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WO2017107973A1 - 一种tpbg抗体及其制备方法、其偶联物和应用 - Google Patents

一种tpbg抗体及其制备方法、其偶联物和应用 Download PDF

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Publication number
WO2017107973A1
WO2017107973A1 PCT/CN2016/111699 CN2016111699W WO2017107973A1 WO 2017107973 A1 WO2017107973 A1 WO 2017107973A1 CN 2016111699 W CN2016111699 W CN 2016111699W WO 2017107973 A1 WO2017107973 A1 WO 2017107973A1
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seq
amino acid
sequence
sequence listing
acid sequence
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French (fr)
Inventor
孙晓岚
张莹
张瑜
胡飞飞
宫世勇
刘礼乐
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Xdcexplorer (shanghai) Co Ltd
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Xdcexplorer (shanghai) Co Ltd
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Priority to US16/065,594 priority Critical patent/US20210198377A1/en
Priority to EP16877783.7A priority patent/EP3395834A4/en
Priority to JP2018533643A priority patent/JP2019508023A/ja
Publication of WO2017107973A1 publication Critical patent/WO2017107973A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/4025—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil not condensed and containing further heterocyclic rings, e.g. cromakalim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68033—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a maytansine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6875—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody being a hybrid immunoglobulin
    • A61K47/6877—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody being a hybrid immunoglobulin the antibody being an immunoglobulin containing regions, domains or residues from different species
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2510/00—Detection of programmed cell death, i.e. apoptosis

Definitions

  • the present invention relates to the field of antibodies, and in particular to a TPBG antibody, a preparation method thereof, a conjugate thereof and use thereof.
  • TPBG trophoblast-specific glycoprotein
  • 5T4 trophoblast-specific glycoprotein
  • the human TPBG protein has a molecular weight of about 72 kDa and contains 420 amino acids. Its N-terminal oligosaccharide structure is diverse, prevents protein hydrolysis, and interacts with other molecules during cell membrane signaling.
  • the TPBG protein contains a total of seven repeat leucine domains (LRRs) that are involved in protein-protein interactions.
  • the trophoblast is a special layer of embryonic stem cells between the placenta and the fetus.
  • TPBG is widely expressed in various trophoblast cells during embryonic development. For normal adult tissues, TPBG is only expressed in a limited number of epithelial cells. However, TPBG is expressed in many cancer cells, such as uterine cancer, colon cancer, gastric cancer, ovarian cancer, oral cancer, prostate cancer, lung cancer or kidney cancer tissues, and is detected in colon cancer, gastric cancer or ovarian cancer. There is evidence that the expression of TPBG is associated with a low cure rate for cancer. In tissues of non-small cell lung cancer, kidney cancer or pancreatic cancer, the expression of TPBG is as high as 95% or more.
  • TPBG epithelial cell cadherin
  • EMT epithelial mesenchymal transition
  • TPBG colocalization of TPBG with CXCR4 in the cell membrane indicates that it can induce the binding of its ligand CXCL12 chemokine and promote the spread of inflammation and tumor.
  • CXCL12 binds to another receptor, CXCR7, which inhibits chemotactic responses and facilitates cell growth and survival.
  • the Wnt/b-catenin signaling pathway plays a very important role in development and cell regeneration.
  • TPBG inhibits Wnt signaling pathway by inhibiting endocytosis of LRP6 and Wnt receptors, thereby inhibiting cell adhesion and cytoskeletal formation, and promoting tumorigenesis. Migration and diffusion.
  • TPBG is in breast and gastric cancer cells Participation in the non-canonical Wnt pathway also promotes the migration and infiltration of cancer cells.
  • the antibody drug-conjugated drug is an antibody drug conjugate formed by coupling an antibody and a high-efficiency small molecule drug through a linker, and enables a highly toxic small molecule drug to specifically recognize a target protein on a cancer cell, thereby specifically killing the cancer. cell.
  • antibody-based immunotherapy and chemical-based chemotherapy have been the two major treatment strategies for clinical cancer treatment.
  • Antibodies target antigens overexpressed by tumor cells, and a variety of therapeutic monoclonal antibodies have achieved great clinical success.
  • therapeutic antibodies have good targeting properties, the killing effect is limited.
  • small molecule chemical drugs have an effective killing effect on cancer cells, they also cause the same damage to non-cancer cells.
  • a new generation of antibody drug conjugates which utilizes the specific binding ability of antibodies to target cells, delivers highly cytotoxic chemical drugs, and achieves targeted and efficient killing of cancer cells.
  • ADC drugs With the advent of new chemical ligation technology, antibody drug-conjugated drugs began to enter clinical research in the late 1980s, and two ADC drugs have been approved for FDA approval.
  • ADC drugs involves: screening of drug targets, preparation of recombinant antibodies, development of linker technology, and screening optimization of high cytotoxic compounds.
  • TPBG is a candidate for ADC drugs.
  • the technical problem to be solved by the present invention is to overcome the deficiencies of the current lack of TPBG antibodies, and to provide a TPBG antibody with high affinity and specificity, a preparation method and application thereof, and the TPBG antibody and human source, mouse source or food.
  • the crab-derived TPBG protein has a high affinity.
  • the present invention also provides a conjugate of a pharmaceutically active ingredient comprising the TPBG antibody and a small molecule compound coupled thereto and having antitumor function, the conjugate being capable of entering cells and being positive for TPBG
  • the cells are cytotoxic and can be used in the preparation of drugs such as tumors.
  • the present invention uses a human TBPG protein or a recombinant cell strain overexpressing a human TBPG protein as an immunogen, and adopts a conventional hybridoma preparation technique (Kohler and Milstein, Nature, 1975, 256: 495) through a series of adjustments and improvements. , obtaining a lead antibody of the TPBG antibody. Further, by preliminary production, purification and assay of the lead antibody, a TPBG antibody having high affinity to a protein such as a human TBPG protein is obtained. The TPBG antibody is conjugated with a small molecule compound such as MMAF, which is capable of entering cells and has excellent cytotoxic killing effect on TPBG positive cells. The amino acid sequence of the heavy chain variable region of the obtained TPBG antibody and the light chain variable region of the TPBG antibody was then sequenced by molecular biological methods.
  • the present invention provides an isolated protein comprising one or more of a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3 of a TPBG antibody, and/or a light chain CDR1, a light chain CDR2 and a light chain of a TPBG antibody CDR3
  • a heavy chain CDR1 is SEQ ID No. 2, SEQ ID No. 10, SEQ ID No. 18, SEQ ID No. 26, SEQ ID No. in the sequence listing. 34.
  • the amino acid sequence of the heavy chain CDR2 is SEQ ID No.
  • amino acid sequence of the light chain CDR2 is SEQ ID No. 7, SEQ ID in the sequence listing. No. 15, SEQ ID No. 23, SEQ ID No. 31, SEQ ID No. 39, SEQ ID No. 47, SEQ ID No. 55, SEQ ID No. 63, SEQ ID No. 71 or SEQ ID No.
  • amino acid sequence of the light chain CDR3 is SEQ ID No. 8, SEQ ID No. 16, SEQ ID No. 24, SEQ ID No. 32, SEQ ID No. 40, SEQ ID No. in the sequence listing.
  • amino acid sequence of the heavy chain CDR1 and SEQ ID No. 2 SEQ in the sequence listing ID No. 10, SEQ ID No. 18, SEQ ID No. 26, SEQ ID No. 34, SEQ ID No. 42, SEQ ID No. 50, SEQ ID No. 58, SEQ ID No. 66 or SEQ ID No.
  • the amino acid sequence shown in .74 has an amino acid sequence of at least 80% sequence homology; the amino acid sequence of the heavy chain CDR2 is as SEQ ID No. 3, SEQ ID No. 11, SEQ ID No in the sequence listing. 19.
  • amino acid sequence of 3 is as SEQ ID No. 4, SEQ ID No. 12, SEQ ID No. 20, SEQ ID No. 28, SEQ ID No. 36, SEQ ID No. 44, SEQ ID No. 52 in the sequence listing.
  • amino acid sequence of SEQ ID No. 60, SEQ ID No. 68 or SEQ ID No. 76 is represented by an amino acid sequence having at least 80% sequence homology; the amino acid sequence of the light chain CDR1 is as shown in the sequence listing SEQ ID No. 6, SEQ ID No.
  • amino acid sequence of ID No. 70 or SEQ ID No. 78 is represented by an amino acid sequence having at least 80% sequence homology; the amino acid sequence of the light chain CDR2 is as SEQ ID No. 7, SEQ in the sequence listing. ID No. 15, SEQ ID No. 23, SEQ ID No. 31, SEQ ID No. 39, SEQ ID No. 47, SEQ ID No. 55, SEQ ID No. 63, SEQ ID No. 71 or SEQ ID No.
  • the amino acid sequence shown in .79 is represented by an amino acid sequence having at least 80% sequence homology; the amino acid sequence of the light chain CDR3 is as SEQ ID No. 8, SEQ ID No. 16, SEQ ID No in the sequence listing. .24, SEQ ID N O.32, SEQ ID No. 40, SEQ ID No. 48, SEQ ID No. 56, The amino acid sequence represented by SEQ ID No. 64, SEQ ID No. 72 or SEQ ID No. 80 is represented by an amino acid sequence having at least 80% sequence homology.
  • the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 2 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID No. 3 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR3 The sequence is shown in SEQ ID No. 4 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 10 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID No. 11 of the Sequence Listing.
  • the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 12 of the sequence listing
  • the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No.
  • amino acid sequence of the heavy chain CDR2 is as in the sequence listing.
  • SEQ ID No. 19 and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 20 of the Sequence Listing;
  • the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 26 of the Sequence Listing,
  • the amino acid sequence of the CDR2 of the chain is shown in SEQ ID No. 27 of the Sequence Listing, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 28 of the Sequence Listing;
  • the amino acid sequence of the CDR1 of the heavy chain is SEQ ID No.
  • the amino acid sequence of the heavy chain CDR2 is as shown in SEQ ID No.
  • amino acid sequence of CDR3 is shown in SEQ ID No. 36 of the Sequence Listing; the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 42 of the Sequence Listing, and the amino acid sequence of the heavy chain CDR2 is SEQ ID No. 43 of the Sequence Listing. As shown, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 44 of the Sequence Listing; the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 50 of the Sequence Listing, and the amino acid sequence of the heavy chain CDR2 As shown in the Sequence Listing SEQ ID No. 51, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No.
  • the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID No. 58 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID No. 59 of the sequence listing
  • the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 60 of the sequence listing
  • the amino acid sequence of the heavy chain CDR1 is as in the sequence listing.
  • SEQ ID No. 66 the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID No. 67 of the Sequence Listing
  • the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 68 of the Sequence Listing
  • the amino acid sequence of the CDR1 of the chain is shown in SEQ ID No.
  • amino acid sequence of the heavy chain CDR2 As shown in SEQ ID No. 75 of the Sequence Listing, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID No. 76 of the Sequence Listing; the amino acid sequence of the CDR1 of the light chain is shown in SEQ ID No. 6 of the Sequence Listing.
  • the amino acid sequence of the light chain CDR2 is shown in SEQ ID No. 7 of the sequence listing, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 8 of the sequence listing; the amino acid sequence of the light chain CDR1 is as in the sequence listing.
  • amino acid sequence of the light chain CDR2 is shown in SEQ ID No.
  • the amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 22 of the Sequence Listing, the amino acid sequence of the light chain CDR2 is shown in SEQ ID No. 23 of the Sequence Listing, and the amino acid sequence of the light chain CDR3 is as SEQ ID NO:
  • the amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 30 of the Sequence Listing, the amino acid sequence of the light chain CDR2 is shown in SEQ ID No. 31 of the Sequence Listing, and the light chain is represented by ID No. Amino acid sequence of CDR3 SEQ ID No.
  • amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 38 of the sequence listing
  • amino acid sequence of the light chain CDR2 is shown in SEQ ID No. 39 of the sequence listing
  • the amino acid sequence of the CDR3 of the chain is shown in SEQ ID No. 40 of the Sequence Listing
  • amino acid sequence of the CDR1 of the light chain is shown in SEQ ID No. 46 of the Sequence Listing
  • amino acid sequence of the CDR2 of the light chain is SEQ ID No. of the Sequence Listing. 47
  • amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 48 of the Sequence Listing
  • amino acid sequence of the light chain CDR1 is shown in SEQ ID No.
  • amino acid sequence of the light chain CDR2 is shown in SEQ ID No. 55 of the Sequence Listing, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 56 of the Sequence Listing; the amino acid sequence of the CDR1 of the light chain is shown in SEQ ID No. 62 of the Sequence Listing.
  • the amino acid sequence of the light chain CDR2 is shown in SEQ ID No. 63 of the sequence listing, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 64 of the sequence listing; the amino acid sequence of the light chain CDR1 is as follows. As shown in the list of SEQ ID No. 70, the amino acid sequence of the light chain CDR2 is shown in SEQ ID No.
  • amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 72 of the sequence listing; or the amino acid sequence of the light chain CDR1 is as shown in SEQ ID No. 78 of the sequence listing, and the amino acid sequence of the light chain CDR2 is as The sequence listing is shown in SEQ ID No. 79, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID No. 80 of the Sequence Listing.
  • the present invention also provides an isolated protein comprising a heavy chain variable region of a TPBG antibody and/or a light chain variable region of a TPBG antibody, the amino acid sequence of the heavy chain variable region being SEQ ID No. in the Sequence Listing. 1. SEQ ID No. 9, SEQ ID No. 17, SEQ ID No. 25, SEQ ID No. 33, SEQ ID No. 41, SEQ ID No. 49, SEQ ID No. 57, SEQ ID No. 65 or SEQ ID No. 73; the amino acid sequence of the light chain variable region is SEQ ID No. 5, SEQ ID No. 13, SEQ ID No. 21, SEQ ID No. 29, SEQ ID No. in the sequence listing. 37. SEQ ID No. 45, SEQ ID No. 53, SEQ ID No. 61, SEQ ID No. 69 or SEQ ID No. 77.
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 1 of the Sequence Listing, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 5 of the Sequence Listing;
  • the amino acid sequence of the variable region of the chain is shown in SEQ ID No. 9 of the Sequence Listing, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID No. 13 of the Sequence Listing;
  • the amino acid sequence of the variable region of the heavy chain The sequence of the light chain variable region is shown in SEQ ID No. 21;
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 25, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.
  • the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 33 of the Sequence Listing, and The amino acid sequence of the light chain variable region is set forth in SEQ ID No. 37 of the Sequence Listing; the amino acid sequence of the heavy chain variable region is set forth in SEQ ID No. 41 of the Sequence Listing, and the amino acid of the light chain variable region The sequence is shown in SEQ ID No. 45 of the Sequence Listing; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 49 of the Sequence Listing, and The amino acid sequence of the variable region of the chain is shown in SEQ ID No. 53 of the Sequence Listing; the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID No.
  • amino acid sequence of the variable region of the light chain As shown in the Sequence Listing SEQ ID No. 61; the amino acid sequence of the heavy chain variable region is set forth in SEQ ID No. 65 of the Sequence Listing.
  • the amino acid sequence of the light chain variable region is shown in SEQ ID No. 69 of the sequence listing; or the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 73 of the sequence listing, and The amino acid sequence of the light chain variable region is shown in SEQ ID No. 77 of the Sequence Listing.
  • the number in Table 1 is the sequence number in the sequence listing, such as the amino acid sequence of the heavy chain protein variable region of 12B12C7C3 is SEQ ID No. 1, and the amino acid sequence of CDR1 in the heavy chain protein variable region of 12B12C7C3 is SEQ ID No. 2.
  • the protein further comprises an antibody heavy chain constant region and/or an antibody light chain constant region
  • the antibody heavy chain constant region is conventional in the art, preferably a mouse-derived antibody heavy chain constant region or The human antibody heavy chain constant region, more preferably the human antibody heavy chain constant region.
  • the antibody light chain constant region is conventional in the art, preferably a mouse-derived light chain antibody constant region or a human antibody light chain constant region, more preferably a human antibody light chain constant region.
  • the protein is a protein conventional in the art, preferably a TPBG antibody, more preferably an antibody full-length protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, or a single chain antibody (single chain)
  • a single chain antibody single chain
  • scFv antibody fragment
  • sdAb single domain antibody
  • signle-domain antibody single-domain antibody
  • monoclonal antibodies or polyclonal antibodies prepared by the above antibodies can be developed by a variety of pathways and techniques, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc.
  • the mainstream is the preparation of monoclonal antibodies from wild-type or transgenic mice by hybridoma technology.
  • the full-length antibody protein is a conventional full-length antibody of the art, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region.
  • the heavy chain variable region and the light chain variable region of the protein and the human heavy chain constant region and the human light chain constant region constitute a full human antibody full length protein.
  • the full length protein of the antibody is IgG1, IgG2, IgG3 or IgG4.
  • the single-chain antibody is a conventional single-chain antibody in the art, which comprises a heavy chain variable region, a light chain variable region, and a short peptide of 15-20 amino acids.
  • the antigen-antibody binding domain protein fragment is a conventional antigen-antibody binding domain protein fragment of the art comprising a light chain variable region, a light chain constant region, and an Fd segment of a heavy chain constant region.
  • the antigen-antibody binding domain protein fragments are Fab and F(ab')2.
  • the single domain antibodies are conventional single domain antibodies in the art, including heavy chain variable regions and heavy chain constant regions.
  • the single region antibodies are conventional single region antibodies of the art which include only heavy chain variable regions.
  • the preparation method of the protein is a conventional preparation method in the art.
  • the preparation method is preferably obtained by isolation from an expression transformant recombinantly expressing the protein or by artificially synthesizing a protein sequence.
  • the method for isolating the expression transformant which recombinantly expresses the protein is preferably obtained by cloning a nucleic acid molecule encoding the protein and having a point mutation into a recombinant vector, and transforming the resulting recombinant vector into a transformant to obtain a recombinant expression.
  • the transformant can be isolated and purified by culturing the resulting recombinant expression transformant to obtain the protein.
  • the invention also provides a nucleic acid encoding the protein described above.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is SEQ ID No. 81, SEQ ID No. 83, SEQ ID No. 85, and SEQ ID No. 87.
  • the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No. 82, SEQ ID No. 84, SEQ ID No. 86, and SEQ ID No. 88, Sequence Listing SEQ ID No. 90, Sequence Listing SEQ ID No. 92, Sequence Listing SEQ ID No. 94, Sequence Listing SEQ ID No. 96, Sequence Listing SEQ ID No. 98 or Sequence Listing SEQ ID No. 100 is shown.
  • nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 81 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in the Sequence Listing.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 83, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in ID No. 82.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No.
  • nucleoside of the nucleic acid encoding the light chain variable region is shown in the Sequence Listing.
  • the acid sequence is shown in SEQ ID No. 86 of the Sequence Listing; the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 87 of the Sequence Listing, and the nucleic acid encoding the variable region of the light chain
  • the nucleotide sequence is shown in SEQ ID No. 88 of the sequence listing; encoding the heavy chain can be The nucleotide sequence of the nucleic acid of the variable region is shown in SEQ ID No.
  • nucleotide sequence of the nucleic acid encoding the variable region of the light chain is as shown in SEQ ID No. 90 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid of the heavy chain variable region is shown in SEQ ID No. 91 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is shown in SEQ ID No. 92 of the Sequence Listing;
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 93 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is SEQ ID No.
  • nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 95 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in the Sequence Listing SEQ ID.
  • the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is shown in SEQ ID No. 97 of the Sequence Listing, and the nucleotide sequence of the nucleic acid encoding the light chain variable region is as shown in No. Listed as SEQ ID No. 98; or, the nucleotide sequence of the nucleic acid encoding the heavy chain variable region is as shown in SEQ ID No. 99 of the sequence listing, and encodes the light chain
  • the nucleotide sequence of the nucleic acid of the variable region is shown in SEQ ID No. 100 of the Sequence Listing.
  • the number in Table 2 is the sequence number in the sequence listing, and the nucleotide sequence of the variable region of the heavy chain protein encoding 12B12C7C3 is SEQ ID No. 81.
  • nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 12B12C7C3 is from position 91 to position 105 in SEQ ID No. 81 of the sequence listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 12B12C7C3 is from positions 151 to 198 in SEQ ID No. 81 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 12B12C7C3 is 295th to 327th in SEQ ID No. 81 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 12B12C7C3 is from positions 70 to 114 of SEQ ID No. 82 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 12B12C7C3 is from positions 157 to 180 in SEQ ID No. 82 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 12B12C7C3 is 277th to 303th in SEQ ID No. 82 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 5G4H10G5 is the 91st to 105th positions in SEQ ID No. 83 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 5G4H10G5 is from positions 148 to 198 in SEQ ID No. 83 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 5G4H10G5 is from positions 295 to 330 of SEQ ID No. 83 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 5G4H10G5 is from positions 70 to 102 of SEQ ID No. 84 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 5G4H10G53 is 148th to 168th in SEQ ID No. 84 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 5G4H10G5 is from position 265 to position 288 in SEQ ID No. 84 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 37H9C5G2 is from positions 91 to 105 of SEQ ID No. 85 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 37H9C5G2 is from positions 151 to 198 in SEQ ID No. 85 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 37H9C5G2 is from positions 295 to 327 in SEQ ID No. 85 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 37H9C5G2 is from positions 70 to 102 of SEQ ID No. 86 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 37H9C5G2 is 148th to 168th in SEQ ID No. 86 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 37H9C5G2 is from positions 265 to 291 in SEQ ID No. 86 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 39A11G5F2 is SEQ ID No. 87 of the Sequence Listing. 91st to 105th in the middle;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 39A11G5F2 is from positions 151 to 198 of SEQ ID No. 87 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 39A11G5F2 is 295th to 315th in SEQ ID No. 87 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 39A11G5F2 is from positions 70 to 102 of SEQ ID No. 88 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 39A11G5F2 is 148th to 168th in SEQ ID No. 88 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 39A11G5F2 is from positions 265 to 291 in SEQ ID No. 88 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 52C9E9F6 is position 91 to 105 in SEQ ID No. 89 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 52C9E9F6 is from positions 151 to 198 in SEQ ID No. 89 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 52C9E9F6 is 295th to 315th in SEQ ID No. 89 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 52C9E9F6 is from positions 70 to 102 of SEQ ID No. 90 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 52C9E9F6 is 148th to 168th in SEQ ID No. 90 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 52C9E9F6 is from positions 268 to 291 in SEQ ID No. 90 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 28D4E6A9 is from positions 91 to 105 of SEQ ID No. 91 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 28D4E6A9 is from positions 148 to 198 of SEQ ID No. 91 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 28D4E6A9 is from positions 295 to 327 of SEQ ID No. 91 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 28D4E6A9 is from positions 70 to 102 of SEQ ID No. 92 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 28D4E6A9 is from positions 148 to 168 of SEQ ID No. 92 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 28D4E6A9 is from positions 265 to 291 in SEQ ID No. 92 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 36A10D8B12 is from positions 91 to 108 of SEQ ID No. 93 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 36A10D8B12 is from positions 154 to 198 of SEQ ID No. 93 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 36A10D8B12 is from positions 295 to 324 of SEQ ID No. 93 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 36A10D8B12 is from positions 70 to 102 of SEQ ID No. 94 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 36A10D8B12 is 148th to 168th in SEQ ID No. 94 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 36A10D8B12 is position 265 to 291 of SEQ ID No. 94 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 99E12C7H1 is from positions 91 to 105 of SEQ ID No. 95 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 99E12C7H1 is from positions 151 to 198 in SEQ ID No. 95 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 99E12C7H1 is from positions 295 to 318 of SEQ ID No. 95 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 99E12C7H1 is from positions 70 to 102 of SEQ ID No. 96 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 99E12C7H1 is from positions 148 to 168 in SEQ ID No. 96 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 99E12C7H1 is from position 265 to position 291 in SEQ ID No. 96 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 103E2E9C2 is position 91 to 105 in SEQ ID No. 97 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 103E2E9C2 is SEQ ID No. 97 of the Sequence Listing. 151st to 198th;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 103E2E9C2 is from positions 295 to 324 of SEQ ID No. 97 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 103E2E9C2 is from positions 70 to 105 of SEQ ID No. 98 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 103E2E9C2 is from positions 151 to 171 in SEQ ID No. 98 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 103E2E9C2 is from positions 268 to 294 in SEQ ID No. 98 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 106D5G3D10 is from position 91 to position 105 in SEQ ID No. 99 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 106D5G3D10 is from positions 151 to 198 in SEQ ID No. 99 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 106D5G3D10 is from positions 295 to 318 of SEQ ID No. 99 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 106D5G3D10 is the 70th to 99th positions in SEQ ID No. 100 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 106D5G3D10 is from positions 145 to 165 in SEQ ID No. 100 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 106D5G3D10 is position 262 to 294 in SEQ ID No. 100 of the Sequence Listing.
  • the preparation method of the nucleic acid is a preparation method conventional in the art, and preferably includes the steps of obtaining a nucleic acid molecule encoding the above protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above protein by artificial total sequence synthesis. .
