WO2024165049A1 - 抗cdh6的抗体及其用途 - Google Patents

抗cdh6的抗体及其用途 Download PDF

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Publication number
WO2024165049A1
WO2024165049A1 PCT/CN2024/076687 CN2024076687W WO2024165049A1 WO 2024165049 A1 WO2024165049 A1 WO 2024165049A1 CN 2024076687 W CN2024076687 W CN 2024076687W WO 2024165049 A1 WO2024165049 A1 WO 2024165049A1
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seq
antibody
antigen
sequence
binding fragment
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English (en)
French (fr)
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柴晓鹃
付雅媛
唐任宏
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Shandong Simcere Biopharmaceutical Co Ltd
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Shandong Simcere Biopharmaceutical Co Ltd
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Priority to AU2024218944A priority Critical patent/AU2024218944A1/en
Priority to CN202480011097.8A priority patent/CN120659810A/zh
Priority to KR1020257029935A priority patent/KR20250143344A/ko
Priority to JP2025546299A priority patent/JP2026506623A/ja
Priority to EP24752903.5A priority patent/EP4663661A1/en
Publication of WO2024165049A1 publication Critical patent/WO2024165049A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K16/2896—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the fields of bioengineering and biomedicine, and mainly to an anti-CDH6 antibody or an antigen-binding fragment thereof, its encoding nucleic acid, expression vector and expression cell, preparation method, pharmaceutical composition, and their use in treating diseases, such as treating tumors.
  • Cadherins play an important role in tissue homeostasis and are primarily responsible for cell-cell adhesion during embryogenesis, tissue morphogenesis, differentiation, and tumorigenesis. Any dysfunction or instability of the cadherin-catenin complex may lead to tumor progression.
  • CDH6 is a type II classical cadherin, also known as K-cadherin.
  • CDH6 is a single transmembrane protein consisting of 790 amino acids, and its extracellular domain is divided into five regions. Studies have found that CDH6 is highly expressed in tumor tissues such as renal cancer, ovarian cancer, and thyroid cancer, but is rarely expressed in normal tissues (Cancer Discov; 2017, 7(9): 1030-45; the contents of which are incorporated herein by reference). Like other members of the cadherin superfamily, CDH6 protein is localized to the basolateral membrane of epithelial cells and mediates calcium-dependent cell-cell adhesion, with the characteristic of rapid internalization. Therefore, as a highly recognizable tumor-specific marker, CDH6 has become an attractive target in tumor therapy and can be developed for the treatment of cancers such as ovarian cancer and renal cancer.
  • the present invention provides antibodies or antigen-binding fragments that specifically bind to CDH6, nucleic acids encoding these antibodies and antigen-binding fragments thereof, pharmaceutical compositions and kits containing the antibodies and antigen-binding fragments thereof, and the preparation of drugs that can be used to treat tumors and the like.
  • the present invention provides an antibody or antigen-binding fragment that specifically binds to CDH6, wherein the antibody or antigen-binding fragment specifically binds to one or more amino acid sequence fragments in the EC1 region, EC1-EC2 region, EC2 region, EC2-EC3 region, EC3 region, EC4 region or EC5 region of the extracellular region of the CDH6 protein; preferably, the antibody or antigen-binding fragment specifically binds to one or more amino acid sequence fragments shown in any one of SEQ ID NOs: 168-172; preferably, the antibody or antigen-binding fragment has endocytosis activity.
  • the present invention provides an antibody or antigen-binding fragment that specifically binds to CDH6, wherein the antibody or antigen-binding fragment comprises:
  • the HCDR1, HCDR2 and HCDR3 have any sequence combination selected from the following or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the sequence combination:
  • the LCDR1, LCDR2 and LCDR3 have a sequence combination selected from any of the following sequence combinations or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the sequence combination:
  • the substitution is a conservative amino acid substitution.
  • the antibody or antigen-binding fragment comprises a heavy chain CDRs and light chain CDRs combination selected from the following: VH1+VL1, VH2+VL2, VH3+VL3, VH4+VL4, VH5+VL5, VH6+VL6, VH7+VL7, VH8+VL8, VH9+VL9, VH10+VL10, VH11+VL11, VH12+VL12, VH13+VL13, or VH14+VL14, as well as a CDRs combination having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the sequence of the heavy chain and light chain CDRs combination;
  • the substitution is a conservative amino acid substitution.
  • the framework regions of the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment are derived from a human germline heavy chain template and a human germline light chain template, wherein:
  • the framework region sequence is derived from the combined sequence of human germline heavy chains IGHV1-69*02 and IGHJ6*01; it comprises the FR1, FR2, and FR3 regions of IGHV1-69*02 as shown in SEQ ID NO: 143 and the FR4 region of IGHJ6*01 as shown in SEQ ID NO: 144;
  • the framework region sequence is derived from the combined sequence of human germline heavy chains IGHV1-46*01 and IGHJ6*01; it comprises the FR1, FR2, and FR3 regions of IGHV1-46*01 as shown in SEQ ID NO: 156 and the FR4 region of IGHJ6*01 as shown in SEQ ID NO: 144;
  • the framework region sequence is derived from the combined sequence of human germline light chain IGKV4-1*01 and IGKJ4*01; it includes the FR1, FR2, and FR3 regions of IGKV4-1*01 as shown in SEQ ID NO: 140 and the FR4 region of IGKJ4*01 as shown in SEQ ID NO: 142;
  • the framework sequence is derived from the combined sequence of human germline light chains IGKV1-NL1*01 and IGKJ2*01; it comprises the FR1, FR2, and FR3 regions of IGKV1-NL1*01 as shown in SEQ ID NO: 153 and the FR4 region of IGKJ2*01 as shown in SEQ ID NO: 155; or,
  • the framework region sequence is derived from the combined sequence of human germline light chains IGKV2-28*01 and IGKJ2*01; it includes the FR1, FR2, and FR3 regions of IGKV2-28*01 shown in SEQ ID NO: 154 and the FR4 region of IGKJ2*01 shown in SEQ ID NO: 155.
  • the framework region of the antibody or antigen-binding fragment further comprises one or more mutations selected from the following group according to the Kabat numbering system, wherein:
  • the framework region of the heavy chain variable region comprises: G27Y, S30T, A40R, I70L or A72V; preferably comprises G27Y and A72V; or preferably comprises G27Y, S30T and A72V; or preferably comprises G27Y, S30T, I70L and A72V; or preferably comprises G27Y, S30T, A40R and A72V;
  • the framework region of the heavy chain variable region comprises: G42R, M70L, R72V or T74K; preferably comprises R72V and T74K; or preferably comprises M70L, R72V and T74K; or preferably comprises G42R, R72V and T74K;
  • the framework region of the heavy chain variable region includes: V2I, V5Q, R44G, I70L, R72V, Y95L or R98S; preferably includes R72V and R98S; or preferably includes V2I, R72V and R98S; or preferably includes V2I, I70L, R72V and R98S; or preferably includes R44G, R72V and R98S; or preferably includes V5Q, R72V and R98S; or preferably includes R72V, Y95L and R98S;
  • the framework region of the light chain variable region includes: P44S, A47P or G72E; preferably includes G72E; or preferably includes P44S and G72E; or preferably includes A47P and G72E;
  • the framework region of the light chain variable region includes: Q42K, A43S, K45Q, L48V, I48V or T85R; preferably includes A43S; or preferably includes A43S and L48V; or preferably includes A43S, K45Q and L48V; or preferably includes A43S, L48V and T85R; or preferably includes Q42K and I48V; or,
  • the framework region of the light chain variable region includes: K42G, P43T, P44F or Y49S; preferably includes P44F and Y49S; or preferably includes K42G and Y49S; or preferably includes P43T and Y49S.
  • the antibody or antigen-binding fragment comprises:
  • the heavy chain variable region has a sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 136, 137, 138, 139, 150, 151, 152, 160, 161, 162, 163, 164 or 165;
  • the light chain variable region has a sequence as shown in SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 133, 134, 135, 145, 146, 147, 148, 149, 157, 158 or 159;
  • the heavy chain variable region and the light chain variable region are selected from the following groups:
  • VH and VL combination having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity compared to any of the sequence combinations of (1) to (59) above.
  • the dissociation constant (KD) of the antibody or antigen-binding fragment binding to human CDH6 protein is no greater than 2 ⁇ 10 -7 M.
  • the antibody or antigen-binding fragment is:
  • the antibody or antigen-binding fragment is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific antibody (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, a full-length antibody, an antibody fragment, a naked antibody, a conjugated antibody, a humanized antibody, a fully human antibody, Fab, Fab', F(ab')2, Fd, Fv, scFv, a diabody or a single domain antibody.
  • the antibody comprises the sequence of the constant region of any one of human or mouse antibodies IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; preferably, it comprises the sequence of the constant region of human or mouse antibodies IgG1, IgG2, IgG3 or IgG4.
  • the antigen-binding fragment is selected from one or more of F(ab)2, Fab', Fab, Fv, scFv, bispecific antibodies, nanobodies and antibody minimum recognition units.
  • the antibody or antigen-binding fragment of the present invention is further coupled with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from a radioactive isotope, a chemotherapeutic drug or an immunomodulator, and the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent marker, a chemiluminescent marker, an ultrasound contrast agent or a photosensitizer.
  • the therapeutic agent is selected from a radioactive isotope, a chemotherapeutic drug or an immunomodulator
  • the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent marker, a chemiluminescent marker, an ultrasound contrast agent or a photosensitizer.
  • the present invention further provides a multispecific antigen-binding molecule, comprising a first antigen-binding moiety and a second antigen-binding moiety, wherein the first antigen-binding moiety is the antibody or antigen-binding fragment according to any one of the first or second aspects, and the second antigen-binding moiety specifically binds to an antigen other than CDH6 or binds to a CDH6 antigen epitope different from that of the first antigen-binding moiety;
  • the other antigens are selected from CD3, CD7, CD16, CD16A, CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD66 (a-d), CD74, CD80, CD126, CD138, BCMA, HLA-DR, HER2, VEGF, P1GF, HER3/ERBB3, HER4/ERBB4, IL-2, IL-6, PD-1, PD-L1, TRAIL-R1 or TRAIL-R2;
  • the multispecific antigen-binding molecule is a bispecific antibody, a trispecific antibody or a tetraspecific antibody.
  • the present invention also provides a chimeric antigen receptor (CAR), which comprises at least an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, and the extracellular antigen binding domain comprises the CDH6 antibody or antigen binding fragment described in the first or second aspect above.
  • CAR chimeric antigen receptor
  • the present invention further provides an immune effector cell, wherein the immune effector cell comprises the chimeric antigen receptor described in the fifth aspect or comprises a nucleic acid fragment encoding the chimeric antigen receptor described in the fifth aspect;
  • the immune effector cells are selected from T cells, NK cells (natural killer cells), NKT cells (natural killer T cells), monocytes, macrophages, dendritic cells or mast cells;
  • the T cells can be selected from inflammatory T cells, cytotoxic T cells, regulatory T cells (Treg) or helper T cells;
  • the immune effector cells are allogeneic immune effector cells or autologous immune cells.
  • the present invention also provides an isolated nucleic acid molecule, which encodes the antibody, antigen-binding fragment, or any combination described in the first and second aspects, the antibody or antigen-binding fragment coupled with a tracer described in the third aspect, the multispecific antigen-binding molecule of the fourth aspect, or the chimeric antigen receptor of the fifth aspect.
  • the present invention also provides an expression vector comprising the isolated nucleic acid molecule described in the seventh aspect.
  • the present invention provides an isolated host cell of the isolated nucleic acid molecule of the seventh aspect, or the expression vector of the eighth aspect; preferably, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from a mammalian cell, a yeast cell, an insect cell, Escherichia coli and/or Bacillus subtilis; more preferably Preferably, the host cell is selected from Expi293 or CHO cells.
  • the present invention provides a method for preparing the antibody or antigen-binding fragment described in the first or second aspect, the antibody or antigen-binding fragment coupled with a tracer described in the third aspect, or the multispecific antigen-binding molecule described in the fourth aspect, by producing the host cell described in the ninth aspect under appropriate conditions, and isolating the antibody or antigen-binding fragment or the multispecific antigen-binding molecule.
  • the present invention provides a method for preparing the immune effector cells described in the sixth aspect, the method comprising introducing a nucleic acid fragment encoding the CAR described in the fifth aspect into the immune effector cells, and optionally, the method also comprises starting the immune effector cells to express the CAR described in the fifth aspect.
  • the present invention provides a pharmaceutical composition, which comprises the antibody or antigen-binding fragment described in the first or second aspect, the antibody or antigen-binding fragment coupled with a tracer described in the third aspect, the multispecific antigen-binding molecule described in the fourth aspect, the chimeric antigen receptor described in the fifth aspect, the immune effector cell described in the sixth aspect, the isolated nucleic acid molecule described in the seventh aspect, the expression vector described in the eighth aspect, the cell described in the ninth aspect, or a product prepared by the method described in the tenth or eleventh aspect; preferably, the composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant; preferably, the pharmaceutical composition further comprises an additional anti-tumor agent.
  • the present invention provides the use of the antibody or antigen-binding fragment described in the first or second aspect, the antibody or antigen-binding fragment coupled with a tracer described in the third aspect, the multispecific antigen-binding molecule described in the fourth aspect, the chimeric antigen receptor described in the fifth aspect, the immune effector cell described in the sixth aspect, the isolated nucleic acid molecule described in the seventh aspect, the expression vector described in the eighth aspect, the cell described in the ninth aspect, or the product prepared by the method described in the tenth or eleventh aspect, or the pharmaceutical composition described in the twelfth aspect in the preparation of a medicament for preventing and/or treating a tumor disease; preferably, the tumor disease is selected from a solid tumor, preferably, the solid tumor is a solid tumor expressing CDH6 protein, and more preferably, the solid tumor is selected from renal cancer, ovarian cancer, thyroid cancer, bile duct cancer, lung cancer, glioblastoma, mesothelioma, uter
  • the present invention provides a method for preventing and/or treating a tumor disease, comprising administering to a patient in need thereof an effective amount of the antibody or antigen-binding fragment described in the first or second aspect, the antibody or antigen-binding fragment coupled with a tracer described in the third aspect, the multispecific antigen-binding molecule described in the fourth aspect, the chimeric antigen receptor described in the fifth aspect, the immune effector cell described in the sixth aspect, the isolated nucleic acid molecule described in the seventh aspect, the expression vector described in the eighth aspect, the cell described in the ninth aspect, or the product prepared by the method described in the tenth or eleventh aspect, or the pharmaceutical composition described in the twelfth aspect; preferably, the tumor disease is selected from solid tumors, preferably, the solid tumor is a solid tumor expressing CDH6 protein, and more preferably, the solid tumor is selected from renal cancer, ovarian cancer, thyroid cancer, bile duct cancer, lung cancer, glioblastoma, meso
  • the present invention provides the antibody or antigen-binding fragment described in the first or second aspect, the antibody or antigen-binding fragment coupled with a tracer described in the third aspect, the multispecific antigen-binding molecule described in the fourth aspect, the chimeric antigen receptor described in the fifth aspect, the immune effector cell described in the sixth aspect, the isolated nucleic acid molecule described in the seventh aspect, the expression vector described in the eighth aspect, the cell described in the ninth aspect, or the product prepared by the method described in the tenth or eleventh aspect, or the pharmaceutical composition described in the twelfth aspect for use in and/or treatment of tumor diseases; preferably, the tumor disease is selected from solid tumors, preferably, the solid tumor is a solid tumor expressing CDH6 protein, and more preferably, The solid tumor is selected from renal cancer, ovarian cancer, thyroid cancer, bile duct cancer, lung cancer, glioblastoma, mesothelioma, uterine cancer, pancreatic cancer, bile
  • the present invention provides a kit comprising the antibody or antigen-binding fragment described in the first or second aspect above, the antibody or antigen-binding fragment coupled to a tracer described in the third aspect, the multispecific antigen-binding molecule described in the fourth aspect, the chimeric antigen receptor described in the fifth aspect, the immune effector cell described in the sixth aspect, the isolated nucleic acid molecule described in the seventh aspect, the expression vector described in the eighth aspect, the cell described in the ninth aspect, or the product prepared by the method described in the tenth or eleventh aspect, or the pharmaceutical composition described in the twelfth aspect, and instructions for use.
  • the present invention aims to develop a monoclonal antibody that specifically binds to CDH6, which has better binding activity and internalization ability, and can be used for the subsequent development of drugs such as CDH6-ADC, and for the treatment of diseases such as ovarian cancer and renal cancer.
  • antibody refers to an immunoglobulin molecule that specifically binds to or is immunoreactive with a target antigen, including polyclonal, monoclonal, genetically engineered and other modified forms of antibodies (including but not limited to chimeric antibodies, humanized antibodies, fully human antibodies, heterojunction antibodies (e.g., bispecific, trispecific and tetraspecific antibodies, diabodies, triabodies and tetrabodies, antibody conjugates) and antigen-binding fragments of antibodies (including, for example, Fab', F(ab')2, Fab, Fv, rIgG and scFv fragments).
  • mAb monoclonal antibody
  • mAb monoclonal antibody
  • Fab and F(ab')2 fragments which lack the Fc fragment of an intact antibody (cleared more quickly from the animal circulation) and therefore lack Fc-mediated effector functions (see Wahl et al., J. Nucl. Med. 24:316, 1983; the contents of which are incorporated herein by reference).
  • antibodies herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, ostriches, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g. sharks).
  • the term "monospecific” herein refers to having one or more binding sites, wherein each binding site binds to the same epitope of the same antigen.
  • multispecific herein refers to having at least two antigen binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of different antigens.
  • terms such as “bispecific”, “trispecific”, “tetraspecific” and the like refer to the number of different epitopes to which an antibody/antigen binding molecule can bind.
  • Fully-length antibody “intact antibody,” and “intact antibody” are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody.
  • the term "antigen-binding fragment” refers to one or more antibody fragments that retain the ability to specifically bind to a target antigen.
  • the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody.
  • An antibody fragment can be a Fab, F(ab')2, scFv, SMIP, diabody, triabody, affibody, nanobody, aptamer or domain antibody.
  • binding fragments encompassed by the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb comprising the VH and VL domains; (vi) a dAb fragment consisting of the VH domain (Ward et al., Nature 341:544-546, 1989; the contents of which are incorporated herein by reference); (vii) A dAb consisting of a VH or VL domain; (viii) an isolated complementary determining region (CDR); and (ix)
  • the two domains of the Fv fragment, VL and VH are encoded by independent genes, the two domains can be joined by a linker using recombinant methods that enables it to be made into a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called a single-chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; the contents of which are incorporated herein by reference).
  • scFv single-chain Fv
  • Antigen-binding fragments can be produced by recombinant DNA techniques, by enzymatic or chemical cleavage of intact immunoglobulins, or in some embodiments by chemical peptide synthesis procedures known in the art.
  • CDH6 also known as “cadherin 6” refers to a cell adhesion molecule, which is a member of the cadherin family of cell-cell adhesion molecules.
  • CDH6 is a single transmembrane protein composed of 790 amino acids, which is classified as a type II cadherin family, and the protein has an N-terminal extracellular and C-terminal intracellular domain.
  • the CDH6 protein used in the present invention can be directly purified from a CDH6-expressing cell of a human or non-human mammal (e.g., a rat, a mouse, or a monkey), and can then be used, or a cell membrane fraction of the above-mentioned cell can be prepared and can be used as a CDH6 protein.
  • CDH6 can also be obtained by in vitro synthesis or by genetic manipulation to allow host cells to produce CDH6.
  • CDH6 protein can be obtained, specifically, CDH6 cDNA is incorporated into a vector capable of expressing CDH6 cDNA, and then CDH6 is synthesized in a solution containing enzymes, substrates, and energy materials required for transcription and translation, or by transforming host cells of other prokaryotes or eukaryotes, thereby allowing them to express CDH6.
  • Cells expressing CDH6 or cell lines expressing CDH6 based on the above genetic manipulation can also be used to present CDH6 protein.
  • an expression vector into which CDH6 cDNA has been incorporated can be directly administered to an animal to be immunized, and CDH6 can be expressed in the animal thus immunized.
  • the term "bispecific antibody” refers to an antibody, typically a human or humanized antibody, having monoclonal binding specificities for at least two different antigens.
  • one of the binding specificities can be detected for an antigenic epitope of CDH6, and the other can be detected for another antigenic epitope of CDH6 or any other antigen other than CDH6, such as a cell surface protein, a receptor, a receptor subunit, a tissue-specific antigen, a virus-derived protein, a virus-encoded envelope protein, a bacterial-derived protein, or a bacterial surface protein.
  • chimeric antibody refers to an antibody having variable sequences of an immunoglobulin derived from one source organism (e.g., rat or mouse) and constant regions of an immunoglobulin derived from a different organism (e.g., human). Methods for producing chimeric antibodies are known in the art.
  • CDR complementarity determining region
  • FR framework region
  • the amino acid position representing the hypervariable region of an antibody can vary depending on the context and various definitions known in the art.
  • variable domains of the natural heavy and light chains each include four framework regions that are primarily in a sheet configuration, which are connected by three CDRs (CDR1, CDR2, and CDR3), which form a loop connecting the sheet structure, and in some cases form a part of the sheet structure.
  • CDR1, CDR2, and CDR3 CDRs
  • the CDRs in each chain are sequentially connected by the FR region.
  • FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 are closely held together and contribute to the formation of the antigen binding site of the antibody with the CDRs from other antibody chains (see Kabat et al., Sequences of Protein of Immunological Interest, National Institute of Health, Bethesda, Md.
  • CDR1-VH, CDR2-VH and CDR3-VH refer to the first CDR, the second CDR and the third CDR of the heavy chain variable region (VH), respectively, and these three CDRs constitute the CDR combination (VHCDR combination) of the heavy chain (or its variable region);
  • CDR1-VL, CDR2-VL and CDR3-VL refer to the first CDR, the second CDR and the third CDR of the light chain variable region (VL), respectively, and these three CDRs constitute the CDR combination (VLCDR combination) of the light chain (or its variable region).
  • the term "monoclonal antibody” refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone), without limitation to the method by which the antibody is produced.
  • VH refers to the variable region of an immunoglobulin heavy chain (including the heavy chain of Fv, scFv or Fab) of an antibody.
  • VL refers to the variable region of an immunoglobulin light chain (including the light chain of Fv, scFv, dsFv or Fab).
  • heavy chain constant region herein refers to the carboxyl terminal portion of the heavy chain of an antibody, which is not directly involved in the binding of the antibody to the antigen, but exhibits effector functions, such as interactions with Fc receptors, which have a more conservative amino acid sequence relative to the variable domains of the antibody.
  • the "heavy chain constant region” comprises at least one of the following: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or a variant or fragment thereof.
  • the “heavy chain constant region” includes a “full-length heavy chain constant region” and a “heavy chain constant region fragment”, the former having a structure substantially similar to that of a natural antibody constant region, while the latter only includes “a portion of a full-length heavy chain constant region”.
  • a typical "full-length antibody heavy chain constant region” consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include a CH1 domain.
  • a typical "heavy chain constant region fragment” can be selected from a CH1, Fc or CH3 domain.
  • light chain constant region refers to the carboxyl terminal portion of the antibody light chain, which is not directly involved in the binding of the antibody to an antigen, and the light chain constant region can be selected from a constant kappa domain or a constant lambda domain.
  • Fc refers to the antibody carboxyl terminal portion formed by papain hydrolysis of intact antibodies, typically comprising the CH3 and CH2 domains of antibodies.
  • the Fc region includes, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions.
  • the boundaries of the Fc region of immunoglobulin heavy chains can vary slightly, the Fc region of human IgG heavy chains is generally defined as extending from the amino acid residue at Cys226 position or from Pro230 to its carboxyl terminal.
  • the C-terminal lysine in the Fc region (residue 447 according to the EU numbering system) can be, for example, removed during the production or purification of antibodies, or by recombinant engineering of nucleic acids encoding antibody heavy chains, and therefore, the Fc region may or may not include Lys447.
  • humanized antibody refers to a non-human antibody that has been genetically engineered, and its amino acid sequence has been modified to improve the homology with the sequence of a human antibody.
  • CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and/or constant region) comes from a human immunoglobulin (recipient antibody).
  • donor antibody non-human antibody
  • variable region FR and/or constant region comes from a human immunoglobulin (recipient antibody).
  • humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc.
  • Fully human antibody refers to an antibody having a variable region in which both FR and CDR are derived from human germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, "fully human antibodies” herein are not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been transplanted to human framework sequences.
  • another mammalian species e.g., mouse
  • naked antibody herein refers to an antibody that is not connected, fused or conjugated to another agent or molecule (e.g., a label or drug), a peptide or a polypeptide.
  • a naked antibody expressed by a mammalian host cell can be glycosylated by the glycosylation machinery (e.g., glycosylase) of the host cell.
  • the naked antibody when expressed by a host cell that does not have its own glycosylation machinery (e.g., glycosylase), the naked antibody is not glycosylated.
  • the naked antibody is a complete antibody, while in other embodiments, the naked antibody is an antigen-binding fragment of a complete antibody, such as a Fab antibody.
  • conjugated antibody refers to an antibody that can be associated with a pharmaceutically acceptable carrier or diluent, which can be a monoclonal antibody, a chimeric antibody, a humanized antibody or a human antibody.
  • diabody herein refers to a bivalent, bispecific antibody that can bind to different epitopes on the same or different antigens.
  • minimum antibody recognition unit refers to the smallest unit of an antigen that an antibody can recognize in an antigen-antibody binding reaction.
