WO2024251260A1 - 结合Nectin-4的抗体及其用途 - Google Patents

结合Nectin-4的抗体及其用途 Download PDF

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Publication number
WO2024251260A1
WO2024251260A1 PCT/CN2024/098089 CN2024098089W WO2024251260A1 WO 2024251260 A1 WO2024251260 A1 WO 2024251260A1 CN 2024098089 W CN2024098089 W CN 2024098089W WO 2024251260 A1 WO2024251260 A1 WO 2024251260A1
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Prior art keywords
nectin
seq
antibody
sequence
variable region
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French (fr)
Inventor
方鹏
谭小钉
游猛
曹雨霞
王璐
奚钊
施磊
朱晓红
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Jiangsu Mabwell Health Pharmaceutical R & D Co Ltd
Mabwell Shanghai Bioscience Co Ltd
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Jiangsu Mabwell Health Pharmaceutical R & D Co Ltd
Mabwell Shanghai Bioscience Co Ltd
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Priority to EP24818787.4A priority Critical patent/EP4725964A1/en
Publication of WO2024251260A1 publication Critical patent/WO2024251260A1/zh
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
    • G01N33/5759—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites involving compounds localised on the membrane of tumour or cancer cells
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    • 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
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
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    • G01N2800/7023—(Hyper)proliferation
    • G01N2800/7028—Cancer

