Anti-TRPV6 monoclonal antibody and its applications

Abstract

The hybridoma cell lines 12CT 9.5.1, 16CT 18.1.1.2 and 16CT 4.1.1.2 are obtained by selecting TRPV6 protein characteristic polypeptide antigen, immunizing mice and screening, and the cells of this series secrete monoclonal antibodies against TRPV6, which can specifically recognize TRPV6 protein and tumor tissue. The antibodies of this series can detect the TRPV6 level in cells by immunohistochemistry (IHC), enzyme-linked immunosorbent (ELISA), immunofluorescence (IF), chemiluminescence (CLIA), immunoturbidimetric (TIA) or Western Blot methods artificially or automatically, thereby being used in the diagnosis method of tumor tissue, which belongs to the field of bioassay.

Description

[Technical field]

[0001] The present invention relates to a series of antibodies that can recognize human TRPV6 protein, and hybridoma cell lines that can secrete said series of antibodies. Specifically, the present invention provides a series of monoclonal antibodies that specifically bind to TRPV6 molecules in tumor cell membrane / plasma, and determines the amino acid sequences of the heavy and light chain variable regions of said antibodies and the DNA sequences that code for said variable regions, and said antibodies or their antigen fragments can be artificially or automatically used to detect the TRPV6 level in cells by immunohistochemistry (IHC), enzyme-linked immunosorbent (ELISA), immunofluorescence (IF), chemiluminescence (CLIA), immunoturbidimetric (TIA) or Western Blot methods, thereby being used in the diagnosis method of these tumors, which belongs to the field of bioassays. [Background technology]

[0002] Transient receptor potential cation channel subfamily V member 6, or TRPV6, is a highly selective transmembrane channel for calcium ions, which mediates the active transport of calcium ions from outside the cell to inside the cell. TRPV6 is expressed in normal human kidneys, stomach and intestinal tracts, pancreas, mammary glands, salivary glands, etc., but is mainly expressed in intestinal epithelial cells, and is involved in the transport of calcium ions into cells. Therefore, when the number or function of TRPV6 channels changes, it can cause changes in calcium ion regulation, and further cause abnormalities in the structure or function of related tissues and organs.

[0003] Compared with normal tissues, the expression of TRPV6 is significantly increased in malignant tumors such as breast cancer, cholangiocarcinoma, ovarian cancer, lung squamous cell carcinoma, and prostate cancer, and its abnormal expression may be related to the formation and progression of tumors. Prevarskaya's team found that calcium ion uptake in human prostate cancer LNCap cells, which highly express TRPV6, is mediated by TRPV6, and when the level of TRPV6 protein is reduced by siRNA treatment, the number of cells in the S phase is reduced and cell proliferation is inhibited. Lidocaine can also be used to treat human breast cancer MDA-MB-231 cells, prostate cancer PC-3 cells, and ovarian cancer ES-2 cells to reduce the levels of TRPV6 mRNA and protein, which inhibits cell proliferation and at the same time reduces the rate of calcium ion entry into the cells, indicating that the inhibition of cancer cells may be related to the reduction of calcium ions and may also be related to the level of TRPV6. As can be seen, TRPV6 is closely related to many types of human tumors and may be related to the regulation of calcium ion concentration changes inside and outside the cells.

[0004] As a tumor cell marker, its detection is of great significance for pathological diagnosis and the use of corresponding targeted drugs. At present, there is no anti-TRPV6 antibody for diagnosis on the market, and the antibody of this patent can fill this gap. Summary of the Invention

[0005] According to one aspect of the present application, there is provided an antibody or antigen-binding fragment thereof that specifically binds to TRPV6, wherein the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in a heavy chain variable region as follows: a heavy chain variable region shown in SEQ ID NO:25, a heavy chain variable region shown in SEQ ID NO:27, or a heavy chain variable region shown in SEQ ID NO:29. In some embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3 selected from the group consisting of HCDR1 as set forth in SEQ ID NO:7, HCDR2 as set forth in SEQ ID NO:8, HCDR3 as set forth in SEQ ID NO:9, HCDR1 as set forth in SEQ ID NO:13, HCDR2 as set forth in SEQ ID NO:14, HCDR3 as set forth in SEQ ID NO:15, or HCDR1 as set forth in SEQ ID NO:19, HCDR2 as set forth in SEQ ID NO:20, and HCDR3 as set forth in SEQ ID NO:21.

[0006] In some embodiments, the antibody or antigen-binding fragment thereof further comprises three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the following light chain variable region: the light chain variable region depicted in SEQ ID NO:26, the light chain variable region depicted in SEQ ID NO:28, or the light chain variable region depicted in SEQ ID NO:30. In some embodiments, the antibody or antigen-binding fragment thereof further comprises three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3 selected from the group consisting of LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:17, and LCDR3 shown in SEQ ID NO:18, LCDR1 shown in SEQ ID NO:22, LCDR2 shown in SEQ ID NO:23, and LCDR3 shown in SEQ ID NO:24.

[0007] In some embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and three light chain complementarity determining regions LCDR1, LCDR2 and LCDR3 selected from the group consisting of HCDR1 as set forth in SEQ ID NO:7, HCDR2 as set forth in SEQ ID NO:8, HCDR3 as set forth in SEQ ID NO:9, LCDR1 as set forth in SEQ ID NO:10, LCDR2 as set forth in SEQ ID NO:11 and LCDR3 as set forth in SEQ ID NO:12, HCDR1 as set forth in SEQ ID NO:13, HCDR2 as set forth in SEQ ID NO:14, HCDR3 as set forth in SEQ ID NO:15, LCDR1 as set forth in SEQ ID NO:16, LCDR2 as set forth in SEQ ID NO:17 and LCDR3 as set forth in SEQ ID NO:18, HCDR1 as set forth in SEQ ID NO:19, HCDR2 as set forth in SEQ ID NO:20, HCDR3 as set forth in SEQ ID NO:21, HCDR1 as set forth in SEQ ID NO:22, HCDR2 as set forth in SEQ ID NO:23, HCDR1 as set forth in SEQ ID NO:24, HCDR3 as set forth in SEQ ID NO:25, HCDR1 as set forth in SEQ ID NO:26, HCDR2 as set forth in SEQ ID NO:27 and HCDR3 as set forth in SEQ ID NO:28. HCDR3 shown in SEQ ID NO:21, LCDR1 shown in SEQ ID NO:22, LCDR2 shown in SEQ ID NO:23 and LCDR3 shown in SEQ ID NO:24.

[0008] In some embodiments, the antibody or antigen-binding fragment thereof comprises an HCDR1 as set forth in SEQ ID NO:7, an HCDR2 as set forth in SEQ ID NO:8, an HCDR3 as set forth in SEQ ID NO:9, an LCDR1 as set forth in SEQ ID NO:10, an LCDR2 as set forth in SEQ ID NO:11, and an LCDR3 as set forth in SEQ ID NO:12.

[0009] In some embodiments, the antibody or antigen-binding fragment thereof comprises an HCDR1 as set forth in SEQ ID NO:13, an HCDR2 as set forth in SEQ ID NO:14, an HCDR3 as set forth in SEQ ID NO:15, an LCDR1 as set forth in SEQ ID NO:16, an LCDR2 as set forth in SEQ ID NO:17, and an LCDR3 as set forth in SEQ ID NO:18.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof comprises an HCDR1 as set forth in SEQ ID NO:19, an HCDR2 as set forth in SEQ ID NO:20, an HCDR3 as set forth in SEQ ID NO:21, an LCDR1 as set forth in SEQ ID NO:22, an LCDR2 as set forth in SEQ ID NO:23, and an LCDR3 as set forth in SEQ ID NO:24.

[0011] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region V selected from the group consisting of: H i.e., a heavy chain variable region set forth in SEQ ID NO:25, a heavy chain variable region set forth in SEQ ID NO:27, or a heavy chain variable region set forth in SEQ ID NO:29.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a light chain variable region V selected from the group consisting of: L i.e., a light chain variable region set forth in SEQ ID NO:26, a light chain variable region set forth in SEQ ID NO:28, or a light chain variable region set forth in SEQ ID NO:30.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region V selected from the group consisting of: H and the light chain variable region V L a heavy chain variable region set forth in SEQ ID NO:25 and a light chain variable region set forth in SEQ ID NO:26; a heavy chain variable region set forth in SEQ ID NO:27 and a light chain variable region set forth in SEQ ID NO:28; or a heavy chain variable region set forth in SEQ ID NO:29 and a light chain variable region set forth in SEQ ID NO:30.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO:25 and a light chain variable region set forth in SEQ ID NO:26.

[0015] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO:27 and a light chain variable region set forth in SEQ ID NO:28.

[0016] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO:29 and a light chain variable region set forth in SEQ ID NO:30.

[0017] In some embodiments, the antibody or antigen-binding fragment thereof specifically recognizes or binds to a protein having the amino acid sequence shown in SEQ ID NO:2.

[0018] In some embodiments, the Kd value of the antibody or antigen-binding fragment thereof and the protein having the amino acid sequence set forth in SEQ ID NO:2 is 10 -7 ~10 -10 In some embodiments, the Kd value is 10 -7 ~10 -10 M, 10 -8 ~10 -10 M or 10 -9 ~10 -10 In some embodiments, the Kd value of the antibody or antigen-binding fragment thereof and the protein having the amino acid sequence set forth in SEQ ID NO:2 is 10 -7 In some embodiments, the Kd value is less than 10 -7 Less than M, 10 -8 Less than M, 10 -9 Less than M or 10 -10 In some embodiments, the Kd value is detected by surface plasmon resonance.