  • nanoantibody in this article refers to the natural heavy chain antibody lacking light chain in camels. Cloning its variable region can obtain a single domain antibody consisting only of the heavy chain variable region, also known as VHH (Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment
  • percent (%) sequence identity refers to the percentage of amino acid (or nucleotide) residues of a candidate sequence that are identical to the amino acid (or nucleotide) residues of a reference sequence after aligning sequences and introducing gaps (if necessary) to achieve maximum percent sequence identity (e.g., for optimal alignment, gaps can be introduced in one or both of the candidate and reference sequences, and for the purpose of comparison, non-homologous sequences can be ignored).
  • alignment can be achieved in a variety of ways well known to those skilled in the art, such as using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAIi) software.
  • a reference sequence for comparison with a candidate sequence can show that the candidate sequence exhibits sequence identity from 50% to 100% over the full length of the candidate sequence or a selected portion of the continuous amino acid (or nucleotide) residues of the candidate sequence.
  • the length of the candidate sequence compared for comparison purposes can be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the length of the reference sequence.
  • conservative amino acid generally refers to amino acids belonging to the same class or having similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity).
  • amino acids within each of the following groups are conservative amino acid residues
  • the replacement of amino acid residues within the group is a replacement of conservative amino acids:
  • Acidic amino acids Asp (D) and Glu (E);
  • Aromatic amino acids Phe (F), Tyr (Y) and Trp (W).
  • Kabat numbering system herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991; which is incorporated herein by reference).
  • the term "specific binding” refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biomolecules that are specifically recognized, for example, by an antibody or its antigen-binding fragment.
  • An antibody or its antigen-binding fragment that specifically binds to an antigen will bind to the antigen with a KD of less than 100 nM.
  • an antibody or its antigen-binding fragment that specifically binds to an antigen will bind to the antigen with a KD of up to 100 nM (e.g., between 1 pM and 100 nM).
  • An antibody or its antigen-binding fragment that does not show specific binding to a specific antigen or its epitope will show a KD of greater than 100 nM (e.g., greater than 500 nM, 1 ⁇ M, 100 ⁇ M, 500 ⁇ M, or 1 mM) to the specific antigen or its epitope.
  • a variety of immunoassays can be used to select antibodies that specifically immunoreact with a specific protein or carbohydrate.
  • solid phase ELISA immunoassays are routinely used to select antibodies that specifically immunoreact with proteins or carbohydrates.
  • antibody conjugate refers to a coupling/conjugate formed by chemically bonding an antibody molecule directly or through a linker to another molecule, such as an antibody-drug conjugate (ADC), in which the drug molecule is the other molecule.
  • ADC antibody-drug conjugate
  • nucleic acid herein includes any compound and/or substance comprising a polymer of nucleotides.
  • Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group.
  • cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U) a sugar (i.e., deoxyribose or ribose) and a phosphate group.
  • nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed as 5' to 3'.
  • nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules.
  • Nucleic acid molecules can be linear or cyclic.
  • nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms.
  • nucleic acid molecules as described herein may contain naturally occurring or non-naturally occurring nucleotides.
  • nucleic acid molecules also encompass DNA and RNA molecules that are suitable as vectors for directly expressing the antibodies of the present invention in vitro and/or in vivo, for example in a host or patient.
  • DNA e.g., cDNA
  • RNA e.g., mRNA
  • mRNA can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule so that the mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi: 10.1038/nm.4356 or EP2 101 823B1; the contents of which are incorporated herein by reference).
  • vector includes nucleic acid vectors, such as DNA vectors (such as plasmids), RNA vectors, viruses or other suitable replicons (such as viral vectors).
  • DNA vectors such as plasmids
  • RNA vectors such as RNA vectors
  • viruses or other suitable replicons such as viral vectors.
  • a variety of vectors have been developed for delivering polynucleotides encoding exogenous proteins into prokaryotic or eukaryotic cells.
  • the expression vectors of the present invention contain polynucleotide sequences and additional sequence elements, such as for expressing proteins and/or integrating these polynucleotide sequences into the genome of mammalian cells.
  • Certain vectors that can be used to express the antibodies and antibody fragments of the present invention include plasmids containing regulatory sequences (such as promoter and enhancer regions) that direct gene transcription.
  • kits for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5'
  • the expression vector of the present invention may further comprise a polynucleotide encoding a marker for selecting cells containing the vector. Examples of suitable markers include genes encoding antibiotic resistance such as ampicillin, chloramphenicol, kanamycin or nourseothricin.
  • host cell herein refers to a cell into which an exogenous nucleic acid is introduced, including the offspring of such a cell.
  • Host cells include “transformants” and “transformed cells”, which include primary transformed cells and offspring derived therefrom, without considering the number of passages. Offspring may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant offspring having the same function or biological activity as screened or selected in the initially transformed cell are included herein.
  • composition refers to a preparation which is in a form which permits the biological activity of the active ingredient contained therein to be effective, and which contains no additional ingredients which are unacceptably toxic to a subject to which the pharmaceutical composition is administered.
  • the terms "subject,” “object,” and “patient” refer to an organism that is being treated for a particular disease or condition as described herein, such as cancer or an infectious disease.
  • subjects and patients include mammals such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the Bovidae family (such as cattle, bison, buffalo, elk, and yaks, etc.), sheep, and horses, etc., that are being treated for a disease or condition (e.g., a cell proliferative disorder such as cancer or an infectious disease).
  • treatment refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or lesions in the treated subject, such as the progression of a cell proliferative disorder (such as cancer or an infectious disease).
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial or complete), whether detectable or undetectable.
  • Subjects in need of treatment include subjects who already have a condition or disease, as well as subjects who are susceptible to a condition or disease or subjects for whom a condition or disease is to be prevented.
  • terms such as slow down, alleviate, weaken, alleviate, and relieve their meanings also include situations such as elimination, disappearance, and non-occurrence.
  • the term "effective amount” refers to an amount of a therapeutic agent that is effective in preventing or alleviating a disease condition or the progression of the disease when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject. "Effective amount” also refers to an amount of a compound sufficient to relieve symptoms, such as to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevent, or alleviate these conditions.
  • a therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to the combined amount of the active ingredients that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously.
  • appropriate conditions refers to conditions suitable for culturing various host cells, wherein the host cells include eukaryotic cells and prokaryotic cells.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.
  • cancer and tumor are not mutually exclusive when referred to herein.
  • anti-tumor agent refers to anti-tumor drugs, which are a class of drugs for treating tumor diseases, such as chemotherapeutic drugs, biological agents, etc.
  • EC50 refers to the half-maximal effective concentration, which includes the antibody concentration that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the antibody concentration at which 50% of its maximal effect is observed, and can be measured by methods known in the art.
  • Figure 1-2 shows the binding activity of hCDH6-1 humanized antibody and human CDH6 protein.
  • Figures 3 and 4 show the binding activity of hCDH6-1 humanized antibody to OVCAR3 cells.
  • Figure 5-6 shows the binding activity of hCDH6-6 humanized antibody and human CDH6 protein.
  • Figures 7 and 8 show the binding activity of hCDH6-6 humanized antibody to OVCAR3 cells.
  • Figures 9-11 show the binding activity of hCDH6-14 humanized antibody with human CDH6 protein.
  • FIG. 12-FIG 13 show the binding activity of hCDH6-14 humanized antibody to PA-1 cells.
  • mice Five SJL mice (mouse numbers #2401-2405) or five MRL/lpr mice (mouse numbers #2136-2140) aged 6-8 weeks (purchased from Shanghai Slake Laboratory Animal Co., Ltd.) were used for immunization. The mice were raised under SPF conditions.
  • human CDH6 extracellular domain protein with Fc tag (hCDH6 ECD-hFc, conventional synthesis, hCDH6 ECD sequence is derived from Uniprot#P55285 (19-615), as shown in the following SEQ ID NO: 1, conventional synthesis) was mixed with Titer max (purchased from Sigma, catalog number T2684) and oligonucleotide CPG (ODN 1826, synthesized from Shanghai Shenggong Biological) and injected into the foot pad and back after emulsification.
  • hCDH6 ECD-hFc protein was mixed with Imject Alum (purchased from Thermo fisher, catalog number 77161) and CPG and injected into the abdominal cavity.
  • Each mouse was injected with 50 ⁇ g antigen.
  • hCDH6 ECD-hFc protein, Imject Alum and CPG were mixed and injected into the foot pad and back.
  • hCDH6 ECD-hFc protein, Titer max and CPG were mixed and emulsified and injected into the back.
  • Subsequent booster immunizations were performed alternately in the same manner as the first and second booster immunizations, with 25 ⁇ g of antigen injected into each mouse each time, and an interval of 7 days between each immunization.
  • Blood was collected from mice on the 5th day after the second and fourth booster immunizations, and the serum was separated and the titer of specific antibodies in the serum was determined using the enzyme-linked immunosorbent assay (ELISA) method.
  • ELISA enzyme-linked immunosorbent assay
  • the ELISA detection method is as follows: dilute the human CDH6 extracellular region protein with a his tag (hCDH6 ECD-his, conventionally synthesized, the hCDH6 ECD sequence is derived from Uniprot#P55285 (19-615), as shown in SEQ ID NO: 1) with PBS to a final concentration of 1 ⁇ g/mL, add 100 ⁇ l per well to a 96-well ELISA plate, and incubate overnight at 4°C.
  • TB2 refers to the mouse serum on the 5th day after 5 immunizations (primary immunization + 4 booster immunizations), and the data in the table are OD450nm values.
  • mice Three 6-8 week old SJL mice (mouse numbers #2453, 2454, 2456) or five MRL/lpr mice (mouse numbers #2448-2452) (purchased from Shanghai Slake Laboratory Animal Co., Ltd.) were used for immunization. The mice were raised under SPF conditions.
  • HEK293T cells overexpressing human CDH6 protein HEK293T-hCDH6, conventional construction, HEK293T cells purchased from the Chinese Academy of Sciences Cell Bank, catalog number SCSP-502
  • Titer max purchased from Sigma, catalog number T2684
  • mice were immunized with hCDH6 ECD-hFc protein (protein source is the same as 1.1.1) after 5 cell immunizations (primary immunization + 4 booster immunizations).
  • the titer of specific antibodies in the serum was determined by the enzyme-linked immunosorbent assay (ELISA) method, and the results of the ELISA experiment are shown in Table 2.
  • ELISA enzyme-linked immunosorbent assay
  • Table 2 After 5 cell immunizations and 3 protein immunizations, the titer of the serum of the immunized mice binding to the human CDH6 protein was high, showing an antigen-antibody reaction.
  • TB4 refers to the mouse serum on the 5th day after 8 immunizations, and the data in the table are OD450nm values.
  • the cells were washed 3 times with DMEM basal medium (purchased from Gibco, catalog number 10569044) at 1500 rpm/min, and then mixed with mouse myeloma cells SP2/0 (purchased from ATCC, catalog number CRL-1581) at a ratio of 2:1 in the number of live cells, and cell fusion was performed using the electrofusion method (the instrument is BTX 2001+).
  • DMEM basal medium purchased from Gibco, catalog number 10569044
  • mouse myeloma cells SP2/0 purchased from ATCC, catalog number CRL-1581
  • the fused cells were diluted into DMEM medium containing 20% (w/w) fetal bovine serum (purchased from ExCell Bio, catalog number FND500), 1 ⁇ HAT (purchased from Sigma, catalog number H0262-10VL), bovine insulin (purchased from Yeason, catalog number 40107ES25), and NEAA (purchased from Gibco, catalog number 11140050), and then added into a 96-well cell culture plate at 5 ⁇ 10 4 cells/200 ⁇ L per well, and cultured in a 5% (v/v) CO 2 , 37°C incubator.
  • fetal bovine serum purchasedd from ExCell Bio, catalog number FND500
  • 1 ⁇ HAT purchasedd from Sigma, catalog number H0262-10VL
  • bovine insulin purchased from Yeason, catalog number 40107ES25
  • NEAA purchased from Gibco, catalog number 11140050
  • the supernatant of the fusion plate was screened by ELISA to determine the binding activity to human CDH6 protein; for the supernatant of the positive clone, the binding activity to monkey CDH6 protein, mouse CDH6 protein and cells overexpressing human CDH6 was determined by ELISA. According to the screening results, the positive clones that met the conditions were selected and subcloned using semi-solid culture medium (purchased from Stemcell, catalog number 03810).
  • the grown clones were picked one by one into a 96-well culture plate and cultured in a medium containing 10% (w/w) fetal bovine serum, 1 ⁇ HT (Purchased from Sigma, catalog number H0137-10VL) in DMEM medium, and after 1 day, the ELISA method was used for preliminary screening, and the monoclonal clones with human CDH6 protein binding activity were selected and expanded to 24-well plates for further culture. After 3 days, the culture supernatant was further tested to evaluate its binding activity to monkey CDH6 protein and mouse CDH6 protein, as well as its endocytosis activity on cells overexpressing human CDH6.
  • the best clone was selected and expanded in DMEM medium containing 10% (w/w) FBS at 37°C and 5% (v/v) CO2.
  • the best hybridoma cells were obtained by freezing in liquid nitrogen and can be used for subsequent antibody production and purification.
  • the hybridoma clones with better activity in Example 1 were selected for sequencing.
  • the sequences of the positive control anti-human CDH6 antibodies DS-H01L02 and NOV0712 were derived from patent WO2018212136A1, and the corresponding sequence information is shown in Tables 3 and 4 below, where Table 3 is the VH and VL sequences of the mouse monoclonal antibody, and Table 4 is the CDRs (KABAT analysis) of the mouse monoclonal antibody molecule.
  • heavy chain (HC) and light chain (LC) plasmids were constructed (the nucleic acid sequences encoding antibody VH and VL were recombined into an expression vector with a signal peptide (SEQ ID NO: 132, MGWSWILLFLLSVTAGVHS) and heavy chain constant region/light chain constant region sequences to obtain a recombinant plasmid expressing VH-CH1-Fc/VL-CL).
  • the heavy chain and light chain plasmids corresponding to the antibody and the transfection reagent PEI (purchased from Polysciences, catalog number: 24765-1) were added to OPTI-MEM (purchased from Gibco, catalog number: 11058021) and mixed well, then allowed to stand for 15 minutes, added to Expi293 cells (purchased from Thermofisher, catalog number: A14527), and cultured in a shaking incubator at 5% CO 2 , 120 rpm, and 37° C.
  • OPM-293 ProFeed purchased from Shanghai Aopumai, catalog number: F081918-001
  • glucose purchasedd from Sigma, catalog number: G7528
  • the cell supernatant was collected and purified with Protein A (purchased from GE, catalog number: 28985254).
  • the eluted sample was dialyzed into PBS buffer (pH 7.4) to obtain chimeric antibodies and positive control antibodies.
  • Human CDH6 protein (hCDH6 ECD-his, conventional synthesis), monkey CDH6 protein (cynoCDH6 ECD-his, conventional synthesis, cynoCDH6 ECD sequence derived from Uniprot#A0A2K5TW62 (19-615)) and mouse CDH6 protein (mCDH6 ECD-his, conventional synthesis, mCDH6 ECD sequence derived from Uniprot#P97326 (19-615)) were diluted with PBS to a final concentration of 1 ⁇ g/mL, and 100 ⁇ l was added to each well of a 96-well ELISA plate.
  • the horseradish peroxidase-labeled secondary antibody Peroxidase AffiniPure Goat Anti-Human IgG, Fc ⁇ fragment specific (purchased from Jackson Immuno, catalog number 109-035-098) was diluted with 1% BSA 1:5000, and 50 ⁇ l was added to each well of the 96-well plate. After incubation at 37°C for 1 hour, the plate was washed 5 times with plate washing buffer. TMB substrate (purchased from Seracare, catalog number 5120-0077) was added to each well, and after incubation at room temperature for 10 minutes, 50 ⁇ l of stop solution (1.0N HCl) was added to each well. The OD450nm value was read using an enzyme reader (Biotek). The data was fitted and the EC50 value was calculated using GraphPad Prism software. The ELISA results are shown in Table 5. The chimeric antibodies tested had strong binding to human CDH6 protein, monkey CDH6 protein and mouse CDH6 protein.
  • OVCAR3 cells with high CDH6 expression and PA-1 cells with moderate CDH6 expression were used to detect the binding activity of antibodies to cells.
  • OVCAR3 cells purchased from ATCC, #HTB-161 or PA-1 cells (purchased from Nanjing Kebai Biotechnology Co., Ltd., #CBP60800) were cultured to 90% confluence, the culture medium was aspirated, washed once with PBS, and then treated and collected with enzyme-free cell dissociation solution (Versene, purchased from Gibco, catalog number 15040-066).
  • the cells were washed once with PBS, After counting the cells, resuspend the cells to 2 ⁇ 10 6 cells/mL in FACS buffer (PBS + 2% FBS) and add 50 ⁇ l per well to a 96-well plate. Dilute the chimeric antibody in FACS buffer (PBS + 2% FBS) and add 50 ⁇ l per well to a 96-well plate and incubate at 4°C for 1 hour. Wash three times with PBS and dilute the secondary antibody Alexa Fluor 5000 in FACS buffer at 1:800.
  • OVCAR3 cells were cultured to 90% confluence, the medium was aspirated, and the cells were washed once with PBS. Then, the cells were treated and collected with enzyme-free cell dissociation solution (Versene, purchased from Gibco, catalog number 15040-066). The cells were washed once with PBS, and after cell counting, they were resuspended to 2 ⁇ 10 6 cells/mL with FACS buffer (PBS+2% FBS), and 50 ⁇ l was added to two 96-well plates per well. The chimeric antibody was diluted to 10 ⁇ g/ml with FACS buffer (PBS+2% FBS), and 50 ⁇ l was added to two 96-well plates per well, and incubated at 4°C for 1 hour.
  • the affinity of antibody and antigen was determined by multi-cycle kinetic method using BIAcore 8K instrument.
  • the antibody to be tested was first captured with Protein A chip (purchased from GE, product number: 29-1275-56), and then the antigen human CDH6 protein (hCDH6-his, conventional synthesis) was injected.
  • the binding and dissociation processes of the antibody and antigen protein were recorded, and finally the chip was regenerated with Glycine pH1.5.
  • the mobile phase was HBS-EP+ (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20, 1mM Ca), with a flow rate of 30 ⁇ L/min, binding time of 240s, dissociation time of 300s, regeneration time of 30s, detection temperature of 25°C, and antigen concentration of 31.25-1000nM.
  • Biacore 8K analysis software version 2.0 was used to analyze the data according to the 1:1 binding model, and the antibody-antigen binding kinetic parameters were fitted, including the binding rate constant ka, the dissociation rate constant kd, and the equilibrium dissociation constant KD. The results are shown in Table 8.
  • the affinity value of the control antibody DS-H01L02 could not be detected.
  • the antibodies not shown in the table had affinity values that could not be detected.
  • the heavy chain and light chain variable region germline genes with high homology to the mouse antibody were selected as templates, and the CDRs of the mouse antibody divided according to Kabat were transplanted into the corresponding human templates to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the buried residues, the residues that directly interact with the CDR region, and the residues in the framework region that have an important influence on the conformation of VL and VH are back-mutated to obtain humanized monoclonal antibodies.
  • the humanized light chain templates of the mouse antibody CDH6-1 are IGKV4-1*01/IGKV1-33*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-69*02 and IGHJ6*01.
  • the CDRs of the mouse antibody CDH6-1 are transplanted into their humanized templates respectively, and the CDR sequences remain unchanged, so as to obtain the corresponding humanized antibody hCDH6-1.
  • the key amino acids in the FR region sequence of the humanized antibody hCDH6-1 are reversely mutated to the corresponding amino acids of the mouse antibody to ensure the original affinity.
  • the specific reverse mutation design is shown in Table 9.
  • Graft represents the implantation of mouse antibody CDR into human germline template FR sequence
  • G72E represents the mutation of G at position 72 of Graft to E, and so on.
  • the numbering of the back-mutated amino acids is the natural sequence numbering.
  • amino acid sequence of hCDH6-1.VL1 is shown in SEQ ID NO: 133:
  • amino acid sequence of hCDH6-1.VL2 is shown in SEQ ID NO: 134:
  • amino acid sequence of hCDH6-1.VH1 is shown in SEQ ID NO: 136:
  • amino acid sequence of hCDH6-1.VH2 is shown in SEQ ID NO: 137:
  • amino acid sequence of hCDH6-13.VH3 is shown in SEQ ID NO: 138:
  • amino acid sequence of hCDH6-1.VH4 is shown in SEQ ID NO: 139:
  • amino acid sequence of the human germline light chain template IGKV4-1*01 is shown in SEQ ID NO: 140:
  • amino acid sequence of the human germline light chain template IGKV1-33*01 is shown in SEQ ID NO: 141:
  • amino acid sequence of the human germline light chain template IGKJ4*01 is shown in SEQ ID NO: 142:
  • the amino acid sequence of the human germline heavy chain template IGHV1-69*02 is shown in SEQ ID NO: 143:
  • amino acid sequence of the human germline heavy chain template IGHJ6*01 is shown in SEQ ID NO: 144:
  • variable regions of each antibody were selected for cross-combination, and finally multiple hCDH6-1 humanized antibodies were obtained.
  • the amino acid sequences of the variable regions of each antibody are as follows:
  • heavy chain (HC) and light chain (LC) plasmids were constructed (the nucleic acid sequences encoding antibody VH and VL were recombined into an expression vector with a signal peptide (SEQ ID NO: 132, MGWSWILLFLLSVTAGVHS) and a heavy chain constant region/light chain constant region sequence to obtain a recombinant plasmid expressing VH-CH1-Fc/VL-CL).
  • SEQ ID NO: 132 MGWSWILLFLLSVTAGVHS
  • a heavy chain constant region/light chain constant region sequence to obtain a recombinant plasmid expressing VH-CH1-Fc/VL-CL.
  • hCDH6-1 humanized antibody was obtained, and the binding activity of the humanized antibody was detected by ELISA and FACS (methods refer to 2.2, 2.3).
  • the results are shown in Table 11 and Figures 1 to 4.
  • the humanized light chain templates of the mouse antibody CDH6-6 are IGKV1-NL1*01/IGKV2-28*01 and IGKJ2*01, and the humanized heavy chain templates are IGHV1-46*01 and IGHJ6*01.
  • the CDRs of the mouse antibody CDH6-6 are transplanted into their humanized templates respectively, and the CDR sequences remain unchanged, so as to obtain the corresponding humanized version hCDH6-6.
  • the key amino acids in the FR region sequence of the humanized antibody hCDH6-6 are reverse mutated to the corresponding amino acids of the mouse antibody to ensure the original affinity.
  • the specific reverse mutation design is shown in Table 13.
  • amino acid sequence of hCDH6-6.VL1 is shown in SEQ ID NO: 145:
  • amino acid sequence of hCDH6-6.VL2 is shown in SEQ ID NO: 146:
  • amino acid sequence of hCDH6-6.VL3 is shown in SEQ ID NO: 147:
  • amino acid sequence of hCDH6-6.VL4 is shown in SEQ ID NO: 148:
  • amino acid sequence of hCDH6-6.VL5 is shown in SEQ ID NO: 149:
  • amino acid sequence of hCDH6-6.VH1 is shown in SEQ ID NO: 150:
  • amino acid sequence of hCDH6-6.VH2 is shown in SEQ ID NO: 151:
  • amino acid sequence of hCDH6-6.VH3 is shown in SEQ ID NO: 152:
  • amino acid sequence of the human germline light chain template IGKV1-NL1*01 is shown in SEQ ID NO: 153:
  • the amino acid sequence of the human germline light chain template IGKJ2*01 is shown in SEQ ID NO: 155:
  • amino acid sequence of the human germline heavy chain template IGHV1-46*01 is shown in SEQ ID NO: 156:
  • amino acid sequence of the human germline heavy chain template IGHJ6*01 is shown in SEQ ID NO: 144:
  • heavy chain (HC) and light chain (LC) plasmids were constructed (the nucleic acid sequences encoding antibody VH and VL were recombined into an expression vector with a signal peptide (SEQ ID NO: 132, MGWSWILLFLLSVTAGVHS) and a heavy chain constant region/light chain constant region sequence to obtain a recombinant plasmid expressing VH-CH1-Fc/VL-CL).
  • SEQ ID NO: 132 MGWSWILLFLLSVTAGVHS
  • a heavy chain constant region/light chain constant region sequence to obtain a recombinant plasmid expressing VH-CH1-Fc/VL-CL.
  • hCDH6-6 humanized antibody was obtained, and the binding activity of the humanized antibody was detected by ELISA and FACS (see 2.2 and 2.3 for the method).
  • the results are shown in Table 15 and Figures 5 to 8.
  • the humanized light chain templates of the mouse antibody CDH6-14 are IGKV1-33*01/IGKV4-1*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-3*01 and IGHJ6*01.
  • the CDRs of the mouse antibody CDH6-14 are transplanted into their humanized templates respectively, and the CDR sequences remain unchanged, so as to obtain the corresponding humanized version hCDH6-14.
  • the key amino acids in the FR region sequence of the humanized antibody of hCDH6-14 are reverse mutated to the corresponding amino acids of the mouse antibody to ensure the original affinity.
  • the specific reverse mutation design is shown in Table 17.
  • amino acid sequence of hCDH6-14.VL1 is shown in SEQ ID NO: 157:
  • amino acid sequence of hCDH6-14.VL2 is shown in SEQ ID NO: 158:
  • amino acid sequence of hCDH6-14.VL3 is shown in SEQ ID NO: 159:
  • amino acid sequence of hCDH6-14.VH5 is shown in SEQ ID NO: 164:
  • amino acid sequence of hCDH6-14.VH6 is shown in SEQ ID NO: 165:
  • amino acid sequence of the human germline light chain template IGKV1-33*01 is shown in SEQ ID NO: 141:
  • amino acid sequence of the human germline light chain template IGKV4-1*01 is shown in SEQ ID NO: 140:
  • amino acid sequence of the human germline light chain template IGKJ4*01 is shown in SEQ ID NO: 142:
  • amino acid sequence of the human germline heavy chain template IGHV1-3*01 is shown in SEQ ID NO: 166:
  • amino acid sequence of the human germline heavy chain template IGHJ6*01 is shown in SEQ ID NO: 144:
  • heavy chain (HC) and light chain (LC) plasmids were constructed (the nucleic acid sequences encoding antibody VH and VL were recombined into an expression vector with a signal peptide (SEQ ID NO: 132, MGWSWILLFLLSVTAGVHS) and a heavy chain constant region/light chain constant region sequence to obtain a recombinant plasmid expressing VH-CH1-Fc/VL-CL).
  • SEQ ID NO: 132 MGWSWILLFLLSVTAGVHS
  • a heavy chain constant region/light chain constant region sequence to obtain a recombinant plasmid expressing VH-CH1-Fc/VL-CL.
  • hCDH6-14 humanized antibody was obtained, and the binding activity of the humanized antibody was detected by ELISA and FACS (see 2.2 and 2.3 for the method).
  • the results are shown in Table 19 and Figures 9 to 13.
  • Some antibodies were selected for endocytic activity detection, and the method for detecting endocytic activity of humanized antibodies in OVCAR3 cells was the same as 2.4. The results are shown in Table 20, and the humanized antibodies tested all had good endocytic activity.
  • the full-length human CDH6 protein consists of 790 amino acids (SEQ ID NO: 167), of which the extracellular region includes EC1 (SEQ ID NO: 168), EC2 (SEQ ID NO: 169), EC3 (SEQ ID NO: 170), EC4 (SEQ ID NO: 171) and EC5 (SEQ ID NO: 172).
  • the sequences after removing these five regions were constructed into the pcDNA3.1 vector (purchased from Youbao Bio, catalog number VT1001), and transfected into HEK293T cells (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number SCSP-502), and five CDH6 truncated cell lines were obtained: HEK293T-hCDH6-EC1deletion, HEK293T-hCDH6-EC2deletion, HEK293T-hCDH6-EC3deletion, HEK293T-hCDH6-EC4deletion and HEK293T-hCDH6-EC5deletion.
  • Human CDH6 protein full length sequence (Uniprot#P55285) SEQ ID NO: 167
  • the binding activity of CDH6 chimeric antibody to these five cell lines was detected by FACS.
  • the results are shown in Table 21. Based on the test results, the binding epitopes of the antibodies can be inferred: CHI-CDH6-1 and CHI-CDH6-7 do not bind to EC1-deletion cells, indicating that their binding epitopes are EC1; CHI-CDH6-5 does not bind to EC1-deletion cells and binds very weakly to EC2-deletion cells, indicating that its binding epitope may be between EC1-EC2, CHI-CDH6-4 does not bind to EC2-deletion cells, indicating that its binding epitope is EC2; CHI-CDH6-14 binds very weakly to EC2-deletion and EC3-deletion cells, indicating that its binding epitope may be between EC2-EC3; CHI-CDH6-3 binds very weakly to EC3-deletion cells, indicating that its binding

