WO2012113266A1 - 识别肿瘤起始细胞的抗体和抗原及其应用 - Google Patents

识别肿瘤起始细胞的抗体和抗原及其应用 Download PDF

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WO2012113266A1
WO2012113266A1 PCT/CN2012/000227 CN2012000227W WO2012113266A1 WO 2012113266 A1 WO2012113266 A1 WO 2012113266A1 CN 2012000227 W CN2012000227 W CN 2012000227W WO 2012113266 A1 WO2012113266 A1 WO 2012113266A1
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Prior art keywords
cancer
tumor
cells
cacna2d1
antigen
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PCT/CN2012/000227
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English (en)
French (fr)
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张志谦
赵威
王力民
韩海勃
邢宝才
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北京市肿瘤防治研究所
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Application filed by 北京市肿瘤防治研究所 filed Critical 北京市肿瘤防治研究所
Priority to KR1020177032217A priority Critical patent/KR101885705B1/ko
Priority to US14/001,105 priority patent/US10174123B2/en
Priority to CN201280010033.3A priority patent/CN103562403B/zh
Priority to KR1020137024482A priority patent/KR101797090B1/ko
Priority to BR112013021350A priority patent/BR112013021350A2/pt
Priority to JP2013553769A priority patent/JP6084170B2/ja
Priority to EP12749191.8A priority patent/EP2682475B1/en
Publication of WO2012113266A1 publication Critical patent/WO2012113266A1/zh
Priority to US16/238,752 priority patent/US20200040097A1/en

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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/18—Growth factors; Growth regulators
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    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07—ORGANIC CHEMISTRY
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    • 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
    • C07K14/475—Growth factors; Growth regulators
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • 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
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • 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

Definitions

  • the present invention relates to the field of medical biotechnology, and in particular, the present invention relates to a method for finding a marker for tumor initiation cells and/or a method for treating a target molecule, and a marker for targeting the tumor initiation cell and/or a therapeutic target molecule. Preparation and screening methods for tumor therapeutic drugs.
  • the present invention also relates to a monoclonal antibody or antigen-binding fragment thereof for use in tumor diagnosis, prognosis and treatment, a diagnostic kit or pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof, and the monoclonal antibody or The use of antigen-binding fragments in the diagnosis, prognosis or treatment of tumors and the like.
  • the invention also relates to the use of the antigen to which the antibody is directed or the target molecule thereof to be used as a marker for tumor initiation cells (or tumor stem cells) for the diagnosis or therapeutic effect prediction of cancer, and as a reagent for tumor treatment And molecular targets for drug development.
  • Background technique the use of the antigen to which the antibody is directed or the target molecule thereof to be used as a marker for tumor initiation cells (or tumor stem cells) for the diagnosis or therapeutic effect prediction of cancer, and as a reagent for tumor treatment And molecular targets for drug development.
  • tumor stem cells are the name of a class of cells with less content, unlimited self-renewal ability, and can drive tumor formation and growth. They are named because they are similar to normal stem cells, but it does not mean that cancer stem cells must be derived from the corresponding normal. Stem cells are inextricably linked to normal stem cells. Some people call them tumor-initiating cells (TICs) or tumor-propagating cells (TPCs) to avoid misunderstanding.
  • TICs tumor-initiating cells
  • TPCs tumor-propagating cells
  • Tumor stem cells exhibit strong tumorigenic/cancer ability (100 or even several cells can form tumor/cancer in immunodeficient mice), drug resistance and invasive growth characteristics, and they also differentiate into The potential of tumorigenic cells. The presence of cancer stem cells is thought to be the underlying cause of tumorigenesis, development, and treatment failure.
  • liver cancer stem cells Since the first isolation and identification of cancer stem cells in the blood, the presence of cancer stem cells has been confirmed in solid tumors such as brain, breast, prostate and colon cancer.
  • liver cancer several research groups have also used different strategies to find liver cancer stem cells from cultured cell lines or clinical specimens. For example, side population (SP) cells isolated from Huh7 cell line can be used in continuous animal inoculation experiments. Tumor formation, CD133 in Huh7 and PLC8024 cell lines has also been reported as a marker for liver cancer stem cells.
  • CD90, EpCAM, OV6, CD133/ALDH were also used to successfully isolate stem cells from liver cancer.
  • tumor cells with stem cell characteristics have been identified by different routes, the biological characteristics of various types of cells isolated from the same marker are different because the tumor stem cell marker specificity found so far is not very strong. The differences are sometimes large, so there has been debate about the existence of cancer stem cells and the nature of these cells. In fact, the tumor is fine Cells, or tumor-initiating cells, and even tumor-delivering cells are only an operational term, and the biological nature of this remains to be further revealed.
  • tumor tissues that cells that are highly tumorigenic and resistant to chemoradiotherapy in immunodeficient animals, whether they are called cancer stem cells, tumor-initiating cells or tumor-delivering cells, they are The underlying cause of treatment failure and recurrence in cancer patients.
  • tumor-initiating cells Although low in proportion, survived and gradually grew and migrated due to conventional radiotherapy and chemotherapy, leading to tumors. Recurrent metastasis.
  • Drugs designed to remove tumor-initiating cells can fundamentally block the "seeds" of tumor recurrence and metastasis, alone or in combination with conventional surgery, radiotherapy and chemotherapy, etc., which is expected to bring new dawn to the complete conquest of tumors.
  • These cells should also be the target cells for tumor diagnosis and prognosis.
  • CD133-positive cancer stem cells inhibit the occurrence of apoptosis because of the high expression of IL-4, and treatment of CD133-positive cancer stem cells with IL-4 inhibitors can increase their sensitivity to drugs.
  • Drug development for cancer stem cells can target key molecules in the signaling pathways such as self-renewal, drug resistance or invasive growth of cancer stem cells, surface markers, and even the microenvironment on which they exist and grow.
  • the discovery of molecules in key links depends on the isolation and identification of cancer stem cells and the in-depth disclosure of the nature and regulatory mechanisms of malignant biological properties. It should be noted that the expression of tumor stem cell-associated molecules is generally lower compared to the so-called tumor-associated antigens. Therefore, the discovery of tumor stem cell-associated molecules is primarily dependent on their specific expression in the tumor stem cell-like population rather than overexpression.
  • Monoclonal antibody technology has become a routine technique for preparing antibodies against specific antigens or specific cells. Many antibodies are directly or genetically engineered into murine-human chimeric or even fully humanized antibodies for the treatment of a variety of clinical diseases, including malignancies. To date, antibody-mediated therapeutic experiments related to cancer stem cells include CD44, p-glycoprotein 1 (H-Plycoprotein 1), hyaluronic acid receptor, EpCAM, CD326, CXCR4, IL-4, DLL4, ALDH, and the like.
  • Another object of the invention is also to provide a method of diagnosis, treatment and prevention of a tumor or a disease or condition associated with the CACNA2D1 protein.
  • the present invention provides a method for finding, identifying or confirming markers of tumor-initiating cells and/or therapeutic targets/molecular targets by the step of immunizing animals using cells derived from recurrent tumors and enriched in tumor-initiating cells. .
  • the invention also provides a method of diagnosing, treating and preventing a tumor, comprising using the method of the present invention for finding, identifying or confirming a marker of a tumor-initiating cell and/or a method for treating a target/molecular target, Identifying or identifying markers of tumor-initiating cells and/or therapeutic targets/molecular targets to develop and/or screen for gene expression or/and protein activity, or targeting, that reduces the marker or target/molecular target.
  • a therapeutically effective amount of at least one antibody or antigen-binding fragment, single- or double-stranded oligonucleotide, nucleic acid, short peptide or small molecule compound drug that is developed or screened is administered.
  • the invention also provides monoclonal antibodies or antigen-binding fragments thereof for use in the diagnosis or treatment of a tumor or a disease or condition associated with the CACNA2D1 protein.
  • the invention also provides the use of a monoclonal antibody or antigen-binding fragment thereof for diagnosis, prognosis, and treatment of a tumor or a disease or condition associated with a CACNA2D1 protein.
  • a voltage-dependent calcium channel protein ⁇ 2 ⁇ subunit 1 (calcium-channel, voltage-dependent, ⁇ 2/ ⁇ subunit 1, abbreviated as CACNA2D1, GenBank SEQ ID NO: NM - 000722.2) is a molecule of a tumor-initiating cell. Markers and molecular targets or targets for treatment. It can be used for the development of diagnostic reagents and therapeutic drugs for nucleic acids or proteins of the CACNA2D1 gene.
  • the inventors of the present invention cultured cell lines Hep-11 (original liver cancer tissue source) and Hep-12 (recurrent liver cancer tissue source) from the primary and recurrent foci of the same hepatocellular carcinoma patient.
  • the inventors of the present invention found that most (more than 80%) of the relapsed source of Hep-12 cells possess tumor-initiating cell characteristics, whereas primary Hep-11 cells were inoculated with 10 7 cells for 6 months. Tumor formation.
  • the inventors of the present invention found that a hybridoma was prepared by immunizing balb/c mice with Hep-12 cells, and an antibody 1B50-1 obtained specifically recognized Hep-12 cells.
  • the inventors of the present invention found that 1B50-1 antibody-positive cells were sorted by flow cytometry from 5 hepatoma cell lines and 4 clinical liver cancer specimens, and 100-1000 cells were sufficient to form tumors under the skin of NOD/SCID mice.
  • the inventors of the present invention found that the antibody was injected intraperitoneally
  • the NOD/SCID mice of Hep-12 and Huh7 tumors inhibited the growth of xenografts in a dose-dependent manner, and the tumor inhibition rates were 80.4% and 65.5%, respectively, which were compared with the control IgG group.
  • Immunoprecipitation/mass spectrometry indicated that the antigen recognized by the 1B50-1 antibody was a 150 kd ion channel protein.
  • the inventors of the present invention found that inhibition of the expression of the gene by RNA interference can also inhibit the growth of Hep-12 cells in nude mice.
  • the invention provides methods for finding, identifying or confirming markers of tumor-initiating cells and/or therapeutic targets or molecular targets.
  • the invention also provides methods for finding, identifying or validating molecular targets that can be used for the isolation, identification and treatment of tumor-initiating cells.
  • the method comprises the step of immunizing an animal using a relapsed tumor source, a cell rich in tumor initiating cells.
  • the tumor may be liver cancer, colon cancer, rectal cancer, kidney cancer, esophageal cancer, gastric cancer, lung cancer.
  • the tumor is liver cancer
  • the cell derived from the recurrent tumor is a Hep-12 cell rich in tumor-initiating cells.
  • the method of the present invention for finding a tumor-initiating cell marker and/or a therapeutic target further comprises using Hep-12 cells and Hep-11 cells derived from hepatocellular carcinoma of the same liver cancer patient recurrence and primary liver cancer, respectively. As a screening cell pair.
  • the present invention provides a method of treating a tumor, comprising
  • markers and/or therapeutic targets/molecular targets eg CACNA2D1
  • markers and/or therapeutic targets/molecular targets eg CACNA2D1
  • reduced markers or targets/molecular targets Gene expression or/and protein activity, or antibody or antigen-binding fragment, single- or double-stranded oligonucleotide, nucleic acid, short peptide or small that causes cytotoxicity after targeting the marker or therapeutic target/molecular target Molecular compound drug;
  • the above step 1) can prepare a hybridoma by immunizing an animal (such as a balb/c mouse) with a cell derived from a recurrent tumor source and rich in tumor-initiating cells (such as a Hep-12 cell), A monoclonal antibody (such as 1B50-1) is obtained, and a cell (such as Hep-12 cell) which can be specifically recognized by the antibody (such as 1B50-1) is used as a candidate cell for identifying and confirming tumor stem cells.
  • the anti-obstruction was confirmed by animal experiments (such as whether 100-1000 cells can form tumors subcutaneously in NOD/SCID mice), gene expression and cell differentiation. Whether the body-positive cells have tumor stem cell characteristics.
  • the monoclonal antibody (e.g., 1B50-1) obtained by the method of the present invention specifically recognizes an antigen in a tumor stem cell, which is a marker and/or a therapeutic target/molecular target of the tumor-initiating cell to be sought by the present invention.
  • the inventors of the present invention have discovered and confirmed that the CACNA2D1 protein is a marker for tumor-initiating cells, a therapeutic target or a molecular target for tumors/cancers.
  • the gene expression or/and protein activity of the tumor initiation cell marker, the therapeutic target or the molecular target, which is developed or screened in the above step 2); or targeting the marker, the therapeutic target Or an antibody or antigen-binding fragment which causes a cytotoxic reaction after a molecular target can be used to find, identify or confirm a tumor-initiating cell antigen (marker, tumor/cancer therapeutic target or molecular target) as obtained in step 1) above.
  • a monoclonal antibody (such as 1B50-1) may also be a marker for a tumor-initiating cell found, identified or confirmed, a tumor/cancer therapeutic target or a molecular target (eg, CACNA2D1 protein/antigen) Or other antibodies screened.
  • a novel antibody against the antigen and a derivative thereof can be obtained by various antibody preparation techniques. These techniques may include, but are not limited to, expression of the CACNA2D1 antigen by various expression systems or purification of the antigen, immunization of mice, rabbits or other animals (including genetically engineered animal lines) to obtain antisera specific for the antigen; The spleen cells of the immunized mouse are fused with the myeloma SP2/0 cells, and the monoclonal antibody specific for the CACNA2D1 antigen is specifically screened by the CACNA2D1 antigen; further including the gene for cloning the variable region of the antigen mouse monoclonal antibody, directly or Humanized chimeric or monoclonal antibodies obtained by humanized transformation and expression of the constant region coding gene of the corresponding human antibody, expressed by prokaryotic and/or eukaryotic expression system. Human phage antibody
  • the CACNA2D1 protein can be highly sensitively quantified by using the antibody of the present invention.
  • the monoclonal antibody or antigen-binding fragment thereof of the present invention can be used for diagnosing a tumor or various diseases associated with the CACNA2D1 protein by using a method of quantifying the CACNA2D1 protein in vivo.
  • the invention also provides a method for diagnosing a tumor or a disease or condition associated with a CACNA2D1 protein, the method comprising administering to a subject in need thereof an effective amount of at least one monoclonal antibody of the invention or antigen binding thereof Fragment.
  • the dosage required for such in vivo diagnosis can be less than that required for therapeutic applications and can be determined by one of ordinary skill in the art through routine procedures.
  • the monoclonal antibody or antigen-binding fragment thereof of the present invention can also be used for specifically detecting a CACNA2D1 protein present in a test solution such as a body fluid, tissue or the like.
  • the present invention provides a monoclonal antibody or antigen-binding fragment thereof which specifically recognizes a voltage-dependent calcium channel protein ⁇ 2 ⁇ subunit 1 (CACNA2D1).
  • the monoclonal antibody or antigen-binding fragment thereof that specifically recognizes CACNA2D1 comprises: (i) a heavy chain (hereinafter abbreviated as H-chain), the heavy chain variable region comprising a complementarity determining region (CDR) CDRH1 (SEQ ID NO: 1), CDRH2 (SEQ ID NO: 2) and CDRH3 (SEQ ID NO: 3);
  • L-chain a light chain (hereinafter abbreviated as L-chain), the light chain variable region comprising a complementarity determining region CDRL1 (SEQ ID NO: 4), CDRL2 (Sequence ⁇ IJ 5 ) and CDRL3 (SEQ ID NO: 6); or
  • the above monoclonal antibody or antigen-binding fragment thereof has one or more of the following characteristics: (1) binding to CACNA2D1 protein; (2) identifying positive cells having tumor initiating cell characteristics; Increasing the growth of tumor cells expressing CACNA2D1 in animals.
  • the above tumor may be liver cancer, colon cancer, rectal cancer, kidney cancer, esophageal cancer, gastric cancer, lung cancer, breast cancer, prostate cancer or other tumors highly expressing the CACNA2D 1 gene.
  • the monoclonal antibody or antigen-binding fragment thereof of the invention comprises a heavy chain, the heavy chain variable region comprising at least 70%, 75%, 80 of the amino acid sequences of CDRH1, CDRH2 and CDRH3 %, 85%, 90%, 95%, or 99% identical amino acid sequence.
  • the monoclonal antibody or antigen-binding fragment thereof of the invention comprises a light chain, the light chain variable region comprising at least 70%, 75%, 80% of the amino acid sequence of CDRLK CDRL2 and CDRL3 , 85%, 90%, 95%, or 99% identical amino acid sequence.
  • the antibody or antigen-binding fragment thereof may further comprise both the heavy and light chains described above.