[0019] In some embodiments, the antigen-binding fragment is selected from a F(ab')2, a Fab', a Fab, an Fv, a scFv, a dsFv, or a dAb.

[0020] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and / or a light chain constant region. In some embodiments, the heavy chain constant region is derived from mouse IgG1 or mouse IgG2a. In some embodiments, the heavy chain constant region is derived from human IgG1.

[0021] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a conjugate moiety. In some embodiments, the conjugate moiety is a therapeutic agent, a radioisotope, a detectable label, a pharmacokinetically modified moiety, or a purified moiety. The conjugate moiety is directly linked or linked via a linker.

[0022] According to one aspect, the present application further provides an isolated nucleic acid, which encodes an antibody or antigen-binding fragment thereof that specifically binds to TRPV6, as described herein.

[0023] In some embodiments, the nucleic acid comprises one or both of the nucleic acid sequence set forth in SEQ ID NO:31 or a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:31 and the nucleic acid sequence set forth in SEQ ID NO:32 or a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:32. In some embodiments, the nucleic acid comprises one or both of the nucleic acid sequence set forth in SEQ ID NO:33 or a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:33 and the nucleic acid sequence set forth in SEQ ID NO:34 or a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:34. In some embodiments, the nucleic acid comprises one or both of the nucleic acid sequence set forth in SEQ ID NO:35 or a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:35 and the nucleic acid sequence set forth in SEQ ID NO:6 or a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:6.

[0024] According to one aspect, the present application further provides a vector, which comprises a nucleic acid sequence described herein.

[0025] According to one aspect, the present application further provides a host cell, which comprises the vector described herein.

[0026] According to one aspect, the present application further provides a multispecific antibody, which comprises an antibody or antigen-binding fragment thereof described herein and one or more antibodies or antigen-binding portions thereof that specifically bind to other antigens.

[0027] According to one aspect, the present application further provides an antibody conjugate, which comprises an antibody or an antigen-binding fragment thereof described herein, or a multispecific antibody described herein.

[0028] According to one aspect, the present application further provides a hybridoma cell line secreting an anti-TRPV6 monoclonal antibody, the hybridoma cell line being 12CT9.5.1, 16CT4.1.1.2 and / or 16CT18.1.1.2, preserved in the China Center for Typical Culture Collection, where the preservation number of 12CT9.5.1 is CCTCC NO: C202119, the preservation number of 16CT4.1.1.2 is CCTCC NO: C202124, and the preservation number of 16CT18.1.1.2 is CCTCC NO: C202125.

[0029] According to one aspect, the present application further provides a pharmaceutical composition, comprising an antibody or antigen-binding fragment thereof described herein, an isolated nucleic acid described herein, a vector described herein, a host cell described herein, a multispecific antibody described herein, or an antibody conjugate described herein, and a pharma- ceutically acceptable vector.

[0030] According to one aspect, the present application further provides a reagent for analyzing and detecting TRPV6 in a sample from a subject, the reagent comprising one or more antibodies or antigen-binding fragments thereof described herein. In some embodiments, the reagent is used to analyze and detect TRPV6 in a sample from a subject.

[0031] According to one aspect, the present application further provides a method for detecting TRPV6 in a sample from a subject, the method comprising: (1) contacting the sample with an antibody or antigen-binding fragment described in the present application; (2) optionally removing unbound antibody or antibody fragment; and (3) detecting antibody or antibody fragment bound to TRPV6 in the sample.

[0032] In some embodiments, in step (1) of the method for detecting TRPV6 in a sample from a subject, an antibody or antigen-binding fragment described herein is immobilized on a substrate. In some embodiments, in step (1) of the method for detecting TRPV6 in a sample from a subject, the sample is immobilized on a substrate.

[0033] In some embodiments, step (3) of the method for detecting TRPV6 in a sample from a subject further comprises using a secondary antibody, optionally the secondary antibody capable of directly or indirectly binding to TRPV6 in the sample, optionally the epitope of the secondary antibody is different from the epitope of an antibody or antigen-binding fragment described in the present application, optionally the epitope of the secondary antibody is the same as the epitope of an antibody or antigen-binding fragment described in the present application, optionally the binding of the epitope of the secondary antibody to TRPV6 is not affected by the antibody or antigen-binding fragment described in the present application, and optionally the binding of the epitope of the secondary antibody to TRPV6 is affected by the antibody or antigen-binding fragment described in the present application.

[0034] In some embodiments, step (3) of the method for detecting TRPV6 in a sample from a subject further comprises using a secondary antibody, optionally capable of binding directly or indirectly to an antibody or antigen-binding fragment described herein, and optionally, the secondary antibody is capable of binding to a constant region portion of an antibody or antigen-binding fragment described herein.

[0035] In some embodiments, the secondary antibody is coupled to a detection molecule, optionally comprising an enzyme, a fluorescent label and biotin. In some embodiments, the detection molecule is an enzyme, optionally comprising horseradish peroxidase, alkaline phosphatase or a derivative thereof. In some embodiments, the detection molecule is fluorescein, optionally comprising FITC, rhodamine, Texas Red, phycoerythrin or Dylight. In some embodiments, the detection molecule is biotin or a derivative thereof.

[0036] In some embodiments, the method of detecting TRPV6 in a sample from a subject further comprises quantifying antibody bound to TRPV6 in the sample. In some embodiments, the method is immunohistochemistry (IHC), immunofluorescence (IF), enzyme-linked immunosorbent (ELISA), chemiluminescent immunoassay (CLIA), turbidimetric inhibition immunoassay (TIA), or Western Blot. In some embodiments, the method can assess the proportion of cells in the sample that express TRPV6 on their cell surface.

[0037] In some embodiments, in the method of detecting TRPV6 in a sample from a subject, the subject is a human.

[0038] According to one aspect, the present application further provides a method for determining the formation or risk of formation of a subject disease, assessing the progression or prognosis of a subject disease, or predicting or monitoring the subject's response to treatment in a subject undergoing treatment for a disease, the method comprising detecting TRPV6 in a sample derived from the subject by a method for detecting TRPV6 in a sample derived from the subject described in the present application.

[0039] In some embodiments, a method for determining the formation or risk of formation of a subject disease, assessing the progression or prognosis of a subject disease, or predicting or monitoring the response of the subject to treatment in a subject undergoing treatment for a disease comprises assessing the proportion of cells in the sample that express TRPV6 on the cell surface, and if the proportion exceeds a predetermined threshold, the subject is more likely to have the disease or to benefit from treatment, where the threshold is 10%. In some embodiments, the treatment is a targeted drug treatment.

[0040] In some embodiments, the disease described in the present application is a cancer disease. In some embodiments, the cancer disease is breast cancer, ovarian cancer, endometrial cancer, bile duct cancer, gastric cancer, esophageal cancer, lung cancer, intestinal cancer, pancreatic cancer.

[0041] According to one aspect, the present application further provides a kit, the kit comprising an antibody or antigen-binding fragment thereof described in the present application, and optionally further comprising a reagent for detecting the binding status of the antibody or antigen-binding fragment thereof to TRPV6.

[0042] In some embodiments, the kits described herein are used to diagnose the formation or risk of formation of a disease in a subject, assess the progression or prognosis of a disease in a subject, or predict or monitor the response of said subject to treatment in a subject undergoing treatment for a disease. In some embodiments, the kits described herein are used for pathological diagnosis by immunohistochemistry. In some embodiments, the kits described herein are used for pathological diagnosis by immunohistochemistry of tumor tissue. In some embodiments, the disease is a cancer disease, and in some embodiments, the cancer disease is breast cancer, ovarian cancer, endometrial cancer, bile duct cancer, gastric cancer, esophageal cancer, lung cancer, intestinal cancer, pancreatic cancer.

[0043] According to one aspect, the present application further provides a method for producing an antibody that specifically binds to TRPV6, the method comprising: (1) coupling the amino acid sequence shown in SEQ ID NO:3 with a vector protein to obtain a TRPV6 antigen peptide; (2) immunizing a mouse with the TRPV6 antigen peptide obtained by step (1) as an immunogen; (3) obtaining a positive hybridoma cell line that highly secretes an antibody that specifically binds to TRPV6 through cell fusion and clonal screening of the immunized peptide; and (4) obtaining an antibody that specifically binds to TRPV6.

[0044] In some embodiments, the vector protein is a keyhole limpet hemocyanin (KLH) protein.

[0045] In some embodiments, the amino acid sequence shown in SEQ ID NO:3 has one additional Cys at its N-terminus and is coupled to the vector protein via a free thiol group.

[0046] In some embodiments, the hybridoma cell lines described in the present application are the murine hybridoma cell lines 12CT 9.5.1, 16CT 18.1.1.2 and 16CT 4.1.1.2.

[0047] According to one aspect, the present application further provides the use of the antibody or antigen-binding fragment thereof described in the present application, the isolated nucleic acid described in the present application, the vector described in the present application, the host cell described in the present application, the multispecific antibody described in the present application, the antibody conjugate described in the present application and the pharmaceutical composition described in the present application in medicine for treating and / or preventing and / or diagnosing a disease associated with TRPV6 levels.