Definitions

  • the present invention relates to the field of antibody medicine. Specifically, the present invention relates to an antibody or an antigen-binding fragment thereof that specifically binds to nectin-4 and a kit containing the antibody or antigen-binding fragment. In addition, the present invention relates to a nucleic acid encoding the antibody and a host cell containing the nucleic acid, as well as a method for preparing the antibody. The present invention also relates to the diagnostic and prognostic uses of the antibody that binds to nectin-4.
  • Nectin-4 (also known as “poliovirus receptor-like molecule 4 (PVRL4)) is a type I transmembrane glycoprotein with a molecular weight of about 66KD. It is a member of the Nectin family in the immunoglobulin superfamily. Its extracellular domain is composed of three Ig-like domains (VCC type) and participates in the formation and maintenance of adhesion junctions together with cadherins.
  • VCC type Ig-like domains
  • Nectin-4 is specifically expressed in embryos, placentas, and tumor cells, but is not expressed or expressed at very low levels in normal tissues and organs. It is a classic tumor marker in clinical practice and an ideal target for antibody-drug conjugates (ADCs).
  • ADCs antibody-drug conjugates
  • Antibody-drug conjugates targeting nectin-4 bind to nectin-4 overexpressed on the surface of tumor cells to form ADC-receptor conjugates; then, ADCs enter cells through target-mediated endocytosis, and are then cut off by cathepsins in lysosomes, releasing toxic small molecules and achieving specific killing of tumor cells.
  • PADCEV TM targeting nectin-4 (alias: enfortumab vedotin-ejfv; Chinese name: Vitin-Enfortumab) is an ADC drug, which is a human IgG1 monoclonal antibody targeting nectin-4 protein, coupled with the cytotoxic agent MMAE (monomethyl auristatin E, a microtubule disruptor) (Pia M. Challita-Eid et al. (2016) Cancer Res. 76 (10): 3003-13). It has been approved by the FDA for the treatment of locally advanced or metastatic urothelial carcinoma. However, PADCEV TM targeting nectin-4 is only used for therapeutic applications and cannot be used for immunohistochemistry (IHC) staining, so it has no diagnostic value.
  • IHC immunohistochemistry
  • anti-nectin-4 antibodies that specifically recognize nectin-4, which can detect the presence and/or expression level of nectin-4 in a sample with high sensitivity (e.g., by IHC detection), thereby diagnosing a disease or evaluating the prognosis, which is of great significance for improving the survival rate of cancer patients, prolonging the survival period, avoiding excessive chemotherapy, and improving the quality of life. Therefore, the use of anti-nectin-4 antibodies with good specificity and high sensitivity in diagnosis and prognosis has extremely high application value.
  • the present invention provides an anti-nectin-4 antibody with good specificity and high sensitivity, which can reliably and sensitively detect nectin-4.
  • the antibody or antigen-binding fragment specifically binding to nectin-4 of the present invention has one or more of the following characteristics:
  • Nectin-4 In the ELISA assay, it has the ability to specifically bind only to Nectin-4, and has no specific binding to Nectin family members Nectin-1, Nectin-2, Nectin-3, Nectin-like-1, Nectin-like-2, Nectin-like-3, Nectin-like-4, and Nectin-like-5;
  • (c) can specifically detect Nectin-4 protein when measured by Western blotting; and when an excess of Nectin-4 protein is added, the binding of Nectin-4 protein to the membrane is inhibited; and/or
  • tissues are selected from tonsils, salivary glands, brain, cerebellum, placenta, bladder, lung, breast, esophagus, larynx, thymus, heart, stomach, small intestine, colon, rectum, ureter, ovary, fallopian tube, cervix, endometrium, skin, kidney, prostate, pancreas, thyroid, spleen and liver.
  • the present invention provides an antibody or antigen-binding fragment that specifically binds to nectin-4, comprising a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:5 or a variant of HCDR1 as shown in SEQ ID NO:5 with no more than 2 amino acid changes, HCDR2 as shown in SEQ ID NO:6 or a variant of HCDR2 as shown in SEQ ID NO:6 with no more than 2 amino acid changes, and HCDR3 as shown in SEQ ID NO:7 or a variant of HCDR3 as shown in SEQ ID NO:7 with no more than 2 amino acid changes;
  • the light chain variable region comprises LCDR1 as shown in SEQ ID NO:8 or a variant of LCDR1 as shown in SEQ ID NO:8 with no more than 2 amino acid changes, LCDR2 as shown in SEQ ID NO:9 or a variant of LCDR2 as shown in SEQ ID NO:9 with no more than 2 amino acid changes, and LCDR3 as shown in SEQ ID NO:10 or a variant of LCDR3 as shown in SEQ ID NO:10 with no more than 2 amino acid changes;
  • the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:15 or a variant of HCDR1 shown in SEQ ID NO:15 with no more than 2 amino acid changes, HCDR2 shown in SEQ ID NO:16 or a variant of HCDR2 shown in SEQ ID NO:16 with no more than 2 amino acid changes, and HCDR3 shown in SEQ ID NO:17 or a variant of HCDR3 shown in SEQ ID NO:17 with no more than 2 amino acid changes;
  • the light chain variable region comprises LCDR1 shown in SEQ ID NO:18 or a variant of LCDR1 shown in SEQ ID NO:18 with no more than 2 amino acid changes, LCDR2 shown in SEQ ID NO:19 or a variant of LCDR2 shown in SEQ ID NO:19 with no more than 2 amino acid changes, and LCDR3 shown in SEQ ID NO:20 or a variant of LCDR3 shown in SEQ ID NO:20 with no more than 2 amino acid changes; or
  • the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:25 or a variant of HCDR1 shown in SEQ ID NO:25 with no more than 2 amino acid changes, HCDR2 shown in SEQ ID NO:26 or a variant of HCDR2 shown in SEQ ID NO:26 with no more than 2 amino acid changes, and HCDR3 shown in SEQ ID NO:27 or a variant of HCDR3 shown in SEQ ID NO:27 with no more than 2 amino acid changes;
  • the light chain variable region comprises LCDR1 shown in SEQ ID NO:28 or a variant of LCDR1 shown in SEQ ID NO:28 with no more than 2 amino acid changes, LCDR2 shown in SEQ ID NO:29 or a variant of LCDR2 shown in SEQ ID NO:29 with no more than 2 amino acid changes, and LCDR3 shown in SEQ ID NO:30 or a variant of LCDR3 shown in SEQ ID NO:30 with no more than 2 amino acid changes.
  • the amino acid changes are conservative amino acid modifications.
  • the antibody or antigen-binding fragment of the present invention that specifically binds to nectin-4 comprises
  • a heavy chain variable region and a light chain variable region wherein the heavy chain variable region comprises a sequence of SEQ ID NO:3, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises a sequence of SEQ ID NO:4, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;
  • a heavy chain variable region and a light chain variable region wherein the heavy chain variable region comprises the sequence of SEQ ID NO:13, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain variable region comprises the sequence of SEQ ID NO:14, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or
  • a heavy chain variable region and a light chain variable region wherein the heavy chain variable region comprises a sequence of SEQ ID NO: 23 or has at least
  • the light chain variable region comprises a sequence of SEQ ID NO:24, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain variable region comprises a sequence of SEQ ID NO:24, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.
  • the antigen-binding fragment of the antibody of the present invention that specifically binds to nectin-4 is Fab, Fab', F(ab') 2 , Fv, single-chain Fv, single-chain Fab, or diabody.
  • the antibody or antigen-binding fragment of the present invention that specifically binds to nectin-4 comprises
  • SEQ ID NO:11 or a heavy chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto
  • SEQ ID NO:12 or a light chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; for example, the heavy chain sequence set forth in SEQ ID NO:11 and the light chain sequence set forth in SEQ ID NO:12; or
  • SEQ ID NO:21 or a heavy chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto
  • SEQ ID NO:22 or a light chain sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; for example, the heavy chain sequence set forth in SEQ ID NO:21 and the light chain sequence set forth in SEQ ID NO:22.
  • the antibody or antigen-binding fragment that specifically binds to nectin-4 of the present invention is a mouse antibody or a rabbit antibody.
  • the antibody or antigen-binding fragment of the present invention that specifically binds to nectin-4 is an IgG class antibody.
  • the present invention provides a nucleic acid encoding the anti-nectin-4 antibody or antigen-binding fragment described in the first aspect above, a vector (preferably, an expression vector) comprising the nucleic acid, and a host cell comprising the nucleic acid or the vector.
  • a vector preferably, an expression vector
  • the host cell is prokaryotic or eukaryotic, for example, selected from Escherichia coli cells, yeast cells, mammalian cells, or other cells suitable for preparing antibodies or antigen-binding fragments thereof.
  • the host cell is a HEK 293 cell or a CHO cell.
  • the present invention provides a method for preparing the anti-Nectin-4 antibody or its antigen-binding fragment described in the first aspect, the method comprising culturing the host cell described in the second aspect under conditions suitable for expressing a nucleic acid encoding the anti-Nectin-4 antibody or its antigen-binding fragment described in the first aspect, and recovering the expressed anti-Nectin-4 antibody or its antigen-binding fragment from the culture.
  • the present invention provides a kit comprising the anti-nectin-4 antibody or antigen-binding fragment thereof described in the first aspect.
  • the present invention provides a use of the anti-nectin-4 antibody or antigen-binding fragment thereof described in the first aspect for detecting the level of nectin-4 in a sample.
  • the present invention provides a method for diagnosing cancer in a subject using the anti-nectin-4 antibody or antigen-binding fragment thereof according to the first aspect.
  • the present invention provides a method for using the anti-Nectin-4 antibody or antigen-binding fragment thereof described in the first aspect to determine whether a subject uses an anti-Nectin-4 antibody or an anti-Nectin-4 antibody-drug conjugate (e.g., a drug having a therapeutic effect).
  • the present invention provides a method for prognosing a subject suffering from cancer using the anti-Nectin-4 antibody or its antigen-binding fragment described in the first aspect above, wherein the subject suffering from cancer is a subject suffering from cancer who has been treated with an anti-tumor agent (e.g., an anti-Nectin-4 antibody or an anti-Nectin-4 antibody-drug conjugate (e.g., a therapeutic anti-Nectin-4 antibody or its antigen-binding fragment)).
  • an anti-tumor agent e.g., an anti-Nectin-4 antibody or an anti-Nectin-4 antibody-drug conjugate (e.g., a therapeutic anti-Nectin-4 antibody or its antigen-binding fragment).
  • Figure 1 Shows the binding of antibody MM11, antibody R012, antibody R036 to Nectin family proteins (Nectin-1, Nectin-2, Nectin-3, Nectin-4, Nectin-like-1, Nectin-like-2, Nectin-like-3, Nectin-like-4, Nectin-like-5) detected by ELISA.
  • Nectin family proteins Nectin-1, Nectin-2, Nectin-3, Nectin-4, Nectin-like-1, Nectin-like-2, Nectin-like-3, Nectin-like-4, Nectin-like-5) detected by ELISA.
  • FIG. 2 Flow cytometry (FACS) was used to detect the binding of antibody MM11, antibody R012, and antibody R036 to nectin-4 expressing cells.
  • Blank means that only the secondary antibody was added to human prostate cancer cells (PC-3), and antibody MM11, antibody R012, or antibody R036 as the primary antibody was not added; NC means only PC-3 cells and blank buffer were added; "PC-3-Nectin4" means PC-3 cell line expressing nectin-4.
  • Figure 3 shows the reactivity of antibody MM11, antibody R012, and antibody R036 with cell lysates expressing nectin-4 detected by Western blotting.
  • lane M protein molecular weight marker
  • lane 1 PC-3 cell lysate
  • lane 2 PC-3-nectin-4 cell lysate.
  • the upper panels of Figure 3 show that antibody MM11, antibody R012, and antibody R036 react with cell lysates expressing nectin-4, and do not react with cell lysates not expressing nectin-4; in the lower panels of Figure 3, "MM11 (Inhibition)” and “R012 (Inhibition)” represent the results after cell lysates were incubated with "MM11 antibody” or “R012 antibody” respectively after transfer to the membrane, and an excess of recombinant nectin-4 protein was added; "Gapdh” represents the reaction of cell lysates incubated with anti-glyceraldehyde-3-phosphate dehydrogenase antibody after transfer to the membrane, which serves as an internal reference for Western blotting.
  • Figure 4 shows the immunohistochemical staining results of nectin-4 rabbit monoclonal antibodies R012 and R036 and nectin-4 rabbit monoclonal antibody EPR15613-68 as a positive control and representative normal human tissues (scale bar: 200 ⁇ m).
  • Figure 5 shows the results of immunohistochemical staining of nectin-4 rabbit monoclonal antibodies R012 and R036 and nectin-4 rabbit monoclonal antibody EPR15613-68 as a positive control on human bladder cancer tissues (scale bar: 200 ⁇ m).
  • FIG6 shows the immunohistochemical staining results of nectin-4 rabbit monoclonal antibodies R012 and R036 and nectin-4 rabbit monoclonal antibody EPR15613-68 as a positive control on human lung cancer tissues (scale bar: 200 ⁇ m).
  • FIG. 7 shows the immunohistochemical staining results of nectin-4 rabbit monoclonal antibodies R012 and R036 and nectin-4 rabbit monoclonal antibody EPR15613-68 as a positive control on human breast cancer tissues (scale bar: 200 ⁇ m).
  • FIG8 shows the immunohistochemical staining results of Nectin-4 mouse monoclonal antibody MM11 and Nectin-4 mouse monoclonal antibody M22-321b41.1 as a positive control on representative normal human tissues (scale bar: 100 ⁇ m).
  • FIG. 9 shows the immunohistochemical staining results of Nectin-4 mouse monoclonal antibody MM11 and Nectin-4 mouse monoclonal antibody M22-321b41.1 as a positive control on human prostate cancer tissues (scale bar: 50 ⁇ m).
  • FIG. 10 Nectin-4 mouse monoclonal antibody MM11 and Nectin-4 mouse monoclonal antibody M22-321b41.1 as a positive control Results of immunohistochemical staining on human lung cancer tissues (scale bar: 50 ⁇ m).
  • FIG. 11 shows the immunohistochemical staining results of Nectin-4 mouse monoclonal antibody MM11 and Nectin-4 mouse monoclonal antibody M22-321b41.1 as a positive control on human triple-negative breast cancer tissues (scale bar: 50 ⁇ m).
  • antibody refers to an immunoglobulin molecule that binds an antigen.
  • Embodiments of antibodies include monoclonal antibodies, polyclonal antibodies, or chimeric antibodies.
  • Antibodies can belong to any class (e.g., IgG, IgE, IgM, IgD, IgA).
  • Exemplary antibodies of the present disclosure are immunoglobulin G (IgG) type antibodies comprising the following four polypeptide chains cross-linked by interchain disulfide bonds: two heavy chains (HC) and two light chains (LC).
  • the amino terminal portion of each of the four polypeptide chains includes a variable region of about 100-125 or more amino acids that is primarily responsible for antigen recognition.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region.
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region.
  • VH region and VL region can be further subdivided into hypervariable regions called complementary determining regions (CDRs), with more conservative regions interspersed therebetween, called framework regions (FRs).
  • CDRs are exposed on the surface of the protein and are important regions of the antigen-binding specificity of antibodies.
  • Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDRs of the heavy chain are called “HCDR1, HCDR2 and HCDR3" and the three CDRs of the light chain are called “LCDR1, LCDR2 and LCDR3".
  • CDRs contain most of the residues that form specific interactions with antigens.
  • Amino acid residues can be assigned to CDRs according to well-known numbering schemes, including those described in Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md.
  • the boundaries of the CDRs of the variable regions of the same antibody obtained based on different numbering schemes may differ. Differences. That is, the CDR sequences of the same antibody variable region defined under different numbering schemes are different. Therefore, when referring to antibodies defined by specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different numbering schemes (e.g., different numbering scheme rules or combinations).
  • CDRs of the antibodies of the present invention can be manually evaluated to determine the boundaries according to any numbering scheme or combination thereof in the art. Unless otherwise specified, in the present invention, the term “CDR” or “CDR sequence” encompasses CDR sequences determined in any of the above ways.
  • Antigen binding fragment is a part or segment of an intact or complete antibody having fewer amino acid residues than the intact or complete antibody, which can bind to antigen or compete with the intact antibody (i.e., the intact antibody from which the antigen binding fragment is derived) for antigen binding.
  • Antigen binding fragments can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen binding fragments include, but are not limited to, Fab, Fab', F(ab') 2 , Fv, single-chain Fv (scFv), single-chain Fab, diabody, single domain antibody (sdAb, nanobody), camelid Ig, Ig NAR, F(ab)' 3 fragment, bi-scFv, (scFv) 2 , minibody, bifunctional antibody, trifunctional antibody, tetrafunctional antibody, disulfide-stabilized Fv protein ("dsFv”).
  • the term also includes genetically engineered forms, such as chimeric antibodies (e.g., humanized murine antibodies), heterojunction antibodies (e.g., bispecific antibodies), and antigen binding fragments thereof.
  • chimeric antibodies e.g., humanized murine antibodies
  • heterojunction antibodies e.g., bispecific antibodies
  • antigen binding fragments thereof see also: Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby,
  • Nectin family proteins and “Nectin family” are used interchangeably and are cell adhesion molecules that form physical connections between adjacent cells to achieve intercellular communication, migration and other important cellular processes.
  • Nectin family proteins include at least Nectin-1, Nectin-2, Nectin-3, Nectin-4, Nectin-like-1, Nectin-like-2, Nectin-like-3, Nectin-like-4, and Nectin-like-5.
  • the expression of Nectin-4 protein in healthy humans is mainly limited to placenta and embryonic tissues. Compared with healthy adult tissues, many types of tumor cells have high expression of Nectin-4 protein.
  • Nectin-4 protein is a tumor-associated antigen.
  • binding or “specific binding” used when referring to antigens and antibodies means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions.
  • the ability of an antibody to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA), SPR or biofilm interferometry or other conventional binding assays known in the art.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences may be discarded for comparison purposes).