  • an antibody or antigen-binding fragment thereof of the invention may further comprise one or more of at least 70%, 75%, 80%, 85%, 90%, 95% in combination with any of the above CDRs or CDRs. , or the CDR of the 99% identical amino acid sequence.
  • the present invention provides a monoclonal antibody or antigen-binding fragment thereof for use in diagnosis, prognosis, and treatment of a tumor or a disease or condition associated with CACNA2D1 protein, including the above monoclonal antibody or Antigen-binding fragment.
  • the above tumor or disease or condition may be liver cancer, colon cancer, rectal cancer, kidney cancer, esophageal cancer, gastric cancer, lung cancer, breast cancer, prostate cancer or other tumors highly expressing the CACNA2D1 gene.
  • the present invention provides a hybridoma cell line which is a mouse hybridoma deposited under the Chinese Microbial Culture Collection Management Committee General Microbiology Center on December 08, 2010 (Accession No. CGMCC No. 4416).
  • the monoclonal antibody is a monoclonal antibody produced by a hybridoma cell line (Accession No. CGMCC No. 4416), 1B50-1.
  • Further embodiments of the invention also include, but are not limited to, the production of antibody fragments that recognize a specific epitope of an antigen based on the above antibodies by known techniques.
  • proteolytic cleavage by immunoglobulin molecules, Fab and F(ab') 2 fragments are produced using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 fragments).
  • the F(ab') 2 fragment comprises the entire L-chain and H-chain variable regions, the CH1 region and the hinge region.
  • nucleic acid encoding the immunoglobulin can be chemically synthesized, or from a suitable source (eg, an antibody cDNA library, or from any tissue or cell expressing the antibody, eg,
  • a suitable source eg, an antibody cDNA library, or from any tissue or cell expressing the antibody, eg,
  • the isolated antibody-expressing hybridoma cell-isolated nucleic acid, preferably mR A, or a cDNA library produced therefrom obtains the immunoglobulin encoded by PCR amplification using synthetic primers that match the 3' and 5' ends of the sequence.
  • the nucleic acid encoding the immunoglobulin is obtained from a nucleic acid such as a cDNA library by screening a nucleic acid for the protein or by an oligonucleotide probe for a specific gene sequence.
  • the nucleic acid produced by, for example, PCR amplification can then be cloned into a vector that is replicable and can be expressed in different expression systems (prokaryotic and eukaryotic expression systems, cell-free translation systems, etc.) using any method well known in the art. Expression produces a genetically engineered antibody or antigen-binding fragment that is similar to the biological activity of the antibody of the invention.
  • the antibody or antigen-binding fragment thereof of the invention may also be produced by any other method known in the art for the synthesis of antibodies, for example by recombinant expression techniques or by chemical synthesis.
  • the invention also provides a pharmaceutical composition comprising a monoclonal antibody of the invention or an antigen binding fragment thereof.
  • the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.
  • the invention provides a pharmaceutical composition for treating a tumor or a disease or condition associated with a CACNA2D1 protein comprising a monoclonal antibody or antigen-binding fragment thereof of the invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention may further comprise other active compounds which may add or synergistically improve the above-mentioned diseases or conditions.
  • the active compounds include, but are not limited to, other chemotherapeutic compounds, toxins that treat the disease or condition.
  • the active compound also includes small molecule compounds and other antibodies or antigen-binding fragments thereof.
  • the above tumor or the disease or condition associated with the CACNA2D1 protein may be liver cancer, colon cancer, rectal cancer, kidney cancer, esophageal cancer, gastric cancer, lung cancer, breast cancer, prostate cancer or other tumors highly expressing the CACNA2D1 gene.
  • compositions for oral or parenteral administration are prepared in unit dosage forms suitable for the dosage of the active ingredient.
  • unit dosage forms include, for example, tablets, pills, capsules, injections, suppositories, and the like.
  • kits for the CACNA2D1 gene or protein can be provided.
  • the kit can be used for diagnosing or treating or preventing a tumor or a disease or condition associated with CACNA2D1 protein, and the kit comprises the monoclonal antibody or antigen-binding fragment thereof described above, or A nucleic acid of CACNA2D1, such as DNA, mRNA.
  • the kit can be used for the diagnosis, treatment or prevention of, for example, liver cancer, colon cancer, rectal cancer, kidney cancer, esophageal cancer, gastric cancer, lung cancer, breast cancer, prostate cancer or other tumors highly expressing the CACNA2D1 gene.
  • RT-PCR can be used at the mRNA level, and detection of antigenic proteins can be carried out by using specific antibodies or specific binding of small molecules using well-known techniques such as enzyme-linked immunosorbent assay, flow. Techniques such as cell analysis and immunohistochemistry (cytochemistry) are used for detection.
  • the sample used can be derived from the patient's blood, tissue, exfoliated cells, and the like.
  • the invention provides the use of a monoclonal antibody or antigen-binding fragment thereof as described herein for the manufacture of a medicament for the diagnosis, treatment or prevention of a tumor or a disease or condition associated with a CACNA2D1 protein.
  • the tumor or disease or condition is liver cancer, colon cancer, rectal cancer, kidney cancer, esophageal cancer, gastric cancer, lung cancer, breast cancer, prostate cancer or other tumors highly expressing the CACNA2D1 gene.
  • the monoclonal antibody or antigen-binding fragment thereof of the present invention can be used as a diagnostic agent for cancer (for example, whether a patient's tissue or a tumor patient's serum contains tumor-initiating cells and a prognostic and therapeutic sensitivity prediction) and other CACNA2D1 protein-related diseases or Diagnosis of the condition.
  • the monoclonal antibodies or antigen-binding fragments thereof of the present invention are capable of specifically recognizing the CACNA2D1 antigen, and thus can be used to quantify the CACNA2D1 protein in a test solution, for example, by conventional techniques in the art such as sandwich immunoassays, competitive assays, Quantitative measurement such as immunoassay.
  • the invention provides a method for treating a tumor or a disease or condition associated with a CACNA2D1 protein, the method comprising administering to a subject in need thereof a therapeutically effective amount of at least one monoclonal of the invention An antibody or antigen-binding fragment thereof.
  • the invention provides a method for preventing a tumor or a disease or condition associated with a CACNA2D1 protein, the method comprising administering to a subject in need thereof a prophylactically effective amount of at least one monoclonal of the invention An antibody or antigen-binding fragment thereof.
  • the monoclonal antibody or antigen-binding fragment thereof of the present invention can be combined with other drugs which enhance the biological action of the monoclonal antibody or antigen-binding fragment thereof.
  • therapeutic agents include another antibody, a cytotoxin that inhibits cell growth or kills cells, a radioactive element, and/or other therapeutic agents including anti-inflammatory agents and antibiotics.
  • the monoclonal antibody or antigen-binding fragment thereof of the present invention can be administered orally or parenterally as a liquid preparation or a pharmaceutical composition of a suitable dosage form by mixing it in a suitable solvent.
  • the monoclonal antibody or antigen-binding fragment thereof of the present invention may be mixed with a pharmaceutically acceptable carrier, diluent or excipient to prepare a pharmaceutical composition suitable for oral or parenteral administration.
  • a pharmaceutically acceptable carrier such as a pharmaceutically acceptable carrier, diluent or excipient.
  • the monoclonal antibodies or antigen-binding fragments thereof or pharmaceutical compositions thereof, such as liposome encapsulation, microparticles, microcapsules and the like can be administered by a variety of delivery systems known per se. Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the compound can be administered by any route, such as by infusion or bolus injection.
  • Administration is by a route of absorption through the epithelium or skin mucosa (e.g., oral mucosa or rectum, etc.) and the compound can be administered with other bioactive agents.
  • Administration can be systemic or topical.
  • the monoclonal antibody or antigen-binding fragment thereof of the present invention is administered by intravenous administration.
  • the monoclonal antibodies of the invention may also be administered by topical administration in the area in need of treatment.
  • the composition is made by methods known to the public and comprises a carrier, diluent or excipient conventionally used in the field of pharmaceutical formulations. Examples of carriers or excipients for tablets are lactose, starch, sucrose, magnesium stearate and the like.
  • the injectable preparation may include a dosage form for intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, drip, and the like. These injectable preparations can be prepared by a known method.
  • the injectable preparation can be prepared, for example, by dissolving, suspending or emulsifying the above antibody or a salt thereof in a conventional sterile aqueous or oily medium for injection.
  • the aqueous medium for injection may be, for example, physiological saline, an isotonic solution containing glucose and other auxiliaries, etc., which may be used in combination with a suitable solubilizing agent such as an alcohol (e.g., ethanol), a polyhydric alcohol.
  • a suppository for rectal administration can be prepared by mixing the above antibody or a salt thereof with a suppository with a conventional base.
  • a method of producing and screening a therapeutic agent for a tumor or a disease or condition associated with CACNA2D1 protein comprising using CACNA2D1 as a target, developing and screening for reducing gene expression or/and protein activity thereof Or an antibody or antigen-binding fragment, a single- or double-stranded oligonucleotide, a nucleic acid, a short peptide or various small molecule compound drugs that cause a cytotoxic reaction after targeting the molecule.
  • the above invention is characterized in that hepatocellular carcinoma is the main cancer, and further includes gastric cancer, colon cancer, rectal cancer, renal cancer, esophageal cancer, lung cancer, breast cancer, prostate cancer and the like.
  • the molecule-specific inhibitor-small molecule compound can be easily obtained by computer simulation or screening of an existing library of lead compounds.
  • interfering RNAs - ShRNA1 (SEQ ID NO: 7) and ShRNA2 (SEQ ID NO: 8) directed against the CACNA2D1 gene are designed and synthesized, and a lentiviral expression vector is packaged to package a lentivirus.
  • This virus inhibits the expression of this gene and has a significant inhibitory effect on the growth of Hep-12 cells in immunodeficient animals.
  • these shRNA sequences can be provided by chemical synthesis and modification to obtain different single-stranded RNAs with the matching sequence as the core, or by using different promoters, cloning vectors and the like. Core sequence.
  • tumor-initiating cells “tumor stem cells”, and “tumor-transmitting cells” are used interchangeably herein.
  • the terminology of these terms is only a reference to a class of cells in the actual application process. The specific biological characteristics have yet to be revealed by further research. However, this type of cells generally have, but are not limited to, the following Points: (1) Highly malignant: Very few cells (100 or less) can form tumors in immunodeficient mice, and this tumorigenicity is relatively stable; (2) Tolerance to conventional treatment : Resistant to conventional chemoradiotherapy, not sensitive.
  • antibody refers to an antibody molecule capable of specifically binding to a polypeptide of interest or a target sequence, and encompasses an intact antibody molecule or a fragment thereof, including an antigen-binding fragment thereof.
  • antigen-binding fragment refers to any antibody fragment that retains the ability to specifically bind to a protein, polypeptide or sequence of interest, including single chain antibodies, Fab fragments, F(ab') 2 fragments, disulfide.
  • a linked-chained single-chain antibody (sFv) and a fragment comprising any of the light chain variable region (VL) and/or heavy chain variable region (VH) or a fragment comprising a CDR that specifically binds a protein, polypeptide or sequence of interest Various methods of obtaining the above antibody fragments are well known in the art.
  • CDRs complementarity determining regions
  • the term "specifically recognizes” as used in the present invention refers to the ability of an antibody or antigen-binding fragment thereof to specifically bind to a protein, polypeptide or sequence of interest.
  • the antibody does not bind specifically to other polypeptides or proteins.
  • an antibody or antigen-binding fragment thereof that specifically recognizes CACNA2D1 does not cross-react with other antigens.
  • an effective amount means "therapeutically effective antiproliferative amount” or “prophylactically effective antiproliferative amount”.
  • the term includes an amount effective to achieve a desired result, e.g., sufficient to treat a cell proliferative disorder, in terms of dosage and necessary time period.
  • An effective amount of a compound of the invention may vary depending on, for example, the disease state, age and weight of the subject, and the ability of the compounds of the invention to elicit a desired response in the subject. The dosage regimen can be adjusted to provide the optimal therapeutic response.
  • An effective amount is also an amount of the therapeutic benefit of the compounds of the invention over any toxic or adverse effects (e.g., side effects).
  • Figure 1 is an immunofluorescence staining of antibody 1B50-1 against hepatoma stem cell-rich Hep-12, showing that the antigen recognized by the lb50-l antibody is localized on the cell membrane. Most Hep-12 cells were positive, while Hep-11 cells were mostly negative.
  • Figure 2 shows that animal inoculation experiments demonstrated that 1B50-1 antibody positive cells have tumor initiating cell characteristics.
  • Figure 2 (A) shows tumors formed by NOB/SCID mice from different sources of 1B50-1 antibody-positive cells;
  • Figure 2 (B) shows tumor eosin formed by 1B50-1 antibody-positive cells in NOD/SCID mice A hematoxylin staining showed that the histomorphology was close to that of the patient from which the 1B50-1 antibody-positive cells were derived.
  • Figure 2 (C) shows that the flow cytometric analysis showed that the purified 1B50-1 antibody-positive cells could differentiate into 1B50.
  • Figure 2 (D) shows real-time quantitative RT-PCR results indicate that 1B50-1 antibody-positive cells overexpress multiple stem cell-associated genes.
  • Figure 3 shows the identification of the antigen CACNA2D1 recognized by the 1B50-1 antibody.
  • Figure 3A Results of immunoprecipitation analysis;
  • Figure 3B Results of transfection of plasmids expressing CACNA2Dl-myc with 1B50-1 antibody-negative cells, showing that Myc and 1B50-1 antibody staining has a co-localization relationship on the cell membrane. Merge is the result of stacking Myc and 1B50-1 antibodies together.
  • Figure 4 shows the results of immunohistochemical staining of 1B50-1 antibody in liver cancer specimens. It was shown that 1B50-1 antibody-positive cells were dispersedly distributed in tumor tissues.
  • Figure 4 (A) is a photograph showing the results of immunohistochemical staining of 1B50-1 antibody in cancer, paracancerous and normal liver tissues. The arrows indicate positive cells.
  • Figure 4 (BE) shows Kaplan-Meier survival curve analysis of 86 staining of 1B50-1 antibody in liver cancer specimens showing surgical marginal tissues (C and E), but not liver cancer tissues (B and D) The presence of 1B50-1 positive cells was negatively correlated with postoperative disease-free survival and overall survival in patients with liver cancer.
  • Figure 4 (F) is a multivariate analysis showing 1B50-1 positive cells in adjacent tissues of surgical margins. Existence is an independent prognostic factor for liver cancer. a chi-square test. Abbreviations: 4y, 4 years; Ca, cancer tissue; CI, confidence interval; DFS, disease-free survival; OS, total survival; PCa, adjacent tissues; RR, relative risk factor.
  • Figure 5 shows the expression of the CACNA2D1 gene in various tumor cell lines.
  • Figure 5A RT-PCR analysis of CACNA2D1 gene expression in different cell lines;
  • Figure 5B Immunofluorescence staining analysis of 1B50-1 antibody in different cell lines.
  • Figure 6 shows the expression analysis of CACNA2D1 in human tumor tissue (T) and matched normal tissues (N).
  • T human tumor tissue
  • N matched normal tissues
  • the left side is the result of Western blot, and the quantified result of the gray scale after scanning the strip is corrected with ⁇ -actin internal parameters.
  • Ca cancerous tissue
  • CaP adjacent tissues.
  • Figure 7 shows that the 1B50-1 antibody has an inhibitory effect on the growth of transplanted tumors of human liver cancer Hep-12 cells with NOD/SCID (non-obese diabetic/severe combined immunodeficiency) mice.
  • Figure 7A Tumors formed by different groups of cells;
  • Figure 7B Growth curves of different groups of tumors in NOD/SCID mice;
  • Figure 7C Weight of different groups of tumors during dissection;
  • Figure 7D Volume of tumors of different groups at anatomy.
  • Figure 8 shows that 1B50-1 antibody has a inhibitory effect on the growth of human liver cancer HuH7 cells in NOD/SCID mice.
  • Figure 8A Tumors formed by different groups of cells;
  • Figure 8B Growth curves of different groups of cells in NOD/SCID mice;
  • Figure 8C Volume of tumors of different groups at anatomy;
  • Figure 8D Weight of tumors of different groups at anatomy.
  • Figure 9 shows inhibition of growth of liver cancer Hep-12 cells in NOD/SCID mice by RNA interference of CACNA2D1 gene.
  • Figure 9A After RNA interference 1B50-1 antibody staining showed a significant decrease in cellular CACNA2D1 protein levels;
  • Figure 9B In vivo tumor growth curve after RNA interference;
  • Figure 9C Tumor volume at anatomy;
  • Figure 9D Tumor weight at dissection.
