WO2017054141A1 - Her2线性表位中和活性单克隆抗体及其应用 - Google Patents

Her2线性表位中和活性单克隆抗体及其应用 Download PDF

Info

Publication number
WO2017054141A1
WO2017054141A1 PCT/CN2015/091127 CN2015091127W WO2017054141A1 WO 2017054141 A1 WO2017054141 A1 WO 2017054141A1 CN 2015091127 W CN2015091127 W CN 2015091127W WO 2017054141 A1 WO2017054141 A1 WO 2017054141A1
Authority
WO
WIPO (PCT)
Prior art keywords
monoclonal antibody
her2
amino acid
acid sequence
sequence
Prior art date
Application number
PCT/CN2015/091127
Other languages
English (en)
French (fr)
Inventor
孙乐
李茂华
张翠娟
张小刚
王慕旸
Original Assignee
北京天成新脉生物技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京天成新脉生物技术有限公司 filed Critical 北京天成新脉生物技术有限公司
Priority to PCT/CN2015/091127 priority Critical patent/WO2017054141A1/zh
Publication of WO2017054141A1 publication Critical patent/WO2017054141A1/zh

Links

Images

Classifications

    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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

Definitions

  • the present invention relates to the field of immunology and antibody preparation techniques, in particular to an anti-HER2 linear epitope neutralizing active monoclonal antibody and its use.
  • Human epidermal growth factor receptor 2 (HER2) is a member of the epidermal growth factor receptor (ErbB2) family and is a transmembrane receptor-like protein with a relative molecular mass of 185,000 and has tyrosine kinase activity.
  • HER2 is an important prognostic factor for breast cancer.
  • HER2-positive (overexpressed or expanded) breast cancer has special clinical features and biological behavior, and the treatment pattern is also very different from other types of breast cancer.
  • the drug acting on the HER-2 target is currently the most commonly used and most effective drug for the treatment of breast cancer, mainly trastuzumab and pertuzumab.
  • Herceptin (trastuzumab for injection), the first antibody against the breast cancer marker HER2, in 1997. It has annual sales of more than $5 billion and is now imitation. A hot spot for pharmacy. In 2012, Genentech introduced an antibody drug against the HER-Domain2 site, which is more effective in combination with Herceptin. In March 2013, Genentech also listed Herceptin and the drug DM1 conjugate, which is also very effective for patients with advanced cancer.
  • HER2 human epidermal growth factor receptor 2
  • the HER2 linear epitope-neutralizing active monoclonal antibody provided by the present invention is a hybridoma cell prepared by conjugated to the KHL carrier protein of the HER2 amino acid sequence 374-398 as an immunogen, and the screening can stabilize the secretion specificity.
  • the polypeptide sequences described above for immunizing mice are:
  • the present invention provides the use of a linear epitope polypeptide of human epidermal growth factor receptor 2 in the preparation of a HER2 linear epitope and an active monoclonal antibody, wherein the amino acid sequence of the polypeptide is Or the sequence has an equivalent functional amino acid sequence formed by substitution, deletion or addition of one or several amino acids.
  • the present invention provides the use of a linear epitope polypeptide of human epidermal growth factor receptor 2 for the preparation of a therapeutic agent for diseases targeting HER2, wherein the amino acid sequence of the polypeptide is Or the sequence has an equivalent functional amino acid sequence formed by substitution, deletion or addition of one or several amino acids.
  • the monoclonal antibody prepared by coupling the above polypeptide sequence with a carrier protein as an immunogen can specifically recognize the polypeptide sequence of the HER2 protein, and 40 positive clones are obtained, and 6 positive clones are screened and further neutralized. Two positive clones were screened for the activity assay, and the two monoclonal antibodies were referred to as clone2H4 and clone2D9, respectively.
  • the present invention provides a HER2 linear epitope neutralizing active monoclonal antibody clone2H4,
  • amino acid sequence of the light chain variable region thereof is set forth in SEQ ID No. 1, or the sequence is substituted, deleted or added with one or several amino acids to form an equivalent amino acid sequence;
  • amino acid sequence of the heavy chain variable region thereof is set forth in SEQ ID No. 2, or the sequence is substituted, deleted or added with one or several amino acids to form an equivalent amino acid sequence.
  • the present invention provides hybridoma cells producing the monoclonal antibody clone2H4.
  • the present invention provides the use of the monoclonal antibody clone2H4 in the preparation of a therapeutic agent for diseases targeting HER2.
  • the invention provides a medicament, a detection reagent or a reagent containing the monoclonal antibody clone 2H4 box.
  • the present invention also provides a HER2 linear epitope neutralizing active monoclonal antibody clone2D9,
  • amino acid sequence of the light chain variable region thereof is set forth in SEQ ID No. 3, or the amino acid sequence of the sequence formed by substitution, deletion or addition of one or several amino acids;
  • amino acid sequence of the heavy chain variable region thereof is set forth in SEQ ID No. 4, or the sequence is substituted, deleted or added with one or several amino acids to form an equivalent amino acid sequence.
  • Hybridoma cells producing the monoclonal antibody clone 2D9 are within the scope of the present invention.
  • the present invention provides the use of the monoclonal antibody clone2D9 in the preparation of a therapeutic agent for diseases targeting HER2.
  • a drug, a detection reagent or a kit containing the monoclonal antibody clone 2D9 is also within the scope of the present invention.
  • the invention also provides a method for preparing an anti-HER2 linear epitope neutralizing active monoclonal antibody clone2H4 or clone2D9, and designing and synthesizing a polypeptide comprising amino acids 374-398 between Domain 2/3 of HER2 extracellular region with HER2 as a target protein. Coupling with the carrier protein KLH as an immunogen, prepared by immunizing mice. Wherein the amino acid sequence of the polypeptide is:
  • the mouse is conjugated to the KLH carrier protein as an immunogen, and hybridized to obtain a sustained and stable secretion of the anti-HER2 monoclonal by cell fusion.
