WO2011103700A1 - 一种全人源抗her2单克隆抗体、其制备方法及用途 - Google Patents

一种全人源抗her2单克隆抗体、其制备方法及用途 Download PDF

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WO2011103700A1
WO2011103700A1 PCT/CN2010/000511 CN2010000511W WO2011103700A1 WO 2011103700 A1 WO2011103700 A1 WO 2011103700A1 CN 2010000511 W CN2010000511 W CN 2010000511W WO 2011103700 A1 WO2011103700 A1 WO 2011103700A1
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antibody
her2
seq
her2 antibody
human anti
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李川
仝昕
阚颖
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BIOMAB PHARMACEUTICALS Ltd
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BIOMAB PHARMACEUTICALS Ltd
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Priority to US13/579,180 priority Critical patent/US8974785B2/en
Priority to BR112012021327A priority patent/BR112012021327B8/pt
Priority to CA2790007A priority patent/CA2790007C/en
Priority to EP10846324.1A priority patent/EP2540745B1/en
Priority to JP2012554185A priority patent/JP5690356B2/ja
Publication of WO2011103700A1 publication Critical patent/WO2011103700A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/14—Drugs for genital or sexual disorders; Contraceptives for lactation disorders, e.g. galactorrhoea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3015—Breast
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation

Definitions

  • the present invention relates to the field of biotechnology, and more particularly, to a fully human monoclonal antibody, a process for its preparation and use. Background technique
  • breast cancer is one of the most common malignancies in women. More than 1 million cases of breast cancer are added each year worldwide, and nearly 400,000 people die each year from breast cancer. In recent years, the incidence of breast cancer has shown a clear upward trend in the world. Whether in high-end or low-endemic areas, the incidence of breast cancer increased at a rate of 5-20%. The growth rate of breast cancer in Asian women has been significantly higher than in Europe and the United States. In China, breast cancer has developed into the first place in the incidence of female malignancies in some cities. The usual treatments for breast cancer include surgery, chemoradiotherapy and endocrine therapy. These conventional treatments have greatly prolonged the patient's survival. However, they have large side effects and their efficacy is difficult to further improve. Tumor-targeted therapy is a new type of cancer treatment that has emerged in recent years, and representative of them are anti-tumor monoclonal antibodies.
  • HER2 human epidermal growth factor receptor 2
  • HER2 human epidermal growth factor receptor 2
  • the anti-HER2 humanized monoclonal antibody specifically binds to HER2, and its anti-tumor mechanism includes the following: specific binding to the extracellular domain of the HER2 receptor to block the constitutive activation of the HER2 homodimer and interfere with HER2
  • a heterodimer is formed with other members of the ErbB family.
  • the anti-HER2 humanized monoclonal antibody (Trastuzumab, trade name Herceptin) has been used in clinical trials as a monotherapy for HER2 overexpressing metastatic breast cancer. These patients have received Failure to treat chemotherapy with one or more chemotherapy regimens for metastases.
  • the drug is also used as a first-line drug in combination with paclitaxel or anthracycline (doxorubicin or epirubicin) plus cyclophosphamide in clinical trials.
  • trastuzumab Herceptin
  • Taxol paclitaxel
  • Taxotere docetaxel
  • the present invention constructs a large-capacity natural human phage antibody library, and a whole human anti-HER2 antibody 3E12 was screened therefrom.
  • the present invention provides a fully human anti-HER2 antibody having a heavy chain variable region amino acid sequence of SEQ ID NO: 6 and a light chain variable region amino acid sequence of SEQ ID NO: 8;
  • the above-mentioned fully human anti-HER2 antibody provided by the present invention has a heavy chain amino acid sequence of
  • SEQ ID NO: 10 the light chain amino acid sequence is represented by SEQ ID NO: 12;
  • the present invention also provides an isolated nucleotide encoding the above-described fully human anti-HER2 antibody; the above-mentioned nucleotide provided by the present invention, wherein the nucleotide encoding the heavy chain variable region of the full human anti-HER2 antibody
  • the sequence of SEQ ID NO: 5 the nucleotide sequence encoding the light chain variable region of the fully human anti-HER2 antibody is shown in SEQ ID NO: 7.
