WO2014146487A1 - 抗细胞表面异位表达的单克隆抗体及其制备方法和用途 - Google Patents

抗细胞表面异位表达的单克隆抗体及其制备方法和用途 Download PDF

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WO2014146487A1
WO2014146487A1 PCT/CN2014/000289 CN2014000289W WO2014146487A1 WO 2014146487 A1 WO2014146487 A1 WO 2014146487A1 CN 2014000289 W CN2014000289 W CN 2014000289W WO 2014146487 A1 WO2014146487 A1 WO 2014146487A1
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monoclonal antibody
gastric cancer
cells
cell
cell surface
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PCT/CN2014/000289
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French (fr)
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陆梅生
刘炳亚
李⋅杰夫瑞
陈雪华
苏庆
刘云成
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上海麦柏星生物科技有限公司
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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
    • C07K16/28—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
    • C07K16/3046—Stomach, Intestines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57446—Specifically defined cancers of stomach or intestine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL

Definitions

  • the present invention relates to the field of biopharmaceuticals, and in particular to a monoclonal antibody, in particular to an ectopic expression of a cell surface of a digestive tract such as gastric cancer.
  • Gastric cancer (Gastric Cancer, GO is the most common digestive system malignancy, the second largest cause of cancer-related death worldwide. China is one of the countries with the highest incidence of gastric cancer in the world, with nearly 400,000 new cases each year. It accounts for about 42% of the world, and many patients are in advanced stage.
  • early screening and diagnosis of gastric cancer mainly rely on endoscopy combined with histopathological biopsy, although in recent years the specificity of this early screening diagnostic method, Sensitivity, accuracy and safety have been improved to some extent, but due to the difficulty of popularization and the existence of many interference factors, the clinical application effect is not very satisfactory. Therefore, most patients with gastric cancer often develop to the advanced stage at the time of initial diagnosis.
  • Molecular targeted therapy targets certain marker molecules that are overexpressed or specifically expressed by tumor cells. Since the 1990s, a variety of molecular targeted therapeutic drugs have entered clinical trials for the treatment of a variety of tumors, and some drugs have shown good results. These drugs mainly include two major categories, namely macromolecular monoclonal antibodies and small molecule tyrosine kinase inhibitors. Because antibodies highly recognize and bind antigens, the antibody-targeted Therapy (ATT) of tumors has a unique charm in the field of cancer therapy.
  • ATT antibody-targeted Therapy
  • Antibody drug targeted therapy for gastric cancer has developed rapidly in recent years. Preclinical studies and clinical studies have shown that the monoclonal antibody Cetuximab targeting epidermal growth factor receptor (EGFR), the monoclonal antibody Trastuzumab targeting epidermal growth factor receptor 2 (EGFR2, HER2), and vascular endothelium Growth factor (VEGF)-targeted monoclonal antibody Bevacizumab (Avastin) has a certain degree of anti-gastric cancer activity, but there are problems of low specificity and poor targeting. The actual clinical treatment effect is not very satisfactory. . The core of research on targeted therapy of gastric cancer is to find antibodies that identify gastric cancer-specific antigens and to prepare natural epitopes against specific antigens.
  • EGFR epidermal growth factor receptor
  • VEGF vascular endothelium Growth factor
  • Brichory et al. first introduced proteomics methods to identify and screen tumor-associated antigens and anti-tumor autoantibodies.
  • this strategy has been widely used in a variety of cancer studies, including gastric cancer, and has revealed numerous possible new molecular markers of gastric cancer, such as Glucose Regulated Protein (GRP78), heat shock. Protein (Heat Shock Protein, HSP27, 70, 60) and Fibrin Peptide A.
  • GRP78 Glucose Regulated Protein
  • Heat shock Protein Protein
  • HSP27 Heat shock Protein
  • 70, 60 heat shock Protein
  • Fibrin Peptide A Fibrin Peptide A.
  • the ideal candidate for targeted therapy of tumor monoclonal antibodies is cell surface specific antigen, and some techniques used in proteomics research are difficult to ensure the natural conformation of the antigen, which limits the molecular markers of gastric cancer identified and screened. And further research applications of the corresponding antibodies.
  • Alkaline phosphatase is a homodimeric protein with a molecular weight of 56 kDa. Each monomer consists of
  • ALP is a zinc-containing glycoproteinase. Human ALP exhibits the highest activity in an alkaline environment at pH 8. 0 and is capable of hydrolyzing a phosphate monoester substrate. Normal ALP is encoded by the pho A gene. It is a secreted protein that synthesizes a monomeric precursor that secretes a signal peptide at the amino terminus in the cytoplasm. The secretion signal peptide directs the precursor to be translocated after transmembrane transport, and the resulting homodimer is secreted extracellularly.
  • ALP has multiple subtypes and is a class of enzymes or isozymes that are capable of dephosphorylating the corresponding substrate.
  • the ALP in normal human serum is mainly from the liver, and the ALP from bone tissue is generally less than half of the total activity.
  • ALP subtypes have placental type (PALP), intestinal type (IALP), germ cell type (GALP), and non-specific tissue type (TNSALP).
  • TNSALP is a secondary isoenzyme of liver, kidney, bone, etc. formed by adding multiple modifications after gene expression;
  • ALP is mainly discharged to the peripheral blood through the liver, thereby increasing serum ALP.
  • ALP mainly exists in the body's bones, liver, kidneys and other tissues.
  • Hepatobiliary diseases including hepatobiliary malignancy, skeletal dysplasia, and ALP increase in pregnant women, but there have been no reports of elevated cases in gastric cancer and other digestive tract tumors, and no subtypes of ALP reported in any literature, patent or conference exchange. It can be expressed atopically on the surface of tumor cells.
  • the present invention provides a monoclonal antibody against ectopic expression on a cell surface, and a preparation method and use thereof, wherein the monoclonal antibody against ectopic expression on the cell surface can effectively inhibit the digestive tract Growth and metastasis of tumor cells.
  • the present invention provides a monoclonal antibody against ectopic expression on the cell surface, wherein the amino acid sequence of the light chain variable region of the Fab segment antigen binding site of the antibody molecule is as shown in SEQ ID NO: 2, and the Fab segment antigen binding site of the antibody molecule
  • the heavy chain variable region amino acid sequence is set forth in SEQ ID NO: 4.
  • the cDNA sequence of the light chain variable region thereof is shown in SEQ ID NO: 1
  • the cDNA sequence of the heavy chain variable region thereof is shown in SEQ ID NO: 3.
  • the above monoclonal antibody that is ectopically expressed on the cell surface is an immunoglobulin of a mouse IgG subtype.
  • the present invention also provides the use of the above monoclonal antibody for the preparation of a medicament for treating or preventing gastrointestinal cancer or metastasis.
  • the digestive tract tumor includes but is not limited to gastric squamous cell carcinoma, gastric adenocarcinoma, small cell carcinoma, adenosquamous carcinoma, carcinoid, esophageal cancer, gastric cancer, duodenal cancer, pancreatic cancer, bile duct Cancer, gallbladder cancer, liver cancer, colon cancer, colorectal cancer.
  • the present invention also provides a detection kit comprising the above monoclonal antibody against ectopic expression on the cell surface. Further, the above detection kit further comprises a label that binds to the above-mentioned monoclonal antibody that is ectopically expressed on the cell surface, and the label is a fluorescent label, or a radioactive label, or an enzyme label.
  • the present invention also provides a pharmaceutical composition comprising the above monoclonal antibody against ectopic expression on the cell surface. Further, the above pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient or mixture.
  • the present invention also provides the above-mentioned monoclonal antibody against ectopic expression on the cell surface in serological, pathological diagnosis, tomographic imaging, or positron emission tomography-tomographic tomography of a digestive tract tumor. Applications.
  • the invention also provides a preparation method of the above-mentioned monoclonal antibody against ectopic expression on the cell surface, which is characterized in that: four kinds of gastric cancer cell lines SGC7901, BGC823, MKN28, MKN45 living cells are combined and mixed in four equal ratios.
  • mice three normal immunizations and one booster immunized mouse spleen cells and mouse myeloma cells SP2/0 were chemically fused by PEG, and were screened to secrete surface antigens capable of binding to the above four gastric cancer cells without
  • the present invention establishes an effective method for high-throughput screening of tumor living cells, which is produced by immunizing mice with a living human gastric cancer cell line and selecting a fusion of a highly immunogenic mouse spleen with a mouse SP2/0 cell line.
  • Antibody hybridomas through high-throughput screening of hybridoma antibody-resistant gastric cancer cell lines and peripheral blood mononuclear cells to normal humans Leukocyte (PBMC) control screening resulted in a monoclonal antibody with high specificity response of this strain MS17-57 to GI tumor cells.
