WO2008128455A1 - Épitopes fonctionnels d'ostéopontine, anticorps monoclonaux contre ces épitopes et leurs utilisations - Google Patents

Épitopes fonctionnels d'ostéopontine, anticorps monoclonaux contre ces épitopes et leurs utilisations Download PDF

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WO2008128455A1
WO2008128455A1 PCT/CN2008/070576 CN2008070576W WO2008128455A1 WO 2008128455 A1 WO2008128455 A1 WO 2008128455A1 CN 2008070576 W CN2008070576 W CN 2008070576W WO 2008128455 A1 WO2008128455 A1 WO 2008128455A1
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antibody
monoclonal antibody
osteopontin
opn
cell
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PCT/CN2008/070576
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English (en)
French (fr)
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Yajun Guo
Jianxin Dai
Hao Wang
Geng Kou
Sheng Hou
Weizhu Qian
Ling Peng
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Shanghai Cp Guojian Pharmaceutical Co. Ltd
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Priority to BRPI0810826A priority Critical patent/BRPI0810826A2/pt
Priority to EP08715312A priority patent/EP2149582A4/en
Priority to US12/597,418 priority patent/US20100151486A1/en
Priority to CN2008800134403A priority patent/CN101679485B/zh
Priority to JP2010504425A priority patent/JP2010525795A/ja
Priority to CA002685182A priority patent/CA2685182A1/en
Publication of WO2008128455A1 publication Critical patent/WO2008128455A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • 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
    • A61K47/6845—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 cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • 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
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1021—Tetrapeptides with the first amino acid being acidic
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70546—Integrin superfamily, e.g. VLAs, leuCAM, GPIIb/GPIIIa, LPAM

Definitions

  • osteopontin a functional epitope of osteopontin, a single gram of the body
  • the present invention is in the field of biotechnology, and more particularly, the present invention discloses a functional epitope of a protein, a monoclonal antibody that specifically binds to this epitope, and its use in the preparation of an antitumor drug. Background technique
  • Tumors are serious diseases that seriously endanger the health of our people. After 5 years of surgical resection, the survival rate can reach 40%, but about half of the patients still have metastasis and recurrence after surgery. How to control the high rate of recurrence after tumor resection to improve the efficacy of cancer patients is a key research topic in international medical research. In-depth study of the mechanism of tumor cell metastasis can help elucidate the molecular mechanism of tumor metastasis and recurrence, understand the signaling pathway that promotes metastasis, find effective links to inhibit metastasis, and provide more effective blocking targets for new drug development and clinical treatment. Survival rate of cancer patients.
  • TGF ⁇ metastatic growth factor a
  • EGF-R epidermal growth factor Receptor
  • MMP-2 matrix metalloproteinase-2
  • uP A urokinase-type plasminogen activator
  • PAI plasminogen activator inhibitor-1
  • ICM-1 intercellular adhesion molecule-1
  • VEGF vascular endothelial growth factor
  • PD-ECGF platelet-derived endothelial growth factor
  • Osteopontin plays a crucial role in tumor cell metastasis (Rangaswami H, Bulbule A, Kundu GC. Osteopontin: role in cell signaling and cancer progression. " Osteopontin Role in cell signal transduction and cancer development" Trends Cell Biol. 2006 February; 16(2): 79-87.)
  • OPN-promoting signaling pathways With the joint efforts of national research groups, the field of OPN-promoting signaling pathways continues to be new. It is reported that the main aspects of promoting tumor cell metastasis function are explained from different angles. The important regulatory role of OPN signaling pathway in promoting tumor cell metastasis has become a research hotspot in the field of international tumor metastasis research.
  • Osteopontin is expressed as an important tumor-promoting glycoprotein signaling molecule in bone tissue, kidney tissue, brain tissue, glandular epithelial cells, vascular smooth muscle cells, activated macrophages, lymphocytes and tumor cells ( Weber GF, Ashkar S, Glimcher MJ, Cantor H. Receptor-ligand interaction between CD44 and osteopontin (Eta-1). "Receptor-ligand interaction between CD44 and osteopontin (Eta-1)" Science (Washington , DC) 1996; 271: 509-12.). In the extracellular matrix of tumor tissue, OPN can activate CD44 on the surface of tumor cells (Miyauchi A, Alvarez J, Greenfield EM, etc.
  • osteopontin and related peptides Recognition of osteopontin and related peptides by an ⁇ v ⁇ 3 integrin stimulates immediate cell signals in osteoclasts.
  • ⁇ ⁇ ⁇ 3 integrin-recognized osteopontin and related peptides stimulate cell signaling in osteoclasts "J Biol Chem 1991; 266:20369 - 7) and integrins (Teramoto H, Castellone MD, Malek RL, Letwin N, Frank B, Gutkind JS, Lee NH.
  • OPN signal is recognized by receptor CD44 and causes tumor cell Rho family small G protein such as Rac activation (Teramoto
  • small G protein conveys extracellular chemotaxis Downstream effector proteins such as the WASP (Wiskott-Aldrich syndrome protein) family member, WASP protein binds to and activates the actin-related protein (Arp2/3) complex, which catalyzes actin (Actin) polymerization, which induces tumor cytoskeletal remodeling and cell membrane protrusion formation, and enhances cell migration ability (Wolf K, Mazo I, Leung H et al. Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of Pericellular proteolysis.
  • WASP Wikott-Aldrich syndrome protein
  • OPN-CD44 downstream pathway can activate PI-3K, and one of the target molecules of PI-3K is Akt kinase.
  • Akt kinase regulates cell cycle, promotes cell survival, and cells anchor non- Dependent growth and cell migration mediate OPN-promoting tumor anti-apoptosis and cell migration (Lin, YH and Yang-Yen, HF (2001)
  • the osteopontin-CD44 survival signal initiation of the phosphatidylinositol 3 -kinase/ Akt signaling pathway.
  • the osteopontin-CD44 survival signal is involved in the activation of the phosphatidylinositol 3-kinase/Akt signal transduction pathway" J.
  • JNK1 differentiation regulates osteopontin-induced nuclear factor-directed kinase/MEKK1-dependent activation protein-1 mediated activation of pro-matrix metalloproteinase-9 J. Biol. Chem. 280 , 19381 - 193912.
  • OPN secreted by tumor cells can promote the expression of vascular endothelial growth factor VEGF through autocrine and paracrine pathways, thereby promoting the proliferation of vascular endothelial cells and the formation of capillaries inside the tumor.
  • Osteopontin Promotes Vascular Endothelial Growth Factor - Dependent Breast Tumor Growth and Angiogenesis via Autocrine and Paracrine Mechani sms .
  • Osteopontin promotes vascular endothelial growth factor-dependent breast tumor growth through autocrine and paracrine mechanisms Angiogenesis” Cancer Res 2008; 68: 152-161).
  • the function of the OPN signaling pathway plays a role in the various regulatory functions of tumor metastasis cells that infiltrate the extracellular matrix, diffuse into the peripheral tissues and organs through the blood or lymphatic circulation, and form metastases. .
  • Blocking the pro-metastatic signaling pathway of OPN by anti-OPN antibody may effectively block the adhesion and migration process of tumor cells, prevent the invasion of tumor cells into the cell matrix, inhibit the formation of tumor capillaries, and thus prevent the development of tumors. Transfer. Summary of the invention
  • the object of the present invention is to provide a monoclonal antibody against a specific functional epitope of osteopontin, thereby achieving the effect of treating tumors.
  • the functional epitope is NAPS. In another preferred embodiment, the functional epitope is located at exon 7 exon 212-215aa of osteopontin.
  • a monoclonal antibody against osteopontin that specifically binds to said functional epitope is provided.
  • the CDR amino acid sequence of the heavy chain variable region of the monoclonal antibody is selected from the group consisting of: GYTFTTYVMH, YINPYNDGSKYNEKFKG and P HYGGSPAY (see, for example, HlA12VHb of Figure 4); CDR amino acids of the light chain variable region
  • the sequences are respectively selected from: RSSQSLVHSNGNTYLH, KVSNRFS and P SQSTHVPWT (see, for example, HlA12VLb of FIG. 4;).
  • the heavy chain variable region amino acid sequence of the monoclonal antibody is selected from the group consisting of: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO:
  • the light chain variable region amino acid sequence is selected from the group consisting of: SEQ ID NO: 6 or SEQ ID NO: 21.
  • the constant region of the monoclonal antibody is selected from the group consisting of: a mouse antibody constant region or a human antibody constant region.
  • the constant region is mouse IgG.
  • the monoclonal antibody is produced by a hybridoma method or a recombinant DNA method, or is isolated from a phage antibody library. In another preferred embodiment, the monoclonal antibody is a chimeric antibody or a humanized antibody.
  • a DNA molecule encoding a monoclonal antibody of the invention.
  • the nucleotide sequence encoding the heavy chain variable region in the DNA molecule is SEQ ID NO: 3 or SEQ ID NO: 18; and the nucleotide sequence encoding the light chain variable region is SEQ ID NO: 5 or SEQ ID NO: 20.
  • a vector comprising the above DNA molecule of the invention.
  • a host cell comprising the vector of the invention, or a DNA molecule of the invention integrated in the genome.
  • the host cell is a prokaryotic cell, preferably a bacterial cell; a lower eukaryotic cell, preferably a yeast cell; or a higher eukaryotic cell, preferably a mammalian cell.
  • an immunoconjugate comprising: (a) a monoclonal antibody of the invention; and (b) a coupling moiety selected from the group consisting of: a drug, a toxin, a cytokine , radionuclides, or enzymes.
  • a monoclonal antibody or immunoconjugate of an anti-osteopontin of the invention for the preparation of an anti-tumor drug.
  • the tumor is selected from the group consisting of: adenocarcinoma, leukemia, lymphoma, melanoma, sarcoma; selected from the group consisting of adrenal gland, gallbladder, bone, bone marrow, brain, breast, bile duct, gastrointestinal tract, heart, kidney , liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, skin, salivary gland, spleen, testis, thymus, thyroid or uterus tumor tissue; central nervous system tumor; ocular tumor, endocrine gland tumor, nerve Endocrine system tumors, gastrointestinal pancreatic endocrine system tumors, reproductive system tumors or head and neck tumors.
  • adenocarcinoma leukemia, lymphoma, melanoma
  • sarcoma selected from the group consisting of adrenal gland, gallbladder, bone, bone marrow, brain, breast, bile duct, gastrointestinal tract, heart, kidney ,
  • the central nervous system tumor is selected from the group consisting of: a glial cell tumor or astrocytoma.
  • the ocular tumor is selected from the group consisting of: basal cell carcinoma, squamous cell carcinoma or melanoma.
  • a pharmaceutical composition comprising a monoclonal antibody or immunoconjugate of an anti-osteopontin of the invention; and a pharmaceutically acceptable carrier are provided.
  • the pharmaceutical composition contains 0.00001 to 99.9 wt%; preferably 0.0001 to 90 wt%, more preferably 0.001 to 75 wt%, more preferably
  • the pharmaceutical composition further comprises other anti-tumor actives selected from the group consisting of: TNF-0
  • a kit for detecting osteopontin comprising: A monoclonal antibody or immunoconjugate of an anti-osteopontin of the invention.
  • a method for detecting the presence or amount of osteopontin in a biological sample comprising the steps of: (i) administering a sample to be tested and an anti-osteopontin of the invention The monoclonal antibody or immunoconjugate is contacted; (ii) detecting whether an antigen-antibody complex is formed, wherein the formation of the antigen-antibody complex means that osteopontin is present in the sample, or the antigen-antibody complex formed by quantitative detection is formed.
  • the amount of the substance reflects the amount of osteopontin in the sample.
  • the sample may be pretreated or untreated, preferably subjected to extraction, purification or concentration.
  • Figure 1 SDS-PAGE electropherogram of human and mouse OPN eukaryotic expression and purification; M represents the standard molecular weight of protein.
  • Figure 2 Western blot assay results for murine anti-hOPN mAb 1A12.
  • Figure 3 Schematic diagram of the molecular mimic structure of humanized antibody MA12; FR region residues are represented by dark gray bands, CDR region residues are represented by light gray bands, and 9 key mouse sources located within 5 A distance around CDR regions Residues in the FR region are indicated by black bats.
  • Figure 4 Alignment of the amino acid sequence of the heavy chain (Figure 4A) and light chain ( Figure 4B) of the humanized antibody hlA12 with related sequences.
  • 1A12VH and 1A12VL represent the variable regions of the heavy chain and the light chain of the murine monoclonal antibody 1A12, respectively; the heavy chain variable region of the human antibody CAA79298. 1 and the light chain variable region of the human antibody BAC01734.
  • hlA12VHa and PhlA12VHb represent different humanized antibody heavy chain variable regions
  • hlA12VLa and hlA12VLb represent different humanized antibody light chain variable regions, respectively;
  • the amino acid identical to the residue corresponding to the human antibody CAA79298.1 or BAC01734.1, the CDR region is indicated in parentheses; the amino acid is numbered according to the Kabat numbering method [EA Kabat, TT Wu, HM Perry, KS Gottesman, C. Foeller, Sequences of Proteins of Immunological Interest, Fifth ed., United States Department of Health and Human Services, Bethesda, MD, 1991.].
  • FIG. 5 Experimental results of antigen binding activity of humanized antibody 1A12.
  • Figure 6 Mouse anti-hOPN mAb 1A12, chimeric antibody C1 A12 and humanized antibody hlA12 blocks tumor cell adhesion.
  • Figure 7 Mouse anti-hOPN mAb 1A12, chimeric antibody C1 A12 and humanized antibody hlA12 inhibits tumor cell invasion of the basement membrane.
  • Figure 8 Murine anti-hOPN mAb 1A12, chimeric antibody C1 A12 and humanized antibody hlA12 inhibits the repair of damaged scratches by tumor cells.
  • Figure 9 Murine anti-hOPN mAb 1A12, chimeric antibody C1 A12 and humanized antibody hlA12 inhibits the formation of tumor cells on soft agar.
  • Figure 10 Effect of murine anti-hOPN mAb 1A12, chimeric antibody clA12 and humanized antibody MA12 on HUVEC cell proliferation.
  • Figure 1 1 Effect of murine anti-hOPN monoclonal antibody 1A12, chimeric antibody clA12 and humanized antibody hlA12 on the formation of capillary tube-like structures in HUVEC.
  • Figure 12 Inhibition of CAM angiogenesis in chicken embryo chorioallantoic membrane by murine anti-hOPN monoclonal antibody 1A12, chimeric antibody C 1 A12 and humanized antibody hlA12.
  • the bars in the figure are PBS, VEGF, left to right, respectively.
  • Figure 13 Effect of murine anti-hOPN mAb 1A12, chimeric antibody C 1 A12 and humanized antibody hlA12 on OPN-induced corneal neovascularization.
  • Figure 14 Inhibition of tumor growth in mice by anti-hOPN monoclonal antibody 1A12.
  • Figure 15 Inhibition of mouse anti-hOPN monoclonal antibody 1A12 on lung metastasis in mice.
  • Figure 16 Effect of murine anti-hOPN mAb 1A12 on vascular density in breast cancer tumor tissues in mice.
  • Figure 17 Comparison of output efficiency after three rounds of anti-OPN mAb 1A12 panning.
  • Figure 18 Anti-OPN mAb 1A12 positive phage ELISA and Western blot identification map
  • Figure 18A is a phage ELISA identification map
  • Figure 18B is a 1A12 positive phage ELISA and Western blot identification map.
  • Figure 19 Align X software sequence analysis of the binding epitope results for anti-OPN mAb 1A12.
  • Figure 20 Comparison of the binding of various sequence epitopes to anti-OPN mAb 1A12.
  • the present inventors After long-term and intensive research, the present inventors obtained monoclonal antibodies against specific functions of osteopontin, and further prepared chimeric monoclonal antibodies and humanized antibodies and clarified the coding sequences thereof. The present inventors have also demonstrated that the monoclonal antibody of the present invention has an inhibitory effect on tumor migration and can be used for the treatment of tumors. On the basis of this, the inventors completed the present invention.
  • the present inventors first cloned human and murine OPN genes by molecular biotechnology, expressed human and murine OPN proteins by eukaryotic cells, and prepared mouse anti-human OPN monoclonal by cell fusion-hybridoma method.
  • Antibody 1A12 the gene of this monoclonal antibody was also cloned and its sequence was determined.
  • the present invention further discloses a method of producing a humanized antibody against osteopontin, which comprises computer-aided design of the amino acid sequence of the humanized antibody MA12.
  • the expression vector is then co-transfected into CHO cells by liposome method, and then screened, cultured and purified.
  • the chimeric monoclonal antibody C1A12 can be obtained by the same method.
  • the inventors conducted a series of experiments using the breast cancer cell line MDA-MB-435 S to verify the inhibition of tumor metastasis by the mouse anti-human OPN monoclonal antibody 1A12, human mouse chimeric antibody C1A12 and humanized antibody hLA12 disclosed in the present invention. effect.
  • the results of cell adherence assay showed that anti-hOPN antibody can effectively block the binding of MDA-MB-435 S cells and hOPN.
  • the results of cell invasion assay showed that anti-hOPN antibody can effectively block the substrate of MDA-MB-435 S in the presence of hOPN.
  • the present inventors also conducted a series of experiments using human vascular endothelial cell HUVEC to verify the inhibitory effect of the mouse anti-human OPN monoclonal antibody 1A12, human mouse chimeric antibody C1A12 and humanized antibody hlA12 on tumor angiogenesis. 3 H incorporation experiments showed that anti-hOPN antibody can effectively inhibit the proliferation of endothelial cells.
  • anti-hOPN antibody can inhibit the formation of blood vessels in vitro by vascular endothelial cells, chicken chorioallantoic membrane (CAM) angiogenesis experiment and rabbit
  • the corneal neovascularization experiment confirmed that the anti-hOPN antibody can inhibit the formation of capillaries in vivo; accordingly, the unrelated antibody as a control has no such effect, thereby indicating that the above anti-hOPN antibody disclosed in the present invention can effectively inhibit the formation of tumor blood vessels.
  • the present inventors also established a mouse model of primary tumor and breast cancer lung metastasis of mouse breast cancer using a breast cancer cell line MDA-MB-435 S, and verified the anti-OPN monoclonal on the model.
  • Antibody 1A12 inhibits tumor progression, inhibits tumor metastasis, and inhibits tumor angiogenesis. Accordingly, the control antibody does not have the above-described effects, thereby demonstrating that the anti-hOPN antibody 1A12 disclosed in the present invention also has an effect of inhibiting tumor development, inhibiting tumor angiogenesis, and blocking tumor metastasis in vivo.