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Abstract

提供了一种特异性结合CDH6(钙粘蛋白6)的抗体及其应用，具体公开了结合CDH6的鼠源和人源化抗体及其制备方法和应用，并且与CDH6蛋白具有较好的亲和力，且具有较佳的内吞活性，因此能够运用于治疗肿瘤等药物的制备中。

Description

抗CDH6的抗体及其用途
本公开要求于2023年2月10日提交中国专利局、申请号为202310109927.8、发明名称为“抗CDH6的抗体及其用途”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本发明涉及生物工程、生物医药领域，主要涉及一种抗CDH6的抗体或其抗原结合片段，其编码核酸、表达载体和表达细胞、制备方法、药物组合物、以及它们用于治疗疾病的用途，例如治疗肿瘤的用途。
背景技术
钙粘蛋白(Cadherins)在组织稳态中起重要作用，主要负责胚胎发生、组织形态发生、分化和肿瘤发生过程中的细胞-细胞粘附。钙粘蛋白-连环蛋白复合物的任何功能障碍或不稳定都可能导致肿瘤进展。
CDH6是一种II型经典钙粘蛋白，又称为K-钙粘蛋白。CDH6为单次跨膜蛋白，共有790个氨基酸组成，胞外区分为5个区域。研究发现CDH6主要在肾癌、卵巢癌和甲状腺癌等肿瘤组织高表达，而在正常组织的表达很少(Cancer Discov；2017,7(9):1030-45；其内容通过引用并入本文)。与钙粘蛋白超家族的其他成员一样，CDH6蛋白定位于上皮细胞的基底外侧膜并介导钙依赖性细胞-细胞粘附，具有快速内化的特性。因此，CDH6作为一种具有高度识别性的肿瘤特异性标志物，已成为肿瘤治疗中一个极具吸引力的靶点，可开发用于治疗卵巢癌和肾癌等癌症。
尽管近些年在卵巢和肾癌的治疗上有一些进展，但仍有大量的临床需求未得到满足。目前所研发的靶向CDH6的药物较少，还未有相关药物获批上市，因此开发该靶点的药物具有广阔的临床应用及治疗前景。
发明内容
本发明提供特异性结合CDH6的抗体或抗原结合片段，编码这些抗体及其抗原结合片段的核酸，包含所述抗体及抗原结合片段的药物组合物和试剂盒，以及它能够用于治疗肿瘤等药物的制备。
在第一个方面，本发明提供了一种特异性结合CDH6的抗体或抗原结合片段，所述抗体或抗原结合片段特异性结合CDH6蛋白胞外区EC1区、EC1-EC2区、EC2区、EC2-EC3区、EC3区、EC4区或EC5区的一个或多个氨基酸序列片段；优选的，所述抗体或抗原结合片段特异性结合SEQ ID NO：168-172任意一个序列中显示的一个或多个氨基酸序列片段；优选地，所述抗体或抗原结合片段具有内吞活性。
在第二个方面，本发明一种特异性结合CDH6的抗体或抗原结合片段，所述抗体或抗原结合片段包含：
(a)SEQ ID NO：2、4、6、8、10、12、14、16、18、20、22、24、26、28、136、137、 138、139、150、151、152、160、161、162、163、164或165任一项所述VH的HCDR1、HCDR2和HCDR3；和/或，(b)SEQ ID NO：3、5、7、9、11、13、15、17、19、21、23、25、27、29、133、134、135、145、146、147、148、149、157、158或159任一项所述VL的LCDR1、LCDR2和LCDR3；
优选地，所述HCDR1-3和/或所述LCDR1-3为根据KABAT、IMGT或Chothia的通行分析方法编码。
在一个具体的实施方案中，所述HCDR1、HCDR2和HCDR3具有选自以下的任意序列组合或者与所述序列组合相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列组合：
和，
所述LCDR1、LCDR2和LCDR3具有选自以下任意序列组合或者与所述序列组合相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列组合：

