WO2022152236A1 - 抗trpv6单克隆抗体及其应用 - Google Patents

抗trpv6单克隆抗体及其应用 Download PDF

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WO2022152236A1
WO2022152236A1 PCT/CN2022/071976 CN2022071976W WO2022152236A1 WO 2022152236 A1 WO2022152236 A1 WO 2022152236A1 CN 2022071976 W CN2022071976 W CN 2022071976W WO 2022152236 A1 WO2022152236 A1 WO 2022152236A1
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antibody
seq
antigen
binding fragment
trpv6
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PCT/CN2022/071976
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English (en)
French (fr)
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黄保华
谭巍
王中波
王伟
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同宜医药（苏州）有限公司
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Priority to CA3206472A priority Critical patent/CA3206472A1/en
Priority to CN202280010293.4A priority patent/CN116802277A/zh
Priority to JP2023542926A priority patent/JP2024505430A/ja
Priority to EP22739112.5A priority patent/EP4293044A1/en
Priority to KR1020237022218A priority patent/KR20230132450A/ko
Publication of WO2022152236A1 publication Critical patent/WO2022152236A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels

Definitions

  • the present invention relates to a series of antibodies that can recognize human TRPV6 protein and a hybridoma cell line that can secrete the series of antibodies.
  • the present invention provides a series of monoclonal antibodies that specifically bind to TRPV6 molecules in tumor cell membranes/plasma, and the amino acid sequences of the variable regions of the heavy and light chains of the antibodies and the DNA sequences encoding the variable regions have been determined , the antibody or its antigenic fragment can be used for immunohistochemistry (IHC), enzyme-linked immunosorbent (ELISA), immunofluorescence (IF), chemiluminescence (CLIA), immunoturbidimetry (TIA) by manual or automated means or Western blotting (Western Blot) method to detect the level of TRPV6 in cells, so as to be used in the method for diagnosing these tumors, which belongs to the field of biological detection.
  • IHC immunohistochemistry
  • ELISA enzyme-linked immunosorbent
  • IF immunofluorescence
  • CLIA chemiluminescence
  • Transient receptor potential cation channel subfamily V member 6 is a highly selective calcium ion transmembrane transport channel that mediates the active transport of calcium ions from extracellular to intracellular .
  • TRPV6 is expressed in normal human kidneys, gastrointestinal tract, pancreas, breast, salivary glands, etc., but is mainly expressed in intestinal epithelial cells, which are involved in the transport of calcium ions into cells, so when the number or function of TRPV6 channels changes , which can cause changes in calcium regulation and further lead to structural or functional abnormalities of related tissues and organs.
  • TRPV6 Compared with normal tissues, the expression of TRPV6 is significantly increased in breast cancer, bile duct cancer, ovarian cancer, lung squamous cell carcinoma, prostate cancer and other malignant tumors, and its abnormal expression may be related to the formation and progression of tumors.
  • the Prevarskaya team found that calcium uptake by human prostate cancer LNCap cells with high expression of TRPV6 is mediated by TRPV6. After siRNA treatment to down-regulate the level of TRPV6 protein, the number of cells in S phase was reduced and cell proliferation was inhibited.
  • TRPV6 is closely related to a variety of human tumors, and may be related to its regulation of intracellular and extracellular calcium ion concentration changes.
  • TRPV6 As a tumor cell marker, the detection of TRPV6 is of great significance for pathological diagnosis and the use of corresponding targeted drugs. At present, there is no anti-TRPV6 antibody that can be used for diagnosis on the market, and the antibody involved in this patent can fill the gap in this aspect.
  • One aspect of the present application provides 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 included in the following heavy chain variable regions and HCDR3: heavy chain variable region shown in SEQ ID NO:25; heavy chain variable region shown in SEQ ID NO:27; or heavy chain variable region shown in SEQ ID NO:29.
  • 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 shown in SEQ ID NO:7, HCDR1 shown in SEQ ID NO:8 HCDR2 shown in SEQ ID NO: 9; HCDR1 shown in SEQ ID NO: 13, HCDR2 shown in SEQ ID NO: 14, HCDR3 shown in SEQ ID NO: 15; or HCDR3 shown in SEQ ID NO: 19 HCDR1 shown in SEQ ID NO: 20, HCDR3 shown in SEQ ID NO: 21.
  • the antibody or antigen-binding fragment thereof further comprises three light chain complementarity determining regions LCDR1, LCDR2 and LCDR3 included in the light chain variable region as follows: light chain variable light chain shown in SEQ ID NO: 26 region; the light chain variable region shown in SEQ ID NO:28; or the light chain variable region shown in SEQ ID NO:30.
  • 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, LCDR1 shown in SEQ ID NO:11 LCDR2 shown in SEQ ID NO: 12 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: 23, LCDR2 shown in SEQ ID NO: 23, and LCDR3 shown in SEQ ID NO: 24.
  • LCDR1, LCDR2 and LCDR3 selected from the group consisting of: LCDR1 shown in SEQ ID NO:10, LCDR1 shown in SEQ ID NO:11 LCDR2 shown in SEQ ID NO: 12 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
  • 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: SEQ ID NO: HCDR1 shown in 7, HCDR2 shown in SEQ ID NO: 8, HCDR3 shown in SEQ ID NO: 9, LCDR1 shown in SEQ ID NO: 10, LCDR2 shown in SEQ ID NO: 11 and SEQ ID NO: LCDR3 shown in 12; HCDR1 shown in SEQ ID NO: 13, HCDR2 shown in SEQ ID NO: 14, HCDR3 shown in SEQ ID NO: 15, LCDR1 shown in SEQ ID NO: 16, SEQ ID NO: LCDR2 shown in 17 and LCDR3 shown in SEQ ID NO: 18; HCDR1 shown in SEQ ID NO: 19, HCDR2 shown in SEQ ID NO: 20, HCDR3 shown in SEQ ID NO: 21, SEQ ID NO: LCDR1 shown in 22,
  • the antibody or antigen-binding fragment thereof comprises HCDR1 set forth in SEQ ID NO:7, HCDR2 set forth in SEQ ID NO:8, HCDR3 set forth in SEQ ID NO:9, SEQ ID NO:10 LCDR1 shown, LCDR2 shown in SEQ ID NO: 11, and LCDR3 shown in SEQ ID NO: 12.
  • the antibody or antigen-binding fragment thereof comprises HCDR1 set forth in SEQ ID NO: 13, HCDR2 set forth in SEQ ID NO: 14, HCDR3 set forth in SEQ ID NO: 15, SEQ ID NO: 16 LCDR1 shown, LCDR2 shown in SEQ ID NO: 17, and LCDR3 shown in SEQ ID NO: 18.
  • the antibody or antigen-binding fragment thereof comprises HCDR1 set forth in SEQ ID NO:19, HCDR2 set forth in SEQ ID NO:20, HCDR3 set forth in SEQ ID NO:21, SEQ ID NO:22 LCDR1 shown, LCDR2 shown in SEQ ID NO: 23, and LCDR3 shown in SEQ ID NO: 24.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH selected from the group consisting of: a heavy chain variable region set forth in SEQ ID NO:25; a heavy chain variable region set forth in SEQ ID NO:27 The heavy chain variable region of ; or the heavy chain variable region shown in SEQ ID NO: 29.
  • the antibody or antigen-binding fragment thereof further comprises a light chain variable region VL selected from the group consisting of: a light chain variable region shown in SEQ ID NO:26; a light chain variable region shown in SEQ ID NO:28 The light chain variable region of ; or the light chain variable region shown in SEQ ID NO: 30.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region VH and a light chain variable region VL selected from the group consisting of a heavy chain variable region set forth in SEQ ID NO:25 and Light chain variable region shown in SEQ ID NO:26; heavy chain variable region shown in SEQ ID NO:27 and light chain variable region shown in SEQ ID NO:28; or SEQ ID NO:29 The variable region of the heavy chain and the variable region of the light chain shown in SEQ ID NO: 30.
  • 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.
  • 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.
  • 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.
  • the antibody or antigen-binding fragment thereof specifically recognizes or binds to a protein having the amino acid sequence set forth in SEQ ID NO:2.
  • the antibody or antigen-binding fragment thereof has a Kd value of 10-7-10-10M with a protein having the amino acid sequence shown in SEQ ID NO:2. In some embodiments, the Kd value is 10-7-10-10M , 10-8-10-10M , or 10-9-10-10M . In some embodiments, the antibody or antigen-binding fragment thereof has a Kd value of less than about 10<" 7 >M with a protein having the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the Kd value is less than about 10-7 M, less than about 10-8 M, less than about 10-9 M, or less than about 10-10 M. In some embodiments, the Kd value is detected by surface plasmon resonance methods.
  • the antigen-binding fragment is selected from F(ab') 2 , Fab', Fab, Fv, scFv, dsFv, or dAb.
  • the antibody or antigen-binding fragment thereof further has a heavy chain constant region and/or a light chain constant region.
  • the heavy chain constant region is derived from murine IgGl or murine IgG2a. In some embodiments, the heavy chain constant region is derived from human IgGl.
  • the antibody or antigen-binding fragment thereof further has a conjugation moiety.
  • the conjugated moiety is a therapeutic agent, a radioisotope, a detectable label, a pharmacokinetic modification moiety, or a purification moiety.
  • the conjugated moieties are attached directly or through a linker, and the conjugated moieties are attached directly or through a linker.
  • the present application also provides an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof that specifically binds to TRPV6 as described in the present application.
  • the nucleic acid comprises 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 One or both of the nucleic acid sequences having at least 90% sequence identity to SEQ ID NO:32.
  • the nucleic acid comprises 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 One or both of the nucleic acid sequences having at least 90% sequence identity to SEQ ID NO:34.
  • the nucleic acid comprises 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 One or both of the nucleic acid sequences having at least 90% sequence identity to SEQ ID NO:6.
  • the present application also provides a vector comprising the nucleic acid sequence described in the present application.
  • the present application also provides a host cell comprising the vector described in the present application.
  • the present application also provides a multispecific antibody comprising the antibody or antigen-binding fragment thereof described herein, and one or more antibodies or antigen-binding portions thereof that specifically bind to other antigens.
  • the present application also provides an antibody conjugate comprising the antibody or antigen-binding fragment thereof described herein or the multispecific antibody described herein.
  • the present application also provides a hybridoma cell line that secretes anti-TRPV6 monoclonal antibody, the hybridoma cell line is 12CT9.5.1, 16CT4.1.1.2 and/or 16CT18.1.1.2, which is preserved in Chinese Type Culture
  • the depository number of 12CT9.5.1 is CCTCC NO: C202119
  • the deposit number of 16CT4.1.1.2 is CCTCC NO: C202124
  • the deposit number of 16CT18.1.1.2 is CCTCC NO: C202125.
  • the application also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described in the application, the isolated nucleic acid described in the application, the vector described in the application, and the host described in the application.
  • the present application also provides a reagent for analyzing and detecting TRPV6 in a sample from a subject, the reagent comprising one or more of the antibodies or antigen-binding fragments thereof described herein.
  • the reagents are used to analyze and detect TRPV6 in a sample from a subject.
  • the application also provides a method for detecting TRPV6 in a sample from a subject, the method comprising: (1) contacting the sample with the antibody or antigen-binding fragment described in the application; (2) Optionally, remove unbound antibodies or antibody fragments; (3) detect antibodies or antibody fragments bound to TRPV6 in the sample.
  • step (1) of the method of 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 of detecting TRPV6 in a sample from a subject, the sample is immobilized on a substrate.
  • step (3) of the method for detecting TRPV6 in a sample from a subject, further comprising using a second antibody optionally, the second antibody can directly or indirectly bind to the TRPV6 in the sample, optionally, the epitope of the second antibody is different from the epitope of the antibody or antigen-binding fragment described in this application, optionally, the epitope of the second antibody is different from the epitope of the second antibody described in this application
  • the epitope of the antibody or antigen-binding fragment is the same.
  • the binding of the epitope of the second antibody to TRPV6 is not affected by the antibody or antigen-binding fragment described in this application. Binding of an epitope of an antibody to TRPV6 is affected by the antibody or antigen-binding fragment described herein.
  • step (3) of the method for detecting TRPV6 in a sample from a subject further comprising the use of a second antibody
  • the second antibody can directly or indirectly bind to the present application
  • the antibody or antigen-binding fragment optionally, the second antibody is capable of binding to the constant region portion of the antibody or antigen-binding fragment described herein.
  • the second antibody is coupled to a detection molecule, optionally, the detection molecule includes an enzyme, a fluorescent label, and biotin.
  • the detection molecule is an enzyme, optionally, the enzyme includes horseradish peroxidase, alkaline phosphatase or derivatives thereof.
  • the detection molecule is fluorescein, optionally, the fluorescein includes FITC, Rhodamine, Texas Red, Phycoerythrin or Dylight.
  • the detection molecule is biotin or a derivative thereof.
  • the method of detecting TRPV6 in a sample from a subject further comprises quantifying antibodies that bind to TRPV6 in the sample.
  • the method is immunohistochemistry (IHC), immunofluorescence (IF) enzyme-linked immunosorbent (ELISA) chemiluminescence (CLIA, chemiluminescent immunoassay), immunoturbidimetric (TIA, Turbidimetric inhibition immunoassay) or Western Blot.
  • the method can assess the proportion of cells in the sample that express TRPV6 on the cell surface.
  • the subject in the method of detecting TRPV6 in a sample from a subject, is a human.
  • the application also provides a method for judging the development or risk of developing a disease in a subject, assessing the progression or prognosis of a disease in a subject, or predicting or monitoring the subject in a subject receiving treatment for the disease
  • a method of response to treatment comprising detecting TRPV6 in a sample from a subject by the methods described herein for detecting TRPV6 in a sample from a subject.
  • determining the development or risk of developing a disease in a subject assessing the progression or prognosis of a disease in a subject, or predicting or monitoring the subject's response to treatment in a subject receiving treatment for a disease
  • the method comprises assessing the proportion of cells expressing TRPV6 on the cell surface in the sample, and if the proportion exceeds a predetermined threshold, the experimenter is considered to have the disease or is more likely to benefit from treatment, wherein the threshold is 10%.
  • the treatment is targeted drug therapy.
  • the disease described herein is cancer.
  • the cancer is breast cancer, ovarian cancer, endometrial cancer, bile duct cancer, gastric cancer, esophageal cancer, lung cancer, bowel cancer, pancreatic cancer.
  • the application also provides a kind of test kit
  • the test kit comprises the antibody or its antigen-binding fragment described in the present application, optionally, further comprises for detecting that the antibody or its antigen-binding fragment is combined with TRPV6 Case reagents.
  • kits described herein are used for diagnosing the development or risk of developing a disease in a subject, assessing the progression or prognosis of a disease in a subject, or predicting or monitoring in a subject receiving treatment for a disease the subject's response to treatment.
  • the kits described herein are used for immunohistochemical pathological diagnosis.
  • the kit described in this application is used for immunohistochemical pathological diagnosis of tumor tissue.
  • the disease is cancer, in some embodiments, the cancer is breast cancer, ovarian cancer, endometrial cancer, bile duct cancer, gastric cancer, esophageal cancer, lung cancer, bowel cancer, pancreatic cancer.
  • the present application also provides a method for preparing an antibody that specifically binds TRPV6, the method comprising: (1) coupling the amino acid sequence shown in SEQ ID NO: 3 with a carrier protein to obtain a TRPV6 antigenic peptide (2) using the TRPV6 antigen peptide obtained by step (1) as an immunogen to immunize mice; (3) screening clones through cell fusion and immune peptides to obtain a positive hybridoma cell that efficiently secretes an antibody that specifically binds to TRPV6 (4) Obtain an antibody that specifically binds to TRPV6.
  • the carrier protein is a keyhole limpet hemocyanin (KLH) protein.
  • KLH keyhole limpet hemocyanin
  • a Cys is added to the N-terminus of the amino acid sequence shown in SEQ ID NO: 3, and is coupled to the carrier protein through a free sulfhydryl group.
  • the hybridoma cell lines described herein are mouse hybridoma cell lines 12CT 9.5.1, 16CT 18.1.1.2, and 16CT 4.1.1.2.
  • the application also provides the antibody or antigen-binding fragment thereof described in the application, the isolated nucleic acid described in the application, the vector described in the application, the host cell described in the application, the multispecific described in the application.
  • the present application also provides a method for treating, preventing or diagnosing a disease, the method comprising administering the antibody or antigen-binding fragment thereof of the present application, the multispecificity of the present application to a subject in need thereof Antibodies, antibody conjugates described herein, and pharmaceutical compositions described herein.
  • FIG. 1A shows that CB-Anti-0001 can specifically detect TRPV6 protein in TRPV6 plasmid-transfected 293T cells by immunocytochemistry (left panel), while not in non-TRPV6 plasmid-transfected 293T cells. TRPV6 protein was detected (right panel).
  • Figure 1B shows that CB-Anti-0002 was able to specifically detect TRPV6 protein in TRPV6 plasmid-transfected 293T cells by immunocytochemistry (left panel), but not in non-TRPV6 plasmid-transfected 293T cells TRPV6 protein was detected (right panel).