  • the above polypeptide sequence is selected from HER2:
  • the HER2 linear epitope provided by the present invention neutralizes the active monoclonal antibodies clone2H4 and clone2D9 with good affinity, and experiments have shown that the affinity EC50 of clone2H4 and clone2D9 and HER2-positive cells SK-BR-3 cells is 1-4 nM.
  • the monoclonal antibody of the present invention recognizes a linear epitope of the sequence between the extracellular domain Domain 2/3 of HER2, which is completely different from the recognition HER2 epitope of pattozumab and trastuzumab.
  • the combination of the two drugs enhances the therapeutic effect and has broad prospects in the treatment of breast cancer.
  • Example 1 is a positive clone for identifying HER2 by indirect ELISA according to Example 1 of the present invention, wherein 600 clones were screened, 178 positive clones were selected, and 40 positive clones were selected for further screening.
  • Example 2 is a SDS-PAGE electrophoresis diagram of a monoclonal antibody in Example 2 of the present invention, wherein M is a protein molecular weight standard (kDa), and clone 2H4 and clone 2D9 are two monoclonal antibodies obtained in the present invention;
  • M is a protein molecular weight standard (kDa)
  • clone 2H4 and clone 2D9 are two monoclonal antibodies obtained in the present invention
  • Figure 3A and Figure 3B are flow cytometry plots of clone 2H4 and clone 2D9 combined with SK-BR-3 cells, respectively, showing binding to SK-BR-3 cell surface.
  • FIG. 4A is a flow cytometry test diagram of clone 2H4 combined with SK-BR-3 cells in Example 4 of the present invention, wherein the inhibition effect of clone 2H4 is 40%;
  • FIG. 4B is a flow cytometry detection diagram of clone 2D9 combined with SK-BR-3 cells. The inhibitory effect of clone 2D9 reached 30%, both showing good neutralizing activity.
  • HER2 was used as the target protein, and a polypeptide sequence (polypeptide of amino acid 266-296 in the extracellular domain of HER2) was selected from the following sequence:
  • the above polypeptide is prepared by chemical synthesis, and the purity requirement is greater than 90%.
  • the polypeptide is coupled to KLH to prepare an immunogen.
  • DMEM medium was purchased from Hyclone, HAT, HT selection medium, and phytol was purchased from sigma.
  • mice Balb/c mice, 8-12 weeks old, female, SPF animals were cultured.
  • Freund's complete adjuvant Freund's incomplete adjuvant was purchased from Sigma, PEG4000 was purchased from Fluka, HRP-goat anti-mouse IgG was purchased from Jackson Immune, and the remaining reagents were domestically analyzed pure products.
  • Basal immunization The antigen was mixed with an equal volume of Freund's complete adjuvant and fully emulsified, subcutaneously injected at a sub-point, and each Balb/c mouse was injected in an amount of 100 ⁇ g per injection.
  • mice collected in a conventional manner were fused with SP2/0 cells at a ratio of 10:1 at 500 g/L of PEG4000.
  • the culture was selected with HAT medium, and 10-15 days after the fusion, the supernatant was subjected to indirect ELISA to screen for hybridoma cell lines secreting anti-HER2 antigen.
  • the resulting positive clones were subcloned using the limiting dilution method.
  • the procedure of the indirect ELISA method was as follows: using 200 ⁇ l of HER2 plate, using immunized mouse serum 1:2000 as a positive control, no cloned growth medium supernatant and normal mouse serum as a negative control, adding 1:2000 HRP per well- Goat anti-mouse IgG 100 ⁇ l, and finally measured 450 nm OD value. If the OD450 value is more than 2 times larger than the negative control, it can be initially determined as a positive clone. A total of 178 positive clones were obtained, from which 40 better positive clones were selected. (see picture 1)
  • Purification antibody titer assay The indirect ELISA method for detecting the purified antibody prepared by the above hybridoma cells has a titer of >0.05 ng/ml.
  • the above hybridoma cell line was further cultured and passaged in DMEM medium containing 10% fetal bovine serum, and after 10 passages, the hybridoma cell line was still able to grow well and stably pass, and the supernatant of the culture solution was still valenceable. It reaches 1:10000 or above.
  • the above results indicate that the obtained hybridoma cell line can be stably passaged, and the monoclonal antibody against HER2 can be stably and stably secreted.
  • 10% dimethyl sulfoxide protection solution (dimethyl sulfoxide can damage the filter and is destroyed by high pressure, so it can not be filtered or autoclaved. It is drug, sterile): 10% dimethyl Sulfoxide, 20% inactivated fetal bovine serum, 70% RPMI-1640 solution.
  • FCS-1640 medium penicillin 100 U/ml, streptomycin 100 ⁇ g/ml.
  • the old medium in the cell culture flask was removed, and 10% FCS-1640 solution was added to suspend the cells. Centrifuge at 1000 r/min for 10 min and remove the supernatant. The cell pellet was made into a suspension with a 10% dimethyl sulfoxide solution to make 1.0 ⁇ 10 7 cells/ml. Sampling, trypan blue staining, counting live cells should be above 95%. The cells were dispensed into ampoules with a syringe, 0.5 ml-1.0 ml per bottle, and the ampoules were sealed. Put 4 ° C 2h. The gaseous portion of the liquid nitrogen tank was drained (-70 ° C) for 15 h. Transfer to the liquid nitrogen fraction.
  • mice were selected and intraperitoneally inoculated with pristane, 0.5 ml per mouse. After 7-10 days, the 16th generation hybridoma cells were inoculated intraperitoneally, and each mouse was 1 ⁇ 10 6 - 2 ⁇ 10 6 . After 5 days interval, the abdomen is obviously enlarged, and when the hand is touched, the skin has a sense of tension, and the ascites can be collected with a 9-gauge needle.
  • the ascites was centrifuged (13,000 r/min for 30 minutes) to remove cellular components and other precipitates, and the supernatant was collected. Purification was carried out with Protein G to Sepharose CL-4B.
  • the upper column was 20 mM PBS buffer, and the column chromatography eluate was pH 2.7, 20 mM glycine buffer to obtain a monoclonal antibody against HER2.
  • the Ig subtype of the antibody produced by the above hybridoma cells was identified by an indirect ELISA using antibodies against various mouse Ig subtypes, wherein clone 2H4 showed the strongest IgG3 signal, and clone2D9 showed the strongest IgG1 signal, based on the subtype identification reagent. In the box description, the results were judged as criteria, Clone2H4 was IgG3, and Clone2D9 was IgG1 subtype. See Table 2.