  • the present inventors further identified a functional epitope of OPN against the action of hOPN antibody 1A12 by phage display technology, and this functional epitope is NAPS, further elucidating the target of OPN molecule. More specifically, the applicant of the present invention presupposes the functional epitope of OPN by panning, phage cloning ELISA and Western blotting of monoclonal antibody epitopes, and further selects the functional epitope of OPN by phage cloning and antibody binding ability analysis. A series of short peptides were synthesized by the most binding clones, and the epitopes were identified by binding experiments of these short peptides with specific antibodies. Monoclonal antibody of the invention and preparation thereof
  • mAb monoclonal antibody
  • monoclonal antibody refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical except for a few naturally occurring mutations that may be present. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (typically having different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are also advantageous in that they are synthesized by hybridoma culture and are not contaminated by other immunoglobulins.
  • the modifier "monoclonal” indicates the identity of the antibody and is obtained from a substantially homogeneous population of antibodies, which should not be construed as requiring any particular method to produce the antibody.
  • antibody or "immunoglobulin” is an isotetrameric glycoprotein of about 150,000 daltons having the same structural features, consisting of two identical light chains (L) and two identical heavy chains.
  • H Composition. Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has a regularly spaced intrachain disulfide bond. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions.
  • VH variable region
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain .
  • VL variable region
  • a particular amino acid residue forms an interface between the variable regions of the light and heavy chains.
  • variable means that certain portions of the variable regions of an antibody differ in sequence, and they form the binding and specificity of various specific antibodies for their particular antigen. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments in the variable region of the light and heavy chains called the complementarity determining region (CDR) or hypervariable region. The more conservative part of the variable region is called the framework region (FR).
  • CDR complementarity determining region
  • FR framework region
  • the variable regions of the native heavy and light chains each comprise four FR regions which are substantially in a ⁇ -sheet configuration and are joined by three CDRs forming a linker, in some cases forming a partial b-fold structure.
  • the CDRs in each chain are closely joined together by the FR region and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH Publ. No. 91-3242, Vol. I, pp. 647-669). (1991)).
  • the constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as antibody-dependent cytotoxicity of the antibodies involved.
  • the "light chain" of a vertebrate antibody can be classified into one of two distinct classes (called ⁇ and ⁇ ) according to the amino acid sequence of its constant region. Immunoglobulins can be classified into different classes based on the amino acid sequence of the heavy chain constant region. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2.
  • the heavy chain constant regions corresponding to different classes of immunoglobulins are referred to as (3 ⁇ 4, ⁇ , ⁇ , ⁇ , and .)
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
  • the heavy chain variable region amino acid sequence of the monoclonal antibody of the present invention is preferably selected from the group consisting of: SEQ ID NO: 4 or SEQ ID NO: 19, and the light chain variable region amino acid sequence is preferably selected from the group consisting of: SEQ ID NO: 6 or SEQ ID NO: 21, and the constant region may be selected from a mouse antibody constant region or a human antibody constant region, such as mouse IgG.
  • the CDR1-3 amino acid region sequences in the heavy chain variable region are each selected from:
  • the CDR1-3 amino acid sequences of the light chain variable region are respectively selected from: RSSQSLVHSNGNTYLH, KVSNRFS and P SQSTHVPWT (see HlA12VLb of Figure 4).
  • Monoclonal antibodies can be made by a variety of methods well known to those skilled in the art. For example, monoclonal antibodies can be made by the hybridoma method (first proposed by Kohler et al, Nature, 256:495 (1975)) or by recombinant DNA methods (U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques such as those described by Clackson et al, Nature, 352:624-628 (1991) and Marks et al, J. Mol. Biol., 222:581-597 (1991;). obtain.
  • the monoclonal antibody of the present invention may also be a chimeric antibody or a humanized antibody.
  • “1A12” means mouse anti-hOPN mAb 1A12
  • " cl A12” means human mouse chimeric anti-hOPN mAb cl A12
  • "hlA12” means humanized anti-hOPN mAb hlA12 unless otherwise specified.
  • the heavy chain variable region amino acid sequence of clA12 is SEQ ID N0.4, the light chain variable region amino acid sequence is SEQ ID NO. 6, and the constant region is human antibody constant region; heavy chain of hlA12
  • the variable region amino acid sequence is SEQ ID N0.19, the light chain variable region amino acid sequence is SEQ ID N0.21, and the constant region is a human antibody constant region.
  • the present invention also includes a monoclonal antibody having the corresponding amino acid sequence of the anti-hOPN monoclonal antibody, a monoclonal antibody having the variable region chain of the anti-hOPN monoclonal antibody, and other proteins or protein pairs having these chains Linked and fusion expression products.
  • the invention encompasses any protein or protein conjugate having a light chain and a heavy chain comprising a hypervariable region (complementarity determining region, CDR) and a fusion expression product (ie, an immunoconjugate and a fusion expression product), as long as The hypervariable regions are identical or at least 90% homologous, preferably at least 95% homologous to the hypervariable regions of the light and heavy chains of the invention.
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokine radionuclides, enzymes and other diagnostic or therapeutic molecules and said anti-hOPN monoclonal antibodies or fragments thereof A conjugate formed by binding.
  • the present invention also encompasses cell surface markers or antigens that bind to said anti-hOPN monoclonal antibodies or fragments thereof.
  • the invention encompasses not only intact monoclonal antibodies, but also immunologically active antibody fragments, such as Fab or (Fab') 2 fragments; antibody heavy chains; antibody light chains.
  • the invention also provides a DNA molecule encoding the anti-hOPN monoclonal antibody or fragment thereof.
  • the sequences of these DNA molecules can be obtained by conventional techniques such as PCR amplification or genomic library screening.
  • the coding sequences of the light and heavy chains can be fused together to form a single chain antibody.
  • a DNA molecule of the invention may comprise a nucleotide sequence encoding a heavy chain variable region of SEQ ID NO: 3 or SEQ ID NO: 18; a nucleotide sequence encoding a light chain variable region of SEQ ID NO 5 or SEQ ID NO: 20.
  • the nucleotide sequence encoding the clA12 heavy chain variable region is set forth in SEQ ID N0.3
  • the light chain variable region amino acid sequence is set forth in SEQ ID N0.5
  • the nucleotide encoding the hLA12 heavy chain variable region The sequence is as shown in SEQ ID NO. 18, and the light chain variable region amino acid sequence is shown in SEQ ID NO. 20
  • the present invention also discloses the expression vector containing the above nucleotide sequence, which is pcDNA3.1/ZEC +) and pcDNA3 .1 (+);
  • the present invention also discloses that the host cell transformed by the above expression vector is COS, CHO cells.
  • the recombination method can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then connecting them.
  • the DNA sequence encoding the anti-hOPN monoclonal antibody of the present invention can be obtained completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequence of the invention by chemical synthesis.
  • the invention also relates to vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • the present invention also provides a hybridoma cell line which produces the anti-hOPN monoclonal antibody of the present invention; preferably, the present invention provides a high titer hybridoma cell line against the hOPN monoclonal antibody.
  • an expression vector comprising a nucleotide sequence encoding a monoclonal antibody of the present invention and an expression control sequence operably linked to the sequence is provided.
  • expression control sequence generally refers to a sequence involved in the control of expression of a nucleotide sequence.
  • Expression control sequences include promoter and termination signals operably linked to a nucleotide sequence of interest. They usually also include the sequences required for proper translation of the nucleotide sequence. "Operatively linked" means that certain portions of a linear DNA sequence are capable of affecting the activity of other portions of the same linear DNA sequence. For example, if a promoter or enhancer increases the transcription of a coding sequence, It is operatively linked to the coding sequence.
  • the DNA sequence encoding the monoclonal antibody of the present invention can be produced by conventional means well known to those skilled in the art.
  • a nucleotide sequence encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody can be artificially synthesized according to the sequence disclosed in the present invention or amplified by PCR.
  • These nucleotide sequences are then inserted into the appropriate expression vector by selection of appropriate cleavage sites by various methods well known in the art, such that they are constant in the heavy chain constant region coding sequence and light chain carried by the expression vector, respectively.
  • the regions are encoded before the sequences and are placed in the same reading frame.
  • the expression vectors used in the present invention are various commercially available expression vectors known to those skilled in the art, such as pPICZo pPIC9K.
  • “Host cells” generally include prokaryotic cells and eukaryotic cells. Examples of commonly used prokaryotic host cells include Escherichia coli, Bacillus subtilis and the like. Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells. In the present invention, mammalian cells are preferred. Mammalian cell lines are commonly used as host cells for expression of eukaryotic cell-derived polypeptides. The propagation of mammalian cells in culture is well known in the art. See Tissue Culture, Academic Press, Kruse and Patterson
  • Preferred mammalian cells are a number of commercially available immortalized cell lines. These immortalized cell lines include, but are not limited to, Chinese hamster ovary (CHO) cells, Vero cells, Hela HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocyte cancer cells (eg Hep) G2) and many other cell lines. They provide post-translational modifications to protein molecules, including correct folding, proper disulfide bond formation, and glycosylation at the correct site.
  • CHO Chinese hamster ovary
  • Vero cells Vero cells
  • Hela HeLa cells Hela HeLa cells
  • BHK baby hamster kidney
  • COS monkey kidney cells
  • Hep human hepatocyte cancer cells
  • heterologous polynucleotides into mammalian cells include dextran-mediated transfection, calcium phosphate precipitation, Polybr ene (l, 5- dimethyl-1,5 Diazo-monomethylene poly(methane bromide) mediates transfection, protoplast fusion, electroporation, liposome-mediated transfection, and direct microinjection of DNA into the nucleus.
  • preferred methods are electroporation or liposome-mediated methods and the like.
  • Invitrogen's liposome assay kit can be used to transfect host cells such as COS, CHO cells, and the like.
  • the resulting host cell is then cultured under conditions suitable for expression of the monoclonal antibody of the invention. Then use conventional immunoglobulin purification steps, such as protein A-Sephar 0Se , hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography.
  • the human anti-HOPN monoclonal antibody of the present invention is purified by conventional separation and purification means well known to the skilled person. Identification, expression and purification of monoclonal antibodies
  • the resulting monoclonal antibodies can be identified by conventional means.
  • the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or an in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the binding affinity of a monoclonal antibody can be determined, for example, by Scatchard analysis by Munson et al, Anal. Biochem., 107: 220 (1980).
  • the anti-hOPN monoclonal antibodies of the invention can be expressed intracellularly, or on the cell membrane, or secreted extracellularly.
  • the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting method;), centrifugation, osmotic sterilizing, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption Chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • Pharmaceutical composition include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting method;), centrifugation, osmotic sterilizing, sonication, ultracentrifugation, molecular sieve chromatography (
  • the present invention also provides a pharmaceutical composition for treating a tumor, which comprises a pharmaceutically effective amount of the monoclonal antibody of the present invention or an immunoconjugate thereof, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means that when the molecular body and composition are suitably administered to an animal or a human, they do not produce an adverse, allergic or other untoward reaction.
  • pharmaceutically acceptable carrier should be compatible with the active substance of the present invention, i.e., it can be blended therewith without substantially reducing the drug combination under normal circumstances. The effect of the object.
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
  • composition of the present invention can be administered orally as well as intravenously, intramuscularly or subcutaneously; preferably, it is administered orally or intravenously.
  • the active ingredient of the monoclonal antibody or immunoconjugate of the present invention is 0.00001 to 99.9% by weight based on the total weight of the composition; preferably 0.0001 to 90% by weight, more preferably 0.001 to 75% by weight, more preferably
  • the balance is a pharmaceutically acceptable carrier and other additives.
  • the pharmaceutical composition of the present invention can be formulated into various dosage forms as needed, and can be administered by a physician in accordance with factors such as the type of patient, age, body weight, and general disease state, mode of administration, and the like.
  • the mode of administration can be, for example, perfusion and other treatments.
  • a safe and effective amount of an anti-hOPN monoclonal antibody or immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually from about 0.1 microgram to 5 milligrams per kilogram of body weight, and in most cases is not More than about 3 mg/kg body weight, preferably about 1-10 micrograms per kilogram body weight - about 1 mg/kg body weight.
  • specific doses should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the monoclonal antibody or immunoconjugate of the present invention can inhibit adhesion and migration of tumors, prevent invasion of tumor cells to a mechanism, and promote apoptosis of tumor cells, and thus the active substance of the present invention can be used for treating many kinds of tumors.
  • Tumors such as (including but not limited to, adenocarcinoma, leukemia, lymphoma, melanoma, sarcoma; from the adrenal gland, gallbladder, bone, bone marrow, brain, breast, bile duct, gastrointestinal tract, heart, kidney, liver, lung, muscle Tumor tissue of the ovary, pancreas, parathyroid gland, penis, prostate, skin, salivary gland, spleen, testis, thymus, thyroid or uterus; tumors of the central nervous system, such as glial cell tumors, or astrocytoma; Tumors (eg basal cell carcinoma, squamous cell carcinoma or melanoma;), endocrine gland tumors, neuroendocrine system tumors, gastrointestinal pancreatic endocrine system tumors, reproductive system tumors or head and neck tumors.
  • Tumors eg basal cell carcinoma, squamous cell carcinoma or melanoma;
  • the pharmaceutical composition of the present invention may further comprise other antitumor active substances, or may be used in combination with other antitumor active substances to obtain a better therapeutic effect.
  • the other anti-tumor active substances include, but are not limited to: TNF-0 TGF- ⁇ , IFN-o vasplatin, endostatin, glycophosphorus mustard, hematoporphyrin, lysine betaine, brucea javanica, Etoposide, dehydrated dulcitol, doxorubicin, tamoxifen, 5-fluorouracil, norcantharidin, difuran fluorouracil, cucurbitacin, harringtonine, oridonin, scorpion A, cloud Zhizhi peptide, cytarabine, carbopol, paclitaxel, lentinan, flutamide, ifosfamide, umbrel, leuprolide, deoxyfluorouridine, lopoplatin, ilino Tecan, letrozole or ten
  • hOPN test kit When two or more drugs are administered in combination, they generally have an effect superior to that of the two drugs alone.
  • the co-administered drug or other formulation does not interfere with the therapeutic activity of the monoclonal antibodies of the invention.
  • the present invention also provides a kit for detecting hOPN comprising the anti-hOPN monoclonal antibody or an active fragment thereof, and an immunoconjugate.
  • the kit of the present invention can be used to detect the presence or absence of hOPN or its content in a biological sample.
  • the detection method comprises the steps of: (a) contacting the sample with an anti-hOPN monoclonal antibody or an immunoconjugate thereof in the kit of the present invention; (b) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex means the presence of osteopontin in the sample or the amount of antigen-antibody complex formed by quantitative detection to reflect the content of hOPN in the sample.
  • the sample may be pretreated or untreated, for example, may be extracted, purified or concentrated.
  • the kit contains a container and a monoclonal antibody of the present invention located in the container, or a test plate with the monoclonal antibody, and instructions for use.
  • the kit may also contain other reagents required for detection, such as buffering. Liquid, indicator, etc. Those skilled in the art can adjust the contents of the kit according to specific needs.
  • the invention is further illustrated by the following examples and experimental examples, which are not to be construed as limiting the invention.
  • the examples do not include a detailed description of conventional methods, such as those used to construct vectors and plasmids, methods for inserting genes encoding proteins into such vectors and plasmids, or methods for introducing plasmids into host cells, and classical cell fusions. And methods for screening and purification of monoclonal antibodies. Such methods are well known to those of ordinary skill in the art and are described in a number of publications, including Sambrook, J., Fritsch, EF and Maniais, T.
  • the human hepatoma cell line LM3 (purchased from Shanghai Zhongshan Hospital) was extracted and extracted with TRISOL kit (INVITROGEN) in minutes; seconds, seconds, seconds, cycles; minutes, and a 963 bp DNA fragment was obtained.
  • the fragment was recovered by a gel recovery kit (Shanghai Shenggong), digested with restriction endonucleases Hind III and Kpn 1 , and recovered by gel electrophoresis, and then ligated with Hind III and ⁇ double-digested plasmid vector to transform After E. coli, a positive clone with an insert was screened.
  • the DNA sequence analysis confirmed that the nucleotide sequence of the ⁇ gene is as shown in SEQ ID NO: 1.
  • Mouse OPN antisense primer (primer 4): AT ICTCGAQ TTAATTGACCTCAGAAGA
  • the human and murine OPN gene fragments of the sequence obtained in Example 2 were digested with the corresponding restriction enzymes, and then loaded into the pPICZ a plasmid.
  • the yeast cells were transfected, single yeast was picked, and the human and murine OPN proteins were induced to express.
  • the supernatant of the yeast cells was collected, purified by anion exchange, molecular sieve purification, and confirmed by human SDS-PAGE. After purification, it was subjected to SDS-PAGE electrophoresis, and the results are shown in Fig. 1.
  • mice After emulsification of 100 ⁇ ⁇ human OPN and equal volume of Freund's adjuvant, intraperitoneal injection of BALB/C mice was immunized, and boosted once every two weeks. The dose was the same as the first immunization. After 3 immunizations, serum was selected. The mice with higher anti-zero antibody titer were isolated from the spleen lymphocytes, and the mouse spleen lymphocytes were fused with NS-1 cells by the classical PEG method. The 96-well plate was coated with 10 ⁇ ⁇ / ⁇ 1 human, and the hybridoma cell line 1A12 stably expressing the anti-hOPN antibody was repeatedly screened by ELISA.
  • a large number of monoclonal cell lines 1A12 were amplified, and 5 ⁇ 10 6 /only BALB/C mice were injected intraperitoneally. The ascites of mice was collected in about 10 days.
  • the anti-hOPN monoclonal antibody was purified by affinity chromatography on a Protein A column.
  • mice After emulsification of 100 ⁇ ⁇ human OPN and equal volume of Freund's adjuvant, intraperitoneal injection of BALB/C mice was immunized, and boosted once every two weeks. The dose was the same as the first immunization. After 3 immunizations, serum was selected. The mice with higher anti-sputum antibody titer were isolated from the spleen lymphocytes, and the mouse spleen lymphocytes were fused with NS-1 cells by the classical PEG method.
  • the 96-well plate was coated with 10 ⁇ ⁇ / ⁇ 1 KLH-WLVPDP (synthesized by Shanghai WI), and the hybridoma cell line 23C3D3 stably expressing the anti-human OPN-specific epitope antibody was repeatedly screened by ELISA.