  • FIG. 1C shows that CB-Anti-0003 was able to specifically detect TRPV6 protein in TRPV6 plasmid-transfected 293T cells by immunocytochemistry (left panel), but not in non-TRPV6 plasmid-transfected 293T cells. TRPV6 protein was detected (right panel).
  • Figure 2A shows the results of immunohistochemical detection of CB-Anti-0001 antibody.
  • Figure 2B shows the results of immunohistochemical detection of CB-Anti-0002 antibody.
  • Figure 2C shows the results of immunohistochemical detection of CB-Anti-0003 antibody.
  • Figure 3 shows the results of IHC comparison experiments of ACC-028 antibody, MABN839 antibody and CB-Anti-0001 antibody against human bladder transitional cell papilloma cells (RT4).
  • Figure 3A shows the IHC results of the ACC-028 antibody
  • Figure 3B shows the IHC results of the MABN839 antibody
  • Figure 3C shows the IHC results of the CB-Anti-0001 antibody. It can be seen that the cell membrane in Figure 3C has a clear and significant staining effect.
  • Figure 4 shows that neither the tissue staining intensity nor the staining ratio of the antibodies stored under different conditions was significantly changed.
  • 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 kept at 37°C for 14 days
  • Fig. 4C For the IHC results of CB-Anti-0001 antibody working solution stored at 37°C for 14 days.
  • the applicant has submitted the hybridoma cell lines (lines) 12CT9.5.1, 16CT4.1.1.2 and 16CT18.1.1.2 to the China Center for Type Culture Collection for preservation.
  • the deposit number of 12CT9.5.1 is CCTCC NO: C202119
  • the deposit number of 16CT4.1.1.2 is CCTCC NO: C202124
  • the deposit number of 16CT18.1.1.2 is CCTCC NO: C202125
  • the deposit address of the trihybridoma cell line is Wuhan, China University
  • the preservation time is January 13, 2021
  • the preservation unit is the Chinese Type Culture Collection.
  • One aspect of the present application discloses an antibody or antigen-binding fragment thereof that specifically binds TRPV6, wherein the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions HCDR1 and HCDR2 included in the following heavy chain variable regions and HCDR3: heavy chain variable region shown in SEQ ID NO:25; heavy chain variable region shown in SEQ ID NO:27; or heavy chain variable region shown in SEQ ID NO:29.
  • 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 shown in SEQ ID NO:7, HCDR1 shown in SEQ ID NO:8 HCDR2 shown in SEQ ID NO: 9; HCDR1 shown in SEQ ID NO: 13, HCDR2 shown in SEQ ID NO: 14, HCDR3 shown in SEQ ID NO: 15; or HCDR3 shown in SEQ ID NO: 19 HCDR1 shown in SEQ ID NO: 20, HCDR3 shown in SEQ ID NO: 21.
  • the antibody or antigen-binding fragment thereof of the present application further comprises the antibody or antigen-binding fragment thereof further comprising three light chain complementarity determining regions LCDR1, LCDR2 and LCDR3 included in the light chain variable region as follows: SEQ The light chain variable region shown in ID NO:26; the light chain variable region shown in SEQ ID NO:28; or the light chain variable region shown in SEQ ID NO:30.
  • 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, SEQ ID NO:11 shown in SEQ ID NO:10 LCDR2 shown 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; SEQ ID NO: 22 LCDR1 shown, LCDR2 shown in SEQ ID NO: 23, and LCDR3 shown in SEQ ID NO: 24.
  • LCDR1, SEQ ID NO:11 shown in SEQ ID NO:10 LCDR2 shown 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
  • SEQ ID NO: 22 LCDR1 shown, LCDR2 shown in SEQ ID NO: 23, and LCDR3 shown in SEQ ID NO: 24.
  • the antibody or antigen-binding fragment thereof of the present application comprises HCDR1 shown in SEQ ID NO:7, HCDR2 shown in SEQ ID NO:8, HCDR3 shown in SEQ ID NO:9, SEQ ID NO: LCDR1 shown in 10, LCDR2 shown in SEQ ID NO: 11, and LCDR3 shown in SEQ ID NO: 12.
  • the antibody or antigen-binding fragment thereof of the present application comprises HCDR1 shown in SEQ ID NO: 13, HCDR2 shown in SEQ ID NO: 14, HCDR3 shown in SEQ ID NO: 15, SEQ ID NO: LCDR1 shown in 16, LCDR2 shown in SEQ ID NO: 17 and LCDR3 shown in SEQ ID NO: 18.
  • the antibody or antigen-binding fragment thereof of the present application comprises HCDR1 shown in SEQ ID NO: 19, HCDR2 shown in SEQ ID NO: 20, HCDR3 shown in SEQ ID NO: 21, SEQ ID NO: LCDR1 shown in 22, LCDR2 shown in SEQ ID NO: 23 and LCDR3 shown in SEQ ID NO: 24.
  • the antibody or antigen-binding fragment thereof of the present application comprises a heavy chain variable region VH selected from the group consisting of: the heavy chain variable region shown in SEQ ID NO: 25; the heavy chain variable region shown in SEQ ID NO: 27 The heavy chain variable region shown; or the heavy chain variable region shown in SEQ ID NO:29.
  • the antibody or antigen-binding fragment thereof further comprises a light chain variable region VL selected from the group consisting of: a light chain variable region shown in SEQ ID NO:26; a light chain variable region shown in SEQ ID NO:28 The light chain variable region of ; or the light chain variable region shown in SEQ ID NO: 30.
  • 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.
  • antibody as used herein includes any immunoglobulin, monoclonal, multivalent, multispecific or bispecific (bivalent) antibody.
  • a native intact antibody contains two heavy chains and two light chains bound to each other by disulfide chains.
  • Each heavy chain of an antibody consists of a variable region ( VH ) and first, second and third constant regions ( CH1 , CH2 , CH3 , respectively), while each light chain of an antibody consists of a variable region.
  • VL variable region
  • CL constant region
  • variable regions in each chain are roughly subdivided into 3 hypervariable regions, called complementarity determining regions (CDRs) (wherein light chain CDRs include LCDR1, LCDR2, LCDR3. Heavy chain CDRs include HCDR1, HCDR2, HCDR3).
  • CDRs complementarity determining regions
  • an "antigen-binding fragment" of an antibody, an “antigen-binding portion” of an antibody, and the like, as used herein, includes any naturally occurring, enzymatically obtainable, synthetic or genetically engineered polypeptide that specifically binds an antigen to form a complex thing.
  • Antigen-binding fragments of antibodies can, for example, be derived from intact antibody molecules using any suitable standard technique, such as proteolytic digestion or involving manipulation and expression encoding antibody variable domains and optionally constant Recombinant genetic engineering of DNA domains.
  • DNA can be sequenced and manipulated chemically or by using molecular biology techniques to, for example, arrange one or more variable and/or constant domains into a suitable configuration, or introduce codons, to generate cysteines residues, modified, added or deleted 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; and (vii) minimal recognition units that mimic the amino acid residues of the hypervariable regions of antibodies (eg, isolated complementarity determining regions (CDRs) such as CDR3 peptides) or FR3-CDR3-FR4 peptides.
  • CDRs complementarity determining regions
  • engineered molecules such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, tribodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, di- Nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variant IgNAR domains are also included within the expression "antigen-binding fragment" as used herein.
  • SMIPs small modular immunopharmaceuticals
  • the CDR boundaries of the antibodies and antigen-binding fragments disclosed in this application can be named or identified by Kabat, Chothia or Al-Lazikani nomenclature.
  • the CDR boundaries of the antibody are determined according to the Kabat database.
  • the three CDRs are separated by flanking contiguous portions called framework regions (FRs, where heavy chain FRs include HFR1, HFR2, HFR3, and HFR4, and light chain FRs include LFR1, LFR2, LFR3, and LFR4), which are more highly than CDRs is conserved and forms a scaffold supporting the hypervariable loop.
  • FRs framework regions
  • VH and VL each comprise 3 CDRs and 4 FRs (human amino acid residues N-terminal to C-terminal) in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4.
  • the constant regions of the heavy and light chains are not involved in antigen binding, but exhibit a variety of effector functions.
  • Mammalian heavy chains are classified as ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , and mammalian light chains are classified as ⁇ or ⁇ .
  • Antibodies can be divided into 5 major classes: IgA, IgD, IgE, IgG and IgM according to the amino acid sequence of their heavy chain constant region and the presence or absence of ⁇ , ⁇ , ⁇ , ⁇ and ⁇ , respectively. Subclasses of several major antibody classes such as IgG1 ( ⁇ 1 heavy chain), IgG2 ( ⁇ 2 heavy chain), IgG3 ( ⁇ 3 heavy chain), IgG4 ( ⁇ 4 heavy chain), IgA1 ( ⁇ 1 heavy chain) or IgA2 ( ⁇ 2 heavy chain) ).
  • 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.
  • the Kd value of the antibody or antigen-binding fragment thereof of the present application and the protein having the amino acid sequence shown in SEQ ID NO: 2 is 10 -7 -10 -10 M. In some embodiments, the Kd value is 10-7-10-10M , 10-8-10-10M , or 10-9-10-10M . In some embodiments, the antibody or antigen-binding fragment thereof has a Kd value of less than about 10<" 7 >M with a protein having the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the Kd value is less than about 10-7 M, less than about 10-8 M, less than about 10-9 M, or less than about 10-10 M. In some embodiments, the Kd value is detected by surface plasmon resonance methods.
  • the antigen-binding fragment of the present application is selected from F(ab') 2 , Fab', Fab, Fv, scFv, dsFv, or dAb.
  • the antibody or antigen-binding fragment thereof of the present application further has a heavy chain constant region and/or a light chain constant region.
  • the heavy chain constant region is derived from murine IgGl or murine IgG2a. In some embodiments, the heavy chain constant region is derived from human IgGl.
  • the antibody or antigen-binding fragment thereof of the present application further has a conjugation moiety.
  • the conjugated moiety is a therapeutic agent, a radioisotope, a detectable label, a pharmacokinetic modification moiety, or a purification moiety.
  • the conjugated moieties are attached directly or via linkers.
  • therapeutic agent can be any pharmaceutical compound used to treat or prevent a disease in a subject, including small molecules with a molecular weight of less than 500, nucleotides (eg, DNA, plasmid DNA, RNA, siRNA ) , antisense oligonucleotides, aptamers, etc.), short peptides, proteins (eg, enzymes).
  • nucleotides eg, DNA, plasmid DNA, RNA, siRNA
  • antisense oligonucleotides eg., antisense oligonucleotides, aptamers, etc.
  • short peptides eg, proteins
  • the radioisotope may be selected from the group consisting of 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
  • detectable label can be any detection molecule that is conjugated, directly or indirectly, to an antibody of the invention, while being suitable for or contributing to the detection of the level of TRPV6.
  • the detection molecules include enzymes, fluorescent labels, and biotin.
  • the enzymes include peroxidase (eg, horseradish peroxidase), alkaline phosphatase, beta-galactosidase, glucoamylase, sugar oxidase, urease, heterocyclic oxidase (eg, yellow oxidase). purine oxidase), malate dehydrogenase or their derivatives.
  • the fluorescent marker can be fluorescein, optionally, the fluorescein includes fluorescein isothiocyanate (FITC), rhodamine, Texas red, phycoerythrin, green fluorescent protein (GFP), Dylight , Cy3 or Cy5.
  • FITC fluorescein isothiocyanate
  • rhodamine rhodamine
  • Texas red rhodamine
  • GFP green fluorescent protein
  • Dylight Cy3 or Cy5.
  • the present application also discloses an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof that specifically binds to TRPV6 as described in the present application.
  • the nucleic acid comprises 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 One or both of the nucleic acid sequences having at least 90% sequence identity to SEQ ID NO:32.
  • the nucleic acid comprises 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 One or both of the nucleic acid sequences having at least 90% sequence identity to SEQ ID NO:34.
  • the nucleic acid comprises 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 One or both of the nucleic acid sequences having at least 90% sequence identity to SEQ ID NO:6.
  • the present application also discloses a vector comprising the nucleic acid sequence described in the present application.
  • the present application also discloses a host cell comprising the vector described in the present application.
  • the present application also discloses a multispecific antibody comprising the antibody or antigen-binding fragment thereof described herein, and one or more antibodies or antigen-binding portions thereof that specifically bind to other antigens.
  • the present application also discloses an antibody conjugate, which comprises the antibody or antigen-binding fragment thereof described in the present application or the multispecific antibody described in the present application.
  • the present application also discloses a hybridoma cell line that secretes anti-TRPV6 monoclonal antibody, and the hybridoma cell line is 12CT9.5.1, 16CT4.1.1.2 and/or 16CT18.1.1.2, which are deposited in China Typical Culture Collection Center, wherein the deposit number of 12CT9.5.1 is CCTCC NO: C202119, the deposit number of 16CT4.1.1.2 is CCTCC NO: C202124, and the deposit number of 16CT18.1.1.2 is CCTCC NO: C202125.
  • the present application also discloses a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described in the present application, the isolated nucleic acid described in the present application, the carrier described in the present application, the A host cell, a multispecific antibody described herein or an antibody conjugate described herein, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means that, within the scope of sound medical judgment, it is suitable for use in contact with cells of humans and other animals without undue toxicity, irritation, allergic response, etc., and It is commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a subject used to deliver an antibody or antigen-binding fragment thereof, isolated nucleic acid, vector, host cell, multispecific antibody or antibody provided herein to a subject
  • a pharmaceutically acceptable solvent, suspension or any other pharmaceutically inert carrier for the conjugate that does not interfere with the antibody or antigen-binding fragment thereof, isolated nucleic acid, vector, host cell, multispecific antibody or antibody conjugate The structure, morphology and properties of associations.
  • Certain such carriers are capable of formulating the antibody or antigen-binding fragment thereof, isolated nucleic acid, vector, host cell, multispecific antibody or antibody conjugate, for example, as tablets, pills, capsules, liquids, gels, syrups Tablets, slurries, suspensions and pastilles for oral ingestion by subjects.
  • Certain such vectors enable the antibody or antigen-binding fragment thereof, isolated nucleic acid, vector, host cell, multispecific antibody or antibody conjugate to be formulated for injection, infusion or topical administration.
  • Pharmaceutically acceptable carriers used in the pharmaceutical compositions provided herein include, but are not limited to, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous carriers (for example, Sodium Chloride Injection, Ringer's Injection, Isotonic Dextrose Injection, Sterile Water Injection, or Dextrose and Lactated Ringer's Injection), non-aqueous vehicles (e.g., fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil or peanut oil), antimicrobials, isotonic agents (eg, sodium chloride or dextrose), buffers (eg, phosphoric acid or citric acid buffers), antioxidants (eg, sodium bisulfate), anesthetics (eg, , procaine hydrochloride), suspending/dispersing agents (eg, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, or polyvinylpyrrolidone), chelating agents (eg, EDTA (ethylenediaminetetraacetic acid)
  • the pharmaceutical composition is an injectable formulation.
  • Injectable preparations include sterile aqueous solutions or dispersions, suspensions or emulsions. In all cases, preparations for injection should be sterile and should be fluid to facilitate injection. Injectable preparations should be stable under the conditions of manufacture and storage and must be protected from contamination by microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Injectable preparations should maintain proper fluidity.
  • Proper fluidity can be maintained, for example, by the use of coatings such as lecithin, by the use of surfactants, and the like.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • the pharmaceutical composition is an oral formulation.
  • Oral formulations include, but are not limited to, capsules, cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), powders, granules, or solutions in aqueous or non-aqueous liquids formulations or suspensions, or as oil-in-water or water-in-oil liquid emulsions, or as elixirs or syrups, or as pastilles (using inert bases such as gelatin and glycerol, or sucrose and acacia) and/or as Mouthwash, etc.
  • the antibodies or antigen-binding fragments thereof In solid dosage forms (eg, capsules, tablets, pills, dragees, powders, granules, etc.) for oral administration, the antibodies or antigen-binding fragments thereof, isolated nucleic acids, vectors, host cells, multispecific antibodies Or antibody conjugates mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium hydrogen phosphate, and/or any one of the following: (1) fillers or bulking agents , for example, starch, lactose, sucrose, glucose, mannitol and/or silicic acid; (2) binders, for example, carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerin; (4) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) solution blockers, such as
  • the antibody or antigen-binding fragment thereof, isolated nucleic acid, vector, host cell, multispecific antibody or antibody conjugate is mixed with any of the following: a pharmaceutically acceptable of emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • liquid dosage forms may contain inert diluents commonly used in the art, for example, water or other solvents, solubilizers and Emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, isopropanol, 1,3-butanediol, oils (especially, cottonseed oil, peanut oil, corn oil, olive oil, castor oil and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol and sorbitan fatty acid esters and mixtures thereof.
  • the oral compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservitol, sorbitan fatty acid esters and mixtures thereof.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweeten
  • 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 particle formulations, and the like.