  • variable region amino acid sequence of the antibody of clone 2H4 and clone 2D9 the amino acid sequence of clone 2H4 is: the light chain is shown in SEQ ID No. 1, and the heavy chain is shown in SEQ ID No. 2.
  • the amino acid sequence of clone 2D9 is: the light chain is shown in SEQ ID No. 3, and the heavy chain is shown in SEQ ID No. 4. The sequence was not shown to have the same sequence, indicating that the obtained sequence is a sequence specific to each of the two clones.
  • SK-BR-3 cells The preparation of SK-BR-3 cells was performed on the first day, and SK-BR-3 cells with good growth were selected, washed three times with sterile PBS, and the cells were digested with 0.1% trypsin for 1-3 minutes, and added with 10 5 ml of DMEM medium of % FBS was obtained, and the cells were obtained after centrifugation. After the cells were counted, the cells were adjusted to 1 ⁇ 10 5 /ml, and 200 ⁇ l per well was plated into 96 wells, and cultured overnight. The next day, the cells were removed from the culture supernatant, washed twice with PBS, and fixed with 95% alcohol for 15 min.
  • the cells were washed twice with sterile water and then blocked with 200 ⁇ l/well of 5% skim milk for 1 h. After washing three times with PBS, add gradient dilution of Clone 2H4, Clone2D9, incubate for 1 h at 37 ° C, wash three times with PBS, add HRP (horseradish peroxidase)-labeled murine secondary antibody (1:2000) for 1 h, then wash with PBS. Five times (the first three times 5 minutes, the last two times 10 minutes) were added to the developer to develop color for 15 minutes to detect OD450.
  • HRP horseradish peroxidase
  • the developer A solution is formulated by adding 1 g of urea peroxide per 1000 mL of water, 10.3 g of citric acid, 35.8 g of Na 2 HPO 4 ⁇ 12H 2 O, Tween-20100 ⁇ L, pH 5; the solution of solution B is added to each 1000 mL of distilled water.
  • Methylbenzidine (TMB) 700 mg (40 mL DMSO dissolved), 10.3 g citric acid, pH 2.4.
  • EC50 values of clone 2H4 and clone 2D9 were both measured to be 1.3 nM, indicating a higher affinity.
  • Cell preparation Take SK-BR-3 cells grown in log phase, aspirate the cell supernatant, and wash once with 5-10 ml PBS. After adding 2 ml of 0.1% trypsin for digestion at 37 ° C for 5 min, the digestion was terminated by adding DMEM containing 10% FBS, and the cells were collected by centrifugation at 1400 rpm for 3 min. After washing the cells three times with DMEM without fetal growth, the cells were resuspended in PBS, centrifuged at 1400 rpm for 3 min, and 20 ug/ml antibody was added and incubated at 4 ° C for 1 h.
  • the cells were collected by centrifugation at 1400 rpm for 3 min, and the cells were washed three times with PBS, and then incubated with FITC-labeled murine secondary antibody (1:100) for 30 min at 4 ° C, and then centrifuged at 400 rpm for 3 min to collect the cells. After washing three times with PBS, the up-flow cytometer detects the fluorescence information.
  • clone 2H4 and clone 2D9 were able to bind to HER2 on SK-BR-3 cells.
  • Inhibition of high-value expression of HER2-cell SK-BR-3 cells Prepare SK-BR-3 cells grown in log phase, aspirate cell supernatant, and wash once in 5-10 ml PBS. After adding 2 ml of 0.1% trypsin for digestion at 37 ° C for 5 min, the digestion was terminated by adding DMEM or F-12 containing 10% FBS, and the cells were collected by centrifugation at 1400 rpm for 3 min. The cells were diluted with 8 ml of DMEM containing 10% FBS, and the cells were counted by trypan blue cell counting, and the cells were diluted with DMEM containing 10% FBS into 2 ⁇ 10 4 / ml. 96 well plates were plated with 100 ⁇ l or 2000/well. , 37 ° C, 5% CO 2 attached for 4 h.
  • Test samples In sterile EP tubes, different concentrations of antibody were added to the wells of the cells at 100 ⁇ l per well. The culture was continued for 4 days, during which the growth of the cells was observed. The cell culture supernatant was removed, and the prepared CCK-8 (1:10 DMEM) 100 ul 37 ° C, 5% CO 2 was further cultured for 1-4 h. The OD450 was read in a microplate reader. If the OD value is not measured for a while, 10 ⁇ L of 0.1 M HCL solution or 1% w/v SDS solution may be added to each well, and the culture plate is covered and stored at room temperature in the dark. The absorbance does not change within 24 hours.
  • clone 2H4 and clone 2D9 were able to bind to SK-BR-3 cells and inhibit their proliferation, wherein clone 2H4 inhibited the increase of SK-BR-3 cells by 40%, and clone 2D9 inhibited SK-BR-3. The value of cells increased by 30%.
  • the present invention provides an anti-HER2 (human epidermal growth factor receptor 2) neutralizing active monoclonal antibody and use thereof. Binding protein informatics and immunology, design and synthesis of polypeptides containing amino acids 374-398 between Domain 2/3 of HER2 extracellular domain, coupled with carrier protein KLH as immunogen, and immunization of mice to obtain multiple hybridoma cells A monoclonal antibody that specifically recognizes HER2 on the surface of breast cancer cells and has neutralizing activity is secreted.
  • HER2 human epidermal growth factor receptor 2
  • Binding protein informatics and immunology, design and synthesis of polypeptides containing amino acids 374-398 between Domain 2/3 of HER2 extracellular domain, coupled with carrier protein KLH as immunogen, and immunization of mice to obtain multiple hybridoma cells
  • a monoclonal antibody that specifically recognizes HER2 on the surface of breast cancer cells and has neutralizing activity is secreted.
  • the monoclonal antibody of the present invention has an affinity for binding to HER2 of 1-4 nM, and after binding to HER2 on the surface of tumor cells, it can effectively inhibit the proliferation of tumor cells and induce endocytosis of HER2, and is an ideal biotargeting therapeutic antibody.
  • the combination of the monoclonal antibody of the invention and trastuzumab or patomycin can enhance the therapeutic effect and has significant market value.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • Mycology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Genetics & Genomics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