  • Figure 10 shows that a single chain antibody expressed by QM-7 cells can bind to CACNA2D1 gene-positive cells. Detailed ways
  • the human hepatoma cell lines Hep-11 and Hep-12 are formed by primary culture of primary and recurrent liver cancer tissues derived from the same liver cancer patient (for detailed background materials on the cell pairs, see: Zhang Z, Xu X, Xing B, Wang Y, Han H, Zhao W. Identification and characterization of tumor-initiating cells with stem-like properties from a recurrent hepatocellular carcinoma [abstract], Proceedings of the 100th Annual Meeting of the American Association for Cancer Research, 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR, 2009.
  • Hepatoma cell line HuH7 Japan Revitalization Society
  • HepG2 ATCC
  • SMMC-772K breast cancer cell lines ZR-75 ATCC
  • MCF-7 cells ATCC
  • MDA-MB-231 ATCC
  • BICR-H1 cells a gift from Professor Huang Xinfu of Beijing Cancer Hospital
  • lung cancer Cells A549 ATCC
  • Calu-3 ATCC
  • Calu6 ATCC
  • PG a gift from Professor Wu Bingquan from Peking University School of Medicine.
  • the esophageal cancer cell lines KYSE150, KYSE510 (reported by Professor Lu Fengmin from the Basic Medical College of Peking University), the gastric cancer cell lines BGC823, MGC803, SGC7901, and the prostatic adenocarcinoma PC3M1E7 and PC3M2B4 are common cell lines and are preserved by the inventors' laboratory.
  • cyclophosphamide cyclophosphamide, Sigma-Aldrich, St Louis, MO
  • Preparation method according to the eighteenth day collecting Hep-11 cells Hep-12 cells, each mouse intraperitoneal inoculation of approximately 2.5> ⁇ 10 6 cells /0.5ml PBS.
  • the immunization was boosted with an equal amount of Hep-12 every three weeks for a total of three times.
  • the spleen was taken 3 days after the last booster to prepare a cell suspension, mixed with 10 8 SP2/0 cells (ATCC), serum-free RPMI 1640
  • the cells were fused with 50% PEG4000 (Sigma-Aldrich) as usual.
  • the cells were resuspended in 1640 medium containing 15% fetal calf serum containing HAT (Sigma-Aldrich), seeded in a 96-well cell culture plate, and cultured in a CO 2 cell incubator.
  • the fusion cells were exchanged with 1640 medium containing 15% calf serum containing HAT.
  • the cell culture supernatant was taken to detect the hybridoma clones secreting specific antibodies according to the growth of the hybridoma clones. .
  • Hybridoma clone detection and screening subclones The hepatoma cell lines Hepl l and Hepl2 cells were inoculated into a 96-well cell culture plate with 15% fetal bovine serum 1640, respectively. When the cells were attached and overgrown, the culture supernatant was discarded. The pre-cooled PBS containing 0.125% glutaraldehyde was added and allowed to stand at room temperature for 5 minutes, and then the cell fixing solution was discarded. Wash three times with PBS, add PBS containing 5% skim milk powder, and block overnight at 4 G C. The blocking solution was discarded, and the hybridoma clone culture supernatant was added thereto, and allowed to stand at room temperature for 1 hour.
  • hybridoma was deposited with the China Microbial Culture Collection Management Committee General Microbiology Center (CGMCC) on December 08, 2010, and the accession number is CGMCC No. 4416.
  • CGMCC China Microbial Culture Collection Management Committee General Microbiology Center
  • Hybridoma clones capable of secreting specific antibody 1B50-1 were expanded and inoculated into the plant
  • Horseradish peroxidase-labeled goat anti-mouse antibody was diluted with 5% skim milk powder in PBS, added to the assay plate, incubated for 1 hour at room temperature, and washed 5 times with PBS.
  • the ELISA substrate reaction solution was added, and the reaction was allowed to stand at room temperature for 30 minutes in the dark, and the reaction was terminated by adding an equal volume of 12.5% sulfuric acid.
  • 1B50-1 hybridoma cells were extracted from the total RNA by Trizol method and reverse transcribed into cDNA by Superscript III (Invitrogen) reverse transcriptase, and the mouse antibody heavy chain variable region was used to contain degenerate base primers at the 5' end.
  • an expression vector for the single-chain antibody directed by the MMP-9 signal peptide was constructed and transfected into skeletal muscle myoblast QM-7. After induction, the medium was incubated with Hep-12 cells. Use MYC-label The antibody was subjected to indirect immunofluorescence cytochemical staining to see if the medium specifically binds to Hep-12 after transfection.
  • the 1B50-1 antibody hybridoma culture supernatant was added, and the incubation was continued for 2 hours at 37 ° C, the culture solution was discarded, and the cells were washed 3 times with PBS.
  • the cultured cells were collected with a cell scraper. After centrifugation, 5 ml of deionized water was added, and the cells were resuspended and sonicated. Add 2X cell lysate and sonicate again.
  • Sepharose 4B - protein G (Pharmacia, Uppsala, Sweden) affinity chromatography beads were added. After 1 hour at room temperature, the column was washed with PBS, and the bound antigen was eluted with 0.1 M glycine hydrochloride buffer (pH 2.5), adjusted to pH 7.0 with 1 M Tris-HCU pH 9.0, and an equal amount of 2x SDS-PAGE sample was added. Buffer, analyzed by SDS-PAGE. Coomassie Brilliant Blue R250 stained the 1B50-1 immunoprecipitated specific band in Hep-12 expression, and subjected to MALDI-TOF mass spectrometry after trypsin digestion.
  • M-MLV-RT rodent leukemia virus reverse transcriptase
  • SYBR Green PCR fluorescent dye mixture Toyobo Co. Ltd., Osaka, Japan
  • CACNA2D1 5'-ACAGCAAGTGGAGTCAATCA-3' 5'-ACTGCTGCGTGCTGATAAGA -3'
  • CEACAM6 5'-GAAATACAGAACCCAGCGAGTGC-3' 5'-CAGTGATGTTGGGGATAAAGAGC-3'
  • KLF4 5'-AAGCCAAAGAGGGGAAGAC-3' 5'-CATCTGAGCGGGCGAATTTC-3'
  • the forward and reverse primers are designed in three segments.
  • the three primer sequences are:
  • the first stretch of the forward primer 5,- CCGgaattcTATGGCTGCTGGCTGCCTGCTGG-3', reverse primer 5,-AACCATTAGGATCGATTGCAAAG-3,;
  • the second forward primer 5,-TGTGTACCTGGATGCATTGGAACTG-3'
  • reverse primer 5'- ACCATCATCCAGAATCACACAATC-3'
  • the third forward primer 5,-AGAGACATATGAGGACAGCTTC-3', reverse primer:
  • the cells were inoculated with a density of 80-90% the next day.
  • the constructed CACNA2D1 mychis/pcDNA3.0 plasmid was transfected into cells using Lipofectamine 2000 (Invitrogen) following the company recommended procedure.
  • Immunofluorescence cytochemical staining analysis of the gene expression was performed 24 and 48 hours after transfection. Cellular immunofluorescence staining
  • the cultured cells were digested with trypsin: EDTA to prepare a single cell suspension. 2 ⁇ 10 6 cells were added to the purified 1B50-1 antibody (1:100 dilution, storage solution 1 mg/ml), incubated at 37 ° C for 1 hour, PBS wash 3 times, FITC-labeled goat anti-mouse II anti-IgG (Jackson ImmunoResearch Laboratories, USA, 0.5 mg/ml; 1:100 dilution) was added, and the effect was 1 hour at 37 ° C. After washing with PBS, under the Idea SP5 laser confocal microscope. Observe, or analyze and sort by Aria flow cytometry.
  • the cells transfected with the CACNA2Dlmychis/pcDNA3.0 plasmid were double-stained with 1B50-1 monoclonal antibody and myc rabbit polyclonal antibody, and the secondary antibody was labeled with rhodamine-labeled goat anti-mouse, FITC-labeled goat anti-rabbit IgG, respectively.
  • Radio Immuno-precipitation assay buffer containing 1 mM PMSF and a complete protease, phosphatase inhibitor cocktail (Roche, Mannheim, Germany). After SDS-polypropylene gel electrophoresis, the electricity is transferred to
  • Immobilon-P membrane (Millipore). After blocking with 5% non-fat dry milk, sequentially with I anti-CACNA2DK Abeam, Cambridge, MA) or internal reference ⁇ -actin (Roche Applied Science) antibody and horseradish peroxidase-labeled goat anti-mouse II antibody ( Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) Reaction, positive signal was detected by ImmobilonTM Western Chemiluminescent HRP substrate (Millipore) chemiluminescence. The strip signal was scanned with a Chemilmager scanner (Alpha lnnotech), and the gray scale of the strip was quantitatively analyzed by AlphaEaseFC software. The relative amount of CACNA2D1 in the sample was calculated by using the internal variable of ⁇ -actin as the internal reference.
  • CACNA 2 D The interfering RNA sequence of CACNA 2 D, a gene designed and synthesized by Origene, was constructed based on a retroviral vector using the U6 promoter as a promoter, wherein sequence 1 is the ACTCAACTGGACAAGTGCCTTAGATGAAG sequence for the CACNA2D1 gene coding sequence 546-574. 2 is AGATGCAAGAAGACCTTGTCACACTGGCA for the gene coding sequence 116-144, and the control sequence is an irrelevant isometric nucleotide sequence randomly synthesized by Origene.
  • the vector containing the above sequence was digested with EcoRI and Sail, respectively, and the fragment containing the U6 promoter and the above oligonucleotide sequence was separated and purified by agarose gel electrophoresis, and the Plenti6-linker vector which was similarly digested (Invitrogen was used by the laboratory)
  • the company's plenti6 vector was digested with Cla I and Age I, and then inserted into the cloning sequence containing the multiple cloning site, and stored in the laboratory.
  • the lentiviral plasmid vector plenti6U6CACNA2DlShRNA-l was obtained and ligated.
  • plenti6U6C ACNA2D 1 ShRNA-2 and control plenti6U6-control The packaging of lentiviruses is strictly in accordance with the method recommended by Invitrogen.
  • the culture supernatant containing the lentiviral particles directly infects Hep-12 cells, and 48 hours later, 6 g/ml of Blasticidin (Invitrogen) is added to screen the infected cells, and the fresh medium is replaced every three days to obtain the cell population infected with the virus. And continued to use 6 g/ml during the experiment.
  • Blasticidin to maintain screening pressure.
  • the infected cells were further examined by RT-PCR and immunofluorescence cytochemical staining for the inhibition of CACNA2D, gene suppression. Inhibition of tumor formation and growth was observed by tumorigenic experiments in immunodeficient animals. Animal tumorigenesis and antibody inhibition test
  • Anti-tumor test of antibodies 4-6 weeks old NOD/SCID mice were inoculated subcutaneously with 2 ⁇ 10 6 liver cancer cells, and the tumors were grown to a size visible to the naked eye (about 0.02-0.03 cm 3 ), 6 in each group, and injected intraperitoneally every other day.
  • PBS control mouse IgG (Nakasugi Jinqiao Biotechnology Co., Ltd., 80 ( ⁇ g/only) and 1B50-1 antibody (200, 400 and 80 ( ⁇ g/only) respectively.
  • Measurement of tumors before each injection with vernier calipers For the long diameter and the short diameter, the tumor volume was calculated according to the formula "tumor volume long diameter X short diameter 2 /2". After a total of 7 doses, the animals were sacrificed every other day, and the formed tumor mass was dissected, and the wet weight and volume were measured.
  • Example 1 1B50-1 antibody recognizes cell membrane antigen, and the positive rate of various liver cancer cell lines is quite different: Hybridomas are prepared by subtractive immunoassay, and negative/positive screening is performed by Hep-1 1 and Hep-12 cell pairs. After three fusion screenings, a total of 37 monoclonal antibodies specific for Hep-12 were obtained in the first round. These antibodies were further subcloned and analyzed, and one of the hybridomas was named 1B50-1. The antibodies produced by its secretion are localized in the cell membrane (Fig. 1). Further, flow cytometry was used to analyze the positive rate of 1B50-1 antibody in different hepatoma cell lines and cells from primary cultures of clinical liver cancer tissue samples. The results are shown in Table 2. The content of 1B50-1 positive cells was higher in Hep-12 cells than the regenerative source and tumor-initiating cells.
  • Example 2 Positive cells recognized by 1B50-1 antibody have tumor initiating cell characteristics
  • Flow cytometry was used to sort 1B50-1 antibody negative/positive cell population from 5 hepatocellular carcinoma cell lines including Hep-11, Hep-12. HuH7, HepG2 and SMMC-7721, and 100 and 1000 cells were injected separately. NOD/SCID mice were subcutaneously (negative and positive cells were injected subcutaneously into different sides of the same mouse). After 12-18 weeks, 100-1000 1B50-1 positive cells were sufficient to form tumors under the skin of mice, while negative cells were not. Long or only small nodules (Table 2, Figure 2). It is suggested that the 1B50-1 antibody-positive cells have the characteristics of tumor-initiating cells.
  • 1B50-1 positive cells were cultured in a medium containing 10% fetal bovine serum, and the percentage of 1B50-1 positive cells was analyzed. It was found that the ratio of 1B50-1+ cells decreased to be close to the 1B50-1 positive rate in maternal cells ( Figure 2C), suggesting that 1B50-1 positive cells can differentiate into 1B50-1 positive and negative cells. These results indicate that 1B50-1 positive cells have tumor stem cell characteristics.
  • the antigen recognized by the 1B50-1 antibody is CACNA2D1
  • Antibody 1B50-1 was immunoprecipitated from Hep-12 cells to a specific band around 150 KD (arrows indicate bands) (left panel of Figure 3). Mass spectrometry indicated that the protein was CACNA2D1.
  • the cDNA of Hep-12 cells was used as a template to amplify the gene by PCR. The gene was ligated into the pcDNA3.0 mychis eukaryotic expression vector by conventional DNA recombination technology. Cells that did not express this gene were double-stained with MYC polyclonal antibody and 1B50-1 antibody.
  • the overall survival was shorter than that of the patient's paracancerous tissue 1B50-1 antibody negative group (Fig. 4).
  • the presence or absence of 1B50-1 antibody-positive cells in the margin tissue can predict the recurrence and prognosis of liver cancer patients, and can be used as an indicator for prognosis evaluation of liver cancer.
  • Cirrhosis of the liver 0.002 0.448 None 27 6 22.2 18 66.7
  • Example 5 Expression of CACNA2D1 gene in gastric cancer, esophageal cancer, breast cancer, lung cancer and other cell lines
  • the CACNA2D1 gene in gastric cancer, lung cancer, breast cancer and prostate cancer cell lines other than liver cancer was firstly analyzed by RT-PCR. The expression was examined and the results were as shown in Fig. 5A. Positive bands were detected in MGC-803, SGC7901 in gastric cancer cells, PG and A549 cells in lung cancer, and BICR-HI and MDA-MB-231 cells in breast cancer. Significantly expressed in the highly metastatic prostate cancer PC3M1E7, but not in the low metastatic PC3M2B4.
  • Example 6 Distribution of 1B50-1 antibody-positive cells in other tumor tumor tissue samples and Western blot analysis of CACNA2D1 protein
  • liver cancer tissues were also applicable to other types of tumors.
  • 10 pairs of clinical colorectal cancer, kidney cancer, lung cancer and esophageal cancer and their matched adjacent normal tissues for immunohistochemical staining were selected 10 pairs of clinical colorectal cancer, kidney cancer, lung cancer and esophageal cancer and their matched adjacent normal tissues for immunohistochemical staining.
  • the distribution of 1B50-1 antibody-positive cells was similar to that of liver cancer tissues.
  • the results are shown in Table 4.
  • the number of 1B50-1 positive cells detected was higher than that of adjacent tissues, but the number of cases was too small for statistical analysis.
  • we performed immunoblot hybridization analysis of commercial tissue samples using commercially available CACNA2D1 antibodies were performed in Fig. 6.
  • CACNA2D1 in some cases of cancer tissues was significantly higher than that in adjacent tissues (the high expression rate of CACNA2D1 was different in different cancer tissue types), indicating that the expression of CACNA2D1 was increased after canceration.
  • the high expression of CACNA2D1 is not only suitable for liver cancer, but also widely found in cancer tissues of various tissue types. Therefore, drugs and molecular markers against CACNA2D1 are not limited to liver cancer.
  • Type of tissue Total number of cases Positive number of cancerous tissues Number of positive cases of adjacent tissues
  • Kidney cancer 10 4 1 Kidney cancer 10 4 1
  • the growth rate of the two lentivirus-infected cells carrying the CACNA2D1 interfering RNA in the animals was significantly slower than that in the transfected empty vector control group, and the tumor inhibition rates were 57.5% and 59.6%, respectively.