  • the base sequence encoding the amino acid sequence of the above protein may be appropriately introduced with a substitution, deletion, alteration, insertion or addition to provide a homologue of a polynucleotide.
  • a homologue of a polynucleotide of the invention can be made by replacing, deleting or increasing one or more bases encoding a gene of the protein sequence within the range of activity of the antibody.
  • the invention also provides a recombinant expression vector comprising the nucleic acid.
  • the recombinant expression vector can be obtained by a conventional method in the art, that is, the nucleic acid molecule of the present invention is ligated to various expression vectors.
  • the expression vector is a variety of vectors conventional in the art, as long as it can It is sufficient to carry the aforementioned nucleic acid molecule.
  • the vector preferably includes: various plasmids, cosmids, phage or viral vectors, and the like.
  • the present invention also provides a recombinant expression transformant comprising the above recombinant expression vector.
  • the preparation method of the recombinant expression transformant is a preparation method conventional in the art, and preferably, the recombinant expression vector is transformed into a host cell.
  • the host cell is a variety of host cells conventional in the art, as long as it satisfies the stable self-replication of the above recombinant expression vector, and the nucleic acid carried can be efficiently expressed.
  • the host cell is an E. coli TG1 or BL21 cell (expressing a single-chain antibody or a Fab antibody), or a CHO-K1 cell (expressing a full-length IgG antibody).
  • the recombinant expression plasmid of the present invention can be obtained by transforming the aforementioned recombinant expression plasmid into a host cell.
  • the conversion method is a conventional transformation method in the art, preferably a chemical conversion method, a heat shock method or an electrotransformation method.
  • the present invention also provides a method for producing a TPBG antibody, which comprises the steps of culturing the above recombinant expression transformant and obtaining a TPBG antibody from the culture.
  • the present invention provides an immunoconjugate comprising the above-described protein covalently attached to a cytotoxic agent.
  • the above 1 equivalent of the protein is linked to the y equivalent of the cytotoxic agent via the x equivalent linker, and has the structure shown in Formula 1.
  • the integers x and y are each independently preferably from 1 to 2, or from 2 to 4, or from 4 to 8, or from 8 to 20; the ratio of x to y is preferably 1:1.
  • the L is a linker (or crosslinker or coupling agent) conventional in the art.
  • the L contains two functional groups, a group reactive with an antibody, and a group reactive with a drug (for example, an aldehyde or a ketone).
  • the drug is coupled to the protein described above via a linker molecule.
  • the L is released after entering the cell, and includes, but is not limited to, functional groups, active esters, carbonates, carbamates, imine phosphates, anthraquinones, anthraquinones, acetals, orthoesters, amino groups.
  • the L mainly contains the structure represented by Formula 2, which is the remainder corresponding to the leaving group in L after leaving;
  • Sp 2 is a bond, -S- or -O-, provided that when Alk 2 is a bond, Sp 2 is a bond;
  • Z 1 is H, (C 1 -C 5 )alkyl, or (C 1 -C 5 )alkyl, (C 1 -C 5 )alkoxy, (C 1 -C 4 )thioalkoxy , halogen, nitro, -COOR', -CONHR', -O(CH 2 ) n COOR', -S(CH 2 ) n COOR', -O(CH 2 ) n CONHR' or -S(CH 2 )
  • Sp is a linear or branched divalent or trivalent (C 1 -C 18 ) group, a divalent or trivalent aryl or heteroaryl group, a divalent or trivalent (C 3 -C 18 ) naphthenic group Or a heterocycloalkyl group, a divalent or trivalent aryl or heteroaryl-aryl (C 1 -C 18 ) group, a divalent or trivalent cycloalkyl or heterocycloalkyl-alkyl group ( a C 1 -C 18 ) group, or a divalent or trivalent (C 2 -C 18 ) unsaturated alkyl group, wherein the heteroaryl group is preferably a furyl group, a thienyl group, an N-methylpyrrolyl group, a pyridine group Base, N-methylimidazolyl, oxazolyl, pyrimidinyl.
  • Alk 1 is a branched or unbranched (C 1 -C 5 ) alkylene chain
  • Sp 1 is a bond, -S-, -O-, -CONH-, -NHCO- or -NR', wherein R 'As defined above, the condition is that when Alk 1 is a key, Sp 1 is a key;
  • Ar is composed of (C 1 -C 6 )alkyl, (C 1 -C 5 )alkoxy, (C 1 -C 4 )thioalkoxy, halogen, nitro, -COOR', -CONHR', 1, 2 or 3 groups of -O(CH 2 ) n COOR', -S(CH 2 ) n COOR', -O(CH 2 ) n CONHR' or -S(CH 2 ) n CONHR' Substituted 1,2-, 1,3- or 1,4-phenylene, wherein n and R' are as defined above, or Ar is each from C 1 -C 6 )alkyl, (C 1 -C 5 ) Alkoxy, (C 1 -C 4 )thioalkoxy, halogen, nitro, -COOR', -CONHR', -O(CH 2 ) n COOR', -S(CH 2 ) n COOR', -2 , 3, or
  • Z 1 is (C 1 -C 5 )alkyl, or (C 1 -C 5 )alkyl, (C 1 -C 4 )alkoxy, (C 1 -C 4 )thioalkoxy, halogen , nitro, -COOR', -CONHR', -O(CH 2 ) n COOR', -S(CH 2 ) n COOR', -O(CH 2 ) n CONHR' or -S(CH 2 ) n CONHR '1, 2, or 3 groups are optionally substituted phenyl; Alk 2 and Sp 2 are both bonds; and Sp and Q are as defined above only.
  • the above key means a covalent bond.
  • the L is preferably maleimidocaproyl (MC), maleimidocaproyl-L-valine-L-citrulline p-aminobenzyl alcohol (MC-VC-PAB) Or 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC).
  • MC maleimidocaproyl
  • MC-VC-PAB maleimidocaproyl-L-valine-L-citrulline p-aminobenzyl alcohol
  • SMCC 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester
  • the D is a conventional cytotoxic agent in the art, preferably selected from the group consisting of a cytotoxin, a chemotherapeutic agent, a radioisotope, a therapeutic nucleic acid, an immunomodulatory agent, an anti-angiogenic agent, an anti-proliferative pro-apoptotic agent, or a lytic enzyme.
  • the cytotoxin is a cytotoxin conventional in the art and generally refers to an active agent that inhibits or prevents cellular function and/or causes cell destruction.
  • an active agent that inhibits or prevents cellular function and/or causes cell destruction.
  • doxorubicin is selected from the group consisting of doxorubicin, daunorubicin, idarubicin, arubicin, zolubicin, mitoxantrone, epirubicin, carbofurin, nogamycin, Menoril, pirarubicin, valrubicin, cytarabine, gemcitabine, flufluuridine, acitretin, enoxacitabine, azacitidine, deoxyfluorouridine, pentazostat Ding, bromouridine, capecitabine, cladribine, decitabine, fluorouridine, fludarabine, glutathione, puromycin, tegafur, thiazolidine nucleoside, trichoderma , cisplatin, carboplatin, cyclophosphamide, dacarbazine, vinblastine, vincristine, bleomycin, nitrogen mustard, prednisone, procarbazine, methotrex
  • the chemotherapeutic agent is a conventional chemotherapeutic agent in the art, preferably selected from the group consisting of an alkylating agent, an alkyl sulfonate chemotherapeutic agent, an aziridine chemotherapeutic agent, a vinyl amide, and a methyl group.
  • Amine chemotherapeutic agents nitrogen mustard, nitrourea chemotherapeutics, antibiotics, antimetabolites, folic acid chemotherapeutics, purine analogs, pyrimidine analogs, androgens, anti-adrenalin, folic acid supplements, and beauty
  • the alkylating agent is a conventional alkylating agent in the art, preferably selected from the group consisting of thiotepa or cyclophosphamide.
  • the alkyl sulfonate chemotherapeutic agent is a conventional alkyl sulfonate chemotherapeutic agent, preferably selected from the group consisting of busulfan, propylene bromide or pipersulfuron.
  • the aziridine chemotherapeutic agent is a conventional aziridine chemotherapeutic agent in the art, preferably selected from the group consisting of aziridine, carbazone, metopril or uridine.
  • the ethylene amide and methyl melamine chemotherapeutic agents are conventional ethylene amide and methyl melamine chemotherapeutic agents, preferably selected from the group consisting of hexamethylene melamine, triethylene melamine, and triethylene ether. Phosphoramide, triethylene thiophosphoramide or trimethylol melamine.
  • the nitrogen mustard is a conventional nitrogen mustard, preferably selected from the group consisting of chlorambucil, naphthyl mustard, estramustine, ifosfamide, nitrogen mustard, and oxygen nitrogen mustard hydrochloride. Phenylalanine mustard, neonitrogen mustard, fentanyl cholesteryl, prednisolone, tromethamine or uracil mustard.
  • the nitrourea chemotherapeutic agent is a nitrourea chemotherapeutic agent conventional in the art, preferably selected from the group consisting of carmustine, chlorourea, formoterol, lomustine, and nimes Ting or ramustine.
  • the antibiotic is a conventional antibiotic in the art, preferably selected from the group consisting of aclarubicin, actinomycin, aflatoxin, azase, bleomycin, actinomycin c, calicheamicin, Carbofurin, erythromycin, cancer, chromomycin, dactinomycin, daunorubicin, ditoxin, 6-diazo-5-oxo-L-norleucine, Roubicin, epirubicin, ebispercin, idarubicin, fascin, mitomycin, mycophenolic acid, nogamycin, oligomycin, pedromycin, kaimycin , puromycin, triiron adriamycin, rhodamine, streptavidin, streptozotocin, tuberculin, umbrel, statstatin or zorubicin.
  • aclarubicin preferably selected from the group consisting of aclarubicin, actinomycin
  • the antimetabolite is a conventional antimetabolite in the art, preferably selected from methotrexate or 5-fluorouracil (5-FU).
  • the folic acid chemotherapeutic agent is a conventional folic acid chemotherapeutic agent in the art, preferably selected from the group consisting of dimethyl folic acid, pterostilbose or trimethoate.
  • the purine analogs are conventional anthraquinone analogs in the art, preferably selected from fludarabine, 6-oxime, thioxime or thioguanine.
  • the pyrimidine analog is a pyrimidine analog conventional in the art, preferably selected from the group consisting of amphetamine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, Deoxyfluorouridine, enoxabine, fluorouridine or 5-EU.
  • the androgen is a conventional androgen in the art, preferably selected from the group consisting of caprotestosterone, alpha-androstanol propionate, anthracycline, megestrol or testosterone.
  • the anti-adrenalin is a conventional anti-adrenalin in the art, preferably selected from the group consisting of anlumet, mitoxantrone or tromethamine.
  • the folic acid supplement is a conventional folic acid supplement in the art, preferably selected from the group consisting of folinic acid, acetolactone, aldophosphamide glucoside, amino keto valeric acid, amsacrine, amoxicilin, specific life.
  • the maytansinol is a conventional maytansinol in the art, preferably selected from the group consisting of maytansin, anspirin, mitoxantrone, mitoxantrone, moppeldol, diamine nitidine, Pentastatin, egg ammonia, pirarubicin, loxoprost, podophyllin, 2-ethyl hydrazide or procarbazine.
  • the polysaccharide complex is a conventional polysaccharide complex in the art, preferably selected from the group consisting of razuxon, rhizomycin, cilostazol, quercetin, fine-chain oxysporic acid, and triammine oxime 2, 2' , 2"-trichlorotriethylamine, trichothecenes, urethane, vindesine, dacarbazine, mannimostatin, dibromomannitol, dibromodusol, Piperobbromo, gacytosine, cytarabine, cyclophosphamide or thiotepa.
  • the taxane is a taxane conventional in the art, preferably selected from the group consisting of paclitaxel, non-hydrogenated castor oil, paclitaxel, an albumin engineered nanoparticle preparation (American Pharmaceutical Partners, Schaumberg, Illinois), docetaxel, Chlorambucil, gemcitabine, 6-thioguanine, guanidine or methotrexate.
  • the platinum analogs are conventional platinum analogs, preferably selected from the group consisting of cisplatin, carboplatin, vinblastine, etoposide, ifosfamide, mitoxantrone, vincristine, noanto , teniposide, edaroxib, daunorubicin, aminopterin, capecitabine ibandronate, CPT-11, topoisomerase inhibitor RFS 2000 or difluoromethylornithine .
  • the retinoid is a retinoid of the art, preferably retinoic acid.
  • the radioisotope is a conventional radioisotope in the art, preferably it is directly bound to the above protein or is bound to the above protein by a chelating agent. More preferably, it binds directly to the cysteine residue of the protein.
  • the radioisotope is selected from the group consisting of alpha-emitters, beta-emitters and Auger electrons suitable for radiation therapy and positron emitters or gamma-emitters suitable for diagnosis. More preferably, the radioisotope is selected from the group consisting of 18 fluoro, 64 copper, and 65 copper.
  • the therapeutic nucleic acid is a nucleic acid conventional in the art, preferably a gene encoding an immunomodulator, an anti-angiogenic agent, an anti-proliferative agent or a pro-apoptotic agent.
  • the therapeutic agent includes the therapeutic agent, derivatives thereof, and pharmaceutically acceptable salts, acids, and derivatives of the therapeutic agents.
  • the immunomodulatory agent is a conventional immunomodulator in the art, ie, an agent that elicits an immune response, including a humoral immune response (eg, production of an antigen-specific antibody) and a cell-mediated immune response (eg, lymphocyte proliferation).
  • a humoral immune response eg, production of an antigen-specific antibody
  • a cell-mediated immune response eg, lymphocyte proliferation
  • cytokine a cytokine
  • a growth factor eg, a hormone, an anti-hormonal agent, an immunosuppressive agent or a corticosteroid.
  • the cytokine is a cytokine conventional in the art, preferably selected from the group consisting of xanthine, interleukin or interferon.
  • the growth factor is a conventional growth factor in the art, preferably selected from the group consisting of TNF, CSF, GM-CSF or G-CSF.
  • the hormone is a conventional hormone in the art, preferably selected from the group consisting of estrogen, androgen or progestin. More preferably, the estrogen is diethylstilbestrol or estradiol. More preferably, the androgen is testosterone or fluorotestosterone. More preferably, the progestin is megestrol acetate or medroxyprogesterone acetate.
  • the corticosteroids are conventional corticosteroids in the art, preferably selected from prednisone, dexamethasone or cortisone.
  • the antihormonal agent is a conventional antihormonal agent in the art, which can block the action of a hormone on a tumor, inhibit the production of a cytokine, down-regulate the expression of an autoantigen, or mask an immunosuppressant of an MHC antigen. It is preferably selected from the group consisting of an antiestrogenic drug, an antiandrogen or an antinephrine. More preferably, the antiestrogens are selected from the group consisting of tamoxifen, raloxifene, aromatase inhibitory 4(5)-imidazoles, 4-hydroxy tamoxifen, koji Wolfene or toremifene.
  • the antiandrogen is selected from the group consisting of flutamide, nilutamide, bicalutamide, leuprolide or goserelin.
  • the immunosuppressant is a conventional immunosuppressant in the art, preferably selected from the group consisting of 2-amino-6 aryl-5 substituted pyrimidines, azathioprine, cyclophosphamide, bromocriptine, danazol, ammonia Phenyl sulfone, glutaraldehyde, anti-idiotypic antibodies against MHC antigens and MHC fragments, cyclosporin A, steroids such as glucocorticosteroids, streptokinase, TGFb, rapamycin, T cell receptor, T cell receptor Fragment, cytokine receptor antagonist or T cell receptor antibody. More preferably, the cytokine receptor antagonist is selected from the group consisting of an anti-interferon antibody, an anti-IL10 antibody, an anti-TNFa
  • the anti-angiogenic agent is a conventional anti-angiogenic agent in the art, preferably selected from the group consisting of a farnesyl transferase inhibitor, a COX-2 inhibitor, a VEGF inhibitor, a bFGF inhibitor, a steroid sulfatase Inhibitor, interleukin-24, thrombospondin, metallospondin protein, class I interferon, interleukin 12, protamine, angiostatin, laminin, endostatin or prolactin fragment. More preferably, it is 2-methoxyestradiol diaminosulphonate (2-MeOE2bisMATE).
  • the anti-proliferative pro-apoptotic agent is a conventional anti-proliferative pro-apoptotic agent in the art, preferably selected from the group consisting of a PPAR- ⁇ activator, a retinoid, a triterpenoid, an EGF receptor inhibitor, and an end.
  • the PPAR-gamma activators are conventional PPAR-gamma activators in the art, preferably cyclopentenone prostaglandins (cyPGs).
  • the triterpenoids are conventional triterpenoids in the art, preferably selected from the group consisting of cyclobungane, lupin, ursane, zitherane, xylan, dammarane, cucurbitacin, lemon A bitter analog or a triterpenoid.
  • the EGF receptor inhibitor is a conventional EGF receptor inhibitor in the art, preferably selected from the group consisting of HER4, rapamycin or 1,25-dihydroxycholecalciferol (vitamin D).
  • the iron chelate is a conventional iron chelate compound, preferably 3-aminopyridine-2-formaldehyde thiosemicarbazone.
  • the apoptotic protein is a conventional apoptotic protein in the art, preferably the viral protein 3-VP3 of chicken anemia virus.
  • the PI3K-Akt survival pathway signaling inhibitor is a conventional PI3K-Akt survival pathway signaling inhibitor in the art, preferably UCN-01 or geldanamycin.
  • the cytolytic enzyme is a cytolytic enzyme conventional in the art, preferably an RNase.
  • -(L) x -(D) y is:
  • n is from 1 to 10, preferably m is 5.
  • -(L) x -(D) y is:
  • -(L) x -(D) y is:
  • said D is a tubulin synthetase inhibitor, methyl auristatin F (MMAF), and said linker L is maleimidocaproyl (MC), said immunization
  • MMAF methyl auristatin F
  • MC maleimidocaproyl
  • the L is 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester;
  • D is N2'-deacetyl-N2'-3-indolyl-1 Oxopropyl)-maytansin (DM1), the structure of the immunoconjugate is as shown in Formula 4,
  • L is maleimidocaproyl-L-valine-L-citrulline p-aminobenzyl alcohol and D is methyl auristatin E (MMAE), and the structure of the immunoconjugate is as As shown in Equation 5,
  • n is a natural number, preferably an integer of 1 to 20, more preferably 1 to 2, or 2 to 4, or 4 to 8, or 8 to 20.
  • the preparation method of the immunoconjugate is conventional in the art, and the preparation method described in Doronina, 2006, Bioconjugate Chem. 17, 114-124 is preferably employed.
  • the method of preparation produces an immunoconjugate having a minimal low coupling fraction (LCF) of less than 10%.
  • the preparation method comprises the steps of: dialysis of the above protein through a sodium borate buffer having a pH of 6.5 to 8.5, and adding tris(2-carboxyethyl)phosphine (TCEP), wherein the TCEP and the above protein are The molar ratio is 2 to 10, and the mixture is reduced at room temperature for 1 to 4 hours to obtain a reaction liquid A.
  • the reaction solution A was eluted to remove excess protein, and the reaction liquid B was obtained.
  • MC-MMAF was added to the reaction solution B, wherein the molar ratio of the MC-MMAF to the purified TPBG antibody was 5 to 20, and the reaction was carried out at 10 to 37 ° C for 4 hours.
  • the immunoconjugate can be present in any physical form known in the art, preferably a clear solution.
  • the invention also provides a pharmaceutical composition comprising the immunoconjugate described above and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier is a carrier conventional in the art and may be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient.
  • the pharmaceutical excipients are conventional pharmaceutical excipients in the art, preferably including pharmaceutically acceptable excipients, fillers or diluents and the like. More preferably, the pharmaceutical composition comprises from 0.01 to 99.99% of the above protein and from 0.01 to 99.99% of a pharmaceutically acceptable carrier, the percentage being a percentage by mass of the pharmaceutical composition.
  • the pharmaceutical composition is an anti-tumor drug. More preferably, anti-squamous/adenomatous lung cancer (non-small cell lung cancer), invasive breast cancer, colon cancer, rectal cancer, gastric cancer, squamous cervical cancer, invasive endometrial adenocarcinoma, invasive pancreatic cancer, Drugs for ovarian cancer, squamous bladder cancer, choriocarcinoma, bronchial carcinoma, breast cancer, cervical cancer, pancreatic cancer or seminal vesicle cancer.
  • anti-squamous/adenomatous lung cancer non-small cell lung cancer
  • invasive breast cancer colon cancer
  • rectal cancer gastric cancer
  • squamous cervical cancer invasive endometrial adenocarcinoma
  • pancreatic cancer Drugs for ovarian cancer
  • squamous bladder cancer choriocarcinoma
  • bronchial carcinoma breast cancer
  • cervical cancer pancreatic cancer
  • seminal vesicle cancer
  • the administration route of the pharmaceutical composition of the present invention is preferably parenteral administration, injection administration or oral administration.
  • the administration by injection preferably includes intravenous, intramuscular, intraperitoneal, intradermal or subcutaneous injection.
  • the pharmaceutical composition is in various forms conventional in the art, preferably in the form of a solid, semi-solid or liquid, that is, it may be an aqueous solution, a non-aqueous solution or a suspension, more preferably a tablet, a capsule or a granule.
  • the pharmaceutical composition may also be administered as an aerosol or crude spray, i.e., nasally; or intrathecally, intramedullaryly or intraventricularly. More preferably, the pharmaceutical composition may also be administered transdermally, transdermally, topically, enterally, intravaginally, sublingually or rectally.
  • the dosage level of the pharmaceutical composition of the present invention can be adjusted depending on the amount of the composition that achieves the desired diagnosis or treatment result.
  • the administration regimen can also be a single injection or multiple injections, or adjusted.
  • the selected dosage level and regimen will depend on the activity and stability (ie, half-life) of the pharmaceutical composition, the formulation, the route of administration, the combination with other drugs or treatments, the disease or condition to be detected and/or treated, Reasonable adjustments are made to various factors such as the health status of the subject to be treated and the prior medical history.
  • the therapeutically effective dose of the pharmaceutical composition of the invention can be estimated initially in cell culture experiments or animal models such as rodents, rabbits, dogs, pigs and/or primates. Animal models can also be used to determine the appropriate range and route of administration. This can then be used to determine the useful dose and route of administration in a human. In general, the determination and adjustment of the effective amount or dose administered and the assessment of when and how such adjustments are made are known to those skilled in the art.
  • the above proteins, immunoconjugates and/or additional therapeutic or diagnostic agents described above can each be used as a single agent, in any time frame suitable for performing the intended treatment or diagnosis.
  • these single agents can be administered substantially simultaneously (i.e., as a single formulation or over a few minutes or hours) or sequentially.
  • these single agents can be within one year, or within 10, 8, 6, 4 or 2 months, or within 4, 3, 2, or 1 week, Or administered within 5, 4, 3, 2 or 1 day.
  • the present invention provides the use of the above protein for the preparation of an antitumor drug.
  • the present invention provides an application of the above immunoconjugate in the preparation of an antitumor drug.
  • the present invention provides the use of the above pharmaceutical composition for the preparation of an antitumor drug.
  • the present invention provides the use of the above protein for treating tumors.
  • the present invention provides the use of the above immunoconjugate in the treatment of tumors.
  • the present invention provides the use of the above pharmaceutical composition for treating a tumor.
  • the present invention also provides a method for detecting a cell overexpressing a TPBG protein, comprising the steps of: contacting the protein with a sample to be tested in vitro, and detecting the binding of the protein to the sample to be tested.
  • the meaning of the overexpression is conventional in the art.
  • the cells are subjected to flow detection, and the average fluorescence density (MFI) value of the above protein is 3 times or more of the MFI value of the subtype IgG. .
  • the detection mode of the combination is a conventional detection method in the art, preferably FACS detection.
  • TPBG-positive cells of the present invention are cells that overexpress the TPBG protein, such as the NCI-H1568 cell line; otherwise, they are referred to as "TPBG-negative” cells, such as the tumor cell line NCI-H1770.
  • the reagents and starting materials used in the present invention are commercially available.
  • the TPBG antibody of the present invention is a chimeric antibody which has a high affinity with the TPBG protein and which binds to the extracellular region of the TPBG protein receptor at the protein level and the cellular level.
  • the TPBG antibody is coupled with a small molecule compound such as MC-MMAF to obtain a conjugate which is effective for cytotoxic killing of TPBG-positive cells.
  • TPBG antibodies can introduce small molecule compounds, such as MMAF, into the cells by endocytosis, and release small molecule compounds in the cells to play a cytotoxic killing effect. Therefore, the antibody cross-linking agent prepared by the TPBG antibody can effectively kill tumor cells and treat tumors.
  • Figure 1 shows the results of FACS screening of HEK293 cells transfected with human TPBG protein.
  • Figure 2 shows the results of FACS screening of CHOK1 cells transfected with human TPBG protein.
  • Figure 3 shows the results of FACS screening of CHOK1 cells transfected with cynomolgus monkey TPBG protein.
  • Figure 4 shows the results of FACS screening of CHOK1 cells transfected with mouse TPBG protein.