  • PBMC Leukocyte
  • the invention adopts a method for reverse-screening and identifying antigens by using anti-gastric cancer monoclonal antibody, and aims to realize "one-step method" simultaneous preparation of MS17-57 monoclonal antibody specific for natural conformation antigen on gastric cancer cell surface and identification of specific gastric cancer Molecular marker ALP cell membrane expressed protein. Since the conventional antibody screening preparation method is difficult to obtain an antibody that recognizes the natural conformation antigen on the surface of living cells, the present invention employs a "shotgun method" for living cell immunization, a hybridoma technique combined with a high-throughput method for flow cytometry, in living cells. Direct screening of MS17-57-specific antibodies against cell surface natural conformational antigens
  • MS17 57 antibody Identification of MS17 57 antibody and correlation analysis between MS17-57 antibody and clinicopathological parameters of gastric cancer, Western blotting, immunoprecipitation and protein profiling were used to specifically bind the target antigen-protein to the prepared MS17-57 antibody. Quick identification, thus screening and discovering the surface molecular markers of this ALP new gastric cancer cell.
  • the MS17-57 mAb of the present invention is highly specific for the reaction of IALP and/or PALP molecules naturally expressed on the surface of tumor cells having a molecular weight of about 56 kDa.
  • the expressed monoclonal antibody is secreted by a monoclonal antibody hybridoma cell line named MS17-57, and the subtype of the mouse monoclonal antibody belongs to the IgGl heavy weight. Chain and kappa light chain.
  • the present invention aims to realize the screening and identification of new gastric cancer molecular markers while preparing anti-cancer cell surface molecular marker-specific antibodies by the strategy of reversely identifying antigens by antibodies.
  • Monoclonal antibodies are a good proteomics research tool.
  • Traditional monoclonal antibody preparation pathways are hybridoma methods, usually immunized with non-molecular markers, and less high-throughput screening methods using a large number of plates after fusion. The probability of anti-cell antigen native epitope antibodies is low.
  • the present invention uses a similar "shot gun" manner to immunize mice by living cells, using a unique hybridoma high fusion rate method for fusion, large-scale plating (more than 50-60 plates at a time) combined with FACS -HTS high-throughput screening method for direct screening of antibodies against the cell surface with a conformational Epitopes molecular marker at the viable cell level, and the final selection of specific high-affinity monoclonals by review antibody.
  • the technical progress of the present invention is remarkable as compared with the prior art.
  • the present invention is carried out against the prepared anti-gastric cancer Analysis of the correlation between the monoclonal antibody specific to the mixed cell line and the clinicopathological parameters of gastric cancer and other digestive tract tumors, and the functional analysis of the action of MS17-57 monoclonal antibody on the surface of IALP and/or PALP on the surface of tumor cells, and found MS17 -57 monoclonal antibody is produced against the surface PALP/IALP antigen of a group of human gastric cancer cells, and can induce specific and functional biological reactions, specifically recognize gastric cancer and other digestive tract tumors and perform targeted therapy, and can be applied at the same time. In the diagnosis and imaging of digestive tract tumors.
  • the invention establishes a novel method for preparing high-throughput anti-stomach and natural conformation antigen-specific antibodies on the surface of digestive tract solid tumor cells, and obtains MS17-57 monoclonal antibody as a proteomics research tool for reverse screening and identification of gastric cancer.
  • a biologic agent for the treatment of human gastrointestinal tumors can be developed if the monoclonal antibody human/mouse is chimeric or humanized.
  • Figure 1 shows the detection of the titer response of FACS against the normal human PBMC and gastric cancer SGC7901 and BGC823 cell lines after FACS immunization with four mixed gastric cancer cell lines.
  • Figure 2 shows the different degrees of binding reaction of MS17-57 monoclonal antibody to four immunosuppressive gastric cancer cell lines by FACS.
  • Figure 3 shows the FACS detection of MS17-57 monoclonal antibody in combination with normal human PBM (:, fetal gastric mucosal epithelial-transformed cell line GES-1 and gastric cancer cell line AGS).
  • Figure 4 is a FACS assay of the binding reaction of MS17-57 monoclonal antibody to normal human PBMC:, gastric cancer cells MN74, TMK-1, KKLS, ST-8 and ST-9 cell lines, respectively. ⁇
  • Figure 5 shows the ELISA assay for the binding of MS17-57 monoclonal antibody to gastric cancer BGC823 cell membrane extracting protein.
  • Figure 6 shows the ELISA assay for the binding of MS17-57 monoclonal antibody to gastric cancer MKN45 cell membrane extracting protein.
  • Figure 7 is an ELISA assay for the binding reaction of MS17-57 monoclonal antibody to GES-1 cell membrane extracting protein.
  • Figure 8 shows the immunohistochemical reaction of MS17-57 monoclonal antibody in gastric mucosal transformed cells GES-1 and gastric cancer cells BGC823 and MKN45.
  • Figure 9 is a direct and indirect method for MS17-57 monoclonal antibody to extract gastric cancer BGC823 and MKN45 cell membrane target proteins. Immunoprecipitation.
  • Figure 10 is a qRT-PCR reaction of PALP and IALP at various mRNA levels in various gastric cancer cell lines.
  • Figure 11 shows the inhibitory response of MS 17-57 monoclonal antibody to the growth of gastric cancer BGC823 and MKN45 cells, respectively.
  • Figure 12 is a graph showing the inhibitory effect of MS17-57 monoclonal antibody on migration of gastric cancer cells MKN45; A. cell culture control; B. 5 4g/mL MS17-57 monoclonal antibody; C. 10 ⁇ g/ml MS17-57 monoclonal antibody; D. 20 4g/mL MS 17-57 mAb; E. 5 [ig l isotype control monoclonal antibody; F. 20 ⁇ ⁇ ⁇ isotype control monoclonal antibody.
  • Figure 13 is a graph showing the inhibitory effect of MS 17-57 monoclonal antibody on the migration of gastric cancer cell line BGC823; ⁇ . Cell culture control; ⁇ . 5 4g/raL MS17-57 mAb; C. 10 ⁇ g/ l MS17- 57 mAb D. 20 4g/mL MS17-57 mAb; E. 5 isotype control monoclonal antibody; F. 20 ⁇ ⁇ isotype control monoclonal antibody.
  • the figure is the quantitative calculation and comparison of the inhibitory effect of MS17-57 monoclonal antibody on the migration of gastric cancer cells BGC823.
  • Figure 15 shows that MS17-57 monoclonal antibody can inhibit the growth of gastric cancer cell ⁇ 45 in mice;
  • B. isotype control monoclonal antibody against MKN45 cell line tumor growth control group, 4 mice averaged 8.5 tumors per mouse and diameter of 0. 31 Ccm ⁇ C.
  • FIG 16 shows that MS17-57 monoclonal antibody can inhibit the growth of gastric cancer cell line BGC823 in mice;
  • A. MS17-57 monoclonal antibody inhibits the tumor growth of BGCS23 cell line, and 4 mice have an average of 1 mouse per mouse. The size of the tumor is about 0. 27 cm.
  • B. The average of the tumors in the BGC823 cell line. 5 ⁇
  • C. The blank control PBS buffer for the BGC823 cell line growth of the control group, the blank control group of 4 mice, an average of 6.5 tumors per mouse and a diameter of about 0.3 cm.
  • reagents and laboratory instruments used in the following examples are common items in the field and can be purchased on the market or Can be obtained through formal channels.
  • mice were immunized by subcutaneous and intraperitoneal injections, 1 x 10 7 cells per mouse per time, immunized once every other week; after 1 week of the third immunization, mouse serum was taken, flow cytometry high-throughput system ( FACS-HTS was used to detect the response of serum to 4 gastric cancer cells. Healthy volunteer PBMC was used as control cells. [Peripheral Blood Mononuclear Cells (PBMC) were isolated from the peripheral blood of healthy volunteers by Ficoll solution and used as fluorescence flow. Screening of normal human cell antigens by cytometry high-throughput screening (FACS-HTS).
  • FACS-HTS flow cytometry high-throughput screening
  • Pre-fusion booster immunization was performed in mice with a higher serum titer and a lower degree of cross-reactivity with control cell PBMC (the difference between the mean fluorescence intensity values of serum and gastric cancer cells and control cells at the same dilution ratio > 500).
  • the spleen of the boosted mouse was taken, and the spleen cells of the immunoreactive mouse No. 1 were prepared into a single cell suspension in serum-free DMEM medium; under 50% PEG (pH 7.4), Splenocytes were fused with SP2/0 mouse myeloma cells, fused and plated in large batches (50 plates) and cultured for 10 days in HAT selective medium to the formation of antibody hybridoma cell clones.
  • the above four gastric cancer cell lines were collected and mixed for large-scale culture as a screening object, and normal human peripheral blood PBMC cells were isolated as control control cells, and both cells were resuspended in pre-cooled 1.5% BSA/PBS blocking solution.