  • nucleotide sequence encoding the heavy chain of the fully human anti-HER2 antibody is represented by SEQ ID NO: 9
  • nucleotide sequence encoding the light chain of the fully human anti-HER2 antibody is SEQ ID. NO: 11;
  • the present invention also provides an expression vector comprising the above nucleotide, which is pcDNA3.1/ZEO(+) or pcDNA3.1 (+);
  • the invention also discloses a host cell transformed by the above expression vector, which is a CHO-K1 cell; the invention further provides a preparation method of the above-mentioned whole human antibody, which comprises screening a phage human antibody library to obtain a high affinity full human anti-HER2 single chain.
  • Antibody construction of a fully human anti-HER2 intact antibody molecule eukaryotic expression vector; expression of a fully human anti-HER2 intact antibody molecule in CHO cells; purification of a fully human anti-HER2 intact antibody molecule.
  • the use of the above-mentioned fully human antibody as disclosed in the present invention for the preparation of a medicament for treating tumors, wherein the tumor is a HER2 high expression tumor, more specifically a breast cancer.
  • the present invention utilizes the obtained antibody to carry out a series of experiments, and the experimental results show that: with the humanized antibody Trastuzumab (rhumumAb 4D5) and Chinese Patent Application No. 01132225.
  • the invention name is "humanized resistance”
  • 3E12 has higher antibody affinity, stronger HER2 high expression of breast cancer cell proliferation inhibition, apoptosis Induction activity;
  • In vivo anti-tumor experiments show that the antibodies obtained by the present invention can significantly inhibit the growth of tumors.
  • Figure 1 Results of apoptotic anti-HER2 antibody (Figure 1-1 for SK-BR3 cells, Figure 1-2 for BT-474 cells, Figure 1-3 for MCF-7 cells);
  • the reported sequences were designed to amplify the antibody heavy and light chain constant region genes by RT-PCR.
  • the PCR product was purified by agarose gel electrophoresis and cloned into pGEM-T vector (Promega product). After sequencing, it was confirmed that the correct clone was obtained.
  • SEQ ID NO: l and SEQ ID NO: 2 show the nucleotide and amino acid sequences of the heavy chain constant region (C H) of the.
  • SEQ ID NO: 3 and SEQ ID NO: 4 show the light chain constant region (nucleotide sequence and amino acid sequence of QJ, respectively.
  • the correct clones in this example are designated pGEM-T/C H and pGEM-T/C ⁇ (2) Preparation of cDNA
  • RNA from the cells was extracted from isolated human peripheral blood lymphocytes using Trizol reagent (Invitrogen).
  • the cDNA was reverse transcribed using a cDNA reverse transcription kit (Shanghai Shenneng Gaming Biotechnology Co., Ltd.). The above steps are carried out according to the instructions provided by the manufacturer.
  • the sequence of * is at gcc cag ccg gcc atg gcc, on the 5, end of the V H
  • ⁇ 1 J gcc aga acc acc gcc Gcc gga gcc acc acc gcc, in V L Back primer , 5, terminal ⁇ 1 J tec ggc ggc ggt ggt tct ggc gga ggc gga ggc gga tct contains Not I site at the 5, end of the V L
  • a bacterial single-chain antibody library was constructed using a recombinant Phage antibody system kit (Amersham Biosciences), and then the library was panned with a specific antigen.
  • Antibody library construction and panning methods were performed with reference to the recombinant Phage antibody system kit for panning the specific antigen "human HER2 extramembrane region protein, according to the reference (Proc Natl Acad Sci USA, 1992, 89: 4285- Preparation of the method of 4289).
  • An anti-human HER2 single-chain antibody 3E12ScFv was obtained after multiple panning of the antibody library, and the gene sequence was obtained after sequencing.
  • SEQ ID NO: 5 and SEQ ID NO: 6 respectively showed the 3E12 ScFv weight.