  • the monoclonal cell strain was amplified in a large amount, and 5 10 6 per BALB/C mice were intraperitoneally injected. The ascites of mice was collected in about 10 days.
  • the protein A column was used to purify the anti-human OPN specific epitope.
  • Control monoclonal antibody 23C3D3 o Experimental Example 6. Variable region gene cloning and sequencing of 1A12 monoclonal antibody
  • HGSP1 GATACTGTGATCTGTTTG-3 ' (SEQ ID NO: 12)
  • HGSP2 TCGCAGATGAGTCTGGAC-3 ' ( SEQ ID NO: 13)
  • LGSP1 GAGGTTATGACTTTCATAGTCAGC-3 ' SEQ ID NO: 15
  • LGSP3 TCTGGGATAGAAGTTGTTCATGAG-3, ( SEQ ID NO: 17)
  • HGSP1, LGSP1 were used as the first strand cDNA, and under the action of TdT and dCTP, the first strand of the DNA 3' end was added with poly C.
  • the PCR products of VH and VL were obtained by nested PCR using HGSP2, HGSP3 LGSP2 and LGSP3 as 5' primers respectively; the PCR product was loaded into pGEM-T vector; the cloned plasmid was extracted, restriction endonuclease Positive clones were identified and their sequences were determined by sequencing.
  • the nucleotide sequence of the heavy chain variable region of 1A12 mAb is SEQ ID NO 3
  • the amino acid sequence is SEQ ID NO: 4
  • the nucleotide sequence of the light chain variable region of 1A12 mAb is SEQ ID NO 5
  • amino acid The sequence is SEQ ID NO: 6.
  • Example 7 Cloning of human antibody light and heavy chain constant region genes
  • the reported sequences were designed to amplify the antibody heavy and light chain constant region genes using RT-PCR reactions, respectively.
  • the PCR product was purified by agarose gel electrophoresis and cloned into pGEM-T vector. After sequencing, it was confirmed that the correct clone was obtained.
  • SEQ ID NO: 22 and SEQ ID NO: 23 show the nucleotide sequence and amino acid sequence of the heavy chain constant region CCH, respectively.
  • SEQ ID NO: 24 and SEQ ID NO: 25 respectively show light chain The nucleotide sequence and amino acid sequence of the constant region (CL).
  • the correct clones in this example were designated as pGEM-T/CH and pGEM-T/CL.
  • the chimeric antibody heavy chain gene was synthesized by overlapping PCR using 1A12 heavy chain variable region gene 1A12VH and pGEM-T/CH vector as template. The reaction conditions were: 95 °C for 15 minutes; 94 °C for 50 seconds, 58 °C for 50 seconds. , 72 ° C for 50 seconds, 30 cycles; 72 ° C for 10 minutes.
  • the 5' end of the chimeric heavy chain gene contains the restriction enzyme site Hindlll and the signal peptide gene sequence, and the 3' end contains a translation stop codon TAA and a restriction enzyme site EcoR l .
  • the chimeric antibody light chain gene was synthesized by overlapping PCR using 1A12 light chain variable region gene 1A12VL and pGEM-T/CL vector as template.
  • the reaction conditions were: 95 °C for 15 minutes; 94 °C for 50 seconds, 58 °C for 50 seconds. , 72 ° C for 50 seconds, 30 cycles; 72 °C for 10 minutes, the PCR product C1A12VLCL was obtained, the 5' end contains the restriction enzyme site Hindlll and the signal peptide gene sequence, and the 3' end contains the translation stop codon TAA and restriction enzyme sites.
  • Point EcoR I Signal peptide gene sequence TATCCAGAGGA (SEQ ID NO: 26).
  • the PCR product was purified by agarose gel electrophoresis and cloned into pGEM-T vector (Promega product), and positive clones were screened for sequencing. The correct clones were selected and digested with Hind III and EcoR I.
  • the chimeric antibody light chain fragment C1A12VLCL was purified by agarose gel electrophoresis, and the plasmid pcDNA3.1/ZEO(+) vector digested with Hindlll and EcoR I was used. (Invitrogen, USA;); ligated into a chimeric light chain eukaryotic expression vector pcDNA3.1/ZEO(+;> (cl A12VLCL;).
  • Co-transfected COS-1 cells (ATCC CRL) by constructing successful chimeric heavy and light chain expression vector liposomes
  • Accelrys' Insight II package was used to simulate the three-dimensional structure of the 1A12 murine mAb variable region.
  • the template protein of the 1A12 heavy chain and light chain variable region proteins was searched for in the Protein Data Bank (PDB) using the BLAST program.
  • the highest homology antibody, 1PLG was selected as the model template for 1A12, and the 3D structure of 1A12 was modeled using the Insight II program (Fig. 3).
  • Example 10 Design and construction of anti-hOPN humanized antibody MA12
  • the most similar human template for the light chain and heavy chain variable regions of 1A12 was searched in the Genbank database using the BLAST program.
  • the human antibody with the highest homology to the 1A12 heavy chain variable region is the human antibody CAA79298.1 antibody (emb
  • the antibody is BAC01734.1 (dbj
  • a CDR-grafted antibody is constructed, the heavy chain is hlA12Ha, and the light chain is hlA12La.
  • hlA12Ha The variable region sequences of hlA12La and hlA12La are shown in Figure 4.
  • the humanized antibody heavy and light chain variable region genes (hlA12VHa and hlA12VLa) were synthesized by whole gene, and then the humanized antibody heavy chain gene was synthesized by overlapping PCR using hlA12VHa gene and pGEM-T/CH vector as template.
  • the reaction conditions were: 95 ° C for 15 minutes; 94 ° C for 50 seconds, 58 ° C for 50 seconds, 72 ° C for 50 seconds, 30 cycles; 72 ° C for 10 minutes.
  • the 5' end of the humanized heavy chain gene contains the restriction enzyme site Hindlll and the peptide gene sequence, and the 3' end contains the translation stop codon TAA and the restriction enzyme site EcoR I. Signal peptide
  • AGTCATAATATCCAGAGGA (SEQ ID NO: 26) was finally subjected to agarose gel electrophoresis to separate PCR amplification products, and the desired band was recovered and cloned into pGEMT vector, and positive clones were screened for sequencing.
  • the clones with the correct sequencing were digested with Hindlll and EcoR I, and the humanized antibody heavy chain fragment hLA12VHaCH was purified by agarose gel electrophoresis, and the plasmid pcDNA3.1 digested with Hindlll and EcoR I (+X Invitrogen) ;) ligation, construction of the adult-derived heavy chain eukaryotic expression vector pcDNA3.1 (+) (hlA12VHaCH;).
  • Humanized antibody light chain gene was synthesized by overlapping PCR using hlA12VLa gene and pGEM-T/CL vector as template.
  • the reaction conditions were: 95 °C for 15 minutes; 94 °C for 50 seconds, 58 °C for 50 seconds, 72 °C 50 Second, 30 cycles; 72 °C for 10 minutes, the PCR product hlA12VLaCL was obtained, which contained the restriction enzyme site Hindlll and the signal peptide gene sequence at the 5' end, and the 3' end contained the translation stop codon TAA and the restriction enzyme site EcoR l .
  • the signal peptide gene sequence is
  • TCCAGAGGA (SEQ ID NO: 26) was selected and sequenced.
  • the clones were digested with Hindlll and EcoR I.
  • the humanized antibody light chain fragment hLA12VLaCL was purified by agarose gel electrophoresis, and the plasmid pcDNA3 digested with Hindlll and EcoR I.
  • the 1/ZEO(+) vector (product of Invitrogen, USA) was ligated to construct an adult-derived light chain eukaryotic expression vector pcDNA3.1/ZEO(+) (hi A12VLaCL).
  • the successfully constructed humanized heavy and light chain expression vectors were co-transfected into COS-1 cells (ATCC CRL 1650) by liposome method. After 72 hours, the culture supernatant was taken for analysis, and the culture supernatant was determined by ELISA.
  • the ELISA plate was coated with 2 ⁇ ⁇ / ⁇ 1 , and the activity of the transfected humanized antibody specifically binding to hOPN was detected by ELISA, and it was found that the humanized antibody composed of hlA12Ha and hlA12La was compared with the C1 A12 chimeric antibody ( The activity of hlA12Ha/ hlA12La) is almost completely lost. Therefore, in order to obtain high-affinity humanized antibodies, we also need to analyze and revert to FR region murine residues that may affect MA12 antibody binding activity. By analyzing the three-dimensional structure of the modeled MA12 mAb variable region (Fig.
  • SEQ ID NO: 18 and SEQ ID NO: 19 show the variable region nucleotide sequence and amino acid sequence of hlA12Hb, respectively.
  • SEQ ID NO: 20 and SEQ ID NO: 21 show the variable region nucleotide sequence and amino acid sequence of hlA12Lb, respectively.
  • the humanized antibody heavy and light chain variable region genes (hlA12VHb/hlA12VLb;) were synthesized by whole gene, and the light chain expression vector pcDNA3.1/ZEO was constructed in the same manner as the humanized antibody (hi A12Ha/ hi A12La). (+) (hi A12VLbCL) and the heavy chain expression vector pcDNA3.1 (+) (hlA12VHbCH).
  • the light and heavy expression vectors were then co-transfected into COS-1 cells, and the antigen binding activity of the antibody was determined by ELISA.
  • the OPN protein (2 g/ml) was coated on an ELISA plate at 4 ° C overnight, using 2% BSA-PBS. After blocking at 37 °C for 2 h, add the supernatant of hlA12 to be tested, incubate at 37 °C for 2 h, add HRP-rabbit anti-human IgG for binding reaction, incubate for 1 h at 37 °C, and add TMB at 37 °C for 5 min. Finally, the reaction was terminated with H 2 S0 4 and the A450 and A630 values were measured.
  • the light and heavy chain plasmids of the above successfully constructed C1A12 or MA12 were co-transfected into CHO-K1 cells (ATCC CRL-9618) by liposome method, and transfected for 24 hours, the cells were replaced with 600 g/ml G418 and P 250 g. /ml Zeocin's selection medium was screened for resistant clones.
  • the cell culture supernatant was screened for high expression clones by ELISA, and the highly expressed clones were expanded and cultured in serum-free medium, and the chimeric antibody C1A12 or humanized antibody was isolated and purified using a Protein A affinity column (GE product). hlA12, finally quantified by UV absorption.
  • Experimental Example I Anti-hOPN antibody tumor cell effect test
  • MDA-MB-435 sC was purchased from the Shanghai Institute of Cytology, Chinese Academy of Sciences, irrelevant antibody C23C3D3).
  • 96-well plates (Greiner) were coated with 10 mg/ml of hOPN (from Example 3) or BSA (SIGMA) at 4 ° C overnight; blocked with 1% BSA/PBS at 37 ° C for 1 hour to block Non-specific binding sites.
  • MDA-MB-435s cells were digested with 0.2% EDTA, resuspended in 0.25% BSA/DMEM, and adjusted to a cell concentration of 5 X 10 5 cells/ml. 100 ⁇ of cells were added to each well, and 25 g/ml of mouse anti-human antibody 1 ⁇ 12, human mouse chimeric antibody C1A12 and humanized antibody hLA12 were added to the treatment group, and 25 g/ml of irrelevant antibody was added to the control group.
  • the cell invasion assay was performed using the Transwell chamber system CCorning) with a membrane pore size of 8 mm.
  • the upper layer of the chamber was coated with artificial basement membrane matrigel (Matrigel), dried in a fume hood, and hydrated in DMEM at 37 ° C for 1 hour.
  • MDA-MB-435 S cells were digested with 0.2% EDTA, resuspended in 0.25% BSA/DMEM, and adjusted to a cell concentration of 5 X 10 5 cells/ml.
  • 100 ⁇ M cell suspension was added to the upper layer of the chamber (the treatment group was simultaneously added with 25 g/ml of mouse anti-human OPN antibody 1A12, human mouse chimeric antibody clA12 and humanized antibody hLA12, and the control group was added with 25 g/ml of irrelevant antibody. ;), 0.25% BSA/DMEM (with or without OPN;) was added to the lower layer of the chamber and incubated for 24 hours in a 37 ° C cell incubator.
  • the cells in the upper layer of the chamber were scraped off with cotton swabs, and the cells passed through the basement membrane to the lower layer were washed with PBS, fixed with 1% formaldehyde, and stained with 0.5% crystal violet, observed under a microscope at 200 times. Count the number of cells in each field of view.
  • MDA-MB-435 S cells were grown to near saturation (>90%) in 12-well plates, and after washing with PBS, cells were serum-starved overnight in serum-free DMEM. The monolayer cells were streaked with a 10 ⁇ M pipette tip and the rinsed cells were washed away with PBS. 25 g/ml of mouse anti-human OPN antibody 1A12, human mouse chimeric antibody clA12 and humanized antibody hlA12 were added, 25 ⁇ ⁇ / ⁇ 1 irrelevant antibody was added to the control group, and the control group was treated with an irrelevant antibody. After incubating the cells for 24 hours in a 37 ° C cell incubator, the results are expressed as the number of cells crossing the baseline.
  • Soft agar colony formation experiments were performed using a two-layer soft agar system. 2.5% melted agar powder pre-warmed at 37 ° C The DMEM medium was mixed, and a 0.5% agar powder solution was prepared, and then diluted into a 0.3% agar powder solution by DMEM. 500 ⁇ of 0.5% agar powder solution was added to each well of a 24-well cell culture plate, allowed to solidify at 4 ° C, and transferred to a 37 ° C cell incubator for incubation. MDA-MB-435 S cells were digested with 0.2% EDTA and resuspended in 0.3% agar powder solution to adjust the cell concentration to 5 X 10 3 cells/ml.
  • a 500 ⁇ M cell suspension was added to each well of a 24-well plate and allowed to solidify. From the second day, 1A12, clA12 and hlA12 or irrelevant antibody (23C3D3) (25 g/ml) were added to each well of the cell culture plate every other day, and the size of the clone formation was observed after 3 weeks, with >10 The cells were aggregated as one clone.
  • HUVEC cells The proliferation of HUVEC cells was determined by 3H-TdR incorporation assay to determine the protective effect of OPN on cell survival and the inhibition of OPN antibody against OPN.
  • 96-well plates were seeded with 2 X 10 4 HUVEC per well and cultured in complete medium for 24 hours. The cells were treated differently for the following 24 hours:
  • HUVEC was cultured to 80% confluence in M200 medium containing LCGS; one day before the experiment, take out -20
  • the preserved Matrigel is naturally melted at 4 ° C for 3 to 12 hours.
  • the matrigel was dispensed with a pre-cooled tip and a 96-well plate, and the entire process was run on ice.
  • 60 ⁇ M matrigel was added to each well of a 96-well plate, and the bubbles were carefully removed, and the gel was cured by incubating at 37 ° C for 1 hour.
  • Add anti-hOPN antibodies 1A12, clA12 and hlA12 to the treatment group.
  • the cells were cultured for 12 hours in a 37 ° C cell culture incubator; after 6 hours, capillary tube-like structure formation was observed.
  • the substance to be tested/gelatin sponge is added to the surface of the CAM, and the composition of the substance to be tested is as follows: i) PBS; ii) HOPN; iii) human OPN (250 ng); iv) human OPN (250 ng) Ten unrelated control antibody (1 g); v) human OPN (250 ng) ten mouse anti-OPN antibody 1A12 (1 g); vi) human OPN (250 ng) ten human chimeric anti-OPN antibody clA12 (l ⁇ g) ⁇ , vii) Human OPN (250 ng) ten humanized anti-OPN antibody hlA12 (l
  • the Hydron powder was dissolved in absolute ethanol at a final concentration of 12% (w/v), dissolved at 37 °C to prepare a 12% Hydroon mold solution, and stored at room temperature. .
  • sucralfate powder was added to sterile Milli Q water to prepare a suspension with a final concentration of 100 ⁇ ⁇ / ⁇ 1, stored at 4 ° C, and vortexed before mixing.
  • Each sustained-release pellet contains 200 ng of hOPN and 50 ⁇ of ⁇ sucralfate, and the antibody group contains 1 ⁇ ⁇ anti-sputum antibody or unrelated control antibody.
  • Corneal vein anesthesia was performed with 3% pentobarbital sodium (1 ml/kg body weight;), 1% tetracaine was anesthetized with cornea, and corneal thickness was measured with an ultrasonic corneal thickness gauge.
  • the eye drops were coated with chlortetracycline eye ointment.
  • the double-blind observer observed the corneal neovascularization in the rabbit under the slit lamp daily.
  • the slit lamp was observed to end on the 10th day.
  • Measure the longest vessel length (VL) and the vascular clock angle (CN, 30 degrees 1 CN), and calculate according to the following formula:
  • MDA-MB-435 S cells were digested, resuspended in serum-free DMEM, and the cell concentration was adjusted to 5 X 10 7 cells/1 ml.
  • the tumor formation of nude mice was observed once a week.
  • the maximum diameter (length) and its vertical distance (width) of the tumor were measured with a vernier caliper.
  • Tumor growth curves were plotted versus time for changes in tumor volume. Twelve experimental animals in each treatment group, 6 of them observed the size of tumor growth in situ, and terminated by 10 weeks; the other 6 observed spontaneous lung metastasis, and terminated when the primary tumor reached 1000 mm 3 .
  • mice were sacrificed by cervical dislocation. Tumor tissues of each group were taken, 10% neutral formalin was fixed and paraffin-embedded, and tumor tissues and lung tissues were observed after H&E staining. The size and number of lung metastases were observed under a microscope.
  • the mouse in situ tumor tissue was taken, and the liquid nitrogen was frozen after taking the material.
  • the cryostat frozen slicer 8 um was serially sliced. After taking out, it was air-dried for 5 min, fixed at -20 °C for 30 min, and subjected to IHC staining.
  • Rat anti-mouse CD3 1 monoclonal antibody (working concentration 1: 100) was incubated overnight at 4 °C; secondary antibody was goat anti-rat IgG/PE (working concentration 1: 100).
  • Hochest33258 lining photographed under a fluorescence microscope.
  • CD3 1 protein is mainly expressed on the vascular endothelial cell membrane, and both vascular endothelial cells and microvessels have CD31 protein expression. Intratumoral microvasculature was observed with anti-CD31 antibody (Fig. 16). Microscopic observation showed that MVD was significantly less in the anti-OPN antibody 1A12 treated group than in the unrelated control antibody-treated group (P ⁇ 0.01).