  • the p values compared with the control group were 0.0164 and 0.014, respectively.
  • the culture supernatant of the single-chain antibody of the 1B50-1 variable region expressed by QM-7 cells was co-incubated with Hep-12 cells, and MYC-tagged peptide antibody 9E10 was used as the I antibody, FITC-labeled goat anti-mouse II anti-staining , observed under fluoroscopy. It can be seen that the surface of Hep-12 cells can be recognized by MYC-tagged peptide antibody 9E10 (Fig. 10), and the pattern of fluorescent staining is consistent with that of 1B50-1 antibody, indicating that the expressed single-chain antibody has the same binding activity as 1B50-1 antibody.
  • variable region light chain and heavy chain sequences of the cloned 1B50-1 antibody are correct, further antibody drug modification can be carried out by genetic engineering techniques based on these sequences.
  • markers for tumor-initiating cells can be sought, identified or confirmed.
  • Tumors can also be diagnosed, treated and prevented according to markers determined using the methods of the invention.
  • a tumor-specific cell-specific marker can be determined, thereby developing a therapeutic agent for the starting cell, and formulating a diagnosis, treatment, and prognosis strategy, thereby promising a problem of recurrence and metastasis of the tumor. , bringing a new dawn to the complete conquest of the tumor.
  • the monoclonal antibody or its monoclonal fragment specific for the target CACNA2D1 provided by the present invention can be directly used to treat or prevent a tumor or a disease or condition associated with CACNA2D1, or can be used for a pharmaceutical composition or a diagnostic reagent. Preparation of the box.

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Description

识别肿瘤起始细胞的抗体和抗原及其应用 技术领域
本发明属于医药生物技术领域， 具体而言， 本发明涉及寻找肿瘤起始细胞的标志 物和 /或治疗靶分子的方法， 以及靶向该肿瘤起始细胞的标志物和 /或治疗靶分子的肿 瘤治疗药物的制备和筛选方法。 本发明还涉及用于肿瘤诊断， 预后判断和治疗的单克 隆抗体或其抗原结合片段， 包含该单克隆抗体或其抗原结合片段的诊断试剂盒或药物 组合物， 以及所述单克隆抗体或其抗原结合片段在肿瘤等的诊断、 预后判断或治疗中 的应用。 本发明还涉及到该抗体所针对的抗原或其识别的靶分子在作为肿瘤起始细胞 (或称肿瘤干细胞） 的标志物用于癌症的诊断或疗效预测中的应用， 以及作为肿瘤治 疗的试剂和药物开发的分子靶点。 背景技术
很久以前就已认识到肿瘤组织是由功能有差别、 表型各异的异质性的细胞群体组 成。 肿瘤干细胞 (tumor/cancer stem cells, TSC/CSC)学说认为， 肿瘤如正常的组织器官 一样， 是由肿瘤干细胞不断增殖、 分化所形成， 因此呈现异质性。 肿瘤干细胞是肿瘤 组织中含量较少、具有无限自我更新能力、能驱动肿瘤形成和生长的一类细胞的代称， 因与正常干细胞特点相似而得名， 但并非指肿瘤干细胞一定来源于相应的正常干细胞 或与正常干细胞有必然的联系， 有人为避免误会亦称这些细胞为肿瘤起始细胞 (tumor-initiating cell, TIC)或肿瘤传递细胞 (tumor-propagating cell, TPC)。 肿瘤干细胞 表现出很强的动物致瘤 /癌能力 (100 个甚至数个该细胞在免疫缺陷小鼠中即可成瘤 / 癌)、 耐药性和侵袭性生长特性， 其还具有分化成不具致瘤能力细胞的潜能。肿瘤干细 胞的存在被认为是肿瘤发生、 发展和治疗失败的根本原因。
自从血液中第一次分离鉴定出肿瘤干细胞以来， 世界上相继从脑、 乳腺、 *** 和结肠癌等实体肿瘤中证实肿瘤干细胞的存在。 在肝癌中多个研究小组也采用不同的 策略从培养的细胞系或者临床标本中找到肝癌干细胞， 如从 Huh7细胞系中分离到的 侧群 (side population, SP)细胞在动物连续接种实验中可以成瘤， 也有报道 Huh7 和 PLC8024细胞系中 CD133可作为肝癌干细胞的标志物。最近， CD90、 EpCAM、 OV6、 CD133/ALDH也分别被用来成功分离肝癌的干细胞。尽管通过不同的途径已鉴别到具 有干细胞特性的肿瘤细胞， 但由于目前所发现的肿瘤干细胞标志物特异性都不是很 强， 因此不同来源肿瘤用同一标志物分离到的各类型细胞的生物学特性差别有时很 大， 因此对是否存在肿瘤干细胞以及这些细胞的本质一直存在争论。 事实上肿瘤干细 胞，或称为肿瘤起始细胞，甚至肿瘤传递细胞只是一个可操作性词汇 (operational term), 其生物学的本质尚有待进一步揭示。 但是肿瘤组织中存在一类在免疫缺陷动物体内致 瘤性强、 耐受放化疗的细胞已得到共识， 无论这一类细胞是称为肿瘤干细胞、 肿瘤起 始细胞或者是肿瘤传递细胞， 它们是肿瘤患者治疗失败、 复发的根本原因。 这些细胞 的分离鉴定为肿瘤的诊断和治疗提供了新的思路。 过去的治疗主要集中在肿瘤群体中 大量的恶性度较低的细胞， 而那些所谓的肿瘤起始细胞虽然比例很低， 但因为耐受常 规的放化疗却生存下来并逐步生长、 迁移， 导致肿瘤复发转移。 旨在清除肿瘤起始细 胞的药物， 则可从根本上阻断肿瘤复发转移的 "种子"， 单独或与常规手术、 放化疗等 方法结合有望为肿瘤的彻底征服带来新的曙光。 这些细胞亦应成为肿瘤诊断治疗和预 后判断的靶细胞。
目前已经在靶向决定肿瘤干细胞***的实验中取得令人振奋的进展。 例如在 结直肠癌中因为 CD133阳性的肿瘤干细胞高表达 IL-4而抑制细胞凋亡的发生，用 IL-4 的抑制剂处理 CD133 阳性的肿瘤干细胞则可提高其对药物的敏感性。 研究也表明用 CD44的抗体可以清除急性白血病中的肿瘤干细胞从而达到治疗急性白血病目的。
针对肿瘤干细胞的药物开发可以针对肿瘤干细胞自我更新、 耐药或侵袭性生长等 环节信号传导途径中的关键分子、 表面的标志物甚至其赖以存在和生长的微环境 (niche). 对参加这些关键环节的分子的发现有赖于对肿瘤干细胞的分离鉴定和恶性生 物学特性的本质和调控机制的深入揭示。 需要指出的是， 与通常所说的肿瘤相关抗原 相比， 肿瘤干细胞相关分子的表达一般比较低。 因此， 肿瘤干细胞相关分子的发现主 要是依赖于其在肿瘤干细胞样群体的特异表达而不是过表达。
现代药物开发技术的发展， 使得可以针对特定的分子利用计算机模拟设计特异的 分子拮抗剂、 利用现有的前导药物库筛选候选的药物、 制备具有拮抗作用的抗体以及 利用其它分子细胞生物技术如噬菌体展示技术筛选分子的靶向药物。
单克隆抗体技术已成为制备针对特异抗原或特定细胞抗体的常规技术。 许多抗体 被直接或通过基因工程改造成鼠-人嵌合甚至完全人源化的抗体用于临床多种疾病包 括恶性肿瘤的治疗。 目前为止， 抗体介导的针对肿瘤干细胞相关的治疗实验包括 CD44、 p糖蛋白 l(p-glycoprotein 1)、透明质酸受体、 EpCAM、 CD326、 CXCR4、 IL-4、 DLL4、 ALDH等。
本领域需要能够特异识别肿瘤起始细胞的标志物， 以便用于这类细胞的筛选鉴定 和临床诊断、 预后判断和对这类细胞的恶性生物学行为特性及分子调节机制的研究。 同时研制针对肿瘤起始细胞的药物， 以从根本上***， 阻断肿瘤的复发、 转移。 发明内容
本发明的一个目的在于寻找、 鉴别或确认能用于肿瘤起始细胞分离、 鉴定和治疗 的分子靶点。
本发明的另一个目的还在于提供肿瘤或与 CACNA2D1蛋白相关的疾病或病症的 诊断、 治疗和预防方法。
本发明提供了一种通过使用复发性肿瘤来源并富含肿瘤起始细胞的细胞来免疫 动物的步骤来寻找、 鉴别或确认肿瘤起始细胞的标志物和 /或治疗靶标 /分子靶点的方 法。
本发明还提供了一种诊断、 治疗和预防肿瘤的方法， 包括利用本发明所提供的寻 找、 鉴别或确认肿瘤起始细胞的标志物和 /或治疗靶标 /分子靶点的方法， 针对所找到、 鉴定或确认的肿瘤起始细胞的标志物和 /或治疗靶标 /分子靶点来研制和 /或筛选具有降 低该标志物或靶标 /分子靶点的基因表达或 /和蛋白活性、 或靶向该标志物或治疗靶标 / 分子靶点后引起细胞毒性反应的抗体或抗原结合片段、单链或双链寡聚核苷酸、核酸、 短肽或小分子化合物药物， 然后向需要的受试者施用治疗有效量的至少一种所研制或 筛选出的抗体或抗原结合片段、 单链或双链寡聚核苷酸、 核酸、 短肽或小分子化合物 药物。
本发明还提供了针对肿瘤或与 CACNA2D1蛋白相关的疾病或病症的诊断或治疗 用的单克隆抗体或其抗原结合片段。
本发明还提供了单克隆抗体或其抗原结合片段在诊断、 预后判断和***或与 CACNA2D1蛋白相关的疾病或病症中的应用。
本发明的发明人发现电压依赖性钙离子通道蛋白 α2δ 亚基 1 (calcium-channel, voltage-dependent, α2/δ subunit 1， 简称 CACNA2D1 , GenBank序列号 NM— 000722.2) 是一个肿瘤起始细胞的分子标志物和治疗的分子靶点或靶标。可用于针对 CACNA2D1 基因的核酸或蛋白进行诊断试剂和治疗药物的研发。 本发明的发明人从同一肝细胞癌 病人的原发和复发灶的标本中分别培养出细胞系 Hep-11 (原发肝癌组织来源） 和 Hep-12 (复发肝癌组织来源） 。 利用群体和克隆化法本发明的发明人发现大多数 （超 过 80%) 复发来源的 Hep-12细胞具备肿瘤起始细胞特性， 而原发的 Hep-11细胞接种 107细胞 6个月尚未见肿瘤形成。本发明的发明人发现用 Hep-12细胞免疫 balb/c小鼠， 制备杂交瘤， 得到的一个抗体 1B50— 1能特异识别 Hep-12细胞。 本发明的发明人发 现用流式细胞仪从 5个肝癌细胞系和 4例临床肝癌标本分选 1B50-1抗体阳性的细胞， 100— 1000个细胞足以在 NOD/SCID小鼠皮下形成肿瘤， 基因表达和细胞分化研究提 示该抗体阳性细胞具有肿瘤干细胞特性。 本发明的发明人发现用该抗体腹腔注射荷 Hep-12, Huh7瘤的 NOD/SCID 小鼠， 可剂量依赖性抑制移植瘤的生长， 抑瘤率分别 可达 80.4%、 65.5%(以重量为准，与对照 IgG组相比）。免疫沉淀 /质谱分析表明 1B50-1 抗体识别的抗原是一 150 kd的离子通道蛋白。 本发明的发明人发现用 RNA干扰抑制 该基因的表达， Hep-12 细胞的在裸鼠体内的生长亦可以得到抑制。 这些结果提示 1B50-1抗体识别的抗原是一新的肝癌肿瘤起始细胞的标志物和治疗的分子靶点。
根据本发明的一个实施方案， 本发明提供了寻找、 鉴别或确认肿瘤起始细胞的标 志物和 /或治疗靶标或分子靶点的方法。本发明也提供了寻找、鉴别或确认能用于肿瘤 起始细胞分离、 鉴定和治疗的分子靶点的方法。 所述方法包括使用复发性肿瘤来源、 富含肿瘤起始细胞的细胞来免疫动物的步骤。 在本发明的寻找肿瘤起始细胞标志物和 /或治疗靶标 /分子靶点的方法的具体实施方式中， 所述肿瘤可以为肝癌、 结肠癌、 直 肠癌、 肾癌、 食道癌、 胃癌、 肺癌、 乳腺癌、 ***癌或其它高表达 CACNA2D1基 因的肿瘤。 在本发明的另一个实施方式中， 该肿瘤为肝癌， 所述复发性肿瘤来源、 富 含肿瘤起始细胞的细胞是复发性肝癌来源、 富含肿瘤起始细胞的 Hep-12 细胞。 在本 发明的寻找肿瘤起始细胞标志物和 /或治疗靶标的方法中，所述方法进一步包括利用分 别来源于同一肝癌病人复发和原发的肝细胞癌的 Hep-12细胞和 Hep-11细胞作为筛选 细胞对。
根据本发明的一个实施方案， 本发明提供了一种***的方法， 包括
1) 使用复发性肿瘤来源并富含肿瘤起始细胞的细胞 （如 Hep-12细胞） 来免疫动 物 （如 balb/c 小鼠） ， 寻找、 鉴别或确认肿瘤起始细胞的标志物和 /或治疗靶标 / 分子靶点 （如 CACNA2D1 ) ；
2) 针对步骤 1) 所找到、鉴定或确认的肿瘤起始细胞的标志物和 /或治疗靶标 /分子 靶点 （如 CACNA2D1 ) 研制和 /或筛选具有降低该标志物或靶标 /分子靶点的基因 表达或 /和蛋白活性、或靶向该标志物或治疗靶标 /分子靶点后引起细胞毒性反应的 抗体或抗原结合片段、 单链或双链寡聚核苷酸、 核酸、 短肽或小分子化合物药物;
3) 向需要的受试者施用治疗有效量的至少一种步骤 2)所研制或筛选出的抗体或 抗原结合片段、 单链或双链寡聚核苷酸、 核酸、 短肽或小分子化合物药物。 根据本发明的一个实施方案， 上述步骤 1 ) 可通过用复发性肿瘤来源并富含肿瘤 起始细胞的细胞（如 Hep-12细胞）免疫动物（如 balb/c小鼠） ， 制备杂交瘤， 得到的 一个单克隆抗体（如 1B50— 1 )，以该抗体（如 1B50— 1 )能特异识别的细胞（如 Hep-12 细胞） 作为鉴定和确认肿瘤干细胞的候选细胞。 通过动物实验 （如 100— 1000个细胞 能否在 NOD/SCID小鼠皮下形成肿瘤）、基因表达和细胞分化等研究和实验确认该抗 体阳性细胞是否具有肿瘤干细胞特性。通过本发明的方法得到的单克隆抗体（如 1B50 - 1 ) 在肿瘤干细胞中特异识别的抗原即为本发明所要寻找的肿瘤起始细胞的标志物 和 /或治疗靶标 /分子靶点。 根据本发明的一个具体实施方案， 本发明的发明人发现并 确认 CACNA2D1蛋白是一个肿瘤起始细胞的标志物、 肿瘤 /癌症的治疗靶标或分子靶 点。