[0048] According to one aspect, the present application further provides a method of treating, preventing or diagnosing a disease, the method comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof described herein, a multispecific antibody described herein, an antibody conjugate described herein, and a pharmaceutical composition described herein. The drawings form a part of this specification and illustrate several aspects of the present disclosure, and the present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments described herein. [Brief description of the drawings]

[0049] [Figure 1A] It has been shown that CB-Anti-0001 can chemically specifically detect TRPV6 protein in TRPV6 plasmid-transfected 293T cells via immune cells (left panel), but has no indication of TRPV6 protein detection in non-TRPV6 plasmid-transfected 293T cells (right panel). [Figure 1B] It has been shown that CB-Anti-0002 can chemically specifically detect TRPV6 protein in TRPV6 plasmid-transfected 293T cells via immune cells (left panel), but has no indication of TRPV6 protein detection in non-TRPV6 plasmid-transfected 293T cells (right panel). [Figure 1C]It has been shown that CB-Anti-0003 can chemically specifically detect TRPV6 protein in TRPV6 plasmid-transfected 293T cells via immune cells (left panel), but has no indication of TRPV6 protein detection in non-TRPV6 plasmid-transfected 293T cells (right panel). [Figure 2A] The results of immunohistochemical detection using CB-Anti-0001 antibody are shown. [Figure 2B] The results of immunohistochemical detection using CB-Anti-0002 antibody are shown. [Figure 2C] The results of immunohistochemical detection using CB-Anti-0003 antibody are shown. [Diagram 3] The IHC comparative results of ACC-028 antibody, MABN839 antibody and CB-Anti-0001 antibody on human bladder transitional cell papilloma cells (RT4) are shown. Here, Figure 3A is the IHC result of ACC-028 antibody, Figure 3B is the IHC result of MABN839 antibody, and Figure 3C is the IHC result of CB-Anti-0001 antibody. As can be seen from the figure, the cell membrane in Figure 3C has a clear and significant staining effect. [Figure 4] It is shown that there is no significant change in the tissue staining intensity and staining ratio of the antibody stored under different conditions. Here, Figure 4A shows the IHC result of CB-Anti-0001 antibody stock solution stored at 4°C for 14 days, Figure 4B shows the IHC result of CB-Anti-0001 antibody stock solution stored at 37°C for 14 days, and Figure 4C shows the IHC result of CB-Anti-0001 antibody working solution stored at 37°C for 14 days. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] The following description is merely for illustrating various embodiments of the present application. Therefore, the specific embodiments herein should not be interpreted as limitations on the scope of the application. Those skilled in the art can easily obtain multiple equivalent forms and modifications based on the spirit of the present invention and the description in this specification, and these equivalent embodiments should be understood to be included in the scope of the present invention. All documents, including publications, patents, and patent applications, cited in this application are incorporated by reference in their entirety into this application.

[0051] As used herein, the singular forms "a," "one," "an," "an kind," and "the" or "said" include plural inclusions unless the context clearly indicates otherwise.

[0052] As used in this application, terms such as "comprises," "including," "contains," "containing," and "having" are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. As used in this application, the term "comprising" is closed-ended.

[0053] The applicant has deposited hybridoma cell lines (lines) 12CT9.5.1, 16CT4.1.1.2 and 16CT18.1.1.2 in the China Center for Typical Culture Collection. The storage number of 12CT9.5.1 is CCTCC NO: C202119, the storage number of 16CT4.1.1.2 is CCTCC NO: C202124, and the storage number of 16CT18.1.1.2 is CCTCC NO: C202125. The storage address of the three hybridoma cell lines is Wuhan University, China, the storage date is January 13, 2021, and the storage location is the China Center for Typical Culture Collection.

[0054] One aspect of the present application discloses an antibody or antigen-binding fragment thereof that specifically binds to TRPV6, wherein the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in a heavy chain variable region as follows: the heavy chain variable region shown in SEQ ID NO:25, the heavy chain variable region shown in SEQ ID NO:27, or the heavy chain variable region shown in SEQ ID NO:29. In some embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3 selected from the group consisting of HCDR1 as set forth in SEQ ID NO:7, HCDR2 as set forth in SEQ ID NO:8, HCDR3 as set forth in SEQ ID NO:9, HCDR1 as set forth in SEQ ID NO:13, HCDR2 as set forth in SEQ ID NO:14, HCDR3 as set forth in SEQ ID NO:15, or HCDR1 as set forth in SEQ ID NO:19, HCDR2 as set forth in SEQ ID NO:20, and HCDR3 as set forth in SEQ ID NO:21. In some embodiments, the antibody or antigen-binding fragment thereof of the present application further comprises three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the following light chain variable region: the light chain variable region depicted in SEQ ID NO:26, the light chain variable region depicted in SEQ ID NO:28, or the light chain variable region depicted in SEQ ID NO:30. In some embodiments, the antibody or antigen-binding fragment thereof of the present application further comprises three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3 selected from the group consisting of LCDR1 shown in SEQ ID NO:10, LCDR2 shown in SEQ ID NO:11, and LCDR3 shown in SEQ ID NO:12, LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:17, and LCDR3 shown in SEQ ID NO:18, LCDR1 shown in SEQ ID NO:22, LCDR2 shown in SEQ ID NO:23, and LCDR3 shown in SEQ ID NO:24.

[0055] In some embodiments, the antibody or antigen-binding fragment thereof of the present application comprises an HCDR1 as set forth in SEQ ID NO:7, an HCDR2 as set forth in SEQ ID NO:8, an HCDR3 as set forth in SEQ ID NO:9, an LCDR1 as set forth in SEQ ID NO:10, an LCDR2 as set forth in SEQ ID NO:11, and an LCDR3 as set forth in SEQ ID NO:12. In some embodiments, the antibody or antigen-binding fragment thereof of the present application comprises an HCDR1 as set forth in SEQ ID NO:13, an HCDR2 as set forth in SEQ ID NO:14, an HCDR3 as set forth in SEQ ID NO:15, an LCDR1 as set forth in SEQ ID NO:16, an LCDR2 as set forth in SEQ ID NO:17, and an LCDR3 as set forth in SEQ ID NO:18. In some embodiments, the antibody or antigen-binding fragment thereof of the present application comprises an HCDR1 as set forth in SEQ ID NO:19, an HCDR2 as set forth in SEQ ID NO:20, an HCDR3 as set forth in SEQ ID NO:21, an LCDR1 as set forth in SEQ ID NO:22, an LCDR2 as set forth in SEQ ID NO:23, and an LCDR3 as set forth in SEQ ID NO:24.

[0056] In some embodiments, the antibody or antigen-binding fragment thereof of the present application comprises a heavy chain variable region V selected from the group consisting of: H In some embodiments, the antibody or antigen-binding fragment thereof further comprises a light chain variable region V selected from the group consisting of: L i.e., a light chain variable region set forth in SEQ ID NO:26, a light chain variable region set forth in SEQ ID NO:28, or a light chain variable region set forth in SEQ ID NO:30.

[0057] In some embodiments, the antibody or antigen-binding fragment thereof of the present application comprises a heavy chain variable region set forth in SEQ ID NO:25 and a light chain variable region set forth in SEQ ID NO:26. In some embodiments, the antibody or antigen-binding fragment thereof of the present application comprises a heavy chain variable region set forth in SEQ ID NO:27 and a light chain variable region set forth in SEQ ID NO:28. In some embodiments, the antibody or antigen-binding fragment thereof of the present application comprises a heavy chain variable region set forth in SEQ ID NO:29 and a light chain variable region set forth in SEQ ID NO:30.

[0058] The term "antibody" as used in this application includes any immunoglobulin, monoclonal antibody, polyvalent antibody, multispecific antibody or bispecific (bivalent) antibody. A native intact antibody contains two heavy chains and two light chains linked together via disulfide bonds. Each heavy chain of an antibody contains one variable region (V H ) and the first, second and third constant regions (C H1 , C H2 , C H3 Each light chain of an antibody consists of one variable region (V L ) and one constant region (C L ). The light and heavy chain variable regions are involved in antigen binding. The variable region in each chain is broadly divided into three hypervariable regions, called complementarity determining regions (CDRs) (here, the light chain CDR includes LCDR1, LCDR2, and LCDR3, and the heavy chain CDR includes HCDR1, HCDR2, and HCDR3).

[0059] As used herein, an "antigen-binding fragment" of an antibody, an "antigen-binding portion" of an antibody, etc., includes any naturally occurring, enzymatically derived, synthetic, or genetically engineered polypeptide that specifically binds to an antigen to form a complex. An antigen-binding fragment of an antibody may be derived from an intact antibody molecule, for example, by using any suitable standard technique, such as proteolytic digestion, or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the variable and optional constant domains of an antibody. The DNA may be sequenced and manipulated by the use of chemical methods or molecular biology techniques, for example, to rearrange one or more variable and / or constant domains in the appropriate configuration, or to introduce codons to create cysteine ​​residues, modify, add or delete amino acids, etc. Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single chain Fv (scFv) molecules, (vi) dAb fragments, (vii) minimal recognition units consisting of amino acid residues mimicking the hypervariable regions of an antibody (e.g., isolated complementarity determining regions (CDRs), e.g., CDR3 peptides) or FR3-CDR3-FR4 peptides. Other remodeled molecules, such as domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs) and shark mutant IgNAR domains are also included within the expression "antigen-binding fragment" as used herein.