  • SEQ ID NO: 7 and SEQ ID NO: 8 show the nucleotide sequence and amino acid sequence of the 3E12 ScFv light chain variable region VL, respectively.
  • the full human antibody heavy chain gene was synthesized by overlapping PCR under the following conditions: 95 ° C for 15 minutes; 94 ° C for 50 seconds, 58 ° C for 50 seconds, 72 ° C 50 Seconds, 30 cycles; 72°C for 10 minutes.
  • the 5' end of the full human antibody heavy chain gene contains the restriction enzyme site Hindlll and the signal peptide gene sequence, and the 3' end contains the translation stop codon TAA and restriction enzyme sites.
  • the 3E12ScFv gene and pGEM-T/C L vector were used as templates to synthesize the fully humanized antibody light chain gene by overlapping PCR.
  • the reaction conditions were: 95 ° C for 15 minutes; 94 ° C for 50 seconds, 58 ° C for 50 seconds, 72 ° C 50 seconds, 30 cycles; 72 ° C for 10 minutes, the PCR product was obtained, which contained the restriction enzyme site Hindlll and the signal peptide gene sequence at the 5' end, and the translation stop code TAA and the restriction enzyme site EcoR I at the 3' end.
  • the signal peptide gene sequence is
  • the correct clones were selected and digested with Hindlll and EcoR I, and purified by agarose gel electrophoresis to recover the full human antibody light chain fragment.
  • the plasmid pcDNA3.1/ZEO(+) (Invitrogen) vector digested with Hindlll and EcoR1 was ligated to construct a full human light chain eukaryotic expression vector pcDNA3.1/ZEO(+) (3E12V L C L ) .
  • hGHO/1 is prepared according to the method disclosed in Chinese Patent Application No. 01132225.X, filed on November 16, 2001, entitled "Humanized Anti-HER2 Monoclonal Antibody and Its Preparation and Pharmaceutical Composition".
  • Human breast cancer cells SK-BR-3 high expression of HER2, ATCC: HTB-30
  • BT-474 expressed in HER2, ATCC: HTB-20
  • MCF-7 low expression of HER2, ATCC number: HTB-22
  • dilutions of anti-HER2 antibodies including Trastuzumab, hGHO/1, 3E12
  • the cells were washed with Annexin V/PI kit (product of BD) according to the product specification. Percentage of apoptotic cells.
  • the anti-apoptotic assay is shown in Figure 1: The ability of 3E12 antibody to kill SK-BR3 cells is significantly stronger than that of Trastuzumab antibody and hGH0/l (at antibody concentration ⁇ 0.025 nM, P ⁇ 0.05, t-test, same result) It was also confirmed in BT-474 cells (P ⁇ 0.05 at the concentration of antibody concentration ⁇ 0.025 nM, t-test. However, 3E12 antibody killing ability and Trastuzumab antibody and hGHO in MCF-7 cells with low expression of HER2 The /1 antibody is similar. These results show that the 3E12 antibody kills cells with HER2 specificity, and its ability to kill highly expressed cells in HER2 is stronger than that of Trastuzumab antibody and hGHO/1 antibody.
  • SCID mice purchased from Shanghai Slack
  • SCID mice were subcutaneously inoculated with human breast cancer cell line BT-474 highly expressing HER2 at 0 days, and tumor-bearing mice were intraperitoneally injected 0.5, 5 mg when the tumor grew to 0.3 cm 3 . /kg of various anti-HER2 antibodies, 2 times a week, for 3 weeks of continuous treatment.
  • the changes in body weight and tumor size of the mice in each group were observed regularly for a total of 120 days.
  • the anti-tumor therapeutic effect of the anti-HER2 antibody was evaluated.
  • 3E12 antibody is significantly more potent against the growth of HER2-expressing BT-474 breast cancer than Trastuzumab antibody and hGHO (at the dose of 25 mg/kg, 50, 60, 70) , 80, 90, 100, 110, 120 days, P ⁇ 0.05, Mann-Whitney test).