优选地，所述替换为保守氨基酸的替换。
在另一个具体的实施方案中，所述的抗体或抗原结合片段包含选自以下的重链CDRs和轻链CDRs组合：VH1+VL1、VH2+VL2、VH3+VL3、VH4+VL4、VH5+VL5、VH6+VL6、VH7+VL7、VH8+VL8、VH9+VL9、VH10+VL10、VH11+VL11、VH12+VL12、VH13+VL13、或VH14+VL14，以及与所述重链和轻链CDRs组合之序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的CDRs组合；
优选地，所述替换为保守氨基酸的替换。
在另一个具体的实施方案中，所述抗体或抗原结合片段的重链可变区和轻链可变区的框架区来源于人种系重链模板和人种系轻链模板，其中：
(1)框架区序列来源于人种系重链IGHV1-69*02和IGHJ6*01的组合序列；其包含SEQ ID NO：143所示IGHV1-69*02的FR1、FR2、FR3区和SEQ ID NO：144所示IGHJ6*01的FR4区；
(2)框架区序列来源于人种系重链IGHV1-46*01和IGHJ6*01的组合序列；其包含SEQ ID NO：156所示IGHV1-46*01的FR1、FR2、FR3区和SEQ ID NO：144所示IGHJ6*01的FR4区；
(3)框架区序列来源于人种系重链IGHV1-3*01和IGHJ6*01的组合序列；其包含SEQ ID NO：166所示IGHV1-3*01的FR1、FR2、FR3区和SEQ ID NO：144所示IGHJ6*01的FR4区；
(4)框架区序列来源于人种系轻链IGKV4-1*01和IGKJ4*01的组合序列；其包含SEQ ID NO：140所示IGKV4-1*01的FR1、FR2、FR3区和SEQ ID NO：142所示IGKJ4*01的FR4区；
(5)框架区序列来源于人种系轻链IGKV1-33*01和IGKJ4*01的组合序列；其包含SEQ ID NO：141所示IGKV1-33*01的FR1、FR2、FR3区和SEQ ID NO：142所示IGKJ4*01的FR4区；
(6)框架区序列来源于人种系轻链IGKV1-NL1*01和IGKJ2*01的组合序列；其包含SEQ ID NO：153所示IGKV1-NL1*01的FR1、FR2、FR3区和SEQ ID NO：155所示IGKJ2*01的FR4区；或，
(7)框架区序列来源于人种系轻链IGKV2-28*01和IGKJ2*01的组合序列；其包含SEQ ID NO：154所示IGKV2-28*01的FR1、FR2、FR3区和SEQ ID NO：155所示IGKJ2*01的FR4区。
在一个具体的实施方案中，所述的抗体或抗原结合片段的框架区根据Kabat编号系统编号还包括选自下组的一种或多种突变，其中：
(1)重链可变区的框架区包括：G27Y、S30T、A40R、I70L或A72V；优选包括G27Y和A72V；或优选包括G27Y、S30T和A72V；或优选包括G27Y、S30T、I70L和A72V；或优选包括G27Y、S30T、A40R和A72V；
(2)重链可变区的框架区包括：G42R、M70L、R72V或T74K；优选包括R72V和T74K；或优选包括M70L、R72V和T74K；或优选包括G42R、R72V和T74K；
(3)重链可变区的框架区包括：V2I、V5Q、R44G、I70L、R72V、Y95L或R98S；优选包括R72V和R98S；或优选包括V2I、R72V和R98S；或优选包括V2I、I70L、R72V和R98S；或优选包括R44G、R72V和R98S；或优选包括V5Q、R72V和R98S；或优选包括R72V、Y95L和R98S；
(4)轻链可变区的框架区包括：P44S、A47P或G72E；优选包括G72E；或优选包括P44S和G72E；或优选包括A47P和G72E；
(5)轻链可变区的框架区包括：Q42K、A43S、K45Q、L48V、I48V或T85R；优选包括A43S；或优选包括A43S和L48V；或优选包括A43S、K45Q和L48V；或优选包括A43S、L48V和T85R；或优选包括Q42K和I48V；或，
(6)轻链可变区的框架区包括：K42G、P43T、P44F或Y49S；优选包括P44F和Y49S；或优选包括K42G和Y49S；或优选包括P43T和Y49S。
在一个具体的实施方案中，所述的抗体或抗原结合片段包含：
(1)重链可变区具有SEQ ID NO:2、4、6、8、10、12、14、16、18、20、22、24、26、28、136、137、138、139、150、151、152、160、161、162、163、164或165所示序列；
(2)轻链可变区具有SEQ ID NO:3、5、7、9、11、13、15、17、19、21、23、25、27、29、133、134、135、145、146、147、148、149、157、158或159所示序列；
(3)与上述(1)～(2)中任一所述序列相比具有至少90％同一性的氨基酸序列，优选为至少91％、92％、93％、94％、95％、96％、97％、98％、99％同一性。
在另一个具体的实施方案中，所述的重链可变区和轻链可变区选自如下组：
(1)具有SEQ ID NO.2所示的VH和SEQ ID NO.3所示的VL；
(2)具有SEQ ID NO.4所示的VH和SEQ ID NO.5所示的VL；
(3)具有SEQ ID NO.6所示的VH和SEQ ID NO.7所示的VL；
(4)具有SEQ ID NO.8所示的VH和SEQ ID NO.9所示的VL；
(5)具有SEQ ID NO.10所示的VH和SEQ ID NO.11所示的VL；
(6)具有SEQ ID NO.12所示的VH和SEQ ID NO.13所示的VL；
(7)具有SEQ ID NO.14所示的VH和SEQ ID NO.15所示的VL；
(8)具有SEQ ID NO.16所示的VH和SEQ ID NO.17所示的VL；
(9)具有SEQ ID NO.18所示的VH和SEQ ID NO.19所示的VL；
(10)具有SEQ ID NO.20所示的VH和SEQ ID NO.21所示的VL；
(11)具有SEQ ID NO.22所示的VH和SEQ ID NO.23所示的VL；
(12)具有SEQ ID NO.24所示的VH和SEQ ID NO.25所示的VL；
(13)具有SEQ ID NO.26所示的VH和SEQ ID NO.27所示的VL；
(14)具有SEQ ID NO.28所示的VH和SEQ ID NO.29所示的VL；
(15)具有SEQ ID NO.136所示的VH和SEQ ID NO.133所示的VL；
(16)具有SEQ ID NO.137所示的VH和SEQ ID NO.133所示的VL；
(17)具有SEQ ID NO.138所示的VH和SEQ ID NO.133所示的VL；
(18)具有SEQ ID NO.139所示的VH和SEQ ID NO.133所示的VL；
(19)具有SEQ ID NO.136所示的VH和SEQ ID NO.134所示的VL；
(20)具有SEQ ID NO.137所示的VH和SEQ ID NO.134所示的VL；
(21)具有SEQ ID NO.138所示的VH和SEQ ID NO.134所示的VL；
(22)具有SEQ ID NO.139所示的VH和SEQ ID NO.134所示的VL；
(23)具有SEQ ID NO.136所示的VH和SEQ ID NO.135所示的VL；
(24)具有SEQ ID NO.137所示的VH和SEQ ID NO.135所示的VL；
(25)具有SEQ ID NO.138所示的VH和SEQ ID NO.135所示的VL；
(26)具有SEQ ID NO.139所示的VH和SEQ ID NO.135所示的VL；
(27)具有SEQ ID NO.150所示的VH和SEQ ID NO.145所示的VL；
(28)具有SEQ ID NO.150所示的VH和SEQ ID NO.146所示的VL；
(29)具有SEQ ID NO.150所示的VH和SEQ ID NO.147所示的VL；
(30)具有SEQ ID NO.150所示的VH和SEQ ID NO.148所示的VL；
(31)具有SEQ ID NO.150所示的VH和SEQ ID NO.149所示的VL；
(32)具有SEQ ID NO.151所示的VH和SEQ ID NO.145所示的VL；
(33)具有SEQ ID NO.151所示的VH和SEQ ID NO.146所示的VL；
(34)具有SEQ ID NO.151所示的VH和SEQ ID NO.147所示的VL；
(35)具有SEQ ID NO.151所示的VH和SEQ ID NO.148所示的VL；
(36)具有SEQ ID NO.151所示的VH和SEQ ID NO.149所示的VL；
(37)具有SEQ ID NO.152所示的VH和SEQ ID NO.145所示的VL；
(38)具有SEQ ID NO.152所示的VH和SEQ ID NO.146所示的VL；
(39)具有SEQ ID NO.152所示的VH和SEQ ID NO.147所示的VL；
(40)具有SEQ ID NO.152所示的VH和SEQ ID NO.148所示的VL；
(41)具有SEQ ID NO.152所示的VH和SEQ ID NO.149所示的VL；
(42)具有SEQ ID NO.160所示的VH和SEQ ID NO.157所示的VL；
(43)具有SEQ ID NO.160所示的VH和SEQ ID NO.158所示的VL；
(44)具有SEQ ID NO.160所示的VH和SEQ ID NO.159所示的VL；
(45)具有SEQ ID NO.161所示的VH和SEQ ID NO.157所示的VL；
(46)具有SEQ ID NO.161所示的VH和SEQ ID NO.158所示的VL；
(47)具有SEQ ID NO.161所示的VH和SEQ ID NO.159所示的VL；
(48)具有SEQ ID NO.162所示的VH和SEQ ID NO.157所示的VL；
(49)具有SEQ ID NO.162所示的VH和SEQ ID NO.158所示的VL；
(50)具有SEQ ID NO.162所示的VH和SEQ ID NO.159所示的VL；
(51)具有SEQ ID NO.163所示的VH和SEQ ID NO.157所示的VL；
(52)具有SEQ ID NO.163所示的VH和SEQ ID NO.158所示的VL；
(53)具有SEQ ID NO.163所示的VH和SEQ ID NO.159所示的VL；
(54)具有SEQ ID NO.164所示的VH和SEQ ID NO.157所示的VL；
(55)具有SEQ ID NO.164所示的VH和SEQ ID NO.158所示的VL；
(56)具有SEQ ID NO.164所示的VH和SEQ ID NO.159所示的VL；
(57)具有SEQ ID NO.165所示的VH和SEQ ID NO.157所示的VL；
(58)具有SEQ ID NO.165所示的VH和SEQ ID NO.158所示的VL；
(59)具有SEQ ID NO.165所示的VH和SEQ ID NO.159所示的VL；或，
(60)具有与上述(1)～(59)任一序列组合相比具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的VH和VL组合。
在一个具体的实施方案中，所述的抗体或抗原结合片段与人CDH6蛋白结合的解离常数(KD)不大于2×10-7M。
在另一个具体的实施方案中，所述的抗体或抗原结合片段为：
(1)嵌合抗体或其片段；
(2)人源化抗体或其片段；
(3)全人源抗体或其片段；
优选的，所述抗体或抗原结合片段选自单克隆抗体、多克隆抗体、天然抗体、工程化抗体、单特异性抗体、多特异性抗体(例如双特异性抗体)、单价抗体、多价抗体、全长抗体、抗体片段、裸抗体、缀合抗体、人源化抗体、全人源抗体、Fab、Fab’、F(ab’)2、Fd、Fv、scFv、双抗体(diabody)或单域抗体。
在另一个具体的实施方案中，所述抗体包含人或鼠抗体IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD任何其中之一恒定区的序列；优选包含人或鼠抗体IgG1、IgG2、IgG3或IgG4的恒定区的序列。
在另一个具体的实施方案中，所述抗原结合片段选自F(ab)2、Fab’、Fab、Fv、scFv、双特异性抗体、纳米抗体和抗体最小识别单位中的一种或多种。
在第三个方面，本发明的抗体或抗原结合片段进一步还偶联有治疗剂或示踪剂；优选地，所述治疗剂选自放射性同位素、化疗药或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂或光敏剂。
在第四个方面，本发明还提供一种多特异性抗原结合分子，所述多特异性抗原结合分子包含第一抗原结合模块和第二抗原结合模块，所述第一抗原结合模块上述第一或二方面任一项所述的抗体或抗原结合片段，所述第二抗原结合模块特异性结合CDH6以外的其他抗原或结合与第一抗原结合模块不同的CDH6抗原表位；
优选地，所述其他抗原选自CD3、CD7、CD16、CD16A、CD4、CD5、CD8、CD14、CD15、CD19、CD20、CD21、CD23、CD25、CD33、CD37、CD38、CD40、CD40L、CD46、CD52、CD54、CD66(a-d)、CD74、CD80、CD126、CD138、BCMA、HLA-DR、HER2、VEGF、P1GF、HER3/ERBB3,HER4/ERBB4、IL-2、IL-6、PD-1、PD-L1、TRAIL-R1或TRAIL-R2；
优选地，所述多特异性抗原结合分子为双特异性抗体、三特异性抗体或四特异性抗体。
在第五个方面，本发明还提供一种嵌合抗原受体(CAR)，所述嵌合抗原受体至少包含细胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域，所述细胞外抗原结合结构域包含上述第一或二个方面所述CDH6抗体或抗原结合片段。
在第六个方面，本发明还提供一种免疫效应细胞，所述免疫效应细胞包含上述第五个方面所述的嵌合抗原受体或包含编码上述第五个方面所述的嵌合抗原受体的核酸片段；
优选地，所述免疫效应细胞选自T细胞、NK细胞(natural killer cell)、NKT细胞(natural killer T cell)、单核细胞、巨噬细胞、树突状细胞或肥大细胞；所述T细胞可选自，炎性T细胞、细胞毒性T细胞、调节性T细胞(Treg)或辅助性T细胞；
优选地，所述免疫效应细胞为同种异体免疫效应细胞或自体免疫细胞。
在第七个方面，本发明还提供一种分离的核酸分子，所述核酸分子编码上述第一、二个方面所述的抗体、抗原结合片段、或任意组合，第三个方面所述的偶联有示踪剂的抗体或抗原结合片段，第四个方面的多特异性抗原结合分子或第五个方面的嵌合抗原受体。
在第八个方面，本发明还提供了一种包含第七个方面所述的分离的核酸分子的表达载体。
在第九个方面，本发明提供了上述第七个方面所述的分离的核酸分子，或第八个方面所述的表达载体的分离的宿主细胞；优选地，所述宿主细胞是真核细胞或原核细胞；更优选地，所述宿主细胞来源于哺乳动物细胞、酵母细胞、昆虫细胞、大肠杆菌和/或枯草杆菌；更优选 地，所述宿主细胞选自Expi293或CHO细胞。
在第十个方面，本发明提供了一种制备上述第一或二个方面所述的抗体或抗原结合片段、第三个方面所述的偶联有示踪剂的抗体或抗原结合片段、或第四个方面所述的多特异性抗原结合分子的方法，在适当的条件下上述第九个方面所述的宿主细胞，并分离抗体或抗原结合片段或多特异性抗原结合分子。
在第十一个方面，本发明提供了一种制备上述第六个方面所述免疫效应细胞的方法，所述方法包括将编码第五个方面所述CAR的核酸片段导入免疫效应细胞，可选地，所述方法还包括启动所述免疫效应细胞表达第五个方面所述CAR。
在第十二个方面，本发明提供了一种药物组合物，所述药物组合物包含上述第一或二个方面所述的抗体或抗原结合片段、第三个方面所述的偶联有示踪剂的抗体或抗原结合片段、第四个方面所述的多特异性抗原结合分子、第五个方面所述的嵌合抗原受体、第六个方面所述的免疫效应细胞、第七个方面所述的分离的核酸分子、第八个方面所述的表达载体、第九个方面所述的细胞、第十或第十一个方面所述方法制备的产品；优选地，所述组合物还包含药学上可接受的运载体(carrier)、稀释剂或助剂；优选地，所述药物组合物还包含额外的抗肿瘤剂。
在第十三个方面，本发明提供了上述第一或二个方面所述的抗体或抗原结合片段、第三个方面所述的偶联有示踪剂的抗体或抗原结合片段、第四个方面所述的多特异性抗原结合分子、第五个方面所述的嵌合抗原受体、第六个方面所述的免疫效应细胞、第七个方面所述的分离的核酸分子、第八个方面所述的表达载体、第九个方面所述的细胞、或第十或第十一个方面所述方法制备的产品、或第十二个方面所述的药物组合物在制备预防和/或治疗肿瘤疾病的药物中的用途；优选地，所述肿瘤疾病选自实体瘤，优选地，所述实体瘤为表达CDH6蛋白的实体瘤，更优选地，所述实体瘤选自肾癌、卵巢癌、甲状腺癌、胆管癌、肺癌、胶质母细胞瘤、间皮瘤、子宫癌、胰腺癌、胆管癌、威尔姆氏瘤或神经母细胞瘤。
在第十四个方面，本发明提供了一种预防和/或治疗肿瘤疾病的方法，包含向有此需要的患者施用有效量的上述第一或二个方面所述的抗体或抗原结合片段、第三个方面所述的偶联有示踪剂的抗体或抗原结合片段、第四个方面所述的多特异性抗原结合分子、第五个方面所述的嵌合抗原受体、第六个方面所述的免疫效应细胞、第七个方面所述的分离的核酸分子、第八个方面所述的表达载体、第九个方面所述的细胞、或第十或第十一个方面所述方法制备的产品、或第十二个方面所述的药物组合物；优选地，所述肿瘤疾病选自实体瘤，优选地，所述实体瘤为表达CDH6蛋白的实体瘤，更优选地的，所述实体瘤选自肾癌、卵巢癌、甲状腺癌、胆管癌、肺癌、胶质母细胞瘤、间皮瘤、子宫癌、胰腺癌、胆管癌、威尔姆氏瘤或神经母细胞瘤。
在第十五个方面，本发明提供了上述第一或二个方面所述的抗体或抗原结合片段、第三个方面所述的偶联有示踪剂的抗体或抗原结合片段、第四个方面所述的多特异性抗原结合分子、第五个方面所述的嵌合抗原受体、第六个方面所述的免疫效应细胞、第七个方面所述的分离的核酸分子、第八个方面所述的表达载体、第九个方面所述的细胞、或第十或第十一个方面所述方法制备的产品、或第十二个方面所述的药物组合物用于和/或治疗肿瘤疾病；优选地，所述肿瘤疾病选自实体瘤，优选地，所述实体瘤为表达CDH6蛋白的实体瘤，更优选地， 所述实体瘤选自肾癌、卵巢癌、甲状腺癌、胆管癌、肺癌、胶质母细胞瘤、间皮瘤、子宫癌、胰腺癌、胆管癌、威尔姆氏瘤或神经母细胞瘤。
在第十六个方面，本发明提供了一种试剂盒，包含上述第一或二个方面所述的抗体或抗原结合片段、第三个方面所述的偶联有示踪剂的抗体或抗原结合片段、第四个方面所述的多特异性抗原结合分子、第五个方面所述的嵌合抗原受体、第六个方面所述的免疫效应细胞、第七个方面所述的分离的核酸分子、第八个方面所述的表达载体、第九个方面所述的细胞、或第十或第十一个方面所述方法制备的产品、或第十二个方面所述的药物组合物，以及使用说明。
有益效果：本发明旨在开发特异性结合CDH6的单克隆抗体，其具有较佳的结合活性和内化能力，可用于后续CDH6-ADC等药物的开发，并用于治疗卵巢癌和肾癌等疾病。
术语定义和说明
如本文所用，术语“抗体”(Ab)是指与目标抗原特异性结合或具有免疫反应性的免疫球蛋白分子，包括抗体的多克隆、单克隆、基因工程化和其他修饰形式(包括但不限于嵌合抗体，人源化抗体，全人源抗体，异源偶联抗体(例如双特异性、三特异性和四特异性抗体，双抗体，三抗体和四抗体，抗体缀合物)以及抗体的抗原结合片段(包括例如Fab’、F(ab’)2、Fab、Fv、rIgG和scFv片段)。此外，除非另有说明，否则术语“单克隆抗体”(mAb)意指包括能够特异性结合靶蛋白的完整抗体分子以及不完整的抗体片段(例如Fab和F(ab’)2片段，它们缺少完整抗体的Fc片段(从动物循环中更快地清除)，因此缺乏Fc介导的效应功能(effector function)(参见Wahl等人，J.Nucl.Med.24:316,1983；其内容通过引用并入本文)。
本文“抗体”可以来源于任何动物，包括但不限于人和非人动物，所述非人动物可选自灵长类动物、哺乳动物、啮齿动物和脊椎动物，例如骆驼科动物、大羊驼、原鸵、羊驼、羊、兔、小鼠、大鼠或软骨鱼纲(例如鲨)。
本文术语“单特异性”是指具有一个或多个结合位点，其中每个结合位点结合相同抗原的相同表位。
本文术语“多特异性”是指具有至少两个抗原结合位点，所述至少两个抗原结合位点中的每一个抗原结合位点与相同抗原的不同表位或与不同抗原的不同表位结合。因此，诸如“双特异性”、“三特异性”、“四特异性”等术语是指抗体/抗原结合分子可以结合的不同表位的数目。