[0060] The CDR boundaries of the antibodies and antigen-binding fragments disclosed in this application may be named or recognized according to the Kabat, Chothia or Al-Lazikani nomenclature systems (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J Mol. Biol. Dec 5; 186(3): 651-63 (1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196, 901 (1987); Chothia, C. et al., Nature. Dec 21-28; 31(6252): 877-83 (1989); Kabat EA et al., National Institutes of Health, Bethesda, Md. (1991)). In some embodiments, the CDR boundaries of the antibody are determined based on the Kabat database. The three CDRs are flanked by continuous portions of what are called framework regions (FRs, where the heavy chain FRs include HFR1, HFR2, HFR3, and HFR4, and the light chain FRs include LFR1, LFR2, LFR3, and LFR4), which are more highly conserved than the CDRs and form stents to support the hypervariable loops. Thus, the V H and V L Each of the heavy and light chain constant regions contains three CDRs and four FRs arranged in the following order (human amino acid residues from N-terminus to C-terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy and light chain constant regions are not involved in antigen binding, but exhibit various effector functions. Mammalian heavy chains are classified as α, δ, ε, γ, and μ, and mammalian light chains are classified as λ or κ. Antibodies are broadly classified into five classes, IgA, IgD, IgE, IgG, and IgM, based on the amino acid sequence of their heavy chain constant regions, based on the presence or absence of α, δ, ε, γ, and μ, respectively. Subclasses of some major antibody classes include, for example, IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0061] In some embodiments, the antibody or antigen-binding fragment thereof of the present application specifically recognizes or binds to a protein having the amino acid sequence shown in SEQ ID NO:2.

[0062] SEQ ID NO:2: [Table 1]

[0063] In some embodiments, the Kd value of the antibody or antigen-binding fragment thereof of the present application and a protein having the amino acid sequence shown in SEQ ID NO:2 is 10 -7 ~10 -10 In some embodiments, the Kd value is 10 -7 ~10 -10 M, 10 -8 ~10 -10 M or 10 -9 ~10 -10 In some embodiments, the Kd value of the antibody or antigen-binding fragment thereof and the protein having the amino acid sequence set forth in SEQ ID NO:2 is 10 -7 In some embodiments, the Kd value is less than 10 -7 Less than M, 10 -8 Less than M, 10 -9 Less than M or 10 -10 In some embodiments, the Kd value is detected by surface plasmon resonance.

[0064] In some embodiments, the antigen-binding fragment of the application is selected from F(ab')2, Fab', Fab, Fv, scFv, dsFv, or dAb. In some embodiments, the antibody or antigen-binding fragment thereof of the application further comprises a heavy chain constant region and / or a light chain constant region. In some embodiments, the heavy chain constant region is derived from mouse IgG1 or mouse IgG2a. In some embodiments, the heavy chain constant region is derived from human IgG1.

[0065] In some embodiments, the antibody or antigen-binding fragment thereof of the present application further comprises a conjugate moiety. In some embodiments, the conjugate moiety is a therapeutic agent, a radioisotope, a detectable label, a pharmacokinetically modified moiety, or a purified moiety. The conjugate moiety is directly linked or linked via a linker.

[0066] The term "therapeutic agent" as used in this application may refer to any pharmaceutical compound for treating or preventing a disease in a subject, including small molecules with a molecular weight of less than 500, nucleotides (e.g., DNA, plasmid DNA, RNA, siRNA, antisense oligonucleotides, aptamers, etc.), short peptides, and proteins (e.g., enzymes).

[0067] In some embodiments, the radioisotope is selected from the following groups: 3 H, 11 C. 14 C. 18 F, 32 P, 33 P, 35 S, 45 Ti, 47 Sc, 52 Fe, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Sc, 77 As, 86 Y, 89 Sr, 89 Zr, 90 Y, 90 Nb, 94 Tc, 99 Tc, 99 Mo, 105 Pd, 105 Rh, 111 Ag, 111 In, 123 I, 124 I, 125 I, 131 I, 133 Xe, 142 Pr, 143 Pr,149 Pm, 153 Sm, 154 Gd, 155 Gd, 156 Gd, 157 Gd, 158 Gd, 161 Tb, 166 Dy, 169 Er, 175 Lu, 177 Lu, 186 Re, 188 Re, 189 Re, 194 Ir, 198 Au, 199 Au, 211 At, 211 Pb, 212 Bi, 212 Pb, 213 Bi, 223 Ra and 225 Ac.

[0068] The term "detectable label" as used herein may refer to any detection molecule that is suitable or useful for detecting TRPV6 levels while being directly or indirectly coupled to an antibody of the present invention. In some embodiments, the detection molecule comprises an enzyme, a fluorescent label and biotin. The enzyme comprises peroxidase (e.g., horseradish peroxidase), alkaline phosphatase, β-galactosidase, glucoamylase, carbohydrate oxidase, urease, heterocyclic oxidase (e.g., xanthine oxidase), malate dehydrogenase or derivatives thereof. The fluorescent label may be fluorescein, optionally comprising fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin, green fluorescent protein (GFP), Dylight, Cy3 or Cy5.

[0069] According to another aspect, the present application further discloses an isolated nucleic acid, which encodes an antibody or antigen-binding fragment thereof that specifically binds to TRPV6, as described herein. In some embodiments, the nucleic acid comprises one or both of a nucleic acid sequence shown in SEQ ID NO:31 or a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:31 and a nucleic acid sequence shown in SEQ ID NO:32 or a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:32. In some embodiments, the nucleic acid comprises one or both of a nucleic acid sequence shown in SEQ ID NO:33 or a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:33 and a nucleic acid sequence shown in SEQ ID NO:34 or a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:34. In some embodiments, the nucleic acid comprises one or both of the nucleic acid sequence shown in SEQ ID NO:35 or a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:35 and the nucleic acid sequence shown in SEQ ID NO:6 or a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:6.

[0070] According to another aspect, the present application further discloses a vector, which comprises a nucleic acid sequence described in the present application.

[0071] According to another aspect, the present application further discloses a host cell, which comprises the vector described herein.

[0072] According to another aspect, the present application further discloses a multispecific antibody, which comprises an antibody or antigen-binding fragment thereof described in the present application and one or more antibodies or antigen-binding portions thereof that specifically bind to other antigens.

[0073] According to another aspect, the present application further discloses an antibody conjugate, which comprises the antibody or antigen-binding fragment thereof described herein, or the multispecific antibody described herein.

[0074] According to another aspect, the present application further discloses a hybridoma cell line secreting an anti-TRPV6 monoclonal antibody, the hybridoma cell line being 12CT9.5.1, 16CT4.1.1.2 and / or 16CT18.1.1.2, preserved in China Center for Typical Culture Collection, where the preservation number of 12CT9.5.1 is CCTCC NO: C202119, the preservation number of 16CT4.1.1.2 is CCTCC NO: C202124, and the preservation number of 16CT18.1.1.2 is CCTCC NO: C202125.

[0075] According to another aspect, the present application further discloses a pharmaceutical composition, comprising the antibody or antigen-binding fragment thereof described herein, the isolated nucleic acid described herein, the vector described herein, the host cell described herein, the multispecific antibody described herein, or the antibody conjugate described herein, and a pharma- ceutically acceptable vector.

[0076] As used herein, the term "pharmacologically acceptable" means, within the scope of reasonable medical judgment, suitable for contact with the cells of humans and other animals without undue toxicity, irritation, allergic response, and the like, and commensurate with a reasonable benefit / risk ratio.

[0077] The term "pharmaceutically acceptable vector" as used herein refers to a pharmaceutically acceptable solvent, suspending agent, or any other pharmaceutically inert carrier agent for delivering an antibody or antigen-binding fragment thereof, isolated nucleic acid, vector, host cell, multispecific antibody, or antibody complex according to the present application to a subject, which does not affect the structure, form, and properties of the antibody or antigen-binding fragment thereof, isolated nucleic acid, vector, host cell, multispecific antibody, or antibody complex. Some such vectors allow the antibody or antigen-binding fragment thereof, isolated nucleic acid, vector, host cell, multispecific antibody, or antibody complex to be formulated as, for example, a tablet, pill, capsule, liquid, gel, syrup, slurry, suspension, lozenge, etc., for oral administration by a subject. Some such vectors allow the antibody or antigen-binding fragment thereof, isolated nucleic acid, vector, host cell, multispecific antibody, or antibody complex to be formulated as a preparation for injection, infusion, or topical administration.

[0078] Pharmaceutically acceptable vectors for use in pharmaceutical compositions according to the present application can include, for example, pharma- ceutically acceptable liquid, gel, or solid vectors, aqueous carrier agents (e.g., sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection), non-aqueous carrier agents (e.g., fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil), antibacterial agents, isotonicity agents (e.g., sodium chloride or dextrose), buffers (e.g., phosphate or citrate buffers), antioxidants (e.g., sulfate, glycerol, tert-butyl ether ... Examples of suitable ingredients include, but are not limited to, sodium bicarbonate), anesthetics (e.g., procaine hydrochloride), suspending / dispersing agents (e.g., sodium carboxymethylcellulose, hydroxypropylmethylcellulose or polyvinylpyrrolidone), chelating agents (e.g., EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid)), emulsifying agents (e.g., polysorbate 80 (Tween 80)), diluents, adjuvants, excipients, or non-toxic auxiliary substances, other ingredients known in the art, or various combinations thereof. Suitable ingredients may include, for example, fillers, binders, buffers, preservatives, lubricants, flavoring agents, thickening agents, coloring agents, or emulsifying agents.

[0079] In some embodiments, the pharmaceutical composition is an injectable preparation. Injectable preparations include sterile aqueous solutions or dispersions, suspensions or emulsions. Under all circumstances, injectable preparations should be sterile and fluid to facilitate injection. Injectable preparations must be stable under the conditions of production and storage and must be free of contamination by microorganisms such as bacteria and fungi. The vector may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof, and / or vegetable oils. Injectable preparations should maintain a suitable fluidity. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by using a surfactant, and the like. Microbial inhibition can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

[0080] In some embodiments, the pharmaceutical composition is an oral dosage formulation, including, but not limited to, capsules, flat capsules, pills, tablets, lozenges (using a flavored base, typically sucrose and gum arabic or gum tragacanth), powders, granules, or solutions or suspensions in aqueous or non-aqueous liquids, or oil-in-water or water-in-oil liquid emulsions, or elixirs or syrups, or soft lozenges (using an inert base, such as gelatin and glycerin, or sucrose and gum arabic) and / or mouthwashes.