根据本发明的一个实施方案， 上述步骤 2) 所研制或筛选的具有降低肿瘤起始细 胞标志物、 治疗靶标或分子靶点的基因表达或 /和蛋白活性； 或靶向该标志物、 治疗靶 标或分子靶点后引起细胞毒性反应的抗体或抗原结合片段可以是如上述步骤 1 ) 所得 到用来寻找、 鉴别或确认肿瘤起始细胞抗原（标志物、 肿瘤 /癌症的治疗靶标或分子靶 点） 的单克隆抗体 （如 1B50— 1 ) ， 也可以是针对所找到、 鉴定或确认的肿瘤起始细 胞的标志物、 肿瘤 /癌症的治疗靶标或分子靶点（如 CACNA2D1蛋白 /抗原）所研制或 筛选的其他抗体。
根据本发明提供的肿瘤起始细胞的抗原 CACNA2D1 , 可以通过各种抗体制备技 术获得新的针对该抗原的抗体及其衍生物。 这些技术可以包括但不局限于用各种表达 体系表达 CACNA2D1抗原或提纯该抗原， 免疫小鼠、 兔或者其它动物 (包括经遗传改 造的动物品系)后得到的特异针对该抗原的抗血清； 取免疫小鼠的脾脏细胞， 与骨髓瘤 SP2/0细胞融合， 经 CACNA2D1抗原特异筛选特异针对 CACNA2D1抗原的单克隆抗 体； 进一步还包括克隆该抗原鼠单克隆抗体的可变区的基因， 直接或经人源化改造后 与相应的人源抗体的恒定区编码基因正确连接，经原核和 /或真核细胞表达体系表达所 得到的人鼠嵌合或单克隆抗体。 采用 CACNA2D1抗原筛选人噬菌体抗体库， 或用人 的外周血单核细胞嵌合小鼠技术， 亦可获得针对该基因的全人抗体。
根据本发明的一个实施方案， 可以通过使用本发明的抗体对 CACNA2D1 蛋白进 行高灵敏度定量。 通过采用体内定量 CACNA2D1蛋白的方法， 本发明的单克隆抗体 或其抗原结合片段可用于诊断肿瘤或与 CACNA2D1 蛋白相关的各种疾病。 因此， 本 发明还提供了用于诊断肿瘤或与 CACNA2D1蛋白相关的疾病或病症的方法， 所述方 法包括向需要的受试者施用有效量的至少一种本发明的单克隆抗体或其抗原结合片 段。 此体内诊断所需要的剂量可以小于治疗应用所需的剂量， 并且可以由本领域技术 人员通过常规步骤确定。 本发明的单克隆抗体或其抗原结合片段还可以用于特异性检 测测试液例如体液、 组织等中存在的 CACNA2D1蛋白。
根据本发明的一个实施方案， 本发明提供一种特异性识别电压依赖性钙离子通道 蛋白 α2δ 亚基 1 ( CACNA2D1 ) 的单克隆抗体或其抗原结合片段。 该特异性识别 CACNA2D1的单克隆抗体或其抗原结合片段包含： (i) 重链 （下文简称为 H-链） ， 其重链可变区包含互补决定区 （complementarity determining region, CDR) CDRH1 (序列 1 )、 CDRH2 (序列 2)和 CDRH3 (序列 3 )； 或
(ϋ) 轻链（下文简称为 L-链）， 其轻链可变区包含互补决定区 CDRL1 (序列 4) 、 CDRL2 (序歹 IJ 5 ) 和 CDRL3 (序列 6) ； 或
(iii) 上述 (i)和 (ii)两者。
在本发明的一个实施方案中， 上述单克隆抗体或其抗原结合片段具有以下一种或 多种特征： （1) 结合 CACNA2D1蛋白； （2)识别的阳性细胞具有肿瘤起始细胞特征； (3)抑制表达 CACNA2D1的肿瘤细胞在动物体内的生长。 其中上述肿瘤可以是肝癌、 结肠癌、直肠癌、肾癌、食道癌、胃癌、肺癌、乳腺癌、***癌或其它高表达 CACNA2D 1 基因的肿瘤。
在本发明提供的一个实施方案中， 本发明的单克隆抗体或其抗原结合片段包含重 链， 其重链可变区包含与 CDRH1、 CDRH2和 CDRH3的氨基酸序列具有至少 70%、 75%、 80%、 85%、 90%、 95%、 或 99%相同的氨基酸序列。 在本发明提供的一个实施 方案中， 本发明的单克隆抗体或其抗原结合片段包含轻链， 其轻链可变区包含与 CDRLK CDRL2和 CDRL3的氨基酸序列具有至少 70%、 75%、 80%、 85%、 90%、 95%、 或 99%相同的氨基酸序列。 在本发明提供的一个实施方案中， 抗体或其抗原结 合片段还可以包含上述重链和轻链两者。 在另一个实施方案中， 本发明的抗体或其抗 原结合片段还可以包含一个或多个与上述任何 CDR或 CDR的组合具有至少 70%、 75%、 80%、 85%、 90%、 95%、 或 99%相同的氨基酸序列的 CDR。
在本发明的一个实施方案中， 本发明提供一种用于诊断、 预后判断和***或 与 CACNA2D1蛋白相关的疾病或病症的单克隆抗体或其抗原结合片段， 包括上述的 单克隆抗体或其抗原结合片段。 其中上述肿瘤或疾病或病症可以是肝癌、 结肠癌、 直 肠癌、 肾癌、 食道癌、 胃癌、 肺癌、 乳腺癌、 ***癌或其它高表达 CACNA2D1基 因的肿瘤。
本发明提供一种杂交瘤细胞系， 该杂交瘤细胞系是由中国微生物菌种保藏管理委 员会普通微生物中心于 2010年 12月 08日保藏的小鼠杂交瘤 (保藏编号为 CGMCC No. 4416) 。
在本发明的一个具体的实施方案中， 上述单克隆抗体是由杂交瘤细胞系 （保藏编 号为 CGMCC No. 4416) 所产生的单克隆抗体， 1B50-1。
本发明的进一步实施方案还包括但不限于通过已知技术在上述抗体的基础上产 生识别抗原的特异表位的抗体片段。例如，可以通过免疫球蛋白分子的蛋白水解切割， 利用诸如木瓜蛋白酶（以产生 Fab片段）或胃蛋白酶（以产生 F(ab')2片段） 的酶产生 Fab和 F(ab')2片段。 F(ab')2片段包含完整的 L-链和 H-链的可变区、 CH1区和铰链区。 还可以利用已知的公共信息（例如来自 Genbank的信息、 文献或者通过常规克隆和序 列分析） ， 在本抗体核苷酸序列或氨基酸序列基础上获得进一步的衍生序列 （如用不 同的信号肽、 基于生物信息学分析后的人源化改造等） ， 可以化学合成编码所述免疫 球蛋白的核酸， 或者从合适的来源（例如抗体 cDNA文库， 或者由表达所述抗体的任 何组织或细胞， 例如所筛选的表达抗体的杂交瘤细胞分离的核酸， 优选地 mR A， 或 由其产生的 cDNA文库） 利用与序列的 3'和 5'末端匹配的合成引物通过 PCR扩增获 得编码所述免疫球蛋白的核酸或通过针对特定基因序列的寡核苷酸探针筛选来自如 cDNA文库获得编码所述免疫球蛋白的核酸。然后， 可以利用本领域熟知的任何方法， 将通过如 PCR扩增产生的核酸克隆至可复制、并可在不同表达体系（原核和真核表达 体系、 无细胞翻译体系等） 表达的载体内， 表达产生具有与本发明抗体生物活性相近 的基因工程抗体或抗原结合片段。
在本发明提供的一个实施方案中， 还可以通过本领域已知的任何其它用于合成抗 体的方法， 例如通过重组表达技术或通过化学合成， 产生本发明的抗体或其抗原结合 片段。
本发明还提供包含本发明的单克隆抗体或其抗原结合片段的药物组合物。 在本发 明的进一步实施方式中， 该药物组合物还可包含药学上可接受的载体。 此外， 本发明 提供了用于***或与 CACNA2D1蛋白相关的疾病或病症的药物组合物， 其包含 本发明的单克隆抗体或其抗原结合片段和药学上可接受的载体。 在本发明的一个具体 的实施方式中， 本发明的药物组合物还可以进一步包含可以加和或协同地改善上述疾 病或病症的其它活性化合物。 所述活性化合物包括， 但不限于治疗所述疾病或病症的 其它化疗化合物、 毒素。 所述活性化合物还包括小分子化合物和其它抗体或其抗原结 合片段。 具体而言， 上述肿瘤或与 CACNA2D1蛋白相关的疾病或病症可以是肝癌、 结肠癌、直肠癌、肾癌、食道癌、胃癌、肺癌、乳腺癌、***癌或其它高表达 CACNA2D1 基因的肿瘤。
有利地， 将上述用于经口或肠胃外施用的药物组合物制备成适合固定活性成分的 剂量的单位计量剂型。 此类单位剂量剂型包括， 例如片剂、 丸剂、 胶囊、 注射剂、 栓 剂等。
根据本发明的一个实施方案， 可以提供针对 CACNA2D1基因或蛋白的试剂盒。 其中， 该试剂盒可以用于诊断或治疗或预防肿瘤或与 CACNA2D1蛋白相关的疾病或 病症， 并且该试剂盒包括本文上述的单克隆抗体或其抗原结合片段， 或针对 CACNA2D1 的核酸， 例如 DNA、 mRNA。 该试剂盒可用于， 例如肝癌、 结肠癌、 直 肠癌、 肾癌、 食道癌、 胃癌、 肺癌、 乳腺癌、 ***癌或其它高表达 CACNA2D1基 因的肿瘤的诊断、 治疗或预防。根据该领域专业人员共知的知识， 在 mRNA水平可以 采用诸如定量 RT-PCR, 对抗原蛋白的检测可以通过该基因特异的抗体或特异结合小 分子采用共知的技术如酶联免疫吸附、 流式细胞分析、 免疫组化 （细胞化学） 等技术 进行检测。 所用的样品可以来源于患者的血液、 组织、 脱落细胞等。
根据本发明的另外的实施方案， 本发明提供本文所述的单克隆抗体或其抗原结合 片段在制备用于诊断、 治疗或预防肿瘤或与 CACNA2D1 蛋白相关的疾病或病症的药 物中的应用。 其中， 所述肿瘤或疾病或病症为肝癌、 结肠癌、 直肠癌、 肾癌、 食道癌、 胃癌、 肺癌、 乳腺癌、 ***癌或其它高表达 CACNA2D1基因的肿瘤。 本发明的单 克隆抗体或其抗原结合片段可作为诊断剂用于癌症 （例如病人组织或肿瘤病人血清中 是否含有肿瘤起始细胞以及预后判断和治疗敏感性预测） 以及其它 CACNA2D1 蛋白 相关的疾病或病症的诊断。 本发明的单克隆抗体或其抗原结合片段能够特异性识别 CACNA2D1抗原， 并由此可以用于定量测试液中的 CACNA2D1蛋白， 例如通过本领 域的常规技术如夹心免疫测定法、 竞争性测定法、 免疫测定法等定量测定。
另一方面， 本发明提供了用于***或与 CACNA2D1蛋白相关的疾病或病症 的方法， 所述方法包括向有此需要的受试者施予治疗有效量的至少一种本发明的单克 隆抗体或其抗原结合片段。
再一方面， 本发明提供了用于预防肿瘤或与 CACNA2D1蛋白相关的疾病或病症 的方法， 所述方法包括向有此需要的受试者施予预防有效量的至少一种本发明的单克 隆抗体或其抗原结合片段。
对于以上这些诊断、 治疗和预防方法， 本发明的单克隆抗体或其抗原结合片段可 以与增强所述单克隆抗体或其抗原结合片段的生物学作用的其它药物结合。 此类治疗 药物的实例包括另一种抗体、 抑制细胞生长或杀死细胞的细胞毒素、 放射性元素和 / 或包括抗炎剂和抗生素等在内的其它治疗剂。
可以通过将本发明的单克隆抗体或其抗原结合片段混合在合适的溶剂中而直接 作为液体制剂或作为适当剂型的药物组合物经口或肠胃外等施用。
可以将本发明的单克隆抗体或其抗原结合片段与药学上可接受的载体、 稀释剂或 赋形剂混合制备成药物组合物， 以适合于经口或肠胃外给药。 可以通过己知的多种递 送***施用本发明的单克隆抗体或其抗原结合片段或药物组合物， 例如脂质体包封、 微颗粒、 微胶囊等。 给药方法包括， 但不限于皮内、 肌内、 腹膜内、 静脉内、 皮下、 鼻内、硬膜外和经口途径。所述化合物可以通过任何途径施用， 例如通过输注或推注， 通过经上皮或皮肤粘膜 （例如口腔粘膜或直肠等)吸收的途径施用， 并且所述化合物可 以与其它生物活性剂一起施用。 给药可以是全身的或局部的。 优选地， 通过经静脉内 给药施用本发明的单克隆抗体或其抗原结合片段。 本发明的单克隆抗体还可以通过局 部施用在需要治疗的区域来给药。 所述组合物通过公众已知的方法制造， 且包含药物 制剂领域常规使用的载体、稀释剂或赋形剂。用于片剂的载体或赋形剂的实例为乳糖、 淀粉、 蔗糖和硬脂酸镁等。
所述可注射的制剂可以包括用于静脉内注射、 皮下注射、 皮内注射和肌内注射、 滴注等的剂型。 这些可注射的制剂可以通过公知的方法制备。 所述可注射制剂可以通 过， 例如将上述抗体或其盐溶解、 悬浮或乳化在常规注射用无菌含水介质或油性介质 中而制备。 所述注射用含水介质可以为， 例如生理盐水、 含葡萄糖和其它助剂的等渗 溶液等，其可以与适当的增溶剂组合使用，所述增溶剂为例如醇 (如，乙醇)、多元醇 (如， 丙二醇、 聚乙二醇)、 非离子表面活性剂 (例如聚山梨糖醇酯 80)等。 用于直肠给药的栓 剂可以通过将上述抗体或其盐与栓剂用常规基料混合而制备。
根据本发明的一个实施方案， 还提供肿瘤或与 CACNA2D1蛋白相关的疾病或病 症的治疗药物的制备和筛选方法， 该方法包括利用 CACNA2D1作为靶标， 研制和筛 选具有降低其基因表达或 /和蛋白活性、或靶向该分子后引起细胞毒性反应的抗体或抗 原结合片段、 单链或双链寡聚核苷酸、 核酸、 短肽或各种小分子化合物药物。
优选地上述发明方案以肝细胞癌为主要癌种， 还包括胃癌、 结肠癌、 直肠癌、 肾 癌、 食道癌、 肺癌、 乳腺癌、 ***癌等。
另外， 根据本发明提供的肿瘤起始细胞的抗原 CACNA2D1 , 很容易通过计算机 模拟或筛选现有的前导化合物库得到该分子特异的抑制剂-小分子化合物。
根据本发明的一个实施特例， 设计合成了针对 CACNA2D1 基因的干扰 RNA— ShRNAl (序列 7)和 ShRNA2 (序列 8) ， 并装入慢病毒表达载体， 包装慢病毒。 该病 毒抑制该基因的表达， 并对肝癌 Hep-12 细胞在免疫缺陷动物体内的生长具有显著抑 制效应。 根据本领域专业人员所共知的知识， 提供这些 shRNA序列， 可以通过化学 合成和修饰得到以匹配序列为核心的不同单链 RNA， 或采用不同的启动子、克隆载体 等基因重组常用载体携带这些核心序列。
定义
本发明中， 所使用的一些术语具有如下含义：
术语"肿瘤起始细胞"、 "肿瘤干细胞"、 "肿瘤传递细胞"在本文中可以互换使用， 这些术语的称谓只是一个实际应用过程中的对一类细胞的称谓， 有关这一类细胞的具 体生物学特性尚有待更进一步的研究揭示。 但这一类细胞一般具有但不限于以下特 点： （1) 高度恶性： 在免疫缺陷小鼠体内很少量的细胞 (100个甚至更少)即可成瘤， 且 这种致瘤性相对稳定； （2) 对常规治疗的耐受性： 对常规的放化疗表现为抗拒性， 不 敏感。
本发明使用的术语 "抗体 "是指能够特异性地结合目标多肽或目标序列的抗体分 子， 并涵盖完整的抗体分子或其片段， 包括其抗原结合片段。
本发明使用的术语 "抗原结合片段"是指保留了特异性地结合目标蛋白、 多肽或序 列的能力的任何抗体片段， 其包括单链抗体、 Fab 片段、 F(ab')2片段、 二硫键连接的 单链抗体 (sFv)和包含轻链可变区 （VL)和 /或重链可变区 （VH) 中任一种的片段或包 含特异结合目标蛋白、 多肽或序列的 CDR的片段。 本领域熟知获得上述抗体片段的 各种方法。
本发明使用的术语"互补决定区 （complementarity determining regions, CDR) "是 指抗体识别和结合抗原的区域， 直接决定抗体的特异性， 或决定抗体抗原结合活性的 特定氨基酸序列。
本发明使用的术语"特异性地识别 "是指抗体或其抗原结合片段特异性地结合目标 蛋白、 多肽或序列的能力。 该抗体不非特异性地与其它多肽或蛋白结合。 优选地， 特 异性地识别 CACNA2D1的抗体或其抗原结合片段不与其它抗原交叉反应。
术语"有效量 "是指 "治疗有效的抗增殖量"或"预防有效的抗增殖量"。 该术语包括 就剂量和必要的时间周期而言， 有效获得期望的结果例如足以治疗细胞增殖病症的 量。 本发明的化合物的有效量可以根据例如如下的因素而不同： 受试者的疾病状态、 年龄和体重、 及本发明的化合物在受试者中引起期望的响应的能力。 可以调节剂量方 式以提供最佳的治疗反应。 有效量也是本发明的化合物的治疗有益效果胜于任何毒性 或不良作用 （例如副作用） 的量。 附图说明
图 1是针对富含肝癌干细胞的 Hep-12的抗体 1B50-1的免疫荧光染色，显示 lb50-l 抗体所识别的抗原定位在细胞膜上。 多数 Hep-12细胞呈阳性， 而 Hep-11细胞多为阴 性。