  • Aqueous aerosols are prepared by mixing an aqueous solution or suspension of the agent with conventional pharmaceutically acceptable carriers and stabilizers. Carriers and stabilizers vary according to the requirements of the particular pharmaceutical ingredient, but in general they include nonionic surfactants (Tween or polyethylene glycol), oleic acid, lecithin, amino acids, eg, glycine, buffer solutions , salt, sugar or sugar alcohol. Aerosols are usually prepared from isotonic solutions and can be delivered by spray.
  • the pharmaceutical composition may be administered in admixture with one or more other drugs.
  • the pharmaceutical composition comprises at least one other drug.
  • the other drugs are antineoplastics, cardiovascular drugs, anti-inflammatory drugs, antiviral drugs, digestive system drugs, nervous system drugs, respiratory system drugs, immune system drugs, dermatological drugs, metabolic drugs, and the like.
  • the pharmaceutical composition can be administered to a subject in need thereof by a suitable route, including but not limited to oral, injection (eg, intravenous, intramuscular, subcutaneous, intradermal, intracardiac, intrathecal, intrapleural, intraperitoneal injection, etc.), mucosal (eg, intranasal, intraoral administration, etc.), sublingual, rectal, transdermal, intraocular, and pulmonary administration.
  • a suitable route including but not limited to oral, injection (eg, intravenous, intramuscular, subcutaneous, intradermal, intracardiac, intrathecal, intrapleural, intraperitoneal injection, etc.), mucosal (eg, intranasal, intraoral administration, etc.), sublingual, rectal, transdermal, intraocular, and pulmonary administration.
  • the pharmaceutical composition can be administered intravenously, subcutaneously, orally, intramuscularly, or intraventricularly.
  • the present application also discloses a reagent for analyzing and detecting TRPV6 in a sample from a subject, the reagent comprising one or more of the antibodies or antigen-binding fragments thereof described herein.
  • the reagents are used to analyze and detect TRPV6 in a sample from a subject.
  • the present application also discloses a method for detecting TRPV6 in a sample from a subject, the method comprising: (1) contacting the sample with the antibody or antigen-binding fragment described in the present application; (2) ) optionally, removing unbound antibodies or antibody fragments; (3) detecting antibodies or antibody fragments bound to TRPV6 in the sample.
  • detecting can be by determining and measuring the presence or absence of a molecule (eg, a polypeptide, protein, or nucleic acid molecule), or by determining and measuring an interaction between molecules (eg, binding, agonizing or antagonism, etc.), such as protein-protein interactions (eg, between an antibody and an antigen), protein-nucleic acid interactions, or nucleic acid-nucleic acid interactions.
  • a molecule eg, a polypeptide, protein, or nucleic acid molecule
  • an interaction between molecules eg, binding, agonizing or antagonism, etc.
  • protein-protein interactions eg, between an antibody and an antigen
  • protein-nucleic acid interactions eg, between an antibody and an antigen
  • nucleic acid-nucleic acid interactions e.g, antigen, protein-nucleic acid interactions.
  • detection is by determining and measuring the signal of a detection molecule conjugated directly or indirectly to the antibody. Detection can be
  • detection is performed using immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF), chemiluminescence (CLIA), immunoturbidimetry (TIA), or western blotting (WesternBlot).
  • IHC immunohistochemistry
  • ELISA enzyme-linked immunosorbent assay
  • IF immunofluorescence
  • CLIA chemiluminescence
  • TIA immunoturbidimetry
  • WesternBlot western blotting
  • step (1) of the method of detecting TRPV6 in a sample from a subject the antibody or antigen-binding fragment described herein is immobilized on a substrate.
  • the sample in step (1) of the method of detecting TRPV6 in a sample from a subject, is immobilized on a substrate.
  • the sample is a cell or tissue. Samples include, but are not limited to, cytocentrifugation preparations, cytology smears, core biopsies, fine needle aspiration, and/or tissue sections (eg, cryostat tissue sections, paraffin-embedded tissue sections).
  • the sample comprises living cells.
  • the sample comprises tumor cells.
  • the immobilization is performed using a chemical fixative.
  • Histological samples can be prepared using cross-linking fixatives, such as aldehydes, including, for example, formaldehyde, paraformaldehyde, and glutaraldehyde.
  • Tissue samples can be fixed with formaldehyde and embedded in paraffin. It can also be fixed with paraformaldehyde, embedded in temperature-sensitive cryogenic materials, and snap-frozen with liquid nitrogen.
  • Other classes of chemical fixatives include oxidizing agents and alcohol fixatives. It can also be fixed by physical methods.
  • step (3) of the method for detecting TRPV6 in a sample from a subject, further comprising using a second antibody optionally, the second antibody can directly or indirectly bind to the TRPV6 in the sample, optionally, the epitope of the second antibody is different from the epitope of the antibody or antigen-binding fragment described in this application, optionally, the epitope of the second antibody is different from the epitope of the second antibody described in this application
  • the epitope of the antibody or antigen-binding fragment is the same.
  • the binding of the epitope of the second antibody to TRPV6 is not affected by the antibody or antigen-binding fragment described in this application. Binding of an epitope of an antibody to TRPV6 is affected by the antibody or antigen-binding fragment described herein.
  • epitope refers to an antigenic determinant that interacts with a specific antigen-binding site (called a paratope) in the variable region of an antibody molecule.
  • a single antigen can have more than one epitope.
  • different antibodies can bind to different regions on an antigen and can have different biological effects.
  • the epitope can be a conformational epitope or a linear epitope. Conformational epitopes are created by the spatial juxtaposition of amino acids from different segments of a linear polypeptide chain. Linear epitopes are epitopes that arise from adjacent amino acid residues in a polypeptide chain. In certain instances, epitopes may comprise sugar, phosphoryl, or sulfonyl moieties on the antigen.
  • step (3) of the method for detecting TRPV6 in a sample from a subject further comprising the use of a second antibody
  • the second antibody can directly or indirectly bind to the present application
  • the antibody or antigen-binding fragment optionally, the second antibody is capable of binding to the constant region portion of the antibody or antigen-binding fragment described herein.
  • Secondary antibody as used herein is intended to better detect anti-TRPV6 antibodies, and secondary antibodies are generally commercially available.
  • the second antibody described in this application is coupled to a detection molecule, optionally, the detection molecule includes an enzyme, a fluorescent label and biotin.
  • the detection molecule is an enzyme, optionally, the enzyme includes peroxidase (eg, horseradish peroxidase), alkaline phosphatase, beta-galactosidase, glucoamylase , sugar oxidase, urease, heterocycle oxidase (eg, xanthine oxidase), malate dehydrogenase or derivatives thereof.
  • the detection molecule is fluorescein, optionally, the fluorescein includes FITC, Rhodamine, Texas Red, Phycoerythrin, GFP, Cy3, Cy5 or Dylight.
  • the detection molecule is biotin or a derivative thereof.
  • the method of detecting TRPV6 in a sample from a subject further comprises quantifying antibodies that bind to TRPV6 in the sample.
  • the method is immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF), chemiluminescence (CLIA), immunoturbidimetry (TIA), or Western Blot .
  • the method can assess the proportion of cells in the sample that express TRPV6 on the cell surface.
  • the subject in the method of detecting TRPV6 in a sample from a subject, is a human.
  • the application also discloses a method for judging the development or risk of developing a disease in a subject, assessing the progression or prognosis of a disease in a subject, or predicting or monitoring the subject in a subject undergoing treatment for the disease.
  • a method of a subject's response to treatment comprising detecting TRPV6 in a sample from a subject by the methods described herein for detecting TRPV6 in a sample from a subject.
  • determining the development or risk of developing a disease in a subject assessing the progression or prognosis of a disease in a subject, or predicting or monitoring the subject's response to treatment in a subject receiving treatment for a disease
  • the method comprises assessing the proportion of cells expressing TRPV6 on the cell surface in the sample, and if the proportion exceeds a predetermined threshold, the experimenter is considered to have the disease or is more likely to benefit from treatment, wherein the threshold is 10%.
  • the treatment is targeted drug therapy.
  • the terms “benefit”, “benefit” or “beneficial” as used in this application refer to any desired effect, including but not limited to: (1) inhibiting (eg, reducing, slowing, or completely halting) the disease to some extent; progression, including slowing and complete arrest; (2) reducing the number of disease episodes and/or symptoms; (3) reducing lesion size; (4) inhibiting the infiltration of disease cells into adjacent surrounding organs and/or tissues; (5) ) inhibition of disease spread; (6) some relief of one or more symptoms associated with the disease; (7) increased time to disease-free after treatment; (8) decreased autoimmune responses, which may but not necessarily lead to Regression or ablation of disease lesions, eg, progression-free survival; (9) increased overall survival; (10) higher response rates; and/or (11) decreased mortality at a given time point after treatment.
  • predicting or monitoring the subject's response to treatment involves assessing whether the subject's tumor is reduced or worsened. In some embodiments, predicting or monitoring the subject's response to treatment may involve assessing whether and/or the likelihood that the subject will get better after receiving treatment (eg, treatment with a particular drug).
  • the predictive or supervised methods of the present application can be used to make treatment decisions clinically. Following administration of a treatment regimen (eg, a given treatment regimen, including, for example, administration of a given therapeutic drug or combination, surgical intervention, etc.), the methods of prediction or surveillance of the present application are useful in evaluating whether a particular subject will survive long-term. Possibly a valuable tool.
  • the disease described herein is cancer.
  • the cancer is breast cancer, ovarian cancer, endometrial cancer, bile duct cancer, gastric cancer, esophageal cancer, lung cancer, bowel cancer, pancreatic cancer.
  • the cancer is HER2 + breast cancer, triple negative breast cancer, or colorectal cancer.
  • the present application also discloses a kit, which comprises the antibody or its antigen-binding fragment described in the present application, and optionally, further includes a kit for detecting the relationship between the antibody or its antigen-binding fragment and TRPV6 Binding reagents.
  • kits described herein are used for diagnosing the development or risk of developing a disease in a subject, assessing the progression or prognosis of a disease in a subject, or predicting or monitoring in a subject receiving treatment for a disease the subject's response to treatment.
  • the kits described herein are used for immunohistochemical pathological diagnosis.
  • the kit described in this application is used for immunohistochemical pathological diagnosis of tumor tissue.
  • the disease is cancer, in some embodiments, the cancer is breast cancer, ovarian cancer, endometrial cancer, bile duct cancer, gastric cancer, esophageal cancer, lung cancer, bowel cancer, pancreatic cancer. In yet other embodiments, the cancer is HER2+ breast cancer, triple negative breast cancer, or colorectal cancer.
  • the present application also discloses a method for preparing an antibody that specifically binds TRPV6, the method comprising: (1) coupling the amino acid sequence shown in SEQ ID NO: 3 with a carrier protein to obtain TRPV6 antigen (2) using the TRPV6 antigen peptide obtained in step (1) as an immunogen to immunize mice; (3) screening clones through cell fusion and immune peptides to obtain a positive hybridoma that efficiently secretes an antibody that specifically binds to TRPV6 cell line; (4) obtaining an antibody that specifically binds to TRPV6.
  • the carrier protein is a keyhole limpet hemocyanin (KLH) protein.
  • KLH keyhole limpet hemocyanin
  • a Cys is added to the N-terminus of the amino acid sequence shown in SEQ ID NO: 3, and is coupled to the carrier protein through a free sulfhydryl group.
  • SEQ ID NO: 3 PLKPRTNNRTSPRDNTLLQQKLLQEAYMTPK.
  • the hybridoma cell lines described herein are mouse hybridoma cell lines 12CT 9.5.1, 16CT 18.1.1 or 16CT 4.1.1.2.
  • the antibody produced by cell line 12CT 9.5.1 includes CB-Anti-0001 antibody
  • the antibody produced by cell line 16CT4.1.1.2 includes CB-Anti-0002 antibody
  • the antibody produced by cell line 16CT18.1.1 includes CB-Anti- 0003 antibody.
  • the present application also discloses the use of the antibody or antigen-binding fragment thereof, 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. Use in a medicament for the treatment and/or prevention and/or diagnosis of TRPV6-related diseases.
  • the present application also discloses a method for treating, preventing or diagnosing a disease, the method comprising administering to a subject in need thereof the antibody or antigen-binding fragment thereof, the multispecific Antibodies, antibody conjugates described herein, and pharmaceutical compositions described herein.
  • the cross-linked polypeptide in Example 1 was emulsified with Freund's complete adjuvant, and 4 to 6-week-old female Balb/c mice (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were immunized and injected subcutaneously in the abdomen. Mice 6 points, the dose is 60 ⁇ g/mice.
  • the immunization was boosted once every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant at a dose of 30 ⁇ g/only.
  • indirect ELISA (wavelength 450nm) was used to detect the polyclonal antibody titer against the immunogen in the serum of the mice.
  • the mice with the highest titer were immunized by tail vein injection, and the antigen was mixed with normal saline. The dose is 50 ⁇ g/only.
  • a suspension of mouse spleen cells that reached the immune target was prepared, 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, and the cells were agitated. After standing in warm water for 1 minute, 10 mL of serum-free IMDM (Sigma) was added, mixed well, and centrifuged at 1000 rpm for 5 minutes.
  • the size and density of the cloned cell clusters are moderate. Under the dissecting microscope, the round, solid and large clone clusters are drawn into the 96-well culture plate prepared with the medium in advance, and put into 37°C, 5% CO 2 . Cultivated in an incubator.
  • the cells After 3 days, the cells accounted for about 2/3 of the bottom area, and 100 ⁇ L of the supernatant was taken for ELISA screening with immunogens and synthetic polypeptides respectively. Positive clones were completely exchanged, and 200 ⁇ L of complete medium containing feeder cells and 1% HT (Sigma) was added. The second ELISA screening was performed two days later, and the positive clones were transferred to 24-well plates prepared with medium (containing feeder cells and HT). Five days later, 100 ⁇ L of supernatant was taken for the third ELISA screening, and three positive clones with higher titers, 12CT 9.5.1, 16CT 18.1.1.2 and 16CT 4.1.1.2, were screened and transferred to 6-well plates and cell culture. Flasks were expanded and frozen.
  • Log phase cells were washed with serum-free medium and suspended, counted at ⁇ 5x105 /mL, and 1 mL of the suspended cells were injected intraperitoneally into mice previously sensitized with paraffin oil. Collection of ascites was started 7 days later. The removed ascites was centrifuged at 4000 rpm for 10 minutes at 4°C. Carefully aspirate the ascites in the middle, collect it in a centrifuge tube, and store it at 4°C or -20°C.
  • Antibodies were purified from ascites by Protein G (GE) affinity chromatography according to the manufacturer's instructions. The purity was identified by SDS-PAGE gel, and the concentration was determined by Bradford method. Purified antibodies were stored at -20°C.
  • GE Protein G
  • the obtained anti-TRPV6 antibody was immobilized on the SPR chip, and then the immune peptide was diluted to 11.52nM with 1 ⁇ PBST as a diluent, and then diluted 1.3 times, and the final concentration was 4.04nM, a total of 5 concentration gradients.
  • the SPR instrument was used for affinity detection, and the affinity constant of CB-Anti-0001 was calculated to be 5.19x10 -10 after automatic analysis by the instrument.
  • 293T cells transfected with TRPV6 protein were selected, and the recognition specificity of the monoclonal antibody of the present invention was detected by immunocytochemical method.
  • the immunocytochemical experiment process was as follows: TRPV6 plasmid was transfected in 293T cells, and the transfected cells were plated on In a 24-well plate containing slides, cells were cultured in IMDM+10% FBS medium for 72 hours, the supernatant was removed, washed once with PBS, fixed with methanol for 30 seconds, and washed twice with PBS.
  • CB-Anti-0001 antibody, CB-Anti-0002 antibody and CB-Anti-0003 antibody were able to specifically detect TRPV6 protein by immunocytochemistry in 293T cells transfected with TRPV6 plasmid, respectively , while no TRPV6 protein was shown to be detected in 293T cells that were not transfected with TRPV6 plasmid. This result indicates that the obtained anti-TRPV6 antibody can specifically recognize the TRPV6 protein, that is, it has the recognition property.
  • variable region sequence of the antibody heavy chain and light chain Take the cultured hybridoma cell line 1 ⁇ 10 6 , centrifuge to get the supernatant, and entrust the cell pellet to Suzhou Jinweizhi Biotechnology Co., Ltd. to determine the variable region sequence of the antibody heavy chain and light chain.
  • the following shows an exemplary antibody heavy chain variable region Region sequences and antibody light chain variable region sequences.
  • tissue wax blocks were serially sectioned, with a thickness of 5 ⁇ m.
  • the serial sections were unfolded in warm water at 45°C, mounted on a positron-resistant slide, and air-dried at room temperature overnight, then placed in -20°C. Long-term preservation.
  • CB-Anti-0001 antibody, CB-Anti-0002 antibody and CB-Anti-0003 antibody were able to detect TRPV6 protein in tissue sections by immunohistochemistry, respectively.
  • the inventors also recorded the staining of tumor cell membranes by using the H-score scoring method.