[0081] In solid dosage forms for oral administration (e.g., capsules, tablets, pills, dragees, powders, granules, etc.), the antibody or antigen-binding fragment thereof, isolated nucleic acid, vector, host cell, multispecific antibody or antibody conjugate is mixed with one or more pharma- ceutically acceptable vectors, such as sodium citrate or dicalcium phosphate, and / or any one of the following: (1) a filler or extender, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) a binder, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or alginate; (3) a binder, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or alginate; (4) a binder, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or alginate; (5) a binder, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or alginate; (6) a binder, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or alginate; (7) a binder, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or alginate; (8) a binder, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or alginate; (9) a binder, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or alginate; (10) a binder, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, s labia gum, (3) humectants such as glycerin, (4) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, some silicates and sodium carbonate, (5) solution retardants such as paraffin, (6) absorption accelerators such as quaternary ammonium compounds, (7) wetting agents such as acetyl alcohols and monoglycerides, (8) absorbents such as kaolin and bentonite, (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof, and (10) colorants.

[0082] In liquid dosage forms for oral administration, the antibody or antigen-binding fragment thereof, isolated nucleic acid, vector, host cell, multispecific antibody or antibody conjugate is mixed with any one of pharma- ceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the antibody or antigen-binding fragment thereof, isolated nucleic acid, vector, host cell, multispecific antibody or antibody conjugate, the liquid dosage form may contain an inert diluent commonly used in the art, such as water or other solvents, solubilizing agents and emulsifying agents, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, isopropyl alcohol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycols and sorbitan fatty acid esters, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0083] In some embodiments, the pharmaceutical composition is an oral spray formulation or a nasal spray formulation. Spray formulations include, but are not limited to, aqueous aerosols, non-aqueous suspensions, liposome formulations, or solid granule formulations. Aqueous aerosols are prepared by mixing an aqueous solution or suspension of the drug with a pharma- ceutically acceptable general vector and stabilizer. The vector and stabilizer vary based on the needs of the specific drug component, but generally include non-ionic surfactants (Tween or polyethylene glycol), oleic acid, lecithin, amino acids such as glycine, buffer solutions, salts, sugars or sugar alcohols. Aerosols are generally prepared with isotonic solutions and can be delivered by spraying.

[0084] In some embodiments, the pharmaceutical composition may be used in combination with one or more other pharmaceutical agents. In some embodiments, the pharmaceutical composition contains at least one other pharmaceutical agent. In some embodiments, the other pharmaceutical agent is an anti-neoplastic agent, a cardiovascular agent, an anti-inflammatory agent, an anti-viral agent, a gastrointestinal agent, a nervous system agent, a respiratory system agent, an immune system agent, a skin disease agent, a metabolic agent, or the like.

[0085] In some embodiments, the pharmaceutical composition may be administered to a subject in need via a suitable route, including, but not limited to, oral administration, injection (e.g., intravenous, intramuscular, subcutaneous, intradermal, intracardiac, intraspinal, intrapleural, intraperitoneal injection, etc.), mucosal (e.g., intranasal, buccal, etc.), sublingual, rectal, transdermal, ocular, and pulmonary administration. In some embodiments, the pharmaceutical composition may be administered intravenously, subcutaneously, orally, intramuscularly, or intracardiacly.

[0086] According to another aspect, the present application further discloses a reagent for analyzing and detecting TRPV6 in a sample from a subject, the reagent comprising one or more antibodies or antigen-binding fragments thereof described herein. In some embodiments, the reagent is used for analyzing and detecting TRPV6 in a sample from a subject.

[0087] According to another aspect, the present application further discloses a method for detecting TRPV6 in a sample from a subject, the method comprising: (1) contacting the sample with an antibody or antigen-binding fragment described in the present application; (2) optionally removing unbound antibody or antibody fragment; and (3) detecting the antibody or antibody fragment bound to TRPV6 in the sample.

[0088] The term "detection" as used in this application may refer to determining the presence or absence of a molecule (e.g., a polypeptide, protein, or nucleic acid molecule), or determining an interaction between molecules (e.g., binding, agonistic or antagonistic action), such as a protein-protein interaction (e.g., an interaction between an antibody and an antigen), a protein-nucleic acid interaction, or a nucleic acid-nucleic acid interaction. In some embodiments, "detection" and "measurement" include quantitative detection and measurement. In some embodiments, detection is performed by determining and measuring the signal of a detection molecule that is directly or indirectly coupled to an antibody. Detection may be performed by methods well known to those skilled in the art. In some embodiments, detection is performed using immunohistochemistry (IHC), enzyme-linked immunosorbent (ELISA), immunofluorescence (IF), chemiluminescence (CLIA), immunoturbidimetric (TIA), or Western Blot.

[0089] In some embodiments, in step (1) of the method for detecting TRPV6 in a sample from a subject, the antibody or antigen-binding fragment described herein is immobilized on a substrate. In some embodiments, in step (1) of the method for detecting TRPV6 in a sample from a subject, the sample is immobilized on a substrate. In some embodiments, the sample is a cell or tissue. The sample includes, but is not limited to, a cytocentrifuge preparation, a cytological smear, a core biopsy, a fine needle puncture, and / or a tissue section (e.g., a cryostat tissue section, a paraffin-embedded tissue section). In some embodiments, the sample includes a live cell. In some embodiments, the sample includes a tumor cell. In some embodiments, the fixation is performed using a chemical fixative. A histological sample may be prepared using a crosslinking fixative (e.g., an aldehyde, including formaldehyde, paraformaldehyde, and glutaraldehyde). A tissue sample may be fixed with formaldehyde and embedded in paraffin. Paraformaldehyde may be used, embedded in a temperature-sensitive cryogenic material, and flash frozen in liquid nitrogen. Other types of chemical fixatives further include oxidizing agents and alcohol fixatives. Fixation may also be achieved by physical methods.

[0090] In some embodiments, step (3) of the method for detecting TRPV6 in a sample from a subject further comprises using a secondary antibody, optionally the secondary antibody capable of directly or indirectly binding to TRPV6 in the sample, optionally the epitope of the secondary antibody is different from the epitope of an antibody or antigen-binding fragment described in the present application, optionally the epitope of the secondary antibody is the same as the epitope of an antibody or antigen-binding fragment described in the present application, optionally the binding of the epitope of the secondary antibody to TRPV6 is not affected by the antibody or antigen-binding fragment described in the present application, and optionally the binding of the epitope of the secondary antibody to TRPV6 is affected by the antibody or antigen-binding fragment described in the present application.

[0091] The term "epitope" as used in this application refers to an antigenic determinant that interacts with a specific antigen-binding site (called a complementary site) in the variable region of an antibody molecule. A single antigen may have more than one epitope. Thus, different antibodies can bind to different regions on an antigen and have different biological effects. An epitope may be a conformational epitope or a linear epitope. A conformational epitope is formed by the spatial juxtaposition of amino acids from different segments of a linear polypeptide chain. A linear epitope is an epitope formed by adjacent amino acid residues in a polypeptide chain. In some circumstances, an epitope may include a sugar, phosphoryl group, or sulfonyl group moiety on an antigen.

[0092] In some embodiments, the method for detecting TRPV6 in a sample from a subject further comprises using a secondary antibody in step (3), optionally the secondary antibody is capable of binding directly or indirectly to an antibody or antigen-binding fragment described herein, and optionally the secondary antibody is capable of binding to a constant region portion of an antibody or antigen-binding fragment described herein. As used herein, a "secondary antibody" is used to better detect an anti-TRPV6 antibody, and the secondary antibody is generally commercially available. In some embodiments, the secondary antibody described herein is coupled to a detection molecule, optionally the detection molecule comprises an enzyme, a fluorescent label, and biotin. In some embodiments, the detection molecule is an enzyme, and optionally the enzyme comprises a peroxidase (e.g., horseradish peroxidase), alkaline phosphatase, β-galactosidase, glucoamylase, carbohydrate oxidase, urease, heterocyclic oxidase (e.g., xanthine oxidase), malate dehydrogenase, or a derivative thereof. In some embodiments, the detection molecule is fluorescein, optionally comprising FITC, rhodamine, Texas Red, phycoerythrin, GFP, Cy3, Cy5, or Dylight, hi some embodiments, the detection molecule is biotin or a derivative thereof.

[0093] In some embodiments, the method for detecting TRPV6 in a sample from a subject further comprises quantifying the antibody bound to TRPV6 in the sample. In some embodiments, the method is immunohistochemistry (IHC), enzyme-linked immunosorbent (ELISA), immunofluorescence (IF), chemiluminescence (CLIA), immunoturbidimetry (TIA) or Western Blot. In some embodiments, the method can assess the proportion of cells in the sample that express TRPV6 on the cell surface.

[0094] In some embodiments, in the method of detecting TRPV6 in a sample from a subject, the subject is a human.

[0095] According to another aspect, the present application further discloses a method for determining the formation or risk of formation of a subject disease, assessing the progression or prognosis of a subject disease, or predicting or monitoring the response of the subject to treatment in a subject undergoing treatment for a disease, the method comprising detecting TRPV6 in a sample derived from the subject by a method for detecting TRPV6 in a sample derived from the subject described in the present application.