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Description

一种全人源抗 HER2单克隆抗体、 其制备方法及用途 技术领域
本发明涉及生物技术领域， 更具体地，本发明公开了一种全人源单克隆抗 体、 其制备方法及用途。 背景技术
乳腺癌是女性最为常见的恶性肿瘤之一。全世界每年新增 100万以上的乳 腺癌病例， 而且每年近 40万人死于乳腺癌。 近年来， 乳腺癌的发病率在世界 范围内呈明显的上升趋势。无论是在高流行区还是低流行区， 乳腺癌发病率均 以 5~20%的速度上升。 亚洲妇女的乳腺癌发病率的增长趋势已明显高于欧美 国家。 在中国， 乳腺癌在一些城市已发展为女性恶性肿瘤发病的首位。 乳腺癌 通常的治疗手段有手术治疗，放化疗和内分泌治疗等等， 这些常规治疗手段虽 然在很大程度上延长了病人生存期， 然而它们副作用很大， 其疗效难以进一步 提高。肿瘤靶向治疗是近年来兴起的一种新的肿瘤治疗方式， 其中具有代表性 的是抗肿瘤单克隆抗体。
HER2 (人表皮生长因子受体 2, human epidermal growth factor receptor 2 ) 是一种具有酪氨酸激酶活性的跨膜蛋白， 分子量约为 185 KD。 抗 HER2人源 化单克隆抗体可以特异性的和 HER2结合， 其抗肿瘤机制包括以下几点： 特异 结合于 HER2 受体胞外段从而阻断 HER2 同源二聚体的组成性激活并干扰 HER2与其它 ErbB家族成员形成异源二聚体。 介导 HER2受体的内吞和在溶 酶体中的降解； 活化 PTEN (与 tensin同源的磷酸酯酶）阻断 PI3K (磷酸肌醇 3-激酶）信号通路； 调节细胞周期而抑制肿瘤细胞增殖； 促进肿瘸细胞凋亡； 抗肿瘤血管生成； ADCC (抗体依赖细胞介导的细胞毒性作用）作用； 抑制 DNA修复； 增加化疗药物的细胞毒性； 逆转肿瘤细胞对宿主细胞因子杀伤作 用 ό々4氐抗等 ( Pergram M， Ngo D, Application and potential limitations of animal models utilized in the development of trastuzumab (Herceptin®): A case study. Adv Drug Deliv Rev. 2006;58:723-34. )„
抗 HER2人源化单克隆抗体（Trastuzumab, 商品名 Herceptin ) 已被用于 临床试验，作为单药治疗 HER2过度表达的转移性乳腺癌， 这些患者曾接受过 针对其转移灶 1个或 1个以上化疗方案化疗而失败。本药在临床试验中还与紫 杉醇或蒽环类药物（阿霉素或表阿霉素）加环磷酰胺合用作为一线药物， 治疗
HER2过度表达的转移性乳腺癌 （ Merlin JL, Barberi-Heyob M, Bachmann N， In vitro comparative evaluation of trastuzumab (Herceptin) combined with paclitaxel (Taxol) or docetaxel (Taxotere) in HER2-expressing human breast cancer cell lines. Ann Oncol. 2002;13:1743-8. )„ 但 Trastuzumab是一个人源化抗体， 保留了鼠源 CDR区和少量 FR区鼠源残基，仍未能达到全人源化，且存在着亲和力不高的 问题。 发明内容
本发明构建了大容量的天然人源噬菌体抗体库， 从中筛选获得了一株全人 源抗 HER2抗体 3E12。
更具体地， 本发明提供全人源抗 HER2抗体， 其重链可变区氨基酸序列为 SEQ ID NO:6所示， 轻链可变区氨基酸序列为 SEQ ID NO:8所示；
本发明提供的上述全人源抗 HER2抗体， 其重链氨基酸序列为
SEQ ID NO:10所示， 轻链氨基酸序列为 SEQ ID NO:12所示；