  • An H-score of more than 60 was recorded as strong staining, and an H-score of 1-60 was recorded as weak staining, strong staining and weak staining.
  • the total staining was positive; the H-score of 0 was recorded as negative.
  • the levels of TRPV6 in each tumor tissue detected by IHC were counted, and the TRPV6 mRNA levels in the TCGA database were compared and verified to evaluate the specificity of the antibody. The recorded information is listed in Table 4.
  • the results show that using the antibody of the present invention to detect the TRPV6 protein level, the cancer species with a higher positive rate (especially a higher proportion of strong staining) are correspondingly counted in the Cancer Genome Atlas Program (the Cancer Genome Atlas Program, that is, the TCGA database). TRPV6 mRNA levels were also higher (eg, TPM greater than 1). It can be seen that the use of the antibody of the present invention can accurately evaluate the expression of TRPV6 on the cell surface, and then determine whether the detected sample is a cancer cell expressing TRPV6.
  • Table 4 The level of TRPV6 in tumor tissue detected by IHC
  • Embodiment 7 IHC comparative experiment
  • Anti-Human TRPV6 (extracellular) Antibody (Item No. ACC-028, purchased from Alomone Labs, RRID: AB_2756545, referred to herein as "ACC-028 antibody”)
  • antibody “Anti-TrpV6 Antibody, clone 4A5.1” (Cat. No. MABN839, purchased from Sigma-Aldrich Company, referred to herein as "MABN839 antibody”
  • MABN839 antibody CB-Anti-0001 antibody for comparative experiments.
  • CDX tumor model RT4 human bladder transitional cell papilloma cells
  • Hematoxylin was counterstained for 25 seconds, and PBS was back to blue for 30 seconds.
  • the results showed that the detection sensitivity of ACC-028 antibody was low, and specific signals were only detected on a small amount of RT4 cell membranes (see Figure 3A); the specificity of MABN839 antibody was poor, and signals were detected in all tissue cells and interstitial cells ( See FIG. 3B ); in contrast, the CB-Anti-0001 antibody has accurate and clear signal localization, good specificity and high sensitivity (see FIG. 3C ), and is an ideal antibody for IHC application.
  • the stability of the CB-Anti-0001 antibody was verified by the accelerated destruction method, that is, (1) the antibody stock solution was stored at 4°C for 14 days; (2) the antibody stock solution was placed in a constant temperature incubator at 37°C for 14 days; and (3) placing the antibody working solution (the antibody working solution obtained by diluting the antibody stock solution at a ratio of 1:2000 with Zhongshan Jinqiao ZLI-9028 antibody dilution solution) in a 37°C constant temperature incubator for 14 days. Under the same experimental conditions, the aforementioned three types of antibody solutions were used for IHC detection to evaluate the stability of the antibodies under the three treatment methods.
  • FIG. 4A is the IHC of the CB-Anti-0001 antibody stock solution stored at 4°C for 14 days.
  • Fig. 4B shows the IHC results of the CB-Anti-0001 antibody stock solution stored at 37°C for 14 days
  • Fig. 4C shows the IHC results of the CB-Anti-0001 antibody working solution stored at 37°C for 14 days). It can be seen that after the CB-Anti-0001 antibody working solution was stored at 37°C for 14 days, the antibody was still stable and retained good detection ability.

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Abstract

通过选择了一段TRPV6蛋白特征性的多肽抗原免疫小鼠后筛选获得了杂交瘤细胞系12CT 9.5.1、16CT 18.1.1.2和16CT 4.1.1.2，该系列细胞可分泌针对TRPV6的单克隆抗体，并可特异性识别TRPV6蛋白及肿瘤组织。该系列抗体可通过人工或自动化方式，以免疫组织化学(IHC)、酶联免疫吸附(ELISA)、免疫荧光(IF)、化学发光(CLIA)、免疫比浊(TIA)或免疫印迹(Western Blot)方式检测细胞中TRPV6的水平，从而用于对肿瘤组织进行诊断的方法中，属于生物检测领域。

Description

抗TRPV6单克隆抗体及其应用 技术领域
本发明涉及一系列可以识别人TRPV6蛋白的抗体及可分泌该系列抗体的杂交瘤细胞系。具体而言，本发明提供了一系列特异性结合肿瘤细胞膜/浆内TRPV6分子的单克隆抗体，对该抗体重链和轻链可变区的氨基酸序列和编码可变区的DNA序列进行了确定，该抗体或其抗原片段可以用于通过人工或自动化方式，以免疫组织化学(IHC)、酶联免疫吸附(ELISA)、免疫荧光(IF)、化学发光(CLIA)、免疫比浊(TIA)或免疫印迹(Western Blot)方式检测细胞中TRPV6的水平，从而用于对这些肿瘤进行诊断的方法中，属于生物检测领域。
背景技术
瞬时受体阳离子通道亚家族V成员6(transient receptor potential cation channel subfamily V member 6)，即TRPV6，是一种高选择性钙离子跨膜转运通道，介导钙离子由细胞外向细胞内的主动转运。TRPV6在人正常的肾脏、胃肠道、胰腺、乳腺、唾液腺等中有表达，但是主要表达于肠上皮细胞，其参与钙离子向细胞内的转运，因此当TRPV6通道的数量或功能发生改变时，可引起钙离子调节的变化，进一步导致与其相关组织器官的结构或功能异常。
和正常组织相比，TRPV6在乳腺癌、胆管癌、卵巢癌、肺鳞癌、***癌等恶性肿瘤中表达明显升高，其异常表达可能与肿瘤的形成与进展有关。Prevarskaya团队发现高表达TRPV6的人***癌LNCap细胞摄取钙离子是由TRPV6介导的，在经过siRNA处理，调低TRPV6蛋白水平后，其S期细胞数量减少，细胞增殖受到抑制。还有利用利多卡因处理人乳腺癌MDA-MB-231细胞、***癌PC-3细胞和卵巢癌ES-2细胞后，使TRPV6 mRNA和蛋白的水平下调，细胞增殖受到抑制，同时钙离子进入细胞内的速率降低，表明癌细胞的抑制可能与钙离子的减少有关，并且可能与TRPV6水平有关。可见TRPV6与人体多种肿瘤均有密切联系，并且可能与其调节细胞内外钙离子浓度变化有关。
TRPV6作为肿瘤细胞标记物，针对其的检测对于病理诊断和相应靶向药物使用都有重要意义。目前市面上尚未有可用作诊断用途的抗TRPV6抗体，本专利所涉及抗体可以填补该方面的空白。
发明内容
本申请的一个方面提供了一种特异性结合TRPV6的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含如下重链可变区所包括的三个重链互补决定区HCDR1、HCDR2和HCDR3：SEQ ID NO：25所示的重链可变区；SEQ ID NO：27所示的重链可变区；或SEQ ID NO：29所示的重链可变区。在一些实施方式中，所述抗体或其抗原结合片段包含选自下组的三个重链互补决定区HCDR1、HCDR2和HCDR3：SEQ ID NO：7所示的HCDR1、SEQ ID NO：8所示的HCDR2、SEQ ID NO：9所示的HCDR3；SEQ ID NO：13所示的HCDR1、SEQ ID NO：14所示的HCDR2、SEQ ID NO：15所示的HCDR3；或SEQ ID NO：19所示的HCDR1、SEQ ID NO：20所示的HCDR2、SEQ ID NO：21所示的HCDR3。
在一些实施方式中，所述抗体或其抗原结合片段进一步包含如下轻链可变区所包括的三个轻链互补决定区LCDR1、LCDR2和LCDR3：SEQ ID NO：26所示的轻链可变区；SEQ ID NO：28所示的轻链可变区；或SEQ ID NO：30所示的轻链可变区。在一些实施方式中，所述抗体或其抗原结合片段进一步包含选自下组的三个轻链互补决定区LCDR1、LCDR2和LCDR3：SEQ ID NO：10所示的LCDR1、SEQ ID NO：11所示的LCDR2以及SEQ ID NO：12所示的LCDR3；SEQ ID NO：16所示的LCDR1、SEQ ID NO：17所示的LCDR2以及SEQ ID NO：18所示的LCDR3；SEQ ID NO：22所示的LCDR1、SEQ ID NO：23所示的LCDR2以及SEQ ID NO：24所示的LCDR3。
在一些实施方式中，所述抗体或其抗原结合片段包含选自下组的三个重链互补决定区HCDR1、HCDR2和HCDR3以及三个轻链互补决定区LCDR1、LCDR2和LCDR3：SEQ ID NO：7所示的HCDR1、SEQ ID NO：8所示的HCDR2、SEQ ID NO：9所示的HCDR3、SEQ ID NO：10所示的LCDR1、SEQ ID NO：11所示的LCDR2以及SEQ ID NO：12所示的LCDR3；SEQ ID NO：13所示的HCDR1、SEQ ID NO：14所示的HCDR2、SEQ ID NO：15所示的HCDR3、SEQ ID NO：16所示的LCDR1、SEQ ID NO：17所示的LCDR2以及SEQ ID NO：18所示的LCDR3；SEQ ID NO：19所示的HCDR1、 SEQ ID NO：20所示的HCDR2、SEQ ID NO：21所示的HCDR3、SEQ ID NO：22所示的LCDR1、SEQ ID NO：23所示的LCDR2以及SEQ ID NO：24所示的LCDR3。
在一些实施方式中，所述抗体或其抗原结合片段包含SEQ ID NO：7所示的HCDR1、SEQ ID NO：8所示的HCDR2、SEQ ID NO：9所示的HCDR3、SEQ ID NO：10所示的LCDR1、SEQ ID NO：11所示的LCDR2以及SEQ ID NO：12所示的LCDR3。
在一些实施方式中，所述抗体或其抗原结合片段包含SEQ ID NO：13所示的HCDR1、SEQ ID NO：14所示的HCDR2、SEQ ID NO：15所示的HCDR3、SEQ ID NO：16所示的LCDR1、SEQ ID NO：17所示的LCDR2以及SEQ ID NO：18所示的LCDR3。
在一些实施方式中，所述抗体或其抗原结合片段包含SEQ ID NO：19所示的HCDR1、SEQ ID NO：20所示的HCDR2、SEQ ID NO：21所示的HCDR3、SEQ ID NO：22所示的LCDR1、SEQ ID NO：23所示的LCDR2以及SEQ ID NO：24所示的LCDR3。
在一些实施方式中，所述抗体或其抗原结合片段包含选自下组的重链可变区V H和：SEQ ID NO：25所示的重链可变区；SEQ ID NO：27所示的重链可变区；或SEQ ID NO：29所示的重链可变区。
在一些实施方式中，所述抗体或其抗原结合片段进一步包含选自下组的轻链可变区V L：SEQ ID NO：26所示的轻链可变区；SEQ ID NO：28所示的轻链可变区；或SEQ ID NO：30所示的轻链可变区。
在一些实施方式中，所述抗体或其抗原结合片段包含选自下组的重链可变区V H和轻链可变区V L：SEQ ID NO：25所示的重链可变区和SEQ ID NO：26所示的轻链可变区；SEQ ID NO：27所示的重链可变区和SEQ ID NO：28所示的轻链可变区；或SEQ ID NO：29所示的重链可变区和SEQ ID NO：30所示的轻链可变区。
在一些实施方式中，所述抗体或其抗原结合片段包含SEQ ID NO：25所示的重链可变区和SEQ ID NO：26所示的轻链可变区。
在一些实施方式中，所述抗体或其抗原结合片段包含SEQ ID NO：27所示的重链可变区和SEQ ID NO：28所示的轻链可变区。
在一些实施方式中，所述抗体或其抗原结合片段包含SEQ ID NO：29所示的重链可变区和SEQ ID NO：30所示的轻链可变区。
在一些实施方式中，所述抗体或其抗原结合片段特异性识别或结合具有SEQ ID NO：2所示的氨基酸序列的蛋白。
在一些实施方式中，所述抗体或其抗原结合片段与具有SEQ ID NO：2所示的氨基酸序列的蛋白的Kd值为10 -7-10 -10M。在一些实施方式中，所述Kd值为10 -7-10 -10M、10 -8-10 -10M或10 -9-10 -10M。在一些实施方式中，所述抗体或其抗原结合片段与具有SEQ ID NO：2所示的氨基酸序列的蛋白的Kd值小于约10 -7M。在一些实施方式中，所述Kd值小于约10 -7M、小于约10 -8M、小于约10 -9M或小于约10 -10M。在一些实施方式中，Kd值是通过表面等离子共振方法检测的。
在一些实施方式中，所述抗原结合片段是选自F(ab’) 2、Fab’、Fab、Fv、scFv、dsFv或dAb。