本文“全长抗体”、“完好抗体”和“完整抗体”可互换使用，是指其具有基本上与天然抗体结构相似的结构。
如本文所用，术语“抗原结合片段”是指保留特异性结合靶抗原的能力的一个或更多个抗体片段。抗体的抗原结合功能可以由全长抗体的片段执行。抗体片段可以是Fab、F(ab’)2、scFv、SMIP、双抗体、三抗体、亲和体(affibody)、纳米抗体、适体或结构域抗体。涵盖术语抗体的“抗原结合片段”的结合片段的实例包括但不限于：(i)Fab片段，一种由VL、VH、CL和CHl结构域组成的单价片段；(ii)F(ab)2片段，一种包含由二硫键在铰链区连接的两个Fab片段的双价片段；(iii)由VH和CHl结构域组成的Fd片段；(iv)由抗体单臂的VL和VH结构域组成的Fv片段；(V)包含VH和VL结构域的dAb；(vi)由VH结构域组成的dAb片段(Ward等人，Nature 341:544-546,1989；其内容通过引用并入本文)；(vii) 由VH或VL结构域组成的dAb；(viii)分离的互补决定区(CDR)；以及(ix)两个或更多个分离的CDR的组合，所述CDR可以任选地由合成接头连接。此外，虽然Fv片段的两个结构域VL和VH是通过独立的基因编码的，但是这两个结构域可以使用重组方法通过接头接合，该接头能够使其制成其中VL和VH区配对以形成单价分子的单蛋白质链(称为单链Fv(scFv)；参见例如，Bird等人，Science 242:423-426,1988以及Huston等人，Proc.Natl.Acad.Sci.USA 85:5879-5883,1988；其内容通过引用并入本文)。这些抗体片段可以使用本领域技术人员已知的常规技术获得，并且这些片段被筛选用于与完整抗体相同的方式使用。可以通过重组DNA技术、完整免疫球蛋白的酶促或化学裂解、或在一些实施方式中通过本领域已知的化学肽合成程序来产生抗原结合片段。
如本文所用，术语“CDH6”又称“钙粘蛋白6”，是指细胞粘附分子,它是细胞-细胞粘附分子的钙粘蛋白家族的成员。CDH6是由790个氨基酸构成的单次跨膜蛋白，其被分类为II型钙黏着蛋白家族，并且该蛋白具有N-末端细胞外和C-末端细胞内结构域。用于本发明的CDH6蛋白可以直接从人或非人哺乳动物(例如大鼠、小鼠或猴)的表达CDH6的细胞中纯化，并且随后可以使用，或者可以制备上述细胞的细胞膜级分并且可以作为CDH6蛋白使用。或者，CDH6也可以通过在体外合成或通过遗传操作而允许宿主细胞产生CDH6而获得。根据这种遗传操作，可以获得CDH6蛋白，具体地，将CDH6 cDNA并入到能够表达CDH6 cDNA的载体中，并且随后在含有酶、底物和转录和翻译所需的能量材料的溶液中合成CDH6，或者通过转化其他原核生物或真核生物的宿主细胞，从而允许其表达CDH6。基于上述遗传操作的表达CDH6的细胞或表达CDH6的细胞系也可以用于呈递CDH6蛋白。或者，可以直接向待免疫的动物施用已并入CDH6 cDNA的表达载体，并且CDH6可以在如此免疫的动物体内表达。
如本文所用，术语“双特异性抗体”是指对至少两种不同的抗原具有单克隆结合特异性的抗体，其通常是人或人源化的抗体。在本发明中，结合特异性之一可以针对CDH6的抗原表位而被检测，另一个可以针对CDH6的另一个抗原表位或除CDH6外的任何其他抗原，例如针对细胞表面蛋白、受体、受体亚基、组织特异性抗原、病毒来源蛋白、病毒编码的包膜蛋白、细菌来源蛋白或细菌表面蛋白等而被检测。
如本文所用，术语“嵌合”抗体是指以下抗体，其具有源自一种来源生物(如大鼠或小鼠)的免疫球蛋白的可变序列以及源自不同生物体(例如人)的免疫球蛋白的恒定区。用于生产嵌合抗体的方法是本领域已知的。
如本文所用，术语“互补决定区”(CDR)指在轻链和重链可变结构域中均发现的高变区。可变结构域中更高保守性的部分称为框架区(FR)。如本领域所理解的，表示抗体的高变区的氨基酸位置可以根据上下文和本领域已知的各种定义而变化。可变结构域内的一些位置可以被视为杂合高变位置，因为这些位置可以被认为是在一组标准(如IMGT或KABAT)下的高变区之内，而被认为在不同组的标准(如KABAT或IMGT)下的高变区之外。这些位置中的一个或更多个也可以在延伸的高变区中找到。本发明包括在这些杂合高变的位置中包含修饰的抗体。天然重链和轻链的可变结构域各自包含主要采用片层构型的四个框架区，其通过三个CDR(CDR1、CDR2和CDR3)连接，这三个CDR形成连接片层结构的环，并且在一些情况下形成片层结构的一部分。每条链中的CDR通过FR区按顺序 FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4紧密保持在一起，并且与来自其他抗体链的CDR促成了抗体的抗原结合位点的形成(参见Kabat等人，Sequences of Protein sofImmunological Interest,National Institute of Health,Bethesda,Md.1987；其通过引用并入本文)。例如在本文中，CDR1-VH、CDR2-VH和CDR3-VH分别是指重链可变区(VH)的第一个CDR、第二个CDR和第三个CDR，这三个CDR构成了重链(或其可变区)的CDR组合(VHCDR组合)；CDR1-VL、CDR2-VL和CDR3-VL分别是指轻链可变区(VL)的第一个CDR、第二个CDR和第三个CDR，这三个CDR构成了轻链(或其可变区)的CDR组合(VLCDR组合)。
如本文所用，术语“单克隆抗体”是指来源于单个克隆(包括任何真核、原核、或噬菌体克隆)的抗体，而不限于该抗体的产生方法。
如本文所用，术语“VH”是指抗体的免疫球蛋白重链(包括Fv、scFv或Fab的重链)的可变区。术语“VL”是指免疫球蛋白轻链(包括Fv、scFv、dsFv或Fab的轻链)的可变区。
本文术语“重链恒定区”是指抗体重链的羧基端部分，其不直接参与抗体与抗原的结合，但是表现出效应子功能，诸如与Fc受体的相互作用，其相对于抗体的可变结构域具有更保守的氨基酸序列。“重链恒定区”至少包含以下之一：CH1结构域，铰链区，CH2结构域，CH3结构域，或其变体或片段。“重链恒定区”包括“全长重链恒定区”和“重链恒定区片段”，前者具有基本上与天然抗体恒定区基本相似的结构，而后者仅包括“全长重链恒定区的一部分”。示例性地，典型的“全长抗体重链恒定区”由CH1结构域-铰链区-CH2结构域-CH3结构域组成；当抗体为IgE时，其还包括CH4结构域；当抗体为重链抗体时，则其不包括CH1结构域。示例性地，典型的“重链恒定区片段”可选自CH1、Fc或CH3结构域。
本文术语“轻链恒定区”是指抗体轻链的羧基端部分，其不直接参与抗体与抗原的结合，所述轻链恒定区可选自恒定κ结构域或恒定λ结构域。
本文术语“Fc”是指完整抗体经木瓜蛋白水解而成的抗体羧基端部分，典型地，其包含抗体的CH3和CH2结构域。Fc区包括例如天然序列Fc区、重组Fc区和变体Fc区。尽管免疫球蛋白重链的Fc区的边界可以略微变化，但是人IgG重链的Fc区通常被定义为从Cys226位置的氨基酸残基或从Pro230延伸至其羧基末端。Fc区的C末端赖氨酸(根据EU编号系统的残基447)可以例如在抗体的产生或纯化过程中，或通过对编码抗体重链的核酸重组工程化而除去，因此，Fc区可包括或不包括Lys447。
本文术语“人源化抗体”是指，经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留或部分保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力、增强免疫应答的能力等。
本文术语“全人源抗体”是指具有其中FR和CDR二者都源自人种系免疫球蛋白序列的可变区的抗体。此外，如果抗体包含恒定区，则恒定区也源自人种系免疫球蛋白序列。本文全人源抗体可以包括不由人种系免疫球蛋白序列编码的氨基酸残基(例如，通过体外随机或位点特异性诱变或通过体内体细胞突变引入的突变)。然而，本文“全人源抗体”不意图包括其中来源于另一个哺乳动物物种(例如小鼠)的种系的CDR序列已被移植到人框架序列上的抗体。
本文术语“裸抗体”是指不与另一种作用剂或分子(例如标记或药物)、肽或多肽连接、融合或缀合的抗体。在具体的实施方案中，由哺乳动物宿主细胞表达的裸抗体可被宿主细胞的糖基化机器(例如糖基化酶)糖基化。在某些实施方案，当通过不具有其自身糖基化机器(例如糖基化酶)的宿主细胞表达时，裸抗体不被糖基化。在某些实施方案中，裸抗体是完整抗体，而在其它实施方案中，裸抗体是完整抗体的抗原结合片段，例如Fab抗体。
本文术语“缀合抗体”是指可与药学上可接受的载体或稀释剂缔合的抗体，其可为单克隆抗体、嵌合抗体、人源化抗体或人抗体。
本文术语“双抗体”是指二价的双特异性抗体，可以与相同或不同抗原上的不同表位结合。
本文术语“抗体最小识别单位”是指在抗原抗体的结合反应中，抗体所能识别抗原的最小单位。
本文术语“纳米抗体”是指骆驼体内存在天然的缺失轻链的重链抗体，克隆其可变区可以得到只有重链可变区组成的单域抗体，也称为VHH(Variable domain of heavy chain of heavy chain antibody)，它是最小的功能性抗原结合片段
如本文所用，术语“百分比(％)序列一致性”是指在为达到最大百分比序列一致性而比对序列和引入空位(如果需要)(例如，为了最佳比对，可以在候选和参比序列中的一个或两个中引入空位，并且出于比较的目的，可以忽略非同源序列)之后，候选序列的氨基酸(或核苷酸)残基与参比序列的氨基酸(或核苷酸)残基相同的百分比。出于确定百分比序列一致性的目的，可以用本领域技术人员熟知的多种方式来实现比对，例如使用公众可得的计算机软件，如BLAST、ALIGN或Megalign(DNASTAIi)软件。本领域技术人员可以确定用于测量比对的适当参数，包括需要在被比较序列的全长范围实现最大比对的任何算法。例如，用于与候选序列进行比较而比对的参比序列可以显示候选序列在候选序列的全长或候选序列的连续氨基酸(或核苷酸)残基的选定部分上表现出从50％至100％的序列同一性。出于比较目的而比对的候选序列的长度可以是例如参比序列的长度的至少30％(例如30％、40％、50％、60％、70％、80％、90％或100％)。当候选序列中的位置被与在参比序列中的相应位置相同的氨基酸(或核苷酸)残基占据时，则这些分子在那个位置是相同的。
本文术语“保守氨基酸”通常是指属于同一类或具有类似特征(例如电荷、侧链大小、疏水性、亲水性、主链构象和刚性)的氨基酸。示例性地，下述每组内的氨基酸彼此属于保守氨基酸残基，组内氨基酸残基的替换属于保守氨基酸的替换：
(1)酸性氨基酸：Asp(D)和Glu(E)；
(2)碱性氨基酸：Lys(K)、Arg(R)和His(H)；
(3)亲水性不带电荷氨基酸：Ser(S)、Thr(T)、Asn(N)和Gln(Q)；
(4)脂肪族不带电荷氨基酸：Gly(G)、Ala(A)、Val(V)、Leu(L)和Ile(I)；
(5)非极性不带电荷的氨基酸：Cys(C)、Met(M)和Pro(P)；
(6)芳香族氨基酸：Phe(F)、Tyr(Y)和Trp(W)。
本文术语“Kabat编号系统”通常是指由Elvin A.Kabat提出的免疫球蛋白比对及编号系统(参见，例如Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health  Service,National Institutes of Health,Bethesda,Md.,1991；其通过引用并入本文)。
如本文所用，术语“特异性结合”是指一种结合反应，其决定抗原在蛋白质和其他生物分子的一个异质性群体中的存在状况，所述蛋白质和其他生物分子例如被抗体或其抗原结合片段特异性识别。与抗原特异性结合的抗体或其抗原结合片段将以小于100nM的KD与抗原结合。例如，与抗原特异性结合的抗体或其抗原结合片段将以高达100nM(例如，1pM至100nM之间)的KD与抗原结合。不显示与特定抗原或其表位特异性结合的抗体或其抗原结合片段将显示对该特定抗原或其表位的大于100nM(例如，大于500nM、1μM、100μΜ、500μΜ或1mM)的KD。可以使用多种免疫测定方式来选择与特定蛋白或碳水化合物进行特异性免疫反应的抗体。例如，常规地使用固相ELISA免疫测定法来选择与蛋白质或碳水化合物进行特异性免疫反应的抗体。参见，Harlow&Lane，Antibodies,ALaboratory Manual,Cold Spring Harbor Press,NewYork(1988)以及Harlow&Lane,Using Antibodies，A Laboratory Manual,Cold Spring Harbor Press,NewYork(1999)，其描述了可以用于确定特异免疫反应性的免疫测定方式和条件(其通过引用并入本文)。
如本文所用，术语“抗体缀合物”是指抗体分子直接或者通过连接接头与另一个分子化学键合而形成的偶联体/缀合物。例如抗体-药物缀合物(ADC)，其中药物分子就是所述的另一个分子。
本文术语“核酸”包括包含核苷酸的聚合物的任何化合物和/或物质。每个核苷酸由碱基，特别是嘌呤或嘧啶碱基(即胞嘧啶(C)、鸟嘌呤(G)、腺嘌呤(A)、胸腺嘧啶(T)或尿嘧啶(U))、糖(即脱氧核糖或核糖)和磷酸基团组成。通常，核酸分子由碱基的序列描述，由此所述碱基代表核酸分子的一级结构(线性结构)。碱基的序列通常表示为5′至3′。在本文中，术语核酸分子涵盖脱氧核糖核酸(DNA)，包括例如互补DNA(cDNA)和基因组DNA、核糖核酸(RNA)，特别是信使RNA(mRNA)、DNA或RNA的合成形式，以及包含两种或更多种这些分子的混合的聚合物。核酸分子可以是线性的或环状的。此外，术语核酸分子包括有义链和反义链二者，以及单链和双链形式。而且，本文所述的核酸分子可含有天然存在的或非天然存在的核苷酸。非天然存在的核苷酸的例子包括具有衍生的糖或磷酸骨架键合或化学修饰的残基的修饰的核苷酸碱基。核酸分子还涵盖DNA和RNA分子，其适合作为载体用于在体外和/或体内，例如在宿主或患者中，直接表达本发明的抗体。此类DNA(例如cDNA)或RNA(例如mRNA)载体可以是未修饰的或修饰的。例如，可以对mRNA进行化学修饰以增强RNA载体的稳定性和/或被编码分子的表达，从而可以将mRNA注入到受试者内以在体内产生抗体(参见例如Stadler等人，Nature Medicine 2017，published online 2017年6月12日，doi：10.1038/nm.4356或EP2 101 823B1；其内容通过引用并入本文)。
如本文所用，术语“载体”包括核酸载体，例如DNA载体(如质粒)，RNA载体，病毒或其他适合的复制子(例如病毒载体)。已经开发了多种载体用于将编码外源蛋白质的多核苷酸递送到原核或真核细胞中。本发明的表达载体含有多核苷酸序列以及例如用于表达蛋白质和/或将这些多核苷酸序列整合到哺乳动物细胞基因组中的附加序列元件。可以用于表达本发明的抗体和抗体片段的某些载体包括含有指导基因转录的调控序列(如启动子和增强子区域)的质粒。用于表达抗体和抗体片段的其他有用的载体含有多核苷酸序列，其增强这些基因的翻译速率或改善由基因转录产生的mRNA的稳定性或核输出。这些序列元件包括例如5’ 和3’非翻译区、内部核糖体进入位点(IRES)和聚腺苷酸化信号位点，以便指导表达载体上携带的基因的有效转录。本发明的表达载体还可以含有以下多核苷酸，该多核苷酸编码用于选择含有这种载体的细胞的标记。适合的标记的实例包括编码抗生素(如氨苄青霉素、氯霉素、卡那霉素或诺尔丝菌素)抗性的基因。
本文术语“宿主细胞”是指细胞中引入外源核酸的细胞，包括这种细胞的后代。宿主细胞包括“转化体”和“经转化的细胞”，其包括原代的经转化的细胞和来源于其的后代，而不考虑传代的次数。后代在核酸内容物上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括具有与在初始转化的细胞中筛选或选择的相同功能或生物学活性的突变体后代。
本文术语“药物组合物”是指这样的制剂，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不含有对施用所述药物组合物的受试者具有不可接受的毒性的另外的成分。
如本文所用，术语“受试者”、“对象”和“患者”是指接受对如本文所述的特定疾病或病症(如癌症或传染性疾病)的治疗的生物体。对象和患者的实例包括接受疾病或病症(例如细胞增殖性病症，如癌症或传染性疾病)的治疗的哺乳动物，如人、灵长类动物、猪、山羊、兔、仓鼠、猫、狗、豚鼠、牛科家族成员(如家牛、野牛、水牛、麋鹿和牦牛等)、绵羊和马等。
如本文所用，术语“治疗”是指外科手术或药物处理(surgical or therapeutic treatment)，其目的是预防、减缓(减少)治疗对象中不希望的生理变化或病变，如细胞增殖性病症(如癌症或传染性疾病)的进展。有益的或所希望的临床结果包括但不限于症状的减轻、疾病程度减弱、疾病状态稳定(即，未恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和、以及缓解(无论是部分缓解或完全缓解)，无论是可检测的或不可检测的。需要治疗的对象包括已患有病症或疾病的对象以及易于患上病症或疾病的对象或打算预防病症或疾病的对象。当提到减缓、减轻、减弱、缓和、缓解等术语时，其含义也包括消除、消失、不发生等情况。
本文术语“有效量”指单独给予或与另一治疗剂组合给予细胞、组织或对象时能有效防止或缓解疾病病症或该疾病进展的治疗剂用量。“有效量”还指足以缓解症状，例如治疗、治愈、防止或缓解相关医学病症，或治疗、治愈、防止或缓解这些病症的速度增加的化合物用量。当将活性成分单独给予个体时，治疗有效剂量单指该成分。当应用某一组合时，治疗有效剂量指产生治疗作用的活性成分的组合用量，而无论是组合、连续或同时给予。
本文术语“适当的条件”指适合培养各种宿主细胞的条件，其中宿主细胞包括真核细胞和原核细胞。
本文术语“肿瘤”指所有赘生性(neoplastic)细胞生长和增殖,无论是恶性的还是良性的,及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”和“肿瘤”在本文中提到时并不互相排斥。
本文术语“抗肿瘤剂”指抗肿瘤药物，其为治疗肿瘤疾病的一类药物，例如化疗药物、生物制剂等。
本文术语“EC50”是指半最大有效浓度，其包括在指定暴露时间之后诱导基线与最大值之间的半途响应的抗体浓度。EC50本质上代表其中观察到其最大作用的50％的抗体浓度，可通过本领域已知方法测量。
附图说明
除非本发明另外定义，与本发明相关的科学和技术术语应具有本领域普通技术人员所理解的含义。
图1-图2为hCDH6-1人源化抗体与human CDH6蛋白的结合活性。
图3-图4为hCDH6-1人源化抗体与OVCAR3细胞的结合活性。
图5-图6为hCDH6-6人源化抗体与human CDH6蛋白的结合活性。
图7-图8为hCDH6-6人源化抗体与OVCAR3细胞的结合活性。
图9-图11为hCDH6-14人源化抗体与human CDH6蛋白的结合活性。
图12-图13为hCDH6-14人源化抗体与PA-1细胞的结合活性。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
本发明实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
实施例1抗人CDH6鼠源单克隆抗体的制备
1.1动物免疫
1.1.1蛋白免疫
免疫采用6-8周龄的SJL小鼠5只(小鼠编号#2401～2405)或MRL/lpr小鼠5只(小鼠编号#2136～2140)(购自上海斯莱克实验动物有限公司)，小鼠在SPF条件下饲养。初次免疫时，使用带Fc标签的人CDH6胞外区蛋白(hCDH6 ECD-hFc,常规合成，hCDH6 ECD序列来源于Uniprot#P55285(19-615)，下述SEQ ID NO：1所示，常规合成)，与Titer max(购自Sigma，货号T2684)和寡核苷酸酸CPG(ODN 1826,合成自上海生工生物)混合乳化后注射足垫和背部，hCDH6 ECD-hFc蛋白与Imject Alum(购自Thermo fisher,货号77161)和CPG混合后注射腹腔，每只小鼠注射50μg抗原。第一次加强免疫时，使用hCDH6 ECD-hFc蛋白、Imject Alum和CPG混匀后注射足垫和背部。第二次加强免疫时使用hCDH6 ECD-hFc蛋白、Titer max和CPG混合乳化后注射背部。后续的加强免疫按照第一次和第二次加强免疫的方式交替进行，每次每只小鼠注射25μg抗原，每次免疫之间均间隔7天。分别在第二次和第四次加强免疫后的第5天进行小鼠的采血操作，分离血清，使用酶联免疫吸附(ELISA)方法测定血清中特异性抗体的滴度。
hCDH6 ECD，SEQ ID NO：1(Uniprot#P55285(19-615))