提供了一种HER2线性表位中和活性单克隆抗体及其制备方法，包括将含有HER2的第374-398位氨基酸的多肽与载体蛋白偶联免疫小鼠制备获得多株杂交瘤细胞分泌可以特异性识别乳腺癌细胞表面HER2且具有中和活性的单克隆抗体，所述多肽可制备HER2单抗及治疗以HER2为靶标的疾病治疗药物；所述单抗与HER2结合的亲和力介于1-4nM，可与曲妥珠或帕妥珠联合用药。

Description

HER2线性表位中和活性单克隆抗体及其应用 技术领域
本发明涉及免疫学领域及抗体制备技术，具体地说，涉及抗HER2线性表位中和活性单克隆抗体及其应用。
背景技术
人表皮生长因子受体2(HER2)是表皮生长因子受体(ErbB2)家族中的一员，是相对分子质量为185000的跨膜受体样蛋白，具有酪氨酸激酶活性。HER2是重要的乳腺癌预后判断因子，HER2阳性(过表达或扩增)的乳腺癌，其临床特点和生物学行为有特殊表现，治疗模式也与其他类型的乳腺癌有很大的区别。作用于HER-2靶点的药物，是目前治疗乳腺癌最常用最有效的药物，主要有曲妥珠单抗、帕妥珠单抗等。
基因泰克公司早在1997年就上市了第一个针对乳腺癌标志物HER2的抗体药物赫赛汀(Herceptin，注射用曲妥珠单抗)，每年销售额可达50多亿美元，目前成为仿制药的热点。2012年基因泰克又推出一种针对HER-Domain2位点的抗体药，与赫赛汀联合使用治疗效果更佳。2013年3月，基因泰克又上市了赫赛汀和药物DM1偶联物，对癌症晚期病人也有非常好的疗效。如果能开发出与曲妥珠单抗和帕妥珠单抗的抗原位点不同的新的单抗药物，不仅有利于打破垄断，降低现有药物的市场份额，降低癌症治疗成本，还有利于丰富治疗癌症的抗体药物种类，与现有药物联合用药、增加疗效并巩固治疗效果。但目前还没有针对Domain 2/3位点的单抗药物的报道。
发明内容
本发明的目的是提供针对与曲妥珠单抗识别表位不同的人表皮生长因子受体2(HER2)单克隆抗体及其应用。
本发明提供的HER2线性表位中和活性单克隆抗体是以HER2氨 基酸序列第374-398的多肽偶联到KHL载体蛋白作为免疫原免疫小鼠后，制备杂交瘤细胞，筛选能够稳定分泌特异性识别乳腺癌细胞表面HER2且具有生物学活性单克隆抗体的杂交瘤细胞株。上述用于免疫小鼠的多肽序列为：
Figure PCTCN2015091127-appb-000001
本发明提供了人表皮生长因子受体2的线性表位多肽在制备HER2线性表位中和活性单克隆抗体中的应用，所述多肽的氨基酸序列为
Figure PCTCN2015091127-appb-000002
或该序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列。
本发明提供了人表皮生长因子受体2的线性表位多肽在制备以HER2为靶标的疾病治疗药物中的应用，所述多肽的氨基酸序列为
Figure PCTCN2015091127-appb-000003
或该序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列。
本发明将上述多肽序列与载体蛋白偶联作为免疫原制备获得的单克隆抗体能够特异性地识别HER2蛋白的多肽序列，共获得40个阳性克隆，复筛筛选出6个阳性克隆，进一步中和活性试验筛选出2个阳性克隆，将这两个单克隆抗体分别称之为clone2H4和clone2D9。
本发明提供了HER2线性表位中和活性单克隆抗体clone2H4，
i)其轻链可变区的氨基酸序列如SEQ ID No.1所示，或该序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列；以及
ii)其重链可变区的氨基酸序列如SEQ ID No.2所示，或该序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列。
本发明提供了产生单克隆抗体clone2H4的杂交瘤细胞。
本发明提供了单克隆抗体clone2H4在制备以HER2为靶标的疾病治疗药物中的应用。
本发明提供了含有单克隆抗体clone2H4的药物、检测试剂或试剂 盒。
本发明还提供了HER2线性表位中和活性单克隆抗体clone2D9，
i)其轻链可变区的氨基酸序列如SEQ ID No.3所示，或该序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列；以及
ii)其重链可变区的氨基酸序列如SEQ ID No.4所示，或该序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列。
产生单克隆抗体clone2D9的杂交瘤细胞属于本发明的保护范围。