  • the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at this position.
  • the comparison of sequences and calculation of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (available at http://www.gcg.com) that has been integrated into the GAP program in the GCG software package, using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6.
  • the percent identity between two amino acid sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com).
  • the percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller, ((1989) CABIOS, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted remainder table, a gap length penalty of 12, a gap penalty of 4).
  • nucleic acid sequences and protein sequences described herein can further use as a "query sequence" to perform a search against public databases to, for example, identify other family member sequences or related sequences.
  • “conservative modifications” or “conservative amino acid modifications” include substitutions, deletions or additions to a polypeptide sequence that result in substitution of an amino acid with a chemically similar amino acid.
  • Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • Such conservatively modified variants are in addition to and do not exclude the polymorphic variants, interspecies homologs and alleles of the present invention.
  • the following eight groups contain amino acids that are conservative substitutions for each other: 1) Alanine (A), Glycine (G); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
  • the term "conservative amino acid modifications” is used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence.
  • vector means a construct that is capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the genes or sequences in the host cell.
  • vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.
  • host cell refers to cells into which exogenous nucleic acids have been introduced, including the progeny of these cells.
  • Host cells include “transformants” and “transformed cells”, which include the primary transformed cells and the progeny derived therefrom, without considering the number of passages.
  • the progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as the cells screened or selected in the initial transformed cells are included herein.
  • subject refers to an animal, preferably a mammal, more preferably a human, in need of alleviation, prevention, treatment and/or diagnosis of a disease or condition. Mammals also include, but are not limited to, farm animals, racing animals, pets, primates, horses, dogs, cats, mice and rats. The term includes human subjects having a disease or at risk of having a disease.
  • a “biological sample” from a subject refers to a collection of cells, tissues, or body fluids obtained from an individual or subject.
  • the source of the tissue or cell sample can be solid tissue, such as a sample or biopsy sample or puncture sample from a fresh, frozen and/or preserved organ or tissue sample; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time of pregnancy or development of the subject.
  • Tissue samples may contain compounds that are not naturally mixed with tissues in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.
  • tumor samples include, but are not limited to, tumor biopsies, fine needle aspirates, bronchial lavage fluid, pleural fluid (pleural effusion), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples, such as formin. Marin-fixed, paraffin-embedded tumor tissue sections or frozen tumor samples.
  • reference sample refers to the level of a sample, cell, tissue or standard used for comparison purposes.
  • the reference sample, reference cell, reference tissue, control sample, control cell or control tissue is obtained from a healthy and/or non-diseased part of the body of the same subject or individual, and is a healthy and/or non-diseased tissue or cell.
  • the reference sample, reference cell, reference tissue, control sample, control cell or control tissue is obtained from a healthy tissue or cell of an individual who is not the subject.
  • the presence or expression level of Nectin-4 from a "reference sample”, “reference cell”, “reference tissue”, “control sample”, “control cell” or “control tissue” is referred to as the "reference presence or expression level”.
  • primary antibody refers to an antibody that specifically binds to Nectin-4 in a biological sample.
  • secondary antibody refers to an antibody that specifically binds to the primary antibody, thereby forming a bridge between the primary antibody and a subsequent reagent (if a subsequent reagent is required).
  • Immunofluorescence (IF) technology is the earliest developed technology in labeling immunology. It is based on the principle of antigen-antibody reaction. First, the known antigen or antibody is labeled with a fluorescent group, and then the fluorescent antibody (or antigen) is used as a probe to examine the corresponding antigen (or antibody) in the cell or tissue. The antigen-antibody complex formed in the tissue or cell contains labeled fluorescein.
  • the fluorescein When the specimen is observed under a fluorescence microscope, the fluorescein emits bright fluorescence (yellow-green or orange-red) under the irradiation of external excitation light, and the tissue cells where the fluorescence is located can be seen, thereby determining the nature and location of the antigen or antibody, and using quantitative technology to measure the content.
  • IP Immunoprecipitation
  • Co-IP Co-Immunoprecipitation
  • Co-IP is an extension of immunoprecipitation.
  • Co-IP is a method developed by utilizing the specific binding of antigenic proteins and antibodies and the phenomenon that "Protein A/G" of bacterial proteins specifically binds to the Fc fragment of antibodies (immunoglobulins).
  • Protein A/G is usually pre-bound to Argarose beads, and reacted with a solution containing antigenic proteins (such as cell lysate) and antibodies against bait protein X.
  • Prorein A/G on the Argarose beads adsorbs bait protein X through antibodies, and bait protein X separates target protein Y from cell lysate by interacting with target protein Y, thereby achieving the purpose of co-immunoprecipitation.
  • the present invention provides antibodies that specifically bind to nectin-4.
  • the antibodies of the present invention are monoclonal antibodies, for example, they can be derived from any eukaryotic clone, prokaryotic clone or phage clone.
  • Monoclonal antibodies can be produced by hybridoma technology, recombinant technology, phage display technology, B cell clone screening technology, synthetic technology (e.g., CDR transplantation) or other technology combinations known in the art.
  • mice or rabbits or other animals can be immunized with human nectin-4 and the antibodies produced can be recovered, purified, and the amino acid sequence determined using conventional methods well known in the art.
  • phage libraries can be screened to screen thousands of Fab fragments for interaction with human nectin-4 and the resulting interactors can be recovered, purified, and the amino acid sequence determined using conventional methods well known in the art.
  • antibody that binds to nectin-4 protein refers to the antibody of the present invention, which is capable of specifically binding to Nectin-4 protein, thereby the antibody can be used as a diagnostic agent and/or detection agent targeting Nectin-4 protein.
  • the isolated anti-nectin-4 antibodies and antigen-binding fragments of the present invention specifically bind to the nectin-4 protein, and comprise a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:5 or a variant of HCDR1 as shown in SEQ ID NO:5 with no more than 2 amino acid changes, HCDR2 as shown in SEQ ID NO:6 or a variant of HCDR2 as shown in SEQ ID NO:6 with no more than 2 amino acid changes, and HCDR3 as shown in SEQ ID NO:7 or a variant of HCDR3 as shown in SEQ ID NO:7 with no more than 2 amino acid changes;
  • the light chain variable region comprises LCDR1 as shown in SEQ ID NO:8 or a variant of LCDR1 as shown in SEQ ID NO:8 with no more than 2 amino acid changes, LCDR2 as shown in SEQ ID NO:9 or a variant of LCDR2 as shown in SEQ ID NO:9 with no more than 2 amino acid changes, and LCDR3 as shown in SEQ ID NO:10 or a variant of LCDR3 as shown in SEQ ID NO:10 with no more than 2 amino acid changes;
  • the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:15 or a variant of HCDR1 shown in SEQ ID NO:15 with no more than 2 amino acid changes, HCDR2 shown in SEQ ID NO:16 or a variant of HCDR2 shown in SEQ ID NO:16 with no more than 2 amino acid changes, and HCDR3 shown in SEQ ID NO:17 or a variant of HCDR3 shown in SEQ ID NO:17 with no more than 2 amino acid changes;
  • the light chain variable region comprises LCDR1 shown in SEQ ID NO:18 or a variant of LCDR1 shown in SEQ ID NO:18 with no more than 2 amino acid changes, LCDR2 shown in SEQ ID NO:19 or a variant of LCDR2 shown in SEQ ID NO:19 with no more than 2 amino acid changes, and LCDR3 shown in SEQ ID NO:20 or a variant of LCDR3 shown in SEQ ID NO:20 with no more than 2 amino acid changes; or
  • the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:25 or a variant of HCDR1 shown in SEQ ID NO:25 with no more than 2 amino acid changes, HCDR2 shown in SEQ ID NO:26 or a variant of HCDR2 shown in SEQ ID NO:26 with no more than 2 amino acid changes, and HCDR3 shown in SEQ ID NO:27 or a variant of HCDR3 shown in SEQ ID NO:27 with no more than 2 amino acid changes;
  • the light chain variable region comprises LCDR1 shown in SEQ ID NO:28 or a variant of LCDR1 shown in SEQ ID NO:28 with no more than 2 amino acid changes, LCDR2 shown in SEQ ID NO:29 or a variant of LCDR2 shown in SEQ ID NO:29 with no more than 2 amino acid changes, and LCDR3 shown in SEQ ID NO:30 or a variant of LCDR3 shown in SEQ ID NO:30 with no more than 2 amino acid changes;
  • amino acid change is an addition, deletion or substitution of an amino acid, for example, the amino acid change is a conservative amino acid substitution.
  • the CDR is a CDR according to the Kabat numbering scheme.
  • the isolated anti-Nectin-4 protein antibody or antigen-binding fragment of the present invention comprises:
  • a heavy chain variable region and a light chain variable region wherein the heavy chain variable region comprises a sequence of SEQ ID NO:3, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the light chain variable region comprises a sequence of SEQ ID NO:4, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;
  • a heavy chain variable region and a light chain variable region wherein the heavy chain variable region comprises the sequence of SEQ ID NO:13, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain variable region comprises the sequence of SEQ ID NO:14, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or
  • a heavy chain variable region and a light chain variable region wherein the heavy chain variable region comprises a sequence of SEQ ID NO: 23, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the light chain variable region comprises a sequence of SEQ ID NO: 23, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and
  • the variable region comprises the sequence of SEQ ID NO: 24, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.
  • the antibodies of the invention bind to mammalian nectin-4, such as human nectin-4. In some embodiments, the nectin-4 antibodies of the invention bind to one or more extracellular domains of nectin-4.
  • the antibodies of the invention have one or more of the following properties:
  • Nectin-4 In the ELISA assay, it has the ability to specifically bind only to Nectin-4, and has no specific binding to Nectin family members Nectin-1, Nectin-2, Nectin-3, Nectin-like-1, Nectin-like-2, Nectin-like-3, Nectin-like-4, and Nectin-like-5;
  • (c) can specifically detect Nectin-4 protein when measured in Western blotting; and when an excess of Nectin-4 protein is added, the binding of Nectin-4 protein to the membrane is inhibited; and/or
  • tissues are selected from tonsils, salivary glands, brain, cerebellum, placenta, bladder, lung, breast, esophagus, larynx, thymus, heart, stomach, small intestine, colon, rectum, ureter, ovary, fallopian tube, cervix, endometrium, skin, kidney, prostate, pancreas, thyroid, spleen and liver.
  • the present invention provides a nucleic acid encoding any of the above Nectin-4 antibodies or antigen-binding fragments thereof or any of their chains.
  • a vector comprising the nucleic acid is provided.
  • the vector is an expression vector.
  • a host cell comprising the nucleic acid or the vector is provided.
  • the host cell is eukaryotic.
  • the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells) or other cells suitable for preparing antibodies or antigen-binding fragments thereof.
  • the host cell is prokaryotic.
  • the present invention also encompasses nucleic acids that hybridize to the above-described nucleic acids under stringent conditions or nucleic acids encoding polypeptide sequences having one or more amino acid substitutions (eg, conservative substitutions), deletions or insertions compared to the above-described nucleic acids.
  • the vector is an expression vector, such as a eukaryotic expression vector.
  • the vector includes, but is not limited to, a virus, a plasmid, a cosmid, a lambda phage, or a yeast artificial chromosome (YAC).
  • the expression vector can be transfected or introduced into a suitable host cell.
  • a variety of techniques can be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques.
  • protoplast fusion the cells are cultivated in a culture medium and screened for suitable activity. Methods and conditions for culturing the transfected cells produced and for recovering the antibody molecules produced are known to those skilled in the art and can be based on this specification and methods known in the prior art, depending on the specific expression vector and mammalian host cell used. Variation or optimization.
  • the present invention provides a method for preparing a Nectin-4 antibody, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the Nectin-4 antibody or an expression vector comprising the nucleic acid under conditions suitable for expressing the nucleic acid encoding the Nectin-4 antibody, and optionally isolating the Nectin-4 antibody. In a certain embodiment, the method further comprises recovering the Nectin-4 antibody from the host cell or host cell culture.
  • the nucleic acid encoding the Nectin-4 antibody of the present invention is first isolated and inserted into a vector for further cloning and/or expression in a host cell.
  • Such nucleic acids are easily isolated and sequenced using conventional procedures, for example, by using oligonucleotide probes that are capable of specifically binding to the nucleic acid encoding the Nectin-4 antibody of the present invention.
  • the antibodies of the invention prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like.
  • the actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, and these will be apparent to those skilled in the art.
  • the purity of the antibodies of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.
  • the nectin-4 antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activities by a variety of assays known in the art.
  • antibodies of the invention are tested for their antigen binding activity, for example, by known methods such as ELISA, Western blot, FACS, and the like.
  • Cells used in any of the above in vitro assays include cells that naturally express Nectin-4 or cell lines engineered to express Nectin-4.
  • the cell lines engineered to express Nectin-4 are cell lines that do not normally express Nectin-4 but express Nectin-4 after transfection of DNA encoding Nectin-4 into the cells.
  • any of the Nectin-4 antibodies provided herein can be used to detect the presence and/or level of Nectin-4 in a biological sample.
  • the term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads of antibody molecule complexes, ELISA assays.
  • the biological sample comprises cells or tissues.
  • the biological sample is from a hyperproliferative or cancerous lesion.
  • a nectin-4 antibody is provided for use in a diagnostic or detection method to detect and/or assess the presence and/or level of nectin-4 for diagnostic or prognostic purposes, for example, to determine the efficacy of a given treatment regimen.
  • methods of detecting the presence and/or level of Nectin-4 in a biological sample are provided.
  • the method comprises detecting the presence and/or level of Nectin-4 protein in a biological sample.
  • Nectin-4 is human Nectin-4.
  • the method comprises contacting a biological sample with a Nectin-4 antibody as described herein under conditions that allow the Nectin-4 antibody to bind to Nectin-4, and detecting whether a complex is formed between the Nectin-4 antibody and Nectin-4. The formation of a complex indicates the presence of Nectin-4.
  • the method can be an in vitro or in vivo method.
  • the Nectin-4 antibody is used to select a subject suitable for treatment with a therapeutic Nectin-4 antibody, for example, wherein Nectin-4 is a biomarker for selecting the subject.
  • the antibodies of the invention can be used to diagnose cancers or tumors that express Nectin-4 protein, such as evaluating solid tumors expressing Nectin-4 protein in a subject (e.g., sarcomas and carcinomas of multiple organ systems, such as those affecting the esophagus, lung, breast, ovary, lymphoid, gastrointestinal tract (e.g., colon), anus, genital and genitourinary tract (e.g., kidney, urothelial, bladder cell, prostate), pharynx, central nervous system (e.g., brain, neural or glial cells), head and neck, skin (e.g., melanoma), nasopharynx (e.g., differentiated or undifferentiated metastatic or locally recurrent nasopharyngeal carcinoma) and pancreas, as well as adenocarcinomas, including malignancies), hematological cancers (e.g., leukemias), he
  • the cancer is selected from colon cancer, rectal cancer, colorectal cancer, esophageal cancer, skin cancer, urothelial cancer, ovarian cancer, pancreatic cancer, bladder cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute lymphocytic leukemia, multiple myeloma, breast cancer, gastric cancer, hepatocellular carcinoma, non-small cell lung cancer, small cell lung cancer, melanoma, glioblastoma, renal cell carcinoma, prostate cancer.
  • a labeled nectin-4 antibody is provided.
  • Labels include, but are not limited to, labels that are directly detected (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as labels that are indirectly detected, such as enzymes or ligands, for example, by enzymatic reactions or molecular interactions.
  • Exemplary labels include, but are not limited to, radioisotopes 32 P, 14 C, 125 I, 3 H, and 131 I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferase, for example, firefly luciferase and bacterial luciferase (U.S. Pat. No.
  • luciferin 2,3-dihydrophthalazinedione, horseradish peroxidase (HR), alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lytic enzymes, carbohydrate oxidases, for example, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, and enzymes that utilize hydrogen peroxide to oxidize dye precursors such as HR, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and the like.
  • HR horseradish peroxidase
  • alkaline phosphatase alkaline phosphatase
  • ⁇ -galactosidase glucoamylase