本发明还提供了一种分离的核苷酸， 其编码上述的全人源抗 HER2抗体； 本发明提供的上述核苷酸，其中编码全人源抗 HER2抗体重链可变区的核 苷酸序列为 SEQ ID NO: 5所示， 编码全人源抗 HER2抗体轻链可变区的核苷 酸序列为 SEQ ID NO: 7所示；
本发明提供的上述核苷酸，其中编码全人源抗 HER2抗体重链的核苷酸序 列为 SEQ ID NO:9所示，编码全人源抗 HER2抗体轻链的核苷酸序列为 SEQ ID NO: 11所示；
本发明还提供一种表达载体， 含有上述的核苷酸， 为 pcDNA3.1/ZEO(+) 或 pcDNA3.1 (+);
本发明还公开了上述表达载体转化的宿主细胞， 为 CHO-K1 细胞； 本发明进一步提供上述全人源抗体的制备方法，包括从噬菌体人源抗体库 筛选获得高亲和力全人源抗 HER2单链抗体；全人源抗 HER2完整抗体分子真 核表达载体的构建； 全人源抗 HER2完整抗体分子在 CHO细胞中的表达； 全 人源抗 HER2完整抗体分子的纯化。 本发明最后公开的上述的全人源抗体在制备治疗肿瘤药物中的用途，所述 的肿瘤为 HER2高表达肿瘤， 更具体的为乳腺癌。
本发明利用得到的抗体进行了一系列实验， 实验结果表明： 与人源化抗体 Trastuzumab ( rhumAb 4D5 )和中国专利申请号 01132225. X申请日 2001年 11 月 16日发明名称为 《人源化抗 HER2单克隆抗体及其制法和药物组合物》 中 公开的人源化抗体 hGHO/1相比， 3E12具有更高的抗体亲和力， 更强的 HER2 高表达乳腺癌细胞增殖抑制作用、 细胞凋亡诱导活性； 体内抗肿瘤实验结果表 明， 本发明得到的抗体可以明显抑制肿瘤的增长。 附图说明
图 1. 抗 HER2抗体的凋亡实睑结果（其中图 1-1为 SK-BR3细胞， 图 1-2为 BT-474细胞， 图 1-3为 MCF-7细胞 );
图 2. 抗 HER2抗体的的生长抑制实验结果（其中图 2-1为 SK-BR3细胞， 图 2-2为 BT-474细胞， 图 2-3为 MCF-7细胞）；
图 3. 抗 HER2抗体的体内抗肿瘤实验结果。 具体实施方式
以下实施例、 实验例仅仅对本发明进行进一步的说明， 不应理解为对本发 明的限制。
实施例 抗体的制备
( 1 )人抗体轻、 重链恒定区基因的克隆
用淋巴细胞分离液（鼎国生物技术发展公司产品）分离健康人淋巴细胞， 用 Trizol试剂 (Invitrogen公司产品)提取总 RNA， 根据文献（ Cdl,1980,22: 197-207 )和文献（Nucleic Acids Research, 1982, 10: 4071-4079 )报道的序 列分别设计引物采用 RT-PCR反应扩增抗体重链和轻链恒定区基因。 PCR产物 经琼脂糖凝胶电泳纯化回收并克隆到 pGEM-T载体（ Promega公司产品） 中 , 测序验证后确认获得了正确的克隆。 SEQ ID NO:l和 SEQ ID NO:2分别显示了 重链恒定区（CH )的核苷酸序列和氨基酸序列。 SEQ ID NO:3和 SEQ ID NO:4 分别显示了轻链恒定区 (QJ的核苷酸序列和氨基酸序列。 本例中的正确克隆记 作 pGEM-T/CH和 pGEM-T/C^ (2) cDNA的制备
收集 50健康人的外周血各 20ml, 混合， 用淋巴细胞分离液 (医科院天津 血研所生产)分离单个核细胞。 用 Trizol试剂 （ Invitrogen公司）从分离的人外 周血淋巴细胞中提取细胞的总 RNA。 用 cDNA反转录试剂盒（上海申能博彩 生物科技有限公司）反转录出 cDNA。 以上步骤按照厂家提供的说明书进行。
(3)引物设计
参考文献（Immunotechnology, 1998,3:271-278 )设计并合成克隆人抗体重 链可变区（VH)和轻链可变区 (VL)基因的 VHBack, VHFor, VLBack和 ViJor引 物 。 VHBack , VHFor ， VLBack 和 VJFor 的 序 列 见 Immunotechnology, 1998,3 :271-278。 其中在 VHBack引物的 5，端加上含有 Sfi I 位, *的序歹l atg gcc cag ccg gcc atg gcc, 在 VHFor引物的 5，端力口上序歹1 J gcc aga acc acc gcc gcc gga gcc acc acc gcc,在 VLBack引物 ό 5，端力口上序歹1 J tec ggc ggc ggt ggt tct ggc gga ggc gga tct, 在 VLFor引物的 5，端力口上含有 Not I位点的序歹1 J atg egg ccg c。