  • Experimental Example IV Identification of 1A12 monoclonal antibody epitope
  • a random peptide library was used for immunopanning, and the entire process was performed on a 96-well plate. 100 ⁇ ⁇ / ⁇ 1, ⁇ ⁇ /well 1A12 antibody coated at 4 °C overnight, 10% skim milk powder (TBST dilution;) blocked overnight, 1 X TBST (Tween-20 0.1%) washed 6 times; phage random peptide library (; available from NEB, Ph.D.-12 TM peptide phage display kit laboratory phage display peptide Library kit) 4 X 10 10 pfu + ⁇ normal mouse serum, rocked at room temperature for 1 hour.
  • a random peptide library was used for immunopanning, and the whole process was performed on a 96-well plate. 100 ⁇ ⁇ / ⁇ 1, ⁇ ⁇ /well 23C3D3 antibody coated at 4 °C overnight, 10% skim milk powder (TBST dilution;) blocked overnight, 1 X TBST (Tween- 20 0.1%) washed 6 times; phage random peptide library (; Purchased from NEB, Ph.D.-12 TM phage display peptide laboratory kit; >4 X 10 1Q pfu + ⁇ normal mouse serum, shake gently for 1 hour at room temperature l X TBST (Tween-20 0.1%) 15 times It was eluted with glycine-Cl (pH 2.2) containing 1 mg/ml BS A, gently shaken at room temperature for 15 min, and neutralized with 15 ⁇ l Tris-Cl PH 9.1.
  • the ELISA procedure was performed in a 96-well plate, 100 g/ml, 50 ⁇ l/well 1A12 monoclonal antibody coated at 4 ° C overnight, 10% skim milk powder CTBST diluted at 7 ° C for 2 hours, l X TBSTXTween-20 0.1%) washed 5 times Each monoclonal phage amplified supernatant was diluted with 1 ⁇ TBS, both at 5 ⁇ 10 8 ⁇ !/50 ⁇ 1, the control antibody was a mouse anti-human ⁇ monoclonal antibody (Santa Cruz), and the negative control phage was 5F12 (the clone) Is a positive clone of 23C3D3).
  • Figure 18B shows the 1A12 mAb hybridization, and the right panel shows the unrelated antibody 23C3D3 hybridization; the arrow shows the target band; the results show that the positive clone reacts with the antibody is specific, as shown in Figure 18B.
  • Experimental Example IV-4 Sequencing and Sequence Analysis of Antibody Recognition Epitopes

Description

骨桥蛋白的功能表位、 针对该单位的单克降杭体及
它们的应用 技术领域
本发明属于生物技术领域， 更具体地， 本发明公开了一种蛋白的功能表位、 与这个 表位特异性结合的单克隆抗体及其在制备抗肿瘤药物中的用途。 背景技术
肿瘤是严重危害我国人民生命健康的重大疾病， 肿瘤手术切除后 5年， 生存率可达 40%左右， 但仍有一半左右的病人手术后出现转移复发。 如何控制肿瘤切除后很高的转 移复发率以提高肿瘤患者的疗效， 是国际医学研究的重点攻关课题。 深入研究肿瘤细胞 转移的机理有助于阐明肿瘤转移复发的分子机制， 了解促进转移的信号传导通路， 寻找 抑制转移的有效作用环节， 为新药研制和临床治疗提供更有效的阻断靶点， 提高肿瘤病 人的生存率。
肿瘤转移分子机理研究提示多种因素与肿瘤细胞转移密切相关， 如： P16突变、 p53 突变， p21、 c-erbB-2、 mdm-2、 转移生长因子 a (TGF α；)、 表皮生长因子受体 (EGF-R；)、 基质金属蛋白酶 -2(MMP-2)、尿激酶型纤溶酶原激活物 (uP A)及其受体与纤溶酶原激活物 抑制剂 -1(PAI-1)、 细胞间黏附分子 -1(ICAM-1)、 血管内皮生长因子 (VEGF)、 血小板衍化 内皮生长因子（PD-ECGF)等为肝癌侵袭性正相关因子（Yamaguchi H, Wyckoff J, Condeelis J. Cell migration in tumors. "月中瘤中的细胞迁移 "Curr Opin Cell Biol. 2005 ； 17(5):559-64. Huber MA, Kraut N, Beug H. Molecular requirements for epithelial-mesenchymal transition during tumor progression. "月中瘤发展其月间上皮-间质转变 白 分子学要求" Curr Opin Cell Biol. 2005 年 10月； 17(5):548-58.)。
近年来的研究发现骨桥蛋白（Osteopontin, OPN)在肿瘤细胞转移过程中发挥了至关 重要的作用 (Rangaswami H, Bulbule A, Kundu GC. Osteopontin: role in cell signaling and cancer progression. "骨桥蛋白在细胞信号转导和癌症发展中的作用" Trends Cell Biol. 2006年 2月； 16(2):79-87.)， 在各国研究小组共同努力下， OPN促转移信号通路领域不 断有新的报道， 从不同的角度对其促肿瘤细胞转移功能的主要方面进行了解释， OPN信 号通路在促进肿瘤细胞转移方面的重要调控作用已经成为国际肿瘤转移研究领域的研 究热点。
骨桥蛋白作为一种重要的促肿瘤转移糖蛋白信号分子在骨骼组织、 肾脏组织、 大脑 组织、 腺体表皮细胞、 血管平滑肌细胞、 活化的巨噬细胞、 淋巴细胞和肿瘤细胞中均有 表达 (Weber GF, Ashkar S, Glimcher MJ, Cantor H. Receptor-ligand interaction between CD44 and osteopontin (Eta-1). "CD44和骨桥蛋白（Eta-1)间的受体-配体相互作用" Science (Washington, DC) 1996； 271 : 509-12.)。 OPN在肿瘤组织细胞外基质中， 能够通过激活 肿瘤细胞表面的 CD44(Miyauchi A, Alvarez J, Greenfield EM 等 .Recognition of osteopontin and related peptides by an α v β 3 integrin stimulates immediate cell signals in osteoclasts."通过 α ν β 3 整联蛋白识别的骨桥蛋白及相关肽在破骨细胞中剌激细胞信号 " J Biol Chem 1991； 266:20369 - 7)和整联蛋白（Teramoto H, Castellone MD, Malek RL, Letwin N, Frank B, Gutkind JS, Lee NH. Autocrine activation of an osteopontin-CD44-Rac pathway enhances invasion and transformation by H-RasV12.' '骨桥蛋白 -CD44-Rac途径的 自分泌活化促进通过 H-RasV12的转化和侵袭 "Oncogene. 2005年 1月 13 ; 24(3):489-501.) 两大受体下游的信号通路， 促进肿瘤细胞对细胞外基质的降解、 细胞迁移和细胞的抗凋 亡能力， 包括以下几个方面：
OPN信号被受体 CD44识别引起肿瘤细胞 Rho家族小 G蛋白如 Rac的活化 (Teramoto
H等， 同上， Oncogene. 2005 Jan 13 ; 24(3):489-501.)， 小 G蛋白传达细胞外化学趋化信 号至下游的效应蛋白如 WASP(Wiskott - Aldrich syndrome protein, 维 -埃氏综合征蛋白） 家族成员， WASP蛋白结合并活化肌动蛋白相关蛋白 (Arp2/3) 复合物， 后者催化肌动蛋 白 (Actin)的聚合反应， 从而诱导肿瘤细胞骨架重构以及细胞膜突起的形成， 增强细胞的 迁移能力 (Wolf K, Mazo I, Leung H等. Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis. "月中瘤细胞迁移 的代偿机制：细胞外周蛋白水解阻滞后的间质-变形虫样转化 " J Cell Biol 2003 ; 160: 267 - 77.); 在迁移细胞被拉伸的前缘， 活化的 WASP蛋白促使突出的细胞膜形成整联蛋白 依赖的细胞粘附， 诱导基质金属蛋白酶在局部的积累促进细胞外基质的降解 (Nicholson, K M. 禾口 Anderson, N.G. (2002) The protein kinase B/Akt signaling pathway in human malignancy. "人恶性肿瘤中的蛋白激酶 B/Art信号转导途径" Cell. Signal. 14, 381 - 395)； 此外 OPN-CD44下游通路可以激活 PI-3K, 而 PI-3K的靶分子之一就是 Akt激酶， Akt 激酶调控细胞周期的进行，促进细胞存活，细胞锚定非依赖性生长以及细胞迁移等过程， 介导了 OPN 促进肿瘤抗凋亡和细胞迁移功能 (Lin, Y.H.和 Yang- Yen, H.F. (2001) The osteopontin-CD44 survival signal involves activation of the phosphatidylinositol 3 -kinase/ Akt signaling pathway. "骨桥蛋白 -CD44存活信号涉及磷脂酰肌醇 3-激酶 /Akt信 号转导途径的活化" J. Biol. Chem. 276, 46024 - 46030。 Philip, S.禾 P Kundu, G.C.(2003) Osteopontin induces nuclear factorkB mediated promatrix metalloproteinase-2 activation through I kappa B alpha/IKK signaling pathways and curcumin (diferulolylmethane) downregulates these pathways. "骨桥蛋白通过 ΙκΒα/ΙΚΚ信号转导途径和这些途径下游的 酸性黄诱导核因子 kB 介导的前基质金属蛋白酶 -2 活化" J. Biol. Chem. 278, 14487 - 14497)。 OPN被受体 α ν β 3识别后激活 NIK和 MEKK1分别引起下游 NF- κ B禾 P AP-1 的活化入核诱导效应基因 uPA 和 MMPs 表达的上调， 促进了对细胞外基质的降解 (Rangaswami, H.等 (2004) Nuclear factor inducing kinase plays a crucial role in osteopontin induced MAPK/L Ba kinase dependent nuclear factor-κΒ mediated promatrixmetalloproteinase-9 activation. "核因子诱导激酶在骨桥蛋白诱导 MAPK/ iKBa激 酶依赖性核因子 κΒ介导的前基质金属蛋白酶 9活化中起到重要作用 "J. Biol. Chem. 279: 38921 - 38935 , Rangaswami, H.等 (2005) JNK1 differentially regulates osteopontin induced nuclear factor inducing kinase/MEKKl dependent activating protein- 1 - mediated promatrix metalloproteinase-9 activation. "JNK1 分化调节了骨桥蛋白诱导的核因子 导激酶 /MEKK1依赖性活化蛋白 1介导的前基质金属蛋白酶 9活化" J. Biol. Chem. 280, 19381 - 19392)。 同时， 肿瘤细胞分泌的 OPN能够通过自分泌和旁分泌途径促进血管内皮细胞 生长因子 VEGF 的表达， 从而促进血管内皮细胞的增殖和肿瘤内部毛细血管的形成。 (Goutam Chakraborty等 (2008) Osteopontin Promotes Vascular Endothelial Growth Factor - Dependent Breast Tumor Growth and Angiogenesis via Autocrine and Paracrine Mechani sms . "骨桥蛋白通过自分泌和旁分泌机制促进血管内皮生长因子-依赖性乳腺肿 瘤生长和血管发生" Cancer Res 2008 ; 68: 152-161)。 综上所述， OPN信号通路功能的发 挥为肿瘤转移细胞完成对细胞外基质的侵润、 通过血液或者淋巴循环向外周组织器官扩 散和形成转移灶的各个阶段中表现出多方面的重要调控功能。
通过抗 OPN抗体阻断 OPN的促转移信号传导通路，就有可能有效阻断肿瘤细胞的 黏附和迁移过程， 防止肿瘤细胞向细胞基质的侵润， 抑制肿瘤毛细血管形成， 从而防止 肿瘤的发展和转移。 发明内容
本发明的目的正是在于提供针对骨桥蛋白特定功能表位的单克隆抗体，从而达到治 疗肿瘤的效果。
在本发明的第一方面中，提供了一种骨桥蛋白的功能表位，所述功能表位为 ΝΧΡΥ, 其中 X=A或 G， Y= S、 T、 ^^或！1。
在一个优选例中， 所述功能表位为 NAPS。 在另一个优选例中， 所述功能表位位于 骨桥蛋白第 7外显子 212-215aa处。
在本发明的第二方面，提供了一种与所述的功能表位特异性结合的抗骨桥蛋白的单 克隆抗体。
在一个优选实施方式中， 所述单克隆抗体的重链可变区的 CDR氨基酸序列分别选 自： GYTFTTYVMH、 YINPYNDGSKYNEKFKG 禾 P HYGGSPAY (例如可参见图 4 的 HlA12VHb) ; 轻链可变区的 CDR 氨基酸序列分别选自： RSSQSLVHSNGNTYLH、 KVSNRFS禾 P SQSTHVPWT (例如可参见图 4的 HlA12VLb；)。
在另一个优选实施方式中，所述单克隆抗体的重链可变区氨基酸序列选自： SEQ ID
NO:4或 SEQ ID NO: 19， 轻链可变区氨基酸序列选自： SEQ ID NO:6或 SEQ ID NO:21。
在另一优选实施方式中， 所述单克隆抗体的恒定区选自： 小鼠抗体恒定区或人抗体 恒定区。
在另一优选例中， 所述恒定区为小鼠 IgG。 在一个优选例中， 所述单克隆抗体是采 用杂交瘤方法或重组 DNA方法制得的， 或是从噬菌体抗体库中分离获得的。 在另一优 选例中， 所述单克隆抗体是嵌合抗体或人源化抗体。
在本发明的第三方面中， 提供了一种 DNA分子， 其编码本发明的单克隆抗体。 在另一优选例中， 所述 DNA分子中编码重链可变区的核苷酸序列为 SEQ ID NO:3 或 SEQ ID NO: 18；并且编码轻链可变区的核苷酸序列为 SEQ ID NO: 5或 SEQ ID NO:20。
在本发明的第四方面中， 提供了一种载体， 其包含本发明上述的 DNA分子。
在本发明的第五方面中， 提供了一种宿主细胞， 其包含本发明的载体， 或基因组中 整合有本发明的 DNA分子。
在一个优选例中， 所述宿主细胞为原核细胞， 优选细菌细胞； 低等真核细胞， 优选 酵母细胞； 或高等真核细胞， 优选哺乳动物细胞。
在本发明的第六方面中，提供了一种免疫偶联物，其含有：（a)本发明的单克隆抗体； 和 (b)选自下组的偶联部分： 药物、 毒素、 细胞因子、 放射性核素、 或酶。
在本发明的第七方面中，提供了本发明抗骨桥蛋白的单克隆抗体或免疫偶联物在制 备抗肿瘤药物中的用途。