  • binding reaction with 4 gastric cancer cell surface antigens ie, the sample signal peak shift amplitude is greater than one logarithmic value compared with the isotype control signal peak
  • No binding reaction with PBMC cells ie, the sample signal peak shift amplitude is less than 5-10% compared to the isotype control signal peak
  • Table-1 (a), Variable region cDNA sequence of MSI 7-57 mAb light chain (SEQ ID NO: 1): 5 ' -atgtctgcatctccaggggaaaaggtcaccatgacctgcagggccagctcaagtat Gggtctgggacctcttactctctcacaatcagcagtgtggaggctgaagatgctgc
  • variable region amino acid sequence of MS17-57 mAb light chain (SEQ ID NO: 2): MSASPGEKVTMTC 5 J/ m screen QQKSGAPPKLWIY ⁇ ⁇ SG VPARFSGSGSGTSYSLTISSVEAEDAATYYC ⁇ S ⁇ iTFGAGPSWNX Table - 2 (a), the variable region cDNA sequence of the MS17-57 mAb heavy chain (SEQ ID NO: 3):
  • %-well plate On the %-well plate, an average of about 200,000 different gastric cancer cells/100 ⁇ l per well were dispensed with 1% BSA/PBS and added to the U-shaped plate, and the serum of the gastric cancer live cells was continuously diluted 5 times. Dilute, then add 100 ⁇ l per well, mix and react on ice or 4 ° C for 20 minutes. After 2 washes, add 1:333 diluted goat anti-mouse IgGFc-FITC 100 Ml, 4 ° C reaction and After washing, the MFI values of the wells were read on a BD LSR-II fluorescence flow cytometer-HTS machine.
  • the mouse spleen cells were used for the hybridization of MS17-57 monoclonal antibody. Fusion experiment. (As shown in Figure 1).
  • the affinity-purified MS17-57 mAb was subjected to the combined staining reaction of the four immunological gastric cancer cell lines by the U-plate staining method described in Example 3, and was read on the LSR-II FACS instrument. MRI value.
  • the other experimental steps were the same as in Example 3.
  • the affinity-purified MS17-57 monoclonal antibody was subjected to a binding staining reaction to normal human PBMC, gastric cancer cell line GES-1, AGS by the U-plate staining method described in Example 3, and in LSR-II FACS.
  • the MRI value is read on the instrument.
  • MS17-57 monoclonal antibody had high binding reactivity to GES-1 and AGS cell lines, but no binding reaction with normal human PBMC, and the isotype control unrelated monoclonal antibody was a negative control without binding reaction (such as Figure 3).
  • Example 6 The affinity-purified MS17-57 mAb was combined with the normal human PBMC and each gastric cancer cell line by the U-plate staining method described in Figure-1, and was read on the LSR-II FACS instrument. MFI value.
  • the affinity-purified MS17-57 monoclonal antibody and the gastric cancer cell line BGC823 cell membrane extracting protein (previously coated in the Immuno Scientific-Immunlon-II ELISA reaction plate of the United States) were subjected to serial enzyme-binding reaction and read in ELISA plate. The 0D value and plot are read on the instrument.
  • the affinity-purified MS17-57 monoclonal antibody and the gastric cancer cell line MKN45 cell membrane extracting protein were subjected to serial enzyme-binding reaction and read in ELISA plate. The 0D value and plot are read on the instrument.
  • Gastric mucosal transformed cells GES-1 and gastric cancer cells BGC823 and N45 were stained with catalase bound to MS17-57 monoclonal antibody after centrifugation on a cell slide (Cytospin) and showed the distribution of target protein and cell membrane surface results (magnified Immunohistochemical reactions with multiples of 40x and ⁇ ), respectively.
  • MS17-57 monoclonal antibody can bind to the surface of cell membrane of gastric mucosal transformed cells GES-1 and gastric cancer cells BGC823 and MKN45, which is also a proof of the localization of MS17-57 monoclonal antibody target protein (such as Figure 8).
  • Indirect immunoprecipitation is a reaction between MS17-57 monoclonal antibody and gastric cancer cell membrane extracting protein.
  • the Fc end of the antibody specifically binds to Protein-A magnetic beads and washes away non-specifically adsorbed proteins, and then together with SDS-PAGE loading buffer. Heat dissociation; direct immunoprecipitation was directly coupled to activated Dynabeads magnetic beads by Invitrogen, Inc. (Grand Island, NY, USA) using MS17-57 mAb, and the subsequent steps were the same as indirect immunoprecipitation. It has been shown that the immunoprecipitation method for MS17-57 mAb must use a direct method to obtain a specific and clear target band. Subsequent multiple high-sensitivity mass spectrometry confirmed that the corresponding target of MS17-57 monoclonal antibody was PALP and / or IALP protein.
  • RNA expression in various gastric cancer cell lines by qIP-PCR of IALP and PALP, IALP is expressed in different degrees in MKN45, SGC790 AGS and GES-1 cells, but PALP is only in BGC823 cells. The expression of the strain is higher.
  • CCK-8 cell staining counting kit (Cel l Counting Kit-8) from Doj indo (Santa Clara, CA) by each of the 5 replicate CCK-8 staining experiments, different MS17-57 single Anti-dose control was added to each cell well (each cell was controlled at about 500 cells), each plate containing various doses, conditions and 5 replicate wells, and 8 plates were plated. A plate was taken out every day for CCK-8 dye to stain the living cells in the wells. After incubating for 4 hours in a 37° C cell incubator, the 0D value was measured and calculated by using a microplate reader at 0D450nm, thereby indirectly demonstrating the cells. Proliferation.
  • the CCK8 dye test results showed that MS17-57 monoclonal antibody inhibited the growth of cells in gastric cancer BGC823 (Fig. 11A) and MKN45 (Fig. 11B) at two different doses.
  • MS17-57 monoclonal antibody is still very sensitive to the proliferation inhibition of gastric cancer cells, and the concentration of MS17-57 monoclonal antibody at 2 ⁇ 3 4g/mL can significantly inhibit the reaction.
  • the gastric cancer cell line MKN45 was placed in a matrigel-free chamber, and 50,000 cells were plated in each chamber, and then concentration gradients of 20 ⁇ ⁇ , 10 g/mL, 54 g/mL, and medium control solution, respectively.
  • MS17-57 mAb and isotype control antibody were separately added, and the effect of MS17-57 mAb on the migration of gastric cancer cell lines was observed after standing at 37 ° C for 48 hours.
  • the experimental results of the experimental group and the control group showed that MS17-57 monoclonal antibody has the function of inhibiting the migration of gastric cancer cells.
  • MS17-57 monoclonal antibody showed different concentrations of gastric cancer cell MKN45 migration movement in different degrees in the Transwel l chamber, and the homotypic antibody showed a good negative control (as shown in Figure 12).
  • the gastric cancer cell line BGC823 was placed in a chamber containing no Matrigel, and each chamber was plated with 50,000 cells, and then at 20 4 g/mL, 10 g/tnL, 5 g/, respectively.
  • the concentration gradient of mL and medium control solution was added to MS17-57 monoclonal antibody and isotype control antibody, respectively.
  • the effect of MS17-57 monoclonal antibody on the migration of gastric cancer cell lines was observed.
  • the experimental results of the experimental group and the control group showed that the MS17-57 monoclonal antibody inhibited the migration function of gastric cancer cells.
  • MS17-57 monoclonal antibody showed different concentrations of inhibitory effect on the migration movement of gastric cancer cell BGC823 in the Transwel l chamber, and the homologous antibody showed a good negative control (as shown in Figure 13).
  • the Transwell l chamber-transfected gastric cancer cell line BGC823 cells and membranes of different concentrations of MS17-57 mAb were collected and stained, and then the optical density values were read at 0D450 nm, and the results were calculated. This histogram is made after the intra-batch error value.
  • mice In the experimental group of Fig. 15A, mice (nude mice) were injected with 2.0 ml of MS17-57 monoclonal antibody and MKN45 cells mixed with PBS containing 50 g/mL of MS 17-57 monoclonal antibody (100 ⁇ g of antibody protein). And MKN45 cells of lxlO 6 were injected once every two weeks (abdominal and tail veins) 3 times, and the abdominal cavity of the mice was opened and observed for gastric cancer at the 6th week after the initial injection. A tumor that grows in the cell MKN45.
  • FIG. 15B isotype of monoclonal antibody control group
  • mice node mice
  • mice were injected with 2.0 ml of isotype control monoclonal antibody and MKN45 cells mixed with PBS containing 50 g/mL of isotype control monoclonal antibody (100 ⁇ g antibody) Protein) and lxlO 6 MKN45 cells.
  • the injection was performed once every two weeks (abdominal and tail veins) for 3 times.