( 4 )噬菌体抗体库的构建及筛选
采用 （2)中的 cDNA 和 （3)中的引物， 利用 recombinant Phage antibody system试剂盒（ Amersham Biosciences公司 )构建^：菌体单链抗体库， 然后用 特异性抗原对文库进行淘选。 抗体库构建和淘选方法参照 recombinant Phage antibody system试剂盒说明书进行， 用于淘选的特异性抗原 "人 HER2膜外区 蛋白，，按照参考文献（ Proc Natl Acad Sci USA, 1992,89: 4285-4289 )的方法制备。 经多次淘选抗体库后获得了一株抗人 HER2单链抗体 3E12ScFv， 测序后获得 其基因序列。 SEQ ID NO:5和 SEQ ID NO:6分别显示了 3E12 ScFv重链可变区 VH的核苷酸序列和氨基酸序列。 SEQ ID NO:7和 SEQ ID NO:8分别显示了 3E12 ScFv轻链可变区 VL的核苷酸序列和氨基酸序列。
( 5 )全人源抗体在真核细胞中的表达
以 3E12ScFv基因和 pGEM-T/CH为模版， 通过重叠 PCR合成全人源抗体重 链基因， 反应条件为： 95°C 15分钟； 94 °C 50秒， 58°C50秒， 72°C 50秒， 30 个循环； 72°C 10分钟。 并使此全人源抗体重链基因的 5'端含有限制酶位点 Hindlll和信号肽基因序列， 3 '端含有翻译终止密码 TAA和限制酶位点
EcoR l 。 信 号 肽 基 因 序 列 为 GTCATAATATCCAGAGGA )„ 最后琼脂糖凝胶电泳分离 PCR扩增产物， 回收 目的条带并克隆到 pGEM-T载体（Promega公司产品）中， 筛选阳性克隆测序。 挑选测序正确的克隆用 Hindlll和 EcoR l酶切， 经琼脂糖凝胶电泳纯化回收全 人源抗体重链片段 3E12VHCH, 与用 Hindlll和 EcoR I酶切的质粒 pcDNA3.1(+) ( Invitrogen公司）进行连接， 构建成全人源重链真核表达载体 pcDNA3.1 (+)
(3E12VHCH)。
以 3E12ScFv基因和 pGEM-T/CL载体为模板， 通过重叠 PCR合成全人源 化抗体轻链基因， 反应条件为： 95°C 15分钟； 94°C 50秒， 58°C50秒， 72°C 50秒， 30个循环； 72 °C 10分钟，得到 PCR产物，其 5'端含有限制酶位点 Hindlll 和信号肽基因序列， 3 '端含有翻译终止密码 TAA和限制酶位点 EcoR I。 信号 肽基因序列为
GTCATAATATCCAGAGGA )。挑选测序正确的克隆用 Hindlll和 EcoR I酶切， 经琼脂糖凝胶电泳纯化回收全人源抗体轻链片段
Figure imgf000006_0001
与用 Hindlll和 EcoR l酶切的质粒 pcDNA3.1/ZEO(+) ( Invitrogen公司）载体进行连接， 构建 成全人源轻链真核表达载体 pcDNA3.1/ZEO(+) (3E12VLCL)。
于 3.5cm组织培养亚中接种 3 105 CHO-K1 细胞
( ATCC CRL-9618 ) , 细胞培养至 90%-95%融合时进行转染： 取质粒 lO g (质 粒 pcDNA3.1(+)(3E12VHCH)4 g, 质粒 pcDNA3.1/ZEO(+) (3E12VLCL)6 g)和 20μ1 Lipofectamine2000 Reagent ( Invitrogen公司产品）按 Lipofectamine2000 Reagent试剂盒说明书进行转染。 转染进行 24h后细胞换含 60(^g/ml G418