本发明提供了单克隆抗体clone2D9在制备以HER2为靶标的疾病治疗药物中的应用。
含有单克隆抗体clone2D9的药物、检测试剂或试剂盒也属于本发明的保护范围。
本发明还提供抗HER2线性表位中和活性单克隆抗体clone2H4或clone2D9的制备方法，以HER2为靶蛋白，设计合成含有HER2胞外区Domain 2/3之间第374-398位氨基酸的多肽，与载体蛋白KLH偶联作为免疫原，免疫小鼠制备获得。其中，所述多肽的氨基酸序列为：
Figure PCTCN2015091127-appb-000004
使用可以诱发机体产生免疫反应并生成具有中和效应抗体的多肽序列，偶联至KLH载体蛋白作为免疫原免疫小鼠，采用杂交瘤技术经过细胞融合并筛选得到能持续、稳定分泌抗HER2单克隆抗体的杂交瘤细胞株，由该细胞株分泌得到单克隆抗体。
优选地，上述多肽序列选自HER2：
含有
Figure PCTCN2015091127-appb-000005
本发明提供的HER2线性表位中和活性单克隆抗体clone2H4和clone2D9具有良好的亲和力，实验表明，clone2H4和clone2D9与HER2阳性细胞SK-BR-3细胞的亲和力EC50为1-4nM。能够结合到 SK-BR-3细胞表面HER2，有效地抑制乳腺癌细胞的增值生长，且诱导HER2内吞，可以成为非常理想的生物靶向治疗抗体。
此外本发明的单克隆抗体识别的是一个在HER2胞外区Domain2/3之间序列的一个线性表位，与帕妥株单抗和曲妥珠单抗的识别HER2表位完全不同，可以和它们二者联合用药，增强治疗效果，在乳腺癌的治疗中有着广阔的前景。
附图说明
图1为本发明实施例1通过间接ELISA法筛选识别HER2的阳性克隆，其中筛选了600个克隆，阳性克隆178个，挑选较好的40个阳性克隆进行下一步复筛。
图2是本发明实施例2中单抗的SDS-PAGE电泳图，其中M是蛋白分子量标准(kDa)，clone2H4和clone2D9分别为本发明获得的两个单克隆抗体；
图3A和图3B分别为clone2H4和clone2D9结合SK-BR-3细胞流式细胞仪检测图，显示能够结合到SK-BR-3细胞表面。
图4A为本发明实施例4中clone2H4结合SK-BR-3细胞流式细胞仪检测图，clone2H4的抑制效果达到40％；图4B为clone2D9结合SK-BR-3细胞流式细胞仪检测图，clone2D9的抑制效果达到30％，均显示良好的中和活性。
具体实施方式
以下实施例进一步说明本发明的内容，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。
若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1产生clone2H4和clone2D9的杂交瘤细胞系的建立
一、实验材料
1、免疫原：以HER2作为靶蛋白，从中选取一段多肽序列(HER2胞外区Domain 2第266-296位氨基酸的多肽)，序列如下：
Figure PCTCN2015091127-appb-000006
采用化学合成的方式制备上述多肽，纯度要求大于90％。将多肽与KLH偶联制备成免疫原。
2、培养基：DMEM培养基购自Hyclone公司，HAT、HT选择培养基、降植烷购自sigma公司。
3、实验动物：Balb/c小鼠，8-12周龄，雌性，SPF级动物培养。
4、其他材料：福氏完全佐剂、福氏不完全佐剂购自Sigma公司，PEG4000购自Fluka公司，HRP-山羊抗小鼠IgG抗体购自JacksonImmune公司，其余试剂均为国产分析纯产品。
二、杂交瘤细胞系的建立
1、动物免疫
1)基础免疫：将抗原与福氏完全佐剂等体积混合并充分乳化，分点皮下注射，每只Balb/c小鼠每次注射量为100μg。
2)加强免疫：加强免疫采用抗原与福氏不完全佐剂的乳化液。在进行细胞融合前3天，经腹腔注射含150μg抗原的生理盐水溶液。
2、杂交瘤细胞的制备
按常规方法收集小鼠的脾细胞与SP2/0细胞按10:1的比例以500g/L的PEG4000进行融合。用HAT培养液选择培养，融合后10-15天，取上清采用间接ELISA法筛选分泌抗HER2抗原的杂交瘤细胞株。对所得阳性克隆株采用有限稀释法进行亚克隆。间接ELISA法的操作步骤如下：用200μl的HER2包板，用免疫小鼠血清1:2000作为阳性对照，无克隆生长的培养基上清和正常小鼠血清作为阴性对照，每孔加1:2000HRP-山羊抗小鼠IgG 100μl，最后测定450nm OD值。OD450值大于阴性对照2倍以上者，即可初步判定为阳性克隆。共获得了178个阳性克隆，从中挑选出40个较好的阳性克隆。(见图1)
3、杂交瘤细胞系的建立
经过4次亚克隆和间接ELISA或细胞ELISA筛选，得到6株分别针对HER2，识别SK-BR-3细胞的稳定分泌单克隆抗体的杂交瘤细胞系。(见表1)