在一些实施方式中，所述抗体或其抗原结合片段进一步具有重链恒定区和/或轻链恒定区。在一些实施方式中，重链恒定区源于鼠的IgG1或鼠的IgG2a。在一些实施方式中，重链恒定区源于人的IgG1。
在一些实施方式中，所述抗体或其抗原结合片段进一步具有缀合部分。在一些实施方式中，所述缀合部分是治疗剂、放射性同位素、可检测标签、药代动力学修饰部分或纯化部分。所述缀合部分直接连接或者通过连接子连接，所述缀合部分直接连接或者通过连接子连接。
一个方面，本申请还提供了一种分离的核酸，其编码本申请所述的特异性结合TRPV6的抗体或其抗原结合片段。
在一些实施方式中，所述核酸包含SEQ ID NO：31所示的核酸序列或与SEQ ID NO：31具有至少90％序列同一性的核酸序列，和SEQ ID NO：32所示的核酸序列或与SEQ ID NO：32具有至少90％序列同一性的核酸序列中的一者或两者。在一些实施方式中，所述核酸包含SEQ ID NO：33所示的核酸序列或与SEQ ID NO：33具有至少90％序列同一性的核酸序列，和SEQ ID NO：34所示的核酸序列或与SEQ ID NO：34具有至少90％序列同一性的核酸序列中的一者或两者。在一些实施方式中，所述核酸包含SEQ ID NO：35所示的核酸序列或与SEQ ID NO：35具有至少90％序列同一性的核 酸序列，和SEQ ID NO：6所示的核酸序列或与SEQ ID NO：6具有至少90％序列同一性的核酸序列中的一者或两者。
一个方面，本申请还提供了一种载体，其包含本申请所述的核酸序列。
一个方面，本申请还提供了一种宿主细胞，其包含本申请所述的载体。
一个方面，本申请还提供了一种多特异性抗体，其包含本申请所述的抗体或其抗原结合片段，和一个或多个与其他抗原特异性结合的抗体或其抗原结合部分。
一个方面，本申请还提供了一种抗体偶联物，其包含本申请所述的抗体或其抗原结合片段或本申请所述的多特异性抗体。
一个方面，本申请还提供了分泌抗TRPV6单克隆抗体的杂交瘤细胞株，所述杂交瘤细胞株为12CT9.5.1、16CT4.1.1.2和/或16CT18.1.1.2，保藏于中国典型培养物保藏中心，其中12CT9.5.1的保藏编号为CCTCC NO：C202119，16CT4.1.1.2的保藏编号为CCTCC NO：C202124，16CT18.1.1.2的保藏编号为CCTCC NO：C202125。
一个方面，本申请还提供了一种药物组合物，其包含本申请所述的抗体或其抗原结合片段、本申请所述的分离的核酸、本申请所述的载体、本申请所述的宿主细胞、本申请所述的多特异性抗体或本申请所述的抗体偶联物，和药学上可接受的载体。
一个方面，本申请还提供了一种用于分析和检测来自受试者的样品中的TRPV6的试剂，所述试剂包含一种或多种本申请所述的抗体或其抗原结合片段。在一些实施方式中，所述试剂用于分析和检测来自受试者的样品中的TRPV6。
一个方面，本申请还提供了一种检测来自受试者的样品中的TRPV6的方法，所述方法包括：(1)使所述样品与本申请述的抗体或抗原结合片段接触；(2)可选地，移除未结合的抗体或抗体片段；(3)检测与所述样品中的TRPV6结合的抗体或抗体片段。
在一些实施方式中，在检测来自受试者的样品中的TRPV6的方法的步骤(1)中，本申请所述的抗体或抗原结合片段被固定在基板上。在一些实施方式中，在检测来自受试者的样品中的TRPV6的方法的步骤(1)中，所述样品被固定在基板上。
在一些实施方式中，在检测来自受试者的样品中的TRPV6的方法的步骤(3)中，还包括使用第二抗体，可选的，所述第二抗体能够直接或者间接地结合所述样品 中的TRPV6，可选的，所述第二抗体的表位与本申请所述的抗体或抗原结合片段的表位不同，可选的，所述第二抗体的表位与本申请所述的抗体或抗原结合片段的表位相同，可选的，所述第二抗体的表位与TRPV6的结合不受本申请所述的抗体或抗原结合片段的影响，可选的，所述第二抗体的表位与TRPV6的结合受本申请所述的抗体或抗原结合片段的影响。
在一些实施方式中，在检测来自受试者的样品中的TRPV6的方法的步骤(3)中，还包括使用第二抗体，可选的，所述第二抗体能够直接或间接地结合本申请所述的抗体或抗原结合片段，可选的，所述第二抗体能够结合本申请所述的抗体或抗原结合片段的恒定区部分。
一些实施方式中，所述第二抗体与检测分子偶联，可选的，所述检测分子包括酶、荧光标记物和生物素。在一些实施方式中，所述检测分子为酶，可选的，所述酶包括辣根过氧化酶、碱性磷酸酶或其衍生物。在一些实施方式中，所述检测分子为荧光素，可选的，所述荧光素包括FITC、罗丹明、德克萨斯红、藻红蛋白或Dylight。在一些实施方式中，所述检测分子为生物素或其衍生物。
在一些实施方式中，检测来自受试者的样品中的TRPV6的方法进一步包括对与所述样品中的TRPV6结合的抗体进行定量。在一些实施方式中，所述方法为免疫组织化学(IHC)、免疫荧光(IF)酶联免疫吸附(ELISA)化学发光(CLIA，chemiluminescent immunoassay)、免疫比浊(TIA，Turbidimetric inhibition immuno assay)或免疫印迹(WesternBlot)。在一些实施方式中，所述方法可以评估所述样品中在细胞表面上表达TRPV6的细胞的比例。
在一些实施方式中，在检测来自受试者的样品中的TRPV6的方法中，所述受试者为人类。
一个方面，本申请还提供了一种判断受试者疾病的形成或形成的风险、评估受试者疾病的进展或预后、或在接受疾病治疗的受试者中预测或监测所述受试者对治疗的应答的方法，所述方法包括通过本申请所述的检测来自受试者的样品中TRPV6的方法，以检测来自所述受试者的样品中的TRPV6。
在一些实施方式中，判断受试者疾病的形成或形成的风险、评估受试者疾病的进展或预后、或在接受疾病治疗的受试者中预测或监测所述受试者对治疗的应答的 方法包括评估所述样品中在细胞表面上表达TRPV6的细胞的比例，若所述比例超过预定的阈值，则认为所述受试者患有所述疾病或者是更可能从治疗中获益，其中所述阈值为10％。在一些实施方式中，所述治疗是靶向药物治疗。
在一些实施方式中，本申请所述的疾病为癌症。在一些实施方式中，癌症为乳腺癌、卵巢癌、子宫内膜癌、胆管癌、胃癌、食道癌、肺癌、肠癌、胰腺癌。
一个方面，本申请还提供了一种试剂盒，所述试剂盒包含本申请所述的抗体或其抗原结合片段，可选的，进一步包括用于检测所述抗体或其抗原结合片段与TRPV6结合情况的试剂。
在一些实施方式中，本申请所述的试剂盒用于诊断受试者疾病的形成或形成的风险、评估受试者疾病的进展或预后、或在接受疾病治疗的受试者中预测或监测所述受试者对治疗的应答。在一些实施方式中，本申请所述的试剂盒用于免疫组化病理诊断。在一些实施方式中，本申请所述的试剂盒用于肿瘤组织免疫组化病理诊断。在一些实施方式中，所述疾病为癌症，在一些实施方式中，癌症为乳腺癌、卵巢癌、子宫内膜癌、胆管癌、胃癌、食道癌、肺癌、肠癌、胰腺癌。
一个方面，本申请还提供了一种制备特异性结合TRPV6的抗体的方法，所述方法包括：(1)将SEQ ID NO：3所示的氨基酸序列与载体蛋白偶联，以获得TRPV6抗原肽；(2)将通过步骤(1)获得的所述TRPV6抗原肽作为免疫原免疫小鼠；(3)经细胞融合、免疫肽筛选克隆，获得高效分泌特异性结合TRPV6的抗体的阳性杂交瘤细胞系；(4)获得特异性结合TRPV6的抗体。
在一些实施方式中，所述载体蛋白为钥孔虫戚血蓝素(KLH)蛋白。
在一些实施方式中，在SEQ ID NO：3所示的氨基酸序列的N端增加一个Cys，并通过自由巯基和所述载体蛋白偶联。
在一些实施方式中，本申请所述的杂交瘤细胞系为小鼠杂交瘤细胞系12CT 9.5.1、16CT 18.1.1.2和16CT 4.1.1.2。
一个方面，本申请还提供了本申请所述抗体或其抗原结合片段、本申请所述的分离的核酸、本申请所述的载体、本申请所述的宿主细胞、本申请所述的多特异性 抗体、本申请所述的抗体偶联物以及本申请所述的药物组合物在用于治疗和/或预防和/或诊断与TRPV6水平相关的疾病的药物中的用途。
一个方面，本申请还提供了一种治疗、预防或诊断疾病的方法，所述方法包括向有需要的受试者施用本申请所述抗体或其抗原结合片段、本申请所述的多特异性抗体、本申请所述的抗体偶联物以及本申请所述的药物组合物。
附图说明
附图形成本说明书的一部分，以进一步说明本公开的某些方面。通过参考这些附图中的一个或多个，结合本公开中呈现的具体实施方式的详细描述，可以更好地理解本公开。
图1A示出了CB-Anti-0001能够通过免疫细胞化学特异地检测出TRPV6质粒转染的293T细胞中的TRPV6蛋白(左图)，而同时在非TRPV6质粒转染的293T细胞中未显示出检测到TRPV6蛋白(右图)。图1B示出了CB-Anti-0002能够通过免疫细胞化学特异地检测出TRPV6质粒转染的293T细胞中的TRPV6蛋白(左图)，而同时在非TRPV6质粒转染的293T细胞中未显示出检测到TRPV6蛋白(右图)。图1C示出了CB-Anti-0003能够通过免疫细胞化学特异地检测出TRPV6质粒转染的293T细胞中的TRPV6蛋白(左图)，而同时在非TRPV6质粒转染的293T细胞中未显示出检测到TRPV6蛋白(右图)。
图2A示出了CB-Anti-0001抗体的免疫组织化学检测结果。图2B示出了CB-Anti-0002抗体的免疫组织化学检测结果。图2C示出了CB-Anti-0003抗体的免疫组织化学检测结果。
图3显示了ACC-028抗体、MABN839抗体和CB-Anti-0001抗体针对人膀胱移行细胞***瘤细胞(RT4)的IHC对比结果实验。其中附图3A为ACC-028抗体的IHC结果，附图3B为MABN839抗体的IHC结果，附图3C为CB-Anti-0001抗体的IHC结果。可以看到，附图3C中细胞膜有清晰、显著的染色效果。
图4显示了在不同条件下保存的抗体的组织染色强度和染色比例均未出现显著改变。其中附图4A为在4℃下保存14天的CB-Anti-0001抗体原液的IHC结果， 附图4B为在37℃下保存14天的CB-Anti-0001抗体原液的IHC结果，附图4C为在37℃下保存14天的CB-Anti-0001抗体工作液的IHC结果。
发明详述
以下描述只为说明本申请的多种实施方式。因此，此处的具体实施方式不应理解为对申请范围的限制。本领域的技术人员基于本发明的宗旨和本文的描述，可很容易地得出多种等同方式和修改，应理解这样的等同实施方式包含在本发明范围内。在本申请中引用的所有文献，包含公开出版物、专利和专利申请都通过引用的方式全文并入本申请。
除非上下文另外明确指出，否则本文所使用的单数形式“一”、“一个”、“一种”和“该”或“所述”包括复数引用。
用于本申请的诸如“包括”、“包含”、“含有”、“含有”和“具有”等术语是包括性的或开放式的，并且不排除额外的、未叙述的要素或方法步骤。用于本申请的术语“由……组成”是封闭式的。
申请人已将杂交瘤细胞株(系)12CT9.5.1、16CT4.1.1.2和16CT18.1.1.2提交至中国典型培养物保藏中心进行保藏。12CT9.5.1的保藏编号为CCTCC NO：C202119，16CT4.1.1.2的保藏编号为CCTCCNO：C202124，16CT18.1.1.2的保藏编号为CCTCC NO：C202125，三杂交瘤细胞株的保藏地址为中国武汉大学，保藏时间为2021年1月13日，保藏单位为中国典型培养物保藏中心。
本申请的一个方面公开了一种特异性结合TRPV6的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含如下重链可变区所包括的三个重链互补决定区HCDR1、HCDR2和HCDR3：SEQ ID NO：25所示的重链可变区；SEQ ID NO：27所示的重链可变区；或SEQ ID NO：29所示的重链可变区。在一些实施方式中，所述抗体或其抗原结合片段包含选自下组的三个重链互补决定区HCDR1、HCDR2和HCDR3：SEQ ID NO：7所示的HCDR1、SEQ ID NO：8所示的HCDR2、SEQ ID NO：9所示的HCDR3；SEQ ID NO：13所示的HCDR1、SEQ ID NO：14所示的HCDR2、SEQ ID NO：15所示的HCDR3；或SEQ ID NO：19所示的HCDR1、SEQ ID NO：20所示的HCDR2、SEQ ID NO：21所示的HCDR3。在一些实施方式中，本申请的抗体或其抗原结合片段进一步 包含所述抗体或其抗原结合片段进一步包含如下轻链可变区所包括的三个轻链互补决定区LCDR1、LCDR2和LCDR3：SEQ ID NO：26所示的轻链可变区；SEQ ID NO：28所示的轻链可变区；或SEQ ID NO：30所示的轻链可变区。在一些实施方式中，本申请的抗体或其抗原结合片段进一步包含选自下组的三个轻链互补决定区LCDR1、LCDR2和LCDR3：SEQ ID NO：10所示的LCDR1、SEQ ID NO：11所示的LCDR2以及SEQ ID NO：12所示的LCDR3；SEQ ID NO：16所示的LCDR1、SEQ ID NO：17所示的LCDR2以及SEQ ID NO：18所示的LCDR3；SEQ ID NO：22所示的LCDR1、SEQ ID NO：23所示的LCDR2以及SEQ ID NO：24所示的LCDR3。
在一些实施方式中，本申请的抗体或其抗原结合片段包含SEQ ID NO：7所示的HCDR1、SEQ ID NO：8所示的HCDR2、SEQ ID NO：9所示的HCDR3、SEQ ID NO：10所示的LCDR1、SEQ ID NO：11所示的LCDR2以及SEQ ID NO：12所示的LCDR3。在一些实施方式中，本申请的抗体或其抗原结合片段包含SEQ ID NO：13所示的HCDR1、SEQ ID NO：14所示的HCDR2、SEQ ID NO：15所示的HCDR3、SEQ ID NO：16所示的LCDR1、SEQ ID NO：17所示的LCDR2以及SEQ ID NO：18所示的LCDR3。在一些实施方式中，本申请的抗体或其抗原结合片段包含SEQ ID NO：19所示的HCDR1、SEQ ID NO：20所示的HCDR2、SEQ ID NO：21所示的HCDR3、SEQ ID NO：22所示的LCDR1、SEQ ID NO：23所示的LCDR2以及SEQ ID NO：24所示的LCDR3。
在一些实施方式中，本申请的抗体或其抗原结合片段包含选自下组的重链可变区V H和：SEQ ID NO：25所示的重链可变区；SEQ ID NO：27所示的重链可变区；或SEQ ID NO：29所示的重链可变区。在一些实施方式中，所述抗体或其抗原结合片段进一步包含选自下组的轻链可变区V L：SEQ ID NO：26所示的轻链可变区；SEQ ID NO：28所示的轻链可变区；或SEQ ID NO：30所示的轻链可变区。
在一些实施方式中，本申请的抗体或其抗原结合片段包含SEQ ID NO：25所示的重链可变区和SEQ ID NO：26所示的轻链可变区。在一些实施方式中，本申请的抗体或其抗原结合片段包含SEQ ID NO：27所示的重链可变区和SEQ ID NO：28所示的轻链可变区。在一些实施方式中，本申请的抗体或其抗原结合片段包含SEQ ID NO：29所示的重链可变区和SEQ ID NO：30所示的轻链可变区。
用于本申请的术语“抗体”包括任意免疫球蛋白、单克隆抗体、多价抗体、多特异性抗体或双特异性(双价)抗体。一个天然的完整抗体包含通过二硫链相互结合的两条重链和两条轻链。抗体的每条重链由一个可变区(V H)以及第一、第二和第三恒定区(分别为C H1、C H2、C H3)组成，而抗体的每条轻链由一个可变区(V L)以及一个恒定区(C L)组成。轻链和重链的可变区负责抗原结合。每条链中的可变区大致细分为3个高变区，称为互补决定区(CDR)(其中轻链CDR包括LCDR1、LCDR2、LCDR3。重链CDR包括HCDR1、HCDR2、HCDR3)。
用于本申请的抗体的“抗原结合片段”、抗体的“抗原结合部分”等包括任何天然存在的、可酶促获得的、合成的或基因改造的多肽，所述多肽特异性结合抗原形成复合物。抗体的抗原结合片段可以，例如，通过使用任何合适的标准技术从完整的抗体分子衍生出，所述合适的标准技术例如蛋白水解消化或涉及操纵和表达编码抗体可变结构域和任选地恒定结构域的DNA的重组基因工程技术。DNA可以通过化学方法或通过使用分子生物学技术进行测序和操作，从而例如，将一个或多个可变和/或恒定结构域排列成合适的构型，或引入密码子，产生半胱氨酸残基，修饰、添加或删除氨基酸等。抗原结合片段的非限制性实例包括：(i)Fab片段；(ii)F(ab′)2片段；(iii)Fd片段；(iv)Fv片段；(v)单链Fv(scFv)分子；(vi)dAb片段；和(vii)模拟抗体高变区的氨基酸残基组成的最小识别单位(例如，分离的互补决定区(CDR)如CDR3肽)或FR3-CDR3-FR4肽。其他改造的分子，如结构域特异性抗体、单域抗体、域缺失抗体、嵌合抗体、CDR嫁接抗体、双抗体、三抗体、四抗体、微抗体、纳米抗体(例如，单价纳米抗体、二价纳米抗体等)、小模块化免疫药物(SMIP)和鲨鱼变体IgNAR结构域也包括在本文所用的“抗原结合片段”表述内。