图 2显示动物接种实验证明 1B50-1抗体阳性细胞具有肿瘤起始细胞特性。图 2 (A) 显示不同来源的 1B50-1抗体阳性细胞在 NOD/SCID小鼠中形成的肿瘤； 图 2 (B)显 示 1B50-1抗体阳性细胞在 NOD/SCID小鼠中形成的肿瘤伊红一苏木精染色结果表明 组织形态接近于 1B50-1抗体阳性细胞所来源的病人的癌组织形态； 图 2 (C) 显示流 式细胞分析结果表明纯化的 1B50-1抗体阳性细胞可以分化形成 1B50-1抗体阳性和阴 性细胞； 图 2 (D) 显示实时荧光定量 RT-PCR结果表明 1B50-1抗体阳性细胞高表达 多个与干细胞相关的基因。
图 3显示对 1B50-1抗体所识别的抗原 CACNA2D1的鉴定。 图 3A. 免疫沉淀分 析结果； 图 3B. 表达 CACNA2Dl-myc的质粒转染 1B50-1抗体阴性的细胞的结果， 显 示 Myc和 1B50-1抗体染色在细胞膜上具有共定位关系。 合并 （Merge) 为将 Myc和 1B50-1抗体染色叠放在一起的结果。
图 4 是肝癌标本中 1B50-1抗体免疫组化染色结果。显示 1B50-1抗体阳性细胞分 散地分布在肿瘤组织中。图 4 (A) 是癌、癌旁和正常肝组织 1B50-1抗体免疫组化染色 代表结果图片。 箭头指示阳性细胞. 图 4 (B-E) 显示 86对肝癌标本中 1B50-1抗体染 色情况的 Kaplan-Meier生存曲线分析表明手术切缘癌旁组织 （C和 E) ， 而非肝癌组 织（B和 D)， 中 1B50-1阳性细胞的存在与肝癌病人的术后无病生存和总生存期呈负 相关. 图 4 (F) 是多因素分析显示手术切缘癌旁组织中 1B50-1阳性细胞的存在是独立 的肝癌预后不良因素。 a卡方检验。缩写: 4y, 4 年; Ca， 癌组织; CI， 置信区间; DFS, 无 病生存; OS, 总生存; PCa, 癌旁组织; RR，相对危险系数。
图 5显示 CACNA2D1基因在各种肿瘤细胞系中表达。 图 5A. 不同细胞系中 CACNA2D1基因表达的 RT-PCR分析； 图 5B. 不同细胞系中 1B50-1抗体免疫荧光细 胞化学染色分析。
图 6显示 CACNA2D1在人肿瘤组织 （T)和配对癌旁正常组织 （N)标本的表达 分析。 左侧为蛋白质印迹 （Western blot) 结果， 右侧显示的对条带扫描后的灰度用 β-actin内参校正后的定量结果。 Ca: 癌组织； CaP: 癌旁组织。
图 7显示 1B50-1抗体对人肝癌 Hep-12细胞 NOD/SCID (非肥胖糖尿病 /严重联合 免疫缺陷）小鼠移植瘤的生长具有抑制作用。图 7A. 不同组细胞形成的肿瘤；图 7B. 不 同组肿瘤在 NOD/SCID小鼠的生长曲线；图 7C. 解剖时不同组肿瘤的重量；图 7D. 解 剖时不同组肿瘤的体积。
图 8 显示 1B50-1抗体对人肝癌 HuH7细胞 NOD/SCID小鼠移植瘤的生长具有抑 制作用。 图 8A. 不同组细胞形成的肿瘤； 图 8B. 不同组细胞在 NOD/SCID小鼠的生 长曲线； 图 8C. 解剖时不同组肿瘤的体积； 图 8D. 解剖时不同组肿瘤的重量。
图 9 显示 CACNA2D1基因 RNA干扰对肝癌 Hep-12细胞在 NOD/SCID小鼠体内 生长的抑制。 图 9A. RNA干扰后 1B50-1抗体染色显示， 细胞 CACNA2D1蛋白水平 显著降低； 图 9B. RNA干扰后动物体内肿瘤生长曲线； 图 9C. 解剖时瘤体体积； 图 9D. 解剖时瘤体重量。
图 10显示经 QM-7细胞表达的单链抗体可以与 CACNA2D1基因阳性细胞结合。 具体实施方式
通过实施例的方式对本发明作进一步的说明， 但是本发明并不仅局限于以下实施 例。
下述实施例中所使用的实验方法如无特殊说明， 均为常规方法。
下述实施例中所用的材料、 试剂等， 如无特殊说明， 均可从商业途径得到。
试剂材料：
人肝癌细胞系 Hep-11和 Hep-12是由来源于同一肝癌病人原发和复发肝癌组织经 原代培养后建系而成（有关该细胞对的详细背景材料见： Zhang Z, Xu X, Xing B， Wang Y, Han H， Zhao W. Identification and characterization of tumor-initiating cells with stem-like properties from a recurrent hepatocellular carcinoma [abstract], Proceedings of the 100th Annual Meeting of the American Association for Cancer Research, 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR, 2009. Abstract nr 190; Xu XL, Xing BC, Han HB, Zhao W, Hu MH, Xu ZL, Li JY, Xie Y, Gu J, Wang Y，Zhang ZQ. The properties of tumor-initiating cells from a hepatocellular carcinoma patient's primary and recurrent tumor, Carcinogenesis, 2010; 31(2): 167-74. ) ， 肝癌细胞系 HuH7 (日本振兴学会） 、 HepG2 (ATCC), SMMC-772K 乳腺癌细胞系 ZR-75(ATCC)、 MCF-7细胞 （ATCC ) 、 MDA-MB-231 (ATCC ) 、 BICR-H1细胞 （由北京肿瘤医院黄信孚教授惠赠） 、 肺癌 细胞 A549 (ATCC ) 、 Calu-3 (ATCC ) 、 Calu6 (ATCC ) 、 PG (由北京大学医学部 基础医学院吴秉诠教授惠赠） 。 食道癌细胞系 KYSE150、 KYSE510 (由北京大学医 学部基础医学院鲁风民教授惠赠） 、 胃癌细胞系 BGC823、 MGC803、 SGC7901 , 前 列腺癌 PC3M1E7、 PC3M2B4均为常用细胞系， 由本发明人的实验室保存。
临床组织标本来源于北京大学肿瘤医院手术切除标本， 病理类型经病理科大夫确 认。 杂交瘤的制备：
a) 小鼠免疫和细胞融合：用 Hep-1 1和 Hep-12细胞采用差减免疫法 (Brooks, P. C.， Lin, J. M.， French, D. L., and Quigley, J. P.. Subtractive immunization yields monoclonal antibodies that specifically inhibit metastasis. J Cell Biol, 1993, 122, 1351-1359；
Rasmussen, N.， and Ditzel, H. J.. Scanning the cell surface proteome of cancer cells and identification of metastasis-associated proteins using a subtractive immunization strategy. J Proteome Res, 2009, & 5048-5059 )免疫 6周龄雌性 Balb/C小鼠 (北京维通利华实验动物 技术有限公司)。 接近密度饱和的 Hep-11细胞经 PBS涮洗 3遍后， 用细胞刮子收集细 胞， 离心后悬浮在无菌 PBS中 （~5χ 106细胞 /ml) ， 接种在 4一 6周龄雌性 Balb/c小鼠 腹腔内， 每只 0.5ml， 共接种 4只。 分别于接种 Hep-11细胞后第二天、 第 4天腹腔注 射 200mg/Kg体重的环磷酰胺 （cyclophosphamide, Sigma-Aldrich, St Louis, MO) 。 第 十八天时按照收集 Hep-11细胞的方法准备 Hep-12细胞，每只小鼠腹腔接种约 2.5>< 106 细胞 /0.5ml PBS。 每三周用等量的 Hep-12加强免疫， 共加强 3次。 最后一次加强免疫 后 3天取脾脏制备细胞悬液， 与 108 SP2/0细胞 （ATCC) 混合， 无血清 RPMI 1640
(Invitrogen)培养液洗两遍后， 用 50% PEG4000 (Sigma-Aldrich)按照常规操作融合 细胞。 用含 HAT (Sigma-Aldrich) 的 15%小牛血清的 1640培养液重悬细胞， 接种于 96孔细胞培养板，置 CO2细胞孵育箱中培养。 5天后用含 HAT的 15%小牛血清的 1640 培养液给融合细胞换液， 大约于融合后两周， 根据杂交瘤克隆生长情况， 取细胞培养 上清检测筛选分泌特异性抗体的杂交瘤克隆。
b) 杂交瘤克隆检测筛选亚克隆： 将肝癌细胞系 Hepl l、Hepl2细胞分别用 15% 小牛血清 1640培养液接种于 96孔细胞培养板， 待细胞贴壁并长满时， 弃培养上清， 加入预冷的含 0.125%戊二醛的 PBS， 室温静置 5分钟后， 弃去细胞固定液。 用 PBS 洗三遍， 加入含 5%脱脂奶粉的 PBS， 4GC封闭过夜。 弃去封闭液， 加入杂交瘤克隆培 养上清， 室温静置 1小时。 弃上清， 用 PBS洗两遍。 用 5%脱脂奶粉 PBS稀释辣根过 氧化物酶标记的羊抗鼠抗体， 混匀， 加入 96孔板中室温孵育 1小时。 弃上清， PBS 洗 5遍。 加入 ELISA底物反应液， 室温避光反应 30分钟， 加入等体积 12.5%硫酸终 止反应。 置酶标仪中测 492nm波长处的光密度值。挑选对 Hepl l呈阴性， Hepl2呈阳 性反应的杂交瘤克隆， 按有限稀释法亚克隆。 连续三次亚克隆后呈稳定阳性的杂交瘤 克隆扩大培养， 冻存细胞。
经过亚克隆后鉴定， 最终获得 30多个杂交瘤细胞株， 其中的一个杂交瘤命名为 1B50-1。 该杂交瘤于 2010年 12月 08日交中国微生物菌种保藏管理委员会普通微生 物中心 (CGMCC)保藏， 保藏编号为 CGMCC No. 4416。 抗体的生产和纯化
能分泌特异性抗体 1B50-1的杂交瘤克隆经扩大培养， 接种于降植垸预
( Sigma-Aldrich) 处理的雌性 Balb/c小鼠腹腔， 每只小鼠接种 2xl06细胞。 约一周左 右处死小鼠， 取腹水。 按常规操作进行 G蛋白亲和层析 1B50-1抗体纯化。 纯化后的 1B50-1抗体浓度按公式计算： 抗体 (毫克 /毫升) = OD28Q x0.6868。 抗体的纯度用
SDS-PAGE分析， 达到电泳级纯，用于有关实验。 抗体亚型测定
按照 Sigma小鼠单抗亚类测定试齐 !j盒 (Mouse Monoclonal Antibody Isotyping Reagents, Cat# Iso2-lKT, Sigmal-Aldrich, St Louis, MO, USA)说明书推荐的步骤操作对 1B50-1杂交瘤分泌的抗体的亚型进行测定： 用 PBS将亚类特异的抗体 1 : 1000稀释后 加于 96孔检测板， 每孔 100微升， 做平行孔 2个。 37°C孵育 1小时， PBS洗 3遍后， 加入 1B50-1杂交瘤上清， 室温孵育 1小时， PBS洗 3遍。 用含 5%脱脂奶粉的 PBS 稀释辣根过氧化物酶标记的羊抗鼠抗体， 加入检测板， 室温孵育 1小时后， PBS洗 5 遍。 加入 ELISA底物反应液， 室温避光反应 30分钟， 加入等体积 12.5%硫酸终止反 应。
经测定 1B50-1抗体与亚型 IgG3反应强阳性， 而与其它亚型的反应为阴性或弱阳 性， 提示 1B50-1抗体的亚型为 IgG3。 抗体的重链和轻链可变区的克隆、 鉴定
1B50-1杂交瘤细胞用 Trizol法提取细胞总 RNA， 经 Superscript III (Invitrogen公 司)逆转录酶逆转录成 cDNA后， 用鼠源抗体重链可变区 5'端含简并碱基引物
5， -CTTCCGG^ rrCSARGTNMAGCTGSAGSAGTC-3 ' +
5'-CTTCCGG^rrCSARGTNMA GCTGSAGSAGTCWGG-3'与 3，端引物
5'-GGAGG^rCCAGGGACCAAGGGATAGAC AGATGG-3' , 轻链可变区正向弓 I物 5'-GG^GCrCGAYATTGTGMTSACMCARWCTMCA-3 '和反向引物
5 '-TATAG^GCrC4^GCrrGGATGGTGGGAAGATGGATACAGTTGGTC-3 ' PCR分 别扩增 1B50-1抗体重链和轻链可变区。 扩增产物克隆入 PCR-blunt (Invitrogen)载体， 选取阳性克隆双向测序。 用 http:// www.bioinf.org.uk/abs/chothia.html提供的工具对获 得的 1B50-1抗体重链和轻链的氨基酸序列确定 Chothia标准结构域 （Morea V, Tramontano A, Rustici M, Chothia C, Lesk AM. Antibody structure, prediction and redesign. Biophys Chem. 1997 Oct;68(l-3):9-16. Al-Lazikani B, Lesk AM, Chothia C. Standard conformations for the canonical structures of immunoglobulins. JMol Biol. 1997; 273(4):927-48. ) ， 获得重链和轻链的 CDR区的序列。
1B50-1抗体可变区单链抗体表达载体的构建和表达
为验证克隆的可变区， 构建了用 MMP-9信号肽引导的单链抗体的表达载体， 并 转染骨骼肌成肌细胞 QM-7, 诱导分化后取培养基与 Hep-12细胞孵育， 用 MYC-标签 抗体进行间接免疫荧光细胞化学染色， 观察转染后培养基是否可特异与 Hep-12结合。
1B50-1抗体识别抗原鉴定
培养的肝癌 Hep-11和 Hep-12细胞去除培养基后，分别加入 1B50-1抗体杂交瘤培 养上清， 37°C培养箱继续温育 2小时， 弃去培养液， 用 PBS洗细胞 3次， 用细胞刮子 收集培养细胞， 离心后分别加去离子水 5毫升， 重悬细胞， 超声处理。 加 2X细胞裂 解液，再超声。在 4°C下 10000转 /分钟离心 10分钟，取上清，加预平衡的 Sepharose 4B - protein G (Pharmacia, Uppsala, Sweden)亲和层析珠。 室温作用 1小时， PBS流洗层析 柱， 0.1M甘氨酸盐酸缓冲液 (pH2.5)洗脱结合的抗原， 用 lM Tris-HCUpH9.0)调节至 pH7.0， 加入等量 2xSDS-PAGE样品缓冲液， 行 SDS-PAGE分析。 考马斯亮蓝 R250 染色后将 1B50-1免疫沉淀下来的特异在 Hep-12表达的条带切割下来， 胰酶消化后行 MALDI-TOF质谱分析。
CACNA2D1基因表达的逆转录— PCR分析
密度接近饱和的培养细胞弃去培养基， 经 PBS涮洗后， 用 Trizo法提取细胞总 RNAc 3μg细胞总 RNA在如下 20μ1反应体系中逆转录合成第一链 cDNA: l x逆转录缓 冲液 （50 mM Tris-HCl pH 8.3、 75 mM KC1和 3 mM MgCl2)、 20 U RNA酶抑制剂 (Promega, Madison, WI, USA)、 10 mM DTT、 50 mM dNTP、 0.5 oligo-(dT)15 (Promega)和 200 U Moloney 啮齿类白血病病毒逆转录酶（M-MLV-RT; Invitrogen)。 1 μΐ 的 cDNA用针对 CACNA2Di基因的正向引物
5 '-ACAGCAAGTGGAGTCAATCA-3 ' , 反向引物 5，-ACTGCTGCGTGCTGATAAG A-3，在 Taq酶作用下经 94°C预变性 5分钟， 94 °C 45秒， 56°C 45秒， 72°C 1分钟 PCR 反应 25个循环， 72Ό延伸 10分钟。 反应产物经琼脂糖凝胶电泳、 溴化乙锭染色后， 紫外灯下观察并照相。 实时荧光定量 RT-PCR分析