表1
Clone编号 OD值 Clone编号 OD值 Clone编号 OD值
1A7 0.444 1B7 0.244 1E10 0.089
2D11 0.112 2E1 0.119 2E7 0.106
3A10 0.121 3A11 0.238 3E6 0.174
5F4 0.099 5F5 0.093 5F6 0.106
1F8 0.122 1G12 0.772 1G6 0.084
2F3 0.291 2F8 0.174 2G4 0.156
3F1 0.253 3F2 0.139 3H10 0.342
6A9 0.129 6B10 0.16 6B12 0.638
1H10 0.138 2D8 0.085 2D9 1.859
2H4 1.062 2H5 0.12 3A8 0.099
4B7 0.295 4D3 0.113 5B10 0.083
6C1 0.089 6C2 0.094 6G6 0.077
2D10 0.072 NC 0.126    
3A9 0.091 NC 0.132    
5D11 1.268 PC 0.184    
6H10 0.083 PC 0.138    
4、应用上述杂交瘤细胞系所得单抗的效价检测
1)小鼠腹水效价测定：间接ELISA法检测上述杂交瘤细胞制备的腹水效价为：＞1：20000000。
2)纯化抗体效价测定：间接ELISA法检测上述杂交瘤细胞制备的纯化抗体效价为：＞0.05ng/ml。
5、杂交瘤细胞系的传代培养
将上述杂交瘤细胞系在含有10％胎牛血清的DMEM培养基中继续进行培养、传代，培养到10代后，杂交瘤细胞系仍然能够生长良好、稳定传代，培养液上清效价仍然可以达到1:10000以上。
以上结果表明，所得杂交瘤细胞系能够稳定传代，可以持续、稳定分泌抗HER2的单克隆抗体。
获得产生所需的单克隆抗体的杂交瘤细胞后，必须将一部分杂交瘤细胞保存，否则在连续传代的过程中，可能产生突变或染色体的漂移以至丧失固有特性或丢失产生抗体的特性。另外在长期的培养过程中，难免不发生污染以至于毁灭。因此必须冷藏保存一部分。保存方法如下：
(1)材料
1)细胞：取对数生长期的细胞。
2)10％二甲基亚砜保护液(二甲基亚砜能损坏滤器，而又被高压所破坏，所以不能过滤或高压消毒。其本身就是毒品，无菌)：含10％二甲基亚砜、20％灭活胎牛血清，70％RPMI-1640液。
3)20％FCS-1640培养液：含青霉素100U/ml，链霉素100μg/ml。
4)灭菌的2ml安瓶等。
(2)操作方法
去掉细胞培养瓶中的旧的培养液，加入10％FCS-1640液，使细胞悬浮。1000r/min离心10min，去上清。细胞沉淀用10％二甲基亚砜保 护液制成悬液，使成1.0×107细胞/ml。取样，台盼兰染色，计数活细胞，应在95％以上。用注射器将细胞分装安瓶，每瓶0.5ml-1.0ml，熔封安瓶。放4℃2h。放液氮罐气态部分(-70℃)15h。转入液氮部分。
实施例2抗HER2的单克隆抗体的制备
一、抗体制备
选择成年BALB/c小鼠，腹腔接种降植烷，每只小鼠0.5ml。7-10天后腹腔接种第16代杂交瘤细胞，每只小鼠1×106-2×106个。间隔5天后，待腹部明显膨大，以手触摸时，皮肤有紧张感，即可用9号针头采集腹水。
将腹水离心(13000r/min 30分钟)，除去细胞成分和其他的沉淀物，收集上清。用Protein G～Sepharose CL-4B进行纯化，上柱液为20mM的PBS缓冲液，柱层析洗脱液为：pH2.7，20mM的甘氨酸缓冲液，得到抗HER2的单克隆抗体。
二、抗体的鉴定
1、抗体纯度鉴定：
SDS-PAGE电泳鉴定，纯度在95％以上。(见图2)
2、抗体类及亚类鉴定：
采用间接ELISA法，使用抗小鼠各种Ig亚型的抗体鉴定上述杂交瘤细胞产生的抗体的Ig亚型，其中clone 2H4显示IgG3信号最强，clone2D9显示IgG1信号最强，依据亚型鉴定试剂盒说明中结果判定标准，Clone2H4为IgG3，Clone2D9为IgG1亚型。见表2。
表2
  IgG1 IgG2a IgG2b IgG3 IgA IgM
clone2H4 0.045 0.051 0.042 0.294 0.06 0.06
clone2D9 0.823 0.054 0.04 0.047 0.061 0.048
3、clone 2H4和clone 2D9的可变区序列测定