在另一优选例中， 所述肿瘤选自： 腺癌、 白血病、 淋巴瘤、 黑色素瘤、 肉瘤； 选自 来自肾上腺、 胆囊、 骨、 骨髓、 脑、 乳腺、 胆管、 胃肠道、 心脏、 肾脏、 肝脏、 肺、 肌 肉、 卵巢、 胰腺、 甲状旁腺、 阴茎、 前列腺、 皮肤、 唾液腺、 脾脏、 睾丸、 胸腺、 甲状 腺或子宫的肿瘤组织； 中枢神经系统肿瘤； 眼部肿瘤、 内分泌腺肿瘤、 神经内分泌系统 肿瘤、 胃肠道胰腺内分泌系统肿瘤， 生殖系统肿瘤或头颈部肿瘤。
在一个优选例中， 所述中枢神经系统肿瘤选自： 胶质细胞多样性瘤或星细胞瘤。 在 另一优选例中， 所述眼部肿瘤选自： 基底细胞癌、 鳞状细胞癌或黑色素瘤。
在本发明的第八方面中， 提供了一种药物组合物， 其包含本发明的抗骨桥蛋白的单 克隆抗体或免疫偶联物； 以及药学上可接受的载体。
在一个优选例中， 所述药物组合物含有 0.00001〜99.9 wt %； 优选 0.0001-90wt%， 更优选的 0.001-75wt%， 更优选
Figure imgf000005_0001
在另一优选例中，所述药物组合物还包含选自下组的其它抗肿瘤活性物质： TNF-0
TGF-β, IFN-0 血管它丁、 内皮它丁、 甘磷酰芥、 血卟啉、 石蒜碱内铵盐、 鸦胆子乳、 足叶乙甙、 脱水卫矛醇、 阿霉素、 三苯氧胺、 5-氟尿嘧啶、 去甲斑螯素、 双呋喃氟尿嘧 啶、 葫芦素、 三尖杉酯碱、 冬凌草乙素、 马蔺子甲素、 云芝糖肽、 阿糖胞苷、 卡波铂、 紫杉醇、 香菇多糖、 氟他胺、 异环磷酰胺、 乌苯美司、 醋酸亮丙瑞林、 脱氧氟尿苷、 洛 波铂、 依林诺特肯、 来屈唑或替尼泊甙
在本发明的第九方面中，提供了一种用于检测骨桥蛋白的试剂盒，所述试剂盒包含： 本发明的抗骨桥蛋白的单克隆抗体或免疫偶联物。
在本发明的第十方面中，提供了一种检测生物样品中是否存在骨桥蛋白或检测其含 量的方法， 所述方法包括步骤： （i)将待测样品与本发明的抗骨桥蛋白的单克隆抗体或免 疫偶联物接触； （ii)检测是否形成抗原-抗体复合物， 其中， 形成抗原 -抗体复合物就表示 样品中存在骨桥蛋白，或定量检测所形成的抗原 -抗体复合物的量以反映样品中骨桥蛋白 的含量。
在一个优选例中， 所述样品可为经过预处理或未经过预处理， 优选经过提取、 纯化 或浓缩等。
本发明的其它方面由于本文的公开内容， 对本领域的技术人员而言是显而易见的。 附图说明
图 1 :人、 鼠 OPN真核表达纯化后的 SDS-PAGE电泳图； M代表蛋白质标准分子量。 图 2: 鼠抗 hOPN单抗 1A12的蛋白质印迹法测定结果。
图 3 :人源化抗体 MA12的分子模拟结构示意图； FR区残基用深灰色条带表示， CDR 区残基用浅灰色条带表示， 9个位于 CDR区周围 5 A距离内的关键鼠源 FR区残基用黑 色球棒状表示。
图 4 : 人源化抗体 hlA12的重链 (图 4A)和轻链 (图 4B)氨基酸序列与相关序列的比对 图。 其中： 1A12VH和 1A12VL分别表示鼠源单克隆抗体 1A12的重链和轻链的可变区； 选择人抗体 CAA79298. 1 的重链可变区和人抗体 BAC01734. 1 的轻链可变区分别作为人 源化抗体 hlA12重链和轻链的框架区； hlA12VHa禾 P hlA12VHb表示不同的人源化抗体 重链可变区， hlA12VLa和 hlA12VLb分别表示不同的人源化抗体轻链可变区； 破折号 表示与人抗体 CAA79298.1或 BAC01734.1对应残基相同的氨基酸，括弧里表示的是 CDR 区； 氨基酸按照 Kabat 的编号方式进行编号 [E.A. Kabat, T.T. Wu, H.M. Perry, K. S. Gottesman, C. Foeller, Sequences of Proteins of Immunological Interest, Fifth ed., United States Department of Health and Human Services, Bethesda, MD, 1991.]。
图 5 : 人源化抗体 1A12的抗原结合活性实验结果。
图 6: 鼠抗 hOPN单抗 1A12、 嵌合抗体 C1 A12及人源化抗体 hlA12阻断肿瘤细胞黏 附的结果。
图 7: 鼠抗 hOPN单抗 1A12、 嵌合抗体 C1 A12及人源化抗体 hlA12抑制肿瘤细胞对 基底膜侵袭的结果。
图 8 : 鼠抗 hOPN单抗 1A12、 嵌合抗体 C1 A12及人源化抗体 hlA12抑制肿瘤细胞对 损伤划痕的修复。
图 9: 鼠抗 hOPN单抗 1A12、 嵌合抗体 C1 A12及人源化抗体 hlA12抑制肿瘤细胞在 软琼脂上克隆形成的结果。
图 10: 鼠抗 hOPN单抗 1A12、 嵌合抗体 clA12及人源化抗体 MA12对 HUVEC细 胞增殖的影响。
图 1 1 : 鼠抗 hOPN单抗 1A12、 嵌合抗体 clA12及人源化抗体 hlA12对 HUVEC毛 细血管管状样结构生成的影响。
图 12: 鼠抗 hOPN单抗 1A12、 嵌合抗体 C 1 A12及人源化抗体 hlA12对鸡胚尿囊膜 CAM血管生成的抑制， 图示中各柱图从左到右分别为 PBS、 VEGF、 OPN、 OPN+无关 抗体、 0PN+1A12、 OPN+clA12 OPN+hl al2。
图 13 : 鼠抗 hOPN单抗 1A12、 嵌合抗体 C 1 A12及人源化抗体 hlA12对 OPN诱导的 角膜新生血管生成的影响。
图 14 : 鼠抗 hOPN单抗 1A12对小鼠体内肿瘤生长的抑制作用。
图 15 : 鼠抗 hOPN单抗 1A12对小鼠体内乳腺癌肺转移的抑制作用。
图 16 : 鼠抗 hOPN单抗 1A12对小鼠体内乳腺癌肿瘤组织中血管密度的影响。 图 17: 抗 OPN单抗 1A12淘选三轮后的产出效率比较图。
图 18 : 抗 OPN单抗 1A12阳性噬菌体 ELISA和蛋白质印迹鉴定图， 图 18A为噬菌 体 ELISA鉴定图， 图 18B为 1A12阳性噬菌体 ELISA和蛋白质印迹鉴定图。
图 19: Align X软件序列分析抗 OPN单抗 1A12的结合表位结果。
图 20: 各种序列表位与抗 OPN单抗 1A12结合力的比较。
图 21 : 抗 OPN单抗 1A12特异识别表位在 OPN分子上的相对部位。 具体实施方式
本发明人经过长期而深入的研究， 获得了抗骨桥蛋白特定功能表位的单克隆抗体， 并进一步制得了嵌合的单克隆抗体及人源化抗体和明确了其编码序列。 通过研究本发明 人还证明了本发明的单克隆抗体对肿瘤迁移具有抑制作用， 可用于肿瘤的治疗。 在此基 础上， 本发明人完成了本发明。
具体而言， 本发明人首先利用分子生物技术克隆人和鼠 OPN基因， 利用真核细胞表 达了人和鼠的 OPN蛋白，并通过细胞融合-杂交瘤的方法制备了鼠抗人的 OPN单克隆抗 体 1A12, 还对此单克隆抗体的基因进行了克隆并测定了其序列。
本发明进一步公开了抗骨桥蛋白的人源化抗体的制备方法， 包括通过计算机辅助设 计出人源化抗体 MA12 的氨基酸序列。 全基因合成 MA12 的重链和轻链可变区基因并 经基因重组分别与人抗体重、 轻链恒定区基因拼接， 克隆到真核表达载体中， 分别构建 人源化抗体的轻、 重链表达载体， 然后将轻、 重链表达载体用脂质体法共转染 CHO细 胞，然后进行筛选、培养纯化即得。嵌合的单克隆抗体 C1A12可以参照相同的方法获得。
发明人利用乳腺癌细胞株 MDA-MB-435 S 进行了一系列实验验证了本发明公开的鼠 抗人 OPN单克隆抗体 1A12、人鼠嵌合抗体 C1A12及人源化抗体 hlA12对肿瘤转移的抑 制作用。细胞贴壁实验结果显示抗 hOPN抗体可以有效阻断 MDA-MB-435 S细胞和 hOPN 的结合， 细胞侵袭实验结果表明抗 hOPN抗体可有效阻断 MDA-MB-435 S于 hOPN存在 时进行的基底膜侵袭， 损伤划痕实验结果显示抗 hOPN抗体可以有效抑制细胞损伤划痕 的修复， 软琼脂克隆形成实验结果显示抗 hOPN抗体可以抑制 MDA-MB-435 S细胞在软 琼脂上克隆形成的大小， 相应地， 作为对照的无关抗体则没有上述作用， 从而说明了本 发明公开的上述抗 hOPN抗体可有效抑制转移肿瘤灶的形成的作用。
本发明人还利用人血管内皮细胞 HUVEC 进行了一系列实验， 验证本发明的鼠抗人 OPN单克隆抗体 1A12、 人鼠嵌合抗体 C1A12及人源化抗体 hlA12对肿瘤血管生成的抑 制作用。 3H掺入实验显示抗 hOPN抗体可有效抑制内皮细胞的增殖， HUVEC管状结构 实验结果表明抗 hOPN 抗体可以抑制血管内皮细胞在体外形成血管， 鸡胚绒毛尿囊膜 (CAM)血管生成实验和兔角膜新生血管形成实验证实抗 hOPN抗体能够在体内抑制毛细 血管的生成； 相应地， 作为对照的无关抗体则没有上述作用， 从而说明了本发明公开的 上述抗 hOPN抗体可有效抑制肿瘤血管的形成。
同时， 本发明人还利用乳腺癌细胞株 MDA-MB-435 S在小鼠体内建立了小鼠乳腺癌 的原发肿瘤和乳腺癌肺转移动物模型，并在该模型上验证了抗 OPN单克隆抗体 1A12抑 制肿瘤发展、 抑制肿瘤转移以及抑制肿瘤内部血管形成的作用。 相应地， 作为对照的无 关抗体则没有上述作用， 从而说明了本发明公开的抗 hOPN抗体 1A12在体内也具有有 效的抑制肿瘤发展、 抑制肿瘤血管形成和阻断肿瘤转移的作用。
本发明人进一步利用噬菌体展示技术鉴定了抗 hOPN抗体 1A12作用的 OPN的功能 表位， 这个功能表位为 NAPS , 进一步阐明了 OPN分子的作用靶点。 更具体地， 本发明 的申请人通过单克隆抗体抗原表位的淘选、噬菌体克隆 ELISA和蛋白质印迹法鉴定测序 及序列分析推测出 OPN 的功能表位， 进一步通过噬菌体克隆与抗体结合能力分析选择 结合力最强的克隆合成了系列短肽， 通过这些短肽与特异性抗体的结合实验对此表位进 行了鉴定。 本发明的单克隆抗体及其制备
本文所用的术语 "单克隆抗体 (单抗)"指从一类基本均一的群体获得的抗体， 即该群 体中包含的单个抗体是相同的， 除少数可能存在的天然发生的突变外。 单克隆抗体高特 异性地针对单个抗原位点。而且， 与常规多克隆抗体制剂 (通常是具有针对不同决定簇的 不同抗体)不同， 各单克隆抗体是针对抗原上的单个决定簇。 除了它们的特异性外， 单克 隆抗体的好处还在于它们是通过杂交瘤培养来合成的， 不会被其它免疫球蛋白污染。 修 饰语 "单克隆"表示了抗体的特性， 是从基本均一的抗体群中获得的， 这不应被解释成 需要用任何特殊方法来生产抗体。
如本文所用， 术语 "抗体"或 "免疫球蛋白"是有相同结构特征的约 150000道尔顿 的异四聚糖蛋白， 其由两个相同的轻链 (L)和两个相同的重链 (H)组成。 每条轻链通过一 个共价二硫键与重链相连， 而不同免疫球蛋白同种型的重链间的二硫键数目不同。 每条 重链和轻链也有规则间隔的链内二硫键。 每条重链的一端有可变区 (VH)， 其后是多个恒 定区。 每条轻链的一端有可变区 (VL)， 另一端有恒定区； 轻链的恒定区与重链的第一个 恒定区相对， 轻链的可变区与重链的可变区相对。 特殊的氨基酸残基在轻链和重链的可 变区之间形成界面。
如本文所用， 术语 "可变" 表示抗体中可变区的某些部分在序列上有所不同， 它形 成了各种特定抗体对其特定抗原的结合和特异性。 然而， 可变性并不均匀地分布在整个 抗体可变区中。 它集中于轻链和重链可变区中称为互补决定区 (CDR)或超变区中的三个 片段中。 可变区中较保守的部分称为构架区 (FR)。 天然重链和轻链的可变区中各自包含 四个 FR区， 它们大致上呈 β-折叠构型， 由形成连接环的三个 CDR相连， 在某些情况下 可形成部分 b折叠结构。每条链中的 CDR通过 FR区紧密地靠在一起并与另一链的 CDR 一起形成了抗体的抗原结合部位（参见 Kabat等， NIH Publ. No. 91-3242, 卷 I， 647-669页 (1991))。 恒定区不直接参与抗体与抗原的结合， 但是它们表现出不同的效应功能， 例如 参与抗体的依赖于抗体的细胞毒性。
脊椎动物抗体 (免疫球蛋白）的"轻链"可根据其恒定区的氨基酸序列归为明显不同的 两类 (称为 κ和 λ)中的一类。 根据其重链恒定区的氨基酸序列， 免疫球蛋白可以分为不同 的种类。 主要有 5类免疫球蛋白： IgA, IgD, IgE, IgG和 IgM， 其中一些还可进一步分成 亚类（同种型）， 如 IgGl, IgG2, IgG3, IgG4, IgA和 IgA2。对应于不同类免疫球蛋白的重链 恒定区分别称为(¾、 δ、 ε、 γ、 和 。 不同类免疫球蛋白的亚单位结构和三维构型是本领 域人员所熟知的。
例如，本发明单克隆抗体的重链可变区氨基酸序列优选选自： SEQ ID NO:4或 SEQ ID NO: 19, 轻链可变区氨基酸序列优选选自： SEQ ID NO:6或 SEQ ID NO:21， 而恒定区可 选自小鼠抗体恒定区或人抗体恒定区， 例如小鼠 IgG。
在本发明的一个实施方式中， 重链可变区中的 CDR1-3 氨基酸区序列分别选自：
GYTFTTYVMH， YINPYNDGSKYNEKFKG 禾 P HYGGSPAY(可参见 附 图 4 的 HlA12VHb); 轻链可变区的 CDR1-3 氨基酸序列分别选自： RSSQSLVHSNGNTYLH, KVSNRFS禾 P SQSTHVPWT (可参见图 4的 HlA12VLb)。
单克隆抗体可用本领域技术人员熟知的各种方法来制得。 例如， 单克隆抗体可用杂 交瘤方法（由 Kohler等， Nature, 256:495(1975)首先提出）制得， 或用重组 DNA方法 (美国 专利 No. 4,816,567)制得。单克隆抗体也可用例如 Clackson等， Nature, 352:624-628(1991) 禾口 Marks等， J. Mol. Biol., 222:581-597(1991；)所述的技术从噬菌体抗体库中分离获得。
本发明的单克隆抗体也可以是嵌合抗体或人源化抗体。 在本发明中， 如果没有特别 说明， " 1A12 "表示鼠抗 hOPN单抗 1A12， " cl A12"表示人鼠嵌合抗 hOPN单抗 cl A12， " hlA12 "表示人源化抗 hOPN单抗 hlA12。 clA12的重链可变区氨基酸序列为 SEQ ID N0.4, 轻链可变区氨基酸序列为 SEQ ID NO.6， 恒定区为人抗体恒定区； hlA12的重链 可变区氨基酸序列为 SEQ ID N0.19, 轻链可变区氨基酸序列为 SEQ ID N0.21， 恒定区 为人抗体恒定区。
本发明还包括具有所述的抗 hOPN单克隆抗体的相应氨基酸序列的单克隆抗体、 具 有所述的抗 hOPN单克隆抗体可变区链的单克隆抗体， 以及具有这些链的其它蛋白质或 蛋白质偶联物及融合表达产物。 具体地， 本发明包括具有含超变区 (互补决定区， CDR) 的轻链和重链的任何蛋白质或蛋白质偶联物及融合表达产物 (即免疫偶联物及融合表达 产物)， 只要该超变区与本发明的轻链和重链的超变区相同或至少 90%同源性， 较佳地 至少 95%同源性。 如本领域技术人员所知， 免疫偶联物及融合表达产物包括： 药物、 毒 素、 细胞因子 (cytokine) 放射性核素、 酶和其他诊断或治疗分子与所述的抗 hOPN单克 隆抗体或其片段结合的而形成的偶联物。 本发明还包括与所述的抗 hOPN单克隆抗体或 其片段结合的细胞表面标记物或抗原。
本发明不仅包括完整的单克隆抗体， 还包括具有免疫活性的抗体片段， 如 Fab 或 (Fab')2片段； 抗体重链； 抗体轻链。 抗 hOPN单克隆抗体或其片段的编码分子、 含该分子的表达载体及宿主细胞
本发明还提供了编码所述的抗 hOPN单克隆抗体或其片段的 DNA分子。 这些 DNA 分子的序列可以用常规技术， 比如利用 PCR扩增或基因组文库筛选等方法获得。 此外， 还可将轻链和重链的编码序列融合在一起， 形成单链抗体。 例如， 本发明的 DNA分子 可包含编码重链可变区的核苷酸序列 SEQ ID NO:3或 SEQ ID NO: 18； 编码轻链可变区 的核苷酸序列 SEQ ID N0 5或 SEQ ID NO:20。 例如， 编码 clA12重链可变区的核苷酸 序列如 SEQ ID N0.3所示， 轻链可变区氨基酸序列如 SEQ ID N0.5所示； 编码 hlA12 重链可变区的核苷酸序列如 SEQ ID NO.18 所示， 轻链可变区氨基酸序列如 SEQ ID NO.20 所示； 本发明还公开了含有上述核苷酸序列表达载体， 为 pcDNA3.1/ZEC +)和 pcDNA3.1 (+)； 本发明还公开了被上述表达载体转化的宿主细胞为 COS、 CHO细胞。
一旦获得了有关的序列， 就可以用重组法来大批量地获得有关序列。 这通常是将其 克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外， 还可用人工合成的方法来合成有关序列， 尤其是片段长度较短时。 通常， 通 过先合成多个小片段， 然后再进行连接可获得序列很长的片段。
目前， 已经可以完全通过化学合成来得到编码所述的本发明的抗 hOPN单克隆抗体 (或其片段， 或其衍生物)的 DNA序列。 然后可将该 DNA序列引入本领域中已知的各种 现有的 DNA分子 (或如载体)和细胞中。此外， 还可通过化学合成将突变引入本发明蛋白 序列中。
本发明还涉及包含上述的适当 DNA序列以及适当启动子或者控制序列的载体。这些 载体可以用于转化适当的宿主细胞， 以使其能够表达蛋白质。宿主细胞可以是原核细胞， 如细菌细胞； 或是低等真核细胞， 如酵母细胞； 或是高等真核细胞， 如哺乳动物细胞。
本发明还提供了可生产本发明抗 hOPN单克隆抗体的杂交瘤细胞系； 优选的， 本发 明提供了高效价的抗 hOPN单克隆抗体的杂交瘤细胞系。
在获得生产本发明的抗 hOPN单克隆抗体的杂交瘤， 本领域人员可很方便地获知本 发明的抗体的结构 （比如抗体的重链可变区和轻链可变区） ， 然后可通过以下方法来制 备本发明的单克隆抗体。
首先， 提供含有编码本发明单克隆抗体的核苷酸序列以及与该序列操作性相连的表 达调控序列的表达载体。