ELISA检测方法如下：将带his标签的人CDH6胞外区蛋白(hCDH6 ECD-his，常规合成，hCDH6 ECD序列来源于Uniprot#P55285(19-615)，SEQ ID NO：1所示)用PBS稀释到终浓度1μg/mL，以100μl每孔加入96孔ELISA板，4℃孵育过夜。第二天用洗板液(PBS+0.01％(v/v)Tween20)洗板3次，加入封闭液(PBS+0.01％(v/v)Tween20+2％(w/w)BSA)室温封闭2小时。弃去封闭液，用洗板液洗板3次。用1％BSA+PBS稀释小鼠血清，50μl每孔加入96孔ELISA板中。37℃孵育2小时后，用洗板液洗板3次。加入1：5000稀释的辣根过氧化物酶标记的二抗(Peroxidase AffiniPure Goat Anti-Mouse IgG(H+L)，购自Jackson Immuno，货号115-035-003)50μL每孔，37℃孵育1小时后，用洗板液洗板3次。加入TMB底物50μl每孔，室温孵育10分钟后，加入终止液(1.0N HCl)50μl每孔。用酶标仪(Biotek)读取OD450nm数值。
ELISA实验结果如表1所示，经过5次免疫后，所免疫小鼠的血清与人CDH6蛋白结合的滴度都较高，呈现抗原抗体反应。其中TB2指5次免疫(初次免疫+4次加强免疫)后第5天的小鼠血清，表中数据为OD450nm值。
表1 ELISA检测hCDH6 ECD-hFc蛋白免疫后小鼠血清效价
1.1.2细胞和蛋白免疫
免疫采用6-8周龄的SJL小鼠3只(小鼠编号#2453，2454，2456)或MRL/lpr小鼠5只(小鼠编号#2448～2452)(购自上海斯莱克实验动物有限公司)，小鼠在SPF条件下饲养。初次免疫时，使用过表达人CDH6蛋白的HEK293T细胞(HEK293T-hCDH6,常规构建，HEK293T细胞购自中国科学院细胞库，货号SCSP-502)，与Titer max(购自Sigma，货号T2684)混合后腹腔注射，每只小鼠注射5×106个细胞。第一次加强免疫时，使用 HEK293T-hCDH6细胞和CPG混匀后腹腔注射；第二次加强免疫时使用HEK293T-hCDH6细胞和Titer max混合后腹腔注射。后续的加强免疫按照第一次和第二次加强免疫的方式交替进行，每次每只小鼠注射5×106个细胞，每次免疫之间均间隔7天。分别在第二次和第四次加强免疫后的第5天进行小鼠的采血操作，分离血清，使用ELISA方法测定血清中特异性抗体的滴度。根据小鼠滴度，于5次细胞免疫(初次免疫+4次加强免疫)后给予小鼠hCDH6 ECD-hFc蛋白免疫(蛋白来源同1.1.1)。
使用酶联免疫吸附(ELISA)方法测定血清中特异性抗体的滴度，ELISA实验结果如表2所示。经过5次细胞免疫和3次蛋白免疫后，所免疫小鼠的血清与人CDH6蛋白结合的滴度都较高，呈现抗原抗体反应。其中TB4指8次免疫后第5天的小鼠血清，表中数据为OD450nm值。
表2 ELISA检测HEK293T-hCDH6细胞和hCDH6 ECD-hFc蛋白免疫后小鼠血清效价
1.2杂交瘤制备
挑选效价较高的小鼠，于腹腔、足底和背部共注射50μg hCDH6 ECD-hFc蛋白(蛋白来源同1.1.1)，3天后处死小鼠，收集脾细胞和淋巴细胞。1500rpm/min离心后，弃掉上清，在细胞中加入ACK裂解液(购自Gibco,货号A1049201)，裂解细胞中掺杂的红细胞，获得细胞悬液。用DMEM基础培养基(购自Gibco，货号10569044)1500rpm/min清洗细胞3次，然后按活细胞数目2:1与小鼠骨髓瘤细胞SP2/0(购自ATCC，货号CRL-1581)混合，采用电融合方法(仪器为BTX 2001+)进行细胞融合。融合后的细胞稀释到含20％(w/w)胎牛血清(购自ExCell Bio，货号FND500)、1×HAT(购自Sigma，货号H0262-10VL)、牛胰岛素(购自Yeason，货号40107ES25)、NEAA(购自Gibco，货号11140050)的DMEM培养基中，然后按5×104个/200μL每孔加入96孔细胞培养板中，放入5％(v/v)CO2、37℃培养箱中培养。
7天后用ELISA方法筛选融合板上清以确定对人CDH6蛋白的结合活性；对阳性克隆上清，通过ELISA方法确定对猴CDH6蛋白、鼠CDH6蛋白和过表达人CDH6细胞的结合活性。根据筛选结果，挑选符合条件的阳性克隆，用半固体培养基(购自Stemcell，货号03810)进行亚克隆，7天后将长出的克隆逐个挑入96孔培养板中，在含10％(w/w)胎牛血清、1×HT (购自Sigma，货号H0137-10VL)的DMEM培养基中扩大培养，1天后用ELISA方法进行初步筛选，挑选出有人CDH6蛋白结合活性的单克隆扩增到24孔板继续培养。3天后对培养上清进行进一步检测，评价其对猴CDH6蛋白、鼠CDH6蛋白的结合活性以及在过表达人CDH6细胞上的内吞活性。根据24孔板样品检测结果，挑选出最优的克隆，并于含10％(w/w)FBS的DMEM培养基中在37℃、5％(v/v)CO2条件下进行扩大培养，液氮冻存即得最优的杂交瘤细胞，并可用于后续的抗体生产和纯化。
实施例2嵌合抗体的制备和鉴定
2.1嵌合抗体的制备
挑选实施例1中活性较好的杂交瘤克隆进行测序，阳性对照抗人CDH6抗体DS-H01L02和NOV0712序列来源于专利WO2018212136A1，对应的序列信息如下表3和表4所示，其中表3为鼠源单克隆抗体的VH和VL序列，表4为鼠源单克隆抗体分子的CDRs(KABAT分析)。
表3鼠源单克隆抗体和阳性对照抗体的VH和VL序列及嵌合抗体的恒定区序列


表4鼠源单克隆抗体分子及阳性对照抗体的CDRs(KABAT分析)

根据上述序列，构建重链(HC)和轻链(LC)质粒(将编码抗体VH和VL的核酸序列重组至带有信号肽(SEQ ID NO：132，MGWSWILLFLLSVTAGVHS)和重链恒定区/轻链恒定区序列的表达载体上，得到表达VH-CH1-Fc/VL-CL的重组质粒)。将抗体相应的重链、轻链质粒和转染试剂PEI(购自Polysciences，货号：24765-1)加入OPTI-ME M(购自Gibco，货号：11058021)中混匀后静置15min，加入Expi293细胞(购自Thermofisher，货号：A14527)中，放入5％CO2，120rpm，37℃摇床培养。转染第二天，加入OPM-293ProFeed(购自上海奥浦迈，货号：F081918-001)和葡萄糖(购自Sigma，货号:G7528)。转染第六天，收集细胞上清，用Protein A(购自GE，货号：28985254)纯化，洗脱后的样品透析至PBS缓冲液(pH7.4)，得到嵌合抗体和阳性对照抗体。
2.2酶联免疫吸附实验(ELISA)检测嵌合抗体与人CDH6、猴CDH6和鼠CDH6蛋白 的结合
将人CDH6蛋白(hCDH6 ECD-his，常规合成)、猴CDH6蛋白(cynoCDH6 ECD-his，常规合成，cynoCDH6 ECD序列来源于Uniprot#A0A2K5TW62(19-615))和鼠CDH6蛋白(mCDH6 ECD-his，常规合成，mCDH6 ECD序列来源于Uniprot#P97326(19-615))用PBS稀释到终浓度1μg/mL，以100μl每孔加入96孔ELISA板。4℃孵育过夜，第二天用洗板液(PBS+0.01％(v/v)Tween20)洗板3次，加入封闭液(PBS+0.01％(v/v)Tween20+2％(w/w)BSA)室温封闭2小时。弃去封闭液，洗板3次。将嵌合抗体用1％BSA+PBS(购自Cytiva，货号SH30264.01)稀释，50μl每孔加入96孔板中。37℃孵育2小时后，用洗板液洗板3次。将辣根过氧化物酶标记的二抗Peroxidase AffiniPure Goat Anti-Human IgG,Fcγfragment specific(购自Jackson Immuno，货号109-035-098)用1％BSA 1：5000稀释，50μl每孔加入96孔板中，37℃孵育1小时后，用洗板液洗板5次。加入TMB底物(购自Seracare，货号5120-0077)50μl每孔，室温孵育10分钟后，加入终止液(1.0N HCl)50μl每孔。用酶标仪(Biotek)读取OD450nm数值。通过GraphPad Prism软件分析，进行数据拟合，计算EC50值。ELISA结果如表5所示，所测嵌合抗体与人CDH6蛋白、猴CDH6蛋白和鼠CDH6蛋白都有较强的结合。
表5嵌合抗体与人CDH6、猴CDH6和鼠CDH6蛋白的结合活性