本申请中公开的抗体和抗原结合片段的CDR边界可通过Kabat、Chothia或Al-Lazikani命名法命名或识别。(Al-Lazikani，B.，Chothia，C.，Lesk，A.M.，J.Mol.Biol.，273(4)，927(1997)；Chothia，C等，J Mol Biol.Dec 5；186(3)：651-63(1985)；Chothia，C.和Lesk，A.M.，J.Mol.Biol.，196，901(1987)；Chothia，C等，Nature.Dec 21-28；31(6252)：877-83(1989)；Kabat E.A.等，National Institutes of Health，Bethesda，Md.(1991))。在一些实施方式中，根据Kabat数据库确定抗体的CDR边界。三个CDR由被称为框架区(FR，其中重链FR包括HFR1、HFR2、HFR3和HFR4，轻链FR包括LFR1、LFR2、LFR3和LFR4)的侧面连续部分间隔开，框架区比CDR更加高度保守 并形成一个支架支撑超变环。因此，V H和V L各包含按以下顺序排布的3个CDR和4个FR(人氨基酸残基N末端到C末端)：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。重链和轻链的恒定区不参与抗原结合，但表现出多种效应功能。哺乳动物重链分为α、δ、ε、γ和μ，哺乳动物轻链分为λ或κ。抗体根据其重链恒定区的氨基酸序列，根据是否分别存在α、δ、ε、γ和μ，可分为5大类：IgA、IgD、IgE、IgG和IgM。几个主要的抗体分类的亚类如IgG1(γ1重链)、IgG2(γ2重链)、IgG3(γ3重链)、IgG4(γ4重链)、IgA1(α1重链)或IgA2(α2重链)。
在一些实施方式中，本申请的抗体或其抗原结合片段特异性识别或结合具有SEQ ID NO：2所示的氨基酸序列的蛋白。
SEQ ID NO：2：
Figure PCTCN2022071976-appb-000001
在一些实施方式中，本申请的抗体或其抗原结合片段与具有SEQ ID NO：2所示的氨基酸序列的蛋白的的Kd值为10 -7-10 -10M。在一些实施方式中，所述Kd值为10 -7-10 -10M、10 -8-10 -10M或10 -9-10 -10M。在一些实施方式中，所述抗体或其抗原结合片段与具有SEQ ID NO：2所示的氨基酸序列的蛋白的Kd值小于约10 -7M。在一些实施方式中，所述Kd值小于约10 -7M、小于约10 -8M、小于约10 -9M或小于约10 -10M。在一些实施方式中，Kd值是通过表面等离子共振方法检测的。
在一些实施方式中，本申请的抗原结合片段是选自F(ab’) 2、Fab’、Fab、Fv、scFv、dsFv或dAb。在一些实施方式中，本申请的抗体或其抗原结合片段进一步具有重链恒定区和/或轻链恒定区。在一些实施方式中，重链恒定区源于鼠的IgG1或鼠的IgG2a。在一些实施方式中，重链恒定区源于人的IgG1。
在一些实施方式中，本申请的抗体或其抗原结合片段进一步具有缀合部分。在一些实施方式中，所述缀合部分是治疗剂、放射性同位素、可检测标签、药代动力学修饰部分或纯化部分。所述缀合部分直接连接或者通过连接子连接。
用于本申请的术语“治疗剂”可以是用于治疗或预防受试者中的疾病的任意药物化合物，包括分子量小于500小分子、核苷酸(例如，DNA、质粒DNA、RNA、 siRNA、反义寡核苷酸、适体等)、短肽、蛋白(例如，酶)。
在一些实施方式中，放射性同位素可以选自下组： 3H、 11C、 14C、 18F、 32P、 33P、 35S、 45Ti、 47Sc、 52Fe、 59Fe、 62Cu、 64Cu、 67Cu、 67Ga、 68Ga、 75Sc、 77As、 86Y、 89Sr、 89Zr、 90Y、 90Nb、 94Tc、 99Tc、 99Mo、 105Pd、 105Rh、 111Ag、 111In、 123I、 124I、 125I、 131I、 133Xe、 142Pr、 143Pr、 149Pm、 153Sm、 154Gd、 155Gd、 156Gd、 157Gd、 158Gd、 161Tb、 166Dy、 169Er、 175Lu、 177Lu、 186Re、 188Re、 189Re、 194Ir、 198Au、 199Au、 211At、 211Pb、 212Bi、 212Pb、 213Bi、 223Ra和 225Ac。
用于本申请的术语“可检测标签”可以是与本发明的抗体直接或间接地偶联、同时适用于或有助于检测TRPV6的水平的任何检测分子。在一些实施方式中，所述检测分子包括酶、荧光标记物和生物素。所述酶包括过氧化酶(例如，辣根过氧化酶)、碱性磷酸酶、β-半乳糖苷酶、葡糖淀粉酶、糖氧化酶、尿素酶、杂环氧化酶(例如，黄嘌呤氧化酶)、苹果酸脱氢酶或它们的衍生物。所述荧光标记物可以是荧光素，可选的，所述荧光素包括异硫氰酸荧光素(FITC)、罗丹明、德克萨斯红、藻红蛋白、绿色荧光蛋白(GFP)、Dylight、Cy3或Cy5。
另一个方面，本申请还公开了一种分离的核酸，其编码本申请所述的特异性结合TRPV6的抗体或其抗原结合片段。在一些实施方式中，所述核酸包含SEQ ID NO：31所示的核酸序列或与SEQ ID NO：31具有至少90％序列同一性的核酸序列，和SEQ ID NO：32所示的核酸序列或与SEQ ID NO：32具有至少90％序列同一性的核酸序列中的一者或两者。在一些实施方式中，所述核酸包含SEQ ID NO：33所示的核酸序列 或与SEQ ID NO：33具有至少90％序列同一性的核酸序列，和SEQ ID NO：34所示的核酸序列或与SEQ ID NO：34具有至少90％序列同一性的核酸序列中的一者或两者。在一些实施方式中，所述核酸包含SEQ ID NO：35所示的核酸序列或与SEQ ID NO：35具有至少90％序列同一性的核酸序列，和SEQ ID NO：6所示的核酸序列或与SEQ ID NO：6具有至少90％序列同一性的核酸序列中的一者或两者。
另一个方面，本申请还公开了一种载体，其包含本申请所述的核酸序列。
另一个方面，本申请还公开了一种宿主细胞，其包含本申请所述的载体。
另一个方面，本申请还公开了一种多特异性抗体，其包含本申请所述的抗体或其抗原结合片段，和一个或多个与其他抗原特异性结合的抗体或其抗原结合部分。
另一个方面，本申请还公开了一种抗体偶联物，其包含本申请所述的抗体或其抗原结合片段或本申请所述的多特异性抗体。
另一个方面，本申请还公开了分泌抗TRPV6单克隆抗体的杂交瘤细胞株，所述杂交瘤细胞株为12CT9.5.1、16CT4.1.1.2和/或16CT18.1.1.2，保藏于中国典型培养物保藏中心，其中12CT9.5.1的保藏编号为CCTCC NO：C202119，16CT4.1.1.2的保藏编号为CCTCC NO：C202124，16CT18.1.1.2的保藏编号为CCTCC NO：C202125。
另一个方面，本申请还公开了一种药物组合物，其包含本申请所述的抗体或其抗原结合片段、本申请所述的分离的核酸、本申请所述的载体、本申请所述的宿主细胞、本申请所述的多特异性抗体或本申请所述的抗体偶联物，和药学上可接受的载体。
用于本申请的术语“药学上可接受的”指的是在合理的医学判断范围内，其适用于与人和其他动物的细胞接触而没有不适当的毒性、刺激性、过敏反应等，并且与合理的效益/风险比是相称的。
用于本申请的术语“药学上可接受的载体”指的是用于向受试者递送本申请提供的抗体或其抗原结合片段、分离的核酸、载体、宿主细胞、多特异性抗体或抗体偶联物的药学上可接受的溶剂、混悬剂或任意其他药学上惰性的载剂，其不干扰抗体或其抗原结合片段、分离的核酸、载体、宿主细胞、多特异性抗体或抗体偶联物的结构、形态和性质。某些这样的载体能够将抗体或其抗原结合片段、分离的核酸、载体、宿主细胞、多特异性抗体或抗体偶联物配制成例如片剂、丸剂、胶囊剂、液体、凝胶剂、糖 浆剂、浆剂、混悬剂和软锭剂，供受试者口服摄入。某些这样的载体能够将抗体或其抗原结合片段、分离的核酸、载体、宿主细胞、多特异性抗体或抗体偶联物制成注射、输注或局部施用的制剂。
用于本申请提供的药物组合物中的药学上可接受的载体包括但不限于，例如药学上可接受的液体、凝胶或固体载体、水性载剂(例如，氯化钠注射液、林格氏注射液、等渗右旋糖注射液、无菌水注射液或者右旋糖和乳酸林格氏注射液)、非水性载剂(例如，植物来源的固定油、棉籽油、玉米油、芝麻油或花生油)、抗微生物剂、等渗剂(例如，氯化钠或右旋糖)、缓冲剂(例如，磷酸或柠檬酸缓冲剂)、抗氧剂(例如，硫酸氢钠)、麻醉剂(例如，盐酸普鲁卡因)、混悬剂/分散剂(例如，羧甲基纤维素钠、羟丙基甲基纤维素或聚乙烯吡咯烷酮)、螯合剂(例如，EDTA(乙二胺四乙酸)或EGTA(乙二醇四乙酸))、乳化剂(例如，聚山梨醇酯80(吐温-80))、稀释剂、佐剂、赋形剂、或无毒性辅助物质、本领域公知的其他成分或其各种组合。适宜的成分可以包括例如填充剂、粘合剂、缓冲剂、防腐剂、润滑剂、调味剂、增稠剂、着色剂或乳化剂。
在一些实施方式中，药物组合物是注射制剂。注射制剂包括无菌水溶液或分散剂、混悬剂或乳剂。在所有情况下，注射制剂应该是无菌的并且应该是流体以便于注射。注射制剂应在生产和储存条件下保持稳定，并且必须防止细菌和真菌等微生物的污染。载体可以是含有例如水、乙醇、多元醇(例如，甘油、丙二醇和液体聚乙二醇等)及其适宜的混合物和/或植物油的溶剂或分散介质。注射制剂应保持适当的流动性。例如，可以通过使用诸如卵磷脂的包衣、通过使用表面活性剂等保持适当的流动性。可以通过各种抗细菌剂和抗真菌剂实现阻止微生物的作用，例如对羟基苯甲酸酯、氯丁醇、苯酚、山梨酸、硫柳汞等。
在一些实施方式中，药物组合物是口服制剂。口服制剂包括但不限于胶囊、扁胶囊、丸剂、片剂、锭剂(使用调味基质，通常为蔗糖和***胶或黄蓍胶)、粉剂、颗粒剂、或在水性或非水性液体中的溶液剂或混悬剂、或者作为水包油或油包水液体乳剂、或者作为酏剂或糖浆、或者作为软锭剂(使用惰性基质，如明胶和甘油，或者蔗糖和***胶)和/或作为漱口剂等。
在用于口服施用的固体剂型(例如，胶囊、片剂、丸剂、糖衣丸、散剂、颗粒剂等)中，将抗体或其抗原结合片段、分离的核酸、载体、宿主细胞、多特异性抗 体或抗体偶联物的与一种或多种药学上可接受的载体混合，如柠檬酸钠或磷酸氢二钙，和/或下述中的任意一种：(1)填充剂或增量剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和/或硅酸；(2)粘合剂，例如，羧甲基纤维素、藻酸盐、明胶、聚乙烯吡咯烷酮、蔗糖和/或***胶；(3)保湿剂，例如，甘油；(4)崩解剂，例如，琼脂、碳酸钙、马铃薯或木薯淀粉、海藻酸、某些硅酸盐和碳酸钠；(5)溶液阻滞剂，例如，石蜡；(6)吸收促进剂，例如，季铵化合物；(7)润湿剂，例如，乙酰基醇和单硬脂酸甘油酯；(8)吸收剂，例如，高岭土和膨润土；(9)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、月桂基硫酸钠及其混合物；以及(10)着色剂。
在用于口服施用的液体剂型中，将抗体或其抗原结合片段、分离的核酸、载体、宿主细胞、多特异性抗体或抗体偶联物与下述中的任意一种混合：药学上可接受的乳剂、微乳、溶液、混悬剂、糖浆剂和酏剂。除了抗体或其抗原结合片段、分离的核酸、载体、宿主细胞、多特异性抗体或抗体偶联物以外，液体剂型可以含有本领域常用的惰性稀释剂，例如，水或其他溶剂、增溶剂和乳化剂，例如，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、苯甲醇、苯甲酸苄酯、异丙醇、1，3-丁二醇、油(特别是，棉籽油、花生油、玉米油、橄榄油、蓖麻油和芝麻油)、甘油、四氢糠醇、聚乙二醇和脱水山梨醇脂肪酸酯及其混合物。除了惰性稀释剂以外，口服组合物还可以包含佐剂，例如，润湿剂、乳化剂和混悬剂、甜味剂、调味剂、着色剂、增香剂和防腐剂。
在一些实施方式中，药物组合物是口腔喷雾制剂或鼻喷雾制剂。喷雾制剂包括但不限于水性气雾剂、非水性混悬剂、脂质体制剂或固体颗粒制剂等。水性气雾剂通过将药剂的水溶液或混悬液与常规药学上可接受的载体和稳定剂混合制备。载体和稳定剂根据具体药物成分的要求而改变，但是在通常情况下，其包括非离子表面活性剂(吐温或聚乙二醇)、油酸、卵磷脂、氨基酸，例如，甘氨酸、缓冲溶液、盐、糖或糖醇。气雾剂通常由等渗溶液制备，并且能够通过喷雾递送。
在一些实施方式中，药物组合物可以通过与一种或多种其他药物混合使用。在一些实施方式中，药物组合物包含至少一种其他药物。在一些实施方式中，其他药物是抗肿瘤药、心血管药、抗炎药、抗病毒药、消化***药、神经***药、呼吸***药、免疫***药、皮肤病药、代谢药等。
在一些实施方式中，可以通过适宜的途径向有需要的受试者施用药物组合物，包括但不限于口服、注射(例如，静脉内、肌内、皮下、皮内、心内、鞘内、胸膜 内、腹膜内注射等)、粘膜(例如，鼻内、口内施用等)、舌下、直肠、经皮、眼内和肺部施用。在一些实施方式中，药物组合物可以静脉内、皮下、口服、肌内或心室内施用。
另一个方面，本申请还公开了一种用于分析和检测来自受试者的样品中的TRPV6的试剂，所述试剂包含一种或多种本申请所述的抗体或其抗原结合片段。在一些实施方式中，所述试剂用于分析和检测来自受试者的样品中的TRPV6。
另一个方面，本申请还公开了一种检测来自受试者的样品中的TRPV6的方法，所述方法包括：(1)使所述样品与本申请述的抗体或抗原结合片段接触；(2)可选地，移除未结合的抗体或抗体片段；(3)检测与所述样品中的TRPV6结合的抗体或抗体片段。
用于本申请的术语“检测”可以是通过确定和测定分子(例如，多肽、蛋白或核酸分子)的存在或不存在，或是确定和测定分子之间的相互作用(例如，结合、激动或拮抗等作用)，例如蛋白质-蛋白质相互作用(例如，抗体和抗原之间的相互作用)、蛋白-核酸相互作用、或核酸-核酸相互作用。在一些实施方案中，“检测”和“测定”包括定量检测和测定。在一些实施方式中，检测是通过确定和测定与抗体直接或间接偶联的检测分子的信号进行的。可以通过本领域技术人员熟悉的方法进行检测。在一些实施方式中，使用免疫组织化学(IHC)、酶联免疫吸附(ELISA)、免疫荧光(IF)、化学发光(CLIA)、免疫比浊(TIA)或免疫印迹(WesternBlot)进行检测。
在一些实施方式中，在检测来自受试者的样品中的TRPV6的方法的步骤(1)中，本申请所述的抗体或抗原结合片段被固定在基板上。在一些实施方式中，在检测来自受试者的样品中的TRPV6的方法的步骤(1)中，所述样品被固定在基板上。在一些实施方式中，样品是细胞或组织。样品包括但不限于细胞离心制备物、细胞学涂片、核心活检、细针穿刺和/或组织切片(例如，低温恒温器组织切片、石蜡包埋组织切片)。在一些实施方式中，样品包含活细胞。在一些实施方式中，样品包含肿瘤细胞。在一些实施方式中，使用化学固定剂进行所述固定。可以使用交联固定剂(例如醛类，包括例如甲醛、多聚甲醛和戊二醛)制备组织学样品。组织样品可以经甲醛固定并包埋在石蜡中。还可以使用多聚甲醛固定，包埋在温度敏感的低温材料中，并使用液氮速冻。其他类化学固定剂还包括氧化剂和酒精固定剂。还可以通过物理方法进行固定。
在一些实施方式中，在检测来自受试者的样品中的TRPV6的方法的步骤(3)中，还包括使用第二抗体，可选的，所述第二抗体能够直接或者间接地结合所述样品中的TRPV6，可选的，所述第二抗体的表位与本申请所述的抗体或抗原结合片段的表位不同，可选的，所述第二抗体的表位与本申请所述的抗体或抗原结合片段的表位相同，可选的，所述第二抗体的表位与TRPV6的结合不受本申请所述的抗体或抗原结合片段的影响，可选的，所述第二抗体的表位与TRPV6的结合受本申请所述的抗体或抗原结合片段的影响。
用于本申请的术语“表位”是指与抗体分子可变区中的特定抗原结合位点(称为互补位)相互作用的抗原决定簇。单一抗原可以具有超过一个表位。因此，不同抗体可以结合至一个抗原上的不同区域并且可以具有不同的生物作用。表位可以是构象表位或线性表位。构象表位是通过空间并列的来自线性多肽链不同区段的氨基酸产生的。线性表位是由多肽链中的相邻氨基酸残基产生的表位。在某些情况中，表位可以包含抗原上的糖、磷酰基或磺酰基部分。
在一些实施方式中，在检测来自受试者的样品中的TRPV6的方法的步骤(3)中，还包括使用第二抗体，可选的，所述第二抗体能够直接或间接地结合本申请所述的抗体或抗原结合片段，可选的，所述第二抗体能够结合本申请所述的抗体或抗原结合片段的恒定区部分。本文所使用的的“第二抗体”旨在于更好地检测抗TRPV6抗体，第二抗体一般可以通过商购获得。一些实施方式中，本申请所述的第二抗体与检测分子偶联，可选的，所述检测分子包括酶、荧光标记物和生物素。在一些实施方式中，所述检测分子为酶，可选的，所述酶包括过氧化酶(例如，辣根过氧化酶)、碱性磷酸酶、β-半乳糖苷酶、葡糖淀粉酶、糖氧化酶、尿素酶、杂环氧化酶(例如，黄嘌呤氧化酶)、苹果酸脱氢酶或其衍生物。在一些实施方式中，所述检测分子为荧光素，可选的，所述荧光素包括FITC、罗丹明、德克萨斯红、藻红蛋白、GFP、Cy3、Cy5或Dylight。在一些实施方式中，所述检测分子为生物素或其衍生物。