  • the mice were opened in the abdominal cavity and the tumors of gastric cancer cells MKN45 were observed.
  • FIG -15C blank PBS buffer control group
  • Mice (nude mice) were injected with 2.0 ml of a blank control PBS buffer and MKN45 cell mixture, which also contained 1 x 106 MKN45 cells, injected once every two weeks (abdominal and tail vein) 3 times, after the initial injection. At the 6th week, the mouse was opened in the abdominal cavity and the tumor grown by gastric cancer cell N45 was observed.
  • mice In the experimental group of Fig. 16A, mice (nude mice) were injected with 2.0 ml of MS17-57 monoclonal antibody and BGC823 cells mixed with PBS containing 50 ⁇ / ⁇ 3 ⁇ 417-57 monoclonal antibody (100 ⁇ g antibody protein). And 1x106 BGC823 cells were injected once every two weeks (abdominal and tail veins) three times. At the 6th week after the initial injection, the mice were opened intraperitoneally and the tumors of gastric cancer cells BGC823 were observed. In the same type of monoclonal antibody control group of Fig.
  • mice 16B, mice (nude mice) were injected with 2.0 ml of a mixture of the same type of control monoclonal antibody and BGC823 cells, which contained 50 4 g/inL of the isotype control monoclonal antibody (100 ⁇ g antibody protein). ) and 1x106 of BGC823 cells. The injection was performed once every two weeks (abdominal and tail veins) for 3 times. At the 6th week after the initial injection, the mice were opened intraperitoneally and the tumors of gastric cancer cells BGC823 were observed.
  • Figure 16C blank PBS buffer control group, pair Mice (nude mice) were injected with 2.0 ml of a blank control PBS buffer and a BGC823 cell mixture, which also contained lxlO 6 BGC823 cells, once every two weeks (abdominal and tail veins) for 3 times, after the initial injection. At the 6th week, the mouse was opened in the abdominal cavity and the tumor grown by the gastric cancer cell BGC823 was observed.

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Abstract

提供了一种对肿瘤细胞胞外异位表达的PALP和/或IALP抗原具有亲和性的，并可抑制肿瘤细胞生长和迀移的单克隆抗体，其抗体分子Fab段抗原结合部位的轻链可变区的氨基酸序列如SEQ ID NO:2所示，重链可变区的氨基酸序列如SEQ ID NO:4所示。还提供了上述单克隆抗体在制备治疗、预防消化道肿瘤或者消化道肿瘤转移的药物中的用途；含有上述单克隆抗体的检测试剂盒和药物组合物；及上述的单克隆抗体的制备方法。

Description

抗细胞表面异位表达的单克隆抗体及其制备方法和用途 技术领域 本发明属于生物制药领域， 尤其涉及一种单克隆抗体， 具体来说是一种抗胃癌等 消化道肿瘤细胞表面异位表达的碱性磷酸酶的单克隆抗体及其制备方法和用途。 背景技术
胃癌 （Gastric Cancer, GO 是最常见的消化系统恶性肿瘤， 在世界范围内是肿 瘤相关死亡的第二大原因。 中国是世界上胃癌发病率最高的国家之一， 每年新发病例 近 40万， 约占全世界的 42%， 并且很多患者在就诊时己属晚期。 目前， 胃癌的早期筛 査和确诊主要依靠胃镜结合组织病理学活检， 虽然近年来这种早期筛查诊断手段的特 异性、 敏感性、 准确性和安全性均有了一定程度的提高， 但是由于普及困难以及诸多 干扰因素的存在， 临床应用效果并不十分令人满意。 因此， 多数胃癌患者初诊时往往 己发展至晚期， 主要依靠手术治疗、 放疗、 全身化疗， 但是这些传统治疗措施不仅费 用昂贵， 增加了个人、 社会经济负担， 而且治疗的针对性不强， 疗效常常差强人意， 患者的五年生存率通常不超过 10%， 所以胃癌的临床治疗仍不容乐观。近年来， 虽然一 些新的治疗药物以及联合治疗方案的实施使患者的治疗反应率有所提高， 但是在已经 开展的随机临床研究中， 均未达到中位生存期 1年以上的治疗目标。 由于胃癌细胞普 遍对化疗及放疗不敏感， 而且目前尚缺乏有效的胃癌二线治疗方案， 因此近年来新的 胃癌治疗策略以及治疗药物的研发成为基础及临床研究的热点。 2002年 NCI- PRG建议 对肿瘤进行分子层面的定义， 从而有效实现肿瘤患者的个体化治疗、 复发检测以及预 后评估。 在这些新理念的指导下， 胃癌的分子靶向治疗应运而生， 成为新的生物学治 疗方法。
分子靶向治疗是以肿瘤细胞过度表达或者特异性表达的某些标志性分子作为靶 点。 上世纪 90年代至今， 多种分子靶向治疗药物相继进入临床试验用于多种肿瘤的治 疗， 部分药物显示出较好的疗效。 这些药物主要包括两大类， 即大分子单克隆抗体以 及小分子酪氨酸激酶抑制剂。 由于抗体高度特异性识别并结合抗原， 因此肿瘤的抗体 靶向治疗 （Antibody- Targeted Therapy, ATT) 在肿瘤治疗领域彰显出独特魅力。 以 与肿瘤发生发展密切相关的分子作为靶点， 通过单克隆抗体的激动剂（Agonist)或者 竞争性拮抗作用 （Competitive Antagonism), 以及将抗体作为传递药物的载体， 或是 借助某些免疫学机制（如 ADCC) , 实现对肿瘤细胞的特异性抑制、杀伤或对肿瘤生长微 环境的针对性千扰， 因此与传统治疗措施相比， 效果更为明显而毒副作用大大降低。 肿瘤的抗体药物靶向治疗将会成为传统治疗方式的重要补充甚至有可能取代现有的治 疗方法。