本文所用的术语 "表达调控序列" 通常指参与控制核苷酸序列表达的序列。 表达调 控序列包括与目标核苷酸序列操作性相连的启动子和终止信号。 它们通常还包括核苷酸 序列适当翻译所需的序列。 "操作性相连" 是指线性 DNA序列的某些部分能够影响同 一线性 DNA序列其他部分的活性。 例如， 如果启动子或增强子增加了编码序列的转录， 则它与编码序列是操作性相连的。
编码本发明单克隆抗体的 DNA序列可用本领域技术人员熟知的常规手段来制得。例 如，可根据本发明公开的序列人工合成或用 PCR法扩增得到编码该单克隆抗体重链可变 区和轻链可变区的核苷酸序列。 然后， 用本领域熟知的各种方法通过选择合适的酶切位 点将这些核苷酸序列插入合适的表达载体中， 使它们分别在表达载体所携带的重链恒定 区编码序列和轻链恒定区编码序列之前， 并使它们在同一阅读框内。 本发明中所用的表 达载体是本领域技术人员已知的各种市售的表达载体， 例如 pPICZo pPIC9K。
随后， 用上述获得的表达载体转化合适的宿主细胞。 "宿主细胞"一般包括原核细 胞和真核细胞。 常用的原核宿主细胞的例子包括大肠杆菌、 枯草杆菌等。 常用的真核宿 主细胞包括酵母细胞， 昆虫细胞、 和哺乳动物细胞。 在本发明中， 优选哺乳动物细胞。 通常用哺乳动物细胞系来作为表达真核细胞衍生多肽的宿主细胞。 哺乳动物细胞在培养 物中的繁殖是本领域熟知的。 见《组织培养》 ， Academic Press, Kruse and Patterson编辑
(1973), 该文纳入本文作为参考。 较佳的哺乳动物细胞是许多可购得的无限增殖细胞系。 这些无限增殖细胞系包括但不局限于， 中国仓鼠卵巢 (CHO)细胞、 Vero细胞、 Hela海拉 细胞、 幼仓鼠肾 (BHK)细胞、 猴肾细胞 (COS)、 人肝细胞癌细胞 (如 Hep G2)和其它许多 细胞系。 它们为蛋白质分子提供了翻译后修饰， 包括正确的折叠、 正确的二硫键形成以 及正确位点的糖基化。
用表达载体转化宿主细胞的方法有很多种， 所用的转化程序取决于待转化的宿主。 将异源多核苷酸导入哺乳动物细胞中的方法是本领域所知的， 其包括葡聚糖介导的转 染、 磷酸钙沉淀、 Polybrene(l,5-二甲基 -1,5-二氮 ^一亚甲基聚甲溴化物)介导转染、 原生 质体融合、 电穿孔、 脂质体介导转染以及将 DNA直接显微注射到胞核中。 在本发明中， 较佳的方法是电穿孔法或脂质体介导法等。 例如可采用 Invitrogen公司的脂质体法试剂 盒来转染诸如 COS、 CHO细胞等宿主细胞。
然后， 在适合本发明单克隆抗体表达的条件下， 培养转化所得的宿主细胞。 然后用 常规的免疫球蛋白纯化步骤， 如蛋白 A-Sephar0Se、 羟基磷灰石层析、 凝胶电泳、 透析、 离子交换层析、 疏水层析、 分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯 化手段纯化得到本发明的人源抗 HOPN单克隆抗体。 单克隆抗体的鉴定、 表达及纯化
所得单克隆抗体可用常规手段来鉴定。 比如， 单克隆抗体的结合特异性可用免疫沉 淀或体外结合试验 (如放射性免疫测定 (RIA)或酶联免疫吸附测定 (ELISA))来测定。 单克 隆抗体的结合亲和力例如可用 Munson等， Anal. Biochem., 107:220(1980)的 Scatchard分析 来测定。
本发明的抗 hOPN单克隆抗体可在细胞内、 或在细胞膜上表达、 或分泌到细胞外。 如果需要， 可利用其物理的、 化学的和其它特性通过各种分离方法分离和纯化重组的蛋 白。 这些方法是本领域技术人员所熟知的。 这些方法的例子包括但并不限于： 常规的复 性处理、 用蛋白沉淀剂处理 (盐析方法；)、 离心、 渗透破菌、 超声处理、 超离心、 分子筛 层析 (凝胶过滤)、 吸附层析、 离子交换层析、 高效液相层析 (HPLC)和其它各种液相层析 技术及这些方法的结合。 药物组合物
本发明还提供了一种治疗肿瘤的药物组合物， 该组合物含有药学上有效量的本发明 单抗或其免疫偶联物以及药学上可接受的载体。
本文所用的术语 "药学上可接受的" 是指当分子本体和组合物适当地给予动物或人 时， 它们不会产生不利的、 过敏的或其它不良反应。本文所用的 "药学上可接受的载体" 应当与本发明的活性物质相容， 即能与其共混而不会在通常情况下大幅度降低药物组合 物的效果。这些载体是本领域普通技术人员所熟知的。在《雷明顿药物科学》 (Remington's Pharmaceutical Sciences, Mack Pub. Co. , N.J. 1991年） 中可找到关于药学上可接受的 载体的充分讨论。
这类载体包括 (但并不限于)： 盐水、 缓冲液、 葡萄糖、 水、 甘油、 乙醇、 佐剂、 及其 组合。 另外， 这些载体中还可能存在辅助性的物质， 如润湿剂或乳化剂、 pH 缓冲物质 等。
本发明的组合物可通过口服以及静脉内、 肌内或皮下等途径给药； 优选的是口服或 静脉内注射给药。
通常， 在本发明的药物组合物中， 本发明单克隆抗体或免疫偶联物有效成分占组合 物总重量的 0.00001〜99.9% ; 优选 0.0001-90wt%， 更优选的 0.001-75wt%， 更优选
0.01-50wt%。 余量为药学上可接受的载体以及其它添加剂等物质。
本发明的药物组合物可根据需要制成各种剂型， 并可由医师根据患者种类、 年龄、 体重和大致疾病状况、 给药方式等因素确定对病人有益的剂量进行施用。 给药方式例如 可以采用灌注和其它治疗方式。
使用药物组合物时， 是将安全有效量的抗 hOPN单克隆抗体或免疫偶联物施用于哺 乳动物， 其中该安全有效量通常约 0.1微克 -5毫克 /千克体重， 而且在大多数情况下不超 过约 3毫克 /千克体重， 较佳地该剂量是约 1-10微克 /千克体重-约 1毫克 /千克体重。 当 然， 具体剂量还应考虑给药途径、 病人健康状况等因素， 这些都是熟练医师技能范围内 的。
本发明的单克隆抗体或免疫偶联物可抑制肿瘤的黏附和迁移、 防止肿瘤细胞向机制 的侵润、 促进肿瘤细胞的凋亡， 因此本发明的活性物质可用于治疗很多种类的肿瘤， 所 述的肿瘤比如 (包括但不限于 腺癌、 白血病、 淋巴瘤、 黑色素瘤、 肉瘤； 来自肾上腺、 胆囊、 骨、 骨髓、 脑、 乳腺、 胆管、 胃肠道、 心脏、 肾脏、 肝脏、 肺、 肌肉、 卵巢、 胰 腺、 甲状旁腺、 阴茎、 前列腺、 皮肤、 唾液腺、 脾脏、 睾丸、 胸腺、 甲状腺或子宫的肿 瘤组织； 中枢神经系统的肿瘤， 如胶质细胞多样性瘤、 或星细胞瘤； 眼部肿瘤 (例如基底 细胞癌、 鳞状细胞癌或黑色素瘤；)、 内分泌腺肿瘤、 神经内分泌系统肿瘤、 胃肠道胰腺内 分泌系统肿瘤， 生殖系统肿瘤或头颈部肿瘤。
本发明的药物组合物还可包含其它抗肿瘤活性物质， 或可与其它抗肿瘤活性物质联 合使用， 以获得更佳的治疗效果。 所述其它抗肿瘤活性物质包括但不限于： TNF-0 TGF-β, IFN-o 血管它丁、 内皮它丁、 甘磷酰芥、 血卟啉、 石蒜碱内铵盐、 鸦胆子乳、 足叶乙甙、 脱水卫矛醇、 阿霉素、 三苯氧胺、 5-氟尿嘧啶、 去甲斑螯素、 双呋喃氟尿嘧 啶、 葫芦素、 三尖杉酯碱、 冬凌草乙素、 马蔺子甲素、 云芝糖肽、 阿糖胞苷、 卡波铂、 紫杉醇、 香菇多糖、 氟他胺、 异环磷酰胺、 乌苯美司、 醋酸亮丙瑞林、 脱氧氟尿苷、 洛 波铂、 依林诺特肯、 来屈唑或替尼泊甙等。
当两种或两种以上的药物联合给药时， 一般具有优于两种药物分别单独给药的效果。 优选地， 联合施用的药物或其它制剂不干扰本发明单克隆抗体的治疗活性。 hOPN检测试剂盒
本发明还提供了一种检测 hOPN的试剂盒， 它含有所述的抗 hOPN单克隆抗体或其 活性片段、 免疫偶联物。 可采用本发明的试剂盒来检测生物样品中是否存在 hOPN或其 含量， 该检测方法包括步骤： （a)将样品与本发明试剂盒中的抗 hOPN单克隆抗体或其免 疫偶联物接触； （b)检测是否形成抗原-抗体复合物， 其中形成复合物就表示样品中存在 骨桥蛋白或定量检测所形成的抗原-抗体复合物的量以反映样品中 hOPN的含量。所述样 品可为经过预处理或未经过预处理， 例如可经过提取、 纯化或浓缩等。
所述试剂盒含有容器以及位于容器内的本发明的单克隆抗体、 或者带有所述单克隆 抗体的检测板， 以及使用说明书。 该试剂盒中还可含有检测所需的其它试剂， 例如缓冲 液、 指示剂等。 本领域技术人员可根据具体需要对试剂盒的内容物进行调整。 实施例
以下结合实施例、 实验例进一步对本发明进行说明， 这些实施例、 实验例不应理解 为对本发明的限制。 实施例不包括对传统方法的详细描述， 如那些用于构建载体和质拉 的方法， 将编码蛋白的基因插入到这样的载体和质粒的方法或将质粒引入宿主细胞的方 法以及经典的细胞融合和单克隆抗体筛选及纯化的方法等。 这样的方法对于本领域中具 有普通技术的人员是众所周知的， 并且在许多出版物中都有所描述， 包括 Sambrook, J., Fritsch, E. F. 禾口 Maniais, T . (1989) Molecular Cloning: A Laboratory Manual, (《分子 克隆： 实验室指南》第 2版，冷泉港实验室出版社， Cold spring Harbor Laboratory Press；)。 除非另外说明， 否则百分比和份数按重量计算。 本发明实施例或实验例中未标明来源的 原、 辅料均为市售。 实施例 抗 OPN单克隆抗体的制备
实施例 人 OPN cDNA片段的克隆
参照 GENEBANK提供的人 OPN的资料及序列， 合成引物：
有义弓 I物（弓 I物 AGCTTI (Hind
III)(SEQ ID NO :8)
OPN反义引物（引物
(； Kpn I)(SEQ ID NO:9)。
扩增人肝癌细胞株 LM3(购自上海中山医院），用 TRISOL试剂盒 (INVITROGEN)提取 通过 分钟； 秒， 秒， 秒， 个循环； 分钟， 获得 963bp的 DNA片段。 通过凝胶回收试剂盒 (上海生工)回收 片段后， 用限制型内切酶 Hind III和 Kpn l酶切， 凝胶电泳回收后， 与经 Hind III和 Κρη 双酶切的质粒载体连接， 转化大肠杆菌 后， 筛选获得有插入片段的阳性克隆。 DNA序列测定确认， ΟΡΝ基因的核苷酸序列如 SEQ ID NO: l 所示。 实施例 2. 鼠 OPN cDNA片段的克隆
参照 GENEBANK提供的鼠 OPN的资料及序列， 合成引物：
鼠 OPN 有义弓 I物（弓 I物 3): AT IAAGCTTI GG AT G AC G AC G AC A AG AT GAG A A
TTGCAGTGATT (Hind III)(SEQ ID NO: 10)
鼠 OPN反义引物（引物 4): AT ICTCGAQ TTAATTGACCTCAGAAGA
(Kpn I)(SEQ ID NO: 11)。
分离鼠脾脏 T淋巴细胞，用 ConA激活 30小时后，用 TRISOL试剂盒 (INVITROGEN) 提取 RNA，通过 RT-PCR(PROMEGA)PCR反应采用热启动，反应条件： 94°C分钟； 94°C 45秒， 55°C 30秒， 72°C 45秒， 30个循环； 72°C 10分钟。 获得 932bp的 DNA片段。 通过凝胶回收试剂盒 (上海生工)回收片段后， 用限制型内切酶 Hind III和 Kpn l酶切， 凝 胶电泳回收后，与经 Hind III和 Kpn I双酶切的质粒载体连接，转化大肠杆菌 DH10B后， 筛选获得有插入片段的阳性克隆。 DNA序列测定确认。鼠 OPN基因的核苷酸序列如 SEQ ID NO:2所示。 实施例 3. 人、 鼠 OPN的真核细胞表达纯化
将实施例 2所得的序列正确的人、 鼠 OPN基因片段用相应的限制性内切酶酶切回收 后， 装入 pPICZ a质粒中。 转染毕式酵母细胞， 挑取单个克隆酵母， 诱导表达出人、 鼠 OPN蛋白， 收集酵母细胞表达上清， 经阴离子交换、 分子筛纯化， SDS-PAGE证实得到 人、 鼠 OPN纯蛋白。 纯化后经 SDS-PAGE电泳， 结果见图 1。 实施例 4. 鼠抗人 OPN单克隆抗体的筛选与制备
将 100μ§人 OPN和等体积弗氏佐剂乳化后， 腹腔注射 BALB/C小鼠免疫接种， 每二 周后加强免疫一次， 剂量均同第一次免疫， 共免疫 3次后， 选取血清中抗 0ΡΝ抗体滴 度较高小鼠， 分离其脾脏淋巴细胞， 利用经典的 PEG法， 将小鼠脾脏淋巴细胞与 NS-1 细胞进行细胞融合。 用 10μ§/ιη1 人 ΟΡΝ包被 96孔板， 采用 ELISA法反复筛选获得可 稳定表达抗 hOPN抗体的杂交瘤细胞株—— 1A12。 大量扩增单克隆细胞株 1A12,腹腔注 射 BALB/C小鼠 5 X 106/只， 10天左右开始收集小鼠腹水， 利用 Protein A柱， 亲和层析 纯化抗 hOPN的单克隆抗体。
蛋白质印迹试验结果显示， 小鼠抗 hOPN单克隆抗体 1A12不但与人 OPN蛋白特异 结合， 同时与鼠 OPN蛋白也有交叉反应。 结果见图 2。 实施例 5. 鼠抗人 OPN无关对照单克隆抗体 23C3D3的筛选与制备
将 100μ§人 OPN和等体积弗氏佐剂乳化后， 腹腔注射 BALB/C小鼠免疫接种， 每二 周后加强免疫一次， 剂量均同第一次免疫， 共免疫 3次后， 选取血清中抗 ΟΡΝ抗体滴 度较高小鼠， 分离其脾脏淋巴细胞， 利用经典的 PEG法， 将小鼠脾脏淋巴细胞与 NS-1 细胞进行细胞融合。 用 10μ§/ιη1 KLH-WLVPDP(上海业力公司合成)包被 96孔板， 采用 ELISA 法反复筛选获得可稳定表达抗人 OPN 特异表位抗体的杂交瘤细胞株—— 23C3D3。 大量扩增该单克隆细胞株,腹腔注射 BALB/C小鼠 5 106个/只， 10天左右开 始收集小鼠腹水， 利用蛋白 A柱， 亲和层析纯化抗人 OPN特异表位的无关对照单克隆 抗体 23C3D3 o 实验例 6. 1A12单抗的可变区基因克隆和序列测定
收集 1A12的 5 X 106〜1 X 107个杂交瘤细胞；用 TRIzol(Invitrogen目录号： 15596-026) 抽抽提提其其总总 RRNNAA，， 根据小鼠抗体恒定区序列， 设计引物如下：
HGSP1 GATACTGTGATCTGTTTG-3 ' (SEQ ID NO: 12)
HGSP2 TCGCAGATGAGTCTGGAC-3 ' ( SEQ ID NO: 13)
HGSP3 ATGAACACACTCACATTG-3 ' ( SEQ ID NO: 14)
LGSP1 GAGGTTATGACTTTCATAGTCAGC-3 ' ( SEQ ID NO: 15)
LLGGSSPP22: 5 ' -AACACTGTCCAGGACACCATCTCG-3 ' ( SEQ ID NO: 16)
LGSP3 TCTGGGATAGAAGTTGTTCATGAG-3， ( SEQ ID NO: 17)
采用 Invitrogen 5， RACE试剂盒（目录号： 18374-058)， 分别以 HGSP1、 LGSP1为弓 I 物， 合成第一链 cDNA; 在 TdT和 dCTP作用下， 给第一链 CDNA 3 ' 端加 poly C尾； 分别以 HGSP2、 HGSP3 LGSP2、 LGSP3为 5 ' 引物， 通过巢式 PCR得到 VH、 VL的 PCR产物； 将 PCR产物装入 pGEM-T 载体中； 挑取克隆抽提质粒， 限制性内切酶鉴定 得到阳性克隆， 通过测序确定其序列。 1A12单抗的重链可变区的核苷酸序列为 SEQ ID N0 3 , 氨基酸序列为 SEQ ID NO :4, 1A12单抗的轻链可变区的核苷酸序列为 SEQ ID N0 5 , 氨基酸序列为 SEQ ID NO:6。 实施例 7. 人抗体轻、 重链恒定区基因的克隆
用淋巴细胞分离液 (鼎国生物技术发展公司产品)分离健康人淋巴细胞，用 Trizol试剂 (Invitrogen公司产品）提取总 RNA，根据文献 (Cell, 1980,22: 197-207)和文献 (Nucleic Acids Research, 1982， 10： 4071-4079)报道的序列分别设计引物采用 RT-PCR反应扩增抗体重 链和轻链恒定区基因。 PCR产物经琼脂糖凝胶电泳纯化回收并克隆到 pGEM-T载体中， 测序验证后确认获得了正确的克隆。 SEQ ID NO:22和 SEQ ID NO:23分别显示了重链恒 定区 CCH)的核苷酸序列和氨基酸序列。 SEQ ID NO:24和 SEQ ID NO:25分别显示了轻链 恒定区 (CL)的核苷酸序列和氨基酸序列。 将本实施例中的正确克隆记作 pGEM-T/CH和 pGEM-T/CL。 实施例 8. 抗 hOPN嵌合抗体 C1A12的构建
以 1A12重链可变区基因 1A12VH和 pGEM-T/CH载体为模板通过重叠 PCR合成嵌 合抗体重链基因， 反应条件为： 95 °C 15分钟； 94 °C 50秒， 58 °C 50秒， 72 °C 50秒， 30个循环； 72 °C 10分钟。 并使此嵌合重链基因的 5'端含有限制酶位点 Hindlll和信号肽 基因序列， 3 '端含有翻译终止密码 TAA 和限制酶位点 EcoR l 。 信号肽基因序列为 TCCAGAGGA (SEQ ID NO: 26) 0 最后琼脂糖凝胶电泳分离 PCR扩增产物， 回收目的条 带并克隆到 pGEMT 载体中，筛选阳性克隆测序。挑选测序正确的克隆用 Hindlll和 EcoR I酶切，经琼脂糖凝胶电泳纯化回收嵌合抗体重链片段 C1A12VHCH, 与 Hindlll和 EcoR I双酶切的质粒 pcDNA3.1(+) (美国 Invitrogen公司产品)进行连接，构建成嵌合重链真核 表达载体 pcDNA3.1 (+) (clA12VHCH) 0