[0096] In some embodiments, a method for determining the formation or risk of formation of a subject disease, assessing the progression or prognosis of a subject disease, or predicting or monitoring the response of the subject to treatment in a subject undergoing treatment for a disease comprises assessing the proportion of cells in the sample that express TRPV6 on the cell surface, and if the proportion exceeds a predetermined threshold, the subject is more likely to have the disease or to benefit from treatment, where the threshold is 10%. In some embodiments, the treatment is a targeted drug treatment.

[0097] As used herein, the terms "gain," "benefit," or "beneficial" refer to any desired effect, including, but not limited to, (1) inhibiting (e.g., reducing, ameliorating, or completely halting) the progression of the disease to some extent, including slowing and halting it altogether, (2) reducing the incidence and / or number of symptoms of the disease, (3) reducing the size of the lesions, (4) inhibiting the infiltration of disease cells into adjacent surrounding organs and / or tissues, (5) inhibiting the spread of the disease, (6) alleviating to some extent one or more symptoms associated with the disease, (7) increasing disease-free survival after treatment, (8) reducing autoimmune responses, which may, but do not necessarily, cause regression or ablation of disease lesions, e.g., progression-free survival, (9) increasing overall survival, (10) achieving a higher response rate, and / or (11) decreasing mortality at a given time point after treatment.

[0098] In some embodiments, predicting or monitoring the subject's response to treatment includes assessing whether the subject's tumor has remitted or progressed. In some embodiments, predicting or monitoring the subject's response to treatment may include assessing whether the subject has improved and / or the likelihood of improvement after receiving treatment (e.g., treatment with a particular pharmaceutical agent). The prediction or supervision method of the present application may be used in clinical treatment decisions. After implementing a treatment plan (e.g., a given treatment plan, such as administration of a given therapeutic agent or combination, surgical intervention, etc.), the prediction or supervision method of the present application is a useful tool in assessing the likelihood of long-term survival of a particular subject.

[0099] In some embodiments, the disease described in the present application is a cancer disease. In some embodiments, the cancer disease is breast cancer, ovarian cancer, endometrial cancer, bile duct cancer, gastric cancer, esophageal cancer, lung cancer, intestinal cancer, pancreatic cancer. In some embodiments, the cancer disease is HER2 + The cancer is breast cancer, triple-negative breast cancer or colorectal cancer.

[0100] According to another aspect, the present application further discloses a kit, comprising an antibody or an antigen-binding fragment thereof described in the present application, and optionally further comprising a reagent for detecting the binding status of the antibody or antigen-binding fragment thereof with TRPV6.

[0101] In some embodiments, the kits described herein are used to diagnose the formation or risk of formation of a disease in a subject, assess the progression or prognosis of a disease in a subject, or predict or monitor the response of the subject to treatment in a subject undergoing treatment for a disease. In some embodiments, the kits described herein are used for pathological diagnosis by immunohistochemistry. In some embodiments, the kits described herein are used for pathological diagnosis by immunohistochemistry of tumor tissue. In some embodiments, the disease is a cancer disease, and in some embodiments, the cancer disease is breast cancer, ovarian cancer, endometrial cancer, bile duct cancer, gastric cancer, esophageal cancer, lung cancer, intestinal cancer, pancreatic cancer. Also, in some embodiments, the cancer disease is HER2+ breast cancer, triple negative breast cancer, or colorectal cancer.

[0102] According to another aspect, the present application further discloses a method for producing an antibody that specifically binds to TRPV6, the method including: (1) coupling the amino acid sequence shown in SEQ ID NO:3 with a vector protein to obtain a TRPV6 antigen peptide; (2) immunizing a mouse with the TRPV6 antigen peptide obtained by step (1) as an immunogen; (3) obtaining a positive hybridoma cell line that highly secretes an antibody that specifically binds to TRPV6 through cell fusion and clonal screening of the immunized peptide; and (4) obtaining an antibody that specifically binds to TRPV6.

[0103] In some embodiments, the vector protein is a keyhole limpet hemocyanin (KLH) protein.

[0104] In some embodiments, the amino acid sequence shown in SEQ ID NO:3 has one additional Cys at its N-terminus and is coupled to the vector protein via a free thiol group.

[0105] SEQ ID NO:3:PLKPRTNNRTSPRDNTLLQQKLLQEAYMTPK.

[0106] In some embodiments, the hybridoma cell line described in the present application is murine hybridoma cell line 12CT 9.5.1, 16CT 18.1.1, or 16CT 4.1.1.2, where the antibody produced by cell line 12CT 9.5.1 comprises the CB-Anti-0001 antibody, the antibody produced by cell line 16CT4.1.1.2 comprises the CB-Anti-0002 antibody, and the antibody produced by cell line 16CT18.1.1 comprises the CB-Anti-0003 antibody.

[0107] In another aspect, the present application further discloses the use of the antibody or antigen-binding fragment thereof described in the present application, the multispecific antibody described in the present application, the antibody conjugate described in the present application and the pharmaceutical composition described in the present application in medicine for treating and / or preventing and / or diagnosing a disease associated with TRPV6.

[0108] According to another aspect, the present application further discloses a method of treating, preventing or diagnosing a disease, said method comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof described herein, a multispecific antibody described herein, an antibody conjugate described herein, and a pharmaceutical composition described herein. Working Example

[0109] The following examples are used to better illustrate the present invention and should not be understood as limiting the scope of the present invention. All specific compositions, materials and methods described below, in whole or in part, are within the scope of the present invention. These specific compositions, materials and methods are not used to limit the present invention, but to illustrate that certain embodiments are within the scope of the present invention. Those skilled in the art can develop equivalent compositions, materials and methods without departing from the scope of the present invention without additional creativity. It should be understood that various modifications made to the method of the present invention are still included within the scope of the present invention. The inventor intends that such modifications are included within the scope of the present invention. Example 1 Synthesis of antigen polypeptides and preparation of recombinant antigens

[0110] 1. Selection of antigenic polypeptide The protein sequence (shown in SEQ ID NO:2) with the accession number Q9H1D0 and GI:1918017293 in Uniprot and Genbank was subjected to sequence and secondary structure analysis. The full length of the TRPV6 protein is 765 amino acids, and its molecular weight is about 87 kDa. The secondary structure and surface accessibility parameters of the protein were predicted on the online server http: / / www.cbs.dtu.dk / services / NetSurfP / , and according to the analysis of its antigenicity index, the amino acid sequence IFQTEDPEELGHF (SEQ ID NO:4), DGPANYNVDLPF (SEQ ID NO:5) or PLKPRTNNRTSPRDNTLLQQKLLQEAYMTPK (SEQ ID NO:3) was selected as the antigen for chemical synthesis. To facilitate coupling, a cysteine ​​was added to the amino terminus of the polypeptide to provide a thio group.

[0111] 2. Coupling and purification of polypeptides A maleimide-activated keyhole limpet hemocyanin protein kit from Thermo Scientific was selected and operated according to the flow provided by the kit.

[0112] For the polypeptide to be coupled, firstly, the free thio group in the polypeptide was detected using Ellman's reagent as follows: 100μL of Ellman's reagent stock solution was added to a 96-well plate, and then 10μL of the polypeptide solution was added, and its UV absorption value was measured at λ=412nm using a Nano Drop spectrophotometer, if the OD value was >0.15, carry out the next step, if the OD value was <0.15 and >0.05, add more polypeptide until the requirement is met, if the OD value was <0.05, return to the polypeptide synthesis step and carry out quality control again.

[0113] At the start of coupling, 200 μL of deionized water was added to each mcKLH package to prepare a 10 mg / mL KLH solution, 2 mg of hapten was dissolved in 500 μL of Imject EDC coupling buffer, and 500 μL of the polypeptide solution was added to 200 μL of the vector protein solution. 1 mL of deionized water was added to one package of EDC (10 mg), gently shaken until completely dissolved, and 50 μL was taken and added to the mcKLH polypeptide solution, reacted for 2 hours, and then treated with a desalting column to remove uncoupled crosslinker and salts. Example 2 Construction of hybridoma cell lines

[0114] 1. Immunity The crosslinked polypeptide in Example 1 was emulsified with Freund's complete adjuvant and immunized into 4-6 week old female Balb / c mice (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.), and injected subcutaneously into the abdomen at 6 sites per mouse at a dose of 60 μg / mouse. Booster immunization was performed once every 14 days, with the antigen emulsified with Freund's incomplete adjuvant at a dose of 30 μg / mouse. Seven days after the third booster immunization, the anti-immunogen polyclonal antibody titer in the mouse serum was detected by indirect ELISA (wavelength 450 nm), and the mouse with the highest titer was rush immunized by intravenous injection into the tail, with the antigen mixed uniformly with saline at a dose of 50 μg / mouse.

[0115] 2. Cell fusion A splenocyte suspension was prepared from a mouse that had reached the standard of immunity, and mixed with mouse myeloma cells F0 at a ratio of 5:1, and centrifuged at 1500 rpm for 5 minutes. After discarding the supernatant, the centrifuge tube was placed in a 37°C water bath, and 1 mL of PEG1500 (Roche) was slowly added within 1 minute to stir the cells. After leaving it in warm water for 1 minute, 10 mL of serum-free IMDM (Sigma) was added, mixed uniformly, and centrifuged at 1000 rpm for 5 minutes. After discarding the supernatant, 10 mL of serum (PAA) was added, the cells were carefully aspirated and discharged repeatedly, and 5 mL of thymocytes mixed with 10XHAT (Sigma) was added and mixed uniformly. Further, 25 mL of semi-solid medium containing 2.1% nitrocellulose (Sigma) was added, mixed thoroughly and uniformly, and then evenly poured into 20 cell culture dishes. The cell culture dishes were placed in a wet box and cultured in an incubator at 37°C with 5% CO2.