在一些实施方式中，检测来自受试者的样品中的TRPV6的方法进一步包括对与所述样品中的TRPV6结合的抗体进行定量。在一些实施方式中，所述方法为免疫组织化学(IHC)、酶联免疫吸附(ELISA)、免疫荧光(IF)、化学发光(CLIA)、免疫比浊(TIA)或免疫印迹(Western Blot)。在一些实施方式中，所述方法可以评估所述样品中在细胞表面上表达TRPV6的细胞的比例。
在一些实施方式中，在检测来自受试者的样品中的TRPV6的方法中，所述受试者为人类。
另一个方面，本申请还公开了一种判断受试者疾病的形成或形成的风险、评估受试者疾病的进展或预后、或在接受疾病治疗的受试者中预测或监测所述受试者对治疗的应答的方法，所述方法包括通过本申请所述的检测来自受试者的样品中TRPV6的方法，以检测来自所述受试者的样品中的TRPV6。
在一些实施方式中，判断受试者疾病的形成或形成的风险、评估受试者疾病的进展或预后、或在接受疾病治疗的受试者中预测或监测所述受试者对治疗的应答的方法包括评估所述样品中在细胞表面上表达TRPV6的细胞的比例，若所述比例超过预定的阈值，则认为所述受试者患有所述疾病或者是更可能从治疗中获益，其中所述阈值为10％。在一些实施方式中，所述治疗是靶向药物治疗。
用于本申请的术语“获益”、“受益”或“有益”指任何期望的效果，包括但不限于：(1)在某种程度上抑制(如减少、减慢或完全停止)疾病的发展，包括减慢速度和完全停滞；(2)减少疾病发作和/或症状的数量；(3)缩小病变尺寸；(4)抑制疾病细胞浸润到邻近的周围器官和/或组织中；(5)抑制疾病传播；(6)在一定程度上缓解与该疾病有关的一种或多种症状；(7)治疗后无病表现的时间增加；(8)自身免疫反应降低，可能但不一定导致疾病病变消退或消融，例如无进展生存期；(9)增加总生存率；(10)应答率更高；和/或(11)治疗后给定时间点死亡率降低。
在一些实施方式中，预测或监测所述受试者对治疗的应答涉及评估受试者的肿瘤是否减轻或加重。在一些实施方式中，预测或监测所述受试者对治疗的应答可能涉及评估受试者在接受治疗(例如，用特定的药物进行治疗)后是否好转和/或好转的可能性。本申请的预测或监督的方法可以被用于在临床上做出治疗决定。在施用治疗方案(例如，给定的治疗方案，包括例如给定的治疗药物或组合的施用、手术干预等)后，本申请的预测或监督的方法在评价特定受试者能否长期存活的可能性上是有价值的工具。
在一些实施方式中，本申请所述的疾病为癌症。在一些实施方式中，癌症为乳腺癌、卵巢癌、子宫内膜癌、胆管癌、胃癌、食道癌、肺癌、肠癌、胰腺癌。在一些实施方式中，癌症为HER2 +乳腺癌、三阴乳腺癌或结直肠癌。
另一个方面，本申请还公开了一种试剂盒，所述试剂盒包含本申请所述的抗体或其抗原结合片段，可选的，进一步包括用于检测所述抗体或其抗原结合片段与TRPV6结合情况的试剂。
在一些实施方式中，本申请所述的试剂盒用于诊断受试者疾病的形成或形成的风险、评估受试者疾病的进展或预后、或在接受疾病治疗的受试者中预测或监测所述受试者对治疗的应答。在一些实施方式中，本申请所述的试剂盒用于免疫组化病理诊断。在一些实施方式中，本申请所述的试剂盒用于肿瘤组织免疫组化病理诊断。在一些实施方式中，所述疾病为癌症，在一些实施方式中，癌症为乳腺癌、卵巢癌、子宫内膜癌、胆管癌、胃癌、食道癌、肺癌、肠癌、胰腺癌。在又一些实施方式中，所述癌症是HER2+乳腺癌、三阴乳腺癌或结直肠癌。
另一个方面，本申请还公开了一种制备特异性结合TRPV6的抗体的方法，所述方法包括：(1)将SEQ ID NO：3所示的氨基酸序列与载体蛋白偶联，以获得TRPV6抗原肽；(2)将通过步骤(1)获得的所述TRPV6抗原肽作为免疫原免疫小鼠；(3)经细胞融合、免疫肽筛选克隆，获得高效分泌特异性结合TRPV6的抗体的阳性杂交瘤细胞系；(4)获得特异性结合TRPV6的抗体。
在一些实施方式中，所述载体蛋白为钥孔虫戚血蓝素(KLH)蛋白。
在一些实施方式中，在SEQ ID NO：3所示的氨基酸序列的N端增加一个Cys，并通过自由巯基和所述载体蛋白偶联。
SEQ ID NO：3：PLKPRTNNRTSPRDNTLLQQKLLQEAYMTPK。
在一些实施方式中，本申请所述的杂交瘤细胞系为小鼠杂交瘤细胞系12CT 9.5.1、16CT 18.1.1或16CT 4.1.1.2。其中，细胞系12CT 9.5.1产生的抗体包括CB-Anti-0001抗体，细胞系16CT4.1.1.2产生的抗体包括CB-Anti-0002抗体，细胞系16CT18.1.1产生的抗体包括CB-Anti-0003抗体。
另一个方面，本申请还公开了本申请所述抗体或其抗原结合片段、本申请所述的多特异性抗体、本申请所述的抗体偶联物以及本申请所述的药物组合物在用于治疗和/或预防和/或诊断与TRPV6相关的疾病的药物中的用途。
另一个方面，本申请还公开了一种治疗、预防或诊断疾病的方法，所述方法包括向有需要的受试者施用本申请所述抗体或其抗原结合片段、本申请所述的多特异性抗体、本申请所述的抗体偶联物以及本申请所述的药物组合物。
实施例
以下实施例旨在更好地说明本发明，且不应理解为限制本发明的范围。所有下述的特定组合物、材料和方法，其整体或部分，都在本发明的范围内。这些特定的组合物、材料和方法不是为了限制本发明，而只是为说明特定的实施方式在本发明的范围内。本领域技术人员可不添加创造性及不偏离本发明范围而开发出等同的组合物、材料和方法。应理解，在对本发明的方法作出的多种改动可以仍然包含在本发明范围内。发明人意在将这样的变动包含在本发明的范围内。
实施例1抗原多肽的合成和重组抗原的制备
1.抗原多肽选择
针对Uniprot及Genbank中登录号为Q9H1D0和GI：1918017293的蛋白质序列(如SEQ ID NO：2所示)进行序列和二级结构分析。该TRPV6蛋白全长为765个氨基酸，其分子量约为87kDa左右。通过在线服务器http：//www.cbs.dtu.dk/services/NetSurfP/预测该蛋白的二级结构和表面可及性(Surface Accessibility)参数，并通过针对其抗原性指数的分析结果，选择氨基酸序列IFQTEDPEELGHF(SEQ ID NO：4)、DGPANYNVDLPF(SEQ ID NO：5)或PLKPRTNNRTSPRDNTLLQQKLLQEAYMTPK(SEQ ID NO：3)作为抗原并进行化学合成。为便于偶联，在该多肽的氨基端添加了一个半胱氨酸，用于提供硫基。
2.多肽的偶联及纯化
选择Thermo Scientific的马来酰胺活化的钥孔虫戚血蓝素蛋白试剂盒，按照试剂盒提供的流程操作。
对于待偶联的多肽，首先以Ellman试剂检测多肽中自由硫基：在96孔板中加入100μL Ellman试剂储备液，再加入10μL多肽溶液，用Nano Drop分光光度计在λ＝412nm下测其紫外吸收值，如果OD值＞0.15，则进行下一步；若OD值＜0.15且＞0.05，则补加多肽，直至达到要求；若OD值＜0.05，则返回多肽合成步骤重新质控。
开始偶联时，在每个mcKLH包装中加入200μL去离子水，配制成10mg/mL的KLH溶液，在500μL的Imiect EDC偶联缓冲液中溶解2mg半抗原，将500μL多肽溶液加入到200μL的载体蛋白溶液中。将1mL去离子水加入到一个包装的EDC(10mg)中，缓慢摇动至完全溶解，取50μL加入到mcKLH多肽溶液中，反应2小时后，经脱盐柱处理除去未偶联的交联剂和盐类。
实施例2杂交瘤细胞系的建立
1.免疫
将实施例1中交联的多肽用弗氏完全佐剂乳化，免疫4～6周龄雌性Balb/c小鼠(购自北京维通利华实验动物技术有限公司)，腹部皮下注射，每只小鼠6点，剂量为60μg/只。每14天加强免疫一次，抗原使用弗氏非完全佐剂乳化，剂量为30μg/只。第3次加强免疫后7天，以间接ELISA(波长450nm)检测小鼠血清中抗免疫原的多抗效价，效价最高的小鼠以尾静脉注射冲击免疫，抗原用生理盐水混匀，剂量为50μg/只。
2.细胞融合
制备免疫达标的小鼠脾细胞悬液，与小鼠骨髓瘤细胞F0以5：1比例混合，离心1500rpm，5分钟。弃上清后，离心管放入37℃水浴中，在1分钟内缓慢加入1mL的PEG1500(Roche公司)，并搅动细胞。在温水中静置1分钟后，加入10mL无血清的IMDM(Sigma公司)，混匀，离心1000rpm，5分钟。弃上清后，加入10mL血清(PAA公司)，小心的将细胞吹打起来，并加入5mL混合10XHAT(Sigma公司)的胸腺细胞，混匀。再加入25mL含有2.1％硝基纤维素(Sigma公司)的半固体培养基，充分混匀，然后均匀的倒入20个细胞培养皿中。将细胞培养皿放入到湿盒中，放入37℃、5％CO 2培养箱中培养。
3.克隆化及ELISA筛选阳性杂交瘤细胞
融合后7天，克隆细胞团大小密度适中，在解剖镜下，吸取圆、实、大的克隆团，打入事先准备好培养基的96孔培养板中，放入37℃、5％CO 2培养箱中培养。
3天后，细胞量大约占底面积2/3，取100μL上清，用免疫原和合成多肽分别进行ELISA筛选。阳性克隆完全换液，加入200μL的含饲养细胞和1％HT(Sigma公司)的完全培养基。两天后进行第二次ELISA筛选，阳性克隆转入事先准备好培养 基(含饲养细胞和HT)的24孔板培养。五天后取100μL上清，进行第三次ELISA筛选，筛选得到12CT 9.5.1、16CT 18.1.1.2和16CT 4.1.1.2三株效价较高的阳性克隆，并逐次转入6孔板和细胞培养瓶扩大培养并冻存。
实施例3腹水诱生法制备单克隆抗体
1.腹水制备
对数生长期细胞用无血清培养基洗涤并悬起，计数～5x10 5/mL，1mL悬浮的细胞腹腔注射事先用石蜡油致敏的小鼠。7天后开始收集腹水。取出的腹水于4℃下离心4000rpm，10分钟。小心吸出中间的腹水收集于离心管中，4℃或-20℃保存。
2.单克隆抗体的纯化
用Protein G(GE公司)亲和层析法按说明书从腹水中纯化抗体。SDS-PAGE胶鉴定纯度，Bradford法测定浓度。纯化的抗体保存于-20℃。
实施例4单克隆抗体特性鉴定
1.亚型鉴定
用100mL MPBS(pH 7.4)稀释包被羊抗鼠IgG至0.5μg/mL，每孔加100μL，4℃，过夜。倾空液体，用含0.05％Tween的PBS洗3次，每孔加入200μL封闭液(含2％BSA和3％蔗糖的PBS)，37℃下孵育1小时，倾空液体，用PBST清洗3次。每孔加入0.1mL杂交瘤上清，37℃下孵育1小时，倾空液体，用PBST清洗3次。用封闭液1：1000稀释HRP标记的羊抗鼠(κ，λ)抗体或1：2000稀释HRP标记的羊抗鼠(IgM、IgG1、IgG2a、IgG2b、IgG3、IgA)抗体，0.1mL每孔分别加入适当的孔中，37℃下孵育1小时，倾空液体，用PBST清洗3次。每孔加50μL含0.15％ABTS和0.03％H 2O 2的拧檬酸缓冲液(pH 4.0)进行显色反应，10-20分钟内测定405nm波长下的OD值。结果显示，获得的CB-Anti-0001和CB-Anti-0002抗体为IgG1型鼠源单克隆抗体，CB-Anti-0003抗体为IgG2a型鼠源单克隆抗体。
2.亲和常数测定
首先，在SPR芯片上固定获得的抗TRPV6抗体，之后用1×PBST作为稀释液，将免疫肽稀释至11.52nM，之后以1.3倍稀释，最后一个浓度为4.04nM，共 计5个浓度梯度。利用SPR仪器进行亲和力检测，经仪器自动分析，计算出CB-Anti-0001的亲和常数为5.19x10 -10。
3.单抗反应特异性和应用效果
选择转染TRPV6蛋白的293T细胞，用免疫细胞化学的方法检测本发明的单克隆抗体的识别特异性，免疫细胞化学实验过程如下：在293T细胞中转染TRPV6质粒，转染后的细胞铺在含爬片的24孔板中，在IMDM+10％FBS培养基中培养细胞72小时，去除上清，PBS清洗一遍，加入甲醇固定30秒，PBS清洗两遍。用抗体稀释液按照适当稀释比配制抗TRPV6抗体工作液，去除24孔板中的PBS，加入抗体工作液，4度孵育过夜，PBS清洗三遍，滴加二抗(Elivision TMplus Polyer HRP(Mouse/Rabbit)IHC Kit，购自福州迈新生物技术开发有限公司，产品编号KIT-9903)，室温孵育15-20分钟，PBS冲洗3X3分钟(即，冲洗3次，每次持续3分钟)，甩去PBS，用新鲜配置的DAB显色液显色3分钟。苏木素复染25秒，PBS返蓝30秒。按照75％、95％、100％、100％的酒精梯度依次脱水，最后二甲苯透明3分钟，中性树胶封片。
如图1A-1C所示，CB-Anti-0001抗体、CB-Anti-0002抗体和CB-Anti-0003抗体分别能够在转染了TRPV6质粒的293T细胞中通过免疫细胞化学特异地检测出TRPV6蛋白，而同时在未转染TRPV6质粒的293T细胞中未显示出检测到TRPV6蛋白。该结果表明获得的抗TRPV6抗体能特异性地识别TRPV6蛋白，即具有识别特性。
实施例5抗体的可变区序列测定
取培养的杂交瘤细胞系1x10 6，离心取上清，将细胞沉淀委托苏州金唯智生物科技有限公司进行抗体重链和轻链可变区序列测定，如下示出示例性的抗体重链可变区序列和抗体轻链可变区序列。
表1抗体CDR氨基酸序列
Figure PCTCN2022071976-appb-000002
Figure PCTCN2022071976-appb-000003
表2抗体可变区氨基酸序列
Figure PCTCN2022071976-appb-000004
Figure PCTCN2022071976-appb-000005
表3编码抗体可变区的核酸序列
Figure PCTCN2022071976-appb-000006
Figure PCTCN2022071976-appb-000007
实施例6组织切片染色和鉴定
1.切片制备过程
组织蜡块在修片后进行连续切片，厚度定为5μm，将连续切片在45℃的温水中展开，用正电子防脱载玻片裱贴切片，室温风干过夜后，放入-20℃中长期保存。
2.IHC染色及分析
常规二甲苯脱蜡2次，每次15分钟，100％、100％、95％、75％梯度乙醇中水化，每次3分钟，最后自来水冲洗。进行抗原修复，然后将切片放入湿盒中，PBS冲洗3X3分钟。滴加3％H 2O 2孵育10分钟，PBS冲洗3X3分钟。甩去PBS，滴加封闭液(动物非免疫血清)室温孵育60分钟。甩干切片，滴加适当比例稀释的一抗，4℃下孵育过夜，PBS冲洗3X3分钟，滴加二抗(Elivision TM plus Polyer HRP(Mouse/Rabbit)IHC Kit，购自福州迈新生物技术开发有限公司，产品编号KIT-9903)，室温孵育15-20分钟，PBS冲洗3X3分钟，甩去PBS，用新鲜配置的DAB显色液显色3分钟。苏木素复染25秒，PBS返蓝30秒。按照75％、95％、100％、100％的酒精梯度依次脱水，最后二甲苯透明3分钟，中性树胶封片。
3.数据统计
如图2A-2C所示，CB-Anti-0001抗体、CB-Anti-0002抗体和CB-Anti-0003抗体分别能够通过免疫组织化学检测组织切片中的TRPV6蛋白。
此外，发明人还通过采用H-score计分方法记录肿瘤细胞细胞膜染色情况，将H-score为60分以上记录为强染色，H-score为1-60分记录为弱染色，强染色以及弱染色合计为阳性；H-score为0记为阴性。分别统计IHC检测的各肿瘤组织中TRPV6的水平，并于TCGA数据库中TRPV6 mRNA水平进行对比验证，以评估抗体特异性，记录信息列于表4。结果显示，使用本发明的抗体检测TRPV6蛋白水平，阳性率较高(特别是强染色比例较高)的癌种对应地在肿瘤基因组图谱数据库(the Cancer Genome Atlas Program，即TCGA数据库)中统计的TRPV6 mRNA水平也较高(例如，TPM大于1)。由此可见，使用本发明的抗体能够准确的评估细胞表面表达TRPV6的情况，进而判断被检测的样品是否为表达TRPV6的癌细胞。
表4 IHC检测的肿瘤组织中TRPV6的水平
Figure PCTCN2022071976-appb-000008
实施例7 IHC对比实验
将市售抗体“Anti-Human TRPV6(extracellular)Antibody”(货号ACC-028，购于Alomone Labs，RRID：AB_2756545，本文简称“ACC-028抗体”)、抗体“Anti-TrpV6 Antibody，clone 4A5.1”(货号MABN839，购于Sigma-Aldrich公司，本文简称“MABN839抗体”)和CB-Anti-0001抗体进行对比实验。