以 1A12轻链可变区基因 1A12VL和 pGEM-T/CL载体为模板通过重叠 PCR合成嵌合 抗体轻链基因， 反应条件为： 95 °C 15分钟； 94 °C 50秒， 58 °C 50秒， 72 °C 50秒， 30 个循环； 72 °C 10分钟， 得到 PCR产物 C1A12VLCL, 其 5'端含有限制酶位点 Hindlll和 信号肽基因序列， 3 '端含有翻译终止密码 TAA和限制酶位点 EcoR I。 信号肽基因序列 TATCCAGAGGA(SEQ ID NO :26)。 经琼脂糖凝胶电泳纯化回收 PCR 产物并克隆到 pGEM-T载体 （Promega公司产品）中， 筛选阳性克隆测序。 挑选测序正确的克隆用 Hind III和 EcoR I双酶切， 经琼脂糖凝胶电泳纯化回收嵌合抗体轻链片段 C1A12VLCL, 与用 Hindlll和 EcoR I酶切的质粒 pcDNA3.1/ZEO(+)载体 (美国 Invitrogen 公司产品;)进行连 接， 构建成嵌合轻链真核表达载体 pcDNA3. 1/ZEO(+;> (cl A12VLCL；)。
将构建成功的嵌合重链和轻链表达载体脂质体法共转染 COS- 1 细胞 （ATCC CRL
1650)， 72小时后取培养上清进行分析， 采用 ELISA确定培养上清中嵌合抗体 C1A12的 含量： 羊抗人 IgGCFc)包被 ELISA板， 用 2%BSA-PBS于 37°C封闭 2小时， 加入待测的 培养上清和标准品 (Human myeloma IgGl,k) , 37°C 孵育 2小时， 加入 HRP-羊抗人 κ进 行结合反应， 37°C 孵育 1小时， 加入 TMB于 37°C作用 5分钟， 最后用 H2S04终止反 应， 测 OD450值。 实施例 9. 鼠源 1A12单抗可变区 (Fv)三维结构的同源模建
利用 Accelrys公司的 Insight II程序包来模拟 1A12鼠源单抗可变区的三维结构。 首 先， 用 BLAST程序在蛋白质结构数据库 (Protein Data Bank, PDB)中分别搜索 1A12重 链和轻链可变区蛋白的模板蛋白。选取同源性最高的抗体 1PLG作为 1A12的模建模板， 利用 Insight II程序模建出 1A12的三维结构（图 3)。 实施例 10. 抗 hOPN人源化抗体 MA12的设计与构建
用 BLAST程序在 Genbank数据库中分别搜索与 1A12轻链和重链可变区的最相似人 源模板。 与 1A12 重链可变区同源性最高的人源抗体是人抗体 CAA79298.1 抗体 (emb|CAA79298.1 |) , 相似度为 67%， 与 1A12 轻链可变区同源 最高的人源抗体是 BAC01734.1 (dbj |BAC01734.1 |) , 相似度为 81%。 因此， 分别采用 CAA79298.1 和 BAC01734.1作为 1A12重链和轻链人源化的模板。
首先将 1A12 的重链和轻链 CDR 区分别直接移植到人源模板 CAA79298.1 和
BAC01734.1 上， 构成 CDR移植抗体， 重链为 hlA12Ha， 轻链为 hlA12La。 hlA12Ha 和 hlA12La 的可变区序列如图 4 所示。 全基因合成人源化抗体重、 轻链可变区基因 (hlA12VHa和 hlA12VLa)，然后以 hlA12VHa基因和 pGEM-T/CH载体为模板通过重叠 PCR合成人源化抗体重链基因， 反应条件为： 95 °C 15分钟； 94°C 50秒， 58 °C 50秒， 72 °C 50秒， 30个循环； 72°C 10分钟。 并使此人源化重链基因的 5'端含有限制酶位点 Hindlll和， 肽基因序列， 3 '端含有翻译终止密码 TAA和限制酶位点 EcoR I。 信号肽
AGTCATAATATCCAGAGGA(SEQ ID NO:26)最后琼脂糖凝胶电泳分离 PCR扩增产物， 回收目的条带并克隆到 pGEMT 载体中， 筛选阳性克隆测序。 挑选测序正确的克隆用 Hindlll和 EcoR I酶切，经琼脂糖凝胶电泳纯化回收人源化抗体重链片段 hlA12VHaCH， 与用 Hindlll和 EcoR I酶切的质粒 pcDNA3.1 (+X美国 Invitrogen公司产品;)进行连接， 构 建成人源化重链真核表达载体 pcDNA3.1 (+) (hlA12VHaCH；)。
以 hlA12VLa基因和 pGEM-T/CL载体为模板通过重叠 PCR合成人源化抗体轻链基 因， 反应条件为： 95 °C 15分钟； 94 °C 50秒， 58 °C 50秒， 72 °C 50秒， 30个循环； 72 °C 10分钟， 得到 PCR产物 hlA12VLaCL， 其 5'端含有限制酶位点 Hindlll和信号肽基因 序列， ， 3 '端含有翻译终止密码 TAA 和限制酶位点 EcoR l 。 信号肽基因序列为
TCCAGAGGA(SEQ ID NO:26)挑选测序正确的克隆用 Hindlll和 EcoR I酶切， 经琼脂糖 凝胶电泳纯化回收人源化抗体轻链片段 hlA12VLaCL， 与用 Hindlll和 EcoR I酶切的质 粒 pcDNA3.1/ZEO(+)载体 (美国 Invitrogen公司产品)进行连接， 构建成人源化轻链真核 表达载体 pcDNA3.1/ZEO(+) (hi A12VLaCL)。
将构建成功的人源化重链和轻链表达载体用脂质体法共转染 COS-1 细胞 （ATCC CRL 1650)， 72 小时后取培养上清进行分析， 采用 ELISA确定培养上清中人源化抗体 (hlA12Ha/ hlA12La)的含量： 羊抗人 IgG(Fc)包被 ELISA板， 用 2%BSA-PBS于 37°C封 闭 2小时， 加入待测的培养上清和标准品 (Human myeloma IgGl,k)， 37°C 孵育 2小时， 加入 HRP-羊抗人 κ进行结合反应， 37°C 孵育 1小时， 加入 TMB于 37°C作用 5分钟， 最后用 H2S04终止反应， 测 OD450值。
用 2μ§/ιη1 ΟΡΝ包被 ELISA板， 用 ELISA方法检测转染所得人源化抗体特异性结合 hOPN的活性， 结果发现与 C1 A12嵌合抗体相比， hlA12Ha和 hlA12La组成的人源化抗 体 (hlA12Ha/ hlA12La)的活性几乎完全丧失。 因此， 为了获得高亲和力的人源化抗体， 我们还需对可能影响 MA12抗体结合活性的 FR区鼠源残基进行分析和回复突变。 通过 分析模建的 MA12单抗可变区的三维结构（图 3)，我们发现在 CDR区周围 5 A的空间范 围内可能影响原抗体 CDR 构象而又与人源模板中相应位置不同的 FR区残基有 9个， 分别为 L3Leu, L45Lys, L46Leu, H24Ser, H38Lys, H48Ile, H94Ser。 将这些鼠源氨基酸残 基保留在构建的 CDR移植抗体中可得到人源化抗体 ChlA12Hb/ hlA12Lb)。 hlA12Hb和 hlA12Hb的可变区氨基酸序列如图 4所示。 SEQ ID NO: 18和 SEQ ID NO: 19分别显示了 hlA12Hb的可变区核苷酸序列和氨基酸序列。 SEQ ID NO :20和 SEQ ID NO:21分别显示 了 hlA12Lb的可变区核苷酸序列和氨基酸序列。分别全基因合成人源化抗体重、轻链可 变区基因（hlA12VHb/ hlA12VLb；)，并按与人源化抗体 (hi A12Ha/ hi A12La)相同的方法构 建轻链表达载体 pcDNA3.1/ZEO(+) (hi A12VLbCL)和重链表达载体 pcDNA3.1 (+) (hlA12VHbCH)。 然后将轻、 重表达载体共转染 COS-1细胞， 用 ELISA方法测定抗体的 抗原结合活性， OPN蛋白（2 g/ml)包被于 ELISA板， 4°C过夜， 用 2%BSA-PBS于 37°C 封闭 2h， 加入待测的 hlA12培养上清, 37°C 温育 2h， 加入 HRP—兔抗人 IgG进行结合 反应， 37°C 温育 lh， 加入 TMB于 37°C作用 5分钟， 最后用 H2S04终止反应， 测 A450 和 A630值。 MA12特异性结合 OPN结果见图 5， 发现其与 OPN的结合活性与 clA12 嵌合抗体相似， 将这个人源化抗体 (hlA12Hb/ hlA12Lb;>命名为 hlA12。 实施例 1 1. 抗 hOPN人源化抗体 hlA12的稳定表达与纯化
将上述构建成功的 C1A12或 MA12的轻、 重链质粒用脂质体法共转染 CHO-K1 细 胞 (ATCC CRL-9618), 转染进行 24h 后细胞换含 600 g/ml G418 禾 P 250 g/ml Zeocin 的选择培养基筛选抗性克隆。取细胞培养上清用 ELISA检测筛选高表达克隆， 将筛选得 到的高表达克隆用无血清培养基扩大培养， 用 Protein A亲和柱 (GE公司产品)分离纯化 嵌合抗体 C1A12或人源化抗体 hlA12， 最后以紫外吸收法定量。 实验例 I. 抗 hOPN抗体肿瘤细胞作用效果实验
MDA-MB-435 sC购自中国科学院上海细胞学研究所）， 无关抗体 C23C3D3)。
实验例 1-1. 细胞贴壁实验
将 96孔板 (Greiner)以 lO g/ml的 hOPN (来自实施例 3)或 BSA(SIGMA)包被， 4°C过 夜； 用 1%BSA/PBS于 37°C封闭 1小时， 以阻断非特异性结合位点。 MDA-MB-435s细 胞用 0.2%EDTA消化，重悬于 0.25%BSA/DMEM中，调整细胞浓度为 5 X 105个细胞 /ml。 每孔加入 100 μΐ细胞， 处理组同时加入 25 g/ml的鼠抗人 ΟΡΝ抗体 1Α12、 人鼠嵌合抗 体 C1A12和人源化抗体 hlA12， 对照组加入 25 g/ml的无关抗体。 将细胞在 37°C细胞 培养箱中孵育 2小时后， 以 PBS洗 2遍， 洗去未贴壁的细胞。 每孔加入 100 μΐ 1%甲醛 于 4°C固定细胞 10分钟。 PBS洗涤之后， 每孔加入 ΙΟΟμΙ 0.5%的结晶紫室温 30分钟将 细胞染色。 每孔加入 50 μΐ 2%Triton X- 100裂解细胞， 在 OD 595nm处进行读数。
实验结果见图 6，抗 hOPN抗体在 25 g/ml可以有效阻断 MDA-MB-435s细胞和 hOPN 的结合， 1A12、 clA12和 hlA12间无显著差异， 而无关对照抗体则无此作用。 实验例 1-2. 细胞侵袭实验
细胞侵袭实验选用 Transwell小室系统 CCorning)进行， 膜孔径大小为 8 mm。 小室的 上层包被人工基底膜成分基质胶 (Matrigel), 在通风橱内吹干， 用 DMEM于 37 °C， 1小 时对其进行水化。 MDA-MB-435 S细胞用 0.2%EDTA进行消化，重悬于 0.25%BSA/DMEM 中， 调整细胞浓度为 5 X 105细胞 /ml。 在小室的上层加入 100 μΐ细胞悬液 (处理组同时加 入 25 g/ml的鼠抗人 OPN抗体 1A12、 人鼠嵌合抗体 clA12和人源化抗体 hlA12， 对照 组加入 25 g/ml的无关抗体；)， 在小室的下层加入 0.25%BSA/DMEM (；含或不含 OPN；)， 于 37°C细胞培养箱中孵育 24小时。 孵育结束， 用棉拭子将小室上层的细胞刮除， 穿过 基底膜至下层的细胞用 PBS洗涤之后，以 1%甲醛固定之后，以 0.5%的结晶紫进行染色， 200倍显微镜下观察， 计数每个视野下细胞的数量。
实验结果见图 7，抗 hOPN抗体在 25 g/ml即可有效阻断 MDA-MB-435s于 hOPN存 在时进行的基底膜侵袭， 而无关对照抗体则无此作用， 1A12、 C1A12和 MA12之间无显 著差异。 实验例 1-3. 损伤划痕实验
将 MDA-MB-435 S细胞在 12孔板中培养至接近饱和(>90%)， 用 PBS洗涤之后， 以无 血清 DMEM对细胞进行过夜血清饥饿。 用 10 μΐ移液器枪头对单层细胞进行划线， 用 PBS洗去漂起的细胞。加入 25 g/ml的鼠抗人 OPN抗体 1A12、人鼠嵌合抗体 clA12和 人源化抗体 hlA12， 对照组加入 25 μ§/ιη1的无关抗体， 对照组采用无关抗体处理。 将细 胞在 37°C细胞培养箱中孵育 24小时后， 结果以穿越基准线的细胞数表示。
实验结果见图 8， 抗 OPN单抗在 25 g/ml可以有效抑制细胞损伤划痕的修复， 而无 关对照抗体则无此作用。 1A12、 clA12和 hlA12之间无显著差异。 实验例 1-4. 软琼脂克隆形成实验
软琼脂克隆形成实验使用双层软琼脂系统进行。将 2.5%融化的琼脂粉与 37°C预温的 DMEM培养基混合， 配制 0.5%的琼脂粉溶液， 再用 DMEM稀释成浓度为 0.3%琼脂粉 溶液。 在 24孔细胞培养板每孔加入 500 μΐ 0.5%琼脂粉溶液， 置于 4 °C使其凝固后， 转 移至 37 °C细胞培养箱中保温。 MDA-MB-435 S细胞以 0.2%EDTA消化后， 用 0.3%琼脂 粉溶液重悬， 调整细胞浓度为 5 X 103细胞 /ml。 在 24孔板每孔中加入 500 μΐ细胞悬液， 并使其凝固。 从第二日起， 每隔 1 日向细胞培养板的各孔中加入 1A12、 clA12和 hlA12 或无关抗体 (23C3D3)(25 g/ml)处理， 3周后观察克隆形成的大小， 以>10个细胞聚集作 为 1个克隆。
实验结果见图 9， 抗 ΟΡΝ的 1A12抗体可以抑制 MDA-MB-435S细胞在软琼脂上克 隆形成的大小， 而对照抗体则无此作用。 1A12、 clA12和 hlA12之间无显著差异。 说明 该抗体可有效抑制转移肿瘤灶的形成， 且嵌合抗体和人源化抗体保持了鼠源单克隆抗体 的生物学功能。 实验例 II. 抗 OPN抗体抑制血管生成效果实验
实验例 II-1. 3H-TdR掺入法测定人血管内皮细胞 OiUVEC)增殖
通过 3H-TdR掺入法测定 HUVEC细胞增殖， 确定 OPN对细胞生存的保护作用， 以 及抗 OPN抗体对 OPN的保护作用的抑制。 96孔板每孔接种 2 X 104 HUVEC， 完全培养 基培养 24小时。 对细胞进行以下不同处理， 处理时间 24小时：
i. M200 + LCGS (补充培养物）
ii. M200 + 1% BSA
iii. M200 + 1%BSA + OPN
iv. M200 + 1%BSA + OPN + 鼠抗 OPN抗体 1 A12
v. M200 + 1%BSA + OPN + 人鼠嵌合抗 OPN抗体 clA12
vi. M200 + 1%BSA + OPN + 人源化抗 OPN抗体 hi A12
vii. M200 + 1%BSA + OPN + 无关抗体
24小时后： 加入 1 uCi/孔的 1H 胸苷， 37 °C培养 6小时， PBS洗涤 1遍。 冰冷的 10% 三氯乙酸 /H20，4 °C， 10 min，冰 ddH20洗涤 2遍。溶解细胞：每孔加入 0.1 ml 0.5 N NaOH, 0.5% SDS以溶解细胞， 室温 10 min。 每孔加入 0.2 ml 0.5 N HC1， 混匀， 多头细胞收集 器收集。
3H-TdR掺入法测定细胞增殖实验结果见图 10。 实验结果显示， 在 OPN存在的情况 下，其 3H-TdR掺入较 1%BSA处理组，增加了 3.08 ± 0.64倍；而加入抗 OPN抗体 1A12、 C1A12和 hlA12后， 3H-TdR掺入较 OPN组显著降低。 实验结果说明， OPN在血清撤除 情况下， 可以促进 HUVEC的生存， 而这一作用可以被抗 OPN抗体显著抑制 (P<0.01)。 实验例 Π-2. 1A12、 clA12及 MA12对毛细血管管状样结构生成的抑制功能
将 HUVEC在含有 LCGS的 M200培养基中培养至 80%融合； 实验前一天， 取出 -20
°。保存的基质胶， 置于 4°C自然融化， 3~12小时。 用预冷的枪头和 96孔板分装基质胶， 整个过程在冰上操作。 96孔板每孔加入 60 μΐ基质胶， 小心去除气泡， 在 37°C孵育 1小 时使胶固化。 每孔加入 100 μΐ HUVEC (2 X 104/孔)， 轻轻加至基质胶表面， 处理组同时 加入抗 hOPN抗体 1A12、 clA12和 hlA12。 在 37°C细胞培养箱中， 培养 12小时； 6小 时后观察毛细血管管状样结构生成的情况。
HUVEC管状结构实验结果见图 1 1。结果表明： 抗 hOPN抗体可以降低 HUVEC的管 状形成能力， 而对照抗体则无此效果。 1A12、 C1A12及 hlA12之间无显著差异。 实验例 Π-3. 1A12、 clA12及 MA12抑制鸡胚绒毛尿囊膜 (CAM)血管生成实验
来亨种鸡胚， 购自于受精率 >90%的种鸡场。 取 8日龄鸡胚， 消毒后， 用尖头手术刀 片在鸡胚顶端气室中央的位置戳一个小口， 小心的去掉周围的蛋壳和壳膜， 打开一个 1 cm X l cm的小孔， 将待检测物质 /明胶海绵加入至 CAM表面， 待检测物质成分如下： i) PBS ; ii)HOPN; iii)人 OPN(250 ng); iv)人 OPN(250 ng)十无关对照抗体（1 g); v)人 OPN(250 ng)十鼠抗 OPN抗体 1A12(1 g); vi)人 OPN(250 ng)十人鼠嵌合抗 OPN抗体 clA12(l μg)■, vii)人 OPN(250 ng)十人源化抗 OPN抗体 hlA12(l g)。
将待检测物质 /明胶海绵加入至 CAM表面， 远离已形成的致密血管网。 用灭菌透明 胶布封口，继续孵育 7^小时或更长时间。解剖显微镜下观察载体边缘 2 mm内血管数目， 分析血管生成表现， 采用 "血管指数"（Ribatti, D., B. Nico, A. Vacca, 等 The gelatin sponge-chorioallantoic membrane assay.《明胶海绵-续毛膜尿囊膜测试法》 Nat Protoc, 2006. 1(1)： 85-910, 解剖显微镜下以相同放大倍数计数血管， 各组标本血管计数结果以 "均数 士标准差 (X—士 S) "表示， 用多重比较方差分析的 q检验比较各组间差异， 采用 SPSS统 计软件进行。
实验结果如图 12所示， 人 OPN蛋白与 PBS相比， 能显著促进鸡胚 CAM新生血管 的生成， 微血管以明胶海绵为中心， 呈辐射状分布， 差别有显著统计学意义 (P<0.01)。 OPN促进血管生成的能力， 禾 P HOPN的作用相近。抗 OPN抗体 1A12和 2H8对 OPN诱 导的 CAM血管生成有显著的抑制作用 (P<0.01)。 实验例 Π-4. 1A12、 clA12及 MA12抑制兔角膜新生血管形成实验