将两个克隆的细胞提取mRNA，反转录为cDNA，使用可变区通 用引物进行高保真PCR扩增，将PCR产物片段插入到T载体内进行DNA序列测定，并将获得的序列翻译成蛋白质的氨基酸序列。clone 2H4和clone 2D9的抗体的可变区氨基酸序列：clone 2H4的氨基酸序列为：轻链如SEQ ID No.1所示，重链如SEQ ID No.2所示。clone 2D9的氨基酸序列为：轻链如SEQ ID No.3所示，重链如SEQ ID No.4所示。将该序列进行比对后未显示有相同序列，说明所获得的序列为两个克隆各自特异的序列。
实施例3纯化抗体的亲合力实验
一、细胞ELISA法
应用clone2H4和clone2D9单抗做细胞亲和力试验，确定其与HER2阳性SK-BR-3细胞的结合EC50值，见表3。
表3
Figure PCTCN2015091127-appb-000007
检测方法：第一天进行SK-BR-3细胞的铺板准备，选取生长良好的SK-BR-3细胞，无菌PBS洗三次，加入0.1％的胰蛋白酶消化细胞1-3分钟，加入含10％FBS的DMEM培养基5ml，离心后获得细胞，细胞计数后调成1×105/ml，每孔200μl铺到96孔中，过夜培养。第二天，细胞去掉培养上清后，PBS洗2次，加入95％的酒精固定15min。固定细胞后，无菌水清洗细胞2次后加入200μl/well5％的脱脂牛奶封闭1h。PBS清洗三次后，加入梯度稀释的Clone 2H4，Clone2D9，37℃孵育1h后，PBS清洗三次后加入HRP(辣根过氧化物酶)标记的鼠二抗(1:2000)孵育1h后，PBS清洗5次(前三次5min，最后2次10min)加入显色剂显色15min上机检测OD450。
其中显色剂A液配方为每1000mL水中加入过氧化脲1g，10.3g柠檬酸，35.8g Na2HPO4·12H2O，吐温-20100μL，pH5；B液配方为每1000mL蒸馏水中加入四甲基联苯胺(TMB)700mg(40mL DMSO溶解)，10.3g柠檬酸，pH2.4。
检测clone 2H4和clone2D9的EC50值皆达到1.3nM，显示较高的亲和力。
二、流式细胞仪检测
细胞准备：取对数期生长的SK-BR-3细胞，吸除细胞上清，5－10ml PBS清洗一次。加入2ml 0.1％胰酶37℃消化5min，加入含10％FBS的DMEM终止消化，1400转离心3min收集细胞。加入无胎牛秀清的DMEM清洗细胞三次后，PBS重悬细胞后1400转离心3min收集细胞，加入20ug/ml抗体后4℃孵育1h。1400转离心3min收集细胞，PBS清洗细胞三次后，加入FITC标记鼠二抗(1:100)4摄氏度孵育30min后，400转离心3min收集细胞。PBS清洗三次后，上流式细胞仪检测收集荧光信息。
检测如图3A和图3B所示，clone 2H4和clone2D9能够结合SK-BR-3细胞上的HER2。
实施例4纯化抗体的中和活性检测
抑制高表达HER2细胞SK-BR-3细胞的增值细胞准备：取对数期生长的SK-BR-3细胞，吸除细胞上清，5-10ml PBS清洗一次。加入2ml 0.1％胰酶37℃消化5min，加入含10％FBS的DMEM或F-12终止消化，1400转离心3min收集细胞。加入8ml含10％FBS的DMEM稀释细胞，台盼蓝细胞计数法计数细胞，并用含10％FBS的DMEM稀释细胞成2×104/ml.96孔板中每孔铺入100μl即2000/well，37℃，5％CO2贴壁4h。
检测样本：在无菌的EP管中，不同浓度抗体加入到细胞孔中，每孔100μl。继续培养4d，期间观察细胞的生长。去除细胞培养上 清，加入配制好的CCK-8(1:10 DMEM)100ul 37℃，5％CO2继续培养1-4h。在酶标仪中读取OD450。若暂时不测定OD值，可以向每孔中加入10μL 0.1M的HCL溶液或者1％w/v SDS溶液，并遮盖培养板避光保存在室温条件下。24小时内测定，吸光度不会发生变化。
检测结果如图4A和图4B所示，clone 2H4和clone2D9能够结合SK-BR-3细胞且抑制其增值，其中clone2H4抑制SK-BR-3细胞的增值达到40％，clone2D9抑制SK-BR-3细胞的增值达到30％。
工业实用性
本发明提供一种抗HER2(人表皮生长因子受体2)中和活性单克隆抗体及其应用。结合蛋白信息学与免疫学，设计合成含有HER2胞外区Domain 2/3之间第374-398位氨基酸的多肽，与载体蛋白KLH偶联作为免疫原，免疫小鼠制备获得多株杂交瘤细胞分泌可以特异性识别乳腺癌细胞表面HER2且具有中和活性的单克隆抗体。本发明的单抗与HER2结合的亲和力介于1-4nM，结合到肿瘤细胞表面的HER2后，能够有效抑制肿瘤细胞的增殖，诱导HER2内吞，成为非常理想的生物靶向治疗抗体，将本发明的单抗与曲妥珠或帕妥珠联合用药，可增强治疗效果，具有显著的市场价值。
Figure PCTCN2015091127-appb-000008
Figure PCTCN2015091127-appb-000009