  • lytic enzymes carbohydrate oxidases, for example,
  • the sample is obtained prior to treatment with a nectin-4 antibody. In some embodiments, the sample is obtained after the cancer has metastasized. In some embodiments, the sample is a formalin-fixed, paraffin-embedded (FFPE) sample. In some embodiments, the sample is a biopsy (e.g., a core biopsy), a surgical specimen (e.g., a specimen from a surgical resection), or a fine needle aspirate.
  • FFPE formalin-fixed, paraffin-embedded
  • nectin-4 is detected prior to treatment, eg, prior to initiation of treatment; or prior to a treatment after a treatment interval.
  • the present invention provides a method for diagnosing a nectin 4-related disease, such as cancer, in a subject, the method comprising:
  • the biological sample is a tissue sample, a cell sample or a body fluid sample of the subject, for example, a tissue sample, blood, serum, urine, saliva, or tissue fluid sample of a tumor or healthy tissue, for example, a tissue section (e.g., a paraffin section or a frozen section) sample of a tumor or healthy tissue;
  • Nectin-4 and/or detection of an expression level of Nectin-4 in the sample that is significantly higher than a reference level indicates a Nectin-4-related disease, such as cancer, in the subject;
  • Nectin-4 is detected using immunohistochemistry (IHC) method, Western blot assay, fluorescence activated cell sorting (FACS) assay, enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF) assay and/or co-immunoprecipitation (Co-IP).
  • IHC immunohistochemistry
  • FACS fluorescence activated cell sorting
  • ELISA enzyme-linked immunosorbent assay
  • IF immunofluorescence
  • Co-IP co-immunoprecipitation
  • the present invention provides a method for determining whether a subject uses an anti-Nectin-4 antibody or an anti-Nectin-4 antibody.
  • a method for treating a patient with a conjugated drug e.g., the anti-nectin-4 antibody is a therapeutic anti-nectin-4 antibody or an antigen-binding fragment thereof, the method comprising:
  • the biological sample is a tissue sample, a cell sample or a body fluid sample of the subject, for example, a tissue sample, blood, serum, urine, saliva, or tissue fluid sample of a tumor or healthy tissue, for example, a tissue section (e.g., a paraffin section or a frozen section) sample of a tumor or healthy tissue;
  • Detection of Nectin-4 and/or detection of an expression level of Nectin-4 in the sample higher than a reference level indicates that the subject can be treated with an anti-Nectin-4 antibody or an anti-Nectin-4 antibody-drug conjugate (e.g., the anti-Nectin-4 antibody is a therapeutic anti-Nectin-4 antibody or an antigen-binding fragment thereof);
  • Nectin-4 is detected using immunohistochemistry (IHC) method, Western blot assay, fluorescence activated cell sorting (FACS) assay, enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF) assay and/or co-immunoprecipitation (Co-IP).
  • IHC immunohistochemistry
  • FACS fluorescence activated cell sorting
  • ELISA enzyme-linked immunosorbent assay
  • IF immunofluorescence
  • Co-IP co-immunoprecipitation
  • the present invention provides a method of treating cancer in a subject in need thereof, comprising a prior step of diagnosing cancer or determining the eligibility of a patient for treatment with an anti-Nectin-4 antibody, the prior step comprising performing an immunohistochemistry (IHC) assay, an ELISA assay, a flow cytometry (FACS) assay, a Western blot, an immunofluorescence (IF) assay and/or co-immunoprecipitation (Co-IP) on a biological sample from the subject using an anti-Nectin-4 antibody as disclosed herein, for example, the prior step comprises:
  • the biological sample is a tissue sample, cell sample or body fluid sample of a subject, for example, a tissue sample, blood, serum, urine, saliva, or tissue fluid sample of a tumor or healthy tissue, for example, a tissue section (e.g., paraffin section or frozen section) sample of a tumor or healthy tissue;
  • Nectin-4 is detected using immunohistochemistry (IHC) method, Western blot assay, fluorescence activated cell sorting (FACS) assay, enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF) assay and/or co-immunoprecipitation (Co-IP).
  • IHC immunohistochemistry
  • FACS fluorescence activated cell sorting
  • ELISA enzyme-linked immunosorbent assay
  • IF immunofluorescence
  • Co-IP co-immunoprecipitation
  • the present invention provides a method for prognosing a subject with cancer.
  • the anti-tumor agent includes but is not limited to metabolic inhibitors, antibiotic anti-cancer agents, plant alkaloid anti-cancer agents, topoisomerase inhibitors, anti-tumor alkylating agents, monoclonal antibodies, ADCs, etc., the method comprising:
  • an anti-tumor agent e.g., an anti-Nectin-4 antibody or an anti-Nectin-4 antibody-drug conjugate (e.g., the anti-Nectin-4 antibody is a therapeutic anti-Nectin-4 antibody or an antigen-binding fragment thereof)
  • an anti-Nectin-4 antibody or an antigen-binding fragment thereof of the present invention as a capture agent
  • the biological sample is a tissue sample, a cell sample, or a body fluid sample of the subject, e.g., a tissue sample of a tumor or healthy tissue, blood, Serum, urine, saliva, tissue fluid samples, for example, tissue sections (e.g., paraffin sections or frozen sections) of tumors or healthy tissues;
  • Nectin-4 and/or detection of an expression level of Nectin-4 in the sample higher than a reference level indicates a poor prognosis; or, absence of Nectin-4 in the sample or detection of a level of Nectin-4 lower than a reference presence or expression level indicates a good prognosis
  • Nectin-4 is detected using immunohistochemistry (IHC) method, Western blot assay, fluorescence activated cell sorting (FACS) assay, enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF) assay and/or co-immunoprecipitation (Co-IP).
  • IHC immunohistochemistry
  • FACS fluorescence activated cell sorting
  • ELISA enzyme-linked immunosorbent assay
  • IF immunofluorescence
  • Co-IP co-immunoprecipitation
  • the invention also provides a kit comprising an anti-nectin-4 antibody of the invention, and optionally a package insert instructing the use of the kit.
  • a method for treating a tumor comprising: testing a subject (e.g., a sample) (e.g., a subject sample containing cancer cells) for the presence of Nectin-4, thereby determining a Nectin-4 value, comparing the Nectin-4 value with a control value (e.g., the value of Nectin-4 in a sample from a healthy individual), and if the Nectin-4 value is greater than the control value, administering to the subject a therapeutically effective amount of a Nectin-4 antibody (e.g., a therapeutic anti-Nectin-4 antibody), optionally in combination with one or more other therapies, thereby treating the tumor.
  • a subject e.g., a sample
  • a control value e.g., the value of Nectin-4 in a sample from a healthy individual
  • administering to the subject a therapeutically effective amount of a Nectin-4 antibody (e.g., a therapeutic anti-Nect
  • the nectin-4 antibody of the present invention can be prepared by the method described below.
  • the method for preparing the nectin-4 antibody of the present invention is not limited to the method described in this example, and can also be prepared by other methods known in the art.
  • a human nectin-4 fragment for example, a fragment containing the extracellular domain of human nectin-4 (SEQ ID NO: 31) is used as an antigen for immunization.
  • the antigen is applied together with Freund's complete adjuvant (CFA) for the first immunization.
  • Freund's incomplete adjuvant (IFA) is used for the booster immunization.
  • the emulsion containing the antigen is administered subcutaneously.
  • mice were immunized with recombinant Nectin-4 extracellular protein (see UniProt database accession number Q96NY8, 32-349 (SEQ ID NO: 32), prepared using a mammalian cell expression system).
  • the immunized mouse serum had a high titer of anti-Nectin-4 antibodies
  • the mouse spleen was aseptically removed to prepare a spleen cell suspension, which was fused with SP2/0 myeloma cells.
  • the fused cells were resuspended in HAT medium and dispensed into 96-well cell culture plates. The plates were cultured in a 37°C, 5% CO 2 incubator to form hybridoma cell lines.
  • the best Nectin-4 mouse monoclonal antibody in immunohistochemical testing was screened and named MM11.
  • the sequence information of antibody MM11 is as follows: the heavy chain constant region is a mouse IgG1 type antibody sequence, the heavy chain variable region and the light chain variable region are shown in italics and underlined, and the complementarity determining region (CDR) sequence defined according to the KABAT naming system is further shown in bold.
  • CDR complementarity determining region
  • Nectin-4 extracellular protein (see UniProt database accession number Q96NY8, position 32-349) was used to immunize Japanese white rabbits. Mononuclear cells were obtained from the blood of the immunized rabbits, and then total RNA was extracted from the mononuclear cells to generate a cDNA gene library of Nectin-4 rabbit antibodies. A phage library was formed from the cDNA gene library of Nectin-4 rabbit antibodies, and biopanning was performed to screen out Nectin-4 rabbit monoclonal antibodies that performed well in immunohistochemical tests, which were named R012 and R036, respectively.
  • antibody R012 and antibody R036 are as follows, the heavy chain constant region is a rabbit IgG antibody sequence, the heavy chain variable region and the light chain variable region are shown in italics and underlined, and the complementary determining region (CDR) sequence defined according to the KABAT naming system is further shown in bold:
  • Example 2 ELISA method to detect the binding ability of antibodies to nectin-4 and its homologous family proteins
  • an enzyme-linked immunosorbent assay (ELISA) was used to detect whether there was a cross reaction between the antibody and other Nectin family proteins.
  • the nectin family of proteins includes the following 9 proteins: Nectin-1, Nectin-2, Nectin-3, Nectin-4, Nectin-like-1, Nectin-like-2, Nectin-like-3, Nectin-like-4, Nectin-like-5.
  • the specific information is as follows:
  • the ELISA test method was performed as follows:
  • Coating Dilute Nectin-1, Nectin-2, Nectin-3, Nectin-4, Nectin-like-1, Nectin-like-2, Nectin-like-3, Nectin-like-4, and Nectin-like-5 to 5 ⁇ g/ml with coating buffer (pH 9.6 carbonate buffer), coat 96-well plates with 100 ⁇ l/well, and incubate at 2-8°C overnight.
  • coating buffer pH 9.6 carbonate buffer
  • Blocking Take out the plate and pat dry, wash the plate twice with PBST at 200 ⁇ l/well, pat dry. Add 3% BSA/PBST, 200 ⁇ l/well, incubate at room temperature for 1.5h to implement blocking;
  • Sample addition Take out the plate and pat dry, wash the plate twice with PBST at 200 ⁇ l/well, and pat dry. Dilute the nectin-4 antibodies (antibody MM11, antibody R012, antibody R036) prepared in Example 1 to 10 ⁇ g/ml, 1 ⁇ g/ml, 100 ng/ml and 10 ng/ml respectively with 1% BSA/PBST, use 1% BSA/PBST dilution as blank control, add 100 ⁇ l/well to 96-well plate, and incubate at room temperature. Incubate at room temperature for 2 h;
  • Add secondary antibody remove the plate and pat dry, wash the plate 6 times with PBST at 200 ⁇ l/well, pat dry.
  • Dilute goat anti-mouse IgG-HRP Jackson, catalog number: 115-035-003 at a ratio of 1:20000 or dilute goat anti-rabbit IgG (H+L) antibody-HRP (ThermoFisher, catalog number: 31460) at a ratio of 1:10000, 100 ⁇ l/well, incubate at room temperature for 1 hour;
  • Stop the reaction and read the plate Add 1 mol/L H 3 PO 4 at 100 ⁇ l/well to stop the reaction and read the plate. Using 650 nm as the reference wavelength, read and record the absorbance of the well plate at a wavelength of 450 nm (OD450 nm-OD650 nm).
  • Mouse antibody MM11, rabbit antibody R012 and rabbit antibody R036 can specifically bind to the recombinant human nectin-4 protein and have no cross-reaction with other proteins of the same family.
  • the flow cytometry assay was performed as follows:
  • PC-3 Human prostate cancer cells (PC-3) were purchased from ATCC and do not express nectin-4. Human prostate cancer cells (PC-3) were used as a negative control.
  • PC-3-Nectin-4 cells are genetically engineered cell lines constructed by Myvicon, and are PC-3 cell lines expressing Nectin-4. Specifically, the full-length gene of human Nectin-4 (NCBI gene ID: 81607, 1533bp) was knocked into PC-3 cells and a stably transfected cell line with high expression of Nectin-4 was obtained through monoclonal cell screening. Flow cytometry detection confirmed that Nectin-4 was highly expressed on the cell surface, and the cell line was named PC-3-Nectin-4 cells.
  • Antibodies MM11, R012 and R036 were diluted to 5 ⁇ g/ml with PBS, and 200 ⁇ l of antibody dilution solution was taken to resuspend the above cells respectively, and the cells were gently mixed and incubated on ice in the dark for 1 hour.
  • the flow tube was centrifuged at 4°C, 350g for 5 min, the supernatant was discarded, 1 ml of pre-cooled PBS was added to each tube to wash the cells twice, and then 500 ⁇ l of the secondary antibody was added.
  • the secondary antibody was goat anti-mouse IgG Fc-FITC (1:200) (Abcam, catalog number ab150113); for antibody R012 and antibody R036, the secondary antibody was goat anti-rabbit IgG H&L-Alexa 488 (1:2000) (Abcam, Catalog No.
  • the FACS test results are shown in Figure 2.
  • the detection of PC-3-Nectin4 cells using antibody MM11, antibody R012 and antibody R036 all produced high fluorescence signals, while the detection of PC-3 cells had no obvious fluorescence signals.
  • the results showed that Nectin-4 antibodies MM11, R012 and R036 can specifically recognize and bind to Nectin-4 expressed on PC-3-Nectin 4 cells, and do not recognize other proteins on the cells.
  • Example 4 Western blotting to detect the reactivity of antibodies with nectin-4 expressing cell lysates
  • Example 1 Western blotting was used to detect whether the Nectin-4 antibody obtained in Example 1 specifically recognized the Nectin-4 protein. In addition, during Western blotting, an inhibition experiment was performed by adding an excess of Nectin-4 protein to further confirm the specificity of the Nectin-4 antibody obtained in Example 1.
  • the Western blot assay was performed as follows:
  • PC-3 Human prostate cancer cells (PC-3) were purchased from ATCC, and flow cytometry was used to confirm that PC-3 cells did not express nectin-4.
  • PC-3-Nectin-4 cells are genetically engineered cell lines constructed by Myvicon, and are PC-3 cell lines expressing Nectin-4. Specifically, the full-length gene of human Nectin-4 (NCBI gene ID: 81607, 1533bp) was knocked into PC-3 cells and a stably transfected cell line with high expression of Nectin-4 was obtained through monoclonal cell screening. Flow cytometry detection confirmed that Nectin-4 was highly expressed on the cell surface, and the cell line was named PC-3-Nectin-4 cells.
  • Protein extraction Resuscitate PC-3-Nectin 4 cells and PC-3 cells, grow them in cell culture flasks in a cell culture incubator to the appropriate cell density, discard the culture medium, and add pre-cooled PBS to wash twice. Then add RIPA lysis buffer (Radioimmunoprecipitation assay buffer) (Thermo, catalog number 89901) containing protease inhibitors to lyse the cells, scrape the cells with a cell scraper, lyse on ice for 30 minutes, and centrifuge at 12000rpm, 4°C for 10 minutes. Take the supernatant after centrifugation into a new centrifuge tube, determine the protein content by the BCA method and adjust to the appropriate concentration.
  • RIPA lysis buffer Radioimmunoprecipitation assay buffer
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • Electrophoresis The loading amount of the two cell lysates was 4.6 ⁇ g, and the electrophoresis conditions were: 120 V, 80 min;
  • Blocking Incubate the membrane with 5% BSA/TBST at room temperature for 1 hour with shaking;
  • Example 1 diluting the nectin-4 antibodies prepared in Example 1 (antibody MM11, antibody R012, and antibody R036, respectively) to 20 ng/ml with 5% BSA/TBST, adding them as the primary antibody, and incubating the membrane with shaking at 2-8° C. overnight; or
  • nectin-4 antibodies (antibody MM11 and antibody R012) prepared in Example 1 were mixed with 5% BSA/TBST. Dilute to 20 ng/ml and add as the primary antibody; add 200 ⁇ g/ml recombinant human nectin-4 (McVancom, Lot: 20210101) for pre-incubation for 1 h, and then incubate the membrane with shaking at 2-8°C overnight;
  • Color development After the second antibody incubation is completed, wash the membrane three times with TBST, 5 minutes each time, add 1:1 prepared ECL luminescent solution for color development, and collect the luminescent signal.
  • the normal human tissues included tissues and organs from multiple sources; the tumor tissues included: 3 bladder cancers, 2 lung cancers, and 3 breast cancers.
  • Anti-Nectin-4 monoclonal antibody was used to detect the expression of Nectin-4 in tissue samples, and commercial Nectin-4 rabbit monoclonal antibody (purchased from abcam, clone number: EPR15613-68) was used as a positive control. All antibodies were diluted to working concentrations using antibody diluent, which was purchased from Maijie Translational Medicine Research (Suzhou) Co., Ltd., catalog number P010A01. Immunohistochemical staining was detected using a fully automated immunohistochemical staining system (Leica BOND III), and the specific procedures are shown in the table below.
  • Peroxidase inactivation reagent goat anti-rabbit IgG-polymer HRP, DAB color development reagent, and hematoxylin staining solution are all included in Leica's BOND Polymer Refine Detection kit, catalog number DS9800. All reagents have been diluted to working concentrations and can be used directly.
  • the normal human tissues included tissues and organs from multiple sources; the tumor tissues included: 3 cases of prostate cancer, 3 cases of lung cancer, and 3 cases of triple-negative breast cancer tissues.
  • Anti-Nectin-4 monoclonal antibody was used to detect the expression of Nectin-4 in tissue samples, and mouse monoclonal antibody M22-321b41.1 was used as a positive control.
  • M22-321b41.1 is a patented Nectin-4 mouse monoclonal antibody, and the patent publication number is CN111051345A. All antibodies were diluted to working concentrations using antibody diluent.
  • the antibody diluent was purchased from Maijie Translational Medicine Research (Suzhou) Co., Ltd., item number P010A01. Immunohistochemical staining was detected in a fully automatic immunohistochemical staining system (Leica BOND III). The specific procedures are shown in the following table.
  • Peroxidase inactivation reagent rabbit anti-mouse Post-primary, goat anti-rabbit IgG-polymer HRP, DAB color development reagent, and hematoxylin staining solution are all included in Leica's BOND Polymer Refine Detection kit, catalog number DS9800. All reagents have been diluted to working concentrations and can be used directly.
  • the target protein nectin-4 is localized on the cell membrane and/or cytoplasm, which is consistent with the protein localization reported in the literature, and no nonspecific staining is produced.
  • the staining signal of antibody MM11 is significantly stronger than that of antibody M22-321b41.1.