使用与实施例6第2部分相同的步骤，获取CDX肿瘤模型RT4(人膀胱移行细胞***瘤细胞)的切片。滴加ACC-028抗体(稀释比例为1：50)、MABN839抗体(稀释比例为1：50)和CB-Anti-0001抗体(稀释比例为1：2000)，4℃下孵育过夜，PBS冲洗3X3分钟，滴加二抗(Elivision TM plus Polyer HRP(Mouse/Rabbit)IHC Kit，购自福州迈新生物技术开发有限公司，产品编号KIT-9903)，室温孵育15-20分钟，PBS冲洗3X3分钟，甩去PBS，用新鲜配置的DAB显色液显色3分钟。苏木素复染25秒，PBS返蓝30秒。按照75％、95％、100％、100％的酒精梯度依次脱水，最后二甲苯透明3分钟，中性树胶封片，读取染色结果。结果表明，ACC-028抗体检测灵敏度偏低，仅在少量RT4细胞膜上检测到特异性信号(参见附图3A)；MABN839抗体特异性较差，在所有组织细胞和***都检测到信号(参见附图3B)；相比之下，CB-Anti-0001抗体检测信号定位准确清晰、特异性好、灵敏度高(参见附图3C)，是理想的IHC应用抗体。
实施例8稳定性实验
采用加速破坏方法对CB-Anti-0001抗体进行稳定性验证，即分别(1)将抗体原液在4℃下保存14天；(2)将抗体原液放置在37℃恒温孵育箱中孵育14天；以及(3)将抗体工作液(使用中杉金桥ZLI-9028抗体稀释液以1：2000的比例稀释抗体原液后获得该抗体工作液)放置在37℃恒温孵育箱中孵育14天。在相同实验条件下，使用前述三类抗体液进行IHC检测，以评估三种处理方式下抗体的稳定性。
经检测，在不同条件下保存的抗体的组织染色强度和染色比例均未出现显著改变(参见附图4，其中附图4A为在4℃下保存14天的CB-Anti-0001抗体原液的IHC结果，附图4B为在37℃下保存14天的CB-Anti-0001抗体原液的IHC结果，附图4C为在37℃下保存14天的CB-Anti-0001抗体工作液的IHC结果)。可见，CB-Anti-0001抗体工作液在37℃温度下保存14天后，抗体依旧稳定，且保留好的检测能力。

Claims (57)

  1. 一种特异性结合TRPV6的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含如下重链可变区所包括的三个重链互补决定区HCDR1、HCDR2和HCDR3：
    (a)SEQ ID NO：25所示的重链可变区；
    (b)SEQ ID NO：27所示的重链可变区；或
    (c)SEQ ID NO：29所示的重链可变区。
  2. 根据权利要求1所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含选自下组的三个重链互补决定区HCDR1、HCDR2和HCDR3：
    (a)SEQ ID NO：7所示的HCDR1、SEQ ID NO：8所示的HCDR2、SEQ ID NO：9所示的HCDR3；
    (b)SEQ ID NO：13所示的HCDR1、SEQ ID NO：14所示的HCDR2、SEQ ID NO：15所示的HCDR3；或
    (c)SEQ ID NO：19所示的HCDR1、SEQ ID NO：20所示的HCDR2、SEQ ID NO：21所示的HCDR3。
  3. 根据权利要求1所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段进一步包含如下轻链可变区所包括的三个轻链互补决定区LCDR1、LCDR2和LCDR3：
    (a)SEQ ID NO：26所示的轻链可变区；
    (b)SEQ ID NO：28所示的轻链可变区；或
    (c)SEQ ID NO：30所示的轻链可变区。
  4. 根据权利要求3所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段进一步包含选自下组的三个轻链互补决定区LCDR1、LCDR2和LCDR3：
    (a)SEQ ID NO：10所示的LCDR1、SEQ ID NO：11所示的LCDR2以及SEQ ID NO：12所示的LCDR3；
    (b)SEQ ID NO：16所示的LCDR1、SEQ ID NO：17所示的LCDR2以及SEQ ID NO：18所示的LCDR3；或
    (c)SEQ ID NO：22所示的LCDR1、SEQ ID NO：23所示的LCDR2以及SEQ ID NO：24所示的LCDR3。
  5. 根据权利要求1所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含选自下组的三个重链互补决定区HCDR1、HCDR2和HCDR3以及三个轻链互补决定区LCDR1、LCDR2和LCDR3：
    (a)SEQ ID NO：7所示的HCDR1、SEQ ID NO：8所示的HCDR2、SEQ ID NO：9所示的HCDR3、SEQ ID NO：10所示的LCDR1、SEQ ID NO：11所示的LCDR2以及SEQ ID NO：12所示的LCDR3；
    (b)SEQ ID NO：13所示的HCDR1、SEQ ID NO：14所示的HCDR2、SEQ ID NO：15所示的HCDR3、SEQ ID NO：16所示的LCDR1、SEQ ID NO：17所示的LCDR2以及SEQ ID NO：18所示的LCDR3；或
    (c)SEQ ID NO：19所示的HCDR1、SEQ ID NO：20所示的HCDR2、SEQ ID NO：21所示的HCDR3、SEQ ID NO：22所示的LCDR1、SEQ ID NO：23所示的LCDR2以及SEQ ID NO：24所示的LCDR3。
  6. 根据权利要求1所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含选自下组的重链可变区V H：
    (a)SEQ ID NO：25所示的重链可变区；
    (b)SEQ ID NO：27所示的重链可变区；或
    (c)SEQ ID NO：29所示的重链可变区。
  7. 根据权利要求6所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段进一步包含选自下组的轻链可变区V L：
    (a)SEQ ID NO：26所示的轻链可变区；
    (b)SEQ ID NO：28所示的轻链可变区；或
    (c)SEQ ID NO：30所示的轻链可变区。
  8. 根据权利要求1所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含选自下组的重链可变区V H和轻链可变区V L：
    (a)SEQ ID NO：25所示的重链可变区和SEQ ID NO：26所示的轻链可变区；
    (b)SEQ ID NO：27所示的重链可变区和SEQ ID NO：28所示的轻链可变区；或
    (c)SEQ ID NO：29所示的重链可变区和SEQ ID NO：30所示的轻链可变区。
  9. 根据权利要求1至8任一项所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段特异性识别或结合具有SEQ ID NO：2所示的氨基酸序列的蛋白。
  10. 根据权利要求9所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段与具有SEQ ID NO：2所示的氨基酸序列的蛋白的Kd值小于10 -7M。
  11. 根据权利要求10所述的抗体或其抗原结合片段，其中所述Kd值是通过表面等离子共振方法检测的。
  12. 根据权利要求1至11中任一项所述的抗体或其抗原结合片段，其中所述抗原结合片段是选自F(ab’)2、Fab’、Fab、Fv、scFv、dsFv或dAb。
  13. 根据权利要求1至12中任一项所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段进一步具有重链恒定区和/或轻链恒定区。
  14. 根据权利要求13所述的抗体或其抗原结合片段，其中所述重链恒定区源于鼠的IgG1或鼠的IgG2a。
  15. 根据权利要求13所述的抗体或其抗原结合片段，其中所述重链恒定区源于人的IgG1。
  16. 根据权利要求1至15中任一项所述的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段进一步具有缀合部分。
  17. 根据权利要求16所述的抗体或其抗原结合片段，其中所述缀合部分是治疗剂、放射性同位素、可检测标签、药代动力学修饰部分或纯化部分。
  18. 根据权利要求15或16所述的抗体或其抗原结合片段，其中所述缀合部分直接连接或者通过连接子连接。
  19. 一种分离的核酸，其编码权利要求1至18中任一项所述的抗体或其抗原结合片段。
  20. 根据权利要求19所述的核酸，其包含：
    (a)SEQ ID NO：31所示的核酸序列或与SEQ ID NO：31具有至少90％序列同一性的核酸序列，和SEQ ID NO：32所示的核酸序列或与SEQ ID NO：32具有至少90％序列同一性的核酸序列中的一者或两者；
    (b)SEQ ID NO：33所示的核酸序列或与SEQ ID NO：33具有至少90％序列同一性的核酸序列，和SEQ ID NO：34所示的核酸序列或与SEQ ID NO：34具有至少90％序列同一性的核酸序列中的一者或两者；或
    (c)SEQ ID NO：35所示的核酸序列或与SEQ ID NO：35具有至少90％序列同一性的核酸序列，和SEQ ID NO：6所示的核酸序列或与SEQ ID NO：6具有至少90％序列同一性的核酸序列中的一者或两者。
  21. 一种载体，其包含根据权利要求19或20所述的核酸序列。
  22. 一种宿主细胞，其包含根据权利要求21所述的载体。
  23. 一种多特异性抗体，其包含权利要求1至18任一项所述的抗体或其抗原结合片段，和一个或多个与其他抗原特异性结合的抗体或其抗原结合部分。
  24. 一种抗体偶联物，其包含权利要求1至18任一项所述的抗体或其抗原结合片段或权利要求23所述的多特异性抗体。
  25. 分泌抗TRPV6单克隆抗体的杂交瘤细胞株，所述杂交瘤细胞株为12CT9.5.1、16CT4.1.1.2或16CT18.1.1.2，保藏于中国典型培养物保藏中心，其中所述12CT9.5.1保藏编号为CCTCC NO：C202119，所述16CT4.1.1.2保藏编号为CCTCC NO：C202124，所述16CT18.1.1.2保藏编号为CCTCC NO：C202125。
  26. 一种药物组合物，其包含权利要求1至18中任一项所述的抗体或其抗原结合片段、权利要求19-20任一项所述的分离的核酸、权利要求21所述的载体、权利要求22所述的宿主细胞、权利要求23所述的多特异性抗体或权利要求24所述的抗体偶联物，和药学上可接受的载体。
  27. 一种用于分析和检测来自受试者的样品中的TRPV6的试剂，所述试剂包含一种或多种如权利要求1至18所述的抗体或其抗原结合片段。
  28. 根据权利要求27所述的试剂，所述试剂用于分析和检测来自受试者的样品中的TRPV6。
  29. 一种检测来自受试者的样品中的TRPV6的方法，所述方法包括：
    (1)使所述样品与根据权利要求1至18中任一项所述的抗体或抗原结合片段接触；
    (2)可选地，移除未结合的抗体或抗体片段；
    (3)检测与所述样品中的TRPV6结合的抗体或抗体片段。
  30. 根据权利要求29所述的方法，其中所述步骤(1)中的所述根据权利要求1至18中任一项所述的抗体或抗原结合片段被固定在基板上。
  31. 根据权利要求29所述的方法，其中所述步骤(1)中的所述样品被固定在基板上。
  32. 根据权利要求29至31中任一项所述的方法，其中所述步骤(3)包括使用第二抗体，可选的，所述第二抗体能够直接或者间接地结合所述样品中的TRPV6，可选的，所述第二抗体的表位与根据权利要求1至18中任一项所述的抗体或抗原结合片段的表位不同，可选的，所述第二抗体的表位与根据权利要求1至18中任一项所述的抗体或抗原结合片段的表位相同，可选的，所述第二抗体的表位与TRPV6的结合不受根据权利要求1至18中任一项所述的抗体或抗原结合片段的影响，可选的，所述第二抗体的表位与TRPV6的结合受根据权利要求1至18中任一项所述的抗体或抗原结合片段的影响。
  33. 根据权利要求29至31中任一项所述的方法，其中所述步骤(3)包括使用第二抗体，可选的，所述第二抗体能够直接或间接地结合根据权利要求1至18中任一项所述的抗体或抗原结合片段，可选的，所述第二抗体能够结合根据权利要求1至18中任一项所述的抗体或抗原结合片段的恒定区部分。
  34. 根据权利要求32或33所述的方法，其中所述第二抗体与检测分子偶联，可选的，所述检测分子包括酶、荧光标记物和生物素。
  35. 根据权利要求34所述的方法，其中所述检测分子为酶，可选的，所述酶包括辣根过氧化酶、碱性磷酸酶或其衍生物。
  36. 根据权利要求34所述的方法，其中所述检测分子为荧光素，可选的，所述荧光素包括FITC、罗丹明、德克萨斯红、藻红蛋白或Dylight。
  37. 根据权利要求34所述的方法，其中所述检测分子为生物素或其衍生物。
  38. 根据权利要求29至37中任一项所述的方法，其进一步包括对与所述样品中的TRPV6结合的抗体进行定量。
  39. 根据权利要求38所述的方法，其中所述方法为免疫组织化学(IHC)、酶联免疫吸附(ELISA)、免疫荧光(IF)、化学发光(CLIA)、免疫比浊(TIA)或免疫印迹(Western Blot)。
  40. 根据权利要求29至39中任一项所述的方法，其中所述受试者为人类。
  41. 根据权利要求29至40中任一项所述的方法，其用于评估所述样品中在细胞表面上表达TRPV6的细胞的比例。
  42. 一种判断受试者疾病的形成或形成的风险、评估受试者疾病的进展或预后、或在接受疾病治疗的受试者中预测或监测所述受试者对治疗的应答的方法，所述方法包括使用根据权利要求29至41中任一项所述的方法检测来自所述受试者的样品中的TRPV6。
  43. 根据权利要求41所述的方法，所述方法包括评估所述样品中在细胞表面上表达TRPV6的细胞的比例，若所述比例超过预定的阈值，则认为所述受试者患有所述疾病或者更可能从治疗中获益，其中所述阈值为10％，优选的，所述治疗为靶向药物治疗。
  44. 根据权利要求42或43所述的方法，其中所述疾病为癌症。
  45. 根据权利要求44所述的方法，其中所述癌症为乳腺癌、卵巢癌、子宫内膜癌、胆管癌、胃癌、食道癌、肺癌、肠癌、胰腺癌；优选的，所述癌症为HER2 +乳腺癌、三阴乳腺癌或结直肠癌。
  46. 一种试剂盒，所述试剂盒包含权利要求1至18中任一项所述的抗体或其抗原结合片段，可选的，进一步包括用于检测所述抗体或其抗原结合片段与TRPV6结合情况的试剂。
  47. 根据权利要求46所述的试剂盒，其用于诊断受试者疾病的形成或形成的风险、评估受试者疾病的进展或预后、或在接受疾病治疗的受试者中预测或监测所述受试者对治疗的应答。
  48. 根据权利要求46所述的试剂盒，其用于免疫组化病理诊断。
  49. 根据权利要求48所述的试剂盒，其用于肿瘤组织免疫组化病理诊断。
  50. 根据权利要求中47所述的试剂盒，其中所述疾病为癌症。
  51. 根据权利要求48所述的试剂盒，其中所述疾病为乳腺癌、卵巢癌、子宫内膜癌、胆管癌、胃癌、食道癌、肺癌、肠癌、胰腺癌；优选的，所述疾病为HER2 +乳腺癌、三阴乳腺癌或结直肠癌。
  52. 一种制备特异性结合TRPV6的抗体的方法，所述方法包括：
    (1)将SEQ ID NO：3所示的氨基酸序列与载体蛋白偶联，以获得TRPV6抗原肽；
    (2)将通过步骤(1)获得的所述TRPV6抗原肽作为免疫原免疫小鼠；
    (3)经细胞融合、免疫肽筛选克隆，获得高效分泌特异性结合TRPV6的抗体的阳性杂交瘤细胞系；和
    (4)获得特异性结合TRPV6的抗体。
  53. 根据权利要求52所述的方法，其中所述载体蛋白为钥孔虫戚血蓝素(KLH)蛋白。
  54. 根据权利要求52或53所述的方法，其中通过在SEQ ID NO：3所示的氨基酸序列的N端增加一个Cys，并通过自由巯基和所述载体蛋白偶联。
  55. 根据权利要求52至54中任一项所述的方法，其中所述杂交瘤细胞系为小鼠杂交瘤细胞系12CT 9.5.1、16CT 18.1.1.2或16CT 4.1.1.2。
  56. 如权利要求1-18中任一项所述的抗体或其抗原结合片段、权利要求19-20任一项所述的分离的核酸、权利要求21所述的载体、权利要求22所述的宿主细胞、权利要求23所述的多特异性抗体、权利要求24所述的抗体偶联物以及权利要求25所述的药物组合物在用于治疗和/或预防和/或诊断与TRPV6相关的疾病的药物中的用途。
  57. 一种治疗、预防或诊断疾病的方法，所述方法包括向有需要的受试者施用如权利要求1-18中任一项所述的抗体或其抗原结合片段、权利要求19-20任一项所述的分离的核酸、权利要求21所述的载体、权利要求22所述的宿主细胞、权利要求23所述的多特异性抗体、权利要求24所述的抗体偶联物以及权利要求25所述的药物组合物。
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