胃癌的抗体药物靶向治疗近年来发展迅速。 临床前研究以及临床研究显示， 以表 皮生长因子受体（EGFR)为靶点的单抗药物 Cetuximab, 以表皮生长因子受体 2 (EGFR2, HER2) 为靶点的单抗药物 Trastuzumab 以及以血管内皮生长因子 （VEGF) 为靶点的单 抗药物 Bevacizumab (Avastin)均具有一定程度的抗胃癌活性， 但是均存在着特异性 不高， 靶向性不强的问题， 实际临床治疗效果并不十分理想。 胃癌靶向治疗单抗药物 研究的核心是寻找鉴定胃癌特异性抗原以及制备抗特异性抗原天然表位的抗体。 Brichory等首先将蛋白质组学方法引入到鉴定筛选肿瘤相关抗原及抗肿瘤自身抗体领 域。 在目前的后基因组时代， 这一策略在多种肿瘤研究中被广泛应用， 包括胃癌， 并 且揭示出众多可能的新的胃癌分子标志物， 如葡萄糖调节蛋白 （Glucose Regulated Protein, GRP78), 热休克蛋白 (Heat Shock Protein, HSP27、 70、 60) 以及纤维蛋 白肽 A (Fibrin Peptide A)。 但是由于肿瘤单抗靶向治疗的理想候选靶点是细胞表面 特异性抗原， 而且蛋白质组学研究中所采用的某些技术很难保证抗原的天然构象， 限 制了鉴定筛选得到的胃癌分子标志物以及相应抗体的进一步研究应用。 毋庸置疑， 在 天然状态下鉴定胃癌或其它肿瘤细胞表面特异性抗原， 并且实现髙通量制备抗胃癌或 其它肿瘤表面分子标志物的特异性抗体将会为胃癌分子标志物鉴定以及胃癌靶向治疗 单抗药物的研发提供有力工具。 碱性磷酸酶 （ALP) 属于同源二聚体蛋白， 分子量为 56 kDa。 每个单体由
449个氨基酸组成， 完整的 ALP分子呈现典型的 的拓扑结构。 ALP是一 种含锌的糖蛋白酶。 人的 ALP在 pH8. 0的碱性环境呈现最高的活力并能够水解 磷酸单酯化合物底物。 正常 ALP是由 pho A 基因编码的。 它是一种分泌蛋白， 在胞浆内合成氨基末端分泌信号肽的单体前体， 分泌信号肽引导前体跨内膜运 输后被切除， 形成的同源二聚体被分泌至胞外。 ALP有多个亚型并是一类能够将对应底物去磷酸化的酶或同工酶。 正常人 血清中的 ALP主要来自肝脏， 来自骨组织的 ALP—般少于总活性的一半。 目前 己知 ALP的亚型有胎盘型 （PALP)、 肠型 （IALP )、 生殖细胞型 （GALP ) 以及非 特异组织型（TNSALP )。而 TNSALP为基因表达后加上多个修饰所形成的肝、 肾、 骨等次级同工酶；
在人体生理性异常或病理性的情况下， ALP主要经肝脏排出到外周血， 从 而血清 ALP增高。 ALP主要存在机体的骨骼、 肝脏、 肾脏等组织。 肝胆系统疾 病包括肝胆恶性肿瘤、 骨骼发育异常、 孕妇等 ALP会增高， 但是目前还没有报 道在胃癌等消化道肿瘤中升高的报道， 也没有任何文献、 专利或会议交流中报 道 ALP的亚型可以异位性表达在肿瘤细胞表面。
发明内容
为了解决上述技术问题， 本发明提供了一种抗细胞表面异位表达的单克隆抗体及 其制备方法和用途， 所述的这种抗细胞表面异位表达的单克隆抗体能有效的抑制消化 道肿瘤细胞的生长和转移。
本发明提供了一种抗细胞表面异位表达的单克隆抗体，其抗体分子 Fab段抗原结合 部位的轻链可变区氨基酸序列如 SEQ ID NO: 2所示， 其抗体分子 Fab段抗原结合部位 的重链可变区氨基酸序列如 SEQ ID NO: 4所示。
进一步的， 其轻链可变区的 cDNA序列如 SEQ ID NO: 1所示， 其重链可变区的 cDNA 序列如 SEQ ID NO: 3所示。 进一步的， 上述的抗细胞表面异位表达的单克隆抗体是一种鼠源 IgG亚型的免疫球 蛋白。 本发明还提供了上述的单克隆抗体在制备治疗、 预防消化道肿瘤或者转移的药物 中的用途。 进一步的， 所述的消化道肿瘤包括但并不局限于胃鳞状细胞癌、 胃腺癌、 小细胞 癌、 腺鳞癌、 类癌、 食管癌、 胃癌、 十二指肠癌、 胰腺癌、 胆管癌、 胆囊癌、 肝癌、 结肠癌、 结直肠癌。 本发明还提供了一种检测试剂盒， 含有上述的一种抗细胞表面异位表达的单克隆 抗体。 进一步的， 上述的检测试剂盒还含有与上述的抗细胞表面异位表达的单克隆抗体 结合的标记物， 所述的标记物为荧光标记物、 或者放射性标记物、 或者酶标标记物。 本发明还提供了一种药物组合物， 含有上述的一种抗细胞表面异位表达的单克隆 抗体。 进一步的， 上述的药物组合物还包含药学上可接受的载体、 赋形剂或混合剂。 本发明还提供了上述的一种抗细胞表面异位表达的单克隆抗体在消化道肿瘤的血 清学、 病理学诊断、 X线断层显像、 或者正电子发射断层扫描- X线断层显像中的应用。 本发明还提供了上述的一种抗细胞表面异位表达的单克隆抗体的制备方法， 其特 征在于：采用四种等同比例组合（混合）的胃癌细胞株 SGC7901、 BGC823、 MKN28 , MKN45 活细胞多点免疫小鼠， 三次正常免疫和一次加强免疫后的小鼠脾脏细胞与小鼠骨髓瘤 细胞 SP2/0通过 PEG化学融合， 筛选出能够分泌出能够结合上述四种胃癌活细胞表面抗 原而不与人正常外周血单个核细胞相反应的杂交瘤细胞株， 将此杂交瘤细胞株亚克隆 后， 获得所培养的杂交瘤细胞上清液， 经亲和纯化即得到本发明的单克隆抗体。 进一步的， 通过免疫亲和层析方法纯化杂交瘤细胞培养上清液。 本发明建立一个有效的肿瘤活细胞高通量筛选的方法， 通过混合的人胃癌细胞系 活细胞免疫小鼠并选择高免疫反应的小鼠脾脏与小鼠的 SP2/0细胞系融合而产生的抗 体杂交瘤， 在通过对杂交瘤抗体抗胃癌细胞系的高通量筛选和对正常人的外周血单核 白细胞（PBMC)对照筛选从而得到了此株 MS17-57对 GI肿瘤细胞高特异性反应的单克隆 抗体。 本发明釆用了以制备的抗胃癌单抗反向筛选鉴定抗原的方法， 旨在实现 "一步 法" 同时制备特异性抗胃癌细胞表面天然构象抗原的 MS17- 57单抗以及鉴定其特异性 胃癌分子标志物 ALP细胞膜表达蛋白。 由于传统抗体筛选制备方法很难获得识别活细胞表面天然构象抗原的抗体， 所以 本发明采用了 "鸟枪法"活细胞免疫，杂交瘤技术结合流式细胞术高通量检测的方法， 在活细胞水平直接筛选抗细胞表面天然构象抗原的 MS17- 57特异性抗体， 在进行
MS17 57抗体的鉴定及 MS17-57抗体与胃癌临床病理学参数的相关性分析后， 采用蛋白 印迹法、免疫沉淀法和蛋白质谱法完成对所制备的 MS17-57抗体特异性结合靶抗原蛋白 的快捷鉴定， 从而筛选和发现了这个 ALP新的胃癌细胞表面分子标志物。 本发明的 MS17- 57单抗能高度特异性地对分子量约 56kDa的肿瘤细胞表面自然表 达的 IALP和 /或 PALP分子反应。 在本发明自有特殊的单抗杂交瘤筛选设计中， 所表达的 单克隆抗体是由命名为 MS17-57的单抗杂交瘤细胞株分泌产生的， 小鼠单抗的亚型是属 于 IgGl重链和 κ轻链。
本发明通过以抗体反向鉴定抗原的策略， 旨在实现在制备抗胃癌细胞表面分子标 志物特异性抗体的同时筛选鉴定新的胃癌分子标志物。 单克隆抗体是一种良好的蛋白 质组学研究工具， 传统的单抗制备途径是杂交瘤方法， 通常采用非分子标志物免疫， 也较少采用融合后大量铺板的高通量筛选方法， 因此获得抗细胞抗原天然表位抗体的 机率较低。 本发明采用类似 "鸟枪法" （ " shot gun" manner)通过活细胞免疫小鼠， 采用独特的杂交瘤高融合率方法进行融合、 大批量铺板 （每次多于 50-60块板） 结合 FACS-HTS高通量检测筛选的方法， 在活细胞水平上直接筛选抗细胞表面带有构象性表 位 （Conformational Epitopes) 分子标志物的抗体， 并通过复查鉴定最终选定特异性 高亲和力的单克隆抗体。 本发明和已有技术相比， 其技术进步是显著的。 本发明在进行对所制备的抗胃癌 混合细胞株特异性单克隆抗体与胃癌等消化道肿瘤的临床病理学参数相关性分析后， 对 MS17-57单抗相作用于在肿瘤细胞表面的 IALP和 /或 PALP分子作用功能分析， 发现 MS17-57单抗是针对一组人胃癌细胞的表面 PALP/IALP抗原而产生的， 并可诱导特异性、 功效性生物反应， 能够特异性识别胃癌等消化道肿瘤并进行靶向治疗， 同时可以应用 在消化道肿瘤的诊断和影像中。 本发明建立了一套高通量制备抗胃癌及消化道实体瘤 细胞表面天然构象抗原特异性抗体的全新方法， 并以获得的 MS17-57单抗作为蛋白质组 学研究工具， 反向筛选鉴定胃癌等消化道实体肿瘤细胞表面分子靶向标志物。 若将所 述单克隆抗体人 /鼠嵌合化或人源化后可开发用于人胃肠道肿瘤治疗的生物药剂。 附图说明
图 1显示了 FACS对四种混合胃癌细胞株免疫后的小鼠血清与正常人 PBMC、 胃癌的 SGC7901和 BGC823细胞株滴度反应的检测。
图 2显示了 FACS对 MS17-57单抗分别与 4种免疫用的胃癌细胞株不同程度的结合反 应。