注：“/”表示无结合或结合较弱，无法拟合EC50值或EC50>10nM。
2.3流式细胞实验(FACS)检测嵌合抗体与表达CDH6肿瘤细胞的结合
采用CDH6高表达的OVCAR3细胞和CDH6中表达的PA-1细胞检测抗体与细胞的结合活性。将OVCAR3细胞(购自ATCC，#HTB-161)或PA-1细胞(购自南京科佰生物科技有限公司，#CBP60800)培养至90％汇合度，吸尽培养基，用PBS洗涤1次，然后用无酶细胞解离液(Versene，购自Gibco，货号15040-066)处理和收集细胞。用PBS洗涤细胞1次， 进行细胞计数后用FACS缓冲液(PBS+2％FBS)重悬至2×106个细胞/mL，按每孔50μl加入到96孔板中。用FACS缓冲液(PBS+2％FBS)稀释嵌合抗体，按每孔50μl加入到96孔板中，4℃孵育1小时。用PBS洗涤3次，用FACS缓冲液按1：800稀释二抗Alexa647AffiniPure Goat Anti-Human IgG,Fcγfragment specific(购自Jackson Immuno，货号109-605-098)，每孔50μl加入96孔板中，4℃孵育1小时。用PBS离心洗涤2次后用FACS缓冲液重悬细胞。用FACS Canton II(BD公司)检测平均荧光强度(MFI)。通过GraphPad Prism软件分析，进行数据拟合，计算EC50值。FACS结果如表6所示，所测嵌合抗体与OVCAR3细胞和PA-1细胞均有较强结合。
表6嵌合抗体与OVCAR3和PA-1细胞的结合活性
2.4嵌合抗体的内吞活性检测
将OVCAR3细胞培养至90％汇合度，吸尽培养基，用PBS洗涤1次，然后用无酶细胞解离液(Versene，购自Gibco，货号15040-066)处理和收集细胞。用PBS洗涤细胞1次，进行细胞计数后用FACS缓冲液(PBS+2％FBS)重悬至2×106个细胞/mL，按每孔50μl加入到2块96孔板中。用FACS缓冲液(PBS+2％FBS)稀释嵌合抗体至10μg/ml，按每孔50μl分别加入到2块96孔板中，4℃孵育1h。用PBS洗涤细胞3次后，加入FACS缓冲液重悬细胞。1块96板置于4℃孵育1h；另1块96孔板置于37℃孵育1h。用PBS洗涤细胞3次后，每孔加入二抗Alexa647AffiniPure Goat Anti-Human IgG,Fcγfragment specific(购自Jackson Immuno，货号109-605-098)，4℃孵育1小时。用PBS离心洗涤2次后用FACS缓冲液重悬细胞。用FACS Canton II(BD公司)检测平均荧光强度(MFI)。结果如表7所示，所测嵌合抗体均具有较佳的内吞活性。
表7嵌合抗体的内吞活性
2.5 Biacore检测嵌合抗体与人CDH6蛋白的亲和力
应用BIAcore 8K仪器，采用多循环动力学法测定抗体与抗原的亲和力，在每一个循环中，首先用Protein A芯片(购自GE，货号：29-1275-56)捕获待测抗体，然后注入抗原人CDH6蛋白(hCDH6-his，常规合成)，记录抗体和抗原蛋白的结合和解离过程，最后用Glycine pH1.5完成芯片再生，其中流动相为HBS-EP+(10mM HEPES,150mM NaCl,3mM EDTA,0.05％surfactant P20,1mM Ca)，流速30μL/min，结合时间为240s，解离时间为300s，再生时间为30s，检测温度为25℃，抗原浓度为31.25-1000nM。应用Biacore 8K分析软件(版本号2.0)根据1:1binding模型，对数据进行分析，拟合抗体抗原结合动力学参数，包括结合速率常数ka、解离速率常数kd、平衡解离常数KD。结果见表8，对照抗体DS-H01L02的亲和力数值无法检测到，表格中未示出的抗体为亲和力数值无法检测到。
表8嵌合抗体与人CDH6蛋白的亲和力

备注：“/”表示无法检测到数值。
实施例3抗体人源化
通过比对IMGT(http://imgt.cines.fr)人类抗体重轻链可变区种系基因数据库，分别挑选与鼠源抗体同源性高的重链和轻链可变区种系基因作为模板，将根据Kabat划分的鼠源抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。基于抗体的三维结构，对包埋残基、与CDR区有直接相互作用的残基以及对VL和VH的构象有重要影响的构架区的残基进行回复突变，即得到人源化单克隆抗体。
3.1 CDH6-1的人源化
鼠源抗体CDH6-1的人源化轻链模板为IGKV4-1*01/IGKV1-33*01和IGKJ4*01，人源化重链模板为IGHV1-69*02和IGHJ6*01，将鼠源抗体CDH6-1的CDR分别移植到其人源模板中，CDR序列不变，即获得对应的人源化抗体hCDH6-1。根据需要，将hCDH6-1的人源化抗体的FR区序列中关键氨基酸进行回复突变为鼠源抗体对应的氨基酸，以保证原有的亲和力。具体回复突变设计见表9。
表9 hCDH6-1的人源化抗体回复突变设计
注：Graft代表将鼠源抗体CDR植入人种系模板FR区序列；G72E表示将Graft第72位G突变成E，其它依此类推。回复突变氨基酸的编号为自然顺序编号。
hCDH6-1人源化抗体可变区具体序列如下(突变以粗体示出)：
hCDH6-1.VL1氨基酸序列如SEQ ID NO：133所示：
hCDH6-1.VL2氨基酸序列如SEQ ID NO：134所示：
hCDH6-1.VL3氨基酸序列如SEQ ID NO：135所示：
hCDH6-1.VH1氨基酸序列如SEQ ID NO：136所示：
hCDH6-1.VH2氨基酸序列如SEQ ID NO：137所示：

hCDH6-13.VH3氨基酸序列如SEQ ID NO：138所示：
hCDH6-1.VH4氨基酸序列如SEQ ID NO：139所示：
人种系轻链模板IGKV4-1*01氨基酸序列如SEQ ID NO：140所示：
人种系轻链模板IGKV1-33*01氨基酸序列如SEQ ID NO：141所示：
人种系轻链模板IGKJ4*01氨基酸序列如SEQ ID NO：142所示：
人种系重链模板IGHV1-69*02氨基酸序列如SEQ ID NO：143所示：
人种系重链模板IGHJ6*01氨基酸序列如SEQ ID NO：144所示：
从上述hCDH6-1的人源化抗体轻链和重链可变区的回复突变设计中，选择不同的轻链和重链序列进行交叉组合，最终获得多个hCDH6-1人源化抗体，各抗体的可变区氨基酸序列如下：
表10 hCDH6-1抗体可变区对应氨基酸序列
根据上述序列，构建重链(HC)和轻链(LC)质粒(将编码抗体VH和VL的核酸序列重组至带有信号肽(SEQ ID NO：132，MGWSWILLFLLSVTAGVHS)和重链恒定区/轻链恒定区序列的表达载体上，得到表达VH-CH1-Fc/VL-CL的重组质粒)。按实施例2中的表达纯化方法，得到hCDH6-1人源化抗体，并用ELISA和FACS检测人源化抗体的结合活性(方法参见2.2,2.3)，结果如表11和图1-图4所示，hCDH6-1人源化抗体与人CDH6蛋白和OVCAR3细胞的结合活性与嵌合抗体相当。
表11 hCDH6-1人源化抗体与人CDH6蛋白和OVCAR3细胞的结合
挑选回复突变较少的4个hCDH6-1人源化抗体，用Biacore进一步确认与人CDH6蛋白的亲和力，结果如表12所示，挑选的hCDH6-1人源化抗体与嵌合抗体亲和力相当。
表12 hCDH6-1人源化抗体与人CDH6蛋白的亲和力
3.2 CDH6-6的人源化
鼠源抗体CDH6-6的人源化轻链模板为IGKV1-NL1*01/IGKV2-28*01和IGKJ2*01，人源化重链模板为IGHV1-46*01和IGHJ6*01，将鼠源抗体CDH6-6的CDR分别移植到其人源模板中，CDR序列不变，即获得对应的人源化版本hCDH6-6。根据需要，将hCDH6-6的人源化抗体的FR区序列中关键氨基酸进行回复突变为鼠源抗体对应的氨基酸，以保证原有的亲和力。具体回复突变设计见表13。
表13 hCDH6-6的人源化抗体回复突变设计

注：Graft代表将鼠源抗体CDR植入人种系模板FR区序列；A43S表示将Graft第43位A突变成S，其它
依此类推。回复突变氨基酸的编号为自然顺序编号。
hCDH6-6人源化抗体可变区具体序列如下(突变以粗体示出)：
hCDH6-6.VL1氨基酸序列如SEQ ID NO：145所示：
hCDH6-6.VL2氨基酸序列如SEQ ID NO：146所示：
hCDH6-6.VL3氨基酸序列如SEQ ID NO：147所示：
hCDH6-6.VL4氨基酸序列如SEQ ID NO：148所示：
hCDH6-6.VL5氨基酸序列如SEQ ID NO：149所示：
hCDH6-6.VH1氨基酸序列如SEQ ID NO：150所示：
hCDH6-6.VH2氨基酸序列如SEQ ID NO：151所示：
hCDH6-6.VH3氨基酸序列如SEQ ID NO：152所示：
人种系轻链模板IGKV1-NL1*01氨基酸序列如SEQ ID NO：153所示：
人种系轻链模板IGKV2-28*01氨基酸序列如SEQ ID NO：154所示：
人种系轻链模板IGKJ2*01氨基酸序列如SEQ ID NO：155所示：
人种系重链模板IGHV1-46*01氨基酸序列如SEQ ID NO：156所示：
人种系重链模板IGHJ6*01氨基酸序列如SEQ ID NO：144所示：
分别从上述hCDH6-6的人源化抗体轻链和重链可变区的回复突变设计中，选择不同的轻链和重链序列进行交叉组合，最终获得多种hCDH6-6人源化抗体，各抗体的可变区氨基酸序列如下：
表14 hCDH6-6抗体可变区对应氨基酸序列
根据上述序列，构建重链(HC)和轻链(LC)质粒(将编码抗体VH和VL的核酸序列重组至带有信号肽(SEQ ID NO：132，MGWSWILLFLLSVTAGVHS)和重链恒定区/轻链恒定区序列的表达载体上，得到表达VH-CH1-Fc/VL-CL的重组质粒)。按实施例2中的表达纯化方法，得到hCDH6-6人源化抗体，并用ELISA和FACS检测人源化抗体的结合活性(方法参见2.2，2.3)，结果如表15和图5-图8所示，hCDH6-6人源化抗体与人CDH6蛋白和OVCAR3细胞的结合活性与嵌合抗体相当。
表15 hCDH6-6人源化抗体与人CDH6蛋白和OVCAR3细胞的结合
挑选回复突变较少的4个hCDH6-6人源化抗体，用Biacore进一步确认与人CDH6蛋白的亲和力，结果如表16所示，挑选的4个hCDH6-6人源化抗体与嵌合抗体亲和力相当。
表16 hCDH6-6人源化抗体与人CDH6蛋白的亲和力

3.3 CDH6-14的人源化
鼠源抗体CDH6-14的人源化轻链模板为IGKV1-33*01/IGKV4-1*01和IGKJ4*01，人源化重链模板为IGHV1-3*01和IGHJ6*01，将鼠源抗体CDH6-14的CDR分别移植到其人源模板中，CDR序列不变，即获得对应的人源化版本hCDH6-14。根据需要，将hCDH6-14的人源化抗体的FR区序列中关键氨基酸进行回复突变为鼠源抗体对应的氨基酸，以保证原有的亲和力。具体回复突变设计见表17。
表17 hCDH6-14的人源化抗体回复突变设计

注：Graft代表将鼠源抗体CDR植入人种系模板FR区序列；P44F表示将Graft第44位P突变成F，
其它依此类推。回复突变氨基酸的编号为自然顺序编号。
hCDH6-14人源化抗体可变区具体序列如下(突变以粗体示出)：
hCDH6-14.VL1氨基酸序列如SEQ ID NO：157所示：
hCDH6-14.VL2氨基酸序列如SEQ ID NO：158所示：
hCDH6-14.VL3氨基酸序列如SEQ ID NO：159所示：
hCDH6-14.VH1氨基酸序列如SEQ ID NO：160所示：
hCDH6-14.VH2氨基酸序列如SEQ ID NO：161所示：
hCDH6-14.VH3氨基酸序列如SEQ ID NO：162所示：
hCDH6-14.VH4氨基酸序列如SEQ ID NO：163所示：
hCDH6-14.VH5氨基酸序列如SEQ ID NO：164所示：
hCDH6-14.VH6氨基酸序列如SEQ ID NO：165所示：
人种系轻链模板IGKV1-33*01氨基酸序列如SEQ ID NO：141所示：
人种系轻链模板IGKV4-1*01氨基酸序列如SEQ ID NO：140所示：
人种系轻链模板IGKJ4*01氨基酸序列如SEQ ID NO：142所示：
人种系重链模板IGHV1-3*01氨基酸序列如SEQ ID NO：166所示：
人种系重链模板IGHJ6*01氨基酸序列如SEQ ID NO：144所示：
分别从上述hCDH6-14的人源化抗体轻链和重链可变区的回复突变设计中，选择不同的轻链和重链序列进行交叉组合，最终获得多个hCDH6-14人源化抗体，各抗体的可变区氨基酸序列如下：
表18 hCDH6-14抗体可变区对应氨基酸序列
根据上述序列，构建重链(HC)和轻链(LC)质粒(将编码抗体VH和VL的核酸序列重组至带有信号肽(SEQ ID NO：132，MGWSWILLFLLSVTAGVHS)和重链恒定区/轻链恒定区序列的表达载体上，得到表达VH-CH1-Fc/VL-CL的重组质粒)。按实施例2中的表达纯化方法，得到hCDH6-14源化抗体，并用ELISA和FACS检测人源化抗体的结合活性(方法参见2.2，2.3)，结果如表19和图9-图13所示，hCDH6-14人源化抗体与人CDH6蛋白和PA-1细胞的结合活性与嵌合抗体相当。
表19 hCDH6-14人源化抗体与人CDH6蛋白和PA-1细胞的结合

3.4人源化抗体的内吞活性检测
选择部分抗体进行内吞活性检测，人源化抗体在OVCAR3细胞中的内吞活性检测方法同2.4。结果如表20所示，所测人源化抗体均具有较佳的内吞活性。
表20人源化抗体的内吞活性
实施例4 CDH6抗体的表位鉴定
人CDH6蛋白全长共有790个氨基酸(SEQ ID NO:167)，其中胞外区包含EC1(SEQ ID NO：168)、EC2(SEQ ID NO：169)、EC3(SEQ ID NO:170)、EC4(SEQ ID NO:171)和EC5(SEQ ID NO:172)。为确认CDH6抗体的结合表位，将这5个区域分别去除后的序列构建到pcDNA3.1载体(购自优宝生物，货号VT1001)上，并转染HEK293T细胞(购自中国科学院细胞库，货号SCSP-502)，得到5个CDH6截短细胞系：HEK293T-hCDH6-EC1deletion，HEK293T-hCDH6-EC2deletion，HEK293T-hCDH6-EC3deletion，HEK293T-hCDH6-EC4deletion和HEK293T-hCDH6-EC5deletion。
人CDH6蛋白全长序列(Uniprot#P55285)SEQ ID NO:167

EC1 SEQ ID NO:168
EC2 SEQ ID NO:169
EC3 SEQ ID NO:170
EC4 SEQ ID NO:171
EC5 SEQ ID NO:172
用FACS检测CDH6嵌合抗体与这5个细胞系的结合活性，结果如表21所示。根据检测结果，可推测抗体的结合表位：CHI-CDH6-1和CHI-CDH6-7与EC1-deletion细胞不结合，说明其结合表位为EC1；CHI-CDH6-5与EC1-deletion细胞不结合且与EC2-deletion细胞结合很弱，说明其结合表位可能在EC1-EC2之间，CHI-CDH6-4与EC2-deletion细胞不结合，说明其结合表位为EC2；CHI-CDH6-14与EC2-deletion和EC3-deletion细胞结合很弱，说明其结合表位可能在EC2-EC3之间；CHI-CDH6-3与EC3-deletion细胞很弱，说明其结合表位为EC3；CHI-CDH6-12与EC3-deletion细胞不结合，说明其结合表位为EC3。
表21 CDH6嵌合抗体与人CDH6的结合表位

注：MFI高于阴性对照hIgG1的MFI～1.5倍以上可认为有结合。

Claims (26)

  1. 一种特异性结合CDH6的抗体或抗原结合片段，所述抗体或抗原结合片段特异性结合CDH6蛋白胞外区EC1区、EC1-EC2区、EC2区、EC2-EC3区、EC3区、EC4区或EC5区的一个或多个氨基酸序列片段；优选的，所述抗体或抗原结合片段特异性结合SEQ ID NO：168-172任意一个序列中显示的一个或多个氨基酸序列片段；优选地，所述抗体或抗原结合片段具有内吞活性。
  2. 一种特异性结合CDH6的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段包含：
    (a)SEQ ID NO：2、4、6、8、10、12、14、16、18、20、22、24、26、28、136、137、138、139、150、151、152、160、161、162、163、164或165任一项所述VH的HCDR1、HCDR2和HCDR3；和/或，(b)SEQ ID NO：3、5、7、9、11、13、15、17、19、21、23、25、27、29、133、134、135、145、146、147、148、149、157、158或159任一项所述VL的LCDR1、LCDR2和LCDR3；
    优选地，所述HCDR1-3和/或所述LCDR1-3为根据KABAT、IMGT或Chothia的通行分析方法编码。
  3. 根据权利要求2所述的抗体或抗原结合片段，其特征在于，所述HCDR1、HCDR2和HCDR3具有选自以下的任意序列组合或者与所述序列组合相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列组合：