( Invitrogen公司产品）和 25(^g/ml Zeocin ( Invitrogen公司产品） 的 DMEM 培养基筛选抗性克隆。 取细胞培养上清用 ELISA检测筛选高表达克隆： 羊抗 人 IgG (Fc) ( KPL公司） 包被于 ELISA板， 4°C过夜，
用 2 % BSA-PBS于 37。C封闭 2h， 加入待测的抗性克隆培养上清或标准品 Human myeloma IgGl,K ( Sigma), 37。C 温育 2h, 加入 HRP -羊抗人 IgG(K)
( (Southern Biotechnology Associates公司）进行结合反应， 37。C 温育 lh， 力口 入 TMB显色液于 37。C作用 5min， 最后用 H2S04终止反应， 测 A45。值。 将筛 选得到的高表达克隆用无血清培养基扩大培养， 用 Protein A亲和柱（ GE公司 产品）分离纯化全人源抗体 3E12。 将纯化抗体用 PBS进行透析， 最后以紫外 吸收法定量。 SEQ ID NO:9和 SEQ ID NO: 10分别显示了全人源抗体 3E12的 重链核苷酸序列和氨基酸序列。 SEQ ID NO: 11和 SEQ ID NO: 12分别显示了全 人源抗体 3E12的轻链核苷酸序列和氨基酸序列。
实猃例
hGHO/1按中国专利申请号 01132225.X申请日 2001年 11月 16日发明名 称为《人源化抗 HER2单克隆抗体及其制法和药物组合物》中公开的方法制备 得到。
抗 HER2抗体的凋亡实验
将人乳腺癌细胞 SK-BR-3 ( HER2高表达， ATCC号： HTB-30)、 BT-474 ( HER2中表达， ATCC号： HTB-20 )及 MCF-7 ( HER2低表达， ATCC号： HTB-22 )分别与不同稀释度的抗 HER2抗体 （包括 Trastuzumab, hGHO/1 , 3E12 )在 37°C培养 20小时， 洗细胞后用 AnnexinV/PI试剂盒（ BD公司产 品）按照产品说明书检测早期凋亡细胞百分率。抗凋亡实验如图 1所示： 3E12 抗体杀伤 SK-BR3细胞的能力明显强于 Trastuzumab抗体和 hGH0/l(在抗体 浓度≥0.025 nM的浓度的时候 , P<0.05， t检验， 同样的结果也在 BT-474细 胞中得到证实（在抗体浓度≥0.025 nM的浓度的时候， P<0.05， t检验。然而， 在 HER2低表达的 MCF-7细胞中， 3E12抗体杀伤能力与 Trastuzumab抗体 和 hGHO/1抗体差不多。 这些结果显示了 3E12抗体杀伤细胞的 HER2特异 性， 且其杀伤 HER2中高表达的细胞的能力要强于 Trastuzumab抗体和 hGHO/1抗体。
抗 HER2抗体的的细胞生长抑制实验
将人乳腺癌细胞 SK-BR-3、 BT-474及 MCF-7细胞分别与不同稀释度的 抗 HER2抗体在 37°C进行孵育， 第五日用 MTT染色法染色读数后计算生长 抑制率。 生长抑制实验如图 2所示： 3E12抗体对 SK-BR3细胞的生长抑制 能力明显强于 Trastuzumab抗体和 hGHO/1 (在抗体浓度≥0.1 nM的浓度的时 候， P<0.05， t检验） ， 同样的结果也在 BT-474细胞中得到证实 （在抗体浓 度≥0.1 nM的浓度的时候， PO.05, t检验）。然而，在 HER2 氐表达的 MCF-7 细胞中， 3E12抗体对细胞的抑制能力与 Trastuzumab抗体和 hGHO/1抗体差 不多。 这些结果显示了 3E12抗体对细胞生长抑制的的 HER2特异性， 且其 抑制 HER2中高表达的细胞的能力要强于 Trastuzumab抗体和 hGHO/1抗体。
抗 HER2抗体的体内抗肿瘤实验
SCID小鼠 （购自上海斯莱克公司） 于 0天时分别皮下接种高度表达表 达 HER2的人乳腺癌细胞 BT-474, 待肿瘤长至 0.3 cm3时给荷瘤小鼠分别腹 腔注射 0.5， 5 mg/kg的各种抗 HER2抗体， 每周 2次， 连续治疗 3周。 定期 观测各组小鼠体重和肿瘤大小的变化， 共观察 120天。 评价抗 HER2抗体的 抗肿瘤治疗效果。 体内抗肿瘤实验如图 3所示： 3E12抗体对高表达 HER2 的 BT-474乳腺癌的的生长抑制能力明显强于 Trastuzumab抗体和 hGHO (在 25 mg/kg的剂量时， 第 50、 60、 70、 80、 90、 100、 110、 120天的时 候， P<0.05, Mann- Whitney检验） 。