[0116] 3. Cloning and screening of positive hybridoma cells by ELISA On the 7th day after fusion, the size and density of the clonal cell clusters were appropriate, and under a stereomicroscope, the round, compact, and large clonal clusters were aspirated and dispensed into a 96-well culture plate with medium prepared in advance, and cultured in an incubator at 37°C with 5% CO2.

[0117] After 3 days, the cell volume occupied approximately 2 / 3 of the bottom area, and 100 μL of the supernatant was taken and ELISA screening was performed using immunogen and synthetic polypeptide, respectively. The medium of the positive clone was completely replaced, and 200 μL of complete medium containing feeder cells and 1% HT (Sigma) was added. After 2 days, a second ELISA screening was performed, and the positive clone was transferred to a 24-well plate with medium (containing feeder cells and HT) prepared in advance and cultured. After 5 days, 100 μL of the supernatant was taken and a third ELISA screening was performed, and three high-titer positive clones, 12CT 9.5.1, 16CT 18.1.1.2, and 16CT 4.1.1.2, were obtained by screening, which were successively transferred to 6-well plates and cell culture flasks for expansion and cryopreservation. Example 3: Preparation of monoclonal antibodies by ascites induction method

[0118] 1. Preparation of Ascites Logarithmic growth phase cells were washed with serum-free medium, resuspended, and counted at ~5x10 5 1 mL of suspended cells at 100 / mL was injected intraperitoneally into mice previously sensitized with paraffin oil. Ascites collection was started 7 days later. The removed ascites was centrifuged at 4000 rpm for 10 minutes at 4°C. The middle part of the ascites was carefully sucked up and placed in a centrifuge tube, and stored at 4°C or -20°C.

[0119] 2. Purification of monoclonal antibodies The antibodies were purified from the ascites using Protein G (GE) affinity chromatography according to the manufacturer's instructions. The purity was identified on SDS-PAGE gel and the concentration was measured by the Bradford method. The purified antibodies were stored at -20°C. Example 4 Monoclonal Antibody Characterization

[0120] 1. Subtype Identification Coating goat anti-mouse IgG was diluted to 0.5 μg / mL in 100 mL of MPBS (pH 7.4), 100 μL was added per well, and incubated overnight at 4 °C. The wells were emptied and washed three times with PBS containing 0.05% Tween, 200 μL of blocking solution (PBS containing 2% BSA and 3% sucrose) was added per well, incubated for 1 h at 37 °C, emptied and washed three times with PBST, and 0.1 mL of hybridoma supernatant was added per well, incubated for 1 h at 37 °C, emptied and washed three times with PBST. The blocking solution was used to dilute the goat anti-mouse (κ, λ) antibody marked with HRP at 1:1000, or the goat anti-mouse (IgM, IgG1, IgG2a, IgG2b, IgG3, IgA) antibody marked with HRP at 1:2000, and 0.1 mL per well was added to the appropriate wells, incubated at 37 °C for 1 h, drained to the last liquid, and washed three times with PBST. 50 μL of citrate buffer (pH 4.0) containing 0.15% ABTS and 0.03% H2O2 was added to each well to perform the color reaction, and the OD value at 405 nm wavelength was measured within 10 to 20 minutes. According to the results, the obtained CB-Anti-0001 and CB-Anti-0002 antibodies are IgG1 type mouse-derived monoclonal antibodies, and the CB-Anti-0003 antibody is an IgG2a type mouse-derived monoclonal antibody.

[0121] 2. Measurement of affinity constants First, the obtained anti-TRPV6 antibody was immobilized on an SPR chip, and then the immunizing peptide was diluted to 11.52 nM with 1 × PBST as the diluent, and then diluted 1.3 times to a final concentration of 4.04 nM, for a total of five concentration gradients. Affinity detection was performed using an SPR instrument, and the affinity constant of CB-Anti-0001 was found to be 5.19 × 10 by automatic analysis of the instrument. -10 It was calculated that:

[0122] 3. Reaction specificity and application effects of monoclonal antibodies 293T cells transfected with TRPV6 protein were selected, and the recognition specificity of the monoclonal antibody of the present invention was detected by the method of immunocytochemistry. The experimental process of immunocytochemistry is as follows: 293T cells were transfected with TRPV6 plasmid, and the transfected cells were placed in a 24-well plate containing a cover slip, and the cells were cultured in a medium of IMDM+10% FBS for 72 hours, the supernatant was removed, washed once with PBS, and fixed for 30 seconds by adding methanol, and washed twice with PBS. The antibody dilution solution was used to prepare anti-TRPV6 antibody working solution according to the appropriate dilution ratio, and the PBS in the 24-well plate was removed, and the antibody working solution was added and incubated overnight at 4 degrees Celsius, washed three times with PBS, and then incubated with a secondary antibody (Elivision TM The sections were then dripped with a drop of plus Polyer HRP (Mouse / Rabbit) IHC Kit (purchased from Fuzhou Minxin Biotechnology Development Co., Ltd., product number KIT-9903) and incubated at room temperature for 15-20 minutes, washed with PBS for 3X3 minutes (i.e., washed three times, lasting 3 minutes each time), discarded the PBS, and developed with freshly prepared DAB developer for 3 minutes. Secondary staining was performed with hematoxylin for 25 seconds, and color development was performed with PBS for 30 seconds. The sections were dehydrated in a 75%, 95%, 100%, and 100% alcohol gradient, and finally cleared with xylene for 3 minutes and mounted with neutral gum.

[0123] As shown in Figure 1A-1C, CB-Anti-0001 antibody, CB-Anti-0002 antibody and CB-Anti-0003 antibody can respectively detect TRPV6 protein specifically by immunocytochemistry in 293T cells transfected with TRPV6 plasmid, but do not show detection of TRPV6 protein in 293T cells not transfected with TRPV6 plasmid.According to the results, the obtained anti-TRPV6 antibody can specifically recognize TRPV6 protein, i.e. has recognition property. Example 5: Measurement of antibody variable region sequences

[0124] Cultured hybridoma cell lines at 1x10 6The cells were collected, centrifuged to remove the supernatant, and the cell pellet was sent to Suzhou Jinweizhi Bio-Technology Co., Ltd. to measure the antibody heavy and light chain variable region sequences. Exemplary antibody heavy chain variable region sequences and antibody light chain variable region sequences are shown below. [Table 2] [Table 3] [Table 4] JPEG2024505430000005.jpg105140 Example 6 Staining and Identification of Tissue Sections

[0125] 1. Sectioning process Tissue paraffin blocks were coarsely cut and serially sectioned to a thickness of 5 μm. Serial sections were spread in warm water at 45°C and attached to positively charged anti-peel slides and dried in the shade at room temperature overnight before long-term storage at -20°C.

[0126] 2. IHC staining and analysis Deparaffinization was performed twice with normal xylene, 15 minutes each time, hydration in gradient ethanol 100%, 100%, 95%, 75%, 3 minutes each time, and finally rinsing with tap water. Antigen retrieval was performed, and the sections were placed in a wet box and rinsed with PBS for 3X3 minutes. 3% H2O2 was added dropwise, incubated for 10 minutes, and rinsed with PBS for 3X3 minutes. PBS was shaken off, blocking solution (animal non-immune serum) was added dropwise, and incubated for 60 minutes at room temperature. The sections were shaken dry, and primary antibodies diluted in the appropriate ratio were added dropwise, incubated overnight at 4°C, rinsed with PBS for 3X3 minutes, and incubated with secondary antibodies (Elivision TMThe sections were then dripped with a drop of IHC Kit (plus Polyer HRP (Mouse / Rabbit) IHC Kit, purchased from Fuzhou Minxin Biotechnology Development Co., Ltd., product number KIT-9903) and incubated at room temperature for 15-20 minutes, washed with PBS for 3X3 minutes, discarded the PBS, and developed with freshly prepared DAB developer for 3 minutes. Secondary staining was performed with hematoxylin for 25 seconds, and color development was performed with PBS for 30 seconds. The sections were dehydrated in a 75%, 95%, 100%, and 100% alcohol gradient, and finally cleared with xylene for 3 minutes and mounted with neutral gum.

[0127] 3. Data Statistics As shown in Figures 2A to 2C, the CB-Anti-0001 antibody, the CB-Anti-0002 antibody, and the CB-Anti-0003 antibody can each detect TRPV6 protein in tissue sections by immunohistochemistry.

[0128] The inventors further recorded the staining status of the cell membrane of tumor cells using the H-score scoring method, recording H-score 60 points or more as strong staining, H-score 1 to 60 points as weak staining, the sum of strong staining and weak staining as positive, and H-score 0 as negative. The levels of TRPV6 in each tumor tissue detected by IHC were statistically analyzed, and the specificity of the antibody was evaluated by comparing and verifying with the TRPV6 mRNA level in the TCGA database. The recorded information is shown in Table 4. According to the results, in the detection of the TRPV6 protein level by the antibody of the present invention, the cancer type with a high positive rate (especially a high ratio of strong staining) also has a correspondingly high TRPV6 mRNA level statistically calculated in the Cancer Genome Atlas Database (the Cancer Genome Atlas Program, i.e., TCGA database) (for example, TPM is greater than 1). As can be seen from this, the expression status of TRPV6 on the cell surface can be accurately evaluated by using the antibody of the present invention, and further, it can be determined whether the test sample is a cancer cell expressing TRPV6. [Table 5] Example 7 Comparative IHC experiment

[0129] Comparative experiments were performed using the commercially available antibody "Anti-Human TRPV6 (extracellular) Antibody" (product number ACC-028, purchased from Alomone Labs, RRID:AB_2756545, abbreviated herein as "ACC-028 antibody"), the antibody "Anti-TrpV6 Antibody, clone 4A5.1" (product number MABN839, purchased from Sigma-Aldrich, abbreviated herein as "MABN839 antibody"), and the CB-Anti-0001 antibody.