Claims (13)

  1. 人表皮生长因子受体2的线性表位多肽在制备HER2线性表位中和活性单克隆抗体中的应用，其特征在于，所述多肽的氨基酸序列为SLAFLPESFDGDPASNTAPLQPEQLQ，或该序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列。
  2. 人表皮生长因子受体2的线性表位多肽在制备多肽疫苗中的应用，其特征在于，所述多肽的氨基酸序列为SLAFLPESFDGDPASNTAPLQPEQLQ，或该序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列。
  3. 人表皮生长因子受体2的线性表位多肽在制备以HER2为靶标的疾病治疗药物中的应用，其特征在于，所述多肽的氨基酸序列为SLAFLPESFDGDPASNTAPLQPEQLQ，或该序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列。
  4. HER2线性表位中和活性单克隆抗体clone2H4，其特征在于，
    i)其轻链可变区的氨基酸序列如SEQ ID No.1所示，或该序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列；以及
    ii)其重链可变区的氨基酸序列如SEQ ID No.2所示，或该序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列。
  5. 产生权利要求4所述单克隆抗体clone2H4的杂交瘤细胞。
  6. 权利要求4所述的单克隆抗体clone2H4在制备以HER2为靶标的疾病治疗药物中的应用。
  7. 含有权利要求5所述单克隆抗体clone2H4的药物、检测试剂或试剂盒。
  8. 一种单克隆抗体-药物偶联物，其特征在于，所述单克隆抗体为权利要求4所述的单克隆抗体clone2H4，所述药物是丝裂霉素或美 登素类。
  9. HER2线性表位中和活性单克隆抗体clone2D9，其特征在于，
    i)其轻链可变区的氨基酸序列如SEQ ID No.3所示，或该序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列；以及
    ii)其重链可变区的氨基酸序列如SEQ ID No.4所示，或该序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列。
  10. 产生权利要求9所述单克隆抗体clone2D9的杂交瘤细胞。
  11. 权利要求9所述单克隆抗体clone2D9在制备以HER2为靶标的疾病治疗药物中的应用。
  12. 含有权利要求9所述单克隆抗体clone2D9的药物、检测试剂或试剂盒。
  13. 一种单克隆抗体-药物偶联物，其特征在于，所述单克隆抗体为权利要求9所述的单克隆抗体clone2D9，所述药物是丝裂霉素或美登素类。
PCT/CN2015/091127 2015-09-29 2015-09-29 Her2线性表位中和活性单克隆抗体及其应用 WO2017054141A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2015/091127 WO2017054141A1 (zh) 2015-09-29 2015-09-29 Her2线性表位中和活性单克隆抗体及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2015/091127 WO2017054141A1 (zh) 2015-09-29 2015-09-29 Her2线性表位中和活性单克隆抗体及其应用

Publications (1)

Publication Number Publication Date
WO2017054141A1 true WO2017054141A1 (zh) 2017-04-06

Family

ID=58422519

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/091127 WO2017054141A1 (zh) 2015-09-29 2015-09-29 Her2线性表位中和活性单克隆抗体及其应用

Country Status (1)

Country Link
WO (1) WO2017054141A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115975035A (zh) * 2022-08-18 2023-04-18 北京诺赛国际医学研究院 干细胞和抗体联合治疗癌症的用途

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101522717A (zh) * 2006-08-04 2009-09-02 阿斯利康(瑞典)有限公司 针对ErbB2的人抗体

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101522717A (zh) * 2006-08-04 2009-09-02 阿斯利康(瑞典)有限公司 针对ErbB2的人抗体

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
S., K. ET AL.: "P185: an Immunodominant Epitope is an Autoantigen Mimotope", JBC, vol. 286, no. 29, 22 July 2011 (2011-07-22), pages 26221 - 26222, XP055374770 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115975035A (zh) * 2022-08-18 2023-04-18 北京诺赛国际医学研究院 干细胞和抗体联合治疗癌症的用途
CN115975035B (zh) * 2022-08-18 2023-08-18 北京诺赛国际医学研究院 干细胞和抗体联合治疗癌症的用途

Similar Documents

Publication Publication Date Title
US11155632B2 (en) Anti-CD47 monoclonal antibody and use thereof
US9951143B2 (en) Antibodies against immunogenic glycopeptides, compositions comprising the same and use thereof
ES2915410T3 (es) Anticuerpo contra LAG-3, fragmento de unión a antígeno del mismo y aplicación farmacéutica del mismo
CN101970489B (zh) 与hGM-CSF结合的单克隆抗体及包含它的药物组合物
TWI758558B (zh) Cd96抗體、其抗原結合片段及醫藥用途
CN102321173B (zh) 人源化巨噬细胞抑制因子1单克隆抗体及其应用
JP2002518347A (ja) 前立腺癌治療のための免疫療法組成物および方法
CN101896503A (zh) 靶向egf受体的单克隆抗体175及其衍生物和用途
JP2010516229A (ja) ヒトサイトメガロウイルス中和抗体およびその使用
JP2009515827A (ja) 治療薬
CN116239698A (zh) 双功能融合蛋白及其医药用途
CN112969716A (zh) 抗pd-1抗体、其抗原结合片段及医药用途
US20100151486A1 (en) Osteopontin functional epitopes, monoclonal antibodies against the epitopes and uses thereof
JP2022023216A (ja) ビメンチン由来のペプチドに特異的に結合する物質を含む皮膚疾患の予防及び治療用組成物
BR112020003628A2 (pt) anticorpo anti-pacap
WO2022166806A1 (zh) 一种基于cd271的新型抗原表位及其应用
CN102219854A (zh) 抗人p1gf蛋白的单克隆抗体及其制备方法和用途
WO2020249041A1 (zh) 靶向lag-3的抗体和双特异性抗体及其用途
US7147854B2 (en) Topical treatment of psoriasis using neutralizing antibodies to interleukin-8
CN112048019B (zh) 抗人cd47单克隆抗体
WO2017054141A1 (zh) Her2线性表位中和活性单克隆抗体及其应用
WO2023078382A1 (zh) 结合gprc5d的抗体及其用途
WO2015165368A1 (zh) 抗her2中和活性单克隆抗体及其应用
CN106554415B (zh) Her2线性表位中和活性单克隆抗体及其应用
RU2717989C1 (ru) Моноклональное антиидиотипическое антитело АИ-G1, обладающее антигенными свойствами морфина

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15905053

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15905053

Country of ref document: EP

Kind code of ref document: A1