  • Figure 5 shows the serum antibody titer of mice after TPBG immunization by ELISA.
  • Figure 6A and Figure 6B show the binding reaction of TPBG antibody to human TPBG-hFc protein by ELISA.
  • Figure 7A and Figure 7B show the binding reaction of TPBG antibody to CHOk1-hTPBG by FACS.
  • Figure 8A and Figure 8B show the binding reaction of TPBG antibody to CHOk1-cTPBG by FACS.
  • Figure 9A and Figure 9B show the binding reaction of TPBG antibody to CHOk1-mTPBG by FACS.
  • Figure 10A and Figure 10B show the binding reaction of TPBG antibody to CHOk1 by FACS.
  • Figure 11A and Figure 11B show the cell killing effect of TPBG antibody-MMAF antibody cross-linking agent on TPBG-positive non-small lung cancer cell line NCI-H1568.
  • Figure 11C shows the cell killing effect of TPBG antibody on TPBG-positive non-small lung cancer cell line NCI-H1568.
  • Figure 12A and Figure 12B show the cell killing effect of TPBG antibody-MMAF antibody cross-linking agent on TPBG-negative non-small lung cancer cell line NCI-H1770.
  • Figure 13 is a graph showing the cytotoxic effect of a TPBG chimeric antibody drug conjugate on a TPBG positive tumor cell line NCI-H1299.
  • Figure 14A is a graph showing changes in tumor volume after treatment of antibody drug conjugates of TPBG chimeric antibody 12B12 and naked anti-treatment.
  • Figure 14B is a graph showing changes in tumor volume after treatment of the antibody drug conjugate of the TPBG chimeric antibody 5G4 and its naked anti-treatment.
  • Figure 14C is a graph showing changes in tumor volume after treatment of the antibody drug conjugate of TPBG chimeric antibody 39A11 and its naked anti-treatment.
  • Figure 14D is a graph showing changes in tumor volume after treatment of antibody drug conjugates of TPBG chimeric antibody 28D4 and their naked antibodies.
  • Figure 14E is a graph showing changes in tumor volume after treatment of the antibody drug conjugate of TPBG chimeric antibody 36A10 and its naked anti-treatment.
  • Figure 15A is a graph showing changes in body weight of mice after treatment of the antibody drug conjugate of TPBG chimeric antibody 12B12 and its naked anti-treatment.
  • Figure 15B is a graph showing changes in body weight of mice after treatment of the antibody drug conjugate of the TPBG chimeric antibody 5G4 and its naked anti-treatment.
  • Figure 15C is a graph showing changes in body weight of the antibody drug conjugate of the TPBG chimeric antibody 39A11 and its naked anti-treatment.
  • Figure 15D is a graph showing changes in body weight of mice after treatment of antibody drug conjugates of TPBG chimeric antibody 28D4 and their naked antibodies.
  • Figure 15E is a graph showing changes in body weight of mice after treatment of the antibody drug conjugate of TPBG chimeric antibody 36A10 and its naked anti-treatment.
  • 16A and 16B are the cell killing effects of the TPBG chimeric antibody 12B12 and its antibody drug conjugate coupled with different small molecule drugs on the TPBG-positive tumor cell line NCI-H1299, respectively.
  • 16C and 16D are the cell killing effects of the TPBG chimeric antibody 12B12 and its antibody drug conjugates coupled with different small molecule drugs on the TPBG-positive tumor cell line NCI-H1568, respectively.
  • 17A and 17B are cell killing effects of TPBG chimeric antibody drug conjugates on TPBG-positive TPBG-expressing 293-hTPBG stably transfected cell lines.
  • Figures 17C and 17D show the cell killing effect of TPBG chimeric antibody drug conjugates on 293 cell lines that do not express TPBG.
  • the room temperature described in the examples is room temperature conventional in the art, and is generally from 10 to 30 °C.
  • the PBS described in the examples was a PBS phosphate buffer, pH 7.2.
  • the pCpC vector purchased from Invitrogen, V044-50 harboring the human IgG Fc fragment (hFc) was cloned and the plasmid was prepared according to established standard molecular biology methods. For a specific method, see Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press).
  • HEK293 cells purchased from Invitrogen
  • polyetherimide PEI polyetherimide PEI
  • FreeStyleTM 293 purchased from Invitrogen
  • the culture supernatant was applied to a Protein A affinity chromatography column (Mabselect Sure, purchased from GE Healthcare), while monitoring the change in UV absorption (A280nm) with an ultraviolet (UV) detector.
  • the protein A affinity column was washed with PBS phosphate buffer (pH 7.2) until the UV absorbance value returned to the baseline, and then eluted with 0.1 M glycine hydrochloride (pH 2.5) to collect affinity from protein A.
  • the hFc-tagged TPBG protein ie, human TPBG-hFc
  • Dialysis was carried out overnight in a refrigerator at 4 ° C with PBS phosphate buffer (pH 7.2).
  • the dialyzed protein was sterile-filtered at 0.22 ⁇ m and stored at -80 ° C to obtain purified immunogen A.
  • Immunogen A requires a series of quality control tests before use, such as detection of protein concentration, purity, molecular weight, biological activity, etc., and found that the immunogen A indicators are good, can be used as an antigen for subsequent preparation of TPBG antibodies.
  • the nucleotide sequence encoding the full-length amino acid sequence of human TPBG (wherein the nucleotide sequence encoding the human TPBG protein is Genebank ID: AAH37161.1 in Genebank) was cloned into the pIRES vector (purchased from Clontech) and prepared. Plasmid. HEK293 cell line and CHOK1 cell line (both purchased from Invitrogen) were transfected with plasmid (PEI, purchased from Polysciences) in DMEM medium containing 0.5% g/w fetal calf serum containing 0.5 ⁇ g/ml.
  • the cells were selectively cultured for 2 weeks, subcloned in a 96-well culture plate by limiting dilution, and placed in a 5% (v/v) CO 2 culture at 37 ° C. After approximately 2 weeks, a partial monoclonal well was selected and amplified to 6 In the orifice plate.
  • the amplified clones were screened by flow cytometry using a known TPBG antibody (purchased from Sigma, Cat. #SAB1404485). The cell line with good growth and high fluorescence intensity is selected, and the monoclonal cell line continues to expand and the liquid nitrogen is frozen, that is, the immunogen B is obtained.
  • the specific selection results are shown in Table 3 and Figure 1, and the IgG subtype control is a mouse IgG control.
  • Table 3 illustrates that a series of HEK293 cell lines positive for TPBG expression have been made.
  • the abscissa is the fluorescence intensity of the cells, and the ordinate is the number of cells.
  • the results in Figure 1 indicate that 5E5E9 is a high-level expression cell line of TPBG in which the anti-TPBG antibody-labeled cells have an average cell fluorescence density of 216 and a mobility of 98.5%.
  • mice 6-8 weeks old BALB/cAnNCrl mice or SJL/JorllcoCrl mice (all purchased from Shanghai Slack Company) were used, and the mice were raised under SPF conditions.
  • the culture was expanded to 90% confluence in T-75 cell culture flasks, the medium was aspirated, washed twice with DMEM basal medium (purchased from Invitrogen), and then with enzyme-free cell dissociation (purchased from Invitrogen) at 37 °C.
  • the cells are collected until the cells are detachable from the wall of the culture dish.
  • the cells were washed twice with DMEM basal medium, and after cell counting, the cells were diluted with phosphate buffer to 2 ⁇ 10 7 cells per mL.
  • Each mouse was intraperitoneally injected with 0.5 mL of cell suspension per immunization.
  • the first and second immunizations were separated by 2 weeks, and each subsequent immunization interval was 3 weeks.
  • each mouse selected was intraperitoneally injected with 100 ⁇ g of purified immunogen A (mouse against immunogen A) or HEK293 stable cell line containing human TBPG.
  • Mice vaccinated against immunogen B mice were sacrificed 5 days later, and spleen cells were collected.
  • NH 4 OH was added to a final concentration of 1% (w/w), and the erythrocytes mixed in the spleen cells were lysed to obtain a spleen cell suspension.
  • the cells were washed three times with DMEM basal medium at 1000 rpm, and then mixed with mouse myeloma cells SP2/0 (purchased from ATCC) at a 5:1 ratio of viable cells, using a high-efficiency electrofusion method (see METHODS IN ENZYMOLOGY). , VOL.220) for cell fusion.
  • the fused cells were diluted into DMEM medium containing 20% (w/w) fetal bovine serum and 1 x HAT. Then, 1 ⁇ 10 5 /200 ⁇ l per well was added to a 96-well cell culture plate, and cultured in a 5% (v/v) CO 2 incubator at 37 °C.
  • the cell fusion plates were screened by ELISA and Acumen (microplate assay), and positive clones with OD 450nm >1.0 in ELISA and MFI values >100 in Acumen were amplified into 24-well plates at 10% (in 10%).
  • the culture medium expanded in a 24-well plate was centrifuged, the supernatant was collected, and the supernatant was subjected to antibody subtype analysis, and the binding activity to the TPBG protein and the TPBG-positive cells was determined by ELISA and FACS (binding activity).
  • the detection methods are respectively referred to the relevant contents in Example 3A and Example 3B, and the mouse-derived TPBG antibody-MMAF indirect cytotoxic killing experiment (see the relevant content in Example 4 for the indirect cytotoxic killing activity detecting method).
  • the hybridoma cells with OD 450nm >1.0 in the ELISA assay, MFI value >50 in the FACS experiment, and the hybridoma cell culture supernatant in the indirect cytotoxic killing assay on the TPBG-positive cells were 50%.
  • select eligible hybridoma cells were subcloned in 96-well plates by limiting dilution in DMEM medium containing 10% (w/w) FBS (purchased from invitrogen) at 37 ° C, 5% (v/v) culture under CO 2 conditions.
  • Initial screening was performed by ELISA and Acumen 10 days after subcloning, and single positive monoclonal clones were selected and expanded into 24-well plates for further culture.
  • the antigen binding positive was determined by FACS and the biological activity was evaluated by the mouse-derived TBPG antibody-MMAF indirect cytotoxic killing assay.
  • the evaluation criteria were OD 450nm >1.0 in the ELISA experiment, MFI value >50 in the FACS experiment and indirect cytotoxic killing experiment.
  • the hybridoma cell culture supernatant has a killing rate of 50% or more for TPBG-positive cells.
  • the best clones were selected and cultured in DMEM medium containing 10% (w/w) FBS (purchased from invitrogen) at 37 ° C, 5% (v/v) CO 2 conditions.
  • the optimal clone is expanded and cultured, and the hybridoma cells of the present invention are obtained by cryopreservation of liquid nitrogen, and can be used for subsequent obtaining of the lead antibody, production and purification of the antibody.
  • Hybridoma cells produce a lower concentration of antibodies, which are only about 1-10 ⁇ g/mL, and the concentration varies greatly. Moreover, the various proteins produced by the cell culture in the medium and the fetal bovine serum components contained in the medium have different degrees of interference with many biological activity analysis methods, so that small-scale (1-5 mg) antibody production purification is required.
  • the hybridoma cells obtained in Example 1 were inoculated into a T-75 cell culture flask and domesticated for 3 passages using a production medium (Hybridoma serum free medium, available from Invitrogen). After the growth state is good, inoculate the cell culture spinner. 200 mL of production medium was added to each of 2 liters of culture spinner flasks, and the inoculated cell density was 1.0 ⁇ 10 5 /mL. The cap was capped and the roller was placed in a bottle shaker in a 37 ° C incubator at 3 rpm.
  • a production medium Hybridoma serum free medium
  • the culture supernatant of the clarified hybridoma cells can be immediately purified or frozen at -30 °C.
  • the TPBG antibody in the obtained culture supernatant (200 mL) was purified with a 2 mL Protein A column (purchased from GE Healthcare).
  • the protein G column was first equilibrated with equilibration buffer (PBS phosphate buffer, pH 7.4), and then the culture supernatant was applied to a Protein A column at a flow rate of 3 mL/min. After loading, the protein G column was washed with equilibration buffer, and the volume of the equilibration buffer was 4 times the volume of the protein A column.
  • equilibration buffer PBS phosphate buffer, pH 7.4
  • the TPBG antibody bound to the Protein A column was eluted with an eluent (0.1 M sodium citrate buffer, pH 3.5), and the elution condition (A 280 nm ultraviolet absorption peak) was monitored with a UV detector.
  • the eluted antibody was collected, neutralized by adding 10% (v/v) 1.0 M Tris-HCl buffer, and immediately dialyzed overnight with PBS phosphate buffer, and the solution was changed once a day for another 3 hours.
  • the dialyzed TPBG antibody was collected, sterile-filtered with a 0.22 ⁇ m filter, and stored aseptically to obtain a purified TPBG antibody.
  • the purified TPBG antibody was assayed for protein concentration (A 280 nm / 1.4), purity, endotoxicity (Lonza kit), and the results are shown in Table 5.
  • Table 5 shows that the endotoxin concentration of the antibody final product was 1.0 EU/mg. Within.
  • ELISA enzyme-linked immunosorbent assay
  • the purified TPBG antibody obtained in Example 2 was reacted with a human TBPG-hFc protein (immunogen A).
  • the purified immunogen A obtained in Example 1 was prepared (for the preparation method, see step 1 (I) of Example 1 and diluted to a final concentration of 1.0 ⁇ g/mL with PBS, and then added to a 96-well ELISA plate at 100 ⁇ L per well. Incubate overnight at 4 ° C, wash the plate twice with washing solution [PBS containing 0.01% (v/v) Tween 20], add blocking solution [containing 0.01% (v/v) Tween 20 and 1% (w/) w) BSA in PBS] was blocked at room temperature for 2 hours. The blocking solution was discarded, and 100 ⁇ L of the purified TPBG antibody obtained in Example 2 was added per well.
  • the pIRES plasmid containing the nucleotide sequence encoding the full-length amino acid sequence of human TBPG described in the step (2) of Example 1 was transfected into the CHOK1 cell line to obtain a CHOK1 stable cell line containing human TPBG (herein referred to as CHOk1- hPD1 stable cell line).
  • a pIRES plasmid carrying a monkey-derived TPBG full-length gene (the preparation method thereof and the pCpC vector carrying the human IgG Fc fragment (hFc) in the step (1) of the preparation of the immunogen A in Example 1)
  • the full-length amino acid sequence of cynomolgus monkey TPBG (Genebank ID: BAE00432.1) was transfected into CHOK1 cell line to obtain a CHOK1 stable cell line containing monkey TPBG (herein referred to as CHOk1-cTPBG stable cell line).
  • the pIRES plasmid derived from the full-length gene of TPBG (the preparation method thereof is the same as the preparation method of the pCpC vector carrying the human IgG Fc fragment (hFc) in the step (1) "Preparation of immunogen A" of Example 1, and the full length of the mouse TPBG
  • the amino acid sequence (Genebank ID: CAA09931.1) was transfected into the CHOK1 cell line to obtain a CHOK1 stable cell line containing mouse TPBG (herein referred to as CHOk1-mTPBG stable cell line).
  • the titer and specificity of the TPBG antibody in serum were determined by FACS.
  • the detection method the method of identifying the HEK293-hTPBG stable cell line in the preparation of the preparation of immunogen B in the step (2) of Example 1 was carried out.
  • the detection results are shown in Table 7 and Figures 2 to 4.
  • the abscissa is the fluorescence intensity of the cells, and the ordinate is the number of cells.
  • CHOK1-hTPBG 3A2F1 is the human TPBG expression cell line used for screening, and the results of FACS screening test are shown in Figure 2;
  • CHOK1-cTPBG 3F13G4 is the cynomolgus monkey TPBG expression cell line used for screening, and its FACS screening test results As shown in Fig. 3;
  • CHOK1-mTPBG 3A3 is a mouse TPBG expressing cell strain for screening, and the results of FACS screening assay are shown in Fig. 4.
  • Table 7 indicate that the CHOk1-hTPBG stable cell line, the CHOk1-cTPBG stable cell line, and The TPBG protein of human, monkey or mouse is overexpressed on the cell membrane of the CHOk1-mTPBG stable cell line, which can be used for screening TPBG antibodies.
  • CHOk1-hTPBG stable cell line, CHOk1-cTPBG stable cell line, CHOK1-mTPBG stable cell line (ie, CHOk1-hTPBG 3A2F1, CHOk1-cTPBG 3F13G4 and CHOk1-mTPBG 3A3 shown in Table 7) and CHOK1 cells were respectively in T-
  • the culture was expanded to 90% confluence in a 75-cell culture flask, the medium was exhausted, and washed twice with HBSS Balanced Salt Solution (purchased from Invitrogen), followed by an enzyme-free cell dissociation solution (Versene solution: purchased Process and collect cells from Life Technology.
  • the cells were washed twice with HBSS buffer, and after cell counting, the cells were diluted to 2 ⁇ 10 6 cells/mL with HBSS buffer, and 10% goat serum blocking solution was added, the percentage being mass percentage, and incubation on ice for 30 minutes. Then, it was washed twice with HBSS buffer.
  • the collected cells were suspended in FACS buffer (HBSS + 1% BSA, the percentage is percentage by mass) to 2 ⁇ 10 6 cells / mL, and added to a 96-well FACS reaction plate at 100 ⁇ L per well, and the addition was carried out.
  • the purified TPBG antibody obtained in Example 2 was sampled at 100 ⁇ L per well and incubated on ice for 2 hours.
  • the cells were washed twice with FACS buffer, and 100 ⁇ l of a fluorescent (Alexa 488)-labeled secondary antibody (purchased from Invitrogen) per well was added and incubated on ice for 1 hour.
  • the cells were washed three times with FACS buffer, and the cells were resuspended by adding 100 ⁇ l of the fixing solution [4% (v/v) paraformaldehyde] per well, and after 10 minutes, the cells were washed twice with FACS buffer.
  • the cells were suspended in 100 ⁇ l of FACS buffer, and the results were detected and analyzed by FACS (FACS Calibur, available from BD). Data analysis by software (CellQuest) gave the average fluorescence density (MFI) of the cells.
  • the data was fitted and analyzed by software (GraphPad Prism 5) to calculate the EC50 value.
  • the analysis results are shown in Table 8 and Figures 7 to 10.
  • the data in Figures 7 to 10 are the average fluorescence density (MFI) of the cells.
  • the data in Table 8 is the EC50 value calculated from the MFI. Table 8 illustrates that TPBG antibodies bind to the TPBG protein on the cell surface.
  • Example 2 After the purified TPBG antibody obtained in Example 2 was dialyzed against sodium borate buffer of pH 6.5-8.5, tris(2-carboxyethyl)phosphine (TCEP) was added, wherein the molar ratio of TCEP to purified TPBG antibody was 2 The mixture was reduced at room temperature for 1 hour to obtain a reaction liquid A.
  • the reaction solution A was desalted (purchased from GE) through a G25 column to remove excess TCEP to obtain a reaction liquid B.
  • MC-MMAF (purchased from Nanjing Lian Ning) was added to the reaction solution B, wherein the molar ratio of the MC-MMAF to the purified TPBG antibody was 5, and the reaction was carried out at room temperature for 4 hours.
  • Cysteine was added to neutralize excess MC-MMAF and desalted through a G25 column to remove excess small molecules.
  • a purified TPBG antibody drug conjugate was obtained (for coupling methods, see Doronina, 2006, Bioconjugate Chem. 17, 114-124).
  • the cytotoxic activity was analyzed by analyzing the parameters such as the crosslinking rate and purity of the drug by HIC.
  • the drug cross-linking rate (DAR) of all antibody conjugates was 8. Wherein, DAR (drug antibody ratio) refers to the average number of small molecule drugs carried on one antibody molecule after antibody coupling.
  • the obtained purified TPBG antibody drug conjugates were separately diluted with complete medium, and 100 ⁇ l of TBPG-positive NCI-H1568 cell line was purchased at 2000 cells/well in 96-well cell culture plates (purchased from ATCC, Cat# CRL5876) After the cell suspension was cultured overnight, 10 ⁇ l of each dilution of the purified TPBG antibody drug conjugate was added to each well, and the culture was continued for 5 days, and then the CellTiter-Glo kit (purchased from Promega, the method of reference was used). Instructions) to measure cell viability.
  • the TPBG-negative tumor cell line NCI-H1770 (purchased from ATCC, item #CRL5893) was used for cell killing activity detection, and the method was the same as above.
  • the results are shown in Table 9 and Figs. 11-12, wherein the EC50 of Table 9 refers to a half effective amount in which the activity of the cells is inhibited after the action of the drug, and the cell killing activity can be reflected by detecting the activity of the cells.
  • 11A and 11B are the cytotoxic activity of the purified TPBG antibody drug conjugate against the TPBG-positive tumor cell line NCI-H1568
  • FIGS. 12A and 12B are purified TPBG antibody drug conjugates to TPBG-negative tumor cells.
  • the cell killing activity of NCI-H1770 was detected.
  • the results indicate that the purified TPBG antibody drug conjugate has a killing effect on TPBG-positive cells.
  • Example 2 the killing effect of the purified TPBG antibody obtained in Example 2 alone on the cells was also examined.
  • the method for detecting the cell killing activity of the TPBG antibody drug conjugate in Example 4 was examined. The results are shown in Table 9 and Figure 11C. The results show that the purified TPBG antibody alone has no significant killing effect on TPBG-positive cells.
  • Example 5 Competitive ELISA assay for analysis of epitope distribution of TPBG antibodies and antigens
  • TPBG antibodies were grouped by competitive ELISA.
  • the purified antibody to be tested was diluted to 1 ⁇ g/mL with PBS, coated with 96-well high-adsorbing enzyme plate at 50 ⁇ L/well, and coated with 250 ⁇ l of blocking solution at 4 ° C overnight [containing 0.01% (v/v) Tween 20 and 1% (w/w) BSA in PBS] was blocked at room temperature for one hour, and 0.05 ⁇ g/mL of biotin-labeled recombinant TPBG protein was added to each well.
  • the competing antibody that is, the purified TPBG antibody obtained in Example 2, having the clone numbers 12B12C7C3, 5G4H10G5, 37H9C5G2, 39A11G5F2, 52C9E9F6, 28D4E6A9, 36A10D8B12, 99E12C7H1, 103E2E9C2 and 106D5G3D10, respectively, and 25-37 Incubate for 1-2 hours at °C.
  • the plate was washed 3 times with a washing solution [PBS containing 0.01% (v/v) Tween 20], and HRP (horseradish peroxidase)-labeled streptavidin (purchased from Sigma) was added. After incubation at 37 ° C for 0.5 hour, the plate was washed 3 times with a washing solution [PBS containing 0.01% (v/v) Tween 20]. After adding 100 ⁇ L of TMB substrate per well and incubating for 30 minutes at room temperature, 100 ⁇ L of each well was added to the stop solution (1.0 N HCl). The A 450 nm value was read using an ELISA plate reader (SpectraMax 384plus, available from Molecular Device) and the results are shown in FIG. The competition rates of the antibodies were calculated based on the A 450 nm values, and the results are shown in Table 10. The higher the value of the competition rate, the closer the antigen surface of the two antibodies is.
  • each column is coated with a antibody at a concentration of 1 ⁇ g/mL;
  • B means that each behavior competes for antibodies, The concentration was 5 ⁇ g/mL.
  • RNA isolation 5 ⁇ 10 7 hybridoma cells obtained in Example 1 were collected by centrifugation, mixed with 1 mL of Trizol, and transferred to a 1.5 mL centrifuge tube, and allowed to stand at room temperature for 5 minutes. 0.2 mL of chloroform was added, shaken for 15 seconds, allowed to stand for 10 minutes, centrifuged at 12000 g for 5 minutes at 4 ° C, and the supernatant was transferred to a new 1.5 mL centrifuge tube. 0.5 mL of isopropanol was added, and the liquid in the tube was gently mixed. After standing at room temperature for 10 minutes, it was centrifuged at 12,000 g for 15 minutes at 4 ° C, and the supernatant was discarded.
  • Reverse transcription and PCR 1 ⁇ g of total RNA was taken, 20 ⁇ L of the system was placed, reverse transcriptase was added, and the reaction was carried out at 42 ° C for 60 minutes, and the reaction was terminated by reaction at 7 ° C for 10 minutes.
  • a 50 ⁇ L PCR system was configured, including 1 ⁇ L of cDNA, 25 pmol of each primer, 1 ⁇ L of DNA polymerase, and a matching buffer system, 250 ⁇ mol dNTPs; a PCR program was set, predenatured at 95 ° C for 3 minutes, denatured at 95 ° C for 30 seconds, and annealed at 55 ° C for 30 seconds.
  • the extension was carried out at 72 ° C for 35 seconds, and after 35 cycles, an additional 5 minutes at 72 ° C to obtain a PCR product.
  • the kit for reverse transcription was PrimeScript RT Master Mix, purchased from Takara, Catalog No. RR036; the kit used for PCR included Q5 super-fidelity enzyme, available from NEB, Cat. No. M0492.
  • PCR product 5 ⁇ L was detected by agarose gel electrophoresis, and the positive samples were purified using a column recovery kit, wherein the recovery kit was Gel & PCR Clean-up, available from MACHEREY-NAGEL, Cat. No. 740609.