Claims

权利要求书
1. 一种全人源抗 HER2抗体， 其具有如 SEQ ID NO:6所示的重链可变区 氨基酸序列， 以及如 SEQ ID NO:8所示的轻链可变区氨基酸序列。
2. 权利要求 1所述的全人源抗 HER2抗体， 其具有如 SEQ ID NO: 10所 示的重链氨基酸序列， 以及如 SEQ ID NO: 12所示的轻链氨基酸序列。
3. 一种分离的核苷酸， 其编码权利要求 1〜2任一所述的全人源抗 HER2 抗体。
4. 权利要求 3所述的核苷酸， 其具有如 SEQ ID NO:5所示的编码全人源 抗 HER2抗体重链可变区的核苷酸序列， 以及如 SEQ ID NO:7所示的编码全 人源抗 HER2抗体轻链可变区的核苷酸序列。
5. 权利要求 4所述的核苷酸， 其具有如 SEQ ID NO:9所示的编码全人源 抗 HER2抗体重链的核苷酸序列， 以及如 SEQ ID NO: 11所示的编码全人源抗 HER2抗体轻链的核苷酸序列。
6. 一种表达载体， 含有权利要求 3~5 任一所述的核苷酸， 为 pcDNA3.1/ZEO(+)或 pcDNA3.1 (+)。
7. 一种转化权利要求 6所述的表达载体的宿主细胞， 为 CHO-K1 细胞。
8. 权利要求 1〜2任一所述的全人源抗 HER2抗体在制备治疗肿瘤药物中 的用途。
9. 权利要求 8所述的用途， 其中肿瘤为 HER2高表达肿瘤。
10. 权利要求 9所述的用途， 其中 HER2高表达肿瘤可为乳腺肿瘤。
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RU2628094C2 (ru) * 2013-05-16 2017-08-14 Абклон Инк. Антитела, способные специфически связываться с her2
EP4269432A1 (en) 2022-04-26 2023-11-01 Universite de Rouen Normandie Production of therapeutic antibodies by the microalgae phaeodactylum tricornutum
WO2023208883A1 (en) 2022-04-26 2023-11-02 Universite De Rouen Normandie Production of therapeutic antibodies by the microalgae phaeodactylum tricornutum

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US8974785B2 (en) 2015-03-10
CN102167742B (zh) 2014-05-14
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JP2013520180A (ja) 2013-06-06
EP2540745A9 (en) 2013-02-27
CA2790007A1 (en) 2011-09-01
CN102167742A (zh) 2011-08-31
EP2540745B1 (en) 2015-09-30
EP2540745A1 (en) 2013-01-02
BR112012021327B1 (pt) 2020-05-05
BR112012021327A2 (pt) 2017-06-20
US20120309942A1 (en) 2012-12-06
EP2540745A4 (en) 2013-09-11
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