细胞总 RNA用啮齿类白血病病毒逆转录酶 （M-MLV-RT; Invitrogen)逆转录成 cDNA后， 用 SYBR Green PCR荧光染料混合物 （Toyobo Co. Ltd., Osaka, Japan)在 ABI7500 PCR仪上进行实时 PCR扩增。 引物顺序列于表 1。用 2_ΔΔα法计算基因表达 的倍数差另 ll( Pfaffl, M. W.. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001 , 29： e45)， 以 GAPDH 作为内参对照。 表 l. PCR 引物序列
基因名称 正向 ( Sense) 反义 (Anti-sense)
Sox2 5'-ACATGAACGGCTGGAGCAAC-3' 5'-AGGAAGAGGTAACCACAGGG-3'
Oct-4 5'-GACAACAATGAAAATCTTCAGGAGA-3' 5'-CTGGCGCCGGTTACAGAACCA-3'
Nanog 5'-TGCCTCACACGGAGACTGTC-3' 5'-TGCTATTCTTCGGCCAGTTG-3'
AFP 5'-ACCATGAAGTGGGTGGAATC-3' 5'-TGGTAGCCAGGTCAGCTAAA-3'
CACNA2D1 5'-ACAGCAAGTGGAGTCAATCA-3' 5'-ACTGCTGCGTGCTGATAAGA -3'
CEACAM6 5'-GAAATACAGAACCCAGCGAGTGC-3' 5'-CAGTGATGTTGGGGATAAAGAGC-3'
CTNNB 5'-TGATGGAGTTGGACATGGCC-3' 5'-CTCATACAGGACTTGGGAGG-3'
KLF4 5'-AAGCCAAAGAGGGGAAGAC-3' 5'-CATCTGAGCGGGCGAATTTC-3'
MDR-1 5'-GCCTGGCAGCTGGAAGACAAATAC-3' 5'-ATGGCCAAAATCACAAGGGTTAGC-3'
ABCG2 5'-GGAGGCCTTGGGATACTTTGAA-3' 5'-GAGCTATAGAGGCCTGGGGATTAC-3'
BM1 5'-AGCAGCAATGACTGTGATGC -3' 5'-CAGTCTCAGGTATCAACCAG -3'
GAPDH 5'-GACCCCTTCATTGACCTCAAC-3' 5'-CTTCTCCATGGTGGTGAAGA-3'
CACNA2D1基因克隆、 表达载体构建
按照 Genebank 中 CACNA2Di基因 (NM— 000722.2; calcium channel,
voltage-dependent, alpha 2/delta subunit 1, 钙离子通道， 电压依赖型， α2δ亚基 1)序列 分三段分别设计正向和反向引物， 三段引物序列分别为：
第一段正向引物： 5，- CCGgaattcTATGGCTGCTGGCTGCCTGCTGG-3'， 反向引 物 5，-AACCATTAGGATCGATTGCAAAG-3，；
第二段正向引物： 5，-TGTGTACCTGGATGCATTGGAACTG-3'， 反向引物： 5'- ACCATCATCCAGAATCACACAATC-3'；
第三段正向引物： 5，-AGAGACATATGAGGACAGCTTC-3'， 反向引物：
GCTG-3O
以 Hep-12 细胞 cDNA为模板，用上述引物 PCR分别扩增涵盖该基因全长的三个 片段， 产物经琼脂糖凝胶电泳纯化后重组进 PCR-blunt载体， 送公司测序确认。 三个 片段经适当酶切和中间载体拼接成完整全长的 CACNA2D,基因， 最后将全长的 CACNA2D!基因克隆至 pcDNA3.0-mychis载体 (为发明人在 Invitrogen公司的 pcDNA3.0的基础上构建而成)。 基因转染
接种细胞以次日密度达 80-90%饱和。 用 Lipofectamine 2000 (Invitrogen) 按照 公司推荐的步骤将构建好的 CACNA2D1 mychis/ pcDNA3.0质粒转染到细胞内。 转染 后 24、 48小时进行该基因表达的免疫荧光细胞化学染色分析。 细胞免疫荧光染色
培养细胞经胰酶: EDTA消化制成单细胞悬液， 取 2χ 106细胞加入纯化的 1B50-1 抗体 (1 : 100 稀释， 储存液 lmg/ml)， 37°C孵育 1小时， PBS洗漆 3次， 加入 FITC标 记的山羊抗小鼠 II抗 IgG (Jackson ImmunoResearch Laboratories, USA, 0.5mg/ml; 1： 100稀释)， 37°C作用 1小时， 经 PBS洗涤后 Idea SP5激光共聚焦显微镜下观察， 或 用 Aria流式细胞检测分析、分选。对转染 CACNA2Dlmychis/ pcDNA3.0质粒的细胞， 则加入 1B50-1单抗和 myc兔多克隆抗体双染， II抗分别用罗丹明标记的山羊抗小鼠、 FITC标记的山羊抗兔 IgG。
免疫组化染色
临床肝癌病人癌旁和癌组织的冰冻切片， 甲醇固定 30秒， 5%脱脂奶粉封闭后， 与 1B50-1单克隆抗体 (1 : 100稀释，含 5% BSA) 4°C孵育过夜，再经 PBS洗涤，与 FITC 标记的山羊抗小鼠 II抗 IgG室温反应 2小时， PBS洗涤， DAPI (1： 2000)染核 5分钟， 2% DABCO甘油封片， Leica SP5 激光共聚焦显微镜下观察。 蛋白质印迹 （Western Blot) 分析
组织或培养细胞用含有 ImM PMSF和完全蛋白酶、磷酸酶抑制剂混合物（Roche, Mannheim, Germany)的放射免疫沉淀缓冲液 (Radio Immuno-precipitation assay buffer; 北京索来宝科技有限公司） 裂解. 行 10% SDS-聚丙烯凝胶电泳后， 电转移至
Immobilon-P膜上（Millipore)。经 5 %脱脂奶粉封闭后，依次与 I抗 CACNA2DK Abeam, Cambridge, MA)或内参 β— actin(Roche Applied Science)的抗体和辣根过氧化物酶标记 的山羊抗小鼠的 II抗 ( Jackson ImmunoResearch Laboratories Inc.， West Grove, PA， USA)反应， 用 Immobilon™ Western Chemiluminescent HRP substrate (Millipore)化学发 光法检测阳性信号。 条带信号用 Chemilmager扫描仪 (Alpha lnnotech)扫描后， 用 AlphaEaseFC软件对条带的灰度进行定量分析， 以 β— actin的灰度做内参计算样品中 CACNA2D1的相对量。
CACNA，D,基因 RNA干扰慢病毒载体的构建、 包装和感染： CACNA2D,基因的干扰 RNA序列由 Origene公司设计和合成，基于逆转录病毒载 体构建，该载体以 U6的启动子作为启动子，其中序列 1为针对 CACNA2D1基因编码 序列 546-574的 ACTCAACTGGACAAGTGCCTTAGATGAAG, 序列 2为针对该基因 编码序列 116-144的 AGATGCAAGAAGACCTTGTCACACTGGCA, 对照序列为 Origene公司随机合成的无关等长核苷酸序列。 含有上述序列的载体分别经 EcoRI和 Sail酶切， 琼脂糖凝胶电泳分离纯化含有 U6启动子和上述寡核苷酸序列的片段， 与 经同样酶切的 Plenti6-linker载体（由本实验室用 Invitrogen公司的 plenti6载体经 Cla I 和 Age I双酶切后加入含有多克隆位点的连接子序列改造而成， 保存于本实验室） 连 接、 经鉴定分别获得慢病毒质粒载体 plenti6U6CACNA2DlShRNA-l、
plenti6U6C ACNA2D 1 ShRNA-2以及对照 plenti6U6-control。 慢病毒的包装严格按照 Invitrogen公司推荐的方法。 含有慢病毒颗粒的培养上清直接感染 Hep-12细胞， 48小 时后加入 6 g/ml的 Blasticidin (Invitrogen) 筛选感染病毒的细胞， 每三天更换新鲜 培养液， 获得经上述病毒感染的细胞群并在实验过程中持续用含有 6 g/ml的
Blasticidin以维持筛选压力。 感染的细胞进一步用 RT-PCR和免疫荧光细胞化学染色 观察对 CACNA2D,基因抑制的效果。用免疫缺陷动物致瘤实验观察对肿瘤成瘤和生长 的抑制作用。 动物致瘤以及抗体抑瘤实验
致瘤实验： 对于评价肿瘤起始细胞的致瘤和自我更新能力的实验， 选用不同来源 经流式细胞仪分选获得的 1B50-1抗体阴性、 阳性不同数量 (104、 103、 102)的细胞与 Matrigel(BD公司)等体积混合接种在 4-6周龄 NOD/SCID小鼠 (北京维通利华动物中 心， SPF级)皮下 (阴性、 阳性细胞接种在同一只小鼠的不同侧)， 每组接种 5只， 每周 观察肿瘤生长情况。对于评价 CACNAzDi基因抑制后动物致瘤、生长特性改变的动物 实验， NOD/SCID小鼠皮下接种 2> θ6细胞。 待肿瘤生长至肉眼可见时， 每三天测量 肿瘤的长径和短径， 按照公式"肿瘤体积=长径 X短径 2/2"计算肿瘤体积大小， 绘制生 长曲线。
抗体抑瘤实验： 4-6周龄 NOD/SCID小鼠皮下接种 2χ 106肝癌细胞， 待肿瘤生长 至肉眼可见大小 (约 0.02-0.03cm3)随机分组， 每组 6只， 隔日腹腔分别注射 PBS、对照 小鼠 IgG (中杉金桥生物技术公司， 80(^g/只)和 1B50-1抗体 (分别为 200、 400和 80(^g/ 只)。 用游标卡尺在每次注射前测量肿瘤的长径和短径， 按照公式"肿瘤体积=长径 X 短径 2/2"计算肿瘤体积大小， 共计给药 7次后， 隔日处死动物， 解剖形成的瘤块， 测 量湿重和体积。 实施效果
实施效果例一： 1B50-1抗体识别细胞膜抗原， 各种肝癌细胞系的阳性率差别较大： 用差减免疫法制备杂交瘤， 经 Hep-1 1和 Hep-12细胞对进行阴性 /阳性筛选， 经 3 次融合筛选， 第一轮共得到 37个潜在特异针对 Hep-12的单克隆抗体。 对这些抗体进 行进一步的亚克隆和初步分析， 其中一个杂交瘤被命名为 1B50-1。其分泌产生的抗体 定位在细胞膜 （图 1 ) 。 进一步用流式细胞仪对不同的肝癌细胞系中以及临床肝癌组 织标本原代培养物来源的细胞中 1B50-1抗体的阳性率进行了分析， 结果见表 2。 除复 发来源、 富含肿瘤起始细胞的 Hep-12细胞中 1B50-1阳性细胞比例较高外， 含量均较 低。
表 2. 1B50-1抗体阳性细胞的含量及其在 NOD/SCID鼠的致瘤能力
1B50-1+细 1B50-1 阳性 1B50-1 阴性
细胞
胞百分数# 103 102 103 102
HuH-7 0.9-2.2 5/5 5/5 3/5* 0/5
Hep-11 0.4-0.7 5/5 1/5 0/5 0/5
Hep-12 92.1-94.8 5/5 5/5 3/5* 0/5
HepG2 0.5-2.1 4/5 4/5 0/5 0/5
SMMC7721 0.5-0.6 5/5 5/5 0/5 0/5 病例 -1 1.7-3.3 5/5 5/5 3/5* 0/5 病例 -2 0.6-2.1 3/5 2/5 0/5 0/5 病例 -3 0.4-1.8 5/5 3/5 5/5* 1/5 病例 -4 0.6-1.3 2/5 0/5 0/5 0/5 2-8 次流式细胞仪分析结果。
*形成的瘤体远小于对应的阳性细胞。 实施效果例二： 1B50-1抗体识别的阳性细胞具有肿瘤起始细胞特性
用流式细胞仪从 5个肝细胞癌细胞系包括 Hep-11、 Hep-12. HuH7、 HepG2和 SMMC-7721分选 1B50-1抗体阴 /阳性细胞群， 选取 100、 1000个细胞分别注射到 NOD/SCID小鼠皮下（阴性和阳性细胞分别注射到同一小鼠的不同侧面皮下），经 12-18 周， 100— 1000个 1B50-1阳性细胞足以在小鼠皮下形成肿瘤， 而阴性细胞不长或只形 成小的结节 （表 2， 图 2) 。 提示 1B50-1抗体阳性细胞具有肿瘤起始细胞的特性。 用 临床肝癌组织经原代培养的细胞进行 1B50-1抗体阳性和阴性细胞分选后的致瘤实验 获得类似的结果（表 2) 。 用 1B50-1抗体阳性 HuH7细胞形成的瘤体经流式细胞仪分 选 1B50-1阳性细胞， 再接种到 NOD/SCID小鼠皮下， 结果 1B50-1+细胞 100% (5/5 ) 可以再形成瘤子， 说明 1B50-1+细胞具有自我更新能力。 用实时荧光定量 RT-PCR分 析 1B50-1阳性和阴性细胞中与干细胞相关的基因表达，结果发现 1B50-1+细胞高表达 Nanog、 Sox-2、 AFP, ABCG2等干细胞相关基因 （图 2D) 。 取 1B50-1阳性细胞在含 有 10%胎牛血清的培养基中培养，再分析 1B50-1阳性细胞的百分率，结果发现 1B50-1+ 细胞的比率下降为接近母系细胞中 1B50-1阳性率（图 2C)， 提示 1B50-1阳性细胞可 以分化为 1B50-1阳性和阴性细胞。 这些结果说明 1B50-1阳性细胞具有肿瘤干细胞特 性。 实施效果例三： 1B50-1抗体识别的抗原是 CACNA2D1
抗体 1B50-1从 Hep-12细胞经免疫沉淀在 150KD左右有一特异条带（箭头指示条 带） （图 3左图） 。 经质谱分析表明该蛋白为 CACNA2D1。 以 Hep-12细胞的 cDNA 为模板， 用 PCR方法扩增该基因， 步将该基因用常规 DNA重组技术在 C一端添加 MYC标签肽编码序列， 装入 pcDNA3.0mychis真核细胞表达载体， 转染不表达该基因 的细胞， 用 MYC多抗和 1B50-1抗体进行双染， 结果发现转染后的细胞 MYC标签肽 抗体和 1B50-1抗体在细胞表面分布一致， 证明 1B50-1抗体识别的就是 CACNA2D1 基因 (图 3右图)。 实施效果例四： CACNA2D1基因在肝癌临床标本中的表达
选取临床肝细胞癌病例共 86例病人的新鲜标本的癌组织及配对的癌旁组织，进行 冰冻切片。 固定后用 1B50-1抗体进行免疫荧光组织化学染色， 结果如图 4和表 2所 示， 在 72.1%的病例 （62例 /86例） 中， 1B50-1抗体阳性细胞散在地分布在癌组织中 (图 4) 。 在癌旁组织中阳性细胞的检出率为 46.5% (40/86) ， 低于癌组织的阳性率。 5例完全正常的肝组织（来源于血管瘤手术切除标本）则未检测出 1B50-1阳性细胞存 在。 将癌组织和癌旁组织中 1B50-1抗体阳性检出情况与病人的临床指标进行统计分 析 （表 3 ) ， 结果发现， 癌组织中 1B50-1抗体阳性与年龄、 性别、 肝硬化等指标无关 联， 但癌旁组织中 1B50-1抗体阳性组的病人与肝硬化的出现、 手术后四年以下生存 期以及 2年内复发呈正相关。 Kaplan-meier生存曲线以及多因素 Cox 回归分析结果表 明， 1B50-1抗体阳性细胞的存在预示病人手术后无疾病生存及 龄一别女男
总生存期均短于病人癌旁组织 1B50-1抗体阴性组病人（图 4 ) 。 鉴于癌旁组织实际来 源于肝癌病人手术的切缘， 因此通过检测切缘组织中 1B50-1抗体阳性细胞的存在与 否可预测肝癌病人的复发以及预后情况， 可以作为肝癌预后评价的指标。
表 3. 1病例数B50-1抗体染色与肝癌病人临床指征相关性分析