  • the ligation reaction was carried out: 50 ng of the sample, 50 ng of the T vector, 0.5 ⁇ L of the ligase, 1 ⁇ L of the buffer solution, and 10 ⁇ L of the reaction system, and the reaction product was allowed to react at 16 ° C for half an hour.
  • the ligated kit was T4 DNA ligase, purchased from NEB, catalog number M0402; 5 ⁇ L of the ligation product was added to 100 ⁇ L of competent cells (Ecos 101competent cells, purchased from Yeastern, item number FYE607), ice bath for 5 minutes, and then at 42 ° C. The bath was heat shocked for 1 minute, placed on ice for 1 minute, and then 650 ⁇ L of antibiotic-free SOC medium was added and resuscitated at 200 RPM for 30 minutes on a 37 ° C shaker. 200 ⁇ L of the solution was applied to an antibiotic-containing LB solid medium and incubated overnight at 37 ° C in an incubator.
  • a 30 ⁇ L PCR system was configured using the primers M13F and M13R on the T vector, colony PCR was performed, and the colony was pipetted in a PCR reaction system, and 0.5 ⁇ L of the LB containing 100 nM ampicillin was aspirated. The strain was preserved on a solid petri dish. After the end of the PCR reaction, 5 ⁇ L was taken for agarose gel electrophoresis detection, and the positive samples were sequenced and analyzed [see Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991)]. The sequencing results are shown in Tables 11-12.
  • the number in Table 11 is the sequence number in the sequence listing, for example, the amino acid sequence of the heavy chain protein variable region of 12B12C7C3 is SEQ ID No. 1, and the amino acid sequence of CDR1 in the heavy chain protein variable region of 12B12C7C3 is SEQ. ID No. 2.
  • the number in Table 12 is the sequence number in the sequence listing, and the nucleotide sequence of the variable region of the heavy chain protein encoding 12B12C7C3 is SEQ ID No. 81.
  • nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 12B12C7C3 is from position 91 to position 105 in SEQ ID No. 81 of the sequence listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 12B12C7C3 is from positions 151 to 198 in SEQ ID No. 81 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 12B12C7C3 is 295th to 327th in SEQ ID No. 81 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 12B12C7C3 is from positions 70 to 114 of SEQ ID No. 82 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 12B12C7C3 is from positions 157 to 180 in SEQ ID No. 82 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 12B12C7C3 is 277th to 303th in SEQ ID No. 82 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 5G4H10G5 is the 91st to 105th positions in SEQ ID No. 83 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 5G4H10G5 is from positions 148 to 198 in SEQ ID No. 83 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 5G4H10G5 is from positions 295 to 330 of SEQ ID No. 83 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 5G4H10G5 is from positions 70 to 102 of SEQ ID No. 84 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 5G4H10G53 is 148th to 168th in SEQ ID No. 84 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 5G4H10G5 is from position 265 to position 288 in SEQ ID No. 84 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 37H9C5G2 is from positions 91 to 105 of SEQ ID No. 85 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 37H9C5G2 is from positions 151 to 198 in SEQ ID No. 85 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 37H9C5G2 is from positions 295 to 327 in SEQ ID No. 85 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 37H9C5G2 is in SEQ ID No. 86 of the Sequence Listing. 70th to 102nd;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 37H9C5G2 is 148th to 168th in SEQ ID No. 86 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 37H9C5G2 is from positions 265 to 291 in SEQ ID No. 86 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 39A11G5F2 is from positions 91 to 105 of SEQ ID No. 87 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 39A11G5F2 is from positions 151 to 198 of SEQ ID No. 87 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 39A11G5F2 is 295th to 315th in SEQ ID No. 87 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 39A11G5F2 is from positions 70 to 102 of SEQ ID No. 88 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 39A11G5F2 is 148th to 168th in SEQ ID No. 88 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 39A11G5F2 is from positions 265 to 291 in SEQ ID No. 88 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 52C9E9F6 is position 91 to 105 in SEQ ID No. 89 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 52C9E9F6 is from positions 151 to 198 in SEQ ID No. 89 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 52C9E9F6 is 295th to 315th in SEQ ID No. 89 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 52C9E9F6 is from positions 70 to 102 of SEQ ID No. 90 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 52C9E9F6 is 148th to 168th in SEQ ID No. 90 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 52C9E9F6 is from positions 268 to 291 in SEQ ID No. 90 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 28D4E6A9 is from positions 91 to 105 of SEQ ID No. 91 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 28D4E6A9 is from positions 148 to 198 of SEQ ID No. 91 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 28D4E6A9 is from positions 295 to 327 of SEQ ID No. 91 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 28D4E6A9 is from positions 70 to 102 of SEQ ID No. 92 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 28D4E6A9 is from positions 148 to 168 of SEQ ID No. 92 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 28D4E6A9 is from positions 265 to 291 in SEQ ID No. 92 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 36A10D8B12 is from positions 91 to 108 of SEQ ID No. 93 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 36A10D8B12 is from positions 154 to 198 of SEQ ID No. 93 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 36A10D8B12 is from positions 295 to 324 of SEQ ID No. 93 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 36A10D8B12 is from positions 70 to 102 of SEQ ID No. 94 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 36A10D8B12 is 148th to 168th in SEQ ID No. 94 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 36A10D8B12 is position 265 to 291 of SEQ ID No. 94 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 99E12C7H1 is from positions 91 to 105 of SEQ ID No. 95 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 99E12C7H1 is from positions 151 to 198 in SEQ ID No. 95 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 99E12C7H1 is from positions 295 to 318 of SEQ ID No. 95 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 99E12C7H1 is from positions 70 to 102 of SEQ ID No. 96 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 99E12C7H1 is SEQ ID No. 96 of the Sequence Listing. 148th to 168th;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 99E12C7H1 is from position 265 to position 291 in SEQ ID No. 96 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 103E2E9C2 is position 91 to 105 in SEQ ID No. 97 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 103E2E9C2 is from positions 151 to 198 in SEQ ID No. 97 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 103E2E9C2 is from positions 295 to 324 of SEQ ID No. 97 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 103E2E9C2 is from positions 70 to 105 of SEQ ID No. 98 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 103E2E9C2 is from positions 151 to 171 in SEQ ID No. 98 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 103E2E9C2 is from positions 268 to 294 in SEQ ID No. 98 of the Sequence Listing.
  • the nucleotide sequence of CDR1 in the heavy chain protein variable region encoding 106D5G3D10 is from position 91 to position 105 in SEQ ID No. 99 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the heavy chain protein variable region encoding 106D5G3D10 is from positions 151 to 198 in SEQ ID No. 99 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the heavy chain protein variable region encoding 106D5G3D10 is from positions 295 to 318 of SEQ ID No. 99 of the Sequence Listing;
  • the nucleotide sequence of CDR1 in the light chain protein variable region encoding 106D5G3D10 is the 70th to 99th positions in SEQ ID No. 100 of the Sequence Listing;
  • the nucleotide sequence of CDR2 in the light chain protein variable region encoding 106D5G3D10 is from positions 145 to 165 in SEQ ID No. 100 of the Sequence Listing;
  • the nucleotide sequence of CDR3 in the light chain protein variable region encoding 106D5G3D10 is position 262 to 294 in SEQ ID No. 100 of the Sequence Listing.
  • the TPBG antibody heavy chain variable region and light chain variable region sequences were clarified according to the sequencing results of Example 6.
  • the heavy chain variable region sequences of the leader antibodies obtained in Example 2 and Example 3 were recombined into an expression vector comprising a signal peptide and a human heavy chain antibody IgG1 constant region (wherein the expression vector was purchased from Invitrogen, recombinant step
  • the light chain variable region sequence of the TPBG antibody was also recombined into an expression vector containing the signal peptide and the human antibody light chain kappa constant region, and the recombinant plasmid was obtained and verified by sequencing (sequencing method and implementation)
  • the sequencing method in Example 6 is the same).
  • the recombinant plasmid having a purity increased by mass spectrometry using an alkaline lysis kit was used at a mass of 500 ⁇ g or more, and filtered through a 0.22 ⁇ m filter (purchased from Millopore) for transfection.
  • Antibody purification For continuous production of endotoxin-free chromatography column and ProteinA packing, use 0.1M NaOH for 30min or 5 column volumes of 0.5M NaOH for washing; use at least 1M for long-term unused column and column. Soak for 1 h with NaOH, rinse to neutral with non-endotoxic water, and clean the column with 10 column volumes of 1% Triton X100. Equilibration was performed using 5 column volumes of PBS, and the filtered cell supernatant was applied to the column, and the flow-through was collected as necessary. After the upper column was completed, it was washed with 5 column volumes of PBS.
  • the first segment of the chimeric antibody designation uses the first 3 to 5 characters of the corresponding leader antibody clone number.
  • the chimeric antibody 12B12-MMAF corresponds to the leader antibody clone number 12B12C7C3, and the chimeric antibody 5G4-MMAF
  • the corresponding leader antibody clone number is 5G4H10G5 and the like.
  • the purified TPBG chimeric antibody obtained in Example 7 was coupled with MC-MMAF in the same manner as in Example 4, after dialyzing through a sodium borate buffer having a pH of 6.5 to 8.5, and then adding tris(2-carboxyethyl)phosphine (TCEP), wherein the molar ratio of TCEP to purified TPBG antibody is 2, and reduction is carried out for 1 hour at room temperature to obtain reaction liquid A.
  • Reaction solution A After desalting (purchased from GE) on a G25 column, excess TCEP was removed to obtain a reaction solution B.
  • MC-MMAF was added to the reaction solution B, wherein the molar ratio of the MC-MMAF to the purified TPBG antibody was 5, and the reaction was carried out at room temperature for 4 hours. Cysteine was added to neutralize excess MC-MMAF and desalted through a G25 column to remove excess small molecules.
  • a purified TPBG antibody drug conjugate was obtained (for coupling methods, see Doronina, 2006, Bioconjugate Chem. 17, 114-124).
  • the cytotoxic activity was analyzed by analyzing the cross-linking rate of the drug by HIC, analyzing the purity of the antibody drug conjugate by SEC, and the like.
  • the drug cross-linking rate (DAR) of all antibody conjugates was 3.0-5.0. Wherein, DAR (drug antibody ratio) refers to the average number of small molecule drugs carried on one antibody molecule after antibody coupling.
  • the obtained purified TPBG antibody drug conjugates were separately diluted with complete medium, and 100 ⁇ l of TPBG-positive NCI-H1299 cell line was purchased at 2000 cells/well in 96-well cell culture plates (purchased from ATCC, Cat# CRL5803) After the cell suspension was cultured overnight, 10 ⁇ l of each dilution of the purified TPBG chimeric antibody drug conjugate was added to each well, and the culture was continued for 5 days, and then the CellTiter-Glo kit (purchased from Promega, using method) was used. Refer to the product manual for the detection of cell viability. The results are shown in Table 13 and Fig.
  • IC50 of Table 13 refers to a half effective amount in which the activity of the cells is inhibited after the action of the drug, and the cell killing activity can be reflected by detecting the activity of the cells.
  • Figure 13 is a graph showing the cytotoxic activity of purified TPBG chimeric antibody drug conjugate against TPBG-positive tumor cell line NCI-H1299. The results indicate that the purified TPBG antibody drug conjugate has a killing effect on TPBG-positive cells.
  • NCI-H1299 non-small cell lung cancer cell line, ATCC, CRL-5803
  • the tumor was grown to 200 mm 3 to remove body weight.
  • the tumor was too large and too small, and the mice were randomly divided into several groups according to the tumor volume, with 7 rats in each group.
  • D0 began to inject the antibody into the tail vein, once every 4 days, a total of 4 times, the tumor volume was measured twice a week, the rats were weighed, and the data were recorded.
  • FIG. 14 Volume change map of the tumor after treatment
  • Figure 15 Figure of the weight change after treatment.
  • 14A-E are graphs showing tumor volume changes after chimeric antibody 12B12, chimeric antibody 5G4, chimeric antibody 39A11, chimeric antibody 28D4, antibody antibody conjugate of chimeric antibody 36A10, and naked anti-treatment, respectively.
  • 15A-E are graphs showing changes in body weight of mice after chimeric antibody 12B12, chimeric antibody 5G4, chimeric antibody 39A11, chimeric antibody 28D4, antibody antibody conjugate of chimeric antibody 36A10, and naked nude antibody. The results showed that several ADCs could well inhibit the growth of tumor NCI-H1299 and had no significant effect on the body weight of mice.
  • the purified TPBG chimeric antibodies 12B12 and 28D4 obtained in Example 7 were coupled with MC-MMAF and MC-VC-PAB-MMAE, respectively, in the same manner as in Example 8, after being dialyzed against sodium borate buffer of pH 6.5-8.5.
  • Tris(2-carboxyethyl)phosphine (TCEP) was added, wherein a molar ratio of TCEP to purified TPBG antibody was 2, and reduction was carried out for 1 hour at room temperature to obtain a reaction liquid A.
  • the reaction solution A was desalted (purchased from GE) through a G25 column to remove excess TCEP to obtain a reaction liquid B.
  • MC-MMAF or MC-VC-PAB-MMAE purchased from Nanjing Lianning
  • MC-MMAF or MC-VC-PAB-MMAE purchased from Nanjing Lianning
  • Cysteine is added to neutralize excess MC-MMAF or MC-VC-PAB-MMAE, and the excess small molecule is removed by desalting on a G25 column to obtain a purified TPBG chimeric antibody drug conjugate - ie, a table
  • the cytotoxic activity was analyzed by analyzing the cross-linking rate of the drug by HIC, analyzing the purity of the antibody drug conjugate by SEC, and the like.
  • the purified TPBG chimeric antibodies 12B12 and 28D4 obtained in Example 7 were each coupled to SMCC.
  • the purified TPBG chimeric antibodies 12B12 and 28D4 obtained in Example 7 were dialyzed against a phosphate buffer having a pH of 6.5 to 7.4, and then 4-(N-) was added in the presence of 30% by volume of DMA (dimethylacetamide).
  • DMA dimethylacetamide
  • Maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC) wherein the molar ratio of SMCC to purified TPBG chimeric antibody is 8, and the reaction is carried out at room temperature for 1 hour to obtain a reaction. Liquid A.
  • reaction solution A was desalted (purchased from GE) through a G25 column to remove excess small molecules to obtain a reaction liquid B.
  • a final volume of 10% DMA was added to the reaction solution B, followed by the addition of DM1 (chemical name: N2'-deacetyl-N2'-3-mercapto-1 oxopropyl)-maytansin), in which DM1 was purified.
  • DM1 chemical name: N2'-deacetyl-N2'-3-mercapto-1 oxopropyl
  • the molar ratio of the TPBG antibody was 9, and the reaction was carried out for 3.5 hours at room temperature to obtain a reaction liquid C.
  • the reaction solution C was desalted on a G25 column (purchased from GE) to remove excess small molecules to obtain a purified TPBG chimeric antibody drug conjugate, namely the chimeric antibodies 12B12-DM1, 28D4-DM1 described in the table ( For the coupling method, see US5208020).
  • the cytotoxic activity was analyzed by analyzing the cross-linking rate of the drug by LC-MS, analyzing the purity of the antibody drug conjugate by SEC, and the drug cross-linking rate (DAR) of all chimeric antibody conjugates was 3.0- 5.0.
  • DAR drug antibody ratio
  • DAR drug antibody ratio
  • the obtained purified TPBG chimeric antibody drug conjugates were separately diluted with complete medium, and 100 ⁇ l of TBPG-positive NCI-H1299 cell line was purchased at 2000 cells/well in 96-well cell culture plates (purchased from ATCC, article number #CRL5803), or NCI-H1568 cell line (purchased from ATCC, Cat. #CRL-5876) cell suspension was cultured overnight, and each well was diluted with 10 ⁇ l of different concentrations of purified TPBG chimeric antibody drug conjugate. After the culture was continued for 5 days, the cell viability was measured using a CellTiter-Glo kit (purchased from Promega, using the method of reference).
  • IC50 of Table 15 refers to a half effective amount of inhibition of the activity of the cells after the action of the drug, and the cell killing activity can be reflected by detecting the activity of the cells.
  • 16A and 16B are the detection of the cytotoxic activity of the purified TPBG chimeric antibody 12B12 and its antibody drug conjugate coupled with different small molecule drugs against the TPBG-positive tumor cell line NCI-H1299, respectively; 16D was the detection of the cytotoxic activity of the purified TPBG chimeric antibody 12B12 and its antibody drug conjugate coupled with different small molecule drugs against the TPBG-positive tumor cell line NCI-H1568.
  • the obtained purified TPBG chimeric antibody drug conjugates were separately diluted with complete medium, and 100 ⁇ l of 293-hTPBG stably transfected cell line expressing TPBG was added at 2000 cells/well in a 96-well cell culture plate (for the construction method, see Example 1: Preparation of immunogen B) and 293 cell line not expressing TPBG (purchased from ATCC, Cat. #CRL-1573) After cell culture overnight, 10 ⁇ l of purified TPBG was added to each well. The dilution of the antibody drug conjugate was continued for 5 days, and the cell viability was measured using a CellTiter-Glo kit (purchased from Promega, using the method of reference). The results are shown in Table 16 and Fig.
  • IC50 of Table 16 refers to a half effective amount in which the activity of the cells is inhibited after the action of the drug, and the cell killing activity can be reflected by detecting the activity of the cells.
  • 17A and 17B are assays for the cytotoxic activity of purified TPBG chimeric antibody drug conjugates against TPBG-positive TPBG-expressing 293-hTPBG stably transfected cell lines;
  • Figures 17C and 17D are purified TPBG chimeric antibody drug conjugates. The cell killing activity of the 293 cell line which does not express TPBG was detected.

