.16 times with a maximum concentration of 20ug / ml, for a total of 9 detection concentrations, added to the culture plate, and the same number of reporter cells as the target cells were added and incubated together. Fluorescence signals were collected using a fluorescence microplate reader. The optimal screening concentration of the test sample was determined by the binding index to achieve low background and high sensitivity. The optimal screening concentration was determined to be 0.1μg / mL (Figures 10-11) and used in subsequent experiments.

[0226] 2) Membrane protein screening array: Use the membrane protein screening array AB5000 to screen test samples and determine binding targets.

[0227] To identify binding targets, 4032 human membrane proteins were expressed in HEK-293T cells arranged in individual wells of a 96-well plate. After incubation, the test sample and reporter cells were added to the transfected cells. The optimal screening concentration of the test sample was 0.1 μg / mL. Fluorescence signals were collected using a fluorescence microplate reader. The resulting binding values ​​were then normalized and converted to give a single numerical value (i.e., binding index) for the binding of the test sample to each target protein.

[0228] The membrane protein screening array results for the 2-G01 antibody and the 4-F10 antibody are shown in Figures 12-13.

[0229] For example, taking the top 20 targets with the highest binding index as an example, the target information and binding index are as follows:

[0230] 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 ID: P56159, binding index: 2.01), (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 (UniPort ID: Q99835, binding index: 1.92), RNF19A (UniPort ID: ID: Q9NV58, binding index: 1.91).

[0231] 4-F10 antibodies: 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, binding index: 2.22), CLEC10A (UniPort ID: Q8IUN9, binding index: 2.15), C2CD2L (UniPort ID: O14523, binding index: 2.03), SELL (UniPort ID: P14151, binding index: 1.99), OR1D5 (UniPort ID: P58170, binding index: 1.95), GPR33 (UniPort ID: Q49SQ1, binding index: 1.93), CDHR5 (UniPort ID: (UniPort ID: Q9HBB8, binding index: 1.93), ANKK1 (UniPort ID: Q8NFD2, binding index: 1.91), CPNE7 (UniPort ID: Q9UBL6, binding index: 1.90), KCNH4 (UniPort ID: Q9UQ05, binding index: 1.89), FAM189A2 (UniPort ID: Q15884, binding index: 1.89), DPY19L3 (UniPort ID: Q6ZPD9, binding index: 1.86), PRIMA1 (UniPort ID: Q86XR5, binding index: 1.83), CYYR1 (UniPort ID: Q96J86, binding index: 1.82), PTPRK (UniPort ID: Q15262, binding index: 1.82), DIRAS1 (UniPort ID: Q15263, binding index: 1.83), ID: O95057, binding index: 1.81), CHIC1 (UniPort ID: Q5VXU3, binding index: 1.76).

[0232] 3) Target validation: Use reporter cells to validate newly identified targets.

[0233] To determine the optimal sample concentration for target screening, target cells were transfected with a vector encoding the sample-binding target gene (positive control) and a blank vector (negative control) in a 96-well plate. Following transfection, the sample was serially diluted 3.16-fold, starting at 20 μg / ml, for a total of nine test concentrations. The sample was then added to the plate and incubated with the same number of reporter cells as the target cells. Fluorescence signals were collected using a fluorescence microplate reader.

[0234] The results are shown in Figures 14-15.

[0235] In summary, the antibodies screened in the present application specifically bind to IL13Rα2 and can be used for the detection of IL13Rα2 protein.

[0236] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0237] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. An antibody or an antigen-binding fragment thereof, characterized in that: The antibody or antigen-binding fragment thereof comprises CDR1, CDR2, and CDR3, The amino acid sequence of the CDR1 comprises the amino acid sequence shown in GX1X2FSX3AW (SEQ ID NO: 1) or GTIFSDSF (SEQ ID NO: 2), or comprises an amino acid sequence having more than 90% homology with the amino acid sequence shown in GX1X2FSX3AW (SEQ ID NO: 1) or GTIFSDSF (SEQ ID NO: 2); The amino acid sequence of the CDR2 comprises the amino acid sequence shown in ITSX4GX5TV (SEQ ID NO: 3) or ITSGDITN (SEQ ID NO: 4), or comprises an amino acid sequence having more than 90% homology with the amino acid sequence shown in ITSX4GX5TV (SEQ ID NO: 3) or ITSGDITN (SEQ ID NO: 4); The amino acid sequence of the CDR3 comprises an amino acid sequence as shown in NARPGAVNSY (SEQ ID NO: 5), NSRIYTRSY (SEQ ID NO: 6) or NARPRGLSFSSY (SEQ ID NO: 7), or comprises an amino acid sequence having more than 90% homology with the amino acid sequence as shown in NARPGAVNSY (SEQ ID NO: 5), NSRIYTRSY (SEQ ID NO: 6) or NARPRGLSFSSY (SEQ ID NO: 7); wherein X1 is selected from I, S, F, T or V; X2 is selected from I, T or A; X3 is selected from G, N, S, V, L, A, D, Y or I; X4 is selected from G, A, S, D or T; X5 is selected from S, D, I, T, V or M.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: in: X1 is selected from F or I; X2 is selected from T or I; X3 is selected from S or G; X4 is selected from S or G; X5 is selected from D or S.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that: The amino acid sequence of the CDR1 comprises the amino acid sequence shown in GFTFSSAW (SEQ ID NO: 15), GTIFSDSF (SEQ ID NO: 2) or GIIFSGAW (SEQ ID NO: 17), or comprises an amino acid sequence having more than 90% homology with the amino acid sequence shown in GFTFSSAW (SEQ ID NO: 15), GTIFSDSF (SEQ ID NO: 2) or GIIFSGAW (SEQ ID NO: 17); The amino acid sequence of the CDR2 comprises the amino acid sequence shown in ITSSGDTV (SEQ ID NO: 18), ITSGGSTV (SEQ ID NO: 19) or ITSGDITN (SEQ ID NO: 4), or comprises an amino acid sequence having more than 90% homology with the amino acid sequence shown in ITSSGDTV (SEQ ID NO: 18), ITSGGSTV (SEQ ID NO: 19) or ITSGDITN (SEQ ID NO: 4); The amino acid sequence of the CDR3 comprises the amino acid sequence shown in NARPGAVNSY (SEQ ID NO: 5), NSRIYTRSY (SEQ ID NO: 6) or NARPRGLSFSSY (SEQ ID NO: 7), or comprises an amino acid sequence having more than 90% homology with the amino acid sequence shown in NARPGAVNSY (SEQ ID NO: 5), NSRIYTRSY (SEQ ID NO: 6) or NARPRGLSFSSY (SEQ ID NO: 7).