图 3是 MS17- 57单抗分别与正常人 PBM (：、 胎儿胃粘膜上皮转化细胞株 GES-1和胃癌 细胞株 AGS结合反应的 FACS检测。
图 4是 MS17- 57单抗分别与正常人 PBMC：、 胃癌细胞 MN74、 TMK- 1、 KKLS、 ST- 8和 ST- 9 细胞系结合反应的 FACS检测。 ■
图 5是 MS17- 57单抗与胃癌 BGC823细胞膜抽提蛋白结合的 ELISA检测反应。
图 6是 MS17- 57单抗与胃癌 MKN45细胞膜抽提蛋白结合的 ELISA检测反应。
图 7是 MS17-57单抗与 GES- 1细胞膜抽提蛋白结合反应的 ELISA检测。
图 8是 MS17- 57单抗在胃粘膜转化细胞 GES- 1和胃癌细胞 BGC823、 MKN45的免疫组织 化学反应检测。
图 9是 MS17- 57单抗对胃癌 BGC823和 MKN45细胞膜抽提靶蛋白进行直接法和间接法 的免疫沉淀反应。
图 10是各种胃癌细胞株的 PALP和 IALP在 mRNA水平上的 qRT-PCR反应。
图 11是 MS 17- 57单抗分别对胃癌 BGC823和 MKN45细胞生长增值抑制反应。
图 12是 MS17- 57单抗对胃癌细胞 MKN45迁移运动的抑制作用； A. 细胞培养液对照； B. 5 4g/mL MS17-57单抗； C. 10 ^g/ml MS17-57单抗； D. 20 4g/mL MS 17-57单抗； E. 5 [ig l 同型对照单抗； F. 20 μξΜΐ 同型对照单抗。
图 13是 MS 17- 57单抗对胃癌细胞 BGC823迁移运动的抑制作用； Α. 细胞培养液对照; Β. 5 4g/raL MS17-57单抗; C. 10 ^g/ l MS17- 57单抗; D. 20 4g/mL MS17- 57单抗； E. 5 同型对照单抗； F. 20 μ§ ΐ 同型对照单抗。
图 是 MS17- 57单抗对胃癌细胞 BGC823迁移运动的抑制作用的定量计算和比对。 图 15是 MS17- 57单抗能够抑制胃癌细胞 ΜΚΝ45在小鼠体内肿瘤的生长； A. MS17 - 57 单抗抑制 ΜΚΝ45细胞株肿瘤生长的实验组， 4个小鼠平均每个小鼠有 1. 5个肿瘤并直径在 0. 30 cm大小左右； B. 同型对照单抗对 MKN45细胞株肿瘤生长的对照组， 4个小鼠平均 每个小鼠有 8. 5个肿瘤并直径在 0. 31 cm大小左右； C. 空白对照 PBS缓冲液对 MKN45细胞 株肿瘤生长的对照组，空白对照组 4个小鼠，平均每个小鼠有 9. 0个肿瘤并直径在 0. 28 cm 大小左右。
图 16是 MS17- 57单抗能够抑制胃癌细胞 BGC823在小鼠体内肿瘤的生长； A. MS17 - 57 单抗抑制 BGCS23细胞株肿瘤生长的实验组， 4个小鼠平均每个小鼠有 1个肿瘤并直径在 0. 27 cm大小左右； B. 同型对照单抗对 BGC823细胞株肿瘤生长的对照组， 4个小鼠平均 每个小鼠有 7个肿瘤并直径在 0. 31 cm大小左右； C. 空白对照 PBS缓冲液对 BGC823细胞 株肿瘤生长的对照组， 空白对照组 4个小鼠，平均每个小鼠有 6. 5个肿瘤并直径在 0. 3 cm 大小左右。
具体实施方式
下面实施例采用的试剂和实验器具均为本领域的常用物品， 可以在市场上购买或 者通过正规途径可以获得。
实施例 1
采用四种等比例混合的 4株胃癌细胞（BGC823、 MKN28. MKN45和 SGC7901 , 均 来源于中国科学院上海细胞生物学研究所） 经在含 10% FBS的 RPMI 1640培养基中， 5% C02、 37°C环境下培养后， 收集的混合活细胞在 PBS 缓冲液中作为免疫原， 在 A/J-JAX 小鼠 （购置于南京大学实验动物模式中心， 小鼠来源于 The Jackson Laboratory, 美国）背部皮下及腹腔内注射进行免疫，每次每只小鼠 1 X 107个细胞， 隔周免疫一次；在第 3次免疫 1周后，取小鼠血清，流式细胞术高通量系统 (FACS-HTS ) 检测血清与 4株胃癌细胞的反应情况， 健康志愿者 PBMC作为对照细胞【健康志愿者 外周血经 Ficoll液分离外周单个核细胞 （Peripheral Blood Mononuclear Cells, PBMC) 并作为在荧光流式细胞仪高通量筛选 （FACS- HTS ) 的正常人细胞抗原对照筛 选】。 选择血清效价较高以及与对照细胞 PBMC交叉反应程度较低的小鼠 （同一稀释 比例下， 血清与胃癌细胞及对照细胞的平均荧光强度值的差值〉 500)进行融合前加 强免疫。
取已经加强免疫过的小鼠脾脏，用无血清的 DMEM培养液将免疫反应性比较高的 1号小鼠的脾脏细胞制备成单细胞悬液； 在 50% PEG (pH7. 4) 条件下， 将脾细胞和 SP2/0小鼠骨髓瘤细胞进行融合， 融合后大批量铺板 （50块板） 并用 HAT选择性培 养基培养 10天至抗体杂交瘤细胞克隆的形成。收集及混合大批量培养的上述四种胃 癌细胞株作为筛选对象， 分离正常人外周血 PBMC细胞作为筛选对照细胞， 二种细胞 都分别在预冷的 1. 5% BSA/PBS封闭液中重悬并平均分配 （约每孔 1〜2 x 105细胞） 各加入 51块 96孔 U型板中【总共 102块板， 二个第 51块板分别为阳性（免疫后的 小鼠血清并梯度稀释） 和阴性 （正常小鼠血清并梯度稀释和 HAT选择性培养液）对 照板】。
将 50块抗体杂交瘤细胞融合板 （96孔板） 的上清液 （相当于第一抗体即原始的单 抗）每次 70微升 /孔分别移入相应的筛选板和对照板中并震荡混合， 在冰浴中反应和用 封闭液洗涤后， 再加入 100微升 /孔的 FITC荧光标记的羊抗鼠而进行的细胞荧光染色实 验反应， 由此的荧光流式细胞标记高通量筛选（FACS- HTS ), 先以空白对照孔以及同型 对照孔的细胞调节 FACS参数并作为本底，对两组 96孔 U型板每一孔的细胞样本逐一进行 FACS检测。 同时满足以下两个条件的定为阳性细胞孔： （1 )与 4株胃癌细胞表面抗原有 结合反应 （即与同型对照信号峰相比， 样品信号峰偏移幅度大于一个对数值的）； (2) 与 PBMC细胞无结合反应 （即与同型对照信号峰相比， 样品信号峰偏移幅度小于 5- 10% 的）。 所选择和挑取结合独特的杂交瘤高通量筛选细胞， 经选择性培养基和普通培养基 的过渡、 杂交瘤细胞的亚克隆及多次对抗体杂交瘤细胞培养上清的检测， MS17- 57单抗 所选定的杂交瘤细胞上清的抗体亲和纯化并经 0. 2微米膜过滤后 4'C无菌保存和加入 50%甘油在 - 20°C中长期保存。
实施例 2
用 Qiagen (Valencia, 美国加州） 的 RNeasy 试剂盒从 MS17- 57单抗杂交瘤细胞株 中抽提总 RNA， 用 Invitrogen ( Grand Island , 美国纽约州） 的 Superscript III First-Strand 试剂盒将 mRNA反转录成 MS17-57单抗的 cDNA文库。 利用德国的 Progen Biotechnik公司" Mouse IgG Library Primer Set " (F2010) 试剂盒的 23个引物进行 21个 PCR反应 （不包含 λ轻链的反应）， 所产生的特异轻、 重链产物进行 DNA测序、 氨基 酸多肽序列的翻译和 CDRs (抗原决定族区域） 和 FW (骨架区域） 的辨认。
表 -1 (a), MSI 7- 57单抗轻链的可变区 cDNA序列 (SEQ ID NO: 1 )： 5 ' -atgtctgcatctccaggggaaaaggtcaccatgacctgcagggccagctcaagtat gggtctgggacctcttactctctcacaatcagcagtgtggaggctgaagatgctgc
cacttattactgccagcagttcagtggttcccctatcacgttcggtgctggaccaa
gctggaactga-3 ' 表- 1 (b)， MS17-57单抗轻链的可变区氨基酸序列 (SEQ ID NO: 2)： MSASPGEKVTMTC 5 J/ m屏 QQKSGAPPKLWIY^ ^ SG VPARFSGSGSGTSYSLTISSVEAEDAATYYC^S^iTFGAGPSWNX 表- 2 (a), MS17- 57单抗重链的可变区 cDNA序列 （SEQ ID NO: 3 )：
5' -gaggtccaagctgcagcagtctggaactgaactggtaaagcctggggcttcagtga