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Abstract

本发明提供了一种TPBG抗体、其制备方法及其偶联物和应用。所述TPBG抗体的重链可变区和轻链可变区的氨基酸序列分别如SEQ ID NO: 1和5、SEQ ID NO: 9和13、SEQ ID NO: 17和21、SEQ ID NO: 25和29、SEQ ID NO: 33和37、SEQ ID NO:41和45、SEQ ID NO: 49和53、SEQ ID NO: 57和61、SEQ ID NO: 65和69或者SEQ ID NO: 73和77所示。所述TPBG抗体与MMAF的偶联物对TPBG阳性的细胞有杀伤作用。

Description

一种TPBG抗体及其制备方法、其偶联物和应用
本申请要求申请日为2015年12月24日的中国专利申请CN201510990545.6的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及抗体领域，具体涉及一种TPBG抗体及其制备方法、其偶联物和应用。
背景技术
在对胚胎干细胞滋养层和癌细胞进行比较时发现一种细胞表面分子，滋养层特异性糖蛋白(TPBG，又称为5T4)，是胚胎滋养层表达的特异性蛋白。人类TPBG蛋白的分子量约为72kDa，包含420个氨基酸，其N端低聚糖结构具有多样性，能够防止蛋白质水解，并且在细胞膜信号传导过程中与其他分子有交互作用。TPBG蛋白共包含7个重复的亮氨酸结构域(LRR)，可参与蛋白质与蛋白质的相互作用。
滋养层是胎盘和胎儿之间的一层特殊的胚胎干细胞，TPBG广泛表达于胚胎发育期的各种滋养层细胞。对于正常成人组织中，TPBG只在有限的几种上皮细胞中有表达。但是，TPBG在很多癌细胞中表达，如子宫癌、结肠癌、胃癌、卵巢癌、口腔癌、前列腺癌、肺癌或肾癌组织中都检测到TPBG的表达，在结肠癌、胃癌或卵巢癌中有证据表明TPBG的表达量与癌症的低治愈率相关。而在非小细胞肺癌、肾癌或胰腺癌的组织中，TPBG的表达高达95％以上。
很多研究显示，过表达该TPBG能够促进细胞迁移，同时还能够避免免疫监控。在小鼠纤维原细胞中过表达TPBG能够诱导细胞呈现纺锤体形态，降低细胞粘附。在小鼠正常上皮细胞中，同样发现TPBG能够抑制上皮细胞钙粘蛋白(E-cadherin)，促进细胞迁移。而TPBG在细胞内的部分有抑制细胞骨架形成的功能。同时，TPBG与上皮细胞间质转移(EMT)相关，TPBG作为胚胎干细胞发育的早期标志物，增加细胞间质蛋白酶的活性，干扰肌动蛋白细胞骨架的排列，下调E-cadherin的表达。还有研究发现，TPBG与CXCR4共定位于细胞膜表明，能够诱导其配体CXCL12趋化因子的结合，促进炎症和肿瘤的扩散。在TPBG阴性细胞中，CXCL12结合另一个受体CXCR7，抑制趋化反应，有利于细胞的生长和存活。Wnt/b-catenin信号通路对发育和细胞再生起到非常重要的作用，TPBG通过抑制LRP6与Wnt受体的内吞，抑制Wnt信号通路，从而抑制细胞粘附和细胞骨架的形成，促进肿瘤的迁移和扩散。还有证据表明TPBG在乳腺癌和胃癌细胞 中参与非经典的Wnt通路，同样促进癌细胞的迁移和浸润。
抗体药物偶联药物，是由抗体与高效小分子药物通过连接物偶联形成的抗体药物偶联物，能够使高毒性小分子药物特异识别癌细胞上的靶点蛋白，从而特异性杀死癌细胞。近一百年间，基于抗体的免疫疗法与基于化学药物的化学疗法，一直是临床上癌症治疗的两大治疗策略。抗体以肿瘤细胞过度表达的抗原为靶点，多种治疗性单抗已经在临床上取得了巨大成功。在临床实践中，治疗性抗体虽然具有很好的靶向性，但是杀伤作用存在局限性。小分子化学药物虽然具备对癌细胞的高效杀伤作用，但是对非癌细胞也造成同样的伤害。因此临床上抗体药物以及小分子药物各自的局限性，对药物研发提出了新的要求。新一代抗体药物偶联物，利用抗体对靶细胞的特异结合能力，输送高细胞毒的化学药物，实现对癌细胞的靶向高效杀伤。随着新型化学连接技术的出现，抗体药物偶联药在八十年代末开始进入临床研究，目前已经有2个ADC药物经FDA批准上市。
ADC药物的开发涉及：药物靶点的筛选、重组抗体的制备、连接物技术开发以及高细胞毒性化合物的筛选优化等几个方面。TPBG作为癌细胞特异表达的蛋白，是ADC药物的候选靶点。
发明内容
本发明所要解决的技术问题是为了克服目前缺少TPBG抗体的不足，提供一种亲和力高、特异性强的TPBG抗体及其制备方法和应用，所述的TPBG抗体与人源、小鼠源或食蟹猴源的TPBG蛋白具有高度亲和力。本发明还提供一种药物活性成分的偶联物，其包括所述的TPBG抗体和与其偶联的、具有抗肿瘤的功能的小分子化合物，所述的偶联物能够进入细胞，对TPBG阳性的细胞进行细胞毒杀伤作用，能够运用于治疗肿瘤等药物的制备中。
本发明以人源TPBG蛋白或者过表达人源TPBG蛋白的重组细胞株作为免疫原，采用传统的杂交瘤制备技术(Kohler and Milstein，Nature，1975，256：495)，通过一系列的调整和改进，获得TPBG抗体的先导抗体。再通过对先导抗体的初步生产、纯化和检定，获得具备与人源TPBG蛋白等蛋白具有高度亲和力的TPBG抗体。该TPBG抗体与小分子化合物如MMAF偶联得偶联物，所述偶联物能够进入细胞，对TPBG阳性细胞有优异的细胞毒杀伤作用。然后通过分子生物学方法测序获知所得的TPBG抗体的重链可变区和TPBG抗体的轻链可变区的氨基酸序列。
本发明提供一种分离的蛋白质，其包括TPBG抗体的重链CDR1、重链CDR2和重链CDR3中的一种或多种，和/或，TPBG抗体的轻链CDR1、轻链CDR2和轻链CDR3 中的一种或多种，其中，所述重链CDR1的氨基酸序列如序列表中SEQ ID No.2、SEQ ID No.10、SEQ ID No.18、SEQ ID No.26、SEQ ID No.34、SEQ ID No.42、SEQ ID No.50、SEQ ID No.58、SEQ ID No.66或SEQ ID No.74所示；所述重链CDR2的氨基酸序列如序列表SEQ ID No.3、SEQ ID No.11、SEQ ID No.19、SEQ ID No.27、SEQ ID No.35、SEQ ID No.43、SEQ ID No.51、SEQ ID No.59、SEQ ID No.67或SEQ ID No.75所示；所述重链CDR3的氨基酸序列如序列表中SEQ ID No.4、SEQ ID No.12、SEQ ID No.20、SEQ ID No.28、SEQ ID No.36、SEQ ID No.44、SEQ ID No.52、SEQ ID No.60、SEQ ID No.68或SEQ ID No.76所示；所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.6、SEQ ID No.14、SEQ ID No.22、SEQ ID No.30、SEQ ID No.38、SEQ ID No.46、SEQ ID No.54、SEQ ID No.62、SEQ ID No.70或SEQ ID No.78所示；所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.7、SEQ ID No.15、SEQ ID No.23、SEQ ID No.31、SEQ ID No.39、SEQ ID No.47、SEQ ID No.55、SEQ ID No.63、SEQ ID No.71或SEQ ID No.79所示；所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.8、SEQ ID No.16、SEQ ID No.24、SEQ ID No.32、SEQ ID No.40、SEQ ID No.48、SEQ ID No.56、SEQ ID No.64、SEQ ID No.72或SEQ ID No.80所示；或者，所述重链CDR1的氨基酸序列与如序列表中SEQ ID No.2、SEQ ID No.10、SEQ ID No.18、SEQ ID No.26、SEQ ID No.34、SEQ ID No.42、SEQ ID No.50、SEQ ID No.58、SEQ ID No.66或SEQ ID No.74所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述重链CDR2的氨基酸序列与如序列表中SEQ ID No.3、SEQ ID No.11、SEQ ID No.19、SEQ ID No.27、SEQ ID No.35、SEQ ID No.43、SEQ ID No.51、SEQ ID No.59、SEQ ID No.67或SEQ ID No.75所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述重链CDR3的氨基酸序列与如序列表中SEQ ID No.4、SEQ ID No.12、SEQ ID No.20、SEQ ID No.28、SEQ ID No.36、SEQ ID No.44、SEQ ID No.52、SEQ ID No.60、SEQ ID No.68或SEQ ID No.76所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述轻链CDR1的氨基酸序列与如序列表中SEQ ID No.6、SEQ ID No.14、SEQ ID No.22、SEQ ID No.30、SEQ ID No.38、SEQ ID No.46、SEQ ID No.54、SEQ ID No.62、SEQ ID No.70或SEQ ID No.78所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述轻链CDR2的氨基酸序列与如序列表中SEQ ID No.7、SEQ ID No.15、SEQ ID No.23、SEQ ID No.31、SEQ ID No.39、SEQ ID No.47、SEQ ID No.55、SEQ ID No.63、SEQ ID No.71或SEQ ID No.79所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述轻链CDR3的氨基酸序列与如序列表中SEQ ID No.8、SEQ ID No.16、SEQ ID No.24、SEQ ID No.32、SEQ ID No.40、SEQ ID No.48、SEQ ID No.56、 SEQ ID No.64、SEQ ID No.72或SEQ ID No.80所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示。
较佳地，所述重链CDR1的氨基酸序列如序列表SEQ ID No.2所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.3所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.4所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.10所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.11所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.12所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.18所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.19所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.20所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.26所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.27所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.28所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.34所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.35所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.36所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.42所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.43所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.44所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.50所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.51所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.52所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.58所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.59所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.60所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.66所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.67所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.68所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.74所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.75所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.76所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.6所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.7所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.8所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.14所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.15所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.16所示；或，所述轻链CDR1的氨基酸序列如序列表SEQ ID No.22所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.23所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.24所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.30所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.31所示，且所述轻链CDR3的氨基酸序列如序列表 SEQ ID No.32所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.38所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.39所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.40所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.46所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.47所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.48所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.54所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.55所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.56所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.62所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.63所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.64所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.70所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.71所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.72所示；或，所述轻链CDR1的氨基酸序列如序列表SEQ ID No.78所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.79所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.80所示。
本发明还提供一种分离的蛋白质，其包括TPBG抗体的重链可变区和/或TPBG抗体的轻链可变区，所述重链可变区的氨基酸序列如序列表中SEQ ID No.1、SEQ ID No.9、SEQ ID No.17、SEQ ID No.25、SEQ ID No.33、SEQ ID No.41、SEQ ID No.49、SEQ ID No.57、SEQ ID No.65或SEQ ID No.73所示；所述轻链可变区的氨基酸序列如序列表中SEQ ID No.5、SEQ ID No.13、SEQ ID No.21、SEQ ID No.29、SEQ ID No.37、SEQ ID No.45、SEQ ID No.53、SEQ ID No.61、SEQ ID No.69或SEQ ID No.77所示。
较佳地，所述重链可变区的氨基酸序列如序列表SEQ ID No.1所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.5所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.9所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.13所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.17所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.21所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.25所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.29所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.33所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.37所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.41所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.45所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.49所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.53所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.57所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.61所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.65所 示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.69所示；或，所述重链可变区的氨基酸序列如序列表SEQ ID No.73所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.77所示。
综上所述，上述氨基酸序列的编号如表1所示：
表1 TPBG抗体蛋白序列编号
Figure PCTCN2016111699-appb-000001
其中，表1中的数字即为序列表中序列号，如12B12C7C3的重链蛋白可变区的氨基酸序列为SEQ ID No.1，而12B12C7C3的重链蛋白可变区中CDR1的氨基酸序列为SEQ ID No.2。
较佳地，所述的蛋白质还包括抗体重链恒定区和/或抗体轻链恒定区，所述的抗体重链恒定区为本领域常规，较佳地为小鼠源抗体重链恒定区或人源抗体重链恒定区，更佳地为人源抗体重链恒定区。所述的抗体轻链恒定区为本领域常规，较佳地为小鼠源轻链抗体恒定区或人源抗体轻链恒定区，更佳地为人源抗体轻链恒定区。
所述的蛋白质为本领域常规的蛋白质，较佳地为TPBG抗体，更佳地为抗体全长蛋白、抗原抗体结合域蛋白质片段、双特异性抗体、多特异性抗体、单链抗体(single chain antibody fragment，scFv)、单域抗体(single domain antibody，sdAb)和单区抗体(Signle-domain antibody)中的一种或多种，以及上述抗体所制得的单克隆抗体或多克隆抗体。所述单克隆抗体可以由多种途径和技术进行研制，包括杂交瘤技术、噬菌体展示技术、单淋巴细胞基因克隆技术等，主流是通过杂交瘤技术从野生型或转基因小鼠制备单克隆抗体。
所述的抗体全长蛋白为本领域常规的抗体全长蛋白，其包括重链可变区、轻链可变区、重链恒定区和轻链恒定区。所述的蛋白质的重链可变区和轻链可变区与人源重链恒定区和人源轻链恒定区构成全人源抗体全长蛋白。较佳地，所述的抗体全长蛋白为IgG1、IgG2、IgG3或IgG4。
所述的单链抗体为本领域常规的单链抗体，其包括重链可变区、轻链可变区和15～20个氨基酸的短肽。
所述的抗原抗体结合域蛋白质片段为本领域常规的抗原抗体结合域蛋白质片段，其包括轻链可变区、轻链恒定区和重链恒定区的Fd段。较佳地，所述的抗原抗体结合域蛋白质片段为Fab和F(ab’)2。
所述的单域抗体为本领域常规的单域抗体，其包括重链可变区和重链恒定区。
所述的单区抗体为本领域常规的单区抗体，其仅包括重链可变区。
其中，所述蛋白质的制备方法为本领域常规的制备方法。所述制备方法较佳地为：从重组表达该蛋白质的表达转化体中分离获得或者通过人工合成蛋白质序列获得。所述的从重组表达该蛋白质的表达转化体中分离获得优选如下方法：将编码所述蛋白质并且带有点突变的核酸分子克隆到重组载体中，将所得重组载体转化到转化体中，得到重组表达转化体，通过培养所得重组表达转化体，即可分离纯化获得所述蛋白质。
本发明还提供一种核酸，其编码上述的蛋白质。
较佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.81、序列表SEQ ID No.83、序列表SEQ ID No.85、序列表SEQ ID No.87、序列表SEQ ID No.89、序列表SEQ ID No.91、序列表SEQ ID No.93、序列表SEQ ID No.95、序列表SEQ ID No.97或序列表SEQ ID No.99所示；和/或，编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.82、序列表SEQ ID No.84、序列表SEQ ID No.86、序列表SEQ ID No.88、序列表SEQ ID No.90、序列表SEQ ID No.92、序列表SEQ ID No.94、序列表SEQ ID No.96、序列表SEQ ID No.98或序列表SEQ ID No.100所示。
更佳地，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.81所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.82所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.83所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.84所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.85所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.86所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.87所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.88所示；编码所述重链可 变区的核酸的核苷酸序列如序列表SEQ ID No.89所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.90所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.91所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.92所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.93所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.94所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.95所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.96所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.97所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.98所示；或，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.99所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.100所示。
综上所述，上述核苷酸序列的编号如表2所示：
表2 TPBG抗体基因序列编号
克隆号 重链蛋白可变区 轻链蛋白可变区
12B12C7C3 81 82
5G4H10G5 83 84
37H9C5G2 85 86
39A11G5F2 87 88
52C9E9F6 89 90
28D4E6A9 91 92
36A10D8B12 93 94
99E12C7H1 95 96
103E2E9C2 97 98
106D5G3D10 99 100
其中，表2中的数字即为序列表中序列号，如编码12B12C7C3的重链蛋白可变区的核苷酸序列为SEQ ID No.81。
其中，编码12B12C7C3的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.81中的第91位至第105位；
编码12B12C7C3的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.81中的第151位至第198位；
编码12B12C7C3的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.81中的第295位至第327位；
编码12B12C7C3的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.82中的第70位至第114位；
编码12B12C7C3的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.82中的第157位至第180位；
编码12B12C7C3的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.82中的第277位至第303位；
编码5G4H10G5的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.83中的第91位至第105位；
编码5G4H10G5的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.83中的第148位至第198位；
编码5G4H10G5的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.83中的第295位至第330位；
编码5G4H10G5的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.84中的第70位至第102位；
编码5G4H10G53的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.84中的第148位至第168位；
编码5G4H10G5的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.84中的第265位至第288位；
编码37H9C5G2的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.85中的第91位至第105位；
编码37H9C5G2的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.85中的第151位至第198位；
编码37H9C5G2的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.85中的第295位至第327位；
编码37H9C5G2的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.86中的第70位至第102位；
编码37H9C5G2的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.86中的第148位至第168位；
编码37H9C5G2的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.86中的第265位至第291位。
编码39A11G5F2的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.87 中的第91位至第105位；
编码39A11G5F2的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.87中的第151位至第198位；
编码39A11G5F2的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.87中的第295位至第315位；
编码39A11G5F2的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.88中的第70位至第102位；
编码39A11G5F2的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.88中的第148位至第168位；
编码39A11G5F2的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.88中的第265位至第291位。
编码52C9E9F6的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.89中的第91位至第105位；
编码52C9E9F6的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.89中的第151位至第198位；
编码52C9E9F6的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.89中的第295位至第315位；
编码52C9E9F6的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.90中的第70位至第102位；
编码52C9E9F6的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.90中的第148位至第168位；
编码52C9E9F6的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.90中的第268位至第291位。
编码28D4E6A9的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.91中的第91位至第105位；
编码28D4E6A9的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.91中的第148位至第198位；
编码28D4E6A9的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.91中的第295位至第327位；