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Abstract

提供特异性结合Nectin-4的抗体和抗原结合片段、以及包含所述抗体或抗原结合片段的试剂盒。还涉及编码所述抗体的核酸、包含所述核酸的宿主细胞，以及制备所述抗体的方法。还涉及所述特异性结合Nectin-4的抗体的诊断和预后用途。

Description

结合Nectin-4的抗体及其用途 技术领域
本发明涉及抗体医疗领域。具体而言，本发明涉及特异性结合Nectin-4的抗体或其抗原结合片段以及含有所述抗体或抗原结合片段的试剂盒。此外，本发明涉及编码所述抗体的核酸及包含所述核酸的宿主细胞，以及制备所述抗体的方法。本发明还涉及所述结合Nectin-4的抗体的诊断和预后用途。
背景技术
Nectin-4(也称为“脊髓灰质炎病毒受体样分子4(PVRL4))是一个分子量约为66KD的I型跨膜糖蛋白，属于免疫球蛋白超家族中Nectin家族的成员，其细胞外结构域由三个Ig样结构域(VCC型)组成，与钙粘蛋白一起参与粘附连接的形成和维持。
Nectin-4特异性表达于胚胎、胎盘以及肿瘤细胞中，在正常组织器官中不表达或者表达水平极低，是临床上一类经典的肿瘤标志物，也是抗体偶联药物(ADC)的理想靶点。靶向Nectin-4的抗体偶联药物与肿瘤细胞表面过度表达的Nectin-4结合，形成ADC-受体结合物；然后，ADC经靶点介导的内吞作用进入细胞内，之后在溶酶体中被组织蛋白酶切断连接子，释放出毒性小分子，实现肿瘤细胞的特异性杀灭。靶向Nectin-4的PADCEVTM(别名:enfortumab vedotin-ejfv；中文名:维汀-恩弗妥单抗)是一种ADC药物，该ADC药物由靶向Nectin-4蛋白的人IgG1单克隆抗体恩弗妥单抗(enfortumab)与细胞毒制剂MMAE(monomethyl auristatin E，单甲基奥瑞他汀E，一种微管破坏剂)偶联而成(Pia M.Challita-Eid等(2016)Cancer Res.76(10):3003-13)。已获FDA批准用于治疗局部晚期或转移性尿路上皮癌。但是，靶向Nectin-4的PADCEVTM仅用于治疗应用，且不能用于免疫组化(IHC)染色，因此不具有诊断价值。
本领域需要特异性识别Nectin-4的抗Nectin-4抗体，其能够高灵敏地检测(例如，通过IHC检测)样品中Nectin-4的存在和/或表达水平，由此来诊断疾病或评估预后，这对于提高癌症患者的生存率、延长生存期、避免过度化疗和提高生存质量有着重大意义。因此，在诊断和预后中使用特异性好、灵敏度高的抗Nectin-4抗体具有极高的应用价值。
发明内容
本发明提供了特异性好、灵敏度高的抗Nectin-4抗体，其能够可靠和灵敏地检测Nectin-4。本发明的特异性结合Nectin-4的抗体或抗原结合片段具有以下一个或多个特性：
(a)在ELISA测定法中测量，具有仅与Nectin-4特异性结合的能力，与Nectin家族成员Nectin-1、Nectin-2、Nectin-3、Nectin-like-1、Nectin-like-2、Nectin-like-3、Nectin-like-4、Nectin-like-5均无特异性结合；
(b)在流式细胞术测定法中测量，具有结合细胞上表达的Nectin-4分子的能力；
(c)在Western印迹法中测量，能够特异性检测Nectin-4蛋白；并且在添加过量的Nectin-4蛋白时，与膜上Nectin-4蛋白的结合被抑制；和/或
(d)在免疫组织化学染色中，特异性染色表达Nectin-4分子的组织，例如，所述组织选自扁桃体、唾液腺、大脑、小脑、胎盘、膀胱、肺、乳腺、食道、喉、胸腺、心脏、胃、小肠、结肠、直肠、输尿管、卵巢、输卵管、子宫颈、子宫内膜、皮肤、肾脏、前列腺、胰腺、甲状腺、脾脏和肝脏。
因此，在第一方面，本发明提供了特异性结合Nectin-4的抗体或抗原结合片段，其包含重链可变区和轻链可变区，其中：
(a)所述重链可变区包含SEQ ID NO:5所示的HCDR1或SEQ ID NO:5所示的HCDR1的不超过2个氨基酸变化的变体、SEQ ID NO:6所示的HCDR2或SEQ ID NO:6所示的HCDR2的不超过2个氨基酸变化的变体、和SEQ ID NO:7所示的HCDR3或SEQ ID NO:7所示的HCDR3的不超过2个氨基酸变化的变体；所述轻链可变区包含SEQ ID NO:8所示的LCDR1或SEQ ID NO:8所示的LCDR1的不超过2个氨基酸变化的变体、SEQ ID NO:9所示的LCDR2或SEQ ID NO:9所示的LCDR2的不超过2个氨基酸变化的变体、和SEQ ID NO:10所示的LCDR3或SEQ ID NO:10所示的LCDR3的不超过2个氨基酸变化的变体；
(b)所述重链可变区包含SEQ ID NO:15所示的HCDR1或SEQ ID NO:15所示的HCDR1的不超过2个氨基酸变化的变体、SEQ ID NO:16所示的HCDR2或SEQ ID NO:16所示的HCDR2的不超过2个氨基酸变化的变体、和SEQ ID NO:17所示的HCDR3或SEQ ID NO:17所示的HCDR3的不超过2个氨基酸变化的变体；所述轻链可变区包含SEQ ID NO:18所示的LCDR1或SEQ ID NO:18所示的LCDR1的不超过2个氨基酸变化的变体、SEQ ID NO:19所示的LCDR2或SEQ ID NO:19所示的LCDR2的不超过2个氨基酸变化的变体、和SEQ ID NO:20所示的LCDR3或SEQ ID NO:20所示的LCDR3的不超过2个氨基酸变化的变体；或
(c)所述重链可变区包含SEQ ID NO:25所示的HCDR1或SEQ ID NO:25所示的HCDR1的不超过2个氨基酸变化的变体、SEQ ID NO:26所示的HCDR2或SEQ ID NO:26所示的HCDR2的不超过2个氨基酸变化的变体、和SEQ ID NO:27所示的HCDR3或SEQ ID NO:27所示的HCDR3的不超过2个氨基酸变化的变体；所述轻链可变区包含SEQ ID NO:28所示的LCDR1或SEQ ID NO:28所示的LCDR1的不超过2个氨基酸变化的变体、SEQ ID NO:29所示的LCDR2或SEQ ID NO:29所示的LCDR2的不超过2个氨基酸变化的变体、和SEQ ID NO:30所示的LCDR3或SEQ ID NO:30所示的LCDR3的不超过2个氨基酸变化的变体。
在一些实施方案中，所述氨基酸变化是保守的氨基酸修饰。
在一些实施方案中，本发明的特异性结合Nectin-4的抗体或抗原结合片段包含
(a)重链可变区和轻链可变区，其中重链可变区包含SEQ ID NO:3的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:4的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；
(b)重链可变区和轻链可变区，其中重链可变区包含SEQ ID NO:13的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:14的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；或
(c)重链可变区和轻链可变区，其中重链可变区包含SEQ ID NO:23的序列或与其具有至 少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:24的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列。
在一些实施方案中，本发明的特异性结合Nectin-4的抗体的抗原结合片段是Fab、Fab’、F(ab’)2、Fv、单链Fv、单链Fab或双体抗体(diabody)。
在一些实施方案中，本发明的特异性结合Nectin-4的抗体或抗原结合片段包含
(a)SEQ ID NO:1或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的重链序列，以及SEQ ID NO:2或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的轻链序列；例如，SEQ ID NO:1所示的重链序列，以及SEQ ID NO:2所示的轻链序列；
(b)SEQ ID NO:11或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的重链序列，以及SEQ ID NO:12或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的轻链序列；例如，SEQ ID NO:11所示的重链序列，以及SEQ ID NO:12所示的轻链序列；或
(c)SEQ ID NO:21或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的重链序列，以及SEQ ID NO:22或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的轻链序列；例如，SEQ ID NO:21所示的重链序列，以及SEQ ID NO:22所示的轻链序列。
在一些实施方案中，本发明的特异性结合Nectin-4的抗体或抗原结合片段为鼠抗体或兔抗体。
在一些实施方案中，本发明的特异性结合Nectin-4的抗体或抗原结合片段是IgG类抗体。
在第二方面，本发明提供了编码上述第一方面所述的抗Nectin-4抗体或抗原结合片段的核酸、包含所述核酸的载体(优选地，表达载体)、包含所述核酸或所述载体的宿主细胞。在一些实施方案中，所述宿主细胞是原核的或真核的，例如，选自大肠杆菌细胞、酵母细胞、哺乳动物细胞或适用于制备抗体或其抗原结合片段的其它细胞。在一些实施方案中，所述宿主细胞是HEK 293细胞或CHO细胞。
在第三方面，本发明提供了制备上述第一方面所述的抗Nectin-4抗体或其抗原结合片段的方法，所述方法包括在适于表达编码上述第一方面所述的抗Nectin-4抗体或其抗原结合片段的核酸的条件下培养上述第二方面所述的宿主细胞，以及从培养物回收表达的所述抗Nectin-4抗体或其抗原结合片段。
在第四方面，本发明提供了一种试剂盒，其包含上述第一方面所述的抗Nectin-4抗体或其抗原结合片段。
在第五方面，本发明提供了上述第一方面所述的抗Nectin-4抗体或其抗原结合片段的用途，用于检测样品中的Nectin-4水平。
在第六方面，本发明提供了使用上述第一方面所述的抗Nectin-4抗体或其抗原结合片段来诊断受试者中的癌症的方法。
在一些实施方案中，本发明提供了使用上述第一方面所述的抗Nectin-4抗体或其抗原结合片段来确定受试者使用抗Nectin-4抗体或者抗Nectin-4抗体偶联药物(例如，具有治疗作 用的抗Nectin-4抗体或其抗原结合片段)治疗的资格的方法。
在一些实施方案中，本发明提供了使用上述第一方面所述的抗Nectin-4抗体或其抗原结合片段来预后患有癌症的受试者的方法，其中所述患有癌症的受试者是已经使用抗肿瘤剂(例如，抗Nectin-4抗体或者抗Nectin-4抗体偶联药物(例如，具有治疗作用的抗Nectin-4抗体或其抗原结合片段))治疗后的患有癌症的受试者。
附图说明
结合说明书附图一起阅读时，将更好地理解以下详细描述的本发明的优选实施方案。出于说明本发明的目的，图中显示了目前优选的实施方案。然而，应当理解本发明不限于图中所示实施方案的精确安排和手段。
图1：显示通过ELISA法检测抗体MM11、抗体R012、抗体R036与Nectin家族蛋白(Nectin-1、Nectin-2、Nectin-3、Nectin-4、Nectin-like-1、Nectin-like-2、Nectin-like-3、Nectin-like-4、Nectin-like-5)的结合。
图2：显示通过流式细胞术(FACS法)检测抗体MM11、抗体R012、抗体R036与Nectin-4表达细胞的结合。图中，Blank为对人前列腺癌细胞(PC-3)仅加入第二抗体，不加作为第一抗体的抗体MM11、抗体R012或抗体R036；NC为仅有PC-3细胞和添加空白缓冲液；“PC-3-Nectin4”为表达Nectin-4的PC-3细胞株。
图3：显示通过Western印迹法检测抗体MM11、抗体R012、抗体R036与表达Nectin-4的细胞裂解物的反应性。图中，泳道M：蛋白分子量Marker；泳道1：PC-3细胞裂解液；泳道2：PC-3-Nectin-4细胞裂解液。图3的上方各小图分别显示了抗体MM11、抗体R012、抗体R036与表达Nectin-4的细胞裂解物反应，且不与不表达Nectin-4的细胞裂解物反应；图3的下方小图中“MM11(Inhibition)”和“R012(Inhibition)”表示细胞裂解物在转膜后分别与“MM11抗体”或“R012抗体”孵育，且加入过量的重组Nectin-4蛋白后的结果；“Gapdh”表示细胞裂解物在转膜后与抗甘油醛-3-磷酸脱氢酶抗体孵育的反应，作为Western印迹法的内部参照。
图4：显示Nectin-4兔单克隆抗体R012、R036以及作为阳性对照的Nectin-4兔单克隆抗体EPR15613-68与具有代表性的正常人组织上免疫组织化学染色结果(比例尺为200μm)。
图5：显示Nectin-4兔单克隆抗体R012、R036以及作为阳性对照的Nectin-4兔单克隆抗体EPR15613-68在人体膀胱癌组织上免疫组织化学染色结果(比例尺为200μm)。
图6：显示Nectin-4兔单克隆抗体R012、R036以及作为阳性对照的Nectin-4兔单克隆抗体EPR15613-68在人体肺癌组织上免疫组织化学染色结果(比例尺为200μm)。
图7：显示Nectin-4兔单克隆抗体R012、R036以及作为阳性对照的Nectin-4兔单克隆抗体EPR15613-68在人体乳腺癌组织上免疫组织化学染色结果(比例尺为200μm)。
图8：显示Nectin-4鼠单抗MM11以及作为阳性对照的Nectin-4鼠单抗M22-321b41.1在具有代表性的正常人组织上免疫组织化学染色结果(比例尺为100μm)。
图9：显示Nectin-4鼠单抗MM11以及作为阳性对照的Nectin-4鼠单抗M22-321b41.1在人体前列腺癌组织上免疫组织化学染色结果(比例尺为50μm)。
图10：显示Nectin-4鼠单抗MM11以及作为阳性对照的Nectin-4鼠单抗M22-321b41.1 在人体肺癌组织上免疫组织化学染色结果(比例尺为50μm)。
图11：显示Nectin-4鼠单抗MM11以及作为阳性对照的Nectin-4鼠单抗M22-321b41.1在人体三阴性乳腺癌癌组织上免疫组织化学染色结果(比例尺为50μm)。
具体实施方式
在详细描述本发明之前，应了解，本发明不受限于本说明书中的特定方法及实验条件，因为所述方法以及条件是可以改变的。
I.定义
除非另外限定，否则本文中所用的全部技术与科学术语具有如本发明所属领域的普通技术人员通常理解的相同含义。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
如本文所用，术语“和/或”意指可选项中的任一项或可选项的两项或多项。
在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
如本文所用，术语“抗体”指结合抗原的免疫球蛋白分子。抗体的实施方案包括单克隆抗体、多克隆抗体或嵌合抗体。抗体可以属于任何类(例如，IgG、IgE、IgM、IgD、IgA)。
本公开的示例性抗体是包含由链间二硫键交联的以下四条多肽链组成的免疫球蛋白G(IgG)型抗体：两条重链(HC)和两条轻链(LC)。四条多肽链中每条链的氨基端部分包括主要负责抗原识别的约100-125个或更多个氨基酸的可变区。每条重链由重链可变区(VH)和重链恒定区组成。每条轻链由轻链可变区(VL)和轻链恒定区组成。
VH区和VL区可以进一步细分成称为互补决定区(CDR)的高变区，其间散布较保守的区域，称为构架区(FR)。CDR暴露于蛋白质的表面上并且是抗体的抗原结合特异性的重要区域。每个VH和VL由从氨基端至羧基端按以下顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4排列的三个CDR和四个FR组成。重链的三个CDR称为“HCDR1、HCDR2和HCDR3”并且轻链的三个CDR称为“LCDR1、LCDR2和LCDR3”。CDR含有大部分与抗原形成特异性相互作用的残基。可以根据熟知的编号方案将氨基酸残基归属至CDR，所述方案包括在Kabat(Kabat等人,“Sequences of Proteins of Immunological Interest(具有免疫学意义蛋白质的序列),”National Institutes of Health,Bethesda,Md.(1991))、Chothia(Chothia等人,“Canonical structures for the hypervariable regions of immunoglobulins(免疫球蛋白高变区的规范结构)”,Journal of Molecular Biology,196,901-917(1987)；Al-Lazikani等人,“Standard Conformations for the canonical structures of immunoglobulins(免疫球蛋白规范结构的标准构象)”,Journal of Molecular Biology,273,927-948(1997))、North(North等人,“A New Clustering of Antibody CDR Loop Conformations(抗体CDR环构象的新聚类)”,Journal of Molecular Biology,406,228-256(2011))或IMGT(自www.imgt.org可获得的ImMunoGeneTic国际数据库；参见Lefranc等人,Nucleic Acids Res.1999；27:209-212)中描述的那些。
然而，应该注意，基于不同的编号方案获得的同一抗体的可变区的CDR的边界可能有所 差异。即不同编号方案下定义的同一抗体可变区的CDR序列有所不同。因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的编号方案(例如不同的编号方案规则或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。
本发明抗体的CDR可以根据本领域的任何编号方案或其组合人工地评估确定边界。除非另有说明，否则在本发明中，术语“CDR”或“CDR序列”涵盖以上述任一种方式确定的CDR序列。
“抗原结合片段”是比完整或完全抗体的氨基酸残基数要少的完整或完全抗体的一部分或一段，其能结合抗原或与完整抗体(即与抗原结合片段所来源的完整抗体)竞争结合抗原。可以通过重组DNA技术、或通过酶或化学切割完整的抗体制备抗原结合片段。抗原结合片段包括但不限于Fab、Fab’、F(ab’)2、Fv、单链Fv(scFv)、单链Fab、双体抗体(diabody)、单结构域抗体(sdAb，纳米抗体)、骆驼Ig、Ig NAR、F(ab)'3片段、双-scFv、(scFv)2、微型抗体、双功能抗体、三功能抗体、四功能抗体、二硫键稳定的Fv蛋白(“dsFv”)。所述术语还包括经遗传工程改造的形式，例如嵌合抗体(例如人源化鼠抗体)、杂结合抗体(例如双特异性抗体)和其抗原结合片段。更详细的描述也请参见:皮尔斯目录与手册(Pierce Catalog and Handbook),1994-1995(皮尔斯化学公司(PierceChemical Co.),罗克福德(Rockford)，伊利诺伊州(IL))；Kuby,免疫学杂志,第3版,W.H.弗里曼公司(W.H.Freeman&Co.),纽约,1997。
术语“Nectin家族蛋白”与“Nectin家族”可互换地使用，是细胞粘附分子，将相邻细胞之间形成物理连接以实现细胞间的通信、迁移和其他重要的细胞过程。Nectin家族蛋白至少包括Nectin-1、Nectin-2、Nectin-3、Nectin-4、Nectin-like-1、Nectin-like-2、Nectin-like-3、Nectin-like-4、Nectin-like-5，其中，Nectin-4蛋白在健康人体中的表达主要局限于胎盘和胚胎组织。与健康的成人组织相比，许多类型的肿瘤细胞都有Nectin-4蛋白高表达。Nectin-4蛋白是一种肿瘤相关抗原。
当谈及抗原和抗体时使用的术语“结合”或“特异性结合”意指结合作用对抗原是选择性的并且可以与不想要的或非特异的相互作用区别。抗体与特定抗原结合的能力可以通过酶联免疫吸附测定法(ELISA)、SPR或生物膜层干涉技术或本领域已知的其他常规结合测定法测定。
为确定两个氨基酸序列或两个核酸序列的同一性百分数，将所述序列出于最佳比较目的比对(例如，可以为了最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。在一个优选实施方案中，为比较目的，所比对的参考序列的长度是至少30％、优选地至少40％、更优选地至少50％、60％和甚至更优选地至少70％、80％、90％、100％的参考序列长度。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。在一个优选实施方案中，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在http://www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。在又一个优选的实施方案中，使用GCG软件包中的GAP程序(在 http://www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum 62评分矩阵。
还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4)，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。
额外地或备选地，可以进一步使用本文所述的核酸序列和蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其他家族成员序列或相关序列。
对于多肽序列，“保守性修饰”或“保守的氨基酸修饰”包括对多肽序列的置换、缺失或添加，它们导致某个氨基酸置换为化学上相似的氨基酸。提供功能上相似氨基酸的保守性置换表是本领域熟知的。这类保守性修饰的变体相对于本发明的多态性变体、物种间同源物和等位基因而言是附加的并且不排斥它们。以下8组含有互为保守替换的氨基酸:1)丙氨酸(A)、甘氨酸(G)；2)天冬氨酸(D)、谷氨酸(E)；3)天冬酰胺(N)、谷氨酰胺(Q)；4)精氨酸(R)、赖氨酸(K)；5)异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、缬氨酸(V)；6)苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)；7)丝氨酸(S)、苏氨酸(T)；和8)半胱氨酸(C)、甲硫氨酸(M)(参阅例如，Creighton,Proteins(1984))。在一些实施方案中，术语“保守的氨基酸修饰”用于指不显著影响或改变含有氨基酸序列的抗体的结合特征的氨基酸修饰。
如本文所用，“载体(vector)”表示构建体，其能够将一种或多种所关注的基因或序列递送入宿主细胞并且优选在宿主细胞中表达所述基因或序列。载体的实例包括但不限于病毒载体、裸DNA或RNA表达载体、质粒、粘粒或噬菌体载体、与阳离子凝聚剂相关的DNA或RNA表达载体、包囊化于脂质体中的DNA或RNA表达载体以及某些真核细胞，例如生产细胞。
在本发明中术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可互换使用，并且是指已经引入外源性核酸的细胞，包括这些细胞的子代。宿主细胞包括“转化子”和“转化的细胞”，其包括原代转化细胞以及由此来源的子代，而不考虑传代次数。子代在核酸含量上与亲代细胞可能不完全相同，但可能含有突变。本文包括与在初始转化的细胞中筛选或选择的细胞具有相同功能或生物学活性的突变子代。