agttgtcctgcaaggcttttgactacaccttcacaaactacgatattaactgggtg
aagcagaggcctggacagggacttgagtggattggatggatttatcctggaagtgg
tagtactgaatacggtgagaagttcaaggggaaggccacactgactgcagacaaat tatttctgtgcaagatcgagtaactggtacttcgatgtctggggtatagggaccac
ggtcaccgtctcctcagccaaaacgacacccccatctgactat-3' 表- 2 (b)， MS17- 57单抗重链的可变区氨基酸序列 (SEQ ID NO: 4)：
RSKLQQSGTELVKPGASVKLSCKAFZ?1 層 j¾7 VKQRPGQGLEWIG# 7m¾~ 7
YGEKFKGKATLTADKSSSTVYMLLSSLTAEDSAVYFCAR5"6}W7¾7WGIGTTVTVSSA
KTTPPSDY 表- 1和表- 2的 （a)、 (b) DNA与氨基酸序列是对等的。 在氨基酸序列中的黑斜体和 下划线是表明 CDR区域的位置， 是按 CDR1、 CDR2和 CDR3顺序排列并在它们之间的区域序 列是骨架蛋白序列 （FW)。
实施例 3
在％孔 型板上， 用 1%BSA/PBS调配平均每孔约 20万个不同胃癌细胞 /100微升 而加入 U型板中， 分别将胃癌活细胞免疫后的血清成 5倍滴度连续稀释， 然后每孔分别 加入 100微升， 混匀后冰上或 4°C反应 20分钟， 经 2次洗涤后再加入 1 : 333稀释的羊抗鼠 IgGFc-FITC 100 Ml, 4° C反应和洗涤后， 在 BD公司的 LSR- II荧光流式细胞仪 -HTS机 上读取各孔的 MFI值。
结果表明： 免疫反应性比较高的 1号小鼠血清与胃癌细胞结合的滴度反应明显高于 与正常人 PBMC结合的滴度，此小鼠脾细胞用于 MS17-57单抗杂交瘤产生的融合实验。（如 图 1所示）。
实施例 4
亲和纯化后的 MS17-57单抗经与实施例 3说明所述的 U孔板细胞染色方法， 对四种免 疫用的胃癌细胞株进行结合染色反应， 并在 LSR- II FACS仪上读取 MRI值。 其它实验步 骤与实施例 3相同。
结果显示了 FACS对 MS17- 57单抗分别与 4种免疫用的胃癌细胞株不同程度的结合反 应， 其中 MS17-57单抗对 MKN- 45胃癌细胞株反应最高， 而对 SGC7901细胞反应相对较低， 与其它二个细胞株反应性介于中间 （如图 2所示）。
实施例 5
亲和纯化后的 MS17-57单抗经与实施例 3说明所述的 U孔板细胞染色方法， 对正常人 PBMC、 胃癌细胞株 GES-1、 AGS进行结合染色反应， 并在 LSR- II FACS仪上读取 MRI值。
结果说明 MS17- 57单抗对 GES-1和 AGS细胞株都具有较高的结合反应性，而与正常人 PBMC无结合反应， 同时同型对照的无关单抗都是无结合反应的阴性对照（如图 3所示）。 实施例 6 亲和纯化后的 MS17- 57单抗经与图 -1说明所述的 U孔板细胞染色方法， 对正常人 PBMC和各个胃癌细胞株进行结合染色反应， 并在 LSR-II FACS仪上读取 MFI值。
结果显示了 MS17-57单抗分别与正常人 PBMC、 胃癌细胞 MKN74、 TMK- 1、 KKLS ST- 8 和 ST- 9细胞系的结合反应， 而结果全部呈现阴性即无结合反应 （如图 4所示）。
实施例 7
亲和纯化后的 MS17-57单抗与胃癌细胞株 BGC823细胞膜抽提蛋白 （事先己包被于美 国 Fisher Scientific公司的 Immunlon- II ELISA反应板中）进行系列酶联结合反应， 并在 ELISA读板仪上读取 0D值和作图。
ELISA结果显示了 MS17- 57单抗与胃癌 BGC823细胞膜抽提蛋白有一定程度的结合反 应， 说明 MS17- 57单抗能与降解后的结合靶蛋白反应， 这为后面的抗体免疫共沉淀工作 做了准备实验 （如图 5所示）。
实施例 8
亲和纯化后的 MS17-57单抗与胃癌细胞株 MKN45细胞膜抽提蛋白 （事先已包被于美 国 Fisher Scientific公司的 Iramunlon-Π ELISA反应板中） 进行系列酶联结合反应， 并在 ELISA读板仪上读取 0D值和作图。
ELISA结果显示了 MS17-57单抗与胃癌 MKN45细胞膜抽提蛋白有一定程度的结合反 应。 与图 5的实验一样， MS17- 57单抗能与降解后的结合靶蛋白反应， 这为后面的抗体 免疫共沉淀工作做了准备实验 （如图 6所示）。
实施例 9
亲和纯化后的 MS17-57单抗与胎儿胃粘膜上皮转化细胞株 GES-1膜蛋白膜抽提蛋白 (事先已包被于美国 Fisher Scientific公司的 Immunlon-II ELISA反应板中) 进行系 列酶联结合反应， 并在 ELISA读板仪上读取 0D值和作图。
ELISA结果显示了对 MS17- 57单抗与 GES-1细胞膜抽提蛋白有较低的结合反应（如图 7所示）。 实施例 10
胃粘膜转化细胞 GES-1和胃癌细胞 BGC823、 N45经细胞玻片上离心 （Cytospin) 后在与 MS17- 57单抗结合的过氧化氢酶染色反应并显示靶点蛋白均分布与细胞膜表面 结果 （放大倍数分别为 40x和 ΙΟΟχ ) 的免疫组织化学法反应。
免疫组织化学法检测结果显示了 MS17-57单抗能够结合于胃粘膜转化细胞 GES-1和 胃癌细胞 BGC823、 MKN45的细胞膜表面上， 这也是对 MS17- 57单抗靶蛋白定位的一个佐 证 （如图 8所示）。
实施例 11
间接免疫沉淀法是 MS17-57单抗与胃癌细胞膜抽提蛋白反应， 抗体的 Fc端与 Protein- A磁珠特异性结合并洗去非特异性吸附蛋白，然后通过与 SDS-PAGE上样缓冲液 一起加热解离； 直接免疫沉淀法是用 MS17-57单抗直接偶联于美国 Invitrogen公司 (Grand Island, 美国纽约州） 活化的 Dynabeads磁珠上， 然后的几步反应与间接免疫 沉淀法相同。实践证明对于 MS17-57单抗的免疫沉淀法必须要用直接法才能得到特异而 清晰的靶点条带。 后续的多次高敏质谱分析确定了 MS17-57单抗相应的靶点为 PALP和 / 或 IALP蛋白。
直接免疫沉淀法的 SDS- PAGE变性胶银染色条带显示， MS17- 57单抗在 BGC823细胞株 抽提膜蛋白上只有一条带即 IALP蛋白， 而在 MKN45细胞株抽提膜蛋白上有二条带（即有 IALP条带又有 PALP蛋白条带）， 其它残留重、 轻链抗体分解条带都在正常的位置上。 间 接免疫共沉淀法由于方法的放大步骤和过高的敏感性带来了很强的背景和非特异性。 (如图 9所示， A为非直接免疫沉淀， B为直接免疫沉淀。） 实施例 12
qRT-PCR方法： 总 RNA是用 Qiagei (Valencia, 美国加州） 的 RNeasy 试剂盒从细 胞中提取的。 用 Invitrogen ( Grand Island , 美国纽约州） 的 Superscript- III First- Strand试剂盒将 mRNA反转录成 cDNA。 使用 Applied BioSystems (Foster City, 美国加州）的引物，探针以及 TaqMan ( Invitrogen)基因表达 Master Mix ( Invitrogen) 扩增， 用 St印 OnePlus仪器 （Invitrogen ) 检测相关的基因。 GATOH作为内参基因同时 被检测。
_ Q A Ct sample (gene of interest - GAPDH) / r> ^ C reference (gene
Relative expression ― Δ / Δ
of interest - GAPDH) 通过 IALP和 PALP分别的 qRT-PCR对各种胃癌细胞株的 RNA水平表达的分析， IALP在 MKN45、 SGC790 AGS和 GES- 1细胞有不同程度的表达， 但是 PALP仅在 BGC823细胞株表 达较高。 这些数据与 IP和 MASS的结果有些吻合， 但也存在差异， 毕竟是在二个不同 RNA 和蛋白水平上的观察结果 （如图 10所示）。
实施例 13