编码28D4E6A9的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.92中的第70位至第102位；
编码28D4E6A9的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.92中的第148位至第168位；
编码28D4E6A9的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.92中的第265位至第291位。
编码36A10D8B12的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.93中的第91位至第108位；
编码36A10D8B12的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.93中的第154位至第198位；
编码36A10D8B12的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.93中的第295位至第324位；
编码36A10D8B12的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.94中的第70位至第102位；
编码36A10D8B12的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.94中的第148位至第168位；
编码36A10D8B12的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.94中的第265位至第291位。
编码99E12C7H1的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.95中的第91位至第105位；
编码99E12C7H1的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.95中的第151位至第198位；
编码99E12C7H1的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.95中的第295位至第318位；
编码99E12C7H1的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.96中的第70位至第102位；
编码99E12C7H1的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.96中的第148位至第168位；
编码99E12C7H1的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.96中的第265位至第291位。
编码103E2E9C2的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.97中的第91位至第105位；
编码103E2E9C2的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.97 中的第151位至第198位；
编码103E2E9C2的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.97中的第295位至第324位；
编码103E2E9C2的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.98中的第70位至第105位；
编码103E2E9C2的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.98中的第151位至第171位；
编码103E2E9C2的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.98中的第268位至第294位。
编码106D5G3D10的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.99中的第91位至第105位；
编码106D5G3D10的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.99中的第151位至第198位；
编码106D5G3D10的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.99中的第295位至第318位；
编码106D5G3D10的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.100中的第70位至第99位；
编码106D5G3D10的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.100中的第145位至第165位；
编码106D5G3D10的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.100中的第262位至第294位。
所述核酸的制备方法为本领域常规的制备方法，较佳地，包括以下的步骤：通过基因克隆技术获得编码上述蛋白质的核酸分子，或者通过人工全序列合成的方法得到编码上述蛋白质的核酸分子。
本领域技术人员知晓，编码上述蛋白质的氨基酸序列的碱基序列可以适当引入替换、缺失、改变、插入或增加来提供一个多聚核苷酸的同系物。本发明中多聚核苷酸的同系物可以通过对编码该蛋白序列基因的一个或多个碱基在保持抗体活性范围内进行替换、缺失或增加来制得。
本发明还提供一种包含所述核酸的重组表达载体。
其中所述重组表达载体可通过本领域常规方法获得，即：将本发明所述的核酸分子连接于各种表达载体上构建而成。所述的表达载体为本领域常规的各种载体，只要其能 够容载前述核酸分子即可。所述载体较佳地包括：各种质粒、粘粒、噬菌体或病毒载体等。
本发明还提供一种包含上述重组表达载体的重组表达转化体。
其中，所述重组表达转化体的制备方法为本领域常规的制备方法，较佳地为：将上述重组表达载体转化至宿主细胞中制得。所述的宿主细胞为本领域常规的各种宿主细胞，只要能满足使上述重组表达载体稳定地自行复制，且所携带所述的核酸可被有效表达即可。较佳地，所述宿主细胞为E.coli TG1或BL21细胞(表达单链抗体或Fab抗体)，或者CHO-K1细胞(表达全长IgG抗体)。将前述重组表达质粒转化至宿主细胞中，即可得本发明优选的重组表达转化体。其中所述转化方法为本领域常规转化方法，较佳地为化学转化法，热激法或电转法。
本发明还提供一种TPBG抗体的制备方法，其包括如下步骤：培养上述的重组表达转化体，从培养物中获得TPBG抗体。
本发明提供一种免疫偶联物，其包括共价附着至细胞毒剂的上述的蛋白质。
较佳地，所述的免疫偶联物中，上述的1当量的蛋白质通过x当量接头与y当量的细胞毒剂相连，具有如式1所示的结构，
Ab-(L)x-(D)y
式1
其中，Ab为上述的蛋白质；L为接头；D为细胞毒剂；所述x为本领域常规的交联度，x为自然数，优选1-20的整数；y为0或自然数，优选0-20的整数；x和y各自独立地优选为1～2，或2～4，或4～8，或8～20的整数；x和y的比例优选为1∶1。
所述L是本领域常规的接头(或称交联剂或偶联剂)。所述L包含2个官能团，即与抗体反应的基团，和与药物反应的基团(例如，醛或酮)。
药物经由接头分子与上述的蛋白质偶联。所述L进入细胞后释放，其包括但不限于如下的官能团，活性酯、碳酸盐类、氨基甲酸酯类、亚胺磷酸酯、肟类、腙类、缩醛类、原酸酯类、氨基类、小肽段或核苷酸片段。
较佳地，所述L主要含有式2所示结构，其为L中离去基团离去后对应的剩余部分；
(CO-Alk1-Sp1-Ar-Sp2-Alk2-C(Z1)＝Q-Sp)
式2
其中，Alk1和Alk2独立地是键或分支的或不分支的(C1-C10)亚烷基链；Sp1是-S-、-O-、-CONH-、-NHCO-、-NR’-、-N(CH2CH2)2N-、或-X-Ar’-Y-(CH2)n-Z，其中X、Y和Z是独立的键、-NR’-、-S-或-O-，条件是当n＝0时，Y和Z中的至少一个必须是键， 且Ar’是由(C1-C5)烷基、(C1-C4)烷氧基、(C1-C4)硫代烷氧基、卤素、硝基、-COOR’、-CONHR’、-(CH2)nCOOR’、S(CH2)nCOOR’、-O(CH2)nCONHR’或-S(CH2)nCONHR’的1、2或3个基团任选取代的1，2-、1，3-或1，4-亚苯基，n是0-5的整数，条件是当Alk1是键时，Sp1是键；R’是由-OH、(C1-C4)烷氧基、(C1-C4)硫代烷氧基、卤素、硝基、(C1-C3)二烷基氨基、或(C1-C3)三烷基铵-A的一个或2个基团任选取代的分支的或不分支的(C1-C5)链，其中A是完成盐的药学上可接受的阴离子；Ar是由(C1-C6)烷基、(C1-C5)烷氧基、(C1-C4)硫代烷氧基、卤素、硝基、-COOR’、-CONHR’、-O(CH2)nCOOR’、-S(CH2)nCOOR’、-O(CH2)nCONHR”或-S(CH2)nCONHR’的1、2或3个基团任选取代的1，2-、1，3-或1，4-亚苯基，其中n和R’如上述的定义，或Ar是1，2-、1，3-、1，4-、1，5-、1，6-、1，7-、1，8-、2，3-、2，6-或2，7-亚萘基，其中亚萘基或吩噻嗪各任选地由(C1-C6)烷基、(C1-C5)烷氧基、(C1-C4)硫代烷氧基、卤素、硝基、-COOR’、-CONHR’、-O(CH2)nCOOR’、-S(CH2)nCOOR’、或-S(CH2)nCONHR’的1、2、3或4个基团取代，其中n和R’如上文定义，条件是当Ar是吩噻嗪时，Sp1是仅与氮连接的键；
Sp2是键、-S-或-O-，条件是当Alk2是键时，Sp2是键；
Z1是H、(C1-C5)烷基、或由(C1-C5)烷基、(C1-C5)烷氧基、(C1-C4)硫代烷氧基、卤素、硝基、-COOR’、-CONHR’、-O(CH2)nCOOR’、-S(CH2)nCOOR’、-O(CH2)nCONHR’或-S(CH2)nCONHR’的1、2、或3个基团任选取代的苯基，其中n和R’如上文定义；
Sp是直链或支链二价或三价(C1-C18)基团，二价或三价芳基或杂芳基基团，二价或三价(C3-C18)环烷基或杂环烷基基团，二价或三价芳基或杂芳基-芳基(C1-C18)基团，二价或三价环烷基或杂环烷基-烷基(C1-C18)基团，或二价或三价(C2-C18)不饱和的烷基基团，其中杂芳基优选是呋喃基、噻吩基，N-甲基吡咯基、吡啶基、N-甲基咪唑基、噁唑基、嘧啶基。喹啉基、异喹啉基、N-甲基咔唑基、氨基豆素基、或吩嗪基、并且其中如果Sp是三价基团，那么Sp还可以由低级(C1-C5)二烷基氨基、低级(C1-C5)烷氧基、羟基、或低级(C1-C5)烷硫基任选取代；且，Q是＝NHNCO-、＝NHNCS-、＝NHNCONH-、＝NHNCSNH-或＝NHO-。
优选地，Alk1是分支或不分支的(C1-C5)亚烷基链，Sp1是键、-S-、-O-、-CONH-、-NHCO-或-NR’，其中R’如上文定义，条件是当Alk1是键时，Sp1是键；
Ar是由(C1-C6)烷基、(C1-C5)烷氧基、(C1-C4)硫代烷氧基、卤素、硝基、-COOR’、-CONHR’、-O(CH2)nCOOR’、-S(CH2)nCOOR’、-O(CH2)nCONHR’或-S(CH2)nCONHR’的1、2或3个基团任选取代的1，2-、1，3-或1，4-亚苯基，其中n和R’如上文定 义，或Ar是各自由C1-C6)烷基、(C1-C5)烷氧基、(C1-C4)硫代烷氧基、卤素、硝基、-COOR’、-CONHR’、-O(CH2)nCOOR’、-S(CH2)nCOOR’、-O(CH2)nCONHR’或-S(CH2)nCONHR’的1、2、3或4个基团任选取代的1，2-、1，3-、1，4-、1，5-、1，6-、1，7-、1，8-、2，3-、2，6-或2，7-亚萘基。
Z1是(C1-C5)烷基、或由(C1-C5)烷基、(C1-C4)烷氧基、(C1-C4)硫代烷氧基、卤素、硝基、-COOR’、-CONHR’、-O(CH2)nCOOR’、-S(CH2)nCOOR’、-O(CH2)nCONHR’或-S(CH2)nCONHR’的1、2、或3个基团任选取代的苯基；Alk2和Sp2均为键；且Sp和Q如仅在上文中所定义的。上述的键的含义为共价键。
所述L优选为马来酰亚胺基己酰(maleimidocaproyl，MC)、马来酰亚胺基己酰-L-缬氨酸-L-瓜氨酸对氨基苄醇(MC-VC-PAB)或4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯(SMCC)。
所述D为本领域常规的细胞毒剂，较佳地选自细胞毒素、化学治疗剂、放射性同位素、治疗性核酸、免疫调节剂、抗血管生成剂、抗增殖促凋亡剂或细胞溶解酶。
其中，所述细胞毒素为本领域常规的细胞毒素，一般指抑制或阻止细胞功能和/或导致细胞破坏的活性剂。较佳地选自抗生素、微管蛋白聚合的抑制剂、烷化剂、蛋白合成抑制剂、蛋白激酶抑制剂、磷酸酶抑制剂、拓扑异构酶抑制剂、蛋白激酶、磷酸酶、拓扑异构酶或细胞周期蛋白。更佳地选自多柔比星、柔红霉素、依达比星、阿柔比星、佐柔比星、米托蒽醌、表柔比星、卡柔比星、诺加霉素、美诺立尔、吡柔比星、戊柔比星、阿糖胞苷、吉西他滨、曲氟尿苷、安西他滨、依诺他滨、阿扎胞苷、去氧氟尿苷、喷司他丁、溴尿苷、卡培他滨、克拉屈滨、地西他滨、氟尿苷、氟达拉滨、谷氏菌素、嘌呤霉素、替加氟、噻唑羧胺核苷、阿霉素、顺铂、卡铂、环磷酰胺、达卡巴嗪、长春碱、长春新碱、博来霉素、氮芥、强的松、甲基苄肼、氨甲喋呤、氟尿嘧啶、依托泊苷、泰素、泰素类似物、铂类(如顺铂和卡铂)、丝裂霉素、噻替派、紫杉烷、道诺红菌素、放线菌素、安曲霉素、氮丝氨酸、它莫西芬、多拉司他汀、奥瑞他汀及其衍生物、哈米特林、埃斯波霉素或美登素类化合物，最佳地选自甲基奥瑞他汀E(MMAE)、甲基奥瑞他汀F(MMAF)或N2’-脱乙酰-N2’-3-巯基-1氧代丙基-美登素(DM1)。
其中，所述化学治疗剂为本领域常规的化学治疗剂，较佳地选自烷化剂、烷基磺酸酯类化学治疗剂、氮丙啶类化学治疗剂、乙烯酰胺类和甲基密胺类化学治疗剂、氮芥、硝基脲类化学治疗剂、抗生素、抗代谢物、叶酸类化学治疗剂、嘌呤类似物、嘧啶类似物、雄激素、抗肾上腺素、叶酸补充剂、美登醇、多糖复合物、紫杉烷、铂类似物或类视黄醇，或者，其在药学上可接受的盐、酸和衍生物。
所述的烷化剂为本领域常规的烷化剂，较佳地选自噻替派或环磷酰胺。所述的烷基磺酸酯类化学治疗剂为本领域常规的烷基磺酸酯类化学治疗剂，较佳地选自白消安、英丙舒凡或哌泊舒凡。所述的氮丙啶类化学治疗剂为本领域常规的氮丙啶类化学治疗剂，较佳地选自氮丙唳如、卡巴醌、美妥替哌或乌瑞替派。所述乙烯酰胺类和甲基密胺类化学治疗剂为本领域常规的乙烯酰胺类和甲基密胺类化学治疗剂，较佳地选自六甲蜜胺、三乙撑蜜胺、三亚乙基磷酰胺，三亚乙基硫代磷酰胺或三羟甲蜜胺。所述的氮芥为本领域常规的氮芥，较佳地选自苯丁酸氮芥、萘氮芥、雌氮芥(estramustine)、异环磷酰胺、氮芥、氧氮芥盐酸盐、苯丙氨酸氮芥、新氮芥、苯芥胆甾醇、泼尼氮芥、曲磷胺或尿嘧啶氮芥。所述硝基脲类化学治疗剂为本领域常规的硝基脲类化学治疗剂，较佳地选自卡莫司汀、氯脲菌素、福莫司汀、洛莫司汀、尼莫司汀或雷莫司汀。所述抗生素为本领域常规的抗生素，较佳地选自阿克拉霉素、放线菌素、安曲霉素、氮丝氨酸、博来霉素、放线菌素c、加力车霉素、卡柔比星、洋红霉素、嗜癌素、色霉素、更生霉素、柔红霉素、地托比星、6-重氮基-5-氧代-L-正亮氨酸、多柔比星、表柔比星、依索比星、依达比星、发波霉素、丝裂霉素、霉酚酸、诺加霉素、橄榄霉素、培洛霉素、紫菜霉素、嘌呤霉素、三铁阿霉素、罗多比星、链黑菌素、链脲菌素、杀结核菌素、乌苯美司、静司他丁或佐柔比星。所述的抗代谢物为本领域常规的抗代谢物，较佳地选自氨甲喋呤或5-氟尿嘧啶(5-FU)。所述的叶酸类化学治疗剂为本领域常规的叶酸类化学治疗剂，较佳地选自二甲叶酸、蝶罗呤或三甲曲沙。所述的嘌呤类似物为本领域常规的嘌呤类似物，较佳地选自氟达拉滨、6-巯嘌呤、硫咪嘌呤或硫鸟嘌呤。所述的嘧啶类似物为本领域常规的嘧啶类似物，较佳地选自安西他滨、阿扎胞苷、6-阿扎尿苷、卡莫氟、阿糖胞苷、二脱氧尿苷、去氧氟尿苷、依诺他滨、氟尿苷或5-EU。所述的雄激素为本领域常规的雄激素，较佳地选自卡普睾酮、丙酸甲雄烷酮、环硫雄醇、美雄烷或睾内酯。所述的抗肾上腺素为本领域常规的抗肾上腺素，较佳地选自安鲁米特、米托坦或曲洛司坦。所述的叶酸补充剂为本领域常规的叶酸补充剂，较佳地选自亚叶酸、醋葡全内酯、醛磷酰胺糖苷、氨基酮戊酸、安吖啶、阿莫司汀、比生群、依达曲沙、地磷酰胺、秋水仙胺、地吖醌、依氟鸟氨酸、依利醋铵、埃坡西龙、依托格鲁、硝酸镓、羟基脲、香菇多糖或氯尼达明。所述的美登醇为本领域常规的美登醇，较佳地选自美登素、安丝菌素、米托胍腙、米托蒽醌、莫哌达醇、二胺硝吖啶、喷司他丁、蛋氨氮芥、吡柔比星、洛索蒽醌、鬼臼酸、2-乙基酰肼或丙卡巴肼。所述的多糖复合物为本领域常规的多糖复合物，较佳地选自雷佐生、根霉素、西佐喃、锗螺胺、细交链孢菌酮酸、三亚胺醌2，2′，2″-三氯三乙胺、单端孢霉烯族毒素、乌拉坦、长春地辛、达卡巴嗪、甘露莫司汀、二溴甘露醇、二溴卫矛醇、 哌泊溴烷、gacytosine、阿糖胞苷、环磷酰胺或噻替派。更佳地选自T-2毒素、疣孢菌素A、杆孢菌素A或anguidine。所述紫杉烷为本领域常规的紫杉烷，较佳地选自紫杉醇、无氢化蓖麻油、紫杉醇的白蛋白工程化纳米颗粒制剂(American Pharmaceutical Partners，Schaumberg，Illinois)、多西他赛、苯丁酸氮芥、吉西他滨、6-硫代鸟嘌呤、巯嘌呤或甲氨蝶呤。所述的铂类似物为本领域常规的铂类似物，较佳地选自顺铂、卡铂、长春碱、依托泊苷、异环磷酰胺、米托蒽醌、长春新碱、诺安托、替尼泊苷、依达曲沙、道诺霉素、氨基蝶呤、卡培他滨伊班膦酸盐、CPT-11、拓扑异构酶抑制剂RFS 2000或二氟甲基鸟氨酸。所述的类视黄醇为本领域的类视黄醇，较佳地为视黄酸。
其中，所述放射性同位素为本领域常规的放射性同位素，较佳地，其与上述蛋白质直接结合，或者通过螯合剂与上述蛋白质结合。更佳地，其与所述蛋白质的半胱氨酸残基直接结合。较佳地，所述放射性同位素选自适于放射治疗的α-发射体、β-发射体和俄歇电子以及适于诊断的正电子发射体或γ-发射体。更佳地，所述放射性同位素选自18氟、64铜、65铜。67镓、68镓、77溴、80m溴、95钌、97钌、103钌、105钌、99m锝、107汞、203汞、123碘、124碘、125碘、126碘、131碘、133碘、111铟、113铟、99m铼、105铼、101铼、186铼、188铼、121m碲、99锝、122m碲、125m碲、165铥、167铥、168铥、90钇、213铋、213铅或225锕，或者其衍生的氮化物或氧化物。
其中，所述治疗性核酸为本领域常规的核酸，较佳地为编码免疫调节剂、抗血管生成剂、抗增殖剂或促凋亡剂的基因。所述治疗剂包括所述治疗剂、其衍生物和所述治疗剂在药学上可接受的盐、酸及衍生物。
其中，所述的免疫调节剂为本领域常规的免疫调节剂，即引发免疫应答，包括体液免疫应答(例如抗原特异性抗体的产生)和细胞介导的免疫应答(例如淋巴细胞增殖)的试剂。较佳地选自细胞因子、生长因子、激素、抗激素药、免疫抑制剂或皮质类固醇。所述细胞因子为本领域常规的细胞因子，较佳地选自黄嘌呤、白介素或干扰素。所述的生长因子为本领域常规的生长因子，较佳地选自TNF、CSF、GM-CSF或G-CSF。所述的激素为本领域常规的激素，较佳地选自雌激素、雄激素或孕激素。更佳地，所述的雌激素为己烯雌酚或雌二醇。更佳地，所述的雄激素为睾酮或氟甲睾酮。更佳地，所述的孕激素为乙酸甲地孕酮或乙酸甲羟孕酮。所述的皮质类固醇为本领域常规的皮质类固醇，较佳地选自强的松、地塞米松或化可的松。所述抗激素药为本领域常规的抗激素药，其能阻断激素对肿瘤的作用，抑制细胞因子生产，下调自身抗原表达、或掩蔽MHC抗原的免疫抑制剂。较佳地选自抗雌激素药、抗雄激素药或抗肾上腺素药。更佳地，所述抗雌激素药选自它莫西芬、雷洛昔芬、芳香酶抑制性4(5)-咪唑类、4-羟基它莫西芬、曲 沃昔芬或托瑞米芬。所述抗雄激素药选自氟他胺、尼鲁米特、比卡鲁胺、亮丙瑞林或戈舍瑞林。所述免疫抑制剂为本领域常规的免疫抑制剂，较佳地选自2-氨基-6芳基-5取代的嘧啶类、硫唑嘌呤、环磷酰胺、溴隐亭、达那唑、氨苯砜、戊二醛、针对MHC抗原和MHC片段的抗独特型抗体、环孢菌素A、类固醇例如糖皮质类固醇、链激酶、TGFb、雷帕霉素、T细胞受体、T细胞受体片段、细胞因子受体拮抗剂或T细胞受体抗体。更佳地，所述细胞因子受体拮抗剂选自抗干扰素抗体、抗IL10抗体、抗TNFa抗体或抗IL2抗体。
其中，所述的抗血管生成剂为本领域常规的抗血管生成剂，较佳地选自法尼基转移酶抑制剂、COX-2抑制剂、VEGF抑制剂、bFGF抑制剂、类固醇硫酸酯酶抑制剂、白介素-24、凝血栓蛋白、metallospondin蛋白质、I类干扰素、白介素12、鱼精蛋白、血管他丁、层粘连蛋白、内皮他丁或催乳激素片段。更佳地为2-甲氧基雌二醇二氨基磺酸酯(2-MeOE2bisMATE)。
其中，所述抗增殖促凋亡剂为本领域常规的抗增殖促凋亡剂，较佳地选自PPAR-γ激活剂、类视黄醇、三萜类化合物、EGF受体抑制剂、端粒末端转移酶抑制剂、铁螯合剂、凋亡蛋白、Bcl-2和Bcl-X(L)的抑制剂、TNF-α/FAS配体/TNF相关的凋亡诱导配体及其信号传导的激活物或PI3K-Akt存活途径信号抑制剂。所述PPAR-γ激活剂为本领域常规的PPAR-γ激活剂，较佳地为环戊烯酮前列腺素(cyPGs)。所述三萜类化合物为本领域常规的三萜类化合物，较佳地选自环菠萝蜜烷、羽扇豆烷、乌苏烷、齐敦果烷、木栓烷、达玛烷、葫芦素、柠檬苦素类似物或三萜类化合物。所述EGF受体抑制剂为本领域常规的EGF受体抑制剂，较佳地选自HER4、雷帕霉素或1，25-二羟基胆钙化醇(维生素D)。所述的铁螯合物为本领域常规的铁螯合物，较佳地为3-氨基吡啶-2-甲醛硫代缩氨基脲。所述的凋亡蛋白为本领域常规的凋亡蛋白，较佳地为鸡贫血病病毒的病毒蛋白质3-VP3。所述PI3K-Akt存活途径信号抑制剂为本领域常规的PI3K-Akt存活途径信号抑制剂，较佳地为UCN-01或格尔德霉素。
其中，所述细胞溶解酶为本领域常规的细胞溶解酶，较佳地为RNA酶。
本发明优选地，式1中x＝y＝n；由此
在一个优选实施例中，-(L)x-(D)y，为：
Figure PCTCN2016111699-appb-000002
其中m为1～10，优选m为5。
在一个优选实施例中，-(L)x-(D)y为：
Figure PCTCN2016111699-appb-000003
在一个优选实施例中，-(L)x-(D)y为：
Figure PCTCN2016111699-appb-000004
最佳地，所述D为微管蛋白合成酶抑制剂——甲基奥瑞他汀F(MMAF)，且所述接头L为马来酰亚胺基己酰(maleimidocaproyl，MC)，所述免疫偶联物的结构如式3所示，
Figure PCTCN2016111699-appb-000005
或者，所述L为4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯；D为N2’-脱乙酰-N2’-3-巯基-1氧代丙基)-美登素(DM1)，所述免疫偶联物的结构如式4所示，
Figure PCTCN2016111699-appb-000006
或者，L为马来酰亚胺基己酰-L-缬氨酸-L-瓜氨酸对氨基苄醇，D为甲基奥瑞他汀E(MMAE)，所述免疫偶联物的结构如式5所示，
Figure PCTCN2016111699-appb-000007
其中，n为自然数，优选为1～20的整数，更优选为1～2，或2～4，或4～8，或8～20的整数。
所述的免疫偶联物的制备方法为本领域常规，较佳地采用Doronina，2006，Bioconjugate Chem.17，114-124所记载的制备方法。较佳地，所述的制备方法产生具有最低限度的低偶联级分(LCF)小于10％的免疫偶联物。
更佳地，所述的制备方法包括以下的步骤：将上述蛋白质经过pH 6.5～8.5的硼酸钠缓冲液透析后，加入三(2-羧乙基)膦(TCEP)，其中TCEP与上述蛋白质的摩尔比比率为2～10，室温下还原1～4小时，得反应液A。将反应液A洗脱去除多余的上述蛋白质得反应液B。向反应液B中加入MC-MMAF，其中MC-MMAF与纯化的TPBG抗体的摩尔比比率为5～20，10～37℃下反应4小时。
所述的免疫偶联物能够以本领域所知的任何物理形态而存在，较佳地为澄清溶液。
本发明还提供一种药物组合物，其包括上述的免疫偶联物和药学可接受的载体。
所述的药学可接受的载体为本领域常规的载体，所述的载体可以为任意合适的生理学或药学上可接受的药物辅料。所述的药物辅料为本领域常规的药物辅料，较佳地包括药学上可接受的赋形剂、填充剂或稀释剂等。更佳地，所述的药物组合物包括0.01～99.99％的上述蛋白质和0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。
较佳地，所述的药物组合物是抗肿瘤的药物。更佳地为抗鳞状/腺瘤性肺癌(非小细胞肺癌)、浸润性乳腺癌、结肠癌、直肠癌、胃癌、鳞状宫颈癌、浸润性子宫内膜腺癌、浸润性胰腺癌、卵巢癌、鳞状膀胱癌、绒毛膜癌、支气管癌、乳腺癌、子宫颈癌、胰腺癌或精囊癌的药物。
本发明所述的药物组合物的给药途径较佳地为肠胃外施用、注射给药或口服给药。所述注射给药较佳地包括静脉注射、肌肉注射、腹腔注射、皮内注射或皮下注射等途径。所述的药物组合物为本领域常规的各种剂型，较佳地为固体、半固体或液体的形式，即可以为水溶液、非水溶液或混悬液，更佳的为片剂、胶囊、颗粒剂、注射剂或输注剂等。更佳地为经由血管内、皮下、腹膜内或肌内施用。较佳地，所述药物组合物还可以作为气雾剂或粗喷雾剂施用，即经鼻施用；或者，鞘内、髓内或心室内施用。更佳地，所述的药物组合物还可以透皮、经皮、局部、肠内、阴道内、舌下或经直肠施用。
本发明所述的药物组合物的给药剂量水平可以根据达到所需诊断或治疗结果的组合物量而调整。施用方案也可以为单次注射或多次注射，或进行调整。所选择的剂量水平和方案依赖于包括所述药物组合物的活性和稳定性(即，半衰期)、制剂、施用途径、与其他药物或治疗的组合、待检测和/或治疗的疾病或病症、以及待治疗的受试者的健康状况和先前医疗史等各种因素而进行合理地调整。
对于本发明的所述药物组合物的治疗有效剂量可以最初在细胞培养实验或动物模型例如啮齿类动物、兔、犬、猪和/或灵长类动物中进行估计。动物模型也可以用于测定合适的施用浓度范围和途径。随后可以用于确定在人中施用的有用剂量和途径。一般地，施用有效量或剂量的确定和调整以及何时和如何进行此类调整的评估为本领域技术人员已知。
对于组合疗法，上述蛋白质、上述免疫偶联物和/或另外的治疗或诊断剂可以各自作为单一药剂，在适合于执行预期治疗或诊断的任何时间范围内进行使用。因此，这些单一药剂可以基本上同时(即作为单一制剂或在数分钟或数小时内)或以按顺序连续施用。例如，这些单一药剂可以在一年内，或10、8、6、4或2个月内，或4、3、2、或1周内， 或5、4、3、2或1天内施用。
关于制剂、剂量、施用方案和可测量的治疗结果的另外指导，参见Berkow等人(2000)The Merck Manual of Medical Information(Merck医学信息手册)和Merck&Co.Inc.，Whitehouse Station，New Jersey；Ebadi(1998)CRC Desk Reference of Clinical Pharmacology(临床药理学手册)等著作。
本发明提供一种上述的蛋白质在制备抗肿瘤药物中的应用。
本发明提供一种上述的免疫偶联物在制备抗肿瘤药物中的应用。
本发明提供一种上述的药物组合物在制备抗肿瘤药物中的应用。
本发明提供一种上述的蛋白质在治疗肿瘤中的应用。
本发明提供一种上述的免疫偶联物在治疗肿瘤中的应用。
本发明提供一种上述的药物组合物在治疗肿瘤中的应用。
本发明还提供一种检测过表达TPBG蛋白的细胞的方法，包括如下的步骤：上述的蛋白质与待检样品在体外接触，检测上述的蛋白质与所述待检样品的结合即可。
所述的过表达的含义为本领域常规，较佳地为待检样品中，细胞经过流式检测，上述的蛋白质的平均荧光密度(MFI)值是亚型IgG的MFI值的3倍及以上。
所述结合的检测方式是本领域常规的检测方式，较佳地为FACS检测。
本发明所述的“TPBG阳性”的细胞即为过表达TPBG蛋白的细胞，如NCI-H1568细胞株；反之，则称为“TPBG阴性”的细胞，如肿瘤细胞系NCI-H1770。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：本发明所述的TPBG抗体是一嵌合抗体，其与TPBG蛋白具有高度亲和力，能够在蛋白水平和细胞水平结合TPBG蛋白受体的胞外区。所述的TPBG抗体与如MC-MMAF的小分子化合物偶联后得到一偶联物，所述偶联物能够有效地对TPBG阳性细胞进行细胞毒杀伤作用。此外，TPBG抗体能把小分子化合物，如MMAF，通过内吞作用带入细胞，并在细胞内降解释放小分子化合物，从而起到细胞毒杀伤作用。因此所述的TPBG抗体制备的抗体交联药，能够有效杀伤肿瘤细胞，治疗肿瘤。
附图说明
图1为人TPBG蛋白转染的HEK293细胞FACS筛选检测结果。
图2为人TPBG蛋白转染的CHOK1细胞FACS筛选检测结果。
图3为食蟹猴TPBG蛋白转染的CHOK1细胞FACS筛选检测结果。
图4为小鼠TPBG蛋白转染的CHOK1细胞FACS筛选检测结果。
图5为ELISA检测TPBG免疫后小鼠血清抗体效价情况。
图6A和图6B为ELISA检测TPBG抗体与人TPBG-hFc蛋白的结合反应。
图7A和图7B为FACS检测TPBG抗体与CHOk1-hTPBG的结合反应。
图8A和图8B为FACS检测TPBG抗体与CHOk1-cTPBG的结合反应。
图9A和图9B为FACS检测TPBG抗体与CHOk1-mTPBG的结合反应。
图10A和图10B为FACS检测TPBG抗体与CHOk1的结合反应。
图11A和图11B为TPBG抗体-MMAF抗体交联药对TPBG表达阳性非小肺癌细胞株NCI-H1568的细胞杀伤作用。
图11C为TPBG抗体对TPBG表达阳性非小肺癌细胞株NCI-H1568的细胞杀伤作用。
图12A和图12B为TPBG抗体-MMAF抗体交联药对TPBG表达阴性非小肺癌细胞株NCI-H1770的细胞杀伤作用。
图13为TPBG嵌合抗体药物偶联物对TPBG阳性的肿瘤细胞系NCI-H1299的细胞杀伤作用。
图14A为TPBG嵌合抗体12B12的抗体药物偶联物及其裸抗的治疗后的肿瘤体积变化图。
图14B为TPBG嵌合抗体5G4的抗体药物偶联物及其裸抗的治疗后的肿瘤体积变化图。
图14C为TPBG嵌合抗体39A11的抗体药物偶联物及其裸抗的治疗后的肿瘤体积变化图。
图14D为TPBG嵌合抗体28D4的抗体药物偶联物及其裸抗的治疗后的肿瘤体积变化图。
图14E为TPBG嵌合抗体36A10的抗体药物偶联物及其裸抗的治疗后的肿瘤体积变化图。
图15A为TPBG嵌合抗体12B12的抗体药物偶联物及其裸抗的治疗后的小鼠体重变化图。
图15B为TPBG嵌合抗体5G4的抗体药物偶联物及其裸抗的治疗后的小鼠体重变化图。
图15C为TPBG嵌合抗体39A11的抗体药物偶联物及其裸抗的治疗后的小鼠体重变化图。
图15D为TPBG嵌合抗体28D4的抗体药物偶联物及其裸抗的治疗后的小鼠体重变化图。
图15E为TPBG嵌合抗体36A10的抗体药物偶联物及其裸抗的治疗后的小鼠体重变化图。
图16A和16B分别为TPBG嵌合抗体12B12及其偶联了不同的小分子药的抗体药物偶联物对TPBG阳性的肿瘤细胞系NCI-H1299的细胞杀伤作用。
图16C和16D分别为TPBG嵌合抗体12B12及其偶联了不同的小分子药的抗体药物偶联物对TPBG阳性的肿瘤细胞系NCI-H1568的细胞杀伤作用。
图17A和17B为TPBG嵌合抗体药物偶联物对TPBG阳性的表达TPBG的293-hTPBG稳转细胞株的细胞杀伤作用。
图17C和17D为TPBG嵌合抗体药物偶联物对不表达TPBG的293细胞株的，细胞杀伤作用。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例中所述的室温为本领域常规的室温，一般为10～30℃。
若无特别说明，实施例中所述的PBS为PBS磷酸缓冲液，pH7.2。
实施例1 TPBG抗体的制备
将含有编码人源TPBG蛋白胞外区氨基酸序列32-355(Ser32-Ser355)(其中，编码人源TPBG蛋白的核苷酸序列在Genebank的编号为Genebank ID：AAH37161.1)的核苷酸序列克隆到带有人IgG Fc片段(hFc)的pCpC载体(购自Invitrogen，V044-50)并按已建立的标准分子生物学方法制备质粒。具体方法参见Sambrook，J.，Fritsch，E.F.，and Maniatis，T.(1989).Molecular Cloning：A Laboratory Manual，Second Edition(Plainview，New York：Cold Spring Harbor Laboratory Press)。对HEK293细胞(购自Invitrogen)进行瞬时转染(聚醚酰亚胺PEI，购自Polysciences)并使用FreeStyle TM 293(购自Invitrogen)在37℃下进行扩大培养。4天后收集细胞培养液，离心去除细胞成分，得含TPBG蛋白胞外区的培养上清液。将培养上清液上样到蛋白A亲和层析柱(Mabselect Sure，购自 GE Healthcare)，同时用紫外(UV)检测仪监测紫外吸收值(A280nm)的变化。上样后用PBS磷酸盐缓冲液(pH7.2)清洗蛋白A亲和层析柱直到紫外吸收值回到基线，然后用0.1M甘氨酸盐酸(pH2.5)洗脱，收集从蛋白A亲和层析柱上洗脱下来的带hFc标签的TPBG蛋白(即人源TPBG-hFc)。用PBS磷酸盐缓冲液(pH7.2)在4℃冰箱透析过夜。透析后的蛋白经0.22微米无菌过滤后分装于-80℃保存，即获得纯化的免疫原A。
免疫原A在使用前需要进行一系列质控检测，如检测其蛋白浓度、纯度、分子量、生物活性等，结果发现免疫原A各项指标良好，能够作为抗原进行后续制备TPBG抗体的试验。
(二)、免疫原B的制备
编码人源TPBG全长氨基酸序列的核苷酸序列(其中，编码人源TPBG蛋白的核苷酸序列在Genebank的编号为Genebank ID：AAH37161.1)被克隆到pIRES载体(购自Clontech)并制备质粒。对HEK293细胞系和CHOK1细胞系(均购自Invitrogen)进行质粒转染(PEI，购自Polysciences)后，在含0.5μg/ml的含10％(w/w)胎牛血清的DMEM培养基中选择性培养2周，用有限稀释法在96孔培养板中进行亚克隆，并置于37℃、5％(v/v)CO2培养，大约2周后选择部分单克隆孔扩增到6孔板中。对扩增后的克隆用已知的TPBG抗体(购自Sigma，货号#SAB1404485)经流式细胞分析法进行筛选。选择长势较好、荧光强度较高、单克隆的细胞系继续扩大培养并液氮冻存，即获得免疫原B。具体选择结果如表3和图1所示，IgG亚型对照为小鼠IgG对照。表3说明，已经制得一系列TPBG阳性表达的HEK293细胞系。图1中，横坐标为细胞荧光强度，纵坐标为细胞数。图1的结果说明，5E5E9为TPBG高水平表达细胞株，其中抗TPBG抗体标记的细胞平均细胞荧光密度为216，迁移率为98.5％。