癌旁组织 1B50-1阳性 1 癌组织 1B50-1阳性 1 病例 百分数 值 2 病例 百分数 p值
0.223 0.960
75 33 44 54 72
11 7 63.6 8 72.7
0.733 0.063
0 63 30 47.6 42 66.7
0 23 10 43.5 20 87.0
肝硬化 0.002 0.448 无 27 6 22.2 18 66.7
有 59 34 57.6 44 74.6
肿瘤大小 0.141 0.171
<5 cm 46 18 39.1 36 78.3
>5 40 22 55 26 65
肿瘤大小 0.535 0.109
<3 cm 21 11 52.4 18 85.7
>3 65 29 44.6 44 67.7
脉管癌栓 0.170 0.635 无 64 27 42.2 47 73.4
有 22 13 59.1 15 68.2
生存期 0.00005 0.810
<4年 34 25 73.5 25 73.5
≥4年 52 15 28.8 37 71.2
复发 0.00004 0.893
< 2年 42 29 69.0 30 71.4
>4年 44 1 1 25 32 72.7
合计 86 40 46.5 62 72.1 0.0006
1)只要在 1张切片中找到 1个或以上个 1B50-1抗体染色阳性的细胞即定义为
1B50-1阳性。
2)卡方检验。
* 肿瘤组织与癌旁组织比较。 实施效果例五： CACNA2D1基因在胃癌、 食道癌、 乳腺癌、 肺癌等细胞系中的表达 首先用 RT-PCR对除肝癌外的常见肿瘤胃癌、 肺癌、 乳腺癌和***癌细胞系中 CACNA2D1基因的表达进行了检测， 结果如图 5A所示， 在胃癌细胞中的 MGC-803、 SGC7901 ; 肺癌的 PG和 A549细胞、 乳腺癌的 BICR— HI和 MDA-MB-231细胞中检 测到阳性条带， 高转移性的***癌 PC3M1E7中显著表达， 而低转移的 PC3M2B4 中却未检测到。 这说明虽然并不是所有的细胞都呈阳性， 但在多种肿瘤中的确有一部 分表现为该基因阳性表达， 这与肝癌病例中检测到的结果相似。 而且上述阳性的细胞 都为高转移细胞， 提示该基因可能与肿瘤的转移特性呈正相关。 进一步， 用 1B50— 1 抗体对一些细胞进行免疫荧光细胞化学染色，发现胃癌 SGC7901、食管癌 (KYSE-510、 KYSE-150)和乳腺癌 ZR-75细胞中 CACNA2D1基因蛋白表达阳性， 定位在细胞膜上， 不同细胞系中的阳性细胞数差别很大， 但都只占很小的群体（图 5B) 。 这与肝癌中的 情况相似， 提示我们在肝癌中的发现在多种肿瘤中有普遍性， 有可能用于这些肿瘤诊 断和治疗的分子靶点。 实施效果例六：其他肿瘤肿瘤组织标本中 1B50-1抗体阳性细胞的分布及 CACNA2D1 蛋白免疫印迹 （Western blot) 分析
为进一步探讨在肝癌组织中得到的结果是否亦适用于其它类型的肿瘤， 我们选用 临床结直肠癌、 肾癌、 肺癌和食道癌及其配对癌旁正常组织手术标本各 10对进行免 疫组化染色， 1B50-1抗体阳性细胞的分布与肝癌组织类似， 结果如表 4所示， 在癌组 织中 1B50-1阳性细胞的检出病例高于癌旁组织， 但由于病例太少未进行统计分析。 进一步为确认免疫组化的结果， 我们对临床组织标本用商品化的 CACNA2D1抗体进 行免疫印迹杂交分析。结果如图 6所示， CACNA2D1在一些病例癌组织中的表达明显 高于癌旁组织 （不同癌组织类型中 CACNA2D1高表达率有差别） ， 说明癌变后 CACNA2D1的表达升高。 CACNA2D1的高表达不但适用于肝癌， 亦广泛存在于多种 组织类型的癌组织中。 因此针对 CACNA2D1的药物和分子标志物亦不局限于肝癌。
表 4. 1B50-1抗体阳性细胞在不同肿瘤组织和癌旁组织中的检出率
组织类型 总病例数 癌组织阳性病例数 癌旁组织阳性病例数
结直肠癌 10 8 3
肺癌 10 3 0
肾癌 10 4 1
食管癌 10 1 0 实施效果例七： 1B50-1抗体对肝癌荷瘤小鼠的生长的抑制作用
我们先将肝癌 Hep-12细胞、 HuH7细胞分别接种在免疫缺陷动物皮下， 待肿瘤生 长至 0.02-0.03cm3时， 通过腹腔注射不同剂量的 1B50-1抗体， 结果如表和图， 可见 80(^g/只小鼠剂量组对肝癌细胞系 Hep-12和 Huh7的抑瘤率分别为 80.4%、 65.5% (以 重量计算） （图 7， 8) ， 与对照 PBS处理组和 IgG组相比均有统计学上的显著性差 异， 且存在剂量依赖效应。 实施效果例八： ShRNA抑制 Hep -12细胞中 CACNA2D1的表达可以抑制该细胞在动 物体内的生长
为进一步确认 1B50-1抗体所识别的抗原 CACNA2D1是否是一肿瘤治疗的靶分 子， 我们构建了表达该基因干扰 R A的慢病毒载体 plenti6U6CACNA2DlshRNA-l和 plenti6U6C ACNA2D 1 shRNA-2 , 并以 plenti6U6空载体作对照， 包装成慢病毒后感染 Hep-12细胞， 用 blasticidin (Invitrogen)筛选感染的细胞。 1B50-1抗体免疫荧光细胞 化学染色显示 （图 9A) ， 两个含有 CACNA2D1干扰 R A序列的慢病毒感染后， CACNA2D1蛋白的表达明显抑制，偶在细胞膜上见十分稀疏的阳性荧光点，对照组的 荧光则很强，且可见于大多数的细胞。将上述分别感染不同慢病毒的接种在 NOD/SCID 小鼠皮下， 每组 5只， 每只接种 2χ 106细胞， 观察肿瘤形成情况。 结果如图 9Β所示， 感染携带 CACNA2D1干扰 RNA的两个慢病毒的细胞在动物体内的生长速度显著慢于 转染空载体对照组， 抑瘤率分别达 57.5%和 59.6% (按体重计） ， 经 t检验分析， 与对 照组相比的 p值分别为 0.0164和 0.014。这些结果说明抑制 CACNA2D1基因的表达确 实可以抑制 Hep-12的体内生长， 是一治疗的分子靶点。 实施效果例九： 真核细胞表达的单链抗体可识别 Hen-12细胞
将用 QM-7细胞表达的 1B50-1可变区的单链抗体的培养上清与 Hep-12细胞共孵 育， 用 MYC标签肽抗体 9E10作为 I抗， FITC标记的山羊抗小鼠 II抗染色， 荧光镜 下观察。 可见 Hep-12细胞表面可以被 MYC标签肽抗体 9E10识别 （图 10 ) , 荧光染 色的模式与 1B50-1抗体所染结果一致， 表明所表达的单链抗体具有类似 1B50-1抗体 一样的结合活性， 进一步确认所克隆的 1B50-1抗体的可变区轻链、 重链序列正确， 可以以这些序列为基础经基因工程技术进行进一步的抗体药物改造。 虽然已经对本发明的具体实施方案进行了描述， 但是本领域技术人员应认识到， 在不偏离本发明的范围或精神的前提下可以对本发明进行多种改变与修饰。 因而， 本 发明意欲涵盖落在权利要求书及其同等物范围内的所有这些改变与修饰。 工业应用性
通过本发明， 能够寻找、 鉴别或确认肿瘤起始细胞的标志物。 还可以根据利用本 发明的方法所确定的标志物， 来诊断、 治疗和预防肿瘤。 具体而言， 可按照本发明确 定肿瘤起始细胞特异性的标志物， 由此研制针对起始细胞的治疗药剂， 并制定诊断、 治疗和预后判断策略， 从而有望解决肿瘤易复发、 转移的问题， 为肿瘤的彻底征服带 来新的曙光。 此外， 可以直接使用本发明所提供的特异性识别靶标 CACNA2D1 的单 克隆抗体或其单克隆片段， 来治疗或预防肿瘤或与 CACNA2D1相关的疾病或病症， 或者将其用于药物组合物、 诊断试剂盒的制备。
原件（ 提交 ）
Figure imgf000026_0001
下面的说明与本申请说明书中此处提到的
保藏的微生物或其他生物材料相关：
1 页码 6
-2 行号： 26-28
-3 保藏事项
-3-1 保藏单位名称 CGMCC 中国微生物菌种保藏管理委员会普通微生物中心 -3-2 保藏单位地址 Institute of Microbiology, Chinese Academy of
Sciences, No. 1, West Be i chen Road, Ghaoyang District, Beijing 100101, China
-3-3 保藏曰期 2010年 12月 08日 (08.12.2010)
保藏 ¾ CGMCC 4416
5 本说明是对下列指定国 所有指定国 由受理局填写
4 本表格与国际申请一起收到：
(足或否)
-4- 1 受权官员 由国际局填写 -5 国际局收到本表格日期：
-5-1 受权官员

Claims

权利要求
1.一种寻找、 鉴别或确认肿瘤起始细胞的标志物、 肿瘤治疗的靶标或肿瘤治疗的分子 靶点的方法， 所述方法包括使用复发性肿瘤来源并富含肿瘤起始细胞的细胞来免疫 动物的步骤。
2.—种***的方法， 包括利用如权利要求 1所述方法寻找、 鉴别或确认肿瘤起始 细胞的标志物、 肿瘤治疗的靶标或肿瘤治疗的分子靶点； 针对所找到、 鉴定或确认 的肿瘤起始细胞的标志物、 肿瘤治疗的靶标或肿瘤治疗的分子靶点研制和筛选具有 降低该肿瘤起始细胞的标志物、 肿瘤治疗的靶标或肿瘤治疗的分子靶点的基因表达 或 /和蛋白活性、或靶向该肿瘤起始细胞的标志物、肿瘤治疗的靶标或肿瘤治疗的分 子靶点后引起细胞毒性反应的抗体或其抗原结合片段、 单链或双链寡聚核苷酸、 核 酸、 短肽或小分子化合物药物； 向需要的受试者施用治疗有效量的至少一种所研制 或筛选出的抗体或其抗原结合片段、 单链或双链寡聚核苷酸、 核酸、 短肽或小分子 化合物药物。
3.如权利要求 1或 2所述的方法， 其中所述复发性肿瘤来源并富含肿瘤起始细胞的细 胞是 Hep-12细胞。
4.如权利要求 1或 2所述的方法， 其中所述肿瘤起始细胞的标志物、 肿瘤治疗的靶标 或肿瘤治疗的分子靶点是 CACNA2D1。
5.如权利要求 1-4中任一项所述的方法， 进一步包括利用 Hep-12细胞和 Hep-11细胞 作为筛选细胞对。
6.—种特异性识别电压依赖性钙离子通道蛋白 α2δ亚基 1 (CACNA2D1 ) 的单克隆抗 体或其抗原结合片段， 包含：
(i) 重链， 其包含重链可变区互补决定区 CDRH1 (序列 1 ) 、 CDRH2 (序列 2) 和 CDRH3 (序列 3 ) 的氨基酸序列， 或包含与 CDRH1、 CDRH2和 CDRH3的氨基酸 序列具有至少 70%、 75%、 80%、 85%、 90%、 95%、 或 99%同源性的氨基酸序列； 或
(ii) 轻链， 其包含轻链可变区互补决定区 CDRL1 (序列 4) 、 CDRL2 (序列 5 ) 禾口 CDRL3 (序列 6) 的氨基酸序列， 或包含与 CDRL1、 CDRL2和 CDRL3的氨基酸 序列具有至少 70%、 75%、 80%、 85%、 90%、 95%、 或 99%同源性的氨基酸序列； 或
(iii) 上述 (i)和 (ii)两者。
7.如权利要求书 6所述的单克隆抗体或其抗原结合片段， 具有以下一种或多种特征：
(1) 结合 CACNA2D1蛋白；
(2) 识别的阳性细胞具有肿瘤起始细胞特征；
(3) 抑制表达 CACNA2D1的肿瘤细胞在动物体内的生长。 —种杂交瘤细胞系， 所述杂交瘤细胞系是由中国微生物菌种保藏管理委员会普通微 生物中心 (CGMCC)保藏的小鼠杂交瘤， 保藏编号为 CGMCC No. 4416。
如权利要求书 6或 7所述的单克隆抗体或其抗原结合片段， 其中所述单克隆抗体为 由权利要求 8所述的杂交瘤细胞系所产生的单克隆抗体 1B50-1。
.如权利要求书 7所述的单克隆抗体或其抗原结合片段， 其中所述肿瘤为肝癌、 结 肠癌、直肠癌、肾癌、食道癌、胃癌、肺癌、乳腺癌、***癌或其它高表达 CACNA2D1 基因的肿瘤。
.一种用于治疗或预防与 CACNA2D1蛋白相关的疾病或病症的单克隆抗体或其抗 原结合片段， 包括权利要求 6、 7或 9中任一项所述的单克隆抗体或其抗原结合片 段。
.一种药物组合物， 包含权利要求 6、 7或 9中任一项所述的单克隆抗体或其抗原结 合片段， 以及药学上可接受的载体。
.如权利要求 12所述的药物组合物，其还包含可以加和或协同地改善与 CACNA2D1 蛋白相关的疾病或病症的其它活性化合物。
.如权利要求 13所述的药物组合物，其中所述疾病或病症为肝癌、结肠癌、直肠癌、 肾癌、 食道癌、 胃癌、 肺癌、 乳腺癌、 ***癌或其它高表达 CACNA2D1基因的 肿瘤。
.一种用于诊断、治疗或预防与 CACNA2D1蛋白相关的疾病或病症的试剂盒，包含 如权利要求 6、 7或 9中任一项所述的单克隆抗体或其抗原结合片段， 或针对 CACNA2D1的核酸。
.如权利要求 15所述的试剂盒， 其中所述疾病或病症为肝癌、 结肠癌、 直肠癌、 肾 癌、 食道癌、 胃癌、 肺癌、 乳腺癌、 ***癌或其它高表达 CACNA2D1基因的肿 瘤。
.如权利要求 6、 7或 9中任一项所述的单克隆抗体或其抗原结合片段在制备用于诊 断、 治疗或预防 CACNA2D1蛋白相关的疾病或病症的药物中的应用。
.如权利要求 17所述的应用， 其中所述疾病或病症为肝癌、结肠癌、直肠癌、 肾癌、 食道癌、 胃癌、 肺癌、 乳腺癌、 ***癌或其它高表达 CACNA2D1基因的肿瘤。.一种用于诊断与 CACNA2D1蛋白相关的疾病或病症的方法，包括向需要的受试者 施用至少一种如权利要求 6、 7或 9中任一项所述的单克隆抗体或其抗原结合片段。.一种用于治疗与 CACNA2D1蛋白相关的疾病或病症的方法，包括向需要的受试者 施用治疗有效量的至少一种如权利要求 6、 7或 9中任一项所述的单克隆抗体或其 抗原结合片段。
.一种用于预防与 CACNA2D1蛋白相关的疾病或病症的方法，包括向需要的受试者 施用预防有效量的至少一种如权利要求 6、 7或 9中任一项所述的单克隆抗体或其 抗原结合片段。
.如权利要求 19-21中任一项所述的方法，还包括向所述受试者施用能够增强所述单 克隆抗体或其抗原结合片段的生物学作用的其它药物。
.如权利要求 19-21中任一项所述的方法，其中通过静脉内给药施用所述单克隆抗体 或其抗原结合片段。
.如权利要求 1-2或 19-23中任一项所述的方法，其中所述肿瘤、疾病或病症为肝癌、 结肠癌、 直肠癌、 肾癌、 食道癌、 胃癌、 肺癌、 乳腺癌、 ***癌或其它高表达 CACNA2D1基因的肿瘤。
.如权利要求 1-5中任一项所述的方法，其中所述 Hep-12细胞和 Hep-11细胞来源于 同一肝癌病人复发和原发的肝细胞癌。
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JP2014507150A (ja) 2014-03-27
US10174123B2 (en) 2019-01-08
EP2682475A1 (en) 2014-01-08
EP2682475A4 (en) 2015-01-14
KR20140020922A (ko) 2014-02-19
CN103562403A8 (zh) 2018-01-26
US20200040097A1 (en) 2020-02-06
JP6084170B2 (ja) 2017-02-22
KR101797090B1 (ko) 2017-11-13
BR112013021350A2 (pt) 2016-11-08
KR101885705B1 (ko) 2018-08-06
CN103562403A (zh) 2014-02-05
EP2682475B1 (en) 2019-11-13
KR20170126033A (ko) 2017-11-15
CN102251013A (zh) 2011-11-23
US20140044729A1 (en) 2014-02-13

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