[0130] Sections of CDX tumor model RT4 (human bladder transitional cell papilloma cells) were obtained using the same procedure as in the second part of Example 6. ACC-028 antibody (dilution ratio 1:50), MABN839 antibody (dilution ratio 1:50) and CB-Anti-0001 antibody (dilution ratio 1:2000) were added dropwise, incubated overnight at 4°C, washed with PBS for 3X3 minutes, and then incubated with secondary antibody (Elivision TM The sections were then dripped with a drop of plus Polyer HRP (Mouse / Rabbit) IHC Kit (purchased from Fuzhou Minxin Biotechnology Development Co., Ltd., product number KIT-9903) and incubated at room temperature for 15-20 minutes, washed with PBS for 3X3 minutes, discarded the PBS, and developed with freshly prepared DAB coloring solution for 3 minutes. Secondary staining was performed with hematoxylin for 25 seconds, and color development was performed with PBS for 30 seconds. The sections were dehydrated in a 75%, 95%, 100%, and 100% alcohol gradient, and finally cleared with xylene for 3 minutes, mounted with neutral gum, and the staining results were read. The results showed that the ACC-028 antibody had low detection sensitivity, with only a small amount of specific signal detected on the RT4 cell membrane (see Figure 3A), while the MABN839 antibody had poor specificity, with signals detected in all tissue cells and mesenchymal cells (see Figure 3B). In comparison, the CB-Anti-0001 antibody had accurate and clear positioning of the detection signal, excellent specificity, and high sensitivity (see Figure 3C), making it a preferred antibody for IHC application. Example 8 Stability Study

[0131] The stability of CB-Anti-0001 antibody was verified by accelerated destruction method, that is, (1) the antibody stock solution was stored at 4°C for 14 days, (2) the antibody stock solution was incubated in a 37°C constant temperature incubator for 14 days, and (3) the antibody working solution (obtained by diluting the antibody stock solution at a ratio of 1:2000 with Nakasugi Kanabashi ZLI-9028 antibody dilution solution) was incubated in a 37°C constant temperature incubator for 14 days. Under the same experimental conditions, the above three antibody solutions were used to perform IHC detection to evaluate the antibody stability in the three processing methods.

[0132] According to the detection, the antibodies stored under different conditions had no significant changes in either tissue staining intensity or staining ratio (see FIG. 4, where FIG. 4A is the IHC result of the CB-Anti-0001 antibody stock solution stored at 4°C for 14 days, FIG. 4B is the IHC result of the CB-Anti-0001 antibody stock solution stored at 37°C for 14 days, and FIG. 4C is the IHC result of the CB-Anti-0001 antibody working solution stored at 37°C for 14 days). As can be seen, even after storing the CB-Anti-0001 antibody working solution at 37°C for 14 days, the antibody is still stable and has good detection ability. [Table 6] JPEG2024505430000008.jpg159164

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to TRPV6, the antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein the three heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3 are contained in the following heavy chain variable region: (a) a heavy chain variable region as depicted in SEQ ID NO:25; (b) a heavy chain variable region as set forth in SEQ ID NO: 27; or (c) a heavy chain variable region as set forth in SEQ ID NO: 29; and The three light chain complementarity determining regions LCDR1, LCDR2, and LCDR3 are contained in the light chain variable region: (d) a light chain variable region as depicted in SEQ ID NO: 26; (e) a light chain variable region as set forth in SEQ ID NO: 28; or (f) a light chain variable region as set forth in SEQ ID NO: 30; An antibody or an antigen-binding fragment thereof that specifically binds to TRPV6.

2. The antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and three light chain complementarity determining regions LCDR1, LCDR2 and LCDR3 selected from the group consisting of: (a) an HCDR1 as shown in SEQ ID NO:7, an HCDR2 as shown in SEQ ID NO:8, an HCDR3 as shown in SEQ ID NO:9, an LCDR1 as shown in SEQ ID NO:10, an LCDR2 as shown in SEQ ID NO:11, and an LCDR3 as shown in SEQ ID NO:12; (b) an HCDR1 as set forth in SEQ ID NO:13, an HCDR2 as set forth in SEQ ID NO:14, an HCDR3 as set forth in SEQ ID NO:15, an LCDR1 as set forth in SEQ ID NO:16, an LCDR2 as set forth in SEQ ID NO:17, and an LCDR3 as set forth in SEQ ID NO:18, and (c) the HCDR1 shown in SEQ ID NO: 19, the HCDR2 shown in SEQ ID NO: 20, the HCDR3 shown in SEQ ID NO: 21, the LCDR1 shown in SEQ ID NO: 22, the LCDR2 shown in SEQ ID NO: 23, and the LCDR3 shown in SEQ ID NO: 24, according to claim 1, or an antigen-binding fragment thereof.

3. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region V selected from the group consisting of: Ｈ Including, i.e. (a) a heavy chain variable region as depicted in SEQ ID NO:25; (b) a heavy chain variable region as set forth in SEQ ID NO: 27, and (c) The antibody or antigen-binding fragment thereof according to claim 1, which has a heavy chain variable region as shown in SEQ ID NO:

29.

4. The antibody or antigen-binding fragment thereof further comprises a light chain variable region V selected from the group consisting of: Ｌ Including, i.e. (a) a light chain variable region as depicted in SEQ ID NO: 26; (b) a light chain variable region as set forth in SEQ ID NO: 28, and (c) the light chain variable region as shown in SEQ ID NO: 30,

5. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region V selected from the group consisting of: Ｈ and the light chain variable region V Ｌ and (a) a heavy chain variable region as set forth in SEQ ID NO:25 and a light chain variable region as set forth in SEQ ID NO:26; (b) a heavy chain variable region set forth in SEQ ID NO:27 and a light chain variable region set forth in SEQ ID NO:28; and (c) the antibody or antigen-binding fragment thereof according to claim 1, which has a heavy chain variable region shown in SEQ ID NO: 29 and a light chain variable region shown in SEQ ID NO:

30.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antigen-binding fragment is selected from F(ab')2, Fab', Fab, Fv, scFv, dsFv or dAb.

7. The antibody or antigen-binding fragment thereof of claim 1 , further comprising a conjugate moiety.

8. The antibody or antigen-binding fragment thereof of claim 7, wherein the conjugate moiety is a therapeutic agent, a radioisotope, a detectable label, a pharmacokinetically modified moiety or a purified moiety.

9. An isolated nucleic acid encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8.

10. (a) a nucleic acid sequence as set forth in SEQ ID NO:31, or a nucleic acid sequence having at least 90% sequence identity thereto, and a nucleic acid sequence as set forth in SEQ ID NO:32, or a nucleic acid sequence having at least 90% sequence identity thereto; (b) a nucleic acid sequence as set forth in SEQ ID NO:33 or a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:33 and a nucleic acid sequence as set forth in SEQ ID NO:34 or a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:34; or (c) the nucleic acid of claim 9, comprising a nucleic acid sequence as set forth in SEQ ID NO: 35 or a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO: 35, and a nucleic acid sequence as set forth in SEQ ID NO: 6 or a nucleic acid sequence having at least 90% sequence identity with SEQ ID NO:

6.

11. A vector comprising the nucleic acid sequence of claim 9 or 10.

12. A multispecific antibody comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 and one or more antibodies or antigen-binding portions thereof that specifically bind to other antigens.

13. An antibody complex comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8, or a multispecific antibody according to claim 12.

14. A hybridoma cell line secreting an anti-TRPV6 monoclonal antibody, the hybridoma cell line being 12CT9.5.1, 16CT4.1.1.2 or 16CT18.1.1.2 and stored in the China Center for Typical Culture Collection, wherein the storage number of the 12CT9.5.1 is CCTCC NO: C202119, the storage number of the 16CT4.1.1.2 is CCTCC NO: C202124, and the storage number of the 16CT18.1.1.2 is CCTCC NO: C202125.

15. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8, an isolated nucleic acid according to claim 9 or 10, a vector according to claim 11, a multispecific antibody according to claim 12 or an antibody complex according to claim 13, and a pharma- ceutically acceptable vector.

16. Use of an antibody or an antigen-binding fragment thereof described in any one of claims 1 to 8, or a pharmaceutical composition described in claim 15, in the manufacture of a medicament for determining the formation of a disease in a subject or the risk of forming a disease, assessing the progression or prognosis of a disease in a subject, predicting or monitoring the response of the subject to treatment in a subject undergoing treatment for a disease, or detecting TRPV6 in a sample derived from a subject.

17. The use according to claim 16, wherein the disease is a cancer disease.

18. The cancer disease is breast cancer, ovarian cancer, endometrial cancer, bile duct cancer, gastric cancer, esophageal cancer, lung cancer, intestinal cancer, or pancreatic cancer, and preferably, the cancer disease is HER2 ＋ 18. The use according to claim 17, wherein the cancer is breast cancer, triple negative breast cancer or colorectal cancer.

19. A kit comprising an antibody or an antigen-binding fragment thereof described in any one of claims 1 to 8, and optionally further comprising a reagent for detecting the binding status of the antibody or antigen-binding fragment thereof with TRPV6.

20. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the isolated nucleic acid according to claim 9 or 10, the vector according to claim 11, the multispecific antibody according to claim 12, the antibody complex according to claim 13, and the pharmaceutical composition according to claim 15 in the manufacture of a medicament for treating and / or preventing and / or diagnosing a disease associated with TRPV6.

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