表3人源TPBG蛋白转染的HEK293细胞FACS筛选检测结果
Figure PCTCN2016111699-appb-000008
Figure PCTCN2016111699-appb-000009
(三)、杂交瘤细胞的制备和抗体筛选
A、免疫原A免疫
采用6～8周龄BALB/cAnNCrl小鼠或SJL/JorllcoCrl小鼠(均购自上海斯莱克公司)，小鼠在SPF条件下饲养。初次免疫时，免疫原A用弗氏完全佐剂乳化后腹腔注射0.25mL，即每只小鼠注射50μg免疫原A蛋白。加强免疫时，免疫原A用弗氏不完全佐剂乳化后腹腔注射0.25mL，即每只小鼠注射50微克免疫原A。初次免疫与第一次加强免疫之间间隔2周，以后每次加强免疫之间间隔3周。每次加强免疫1周后采血，用ELISA和FACS检测血清中免疫原A的抗体效价和特异性，结果如图5和表4所示。表4说明，经免疫原A免疫的小鼠的免疫后血清对免疫原A均有不同程度的结合，呈现抗原抗体反应，其中最高稀释度在一百万左右。其中空白对照为1％(w/w)BSA，其中批次指第二次加强免疫后第七天的小鼠血清，表中的数据为OD450nm值。
表4 ELISA检测TPBG蛋白免疫后Balb/c小鼠血清抗体效价
Figure PCTCN2016111699-appb-000010
Figure PCTCN2016111699-appb-000011
B、免疫原B免疫
采用6～8周龄BALB/cAnNCrl小鼠或SJL/JorllcoCrl小鼠(均购自上海斯莱克公司)，小鼠在SPF条件下饲养。含有编码人源TPBG全长氨基酸序列的核苷酸序列的pIRES质粒[参见实施例1步骤(二)]转染HEK293细胞系，得含有人源TPBG的HEK293稳定细胞系(转染使用X-treme GENE HP DNA Transfection Reagent，购自Roche公司，货号Cat#06 366 236 001，并按说明书操作)。在T-75细胞培养瓶中扩大培养至90％汇合度，吸尽培养基，用DMEM基础培养基(购自Invitrogen)洗涤2次，然后用无酶细胞解离液(购自Invitrogen)37℃处理直至细胞从培养皿壁上可脱落，收集细胞。用DMEM基础培养基洗涤2次，进行细胞计数后将细胞用磷酸盐缓冲液稀释至2×107细胞每mL。每只小鼠每次免疫时腹腔注射0.5mL细胞悬液。第一次与第二次免疫之间间隔2周，以后每次免疫间隔3周。除第一次免疫以外，每次免疫1周后采血，用FACS检测血清中抗体效价和特异性。在第二次加强免疫后，FACS检测血清抗体效价达到1∶1000以上。
A～B步骤完成前，将所选择的每只小鼠最后一次免疫腹腔注射100微克纯化的免疫原A(针对免疫原A进行免疫反应的小鼠)或含有人源TPBG的HEK293稳定细胞系(针对免疫原B进行免疫反应的小鼠)，5天后处死小鼠，收集脾细胞。加入NH4OH至终浓度1％(w/w)，裂解脾细胞中参杂的红细胞，获得脾细胞悬液。用DMEM基础培养基1000转每分钟离心清洗细胞3次，然后按照活细胞数目5∶1比率与小鼠骨髓瘤细胞SP2/0(购自ATCC)混合，采用高效电融合方法(参见METHODS IN ENZYMOLOGY，VOL.220)进行细胞融合。融合后的细胞稀释到含20％(w/w)胎牛血清、1×HAT的DMEM培养基中。然后按1×105/200微升每孔加入到96孔细胞培养板中，放入5％(v/v)CO2、37℃培养箱中培养。14天后用ELISA和Acumen(微孔板细胞检测法)筛选细胞融合板上清，将ELISA中OD450nm＞1.0和Acumen中MFI值＞100的阳性克隆扩增到24孔板，在含10％(w/w)HT胎牛血清的DMEM(invitrogen)中，于37℃、5％(v/v)CO2条件下扩大培养。培养3天后取24孔板中扩大培养的培养液进行离心，收集上清液，对上清液进行抗体亚型分析，用ELISA、FACS确定对TPBG蛋白和TPBG阳性细胞的结合活性(结合活性的检测方法分别参见实施例3A和实施例3B中的相关内容)，以及小鼠源TPBG抗体-MMAF间接细胞毒杀伤实验(间接细胞毒杀伤活性检测方法参见实施例4中的相关内容)。
根据24孔板筛选结果，挑选ELISA实验中OD450nm＞1.0、FACS实验中MFI值＞50 和间接细胞毒杀伤实验中杂交瘤细胞培养上清对TPBG阳性细胞杀伤率达到50％的杂交瘤细胞为符合条件的阳性克隆，选择符合条件的杂交瘤细胞用有限稀释法在96孔板进行亚克隆，在含10％(w/w)FBS的DMEM培养基中(购自invitrogen)37℃、5％(v/v)CO2条件下培养。亚克隆后10天用ELISA和Acumen进行初步筛选，挑选单个阳性单克隆扩增到24孔板继续培养。3天后用FACS确定抗原结合阳性并用小鼠源TPBG抗体-MMAF间接细胞毒杀伤实验评估生物活性，评估标准为ELISA实验中OD450nm＞1.0、FACS实验中MFI值＞50和间接细胞毒杀伤实验中杂交瘤细胞培养上清对TPBG阳性细胞杀伤率达到50％及以上。
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(购自invitrogen)在37℃、5％(v/v)CO2条件下将该最优的克隆进行扩大培养，液氮冻存即得本发明杂交瘤细胞，并可用于后续的获得先导抗体、生产和纯化抗体。
实施例2先导抗体的生产和纯化
杂交瘤细胞产生的抗体浓度较低，大约仅1-10μg/mL，浓度变化较大。且培养基中细胞培养所产生的多种蛋白和培养基所含胎牛血清成分对很多生物活性分析方法都有不同程度的干扰，因此需要进行小规模(1-5mg)抗体生产纯化。
将实施例1所得的杂交瘤细胞接种到T-75细胞培养瓶并用生产培养基(Hybridoma serum free medium，购自Invitrogen公司)驯化传代3代。待其生长状态良好，接种细胞培养转瓶。每个2升的培养转瓶中加入200mL生产培养基，接种细胞密度为1.0×105/mL。盖紧瓶盖，将转瓶置于37℃培养箱中的转瓶机上，转速3转/分钟。连续旋转培养14天后，收集细胞培养液，过滤去除细胞，并用0.45μm的滤膜过滤至培养上清液澄清，得澄清的杂交瘤细胞的培养上清液。澄清的杂交瘤细胞的培养上清液可立即进行纯化或于-30℃冻存。
将获得的的培养上清液(200mL)中的TPBG抗体用2mL蛋白A柱(购自GE Healthcare)纯化。蛋白G柱先用平衡缓冲液(PBS磷酸缓冲液，pH7.4)平衡，然后将培养上清液上样到蛋白A柱，控制流速在3mL/分钟。上样完毕后用平衡缓冲液清洗蛋白G柱，平衡缓冲液的体积为蛋白A柱柱床体积的4倍。用洗脱液(0.1M柠檬酸钠缓冲液，pH3.5)洗脱结合在蛋白A柱上的TPBG抗体，用紫外检测器监测洗脱情况(A280nm紫外吸收峰)。收集洗脱的抗体，加入10％(v/v)1.0M Tris-HCl缓冲液中和pH，然后立即用PBS磷酸缓冲液透析过夜，第二天换液1次并继续透析3小时。收集透析后的TPBG抗体，用0.22μm的滤器进行无菌过滤，无菌保存，即得纯化的TPBG抗体。
将纯化的TPBG抗体进行蛋白浓度(A280nm/1.4)、纯度、内毒(Lonza试剂盒)等检 测分析，结果如表5所示，表5说明，抗体最终产品内毒素浓度在1.0EU/mg以内。
表5纯化的TPBG抗体检测分析
Figure PCTCN2016111699-appb-000012
实施例3先导抗体的检定
A、酶联免疫吸附实验(ELISA)检测TPBG抗体与TPBG蛋白的结合
对实施例2所得的纯化的TPBG抗体进行与人源TPBG-hFc蛋白(免疫原A)进行反应。
将实施例1获得的纯化的免疫原A(其制备方法参见实施例1步骤(一)用PBS稀释到终浓度1.0μg/mL，然后以100μL每孔加到96孔ELISA板。用塑料膜封好4℃孵育过夜，第二天用洗板液[含0.01％(v/v)Tween20的PBS]洗板2次，加入封闭液[含0.01％(v/v)Tween20和1％(w/w)BSA的PBS]室温封闭2小时。倒掉封闭液，加入实施例2所得的纯化的TPBG抗体100μL每孔。37℃孵育2小时后，用洗板液[含0.01％(v/v)Tween20的PBS]洗板3次。加入HRP(辣根过氧化物酶)标记的二抗(购自Sigma)，37℃孵育2小时后，用洗板液[含0.01％(v/v)Tween20的PBS]洗板3次。加入TMB底物100μL每孔，室温孵育30分钟后，加入终止液(1.0N HCl)100μL每孔。用ELISA读板机(SpectraMax 384plus，购自Molecular Device)读取A450nm数值，结果如图6和表6所示，表6说明，纯化的TPBG抗体与TPBG重组蛋白在ELISA水平结合。表6中IgG对照为对照小鼠IgG，表中的数据为OD450nm值，Blank的含义为板中只有PBS缓冲液时的OD450nm值。
表6 ELISA检测TPBG抗体与人TPBG-hFc蛋白的结合反应
Figure PCTCN2016111699-appb-000013
B、流式细胞实验(FACS)检测TPBG抗体与TPBG表达细胞的结合
将实施例1步骤(二)中所述含有编码人源TPBG全长氨基酸序列的核苷酸序列的pIRES质粒转染CHOK1细胞株得含人源TPBG的CHOK1稳定细胞株(此处称为CHOk1-hPD1稳定细胞株)。类似地，将带有猴源TPBG全长基因的pIRES质粒(其制备方法与实施例1步骤(一)“免疫原A的制备”中带有人源IgG Fc片段(hFc)的pCpC载体的制备方法相同，食蟹猴TPBG全长氨基酸序列(Genebank ID：BAE00432.1)转染CHOK1细胞株得含猴TPBG的CHOK1稳定细胞株(此处称为CHOk1-cTPBG稳定细胞株)。将带有小鼠源TPBG全长基因的pIRES质粒(其制备方法与实施例1步骤(一)“免疫原A的制备”中带有人源IgG Fc片段(hFc)的pCpC载体的制备方法相同，小鼠TPBG全长氨基酸序列(Genebank ID：CAA09931.1)转染CHOK1细胞株得含小鼠TPBG的CHOK1稳定细胞株(此处称为CHOk1-mTPBG稳定细胞株)。
用FACS检测血清中TPBG抗体的效价和特异性，检测方法参见实施例1步骤(二)“免疫原B的制备”中鉴定HEK293-hTPBG稳定细胞株的方法。检测结果如表7和图2～4所示，图2～4中横坐标为细胞荧光强度，纵坐标为细胞数。其中，CHOK1-hTPBG 3A2F1为用来筛选的人TPBG表达细胞株，其FACS筛选检测结果如图2所示；CHOK1-cTPBG 3F13G4为用来筛选的食蟹猴TPBG表达细胞株，其FACS筛选检测结果如图3所示；CHOK1-mTPBG 3A3为用来筛选的小鼠TPBG表达细胞株，其FACS筛选检测结果如图4所示。表7的结果说明，CHOk1-hTPBG稳定细胞株、CHOk1-cTPBG稳定细胞株和 CHOk1-mTPBG稳定细胞株的细胞膜上分别过表达人、猴或小鼠的TPBG蛋白，其可以用于筛选TPBG抗体。
表7人/猴/小鼠TPBG转染的CHOK1细胞FACS筛选检测结果
Figure PCTCN2016111699-appb-000014
将CHOk1-hTPBG稳定细胞株、CHOk1-cTPBG稳定细胞株、CHOK1-mTPBG稳定细胞株(即表7所示的CHOk1-hTPBG 3A2F1、CHOk1-cTPBG 3F13G4和CHOk1-mTPBG 3A3)以及CHOK1细胞分别在T-75细胞培养瓶中扩大培养至90％汇合度，吸尽培养基，用HBSS缓冲液(Hanks Balanced Salt Solution)(购自Invitrogen)洗涤2次，然后用无酶细胞解离液(Versene solution：购自Life technology公司)处理和收集细胞。用HBSS缓冲液洗涤细胞2次，进行细胞计数后将细胞用HBSS缓冲液稀释至2×106个细胞/mL，加入10％山羊血清封闭液，所述百分比为质量百分比，冰上孵育30分钟，然后用HBSS缓冲液离心洗涤2次。将收集的细胞用FACS缓冲液(HBSS+1％BSA，所述百分比为质量百分比)悬浮至2×106个细胞/mL，按每孔100微升加入到96孔FACS反应板中，加入实施例2所得的纯化的TPBG抗体待测样品每孔100微升，冰上孵育2小时。用FACS缓冲液离心洗涤2次，加入每孔100微升荧光(Alexa 488)标记的二抗(购自Invitrogen)，冰上孵育1小时。用FACS缓冲液离心洗涤3次，加入每孔100微升固定液[4％(v/v)多聚甲醛]重悬细胞，10分钟后用FACS缓冲液离心洗涤2次。用100微升FACS缓冲液悬浮细胞，用FACS(FACS Calibur，购自BD公司)检测和分析结果。通过软件(CellQuest)进行数据分析，得到细胞的平均荧光密度(MFI)。再通过软件(GraphPad Prism5)分析，进行数据拟合，计算EC50值。分析结果如表8以及图7～10所示，图7～10的数据为细胞的平均荧光密度(MFI)。表8中的数据为根据MFI计算得到的EC50值。表8说明，TPBG抗体可结合细胞表面的TPBG蛋白。
表8 FACS分析TPBG抗体与人/猴/小鼠TPBG表达细胞株结合活性
Figure PCTCN2016111699-appb-000015
Figure PCTCN2016111699-appb-000016
实施例4 TPBG抗体药物偶联物的细胞杀伤活性实验
将实施例2所得的纯化的TPBG抗体经过pH 6.5～8.5的硼酸钠缓冲液透析后，加入三(2-羧乙基)膦(TCEP)，其中TCEP与纯化的TPBG抗体的摩尔比比率为2，室温下还原1小时，得反应液A。将反应液A经过G25柱脱盐(购自GE)，去除多余的TCEP，得反应液B。向反应液B中加入MC-MMAF(购自南京联宁)，其中MC-MMAF与纯化的TPBG抗体的摩尔比比率为5，室温下反应4小时。再加入半胱氨酸用以中和多余的MC-MMAF，并通过G25柱脱盐除去多余的小分子。得到纯化的TPBG抗体药物偶联物(偶联方法参见Doronina，2006，Bioconjugate Chem.17，114-124)。通过HIC分析药物的交联率、纯度等参数后，进行细胞毒活性的分析。所有抗体偶联物的药物交联率(DAR)为8。其中，DAR(drug antibody ratio)指抗体偶联后一个抗体分子上携带的小分子药物的平均数量。
将获得的纯化的TPBG抗体药物偶联物分别用完全培养基进行梯度稀释，96孔细胞培养板以2000细胞/孔加入100微升TPBG阳性的NCI-H1568细胞株(购自ATCC，货号#CRL5876)细胞悬液过夜培养后，每孔分别加入10微升不同浓度的纯化的TPBG抗体药物偶联物的稀释液，继续培养5天后，用CellTiter-Glo试剂盒(购自Promega，使用方法参照产品说明书)检测细胞活力。同时选用TPBG阴性的肿瘤细胞系NCI-H1770(购自ATCC，货号#CRL5893)进行细胞杀伤活性检测，方法同上。结果如表9以及图11-12所示，其中表9的EC50指药物作用后，细胞的活性受到抑制的半数有效量，能够通过检测细胞的活性从而反映细胞杀伤活性。其中，图11A和11B为纯化的TPBG抗体药物偶联物对TPBG阳性的肿瘤细胞系NCI-H1568的细胞杀伤活性检测，图12A和12B为纯化的TPBG抗体药物偶联物对TPBG阴性的肿瘤细胞系NCI-H1770的细胞杀伤活性检测。结果说明，纯化的TPBG抗体药物偶联物对TPBG阳性的细胞有杀伤作用。
此外还检测了单独使用实施例2所得的纯化的TPBG抗体对细胞的杀伤作用，使用 同实施例4中TPBG抗体药物偶联物进行细胞杀伤活性检测的方法进行检测。结果如表9以及图11C所示，结果说明，单独的纯化的TPBG抗体对TPBG阳性的细胞并没有显著的杀伤作用。
表9细胞杀伤实验检测纯化的TPBG抗体药物偶联物对TPBG阳性细胞的特异性杀伤作用
Figure PCTCN2016111699-appb-000017
实施例5竞争性ELISA检测分析TPBG抗体与抗原的表位分布
为了鉴定抗体对抗原的结合位点，采用竞争ELISA的方法对TPBG抗体进行分组。
纯化的待测抗体用PBS稀释至1μg/mL，以50μL/孔包被96孔高吸附酶标板，4℃过夜包被后用250微升封闭液[含有0.01％(v/v)Tween20和1％(w/w)BSA的PBS]进行室温一小时封闭，每孔加入0.05μg/mL的生物素标记的重组TPBG蛋白。同时加入5μg/mL的竞争抗体，即实施例2所得的纯化的TPBG抗体，其克隆号分别为12B12C7C3、5G4H10G5、37H9C5G2、39A11G5F2、52C9E9F6、28D4E6A9、36A10D8B12、99E12C7H1、103E2E9C2和106D5G3D10，并于25-37℃孵育1-2小时。用洗板液[含有0.01％(v/v)Tween20的PBS]洗板3次，加入HRP(辣根过氧化物酶)标记的链亲和素(购自Sigma)。 37℃孵育0.5小时后，用洗板液[含有0.01％(v/v)Tween20的PBS]洗板3次。加入TMB底物100μL每孔，室温孵育30分钟后，加入终止液(1.0N HCl)100μL每孔。用ELISA读板机(SpectraMax 384plus，购自Molecular Device)读取A450nm数值，结果如图6所示。根据A450nm数值，计算出抗体相互之间的竞争率，结果如表10所示。竞争率的数值越高，表示两个抗体的抗原表面越是接近。
表10 TPBG抗体相互之间的竞争率
Figure PCTCN2016111699-appb-000018
其中，A的含义是各列为包被抗体，浓度为1μg/mL；B的含义是各行为竞争抗体， 浓度为5μg/mL。
结果说明，12B12C7C3和106D5G3D10可以互相竞争，为相似表位；5G4H10G5和99E12C7H1可以互相竞争，为相似表位；37H9C5G2和36A10D8B12可以互相竞争，为相似表位；39A11G5F2和52C9E9F6可以互相竞争，为相似表位；28D4E6A9和103E2E9C2可以互相竞争，为相似表位。
实施例6轻重链可变区氨基酸序列测定
总RNA分离：通过离心搜集实施例1所得的杂交瘤细胞5×107个，加入1mL Trizol混匀并转移到1.5mL离心管中，室温静置5分钟。加0.2mL氯仿，振荡15秒，静置10分钟后于4℃，12000g离心5分钟，取上清转移到新的1.5mL离心管中。加入0.5mL异丙醇，将管中液体轻轻混匀，室温静置10分钟后于4℃，12000g离心15分钟，弃上清。加入1mL 75％(v/v)乙醇，轻轻洗涤沉淀，4℃，12000g离心5分钟后弃上清，将沉淀物晾干，加入DEPC处理过的H2O溶解(55℃水浴促进溶解10分钟)，即得总RNA。
逆转录与PCR：取1μg总RNA，配置20μL体系，加入逆转录酶后于42℃反应60分钟，于7℃反应10分钟终止反应。配置50μL PCR体系，包括1μL cDNA、每种引物25pmol、1μL DNA聚合酶以及相配的缓冲体系、250μmol dNTPs；设置PCR程序，95℃预变性3分钟，95℃变性30秒，55℃退火30秒，72℃延伸35秒，35个循环后再额外于72℃延伸5分钟，得PCR产物。其中逆转录所用的试剂盒为PrimeScript RT Master Mix，购自Takara，货号RR036；PCR所用的试剂盒包括Q5超保真酶，购自NEB，货号M0492。
克隆与测序：取5μL PCR产物进行琼脂糖凝胶电泳检测，将检测阳性样品使用柱回收试剂盒纯化，其中回收试剂盒为
Figure PCTCN2016111699-appb-000019
Gel&PCR Clean-up，购自MACHEREY-NAGEL，货号740609。进行连接反应：样品50ng，T载体50ng，连接酶0.5μL，缓冲液1μL，反应体系10μL，于16℃反应半小时得连接产物。其中连接的试剂盒为T4DNA连接酶，购自NEB，货号M0402；取5μL连接产物加入100μL的感受态细胞(Ecos 101competent cells，购自Yeastern，货号FYE607)中，冰浴5分钟，而后于42℃水浴热激1分钟，放回冰上1分钟后加入650μL无抗生素SOC培养基，于37℃摇床上以200RPM的速度复苏30分钟。取出200μL涂布于含抗生素的LB固体培养基上于37℃孵箱过夜培养。次日，使用T载体上引物M13F和M13R配置30μLPCR体系，进行菌落PCR，用移液器枪头蘸取菌落于PCR反应体系中吹吸，并吸出0.5μL点于另一块含100nM氨苄青霉素的LB固体培养皿上以保存菌株。PCR反应结束后，取出5μL进行琼脂糖凝胶电泳检测，将阳性样品进行测序和分析[参见Kabat，“Sequences of Proteins of Immunological Interest，”National Institutes of Health，Bethesda，Md.(1991)]。测序结 果如表11～12所示。
表11 TPBG抗体蛋白序列编号
Figure PCTCN2016111699-appb-000020
其中，表11中的数字即为序列表中序列号，如12B12C7C3的重链蛋白可变区的氨基酸序列为SEQ ID No.1，而12B12C7C3的重链蛋白可变区中CDR1的氨基酸序列为SEQ ID No.2。
表12 TPBG抗体基因序列编号
克隆号 重链蛋白可变区 轻链蛋白可变区
12B12C7C3 81 82
5G4H10G5 83 84
37H9C5G2 85 86
39A11G5F2 87 88
52C9E9F6 89 90
28D4E6A9 91 92
36A10D8B12 93 94
99E12C7H1 95 96
103E2E9C2 97 98
106D5G3D10 99 100
其中，表12中的数字即为序列表中序列号，如编码12B12C7C3的重链蛋白可变区的核苷酸序列为SEQ ID No.81。
其中，编码12B12C7C3的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.81中的第91位至第105位；
编码12B12C7C3的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.81中的第151位至第198位；
编码12B12C7C3的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.81中的第295位至第327位；
编码12B12C7C3的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.82中的第70位至第114位；
编码12B12C7C3的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.82中的第157位至第180位；
编码12B12C7C3的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.82中的第277位至第303位；
编码5G4H10G5的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.83中的第91位至第105位；
编码5G4H10G5的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.83中的第148位至第198位；
编码5G4H10G5的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.83中的第295位至第330位；
编码5G4H10G5的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.84中的第70位至第102位；
编码5G4H10G53的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.84中的第148位至第168位；
编码5G4H10G5的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.84中的第265位至第288位；
编码37H9C5G2的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.85中的第91位至第105位；
编码37H9C5G2的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.85中的第151位至第198位；
编码37H9C5G2的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.85中的第295位至第327位；
编码37H9C5G2的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.86中 的第70位至第102位；
编码37H9C5G2的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.86中的第148位至第168位；
编码37H9C5G2的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.86中的第265位至第291位。
编码39A11G5F2的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.87中的第91位至第105位；
编码39A11G5F2的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.87中的第151位至第198位；
编码39A11G5F2的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.87中的第295位至第315位；
编码39A11G5F2的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.88中的第70位至第102位；
编码39A11G5F2的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.88中的第148位至第168位；
编码39A11G5F2的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.88中的第265位至第291位。
编码52C9E9F6的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.89中的第91位至第105位；
编码52C9E9F6的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.89中的第151位至第198位；
编码52C9E9F6的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.89中的第295位至第315位；
编码52C9E9F6的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.90中的第70位至第102位；
编码52C9E9F6的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.90中的第148位至第168位；
编码52C9E9F6的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.90中的第268位至第291位。
编码28D4E6A9的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.91中的第91位至第105位；
编码28D4E6A9的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.91中的第148位至第198位；
编码28D4E6A9的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.91中的第295位至第327位；
编码28D4E6A9的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.92中的第70位至第102位；
编码28D4E6A9的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.92中的第148位至第168位；
编码28D4E6A9的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.92中的第265位至第291位。
编码36A10D8B12的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.93中的第91位至第108位；
编码36A10D8B12的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.93中的第154位至第198位；
编码36A10D8B12的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.93中的第295位至第324位；
编码36A10D8B12的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.94中的第70位至第102位；
编码36A10D8B12的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.94中的第148位至第168位；
编码36A10D8B12的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.94中的第265位至第291位。
编码99E12C7H1的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.95中的第91位至第105位；
编码99E12C7H1的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.95中的第151位至第198位；
编码99E12C7H1的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.95中的第295位至第318位；
编码99E12C7H1的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.96中的第70位至第102位；
编码99E12C7H1的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.96 中的第148位至第168位；
编码99E12C7H1的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.96中的第265位至第291位。
编码103E2E9C2的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.97中的第91位至第105位；
编码103E2E9C2的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.97中的第151位至第198位；
编码103E2E9C2的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.97中的第295位至第324位；
编码103E2E9C2的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.98中的第70位至第105位；
编码103E2E9C2的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.98中的第151位至第171位；
编码103E2E9C2的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.98中的第268位至第294位。
编码106D5G3D10的重链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.99中的第91位至第105位；
编码106D5G3D10的重链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.99中的第151位至第198位；
编码106D5G3D10的重链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.99中的第295位至第318位；
编码106D5G3D10的轻链蛋白可变区中CDR1的核苷酸序列为序列表SEQ ID No.100中的第70位至第99位；
编码106D5G3D10的轻链蛋白可变区中CDR2的核苷酸序列为序列表SEQ ID No.100中的第145位至第165位；
编码106D5G3D10的轻链蛋白可变区中CDR3的核苷酸序列为序列表SEQ ID No.100中的第262位至第294位。
实施例7鼠-人嵌合抗体构建、以及抗体的生产和纯化
1.质粒构建与准备：根据实施例6的测序结果明确了TPBG抗体重链可变区和轻链可变区序列。将实施例2和实施例3所得的先导抗体的重链可变区序列重组到包含信号肽和人源重链抗体IgG1恒定区的表达载体(其中表达载体购买自Invitrogen，重组步骤 也由上海睿智化学完成)中，将TPBG抗体的轻链可变区序列重组到包含信号肽和人源抗体轻链kappa恒定区的表达载体当中，得重组质粒并经测序验证(测序方法与实施例6中测序方法相同)。使用碱裂解法试剂盒(购自MACHEREY-NAGEL)中量抽提高纯度的重组质粒，质量为500μg以上，经0.22μm滤膜(购自Millopore)过滤，供转染使用。
2.细胞转染：在培养基Freestyle 293expression medium(购自Invitrogen)培养293E细胞(购自Invitrogen)。摇床设置为37℃、130RPM和8％CO2(v/v)。Freestyle 293expression medium在转染时添加10％(v/v)F68(购自Invitrogen)至F68终浓度为0.1％(v/v)，得含0.1％(v/v)F68的Freestyle 293表达培养基，即培养基A。取5mL培养基A和200μg/mL PEI(购自Sigma)混匀，得培养基B。取5mL培养基A和100μg/mL步骤(1)所得的重组质粒混匀，得培养基C。5分钟后将培养基B和培养基C合并混匀，静置15分钟，得混合液D。将10mL混合液D缓缓加入100mL含293E细胞的培养基Freestyle 293expression medium中至293E的细胞密度为1.5×106个/mL，边加边振荡，避免PEI过度集中，放入摇床培养。第二天加入蛋白胨至终浓度为0.5％(w/v)。第5～7天，测培养液抗体效价。第6～7天，离心(3500RPM，30分钟)收集上清，经0.22μm滤膜过滤，得滤好的细胞上清液，以供纯化。