在本文中，“受试者”、“个体”或“对象”指需要缓解、预防、治疗和/或诊断疾病或病症的动物，优选哺乳动物，更优选是人。哺乳动物还包括但不限于农场动物、竞赛动物、宠物、灵长类、马、犬、猫、小鼠和大鼠。该术语包括具有疾病或处于具有疾病风险的人受试者。
来自受试者的“生物样品”指从个体或受试者得到的细胞、组织或体液的集合。组织或细胞样品的来源可以是实体组织，像来自新鲜的、冷冻的和/或保存的器官或组织样品或活检样品或穿刺样品；血液或任何血液组分；体液，诸如脑脊液、羊膜液(羊水)、腹膜液(腹水)、或间隙液；来自受试者的妊娠或发育任何时间的细胞。组织样品可能包含在自然界中天然不与组织混杂的化合物，诸如防腐剂、抗凝剂、缓冲剂、固定剂、营养物、抗生素、等等。肿瘤样品的例子在本文中包括但不限于肿瘤活检、细针吸出物、支气管灌洗液、胸膜液(胸水)、痰液、尿液、手术标本、循环中的肿瘤细胞、血清、血浆、循环中的血浆蛋白质、腹水、衍生自肿瘤或展现出肿瘤样特性的原代细胞培养物或细胞系，以及保存的肿瘤样品，诸如福尔 马林固定的、石蜡包埋的肿瘤组织切片样品或冷冻的肿瘤样品。
术语“参考样品”、“参考细胞”、“参考组织”、“对照样品”、“对照细胞”或“对照组织”是指用于比较目的的样品、细胞、组织或标准品水平。在一个实施方案中，参考样品、参考细胞、参考组织、对照样品、对照细胞或对照组织获自同一受试者或个体的机体的健康和/或非患病部分，为健康和/或非患病组织或细胞。在又一个实施方案中，参考样品、参考细胞、参考组织、对照样品、对照细胞或对照组织获自不是受试者的个体的健康组织或细胞。来自“参考样品”、“参考细胞”、“参考组织”、“对照样品”、“对照细胞”或“对照组织”的Nectin-4的存在或表达水平称为“参考存在或表达水平”。
术语“第一抗体”是指特异性结合生物样品中的Nectin-4的抗体。术语“第二抗体”是指特异性结合第一抗体，从而在第一抗体和后续试剂(如果需要后续试剂的话)之间形成桥连的抗体。
免疫荧光(Immunofluorescence，IF)技术是标记免疫技术中发展最早的一种技术。它是根据抗原抗体反应的原理，先将已知的抗原或抗体标记上荧光基团，再用这种荧光抗体(或抗原)作为探针检查细胞或组织内的相应抗原(或抗体)。在组织或细胞内形成的抗原抗体复合物上含有标记的荧光素，利用荧光显微镜观察标本，荧光素受外来激发光的照射而发生明亮的荧光(黄绿色或橘红色)，可以看见荧光所在的组织细胞，从而确定抗原或抗体的性质、定位，以及利用定量技术测定含量。
免疫沉淀(Immunoprecipitation，IP)法是利用抗原抗体特异性反应，对某个特定蛋白纯化富集的方法，主要过程包括捕获和固定。
免疫共沉淀(Co-Immunoprecipitation，Co-IP)是在免疫沉淀的基础上进行的扩展。Co-IP是利用抗原蛋白质和抗体的特异性结合以及细菌蛋白质的“Protein A/G"特异性地结合到抗体(免疫球蛋白)的Fc片段的现象开发出来的方法。通常将Protein A/G预先结合在Argarose珠上，使之与含有抗原蛋白的溶液(如细胞裂解液)及针对诱饵蛋白X的抗体反应后，Argarose珠上的Prorein A/G通过抗体吸附诱饵蛋白X，诱饵蛋白X通过与靶蛋白Y相互作用将靶蛋白Y从细胞裂解液中分离出来，从而达到免疫共沉淀的目的。
II.本发明的特异性结合Nectin-4的抗体
本发明提供了特异性结合Nectin-4的抗体。在一些实施方案中，本发明的抗体是单克隆抗体，例如，其可以来自任何真核克隆、原核克隆或噬菌体克隆。可以通过杂交瘤技术、重组技术、噬菌体展示技术、B细胞克隆筛选技术、合成技术(例如CDR移植)或本领域已知的其他技术组合产生单克隆抗体。
用于产生和纯化抗体的方法是本领域熟知的并且可以在例如Harlow和Lane(1988)Antibodies,A Laboratory Manual,Cold Spring Harbor Laboratory Press,Cold Spring harbor,N.Y,第5-8章和第15章,ISBN 0-87969-314-2中找到。例如，可以用人Nectin-4免疫接种小鼠或兔或其他动物并且可以回收、纯化所产生的抗体，以及使用本领域熟知的常规方法测定氨基酸序列。同样，可以筛选噬菌体文库，从而对数千个Fab片段筛选与人Nectin-4的相互作用并且可以回收、纯化所产生的相互作用物，以及使用本领域熟知的常规方法测定氨基酸序列。
术语“结合Nectin-4蛋白的抗体”、“结合Nectin-4的抗体”、“抗Nectin-4蛋白抗体”、“抗Nectin-4抗体”、“结合Nectin-4蛋白的分离的抗体”、“Nectin-4抗体”和“Nectin-4 蛋白抗体”在本文中可互换地使用，是指这样的本发明的抗体，所述抗体能够以特异地结合Nectin-4蛋白，由此所述抗体可以用作靶向Nectin-4蛋白的诊断剂和/或检测剂。
本发明的分离的抗Nectin-4抗体和抗原结合片段特异性结合Nectin-4蛋白，其包含重链可变区和轻链可变区，其中：
(a)所述重链可变区包含SEQ ID NO:5所示的HCDR1或SEQ ID NO:5所示的HCDR1的不超过2个氨基酸变化的变体、SEQ ID NO:6所示的HCDR2或SEQ ID NO:6所示的HCDR2的不超过2个氨基酸变化的变体、和SEQ ID NO:7所示的HCDR3或SEQ ID NO:7所示的HCDR3的不超过2个氨基酸变化的变体；所述轻链可变区包含SEQ ID NO:8所示的LCDR1或SEQ ID NO:8所示的LCDR1的不超过2个氨基酸变化的变体、SEQ ID NO:9所示的LCDR2或SEQ ID NO:9所示的LCDR2的不超过2个氨基酸变化的变体、和SEQ ID NO:10所示的LCDR3或SEQ ID NO:10所示的LCDR3的不超过2个氨基酸变化的变体；
(b)所述重链可变区包含SEQ ID NO:15所示的HCDR1或SEQ ID NO:15所示的HCDR1的不超过2个氨基酸变化的变体、SEQ ID NO:16所示的HCDR2或SEQ ID NO:16所示的HCDR2的不超过2个氨基酸变化的变体、和SEQ ID NO:17所示的HCDR3或SEQ ID NO:17所示的HCDR3的不超过2个氨基酸变化的变体；所述轻链可变区包含SEQ ID NO:18所示的LCDR1或SEQ ID NO:18所示的LCDR1的不超过2个氨基酸变化的变体、SEQ ID NO:19所示的LCDR2或SEQ ID NO:19所示的LCDR2的不超过2个氨基酸变化的变体、和SEQ ID NO:20所示的LCDR3或SEQ ID NO:20所示的LCDR3的不超过2个氨基酸变化的变体；或
(c)所述重链可变区包含SEQ ID NO:25所示的HCDR1或SEQ ID NO:25所示的HCDR1的不超过2个氨基酸变化的变体、SEQ ID NO:26所示的HCDR2或SEQ ID NO:26所示的HCDR2的不超过2个氨基酸变化的变体、和SEQ ID NO:27所示的HCDR3或SEQ ID NO:27所示的HCDR3的不超过2个氨基酸变化的变体；所述轻链可变区包含SEQ ID NO:28所示的LCDR1或SEQ ID NO:28所示的LCDR1的不超过2个氨基酸变化的变体、SEQ ID NO:29所示的LCDR2或SEQ ID NO:29所示的LCDR2的不超过2个氨基酸变化的变体、和SEQ ID NO:30所示的LCDR3或SEQ ID NO:30所示的LCDR3的不超过2个氨基酸变化的变体；
其中所述氨基酸变化是氨基酸的添加、缺失或取代，例如，所述氨基酸变化是保守氨基酸取代。所述CDR是根据Kabat编号方案的CDR。
在一些实施方案中，本发明的分离的抗Nectin-4蛋白抗体或抗原结合片段包含：
(a)重链可变区和轻链可变区，其中重链可变区包含SEQ ID NO:3的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:4的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；
(b)重链可变区和轻链可变区，其中重链可变区包含SEQ ID NO:13的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:14的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；或
(c)重链可变区和轻链可变区，其中重链可变区包含SEQ ID NO:23的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可 变区包含SEQ ID NO:24的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列。
在一些实施方案中，本发明的抗体结合哺乳动物Nectin-4，例如人Nectin-4。在一些实施方案中，本发明的Nectin-4抗体与Nectin-4的一个或多个胞外结构域结合。
在一些实施方案中，本发明的抗体具有以下一个或多个特性：
(a)在ELISA测定法中测量，具有仅与Nectin-4特异性结合的能力，与Nectin家族成员Nectin-1、Nectin-2、Nectin-3、Nectin-like-1、Nectin-like-2、Nectin-like-3、Nectin-like-4、Nectin-like-5均无特异性结合；
(b)在流式细胞术测定法中测量，具有结合细胞上表达的Nectin-4分子的能力；
(c)在Western印迹法中测量，能够特异性检测Nectin-4蛋白；并且在添加过量的Nectin-4蛋白时，与膜上Nectin-4蛋白的结合被抑制；和/或
(d)在免疫组织化学染色中，特异性染色表达Nectin-4分子的组织，例如，所述组织选自扁桃体、唾液腺、大脑、小脑、胎盘、膀胱、肺、乳腺、食道、喉、胸腺、心脏、胃、小肠、结肠、直肠、输尿管、卵巢、输卵管、子宫颈、子宫内膜、皮肤、肾脏、前列腺、胰腺、甲状腺、脾脏和肝脏。
III.本发明的核酸以及包含其的宿主细胞
在一方面，本发明提供了编码以上任何Nectin-4抗体或其抗原结合片段或其任一条链的核酸。在一个实施方案中，提供了包含所述核酸的载体。在一个实施方案中，载体是表达载体。在一个实施方案中，提供包含所述核酸或所述载体的宿主细胞。在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞(例如CHO细胞或293细胞)或适用于制备抗体或其抗原结合片段的其它细胞。在另一个实施方案中，宿主细胞是原核的。
本发明还涵盖与以上所述核酸在严格性条件下杂交的核酸或与以上所述核酸相比编码具有一个或多个氨基酸取代(例如保守性取代)、缺失或插入的多肽序列的核酸。
在一个实施方案中，提供包含以上所述核酸的一个或多个载体。在一个实施方案中，载体是表达载体，例如真核表达载体。载体包括但不限于病毒、质粒、粘粒、λ噬菌体或酵母人工染色体(YAC)。
一旦已经制备了用于表达的表达载体或DNA序列，则可以将表达载体转染或引入适宜的宿主细胞中。多种技术可以用来实现这个目的，例如，原生质体融合、磷酸钙沉淀、电穿孔、逆转录病毒的转导、病毒转染、基因枪、基于脂质的转染或其他常规技术。在原生质体融合的情况下，将细胞在培养基中培育并且筛选适宜的活性。用于培养所产生的转染细胞和用于回收产生的抗体分子的方法和条件是本领域技术人员已知的并且可以基于本说明书和现有技术已知的方法，根据使用的特定表达载体和哺乳动物宿主细胞变动或优化。
IV.本发明的抗体的生产和纯化
在一个实施方案中，本发明提供了制备Nectin-4抗体的方法，其中所述方法包括在适于表达编码所述Nectin-4抗体的核酸的条件下培养包含编码所述Nectin-4抗体的核酸或包含所述核酸的表达载体的宿主细胞，以及任选地分离所述Nectin-4抗体。在某个实施方案中，所述方法还包括从所述宿主细胞或宿主细胞培养物回收Nectin-4抗体。
为了重组产生本发明的抗体，首先分离编码本发明Nectin-4抗体的核酸，并将所述核酸插入载体，用于在宿主细胞中进一步克隆和/或表达。此类核酸易于使用常规规程分离和测序，例如通过使用能够与编码本发明Nectin-4抗体的核酸特异性结合的寡核苷酸探针进行。
如本文所述制备的本发明的抗体可以通过已知的现有技术如高效液相色谱、离子交换层析、凝胶电泳、亲和层析、大小排阻层析等纯化。用来纯化特定蛋白质的实际条件还取决于净电荷、疏水性、亲水性等因素，并且这些对本领域技术人员是显而易见的。可以通过多种熟知分析方法中的任一种方法确定本发明的抗体的纯度，所述熟知分析方法包括大小排阻层析、凝胶电泳、高效液相色谱等。
V.本发明抗体的活性测定法
可以通过本领域中已知的多种测定法对本文中提供的Nectin-4抗体鉴定，筛选，或表征其物理/化学特性和/或生物学活性。
在一些实施方案中，对本发明的抗体测试其抗原结合活性，例如通过已知的方法诸如ELISA、Western印迹、FACS等来进行。
供任何上述体外测定法使用的细胞包括天然表达Nectin-4的细胞或经改造而表达Nectin-4细胞系。所述经改造而表达Nectin-4细胞系是正常情况下不表达Nectin-4的、将编码Nectin-4的DNA转染入细胞之后表达Nectin-4的细胞系。
VI.用于诊断和检测的方法和试剂盒
在一些实施方案中，本文中提供的任何Nectin-4抗体可以用于检测Nectin-4在生物样品中的存在和/或水平。
术语“检测”用于本文中时，包括定量或定性检测，示例性的检测方法可以涉及免疫组织化学、免疫细胞化学、流式细胞术(例如，FACS)、抗体分子复合的磁珠、ELISA测定法。在一些实施方案中，生物样品包含细胞或组织。在一些实施方案中，生物样品来自过度增生性或癌性病灶。
在一个实施方案中，提供了用于诊断或检测方法中的Nectin-4抗体，出于诊断或评估预后的目的，检测和/或评价Nectin-4的存在和/或水平，例如，旨在确定给定治疗方案的功效。
在一个方面，提供检测Nectin-4在生物样品中的存在和/或水平的方法。在一些实施方案中，所述方法包括检测Nectin-4蛋白在生物样品中的存在和/或水平。
在一些实施方案中，Nectin-4是人Nectin-4。在某些实施方案中，所述方法包括将生物样品与如本文所述的Nectin-4抗体在允许Nectin-4抗体与Nectin-4结合的条件下接触，并检测在Nectin-4抗体和Nectin-4之间是否形成复合物。复合物的形成表示存在Nectin-4。该方法可以是体外或体内方法。在一个实施方案中，Nectin-4抗体被用于选择适合利用治疗用Nectin-4抗体治疗的受试者，例如其中Nectin-4是用于选择所述受试者的生物标志物。
在一个实施方案中，可以使用本发明的抗体诊断表达Nectin-4蛋白的癌症或肿瘤，例如评价对象中表达Nectin-4蛋白的实体瘤(例如，多个器官系统的肉瘤和癌，如侵袭食管、肺、乳房、卵巢、淋巴样、胃肠道的(例如，结肠)、肛门、生殖器和生殖泌尿道(例如，肾、膀胱上皮、膀胱细胞、前列腺)、咽、中枢神经系统(例如，脑、神经的或神经胶质细胞)、头和颈、皮肤(例如，黑素瘤)、鼻咽(例如，分化或未分化的转移性或局部复发性鼻咽癌)和胰的那些癌、以及腺癌，包括恶性肿瘤)、血液学癌(例如，白血病、淋巴瘤、骨髓瘤，例如，多发性骨髓 瘤)的治疗或进展、其诊断和/或分期。在一些实施方案中，用抗Nectin-4抗体诊断的癌可以处于早期、中期或晚期或是转移性癌，例如，包括但不限于表达Nectin-4蛋白的原位癌、转移性癌。在一些实施方案中，癌选自结肠癌、直肠癌、结直肠癌、食管癌、皮肤癌、尿路上皮癌、卵巢癌、胰腺癌、膀胱癌，非霍奇金淋巴瘤、霍奇金淋巴瘤、急性淋巴细胞性白血病、多发性骨髓瘤、乳腺癌、胃癌、肝细胞癌、非小细胞肺癌、小细胞肺癌、黑素瘤、胶质母细胞瘤、肾细胞癌、前列腺癌。
在一些实施方案中，提供标记的Nectin-4抗体。标记包括但不限于被直接检测的标记(如荧光标记、发色团标记、电子致密标记、化学发光标记和放射性标记)，以及被间接检测的标记，如酶或配体，例如，通过酶促反应或分子相互作用。标记的示例性标记包括但不限于，放射性同位素32P、14C、125I、3H和131I，荧光团如稀土螯合物或荧光素及其衍生物，罗丹明及其衍生物，丹酰(dansyl)，伞形酮(umbelliferone)，萤光素酶(luceriferase)，例如，萤火虫萤光素酶和细菌萤光素酶(美国专利号4,737,456)，荧光素，2,3-二氢酞嗪二酮，辣根过氧化物酶(HR)，碱性磷酸酶，β-半乳糖苷酶，葡糖淀粉酶，溶解酶，糖类氧化酶，例如，葡萄糖氧化酶，半乳糖氧化酶，和葡萄糖-6-磷酸脱氢酶，杂环氧化酶如尿酸酶和黄嘌呤氧化酶，以及利用过氧化氢氧化染料前体的酶如HR，乳过氧化物酶，或微过氧化物酶(microperoxidase)，生物素/亲和素，自旋标记，噬菌体标记，稳定的自由基，等等。
在本文中提供的任何发明的一些实施方案中，样品是在用Nectin-4抗体治疗之前获得的。在一些实施方案中，样品是在癌症已经转移之后获得的。在一些实施方案中，样品是福尔马林固定、石蜡包埋(FFPE)的样品。在一些实施方案中，样品是活检(例如芯活检)，手术标本(例如来自手术切除的标本)，或细针吸出物。
在一些实施方案中，在治疗之前，例如，在起始治疗之前；或在治疗间隔后的某次治疗之前检测Nectin-4。
在一些实施方案中，本发明提供了诊断受试者的Nectin 4相关疾病，例如，癌症的方法，所述方法包括：
1)将来自所述受试者的生物样品与作为捕获剂的本发明的抗Nectin-4抗体或其抗原结合片段接触，其中所述生物样品是受试者的组织样品、细胞样品或体液样品，例如，肿瘤或健康组织的组织样品、血液、血清、尿液、唾液、组织液样品，例如，肿瘤或健康组织的组织切片(如，石蜡切片或冰冻切片)样品；
2)检测本发明的抗Nectin-4抗体或其抗原结合片段与来自所述受试者的生物样品的结合；和任选地
3)测定来自所述受试者的生物样品中Nectin-4的存在或表达水平，任选地，将来自所述受试者的生物样品中Nectin-4的存在或表达水平与Nectin-4的参考存在或表达水平进行比较；
检测到Nectin-4和/或检测到所述样品中Nectin-4的表达水平显著高于参考水平指示受试者的Nectin-4相关疾病，例如，癌症；
任选地，其中Nectin-4的存在或表达水平使用免疫组织化学(IHC)方法、Western印迹测定、荧光激活的细胞分选(FACS)测定、酶联免疫吸附测定(ELISA)、免疫荧光(IF)测定和/或免疫共沉淀(Co-IP)检测。
在一些实施方案中，本发明提供了确定受试者使用抗Nectin-4抗体或者抗Nectin-4抗体 偶联药物(例如，所述抗Nectin-4抗体是治疗用抗Nectin-4抗体或其抗原结合片段)治疗的资格的方法，所述方法包括：
1)将来自所述受试者的生物样品与作为捕获剂的本发明的抗Nectin-4抗体或其抗原结合片段接触，其中所述生物样品是受试者的组织样品、细胞样品或体液样品，例如，肿瘤或健康组织的组织样品、血液、血清、尿液、唾液、组织液样品，例如，肿瘤或健康组织的组织切片(如，石蜡切片或冰冻切片)样品；
2)检测本发明的抗体或其抗原结合片段与来自所述受试者的生物样品的结合；和任选地
3)测定来自所述受试者的生物样品中Nectin-4的存在或表达水平，任选地，将来自所述受试者的生物样品中Nectin-4的存在或表达水平与Nectin-4的参考存在或表达水平进行比较；
检测到Nectin-4和/或检测到所述样品中Nectin-4的表达水平高于参考水平指示受试者可以使用抗Nectin-4抗体或者抗Nectin-4抗体偶联药物(例如，所述抗Nectin-4抗体是治疗用抗Nectin-4抗体或其抗原结合片段)治疗；
任选地，其中Nectin-4的存在或表达水平使用免疫组织化学(IHC)方法、Western印迹测定、荧光激活的细胞分选(FACS)测定、酶联免疫吸附测定(ELISA)、免疫荧光(IF)测定和/或免疫共沉淀(Co-IP)检测。
在一些实施方案中，本发明提供了在有需要的受试者中治疗癌症的方法，其包括诊断癌症或确定患者用抗Nectin-4抗体治疗的资格的在先步骤，所述在先步骤包括使用如本文公开的抗Nectin-4抗体对来自受试者的生物样品进行免疫组织化学(IHC)测定、ELISA测定、流式细胞术(FACS)测定、Western印迹、免疫荧光(IF)测定和/或免疫共沉淀(Co-IP)，例如，所述在先步骤包括：
1)将所述生物样品与作为捕获剂的本发明的抗Nectin-4抗体或其抗原结合片段接触，其中所述生物样品是受试者的组织样品、细胞样品或体液样品，例如，肿瘤或健康组织的组织样品、血液、血清、尿液、唾液、组织液样品，例如，肿瘤或健康组织的组织切片(如，石蜡切片或冰冻切片)样品；
2)检测所述抗体或其抗原结合片段与所述生物样品的结合；和任选地
3)测定所述生物样品中Nectin-4的表达，其中将所述生物样品中Nectin-4的表达水平与Nectin-4的参考表达水平进行比较；
任选地，其中Nectin-4的表达水平使用免疫组织化学(IHC)方法、Western印迹测定、荧光激活的细胞分选(FACS)测定、酶联免疫吸附测定(ELISA)、免疫荧光(IF)测定和/或免疫共沉淀(Co-IP)检测。
在一些实施方案中，本发明提供了预后患有癌症的受试者的方法。其中所述患有癌症的受试者已使用抗肿瘤剂治疗，所述抗肿瘤剂包括但不限于代谢抑制剂、抗生素抗癌剂、植物生物碱系抗癌剂、拓扑异构酶抑制剂、抗肿瘤烷化剂、单克隆抗体、ADC等，所述方法包括：
1)将来自使用抗肿瘤剂(例如，抗Nectin-4抗体或者抗Nectin-4抗体偶联药物(例如，所述抗Nectin-4抗体是治疗用抗Nectin-4抗体或其抗原结合片段))治疗后的受试者的生物样品与作为捕获剂的本发明的抗Nectin-4抗体或其抗原结合片段接触，其中所述生物样品是受试者的组织样品、细胞样品或体液样品，例如，肿瘤或健康组织的组织样品、血液、血 清、尿液、唾液、组织液样品，例如，肿瘤或健康组织的组织切片(如，石蜡切片或冰冻切片)样品；
2)检测本发明的抗体或其抗原结合片段与来自所述受试者的生物样品的结合；
3)测定来自所述受试者的生物样品中Nectin-4的存在或表达水平，
检测到Nectin-4和/或检测到所述样品中Nectin-4的表达水平高于参考水平指示不良预后；或者，样品中不存在Nectin-4或检测到Nectin-4水平低于参考存在或表达水平表明预后良好，
任选地，其中Nectin-4的存在或表达水平使用免疫组织化学(IHC)方法、Western印迹测定、荧光激活的细胞分选(FACS)测定、酶联免疫吸附测定(ELISA)、免疫荧光(IF)测定和/或免疫共沉淀(Co-IP)检测。
在一些实施方案中，本发明还提供了包含本发明的抗Nectin-4抗体的试剂盒，以及任选地指导使用该试剂盒的包装插页。
在一些实施方案中，提供了一种治疗肿瘤的方法，所述方法包括：对受试者(例如，样品)(例如，包含癌细胞的受试者样品)检验Nectin-4的存在，因而确定Nectin-4值，将Nectin-4值与对照值(例如健康个体的样品中的Nectin-4的值)比较，并且如果Nectin-4值大于对照值，则向受试者施用治疗有效量的任选地与一种或多种其他疗法组合的Nectin-4抗体(例如，治疗用抗Nectin-4抗体)，由此治疗肿瘤。
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成限制本发明的保护范围。
实施例
实施例1.Nectin-4抗体的制备与测序
本发明的Nectin-4抗体可以通过以下记载的方法来制备。但是，本发明的Nectin-4抗体的制备方法并不受本实施例记载的方法限定，也可以通过本领域已知的其他方法制备。
使用人Nectin-4片段，例如，含有人Nectin-4(SEQ ID NO:31)胞外结构域的片段作为免疫用抗原。抗原首次免疫是与弗氏完全佐剂(CFA)一起应用。加强免疫是使用弗氏不完全佐剂(IFA)。皮下施用含抗原的乳剂。
1.1.通过杂交瘤方法制备：
使用重组Nectin-4胞外蛋白(参见UniProt数据库登记号Q96NY8，第32-349位(SEQ ID NO:32)，使用哺乳动物细胞表达系统制备)免疫Balb/c小鼠，当经免疫小鼠血清具有较高的抗Nectin-4抗体滴度后，无菌取小鼠脾脏，制备脾细胞悬液，并与SP2/0骨髓瘤细胞融合，融合后的细胞用HAT培养基重悬后，分装至96孔细胞培养板。置37℃，5％ CO2培养箱内培养，以形成杂交瘤细胞系。筛选获得免疫组织化学测试最优的Nectin-4鼠单克隆抗体，命名为MM11。
抗体MM11的序列信息如下，重链恒定区为小鼠IgG1型抗体序列，重链可变区和轻链可变区以斜体和下划线示出，根据KABAT命名系统定义的互补决定区(CDR)序列进一步以粗体示出。
重链氨基酸序列