    和，
    所述LCDR1、LCDR2和LCDR3具有选自以下任意序列组合或者与所述序列组合相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列组合：
    优选地，所述替换为保守氨基酸的替换。
  4. 根据权利要求3所述的抗体或抗原结合片段，其特征在于，其包含选自以下的重链CDRs和轻链CDRs组合：VH1+VL1、VH2+VL2、VH3+VL3、VH4+VL4、VH5+VL5、VH6+VL6、VH7+VL7、VH8+VL8、VH9+VL9、VH10+VL10、VH11+VL11、VH12+VL12、VH13+VL13、或VH14+VL14，以及与所述重链CDRs和轻链CDRs组合的序列相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的CDRs组合；
    优选地，所述替换为保守氨基酸的替换。
  5. 根据权利要求1-4任一项所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段的重链可变区和轻链可变区的框架区来源于人种系重链模板和人种系轻链模板，其中：
    (1)框架区序列来源于人种系重链IGHV1-69*02和IGHJ6*01的组合序列；其包含SEQ ID NO：143所示IGHV1-69*02的FR1、FR2、FR3区和SEQ ID NO：144所示IGHJ6*01的FR4区；
    (2)框架区序列来源于人种系重链IGHV1-46*01和IGHJ6*01的组合序列；其包含SEQ ID NO：156所示IGHV1-46*01的FR1、FR2、FR3区和SEQ ID NO：144所示IGHJ6*01的FR4区；
    (3)框架区序列来源于人种系重链IGHV1-3*01和IGHJ6*01的组合序列；其包含SEQ ID NO：166所示IGHV1-3*01的FR1、FR2、FR3区和SEQ ID NO：144所示IGHJ6*01的FR4区；
    (4)框架区序列来源于人种系轻链IGKV4-1*01和IGKJ4*01的组合序列；其包含SEQ ID NO：140所示IGKV4-1*01的FR1、FR2、FR3区和SEQ ID NO：142所示IGKJ4*01的FR4区；
    (5)框架区序列来源于人种系轻链IGKV1-33*01和IGKJ4*01的组合序列；其包含SEQ ID NO：141所示IGKV1-33*01的FR1、FR2、FR3区和SEQ ID NO：142所示IGKJ4*01的FR4区；
    (6)框架区序列来源于人种系轻链IGKV1-NL1*01和IGKJ2*01的组合序列；其包含SEQ ID NO：153所示IGKV1-NL1*01的FR1、FR2、FR3区和SEQ ID NO：155所示IGKJ2*01的FR4区；或，
    (7)框架区序列来源于人种系轻链IGKV2-28*01和IGKJ2*01的组合序列；其包含SEQ ID NO：154所示IGKV2-28*01的FR1、FR2、FR3区和SEQ ID NO：155所示IGKJ2*01的FR4区。
  6. 根据权利要求5所述的抗体或抗原结合片段，其特征在于，根据Kabat编号系统编号，所述抗体或抗原结合片段的重链可变区和轻链可变区的框架区还包括选自下组的一种或多种突变，其中：
    (1)重链可变区的框架区包括：G27Y、S30T、A40R、I70L或A72V；优选包括G27Y和A72V；或优选包括G27Y、S30T和A72V；或优选包括G27Y、S30T、I70L和A72V；或优选包括G27Y、S30T、A40R和A72V；
    (2)重链可变区的框架区包括：G42R、M70L、R72V或T74K；优选包括R72V和 T74K；或优选包括M70L、R72V和T74K；或优选包括G42R、R72V和T74K；
    (3)重链可变区的框架区包括：V2I、V5Q、R44G、I70L、R72V、Y95L或R98S；优选包括R72V和R98S；或优选包括V2I、R72V和R98S；或优选包括V2I、I70L、R72V和R98S；或优选包括R44G、R72V和R98S；或优选包括V5Q、R72V和R98S；或优选包括R72V、Y95L和R98S；
    (4)轻链可变区的框架区包括：P44S、A47P或G72E；优选包括G72E；或优选包括P44S和G72E；或优选包括A47P和G72E；
    (5)轻链可变区的框架区包括：Q42K、A43S、K45Q、L48V、I48V或T85R；优选包括A43S；或优选包括A43S和L48V；或优选包括A43S、K45Q和L48V；或优选包括A43S、L48V和T85R；或优选包括Q42K和I48V；或，
    (6)轻链可变区的框架区包括：K42G、P43T、P44F或Y49S；优选包括P44F和Y49S；或优选包括K42G和Y49S；或优选包括P43T和Y49S。
  7. 根据权利要求1-6任一项所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段包含：
    (1)重链可变区具有SEQ ID NO:2、4、6、8、10、12、14、16、18、20、22、24、26、28、136、137、138、139、150、151、152、160、161、162、163、164或165所示序列；
    (2)轻链可变区具有SEQ ID NO:3、5、7、9、11、13、15、17、19、21、23、25、27、29、133、134、135、145、146、147、148、149、157、158或159所示序列；
    (3)与上述(1)～(2)中任一所述序列相比具有至少90％同一性的氨基酸序列，优选为至少91％、92％、93％、94％、95％、96％、97％、98％、99％同一性。
  8. 根据权利要求7所述的抗体或抗原结合片段，其特征在于，所述重链可变区和轻链可变区选自如下组：
    (1)具有SEQ ID NO.2所示的VH和SEQ ID NO.3所示的VL；
    (2)具有SEQ ID NO.4所示的VH和SEQ ID NO.5所示的VL；
    (3)具有SEQ ID NO.6所示的VH和SEQ ID NO.7所示的VL；
    (4)具有SEQ ID NO.8所示的VH和SEQ ID NO.9所示的VL；
    (5)具有SEQ ID NO.10所示的VH和SEQ ID NO.11所示的VL；
    (6)具有SEQ ID NO.12所示的VH和SEQ ID NO.13所示的VL；
    (7)具有SEQ ID NO.14所示的VH和SEQ ID NO.15所示的VL；
    (8)具有SEQ ID NO.16所示的VH和SEQ ID NO.17所示的VL；
    (9)具有SEQ ID NO.18所示的VH和SEQ ID NO.19所示的VL；
    (10)具有SEQ ID NO.20所示的VH和SEQ ID NO.21所示的VL；
    (11)具有SEQ ID NO.22所示的VH和SEQ ID NO.23所示的VL；
    (12)具有SEQ ID NO.24所示的VH和SEQ ID NO.25所示的VL；
    (13)具有SEQ ID NO.26所示的VH和SEQ ID NO.27所示的VL；
    (14)具有SEQ ID NO.28所示的VH和SEQ ID NO.29所示的VL；
    (15)具有SEQ ID NO.136所示的VH和SEQ ID NO.133所示的VL；
    (16)具有SEQ ID NO.137所示的VH和SEQ ID NO.133所示的VL；
    (17)具有SEQ ID NO.138所示的VH和SEQ ID NO.133所示的VL；
    (18)具有SEQ ID NO.139所示的VH和SEQ ID NO.133所示的VL；
    (19)具有SEQ ID NO.136所示的VH和SEQ ID NO.134所示的VL；
    (20)具有SEQ ID NO.137所示的VH和SEQ ID NO.134所示的VL；
    (21)具有SEQ ID NO.138所示的VH和SEQ ID NO.134所示的VL；
    (22)具有SEQ ID NO.139所示的VH和SEQ ID NO.134所示的VL；
    (23)具有SEQ ID NO.136所示的VH和SEQ ID NO.135所示的VL；
    (24)具有SEQ ID NO.137所示的VH和SEQ ID NO.135所示的VL；
    (25)具有SEQ ID NO.138所示的VH和SEQ ID NO.135所示的VL；
    (26)具有SEQ ID NO.139所示的VH和SEQ ID NO.135所示的VL；
    (27)具有SEQ ID NO.150所示的VH和SEQ ID NO.145所示的VL；
    (28)具有SEQ ID NO.150所示的VH和SEQ ID NO.146所示的VL；
    (29)具有SEQ ID NO.150所示的VH和SEQ ID NO.147所示的VL；
    (30)具有SEQ ID NO.150所示的VH和SEQ ID NO.148所示的VL；
    (31)具有SEQ ID NO.150所示的VH和SEQ ID NO.149所示的VL；
    (32)具有SEQ ID NO.151所示的VH和SEQ ID NO.145所示的VL；
    (33)具有SEQ ID NO.151所示的VH和SEQ ID NO.146所示的VL；
    (34)具有SEQ ID NO.151所示的VH和SEQ ID NO.147所示的VL；
    (35)具有SEQ ID NO.151所示的VH和SEQ ID NO.148所示的VL；
    (36)具有SEQ ID NO.151所示的VH和SEQ ID NO.149所示的VL；
    (37)具有SEQ ID NO.152所示的VH和SEQ ID NO.145所示的VL；
    (38)具有SEQ ID NO.152所示的VH和SEQ ID NO.146所示的VL；
    (39)具有SEQ ID NO.152所示的VH和SEQ ID NO.147所示的VL；
    (40)具有SEQ ID NO.152所示的VH和SEQ ID NO.148所示的VL；
    (41)具有SEQ ID NO.152所示的VH和SEQ ID NO.149所示的VL；
    (42)具有SEQ ID NO.160所示的VH和SEQ ID NO.157所示的VL；
    (43)具有SEQ ID NO.160所示的VH和SEQ ID NO.158所示的VL；
    (44)具有SEQ ID NO.160所示的VH和SEQ ID NO.159所示的VL；
    (45)具有SEQ ID NO.161所示的VH和SEQ ID NO.157所示的VL；
    (46)具有SEQ ID NO.161所示的VH和SEQ ID NO.158所示的VL；
    (47)具有SEQ ID NO.161所示的VH和SEQ ID NO.159所示的VL；
    (48)具有SEQ ID NO.162所示的VH和SEQ ID NO.157所示的VL；
    (49)具有SEQ ID NO.162所示的VH和SEQ ID NO.158所示的VL；
    (50)具有SEQ ID NO.162所示的VH和SEQ ID NO.159所示的VL；
    (51)具有SEQ ID NO.163所示的VH和SEQ ID NO.157所示的VL；
    (52)具有SEQ ID NO.163所示的VH和SEQ ID NO.158所示的VL；
    (53)具有SEQ ID NO.163所示的VH和SEQ ID NO.159所示的VL；
    (54)具有SEQ ID NO.164所示的VH和SEQ ID NO.157所示的VL；
    (55)具有SEQ ID NO.164所示的VH和SEQ ID NO.158所示的VL；
    (56)具有SEQ ID NO.164所示的VH和SEQ ID NO.159所示的VL；
    (57)具有SEQ ID NO.165所示的VH和SEQ ID NO.157所示的VL；
    (58)具有SEQ ID NO.165所示的VH和SEQ ID NO.158所示的VL；
    (59)具有SEQ ID NO.165所示的VH和SEQ ID NO.159所示的VL；或，
    (60)具有与上述(1)～(59)任一序列组合相比具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的VH和VL组合。
  9. 根据权利要求1-8任一项所述的抗体或抗原结合片段，其特征在于，其与人CDH6蛋白结合的解离常数(KD)不大于2×10-7M。
  10. 根据权利要求1-9任一项所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段为：
    (1)嵌合抗体或其片段；
    (2)人源化抗体或其片段；
    (3)全人源抗体或其片段；
    优选的，所述抗体或抗原结合片段选自单克隆抗体、多克隆抗体、天然抗体、工程化抗体、单特异性抗体、多特异性抗体(例如双特异性抗体)、单价抗体、多价抗体、全长抗体、抗体片段、裸抗体、缀合抗体、人源化抗体、全人源抗体、Fab、Fab’、F(ab’)2、Fd、Fv、scFv、双抗体(diabody)或单域抗体。
  11. 根据权利要求1-10任一项的抗体或抗原结合片段，其特征在于，所述抗体包含人或鼠抗体IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE或IgD任何其中之一恒定区的序列；优选 包含人或鼠抗体IgG1、IgG2、IgG3或IgG4的恒定区的序列。
  12. 根据权利要求1-11任一项的抗体或抗原结合片段，其特征在于，所述抗原结合片段选自F(ab)2、Fab’、Fab、Fv、scFv、双特异性抗体、纳米抗体和抗体最小识别单位中的一种或多种。
  13. 根据权利要求1-12任一项所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段进一步还偶联有治疗剂或示踪剂；优选地，所述治疗剂选自放射性同位素、化疗药或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂或光敏剂。
  14. 一种多特异性抗原结合分子，其特征在于，所述多特异性抗原结合分子包含第一抗原结合模块和第二抗原结合模块，所述第一抗原结合模块包含根据权利要求1-13任一项所述的抗体或抗原结合片段，所述第二抗原结合模块特异性结合CDH6以外的其他抗原或结合与第一抗原结合模块不同的CDH6抗原表位；
    优选地，所述其他抗原选自CD3、CD7、CD16、CD16A、CD4、CD5、CD8、CD14、CD15、CD19、CD20、CD21、CD23、CD25、CD33、CD37、CD38、CD40、CD40L、CD46、CD52、CD54、CD66(a-d)、CD74、CD80、CD126、CD138、BCMA、HLA-DR、HER2、VEGF、P1GF、HER3/ERBB3,HER4/ERBB4、IL-2、IL-6、PD-1、PD-L1、TRAIL-R1或TRAIL-R2；
    优选地，所述多特异性抗原结合分子为双特异性抗体、三特异性抗体或四特异性抗体。
  15. 一种嵌合抗原受体(CAR)，其特征在于，所述嵌合抗原受体至少包含细胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域，所述细胞外抗原结合结构域包含根据权利要求1-13任一项所述的CDH6抗体或抗原结合片段。
  16. 一种免疫效应细胞，其特征在于，所述免疫效应细胞包含根据权利要求15所述的嵌合抗原受体或包含编码根据权利要求15所述的嵌合抗原受体的核酸片段；
    优选地，所述免疫效应细胞选自T细胞、NK细胞(natural killer cell)、NKT细胞(natural killer T cell)、单核细胞、巨噬细胞、树突状细胞或肥大细胞；所述T细胞可选自炎性T细胞、细胞毒性T细胞、调节性T细胞(Treg)或辅助性T细胞；
    优选地，所述免疫效应细胞为同种异体免疫效应细胞或自体免疫细胞。
  17. 一种分离的核酸分子，其特征在于，所述核酸分子编码根据权利要求1-13任一项所述的抗体或抗原结合片段、或其任意组合，根据权利要求14所述的多特异性抗原结合分子或根据权利要求15所述的嵌合抗原受体。
  18. 一种包含根据权利要求17所述的分离的核酸分子的表达载体。
  19. 一种包含根据权利要求17所述的分离的核酸分子、或根据权利要求18所述的表达载体的分离的宿主细胞；优选地，所述宿主细胞是真核细胞或原核细胞；更优选地，所述宿主细胞来源于哺乳动物细胞、酵母细胞、昆虫细胞、大肠杆菌和/或枯草杆菌；更优选地，所述宿主细胞选自Expi293或CHO细胞。
  20. 一种制备根据权利要求1-13任一项所述的抗体或抗原结合片段或根据权利要求14所述的多特异性抗原结合分子的方法，其特征在于，在适当的条件下培养的根据权利要求19所述的宿主细胞，并分离抗体或抗原结合片段或多特异性抗原结合分子。
  21. 一种制备根据权利要求16所述的免疫效应细胞的方法，其特征在于，所述方法包括将编码根据权利要求15所述的嵌合抗原受体的核酸片段导入免疫效应细胞，可选地，所述方法还包括启动所述免疫效应细胞表达根据权利要求15所述的嵌合抗原受体。
  22. 一种药物组合物，其特征在于，所述组合物包含根据权利要求1-13任一项所述的抗体或抗原结合片段、根据权利要求14所述的多特异性抗原结合分子、根据权利要求15所述的嵌合抗原受体、根据权利要求16所述的免疫效应细胞、根据权利要求17所述的分离的核酸分子、根据权利要求18所述的表达载体、根据权利要求19所述的细胞，或根据权利要求20或21所述的方法制备的产品；优选地，所述组合物还包含药学上可接受的运载体(carrier)、稀释剂或助剂；优选地，所述药物组合物还包含额外的抗肿瘤剂。
  23. 根据权利要求1-13任一项所述的抗体或抗原结合片段、根据权利要求14所述的多特异性抗原结合分子、根据权利要求15所述的嵌合抗原受体、根据权利要求16所述的免疫效应细胞、根据权利要求17所述的分离的核酸分子、根据权利要求18所述的表达载体、根据权利要求19所述的细胞，根据权利要求20或21所述的方法制备的产品、或根据权利要求22所述的药物组合物在制备预防和/或治疗肿瘤疾病的药物中的用途；
    优选地，所述肿瘤疾病选自实体瘤，优选地，所述实体瘤为表达CDH6蛋白的实体瘤，更优选地，所述实体瘤选自肾癌、卵巢癌、甲状腺癌、胆管癌、肺癌、胶质母细胞瘤、间皮瘤、子宫癌、胰腺癌、胆管癌、威尔姆氏瘤或神经母细胞瘤。
  24. 一种预防和/或治疗肿瘤疾病的方法，包含向有此需要的患者施用有效量的根据权利要求1-13任一项所述的抗体或抗原结合片段、根据权利要求14所述的多特异性抗原结合分子、根据权利要求15所述的嵌合抗原受体、根据权利要求16所述的免疫效应细胞、根据权利要求17所述的分离的核酸分子、根据权利要求18所述的表达载体、根据权利要求19所述的细胞、根据权利要求20或21所述的方法制备的产品、或根据权利要求22所述的药物组合物；
    优选地，所述肿瘤疾病选自实体瘤，优选地，所述实体瘤为表达CDH6蛋白的实体瘤， 更优选地，所述实体瘤选自肾癌、卵巢癌、甲状腺癌、胆管癌、肺癌、胶质母细胞瘤、间皮瘤、子宫癌、胰腺癌、胆管癌、威尔姆氏瘤或神经母细胞瘤。
  25. 根据权利要求1-13任一项所述的抗体或抗原结合片段、根据权利要求14所述的多特异性抗原结合分子、根据权利要求15所述的嵌合抗原受体、根据权利要求16所述的免疫效应细胞、根据权利要求17所述的分离的核酸分子、根据权利要求18所述的表达载体、根据权利要求19所述的细胞、根据权利要求20或21所述的方法制备的产品、或根据权利要求22所述的药物组合物，其特征在于，用于和/或治疗肿瘤疾病；
    优选地，所述肿瘤疾病选自实体瘤，优选地，所述实体瘤为表达CDH6蛋白的实体瘤，更优选地，所述实体瘤选自肾癌、卵巢癌、甲状腺癌、胆管癌、肺癌、胶质母细胞瘤、间皮瘤、子宫癌、胰腺癌、胆管癌、威尔姆氏瘤或神经母细胞瘤。
  26. 一种试剂盒，其包含根据权利要求1-13任一项的抗体或其抗原结合片段、根据权利要求14所述的多特异性抗原结合分子、根据权利要求15所述的嵌合抗原受体、根据权利要求16所述的免疫效应细胞、根据权利要求17所述的分离的核酸分子、根据权利要求18所述的表达载体、根据权利要求19所述的细胞、根据权利要求20或21所述的方法制备的产品、或根据权利要求22所述的药物组合物；任选地，还包含使用说明。
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