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that: The antibody or antigen-binding fragment thereof includes any one of the following groups: A) the CDR1 comprises GFTFSSAW (SEQ ID NO: 15), or comprises an amino acid sequence having more than 90% homology to the amino acid sequence of GFTFSSAW (SEQ ID NO: 15); the CDR2 comprises ITSSGDTV (SEQ ID NO: 18), or comprises an amino acid sequence having more than 90% homology to the amino acid sequence of ITSSGDTV (SEQ ID NO: 18); the CDR3 comprises NARPGAVNSY (SEQ ID NO: 5), or comprises an amino acid sequence having more than 90% homology to the amino acid sequence of NARPGAVNSY (SEQ ID NO: 5); B) the CDR1 comprises GTIFSDSF (SEQ ID NO: 2), or comprises an amino acid sequence having more than 90% homology to the amino acid sequence of GTIFSDSF (SEQ ID NO: 2); the CDR2 comprises ITSGGSTV (SEQ ID NO: 19), or comprises an amino acid sequence having more than 90% homology to the amino acid sequence of ITSGGSTV (SEQ ID NO: 19); the CDR3 comprises NSRIYTRSY (SEQ ID NO: 6), or comprises an amino acid sequence having more than 90% homology to the amino acid sequence of NSRIYTRSY (SEQ ID NO: 6); or, C) The CDR1 comprises GIIFSGAW (SEQ ID NO: 17), or comprises an amino acid sequence having more than 90% homology to the amino acid sequence shown by GIIFSGAW (SEQ ID NO: 17); the CDR2 comprises ITSGDITN (SEQ ID NO: 4), or comprises an amino acid sequence having more than 90% homology to the amino acid sequence shown by ITSGDITN (SEQ ID NO: 4); the CDR3 comprises NARPRGLSFSSY (SEQ ID NO: 7), or comprises an amino acid sequence having more than 90% homology to the amino acid sequence shown by NARPRGLSFSSY (SEQ ID NO: 7).

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that: The antibody or antigen-binding fragment thereof includes 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 antigen-binding fragment thereof according to any one of claims 1 to 5, characterized in that: The antibody or antigen-binding fragment thereof binds to IL13Rα2 protein; Preferably, the IL13Rα2 protein includes IL13Rα2 protein from human or non-human animals.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, characterized in that: The amino acid sequence of the antibody or antigen-binding fragment thereof comprises an amino acid sequence as shown in any one of SEQ ID NOs: 8-9 or 16, or comprises an amino acid sequence having more than 90% homology with an amino acid sequence as shown in any one of SEQ ID NOs: 8-9 or 16.

8. A nucleic acid, characterized in that The nucleic acid encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7; Preferably, the nucleic acid comprises a nucleotide sequence shown in any one of SEQ ID NOs: 10-12 or a nucleotide sequence having more than 90% homology with a nucleotide sequence shown in any one of SEQ ID NOs: 10-12.

9. An expression vector or host cell, characterized in that: The expression vector or host cell comprises the nucleic acid according to claim 8.

10. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises an extracellular antigen binding domain, and the extracellular antigen binding domain comprises the antibody or antigen binding fragment thereof according to any one of claims 1 to 7.

11. The chimeric antigen receptor according to claim 10, characterized in that 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.

12. The chimeric antigen receptor according to claim 11, characterized in that The transmembrane domain is selected from one or a combination of two or more of 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 selected from one or a combination of two or more of 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 the antibody or antigen-binding fragment thereof according to any one of claims 1-7, or the chimeric antigen receptor according to any one of claims 10-14.

16. The immune cell according to claim 15, characterized in that The immune cells are selected from one or more of lymphocytes (such as T cells, B cells, NK cells), dendritic cells, monocytes / macrophages, granulocytes or mast cells; Preferably, the immune cells are CAR-T cells.

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

18. A use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, the nucleic acid according to claim 8, the expression vector or host cell according to claim 9, the chimeric antigen receptor according to any one of claims 10 to 14, or the immune cell according to any one of claims 15 to 16, wherein the use comprises use in preparing a product for diagnosing and / or treating a disease associated with IL13Rα2 expression; Preferably, the diseases associated with IL13Rα2 expression include diseases that require inhibition of IL13Rα2 expression for treatment.

19. The use according to claim 18, characterized in that The 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 cancer, rectal cancer, gastric 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; wherein the leukemia is selected from one or more of acute lymphocytic leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia or chronic myeloid leukemia.

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

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