轻链氨基酸序列
1.2.通过噬菌体展示技术制备：
使用重组Nectin-4胞外蛋白(参见UniProt数据库登记号Q96NY8，第32-349位)免疫日本大耳白兔。自经免疫兔的血液获得单核的细胞，随后，从单核的细胞提取总RNA，产生Nectin-4兔抗体的cDNA基因文库。从Nectin-4兔抗体的cDNA基因文库形成噬菌体文库，实施生物淘洗，筛选到免疫组织化学测试良好的Nectin-4兔单克隆抗体，分别命名为R012和R036。
抗体R012和抗体R036的序列信息如下，重链恒定区为兔IgG抗体序列，重链可变区和轻链可变区以斜体和下划线示出，根据KABAT命名系统定义的互补决定区(CDR)序列进一步以粗体示出：
抗体R012
重链氨基酸序列
轻链氨基酸序列
抗体R036
重链氨基酸序列

轻链氨基酸序列
实施例2.ELISA法检测抗体与Nectin-4及其同家族蛋白的结合力
为了验证实施例1获得的Nectin-4抗体的抗原结合特异性，采用酶联免疫吸附测试法(ELISA)检测了所述抗体与其他Nectin家族蛋白之间是否存在交叉反应。
Nectin家族蛋白包括如下9种蛋白：Nectin-1、Nectin-2、Nectin-3、Nectin-4、Nectin-like-1、Nectin-like-2、Nectin-like-3、Nectin-like-4、Nectin-like-5。具体信息如下：
如下所述实施ELISA测试法：
包被：将Nectin-1、Nectin-2、Nectin-3、Nectin-4、Nectin-like-1、Nectin-like-2、Nectin-like-3、Nectin-like-4、Nectin-like-5用包被缓冲液(pH 9.6的碳酸盐缓冲液)分别稀释至5μg/ml，100μl/孔包被96孔板，2-8℃过夜；
封闭：取出板拍干，用PBST以200μl/孔洗板2次，拍干。加入3％ BSA/PBST，200μl/孔，室温孵育1.5h实施封闭；
加样：取出板拍干，用PBST以200μl/孔洗板2次，拍干。将实施例1制备的Nectin-4抗体(抗体MM11、抗体R012、抗体R036)用1％ BSA/PBST分别稀释至10μg/ml、1μg/ml、100ng/ml和10ng/ml，以1％ BSA/PBST稀释液作为空白对照，以100μl/孔加入96孔板，室 温孵育2h；
加入第二抗体：取出板拍干，用PBST以200μl/孔洗板6次，拍干。按照1:20000比例稀释山羊抗小鼠IgG-HRP(Jackson，目录号：115-035-003)或按照1:10000比例稀释山羊抗兔IgG(H+L)抗体-HRP(ThermoFisher，目录号：31460)，100μl/孔，室温孵育1h；
显色：取出板拍干，用PBST以200μl/孔洗板6次，拍干。以100μl/孔加入TMB底物，室温孵育约5min；
终止反应和读板：以100μl/孔加入1mol/L H3PO4终止反应并读板。以650nm为参比波长，读取并记录波长450nm处孔板的吸光度OD450 nm-OD650 nm。
ELISA测试结果：
ELISA测试结果如表1和图1所示，鼠抗体MM11、兔抗体R012和兔抗体R036与重组人Nectin-4蛋白能够特异性结合，与其他同家族蛋白没有交叉反应。
表1.抗体与Nectin-4蛋白、Nectin同家族蛋白的交叉反应结果