3.抗体纯化：对于连续生产的无内毒素的层析柱和ProteinA填料，使用0.1M NaOH处理30min或者5个柱体积0.5M NaOH冲洗；对于长期未使用的柱料和层析柱至少使用1M NaOH浸泡1h，用无内毒的水冲洗至中性，用10倍柱体积的1％Triton X100对柱料清洗。使用5个柱体积的PBS进行平衡，将过滤好的细胞上清上柱，必要时收集流穿液。上柱完成后，使用5倍柱体积PBS清洗。用5倍柱体积的0.1M pH3.0的Glycine-HCl进行洗脱，收集洗脱液，并用1/10体积的pH8.5的1M Tris-HCl(1.5M NaCl)中和。收获抗体后，在1×PBS中透析过夜，避免内毒素污染。透析结束后，使用分光光度或试剂盒测定浓度，使用HPLC-SEC测定抗体纯度，使用内毒素检测试剂盒(购自Lonza)检测抗体内毒素含量。
下述实施例中嵌合抗体命名中首段字符选用对应的先导抗体克隆号的前3～5位字符，例如嵌合抗体12B12-MMAF对应的先导抗体克隆号为12B12C7C3，嵌合抗体5G4-MMAF对应的先导抗体克隆号为5G4H10G5等等。
实施例8嵌合抗体的体外药效实验
将实施例7所得的纯化的TPBG嵌合抗体与MC-MMAF进行偶联，方法同实施例4，经过pH 6.5～8.5的硼酸钠缓冲液透析后，加入三(2-羧乙基)膦(TCEP)，其中TCEP与纯化的TPBG抗体的摩尔比比率为2，室温下还原1小时，得反应液A。将反应液A 经过G25柱脱盐(购自GE)，去除多余的TCEP，得反应液B。向反应液B中加入MC-MMAF，其中MC-MMAF与纯化的TPBG抗体的摩尔比比率为5，室温下反应4小时。再加入半胱氨酸用以中和多余的MC-MMAF，并通过G25柱脱盐除去多余的小分子。得到纯化的TPBG抗体药物偶联物(偶联方法参见Doronina，2006，BioconjugateChem.17，114-124)。通过HIC分析药物的交联率、通过SEC分析抗体药物偶联物的纯度等参数后，进行细胞毒活性的分析。所有抗体偶联物的药物交联率(DAR)为3.0-5.0。其中，DAR(drug antibody ratio)指抗体偶联后一个抗体分子上携带的小分子药物的平均数量。
将获得的纯化的TPBG抗体药物偶联物分别用完全培养基进行梯度稀释，96孔细胞培养板以2000细胞/孔加入100微升TPBG阳性的NCI-H1299细胞株(购自ATCC，货号#CRL5803)细胞悬液过夜培养后，每孔分别加入10微升不同浓度的纯化的TPBG嵌合抗体药物偶联物的稀释液，继续培养5天后，用CellTiter-Glo试剂盒(购自Promega，使用方法参照产品说明书)检测细胞活力。结果如表13以及图13所示，其中表13的IC50指药物作用后，细胞的活性受到抑制的半数有效量，能够通过检测细胞的活性从而反映细胞杀伤活性。图13为纯化的TPBG嵌合抗体药物偶联物对TPBG阳性的肿瘤细胞系NCI-H1299的细胞杀伤活性检测，结果说明，纯化的TPBG抗体药物偶联物对TPBG阳性的细胞有杀伤作用。
表13细胞杀伤实验检测纯化的TPBG嵌合抗体药物偶联物对TPBG阳性NCI-H1299细胞的特异性杀伤作用
Figure PCTCN2016111699-appb-000021
实施例9嵌合抗体的体内药效实验
将NCI-H1299(非小细胞肺癌细胞株，ATCC，CRL-5803)(5X106个)200μl接种与Balb/c nude小鼠右肋皮下，待7-10天肿瘤长至200mm3后，去除体重、肿瘤过大和过小的，按肿瘤体积将小鼠随机分为几组，每组7只。D0开始尾静脉注射抗体，4天一次，共给药4次， 每周测2次瘤体积，称鼠重，记录数据。肿瘤体积(V)计算公式为：V＝1/2×a×b2；其中a、b分别表示长、宽。分组如表14。
表14 TPBG嵌合抗体及其抗体药物偶联物的体内药效实验
Figure PCTCN2016111699-appb-000022
Figure PCTCN2016111699-appb-000023
结果见图14：治疗后肿瘤的体积变化图，和图15：治疗后的鼠重变化图。其中，图14A-E分别是嵌合抗体12B12，嵌合抗体5G4，嵌合抗体39A11，嵌合抗体28D4，嵌合抗体36A10的抗体药物偶联物及其裸抗的治疗后的肿瘤体积变化图。图15A-E分别是嵌合抗体12B12，嵌合抗体5G4，嵌合抗体39A11，嵌合抗体28D4，嵌合抗体36A10的抗体药物偶联物及其裸抗的治疗后的小鼠体重变化图。结果显示，几种ADC可以很好的抑制肿瘤NCI-H1299的生长，而且对小鼠的体重没有显著的影响。
实施例10偶联不同连接子-毒素的抗体偶联物的体外药效实验
将实施例7所得的纯化的TPBG嵌合抗体12B12和28D4分别与MC-MMAF和MC-VC-PAB-MMAE进行偶联，方法同实施例8，经过pH 6.5～8.5的硼酸钠缓冲液透析后，加入三(2-羧乙基)膦(TCEP)，其中TCEP与纯化的TPBG抗体的摩尔比比率为2，室温下还原1小时，得反应液A。将反应液A经过G25柱脱盐(购自GE)，去除多余的TCEP，得反应液B。向反应液B中加入MC-MMAF或MC-VC-PAB-MMAE(购自南京联宁)，其中MC-MMAF或MC-VC-PAB-MMAE与纯化的TPBG抗体的摩尔比比率为5，室温下反应4小时。再加入半胱氨酸用以中和多余的MC-MMAF或MC-VC-PAB-MMAE，并通过G25柱脱盐除去多余的小分子，得到纯化的TPBG嵌合抗体药物偶联物——即表格中所述的嵌合抗体12B12-MMAF、12B12-MMAE、28D4-MMAF、28D4-MMAE(偶联方法参见Doronina，2006，Bioconjugate Chem.17，114-124)。通过HIC分析药物的交联率、通过SEC分析抗体药物偶联物的纯度等参数后，进行细胞毒活性的分析。
将实施例7所得的纯化的TPBG嵌合抗体12B12和28D4分别与SMCC进行偶联。将实施例7所得的纯化的TPBG嵌合抗体12B12和28D4经过pH 6.5～7.4的磷酸盐缓冲液透析后，在体积比为30％DMA(二甲基乙酰胺)存在下加入4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯(SMCC)，其中SMCC与纯化的TPBG嵌合抗体的摩尔比比率为8，室温下反应1小时，得反应液A。将反应液A经过G25柱脱盐(购自GE)，去除多余的小分子，得反应液B。向反应液B中加入终体积为10％DMA，然后加入DM1(化学名称为N2’-脱乙酰-N2’-3-巯基-1氧代丙基)-美登素)，其中DM1与纯化的TPBG抗体的摩尔比比率为9，室温下反应3.5小时，得反应液C。将反应液C经过G25柱脱盐(购自GE)，去除多余的小分子，得到纯化的TPBG嵌合抗体药物偶联物——即表格中所述的嵌合抗体12B12-DM1、28D4-DM1(偶联方法参见US5208020)。通过LC-MS分析药物的交联率、通过SEC分析抗体药物偶联物的纯度等参数后，进行细胞毒活性的分析，所有嵌合抗体偶联物的药物交联率(DAR)为3.0-5.0。其中，DAR(drug antibody ratio)指抗体偶联后一个抗体分子上携带的小分子药物的平均数量。
将获得的纯化的TPBG嵌合抗体药物偶联物分别用完全培养基进行梯度稀释，96孔细胞培养板以2000细胞/孔加入100微升TPBG阳性的NCI-H1299细胞株(购自ATCC，货号#CRL5803)，或NCI-H1568细胞株(购自ATCC，货号#CRL-5876)细胞悬液过夜培养后，每孔分别加入10微升不同浓度的纯化的TPBG嵌合抗体药物偶联物的稀释液，继续培养5天后，用CellTiter-Glo试剂盒(购自Promega，使用方法参照产品说明书)检测细胞活力。结果如表15以及图16所示，其中表15的IC50指药物作用后，细胞的活性受到抑制的半数有效量，能够通过检测细胞的活性从而反映细胞杀伤活性。其中，图16A和16B分别为纯化的TPBG嵌合抗体12B12及其偶联了不同的小分子药的抗体药物偶联物对TPBG阳性的肿瘤细胞系NCI-H1299的细胞杀伤活性检测；图16C和16D分别为纯化的TPBG嵌合抗体12B12及其偶联了不同的小分子药的抗体药物偶联物对TPBG阳性的肿瘤细胞系NCI-H1568的细胞杀伤活性检测。结果说明，偶联不同的小分子毒素的纯化的TPBG嵌合抗体药物偶联物对TPBG阳性的细胞均具有不同程度的杀伤作用，并且偶联了MC-MMAF的TPBG嵌合抗体药物偶联物具有较低的IC50，说明其细胞杀伤能力最强。
表15细胞杀伤实验检测纯化的TPBG嵌合抗体药物偶联物对TPBG阳性NCI-H1299，NCI-H1568细胞的特异性杀伤作用
Figure PCTCN2016111699-appb-000024
Figure PCTCN2016111699-appb-000025
将获得的纯化的TPBG嵌合抗体药物偶联物分别用完全培养基进行梯度稀释，96孔细胞培养板以2000细胞/孔加入100微升表达TPBG的293-hTPBG稳转细胞株(构建方法参见实施例1：免疫原B的制备)及不表达TPBG的293细胞株(购自ATCC，货号#CRL-1573)细胞悬液过夜培养后，每孔分别加入10微升不同浓度的纯化的TPBG嵌合抗体药物偶联物的稀释液，继续培养5天后，用CellTiter-Glo试剂盒(购自Promega，使用方法参照产品说明书)检测细胞活力。结果如表16以及图17所示，其中表16的IC50指药物作用后，细胞的活性受到抑制的半数有效量，能够通过检测细胞的活性从而反映细胞杀伤活性。图17A和17B为纯化的TPBG嵌合抗体药物偶联物对TPBG阳性的表达TPBG的293-hTPBG稳转细胞株的细胞杀伤活性检测；图17C和17D为纯化的TPBG嵌合抗体药物偶联物对不表达TPBG的293细胞株的细胞杀伤活性检测。结果说明，偶联不同的小分子毒素的纯化的TPBG嵌合抗体药物偶联物对TPBG阳性的细胞有不同程度的杀伤作用，对TPBG阴性的293细胞不具有细胞杀伤作用，说明纯化的TPBG嵌合抗体药物偶联物对TPBG阳性的细胞的杀伤是特异的，并且偶联了MC-MMAF的TPBG嵌合抗体药物偶联物具有较低的IC50，说明其细胞杀伤能力最强。
表16细胞杀伤实验检测纯化的TPBG嵌合抗体药物偶联物对TPBG阳性293-hTPBG和TPBG阴性的293细胞的特异性杀伤作用
Figure PCTCN2016111699-appb-000026
Figure PCTCN2016111699-appb-000027
应理解，在阅读了本发明的上述内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (24)

  1. 一种分离的蛋白质，其特征在于，其包括TPBG抗体的重链CDR1、重链CDR2和重链CDR3中的一种或多种，和/或，TPBG抗体的轻链CDR1、轻链CDR2和轻链CDR3中的一种或多种，
    所述重链CDR1的氨基酸序列如序列表中SEQ ID No.2、SEQ ID No.10、SEQ ID No.18、SEQ ID No.26、SEQ ID No.34、SEQ ID No.42、SEQ ID No.50、SEQ ID No.58、SEQ ID No.66或SEQ ID No.74所示；所述重链CDR2的氨基酸序列如序列表SEQ ID No.3、SEQ ID No.11、SEQ ID No.19、SEQ ID No.27、SEQ ID No.35、SEQ ID No.43、SEQ ID No.51、SEQ ID No.59、SEQ ID No.67或SEQ ID No.75所示；所述重链CDR3的氨基酸序列如序列表中SEQ ID No.4、SEQ ID No.12、SEQ ID No.20、SEQ ID No.28、SEQ ID No.36、SEQ ID No.44、SEQ ID No.52、SEQ ID No.60、SEQ ID No.68或SEQ ID No.76所示；
    所述轻链CDR1的氨基酸序列如序列表中SEQ ID No.6、SEQ ID No.14、SEQ ID No.22、SEQ ID No.30、SEQ ID No.38、SEQ ID No.46、SEQ ID No.54、SEQ ID No.62、SEQ ID No.70或SEQ ID No.78所示；所述轻链CDR2的氨基酸序列如序列表中SEQ ID No.7、SEQ ID No.15、SEQ ID No.23、SEQ ID No.31、SEQ ID No.39、SEQ ID No.47、SEQ ID No.55、SEQ ID No.63、SEQ ID No.71或SEQ ID No.79所示；所述轻链CDR3的氨基酸序列如序列表中SEQ ID No.8、SEQ ID No.16、SEQ ID No.24、SEQ ID No.32、SEQ ID No.40、SEQ ID No.48、SEQ ID No.56、SEQ ID No.64、SEQ ID No.72或SEQ ID No.80所示；
    或者，所述重链CDR1的氨基酸序列与如序列表中SEQ ID No.2、SEQ ID No.10、SEQ ID No.18、SEQ ID No.26、SEQ ID No.34、SEQ ID No.42、SEQ ID No.50、SEQ ID No.58、SEQ ID No.66或SEQ ID No.74所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述重链CDR2的氨基酸序列与如序列表中SEQ ID No.3、SEQ ID No.11、SEQ ID No.19、SEQ ID No.27、SEQ ID No.35、SEQ ID No.43、SEQ ID No.51、SEQ ID No.59、SEQ ID No.67或SEQ ID No.75所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述重链CDR3的氨基酸序列与如序列表中SEQ ID No.4、SEQ ID No.12、SEQ ID No.20、SEQ ID No.28、SEQ ID No.36、SEQ ID No.44、SEQ ID No.52、SEQ ID No.60、SEQ ID No.68或SEQ ID No.76所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；
    所述轻链CDR1的氨基酸序列与如序列表中SEQ ID No.6、SEQ ID No.14、SEQ ID No.22、SEQ ID No.30、SEQ ID No.38、SEQ ID No.46、SEQ ID No.54、SEQ ID No.62、SEQ ID No.70或SEQ ID No.78所示的氨基酸序列至少有80％的序列同源性的氨基酸序列 所示；所述轻链CDR2的氨基酸序列与如序列表中SEQ ID No.7、SEQ ID No.15、SEQ ID No.23、SEQ ID No.31、SEQ ID No.39、SEQ ID No.47、SEQ ID No.55、SEQ ID No.63、SEQ ID No.71或SEQ ID No.79所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示；所述轻链CDR3的氨基酸序列与如序列表中SEQ ID No.8、SEQ ID No.16、SEQ ID No.24、SEQ ID No.32、SEQ ID No.40、SEQ ID No.48、SEQ ID No.56、SEQ ID No.64、SEQ ID No.72或SEQ ID No.80所示的氨基酸序列至少有80％的序列同源性的氨基酸序列所示。
  2. 如权利要求1所述的蛋白质，其特征在于，所述重链CDR1的氨基酸序列如序列表SEQ ID No.2所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.3所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.4所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.10所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.11所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.12所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.18所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.19所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.20所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.26所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.27所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.28所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.34所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.35所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.36所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.42所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.43所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.44所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.50所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.51所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.52所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.58所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.59所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.60所示；所述重链CDR1的氨基酸序列如序列表SEQ ID No.66所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.67所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.68所示；或，所述重链CDR1的氨基酸序列如序列表SEQ ID No.74所示，所述重链CDR2的氨基酸序列如序列表SEQ ID No.75所示，且所述重链CDR3的氨基酸序列如序列表SEQ ID No.76所示；
    所述轻链CDR1的氨基酸序列如序列表SEQ ID No.6所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.7所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID  No.8所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.14所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.15所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.16所示；或，所述轻链CDR1的氨基酸序列如序列表SEQ ID No.22所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.23所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.24所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.30所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.31所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.32所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.38所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.39所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.40所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.46所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.47所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.48所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.54所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.55所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.56所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.62所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.63所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.64所示；所述轻链CDR1的氨基酸序列如序列表SEQ ID No.70所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.71所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.72所示；或，所述轻链CDR1的氨基酸序列如序列表SEQ ID No.78所示，所述轻链CDR2的氨基酸序列如序列表SEQ ID No.79所示，且所述轻链CDR3的氨基酸序列如序列表SEQ ID No.80所示。
  3. 一种分离的蛋白质，其特征在于，其包括TPBG抗体的重链可变区和/或TPBG抗体的轻链可变区，所述重链可变区的氨基酸序列如序列表中SEQ ID No.1、SEQ ID No.9、SEQ ID No.17、SEQ ID No.25、SEQ ID No.33、SEQ ID No.41、SEQ ID No.49、SEQ ID No.57、SEQ ID No.65或SEQ ID No.73所示；所述轻链可变区的氨基酸序列如序列表中SEQ ID No.5、SEQ ID No.13、SEQ ID No.21、SEQ ID No.29、SEQ ID No.37、SEQ ID No.45、SEQ ID No.53、SEQ ID No.61、SEQ ID No.69或SEQ ID No.77所示。
  4. 如权利要求3所述的蛋白质，其特征在于，所述重链可变区的氨基酸序列如序列表SEQ ID No.1所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.5所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.9所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.13所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.17所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.21所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.25所示，且所述轻链可变区的氨基酸序列如序列表SEQ  ID No.29所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.33所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.37所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.41所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.45所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.49所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.53所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.57所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.61所示；所述重链可变区的氨基酸序列如序列表SEQ ID No.65所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.69所示；或，所述重链可变区的氨基酸序列如序列表SEQ ID No.73所示，且所述轻链可变区的氨基酸序列如序列表SEQ ID No.77所示。
  5. 如权利要求1～4中至少一项所述的蛋白质，其特征在于，所述的蛋白质还包括抗体重链恒定区和/或抗体轻链恒定区。
  6. 如权利要求5所述的蛋白质，其特征在于，所述的抗体重链恒定区为人源或小鼠源抗体重链恒定区；所述的抗体轻链恒定区为人源或小鼠源抗体轻链恒定区。
  7. 如权利要求6所述的蛋白质，其特征在于，所述的抗体重链恒定区为人源抗体重链恒定区；所述的抗体轻链恒定区为人源抗体轻链恒定区。
  8. 如权利要求1或3所述的蛋白质，其特征在于，所述的蛋白质是TPBG抗体、单克隆抗体、抗体全长蛋白、抗原抗体结合域蛋白质片段、双特异性抗体、多特异性抗体、单链抗体、单域抗体或单区抗体。
  9. 一种核酸，其特征在于，其编码如权利要求1～7中至少一项所述的蛋白质。
  10. 如权利要求9所述的核酸，其特征在于，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.81、序列表SEQ ID No.83、序列表SEQ ID No.85、序列表SEQ ID No.87、序列表SEQ ID No.89、序列表SEQ ID No.91、序列表SEQ ID No.93、序列表SEQ ID No.95、序列表SEQ ID No.97或序列表SEQ ID No.99所示；和/或，编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.82、序列表SEQ ID No.84、序列表SEQ ID No.86、序列表SEQ ID No.88、序列表SEQ ID No.90、序列表SEQ ID No.92、序列表SEQ ID No.94、序列表SEQ ID No.96、序列表SEQ ID No.98或序列表SEQ ID No.100所示。
  11. 如权利要求10所述的核酸，其特征在于，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.81所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.82所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.83所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.84所示；编码所述 重链可变区的核酸的核苷酸序列如序列表SEQ ID No.85所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.86所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.87所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.88所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.89所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.90所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.91所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.92所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.93所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.94所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.95所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.96所示；编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.97所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.98所示；或，编码所述重链可变区的核酸的核苷酸序列如序列表SEQ ID No.99所示，且编码所述轻链可变区的核酸的核苷酸序列如序列表SEQ ID No.100所示。
  12. 一种包含如权利要求9～11中至少一项所述的核酸的重组表达载体。
  13. 一种包含如权利要求12所述的重组表达载体的重组表达转化体。
  14. 一种TPBG抗体的制备方法，其包括如下步骤：培养如权利要求13所述的重组表达转化体，从培养物中获得TPBG抗体。
  15. 一种免疫偶联物，其特征在于，其包括共价附着至细胞毒剂的、如权利要求1～7中至少一项所述的蛋白质。
  16. 如权利要求15所述的免疫偶联物，其特征在于，1当量如权利要求1～7中至少一项所述的蛋白质通过x当量接头与y当量细胞毒剂相连，其具有式1所示结构，
    Ab-(L)x-(D)y
    式1
    其中，Ab为如权利要求1～7中至少一项所述的蛋白质；L为接头；D为细胞毒剂；x为自然数，优选1-20的整数；y为0或自然数，优选0-20的整数；x和y各自独立地优选为1～2，或2～4，或4～8，或8～20的整数；x和y的比例优选为1∶1。
  17. 如权利要求16所述的免疫偶联物，其特征在于，所述接头L为活性酯、碳酸盐类、氨基甲酸酯类、亚胺磷酸酯、肟类、腙类、缩醛类、原酸酯类、氨基类、小肽段或核苷酸片段，较佳地，所述接头L为马来酰亚胺基己酰(MC)、马来酰亚胺基己酰-L-缬氨酸-L-瓜氨酸对氨基苄醇(MC-VC-PAB)或4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥 珀酰亚胺酯(SMCC)；
    和/或，所述D选自细胞毒素、化学治疗剂、放射性同位素、治疗性核酸、免疫调节剂、抗血管生成剂、抗增殖促凋亡剂或细胞溶解酶，较佳地，选自甲基奥瑞他汀E、甲基奥瑞他汀F或N2’-脱乙酰-N2’-3-巯基-1氧代丙基-美登素。
  18. 如权利要求17所述的免疫偶联物，其特征在于，所述式1中x＝y＝n，所述免疫偶联物的结构如式3、如式4或者如式5所示，
    Figure PCTCN2016111699-appb-100001
    式3中m为1～10，优选m为5(即L为马来酰亚胺基己酰)；D为甲基奥瑞他汀F；
    Figure PCTCN2016111699-appb-100002
    式4中，L为4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯；D为N2’-脱乙酰-N2’-3-巯基-1氧代丙基)-美登素(DM1)；
    Figure PCTCN2016111699-appb-100003
    Figure PCTCN2016111699-appb-100004
    式5中，L为马来酰亚胺基己酰-L-缬氨酸-L-瓜氨酸对氨基苄醇，D为甲基奥瑞他汀E(MMAE)；
    其中，n为自然数，优选为1～20的整数，更优选为1～2，或2～4，或4～8，或8～20的整数。
  19. 一种药物组合物，其特征在于，其包括如权利要求15～18中至少一项所述的免疫偶联物和药学可接受的载体。
  20. 如权利要求19所述的药物组合物，其特征在于，所述的药物组合物包括0.01～99.99％的如权利要求1～7中至少一项所述的蛋白质和0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。
  21. 一种如权利要求1～7中至少一项所述的蛋白质在制备抗肿瘤药物中的应用。
  22. 一种如权利要求15～18中至少一项所述的免疫偶联物在制备抗肿瘤药物中的应用。
  23. 一种如权利要求19～20中至少一项所述的药物组合物在制备抗肿瘤药物中的应用。
  24. 一种检测过表达TPBG蛋白的细胞的方法，其特征在于，包括如下的步骤：如权利要求1～7中至少一项所述的蛋白质与待检样品在体外接触，检测如权利要求1～7中至少一项所述的蛋白质与所述待检样品的结合即可。
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