采用 Doj indo公司 （Santa Clara, 美国加州） 的 CCK-8细胞染色计数试剂盒 （Cel l Counting Kit- 8 ) 通过每个条件 5复孔 CCK- 8染色的细胞增殖实验， 不同的 MS17- 57单抗 剂量控制而加入每个细胞孔中 （每孔细胞控制在 500个细胞左右）， 每块板含有各种不 同剂量、 条件和各 5复孔， 铺 8块板。 每天取出一块板进行 CCK- 8染料对孔内存在的活细 胞进行染色反应， 在 37° C细胞培养箱内孵育 4小时后用酶标仪在 0D450nm下测定 0D值并 计算， 从而间接证明细胞的增殖情况。
CCK8染料检测结果显示了 MS17-57单抗在二个不同剂量下分别对胃癌 BGC823 (如图 11A) 和 MKN45 (如图 11B ) 细胞生长的增值抑制反应。 MS17- 57单抗对胃癌细胞增殖抑 制作用还是非常敏感的， 2〜3 4g/mL的 MS17-57单抗浓度即可有显著抑制反应。
实施例 14
将胃癌细胞株 MKN45铺入不含基质胶的小室内， 每个小室铺入 50, 000个细胞， 然后 分别以 20 μ§ , 10 g/mL、 5 4g/mL和培养基对照液的浓度梯度分别加入 MS17- 57单 抗和同型对照抗体， 37° C静置 48小时后观察 MS17-57单抗抑制胃癌细胞株迁移的作用。 实验组与对照组的实验结果显示 MS17-57单抗具有抑制胃癌细胞的迁移功能。
结果显示了 MS17-57单抗在的 Transwel l小室中对胃癌细胞 MKN45迁移运动有 不同浓度梯度而不同程度的抑制作用， 同型抗体显示了很好的阴性对照（如图 12所 示）。
实施例 15
如实施例 14的实验步骤， 将胃癌细胞株 BGC823铺入不含基质胶的小室内， 每 个小室铺入 50, 000个细胞， 然后分别以 20 4g/mL、 10 g/tnL、 5 g/mL和培养基对 照液的浓度梯度分别加入 MS17- 57单抗和同型对照抗体， 37D C静置 48小时后观察 MS17-57单抗抑制胃癌细胞株迁移的作用。实验组与对照组的实验结果显示 MS17 - 57 单抗具有抑制胃癌细胞的迁移功能。
结果显示了 MS17- 57单抗在的 Transwel l小室中对胃癌细胞 BGC823迁移运动有 不同浓度梯度而不同程度的抑制作用， 同型抗体显示了很好的阴性对照（如图 13所 示）。
实施例 16
如实施例 14的实验步骤， 将不同浓度 MS17- 57单抗作用后的 Transwel l小室穿膜过 的胃癌细胞株 BGC823细胞和膜收集和染色， 然后在 0D450 nm下读取光密度值， 计算结 果和批内误差值后作出此直方图。
结果显示了 MS17- 57单抗在 Transwel l小室中对胃癌细胞 BGC823迁移运动的抑制作 用功能反应并进行了多复孔实验的相对抑制定量计算。结果表明 MS17- 57单抗可以抑制 胃癌细胞 BGC823的迁移运动可达到 20- 30% (如图 14所示）。
实施例 17
图 15A的实验组， 对小鼠 （裸鼠）注射 2. 0毫升 MS17-57单抗和 MKN45细胞混合的 PBS 液， 其包含了 50 g/mL的 MS 17- 57单抗（100微克抗体蛋白）和 lxlO6的 MKN45细胞， 每二 周注射一次 （腹腔和尾静脉） 共 3次， 初次注射后的第 6周将小鼠腹腔打开并观察胃癌 细胞 MKN45所长出的肿瘤。 图- 15B的同型单抗对照组， 对小鼠 （裸鼠） 注射 2. 0毫升同 型对照单抗和 MKN45细胞混合的 PBS液， 其包含了 50 g/mL的同型对照单抗 （100微克抗 体蛋白） 和 lxlO6的 MKN45细胞。 每二周注射一次 （腹腔和尾静脉） 共 3次， 初次注射后 的第 6周将小鼠腹腔打开并观察胃癌细胞 MKN45所长出的肿瘤； 图 -15C的空白 PBS缓冲液 的对照组， 对小鼠 （裸鼠）注射 2. 0毫升空白对照 PBS缓冲液和 MKN45细胞混合液， 其也 包含了 1x106的 MKN45细胞， 每二周注射一次（腹腔和尾静脉）共 3次， 初次注射后的第 6周将小鼠腹腔打开并观察胃癌细胞 N45所长出的肿瘤。
结果显示了 MS17-57单抗能够抑制胃癌细胞 MKN45在小鼠体内肿瘤生长的功能。 实施例 18
图 16A的实验组，对小鼠（裸鼠）注射 2. 0毫升 MS17-57单抗和 BGC823细胞混合的 PBS 液， 其包含了50 §/ 的^¾17-57单抗 （100微克抗体蛋白） 和 1x106的 BGC823细胞， 每 二周注射一次 （腹腔和尾静脉） 共 3次， 初次注射后的第 6周将小鼠腹腔打开并观察胃 癌细胞 BGC823所长出的肿瘤。 图 16B的同型单抗对照组， 对小鼠 （裸鼠） 注射 2. 0毫升 同型对照单抗和 BGC823细胞混合的 PBS液， 其包含了 50 4g/inL的同型对照单抗 （100微 克抗体蛋白） 和 1x106的 BGC823细胞。 每二周注射一次 （腹腔和尾静脉） 共 3次， 初次 注射后的第 6周将小鼠腹腔打开并观察胃癌细胞 BGC823所长出的肿瘤； 图 16C的空白 PBS 缓冲液的对照组， 对小鼠 （裸鼠） 注射 2. 0毫升空白对照 PBS缓冲液和 BGC823细胞混合 液， 其也包含了 lxlO6的 BGC823细胞， 每二周注射一次（腹腔和尾静脉）共 3次， 初次注 射后的第 6周将小鼠腹腔打开并观察胃癌细胞 BGC823所长出的肿瘤。
结果显示了 MS17-57单抗能够抑制胃癌细胞 BGC823在小鼠体内肿瘤生长的功

Claims

权利要求
1. 一种抗细胞表面异位表达的单克隆抗体， 其特征在于： 其抗体分子 Fab段抗原结合 部位的轻链可变区氨基酸序列如 SEQ ID NO: 2所示， 其抗体分子 Fab段抗原结合部 位的重链可变区氨基酸序列如 SEQ ID NO: 4所示。
2. 如权利要求 1所述的抗细胞表面异位表达的单克隆抗体， 其特征在于： 其轻链可变 区的 cDNA序列如 SEQ ID N0: 1所示， 其重链可变区的 cDNA序列如 SEQ ID NO: 3所示。
3. 如权利要求 1所述的一种抗细胞表面异位表达的单克隆抗体， 其特征在于： 是一种 鼠源 IgG亚型的免疫球蛋白。
4. 权利要求 1所述的单克隆抗体在制备治疗、 预防消化道肿瘤的药物中的用途。
5. 权利要求 1所述的单克隆抗体在制备治疗、 预防消化道肿瘤转移药物中的用途。
6. 如权利要求 4或 5所述的用途， 所述的消化道肿瘤包括但并不局限于胃鳞状细胞癌、 胃腺癌、 小细胞癌、 腺鱗癌、类癌、食管癌、 胃癌、十二指肠癌、胰腺癌、 胆管癌、 胆囊癌、 肝癌、 结肠癌、 或者结直肠癌。
7. 一种检测试剂盒， 其特征在于： 含有权利要求 1所述的一种抗细胞表面异位表达的 单克隆抗体。
8. 如权利要求 7所述的一种检测试剂盒， 其特征在于： 还含有与所述的抗细胞表面异 位表达的单克隆抗体结合的标记物，所述的标记物为荧光标记物、或者放射性标记 物、 或者酶标标记物。
9. 一种药物组合物，其特征在于：含有权利要求 1所述的一种抗细胞表面异位表达的 单克隆抗体。
10. 如权利要求 9所述的一种药物组合物， 其特征在于： 还包含药学上可接受的载体、 赋形剂或混合剂。
11. 权利要求 1的所述的一种抗细胞表面异位表达的单克隆抗体在消化道肿瘤的血清 学、 病理学诊断、 或者 X线断层显像、 或者正电子发射断层扫描- X线断层显像中的 应用。
12. 权利要求 1的所述的一种抗细胞表面异位表达的单克隆抗体的制备方法， 其特征在 于： 采用四种等比例混合的胃癌细胞株 SGC7901、 BGC823、 MKN28、 MKN45, 活细胞 多点免疫小鼠，三次正常免疫和一次加强免疫后的小鼠脾脏细胞与小鼠骨髓瘤细胞 SP2/0通过 PEG化学融合，筛选出能够分泌出能够结合上述四种胃癌活细胞表面抗原 而不与人正常外周血单个核细胞相反应的杂交瘤细胞株，将此杂交瘤细胞株亚克隆 后， 获得所培养的杂交瘤细胞上清液， 经亲和纯化即得到本发明的单克隆抗体。
13.如权利要求 12所述的一种抗细胞表面异位表达的单克隆抗体的制备方法， 其特 征在于： 通过免疫亲和层析方法纯化杂交瘤细胞培养上清液。
PCT/CN2014/000289 2013-03-20 2014-03-19 抗细胞表面异位表达的单克隆抗体及其制备方法和用途 WO2014146487A1 (zh)

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