实施例3.流式细胞术检测抗体与Nectin-4表达细胞结合的分析
为了验证实施例1获得的Nectin-4抗体与Nectin-4表达细胞的结合特异性，采用流式细胞术(FACS)检测了所述抗体与Nectin-4表达细胞的结合。
如下所述实施流式细胞术测试法：
细胞处理：
人前列腺癌细胞(PC-3)购自ATCC，其不表达Nectin-4。使用人前列腺癌细胞(PC-3)作为阴性对照。
PC-3-Nectin-4细胞为迈威康公司构建的基因工程改造细胞株，为表达Nectin-4的PC-3细胞株。具体而言，将人Nectin-4全长基因(NCBI基因ID:81607,1533bp)敲入PC-3细胞并经单克隆细胞筛选得到稳定转染的高表达Nectin-4的细胞株，经流式细胞术检测确认细胞表面高表达Nectin-4，并将该细胞株命名为PC-3-Nectin-4细胞。
取生长至对数期的PC-3-Nectin 4细胞和作为阴性对照的PC-3细胞，分别经胰酶消化后，200g离心5min收集细胞，配制成约5×105个/ml的细胞密度，分别吸取1ml细胞悬液至流式管中，即5×105个细胞/流式管，然后4℃，350g离心5min，弃上清，并用预冷的PBS清洗两遍，待用。
抗体样品稀释与孵育：
将抗体MM11、抗体R012和抗体R036用PBS稀释至5μg/ml，分别取200μl抗体稀释液重悬上述各细胞，轻轻混匀，冰上避光孵育，孵育时间为1h。
加入荧光第二抗体：
将流式管于4℃，350g离心5min，弃上清，每管加入1ml预冷PBS洗涤细胞2遍，然后加入500μl第二抗体，对于抗体MM11，第二抗体为山羊抗小鼠IgG Fc-FITC(1:200)(Abcam，货号ab150113)；对于抗体R012和抗体R036，第二抗体为山羊抗兔IgG H&L-Alexa488(1:2000)(Abcam，货号ab150077)，重悬细胞，冰上孵育60min。于4℃，350g离心5min，弃上清，然后每管加入1ml预冷PBS洗涤细胞2遍，重悬于500μl PBS中。
检测：
使用流式细胞仪检测各流式管，根据仪器相关操作进行，结果导入相应保存路径中。
FACS检测结果：
FACS检测结果如图2所示，使用抗体MM11、抗体R012和抗体R036检测PC-3-Nectin4细胞均产生较高的荧光信号，而检测PC-3细胞无明显的荧光信号。结果表明Nectin-4抗体MM11、R012和R036均能够特异性识别并结合PC-3-Nectin 4细胞上表达的Nectin-4，且不识别细胞上的其他蛋白。
实施例4.Western印迹法检测抗体与Nectin-4表达细胞裂解物的反应性
使用Western印迹法检测实施例1获得的Nectin-4抗体是否特异性识别Nectin-4蛋白。此外，在Western印迹法检测时，通过添加过量的Nectin-4蛋白进行抑制实验，进一步确认实施例1获得的Nectin-4抗体的特异性。
如下所述实施Western印迹法测试法：
人前列腺癌细胞(PC-3)购自ATCC，经流式细胞术检测确认PC-3细胞不表达Nectin-4。
PC-3-Nectin-4细胞为迈威康公司构建的基因工程改造细胞株，为表达Nectin-4的PC-3细胞株。具体而言，将人Nectin-4全长基因(NCBI基因ID:81607,1533bp)敲入PC-3细胞并经单克隆细胞筛选得到稳定转染的高表达Nectin-4的细胞株，经流式细胞术检测确认细胞表面高表达Nectin-4，并将该细胞株命名为PC-3-Nectin-4细胞。
蛋白提取：复苏PC-3-Nectin 4细胞和PC-3细胞，在细胞培养箱中分别于细胞培养瓶中生长至合适的细胞密度，弃去培养基，加入提前预冷的PBS清洗两遍。随后加入含蛋白酶抑制剂的RIPA裂解液(Radioimmunoprecipitation assay buffer，放射免疫沉淀法缓冲液)(Thermo，货号89901)裂解细胞，用细胞刮刮下细胞，冰上裂解30min后，12000rpm，4℃，离心10min。取离心后的上清至新的离心管中，BCA法测定蛋白含量并调整至合适的浓度。加入5×上样缓冲液混合均匀后，97℃金属浴加热10min使蛋白完全变性；使用甘油醛-3-磷酸脱氢酶(GAPDH(glyceraldehyde-3-phosphate dehydrogenase))作为Western印迹法的内部参照；
电泳：两株细胞裂解物上样量为4.6μg，电泳条件：120V，80min；
转膜：采用湿法转膜，湿法转膜条件：0.35A，90min；
封闭：使用5％ BSA/TBST室温振荡孵育膜1h；
加入第一抗体：
(1)将实施例1制备的Nectin-4抗体(分别为抗体MM11、抗体R012、抗体R036)用5％BSA/TBST稀释至20ng/ml，作为第一抗体添加，2～8℃振荡孵育膜过夜；或者
(2)将实施例1制备的Nectin-4抗体(分别为抗体MM11、抗体R012)用5％BSA/TBST 稀释至20ng/ml，作为第一抗体添加；并加入200μg/ml重组人源Nectin-4(迈威康公司，Lot：20210101)预孵育1h，然后2～8℃振荡孵育膜过夜；
加入第二抗体：复温30min后，TBST洗膜3次，每次5min，将作为第二抗体的山羊抗小鼠IgG-HRP(Jackson，目录号：115-035-003)用5％BSA/TBST按1:50000比例稀释，室温振荡孵育1h；或者将作为第二抗体的山羊抗兔IgG(H+L)抗体-HRP(ThermoFisher，目录号：31460)用5％BSA/TBST按1:25000比例稀释，室温振荡孵育1h；
显色：第二抗体孵育完成后，TBST洗膜3次，每次5min，加入1:1配好的ECL发光液显色，采集发光信号。
Western印迹法测试结果：
Western印迹法结果如图3所示，其中作为内部参照的GAPDH的检测条带大约在36kDa处，保证了实验结果的准确性和可靠性。在Nectin-4阳性表达的细胞中可以发现与抗体MM11、抗体R012、抗体R036结合的特异性条带，而在Nectin-4阴性表达的细胞中没有特异性条带。
Nectin-4阳性表达的细胞中与抗体MM11、抗体R012结合的特异性条带在加入过量的重组Nectin-4蛋白后消失，说明抗Nectin-4抗体能够特异性与人Nectin-4蛋白结合。
实施例5.Nectin-4兔单克隆抗体的免疫组织化学染色
选择多个肿瘤组织和正常人体组织样本，经中性福尔马林固定后，制备成石蜡组织切片。其中正常人体组织包括多个来源的组织器官；肿瘤组织包括：3例膀胱癌，2例肺癌和3例乳腺癌组织。
使用抗Nectin-4单克隆抗体检测组织样本中Nectin-4的表达情况，以商品化Nectin-4兔单抗(购自abcam，克隆号：EPR15613-68)为阳性对照。所有抗体使用抗体稀释液稀释到工作浓度后使用，抗体稀释液购自迈杰转化医学研究(苏州)有限公司，货号P010A01。在全自动免疫组织化学染色系统(Leica BOND III)检测免疫组织化学染色情况，具体程序如下表所示。
表2.Nectin-4兔单克隆抗体的免疫组织化学染色程序

注：过氧化物酶灭活试剂、山羊抗兔IgG-polymer HRP、DAB显色试剂、苏木素染色液均包含在Leica的BOND Polymer Refine Detection试剂盒中，货号DS9800，所有试剂均已稀释至工作浓度，直接使用即可。
免疫组织化学染色结果如表3、图4、图5、图6和图7所示，在不同Nectin-4表达水平的正常组织和肿瘤组织上，EPR15613-68、R012和R036这三个抗体表现基本一致，靶蛋白Nectin-4定位于细胞膜和/或细胞浆上，符合文献报告的蛋白定位，而且不产生非特异性染色。正常组织中胎盘的滋养层细胞、皮肤的上皮和扁桃体的隐窝上皮呈明显的Nectin-4染色阳性，其余正常组织器官基本呈阴性。大部分肿瘤组织中肿瘤细胞呈Nectin-4阳性，小部分呈Nectin-4阴性，而肿瘤组织中的正常细胞呈Nectin-4阴性。
表3.Nectin-4兔单克隆抗体在正常人体组织上的免疫组织化学染色结果
实施例6.Nectin-4鼠单克隆抗体的免疫组织化学染色
选择多个肿瘤组织和正常人体组织样本，经中性福尔马林固定后，制备成石蜡组织切片。其中正常人体组织包括多个来源的组织器官；肿瘤组织包括：3例前列腺癌，3例肺癌和3例三阴性乳腺癌组织。使用抗Nectin-4单克隆抗体检测组织样本中Nectin-4的表达情况，以鼠单抗M22-321b41.1为阳性对照，M22-321b41.1是已申请专利的Nectin-4鼠单克隆抗体，专利公开号为CN111051345A，所有抗体使用抗体稀释液稀释到工作浓度后使用，抗体稀释液购自迈杰转化医学研究(苏州)有限公司，货号P010A01。在全自动免疫组织化学染色系统(Leica BOND III)检测免疫组织化学染色情况，具体程序如下表所示。
表4.Nectin-4鼠单克隆抗体的免疫组织化学染色程序
注：过氧化物酶灭活试剂、兔抗鼠Post-primary、山羊抗兔IgG-polymer HRP、DAB显色试剂、苏木素染色液均包含在Leica的BOND Polymer Refine Detection试剂盒中，货号DS9800，所有试剂均已稀释至工作浓度，直接使用即可。
免疫组织化学染色结果如表5、图8、图9、图10和图11所示，在正常组织上，抗体M22-321b41.1和抗体MM11表现基本一致，正常组织中部分个体的唾液腺和皮肤上皮呈微弱的Nectin-4染色阳性，扁桃体隐窝上皮和肺部的支气管呈明显的Nectin-4阳性，其余正常组织器官基本为阴性。大部分肿瘤组织中肿瘤细胞呈Nectin-4阳性，而肿瘤组织中的正常细胞呈Nectin-4阴性。在不同Nectin-4表达水平的肿瘤组织上，抗体MM11和抗体M22-321b41.1染色后，靶蛋白Nectin-4均定位于细胞膜和/或细胞浆上，符合文献报告的蛋白定位，而且不产生非特异性染色。在部分肿瘤组织中，尤其是三阴性乳腺癌上，抗体MM11的染色信号明显强于抗体M22-321b41.1。
表5.Nectin-4鼠单克隆抗体在正常人体组织上的免疫组织化学染色结果

以上描述了本发明的示例性实施方案，本领域技术人员应当理解的是，这些公开内容仅是示例性的，在本发明的范围内可以进行各种其它替换、适应和修改。因此，本发明不限于文中列举的具体实施方案。
示例性序列



Claims (14)

  1. 抗Nectin-4抗体或其抗原结合片段，包含重链可变区和轻链可变区，其中：
    (a)所述重链可变区包含SEQ ID NO:5所示的HCDR1；SEQ ID NO:6所示的HCDR2；和SEQ ID NO:7所示的HCDR3；所述轻链可变区包含SEQ ID NO:8所示的LCDR1；SEQ ID NO:9所示的LCDR2；和SEQ ID NO:10所示的LCDR3；
    (b)所述重链可变区包含SEQ ID NO:15所示的HCDR1；SEQ ID NO:16所示的HCDR2；和SEQ ID NO:17所示的HCDR3；所述轻链可变区包含SEQ ID NO:18所示的LCDR1；SEQ ID NO:19所示的LCDR2；和SEQ ID NO:20所示的LCDR3；或
    (c)所述重链可变区包含SEQ ID NO:25所示的HCDR1；SEQ ID NO:26所示的HCDR2；和SEQ ID NO:27所示的HCDR3；所述轻链可变区包含SEQ ID NO:28所示的LCDR1；SEQ ID NO:29所示的LCDR2；和SEQ ID NO:30所示的LCDR3。
  2. 根据权利要求1所述的抗Nectin-4抗体或其抗原结合片段，包含重链可变区和轻链可变区，其中
    (a)重链可变区包含SEQ ID NO:3的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:4的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；
    (b)重链可变区包含SEQ ID NO:13的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:14的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；或
    (c)重链可变区包含SEQ ID NO:23的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:24的序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的序列；
    例如，所述抗Nectin-4抗体或其抗原结合片段包含
    (a)重链可变区包含SEQ ID NO:3的序列，且轻链可变区包含SEQ ID NO:4的序列；
    (b)重链可变区包含SEQ ID NO:13的序列，且轻链可变区包含SEQ ID NO:14的序列；或
    (c)重链可变区包含SEQ ID NO:23的序列，且轻链可变区包含SEQ ID NO:24的序列；
    例如，所述抗Nectin-4抗体或其抗原结合片段包含
    (a)SEQ ID NO:1或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的重链序列，以及SEQ ID NO:2或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的轻链序列；例如，SEQ ID NO:1所示的重链序列，以及SEQ ID NO:2所示的轻链序列；
    (b)SEQ ID NO:11或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的重链序列，以及SEQ ID NO:12或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的轻链序列；例如，SEQ ID NO:11所示的重链序列，以及SEQ ID NO:12所示的轻链序列；或
    (c)SEQ ID NO:21或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的重链序列，以及SEQ ID NO:22或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的轻链序列；例如，SEQ ID NO:21所示的重链序列，以及SEQ ID NO:22所示的轻链序列。
  3. 根据权利要求1或2所述的抗Nectin-4抗体或其抗原结合片段，其中所述抗原结合片段是Fab、Fab’、F(ab’)2、Fv、单链Fv、单链Fab或双体抗体(diabody)。
  4. 根据权利要求1至3中任一项所述的抗Nectin-4抗体或其抗原结合片段，具有以下一个或多个特性：
    (a)在ELISA测定法中测量，具有仅与Nectin-4特异性结合的能力，与Nectin家族成员Nectin-1、Nectin-2、Nectin-3、Nectin-like-1、Nectin-like-2、Nectin-like-3、Nectin-like-4、Nectin-like-5均无特异性结合；
    (b)在流式细胞术测定法中测量，具有结合细胞上表达的Nectin-4分子的能力；
    (c)在Western印迹法中测量，能够特异性检测Nectin-4蛋白；并且在添加过量的Nectin-4蛋白时，与膜上Nectin-4蛋白的结合被抑制；和/或
    (d)在免疫组织化学染色中，特异性染色表达Nectin-4分子的组织，例如，所述组织选自扁桃体、唾液腺、大脑、小脑、胎盘、膀胱、肺、乳腺、食道、喉、胸腺、心脏、胃、小肠、结肠、直肠、输尿管、卵巢、输卵管、子宫颈、子宫内膜、皮肤、肾脏、前列腺、胰腺、甲状腺、脾脏和肝脏。
  5. 分离的核酸，其编码权利要求1至4中任一项所述的抗Nectin-4抗体或其抗原结合片段。
  6. 包含权利要求5所述的核酸的载体，优选地所述载体是表达载体。
  7. 包含权利要求6所述的核酸或权利要求7所述的载体的宿主细胞，优选地，所述宿主细胞是原核的或真核的，更优选的选自大肠杆菌细胞、酵母细胞、哺乳动物细胞或适用于制备抗体或其抗原结合片段的其它细胞，最优选地，所述宿主细胞是293细胞或CHO细胞。
  8. 制备权利要求1至4中任一项所述的抗Nectin-4抗体或其抗原结合片段的方法，所述方法包括在适于表达编码权利要求1至4中任一项所述的抗Nectin-4抗体或其抗原结合片段的核酸的条件下培养权利要求7的宿主细胞，以及从培养基回收表达的所述抗Nectin-4抗体或其抗原结合片段。
  9. 试剂盒，其包含权利要求1至4中任一项所述的抗Nectin-4抗体或其抗原结合片段。
  10. 根据权利要求1至4中任一项所述的抗Nectin-4抗体或其抗原结合片段的用途，用 于制备检测样品中Nectin-4的试剂盒。
  11. 一种用于检测来自受试者的生物样品中的Nectin-4的方法，所述方法包括：
    1)将所述生物样品与捕获剂接触，其中，所述捕获剂为权利要求1至4中任一项所述的抗Nectin-4抗体或其抗原结合片段；
    2)检测所述捕获剂与所述生物样品的结合；和任选地
    3)测定来自所述受试者的生物样品中Nectin-4的存在或表达水平，任选地，将来自所述受试者的生物样品中Nectin-4的存在或表达水平与Nectin-4的参考存在或表达水平进行比较；
    任选地，其中Nectin-4的存在或表达水平使用免疫组织化学(IHC)方法、Western印迹测定、荧光激活的细胞分选(FACS)测定、酶联免疫吸附测定(ELISA)、免疫荧光(IF)测定和/或免疫共沉淀(Co-IP)检测。
  12. 用于确定受试者使用抗Nectin-4抗体或者抗Nectin-4抗体偶联药物治疗的资格的方法，所述方法包括：
    1)将来自所述受试者的生物样品与捕获剂接触，其中，所述捕获剂为权利要求1至4中任一项所述的抗Nectin-4抗体或其抗原结合片段；
    2)检测所述捕获剂与来自所述受试者的生物样品的结合；和任选地
    3)测定来自所述受试者的生物样品中Nectin-4的存在或表达水平，任选地，将来自所述受试者的生物样品中Nectin-4的存在或表达水平与Nectin-4的参考存在或表达水平进行比较；
    任选地，其中Nectin-4的存在或表达水平使用免疫组织化学(IHC)方法、Western印迹测定、荧光激活的细胞分选(FACS)测定、酶联免疫吸附测定(ELISA)、免疫荧光(IF)测定和/或免疫共沉淀(Co-IP)检测。
  13. 用于预后患有癌症的受试者的方法，所述方法包括：
    1)将来自使用抗肿瘤剂(例如，抗Nectin-4抗体或者抗Nectin-4抗体偶联药物)治疗后的受试者的生物样品与捕获剂接触，其中，所述捕获剂为权利要求1至4中任一项所述的抗Nectin-4抗体或其抗原结合片段；
    2)检测所述捕获剂与来自所述受试者的生物样品的结合；和任选地
    3)测定来自所述受试者的生物样品中Nectin-4的存在或表达水平，
    检测到Nectin-4和/或检测到所述样品中Nectin-4的表达水平高于参考水平指示不良预后；或者，样品中不存在Nectin-4或检测到Nectin-4水平低于参考存在或表达水平表明预后良好，
    任选地，其中Nectin-4的存在或表达水平使用免疫组织化学(IHC)方法、Western印迹测定、荧光激活的细胞分选(FACS)测定、酶联免疫吸附测定(ELISA)、免疫荧光(IF)测定和/或免疫共沉淀(Co-IP)检测。
  14. 根据权利要求11－13中任一项所述的方法，其中所述生物样品是受试者的组织样品、细胞样品或体液样品，例如，肿瘤或健康组织的组织样品、血液、血清、尿液、唾液、组织液样品，例如，肿瘤或健康组织的组织切片(如，石蜡切片或冰冻切片)样品。
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