在无菌条件下， 将 Hydron粉剂溶解于无水乙醇中， 终浓度为 12%(w/v)， 37 °C条件下 振荡使其溶解， 配制成为 12%的 Hydron铸型溶液， 保存于室温。 在无菌条件下， 将硫 糖铝粉末加入无菌 Milli Q水中， 配制成为终浓度为 100 μ§/μ1的混悬液， 保存于 4°C， 用前涡旋混匀。 每个缓释小丸含有 200 ng hOPN和 50 μ§硫糖铝， 抗体组同时含有 1 μ§ 抗 ΟΡΝ 抗体或无关对照抗体。 将 12%的 Hydron铸型溶液和 hOPN-硫糖铝 -PBS混合液 以 1 : 1等体积混匀， 涡旋振荡 1 min。 将 5 μΐ混合后的 Hydron-硫糖铝 -hOPN溶液加至 无菌的石蜡膜 (Parafilm)表面， 在超净台下干燥 30 min左右， 使其充分聚合。 用眼科镊 将其塑型成为直径为 2 mm， 大小均匀的圆形药丸。 保存于 -20°C。 按照每组 4只眼睛， 设置分组和对照同实验例 11-3。
以 3%戊巴比妥钠做耳缘静脉麻醉 (1 ml/kg体重；)， 1%丁卡因双眼角膜表面麻醉， 以 超声角膜测厚仪测量角膜厚度。用眼科手术切开刀在角膜中央做长约 3 mm的半厚切口， 用 2 mm宽的巩膜隧道刀向 12点或 3点方向角巩膜缘做潜行隧道，隧道位于角膜基质层 内， 隧道顶端距角膜缘约 l mm。 用显微镊将 Hydron/硫糖铝缓释小丸植入隧道顶端。 术 后眼裂涂金霉素眼药膏。 术后第 1 日起， 双盲观察者每日在裂隙灯下观察兔角膜新生血 管情况。 裂隙灯观察到第 10 日终止。 测量最长血管长度 (VL)及血管时钟角度 (CN， 30 度 = 1 CN)， 按照以下公式计算：
面积（mm2) = 0.2 X J! X VL (mm) X CN (mm)
实验结果如图 13所示。 1A12、 clA12及 hlA12处理组新生血管面积较中 OPN组显 著减少，新生血管长度较无关抗体组显著缩短，且面积显著缩小 (P<0.01)。而 1A12 C1 A12 及 hi A12组间无显著差异。 实验例 III. 鼠抗人 OPN抗体 1A12抑制小鼠体内肿瘤转移及血管形成实验
实验例 III-1. 鼠抗人 OPN抗体 1A12抑制小鼠体内肿瘤转移实验
消化 MDA-MB-435 S细胞，以无血清 DMEM重悬，调整细胞浓度至 5 X 107细胞 /1 ml。 每组 10只雌性裸鼠， 4〜6周龄， 戊巴比妥钠腹腔麻醉。 无菌条件下， 消毒皮肤， 在右 侧第二乳头处做 0.5-mm的切口， 分离皮下组织， 暴露乳腺脂肪垫； 用 25号针头吸取细 胞悬液， 接种体积为 100 μ1(5 Χ 106细胞 /只)注射至乳腺脂肪垫内部， 缝合皮肤。 将动物 随机分为 4组， 从接种肿瘤细胞次日开始， 分别接受如下治疗方案：
• 1A12 : 5 mg/kg, 每周 2次； • 对照抗体： 5 mg/kg， 每周 2次。
每周 1次观察裸鼠成瘤情况，用游标卡尺测量肿瘤的最大直径 (长度)和及其垂直距离 (宽度)， 肿瘤体积的计算公式为： 体积 = 0.52 X长度 X宽度 2。 以肿瘤体积变化对时间绘 制肿瘤生长曲线。 各处理组每组 12只实验动物， 其中 6只观察原位肿瘤生长大小， 至 10周终止； 另外 6只观察自发性肺转移， 至原发肿瘤大小达到 1000 mm3时终止。
接种后第 10周末， 脱颈处死小鼠。 取各组小鼠肿瘤组织， 10%中性福尔马林固定后 石蜡包埋， H&E染色后观察肿瘤组织和肺组织。通过显微镜下观察肺转移灶的大小和数 巨。
根据肿瘤大小对时间绘制肿瘤生长曲线。 结果显示如图 14， 至 10周时， 抗 OPN抗 体组 (1A12)肿瘤大小明显小于无关抗体治疗组 (P<0.05)。 在出现肺转移灶的裸鼠中， 肺 转移灶组织切片见图 15， 从结果可以看出， 抗 OPN抗体 1A12处理后， 不仅肺转移的 数目减少， 而且转移灶较小； 对照抗体处理组的肺组织中， 常见较大的转移灶， 且很多 转移灶融合成为较大的转移灶。 实验例 ΠΙ-2. 鼠抗人 OPN抗体 1A12对肿瘤内部微血管密度 (MVD)的影响
取小鼠原位肿瘤组织， 取材后进行液氮速冻」 恒冷箱冰冻切片机内， 8 um连续切片。 取出后晾干 5 min， -20 °C丙酮固定 30 min， 进行 IHC染色。 大鼠抗小鼠 CD3 1单克隆抗 体 (；工作浓度 1 : 100) , 4 °C孵育过夜； 二抗为羊抗大鼠 IgG/PE (；工作浓度 1 : 100)。 Hochest33258衬染， 荧光显微镜下拍照。
CD3 1蛋白主要表达于血管内皮细胞膜上， 单个血管内皮细胞及微血管都有 CD31蛋 白表达。 用抗 CD31抗体可以观察到肿瘤内微血管（图 16)， 镜下观察结果可见， 抗 OPN 抗体 1A12处理组中， MVD显著少于无关对照抗体处理组 (P<0.01)。 实验例 IV. 1A12单抗抗原表位的鉴定实验
实验例 IV-1. 鼠抗 hOPN单克隆抗体 ΠΑ12)抗原表位的淘选
采用随机肽库免疫淘选法， 整个过程在 96 孔板上进行。 100μ§/ιη1， Ι ΟΟμΙ/孔 1A12 抗体 4 °C包被过夜， 10%脱脂奶粉 (TBST稀释;)封闭过夜， 1 X TBST(Tween-20 0.1%)洗涤 6次； 噬菌体随机肽库 (；购自 NEB,Ph.D.-12TM 噬菌体展示肽实验室试剂盒 Phage Display Peptide Library Kit)4 X 1010 pfu + ΙΟΟμΙ 正常小鼠血清， 室温轻摇 1 小时。 I X TBST(Tween-20 0.1%) 15次； 用含 lmg/ml BSA的甘氨酸 -Cl(Glycine-Cl pH 2.2)洗脱， 室 温轻摇 15min， 用 15μ1 1¾5-。10)11 9.1；)中和。 ΙΟμΙ用于测滴度， 其余扩增。 扩增产物经 PEG/NaCl沉淀，测滴度，同时进行第二轮淘选，相同过程进行第三轮淘选。每次加入 (input) 噬菌体数相同 (4 X 101Q pfu)， 三轮淘选选出（output)噬菌体数分别为 6.9 X 102 pfu、 2.99 X 106 pfu、 1.69 X 108 pfu; 淘选效率分别是第一轮的 4333和 240000倍， 结果表明： 淘选 富集效果明显， 见图 17。 实验例 IV-2. 无关对照抗体 23C3D3对应噬菌体克隆的淘选
采用随机肽库免疫淘选法， 整个过程在 96孔板上进行。 100μ§/ιη1， Ι ΟΟμΙ/孔 23C3D3 抗体 4 °C包被过夜， 10%脱脂奶粉 (TBST稀释;)封闭过夜， 1 X TBST(Tween- 20 0.1%)洗涤 6次； 噬菌体随机肽库 (；购自 NEB,Ph.D.-12TM噬菌体展示肽实验室试剂盒 ;>4 X 101Q pfu + ΙΟΟμΙ正常小鼠血清，室温轻摇 1小时。 l X TBST(Tween-20 0.1%) 15次；用含 lmg/ml BS A 的甘氨酸 -Cl(pH 2.2)洗脱， 室温轻摇 15min， 15μ1 Tris-Cl PH 9.1中和。 ΙΟμΙ用于测滴度， 其余扩增。 扩增产物经 PEG/NaCl沉淀， 测滴度， 同时进行第二轮淘选， 相同过程进行 第三轮淘选。 用 23C3D3抗体包板， ELISA方法检测， 选取阳性反应克隆—— 5F 12, 作 为对照噬菌体。 实验例 IV-3. 噬菌体克隆 ELISA和蛋白质印迹法鉴定
ELISA过程在 96孔板进行， lOO g/ml, 50μ1/孔 1A12单抗 4°C包被过夜， 10%脱脂 奶粉 CTBST稀释 7°C封闭 2小时， l X TBSTXTween-20 0.1%)洗涤 5次； 各单克隆噬菌体 扩增上清用 1 X TBS稀释后， 均以 5 Χ 108ρίΐ!/50μ1， 对照抗体为鼠抗人 ΟΡΝ单克隆抗体 (Santa Cruz),阴性对照噬菌体为 5F12(该克隆为 23C3D3的阳性克隆）。室温结合 1小时， l X TBST(Tween-20 0.1%)洗涤 5次后，每孔加入 200μ1 1： 5000稀释的 HRP标记的抗 -M13 抗体 (Pharmacia #27-9411-01)， 室温震荡作用 1 小时， 1 X TBST(Tween-20 0.1%)洗涤 5 次， 晶美公司 ELISA检测试剂盒 A:B=1 : 1新鲜配置的反应底物 50μ1/孔， 室温 1-5分钟。 2N H2S04终止反应。每个克隆对 1A12和对照抗体 23C3D3均设 3复孔平行检测。 OD450 记录结果表明， 阳性克隆与抗体的反应是特异的。 如图 18A。
蛋白质印迹法过程： 扩增后的噬菌体单克隆上清经 20% PEG/NaCl沉淀纯化后， I X 101Qpfu/泳道 10% SDS-PAGE电泳， 360mA恒流 1小时转至硝酸纤维素膜， 10%脱脂奶 粉 4°C封闭过夜或者室温封闭 2小时； l X TBST(Tween-20 0.1%)洗涤 3次，每次 10分钟； 与 lO g/ml—抗室温反应 1小时， l X TBST(Tween-20 0.1%)洗涤 5次，每次 10分钟； 1 : 1000 稀释 HRP标记的兔抗鼠 IgG (北京中山公司）室温反应 1小时， ECL试剂盒 (Tiangen公司） 反应 1-2分钟， 医用 X射线感蓝胶片压片曝光。 图 18B左图为 1A12单抗杂交， 右图为 无关抗体 23C3D3杂交； 箭头所示为目标条带； 结果显示： 阳性克隆与抗体的反应是特 异的， 如图 18B。 实验例 IV-4. 抗体识别表位的测序及序列分析
单链 DNA提取试剂盒 (上海捷瑞公司)制备模版， -96引物测序， 铬酸盐 (Chromas)读 取序列， 100个阳性克隆有 4个独立序列； AlignX 分析， 结果有一致序列 NXNNAP, 又因为 G与 A均为非极性、 芳香族氨基酸； S、 T、 Ν、 Ρ均为极性， 不带电荷氨基酸； 在抗原 hOPN序列上， 可以找到 NAPS的同源基序， 由此可见， 1A12的可能抗原表位 为： NAPS。 结果见图 19。 实验例 IV-5. 噬菌体克隆与抗体结合能力分析
各个克隆均以 5 X 107pfu投入到包被有抗体的 96孔板， 经过相同条件的淘选 (对照抗 体： 23C3D3 ; 无关对照抗体： 5F12) , 对洗脱后的噬菌体进行滴度测定（参见 blood 2006-04-014639)。 结果显示： hOPN 序列中表位 NAPS , 其中 APS在介导 lA12-hOPN 的结合中起重要作用,单独的 N或 NN不能介导两者的结合， 在表位基序的第二氨基酸 位置 A、 G可以互换而不影响结合能力， 两者均为非极性、 脂肪族氨基酸； 在表位基序 的第四氨基酸位置， 只要是极性、 不带电荷的氨基酸 (如： S、 T、 Ν、 Ρ)就不会影响结合 能力。 该实验进一步证实： 1A12的表位是 NAPS。 结果见图 20。
实验说明 1A12抗体的特异识别表位是 NAPS, 位于人 OPN第七外显子处， 是一个 新的表位， 表位的位置和序列如图 21所示， 氨基酸序列见 SEQ ID NO:7。 在本发明提及的所有文献都在本申请中引用作为参考， 就如同每一篇文献被单独引 用作为参考那样。 此外应理解， 在阅读了本发明的上述讲授内容之后， 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

权 利 要 求
I . 一种骨桥蛋白的功能表位， 其特征在于， 所述功能表位为 NXPY, 其中 X=A或 G, Y= S、 Τ、 ί^ Ρ。
2. 与权利要求 1所述的功能表位特异性结合的抗骨桥蛋白的单克隆抗体。
3. 如权利要求 2所述的单克隆抗体， 其特征在于， 所述单克隆抗体的重链可变区 的 CDR 氨基酸序列分别选 自 ： GYTFTTYVMH、 YINPYNDGSKYNEKFKG 或 HYGGSPAY; 轻链可变区的 CDR 氨基酸序列分别选自： RSSQSLVHSNGNTYLH、 KVSNRFS或 SQSTHVPWT。
4. 如权利要求 2所述的单克隆抗体， 其特征在于， 所述单克隆抗体的重链可变区 氨基酸序列选自： SEQ ID NO:4或 SEQ ID NO: 19， 轻链可变区氨基酸序列选自： SEQ ID NO:6或 SEQ ID NO:21。
5. 如权利要求 2所述的单克隆抗体， 其特征在于， 所述单克隆抗体的恒定区选自： 小鼠抗体恒定区或人抗体恒定区。
6. 一种 DNA分子， 其编码权利要求 2-5中任一项所述的单克隆抗体。
7. 一种载体， 其包含权利要求 6所述的 DNA分子。
8. 一种宿主细胞， 其包含权利要求 7 所述的载体， 或基因组中整合有权利要求 6 所述的 DNA分子。
9. 一种免疫偶联物， 其特征在于， 该免疫偶联物含有：
(a) 权利要求 2所述的单克隆抗体； 和
(b) 选自下组的偶联部分： 药物、 毒素、 细胞因子、 放射性核素、 或酶。
10. 权利要求 2所述的抗骨桥蛋白的单克隆抗体或权利要求 9所述的免疫偶联物在 制备抗肿瘤药物中的用途。
I I . 一种药物组合物， 其包含权利要求 2所述的抗骨桥蛋白的单克隆抗体或权利要 求 9所述的免疫偶联物； 以及药学上可接受的载体。
12. 一种用于检测骨桥蛋白的试剂盒， 所述试剂盒包含： 权利要求 2所述的抗骨桥 蛋白的单克隆抗体或权利要求 9所述的免疫偶联物。
13. 一种检测生物样品中是否存在骨桥蛋白或检测其含量的方法， 所述方法包括步 骤：
(i)将待测样品与权利要求 2所述的抗骨桥蛋白的单克隆抗体或权利要求 9所述的免 疫偶联物接触；
(ii)检测是否形成抗原-抗体复合物，
其中， 形成抗原-抗体复合物就表示样品中存在骨桥蛋白， 或定量检测所形成的抗 原-抗体复合物的量以反映样品中骨桥蛋白的含量。
PCT/CN2008/070576 2007-04-24 2008-03-25 Épitopes fonctionnels d'ostéopontine, anticorps monoclonaux contre ces épitopes et leurs utilisations WO2008128455A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
BRPI0810826A BRPI0810826A2 (pt) 2007-04-24 2008-03-25 epítopos funcionais de osteopontina, anticorpo monoclonal contra os epítopos e usos dos mesmos.
EP08715312A EP2149582A4 (en) 2007-04-24 2008-03-25 OSTEOPONTIN FUNCTIONAL EPITOPES, MONOCLONAL ANTIBODIES AGAINST THESE EPITOPES AND USES THEREOF
US12/597,418 US20100151486A1 (en) 2007-04-24 2008-03-25 Osteopontin functional epitopes, monoclonal antibodies against the epitopes and uses thereof
CN2008800134403A CN101679485B (zh) 2007-04-24 2008-03-25 骨桥蛋白的功能表位、针对该单位的单克隆抗体及它们的应用
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CA002685182A CA2685182A1 (en) 2007-04-24 2008-03-25 Osteopontin functional epitopes, monoclonal antibody against the epitopes and uses thereof

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WO2011021146A1 (en) * 2009-08-20 2011-02-24 Pfizer Inc. Osteopontin antibodies
CN102372778A (zh) * 2010-08-19 2012-03-14 上海抗体药物国家工程研究中心有限公司 抗人vegf和opn双特异性抗体、其制备方法及用途
CN102372778B (zh) * 2010-08-19 2015-06-17 上海抗体药物国家工程研究中心有限公司 抗人vegf和opn双特异性抗体、其制备方法及用途
WO2014148895A1 (en) * 2013-03-18 2014-09-25 Biocerox Products B.V. Humanized anti-cd134 (ox40) antibodies and uses thereof
CN105229032A (zh) * 2013-03-18 2016-01-06 比奥塞罗克斯产品公司 人源化抗cd134(ox40)抗体及其应用
US9790281B2 (en) 2013-03-18 2017-10-17 Biocerox Products, B.V. Humanized anti-CD134 (OX40) antibodies and uses thereof
EP3409690A1 (en) * 2013-03-18 2018-12-05 BiocerOX Products B.V. Humanized anti-cd134 (ox40) antibodies and uses thereof
US10273307B2 (en) 2013-03-18 2019-04-30 Biocerox Products B.V. Humanized anti-CD134 (OX40) antibodies and uses thereof

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