WO2011103702A1 - 一种全人源抗vegf单克隆抗体、其制备方法及用途 - Google Patents

一种全人源抗vegf单克隆抗体、其制备方法及用途 Download PDF

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WO2011103702A1
WO2011103702A1 PCT/CN2010/000513 CN2010000513W WO2011103702A1 WO 2011103702 A1 WO2011103702 A1 WO 2011103702A1 CN 2010000513 W CN2010000513 W CN 2010000513W WO 2011103702 A1 WO2011103702 A1 WO 2011103702A1
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vegf
antibody
seq
fully human
human anti
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PCT/CN2010/000513
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English (en)
French (fr)
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王淑蕙
李川
阚颖
仝昕
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百迈博药业有限公司
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Priority to JP2012554187A priority Critical patent/JP2013520182A/ja
Priority to EP10846326.6A priority patent/EP2540744A4/en
Priority to US13/579,218 priority patent/US8664368B2/en
Priority to BR112012021326A priority patent/BR112012021326B8/pt
Priority to CA2790014A priority patent/CA2790014C/en
Publication of WO2011103702A1 publication Critical patent/WO2011103702A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • 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
    • 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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

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
  • Tumors are diseases that seriously endanger human health in the world today, and rank second in the deaths caused by various diseases. Moreover, in recent years, its incidence has shown a clear upward trend. The treatment of malignant tumors is poor, the rate of late metastasis is high, and the prognosis is poor.
  • the conventional treatment methods such as radiotherapy, chemotherapy and surgical treatment have alleviated the pain and prolonged the survival time to a large extent, but these methods have four limitations, and the curative effect is difficult to further improve.
  • VEGF Vascular endothelial growth factor
  • tumor cells In tumor tissues, tumor cells, tumor-invading macrophages and mast cells can secrete high levels of VEGF, stimulate tumor vascular endothelial cells in a paracrine manner, promote endothelial cell proliferation and migration, induce angiogenesis, and promote tumor growth. And improve vascular permeability, causing fibrin deposition in surrounding tissues, promoting invasion of monocyte and fibroblast endothelial cells, facilitating tumor matrix formation and tumor cells entering neovascularization, promoting tumor metastasis, thereby inhibiting tumor angiogenesis It is considered to be one of the most promising cancer treatment methods.
  • Bevacizumab (Avastin, bevacizumab) is a humanized antibody against human VEGF (Cancer Res 1997; 57:4593-9), which was approved by the US Food and Drug Administration as a first-line treatment for metastatic colorectal cancer in 2004. drug.
  • Bevacizumab is a humanized antibody that retains the murine CDR region and a small number of FR region murine residues, but still fails to achieve full humanization, and there is a problem of low affinity. Summary of the invention
  • the invention constructs a large-capacity natural human phage antibody library, and obtains a whole person from the selected one.
  • the present invention provides a fully human anti-VEGF antibody, wherein the heavy chain variable region amino acid sequence is represented by SEQ ID NO: 6, and the light chain variable region amino acid sequence is represented by SEQ ID NO: 8;
  • the whole human anti-VEGF antibody of the invention wherein the heavy chain amino acid sequence is represented by SEQ ID NO: 10, and 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-VEGF antibody; the nucleotide of the present invention, wherein the nucleotide sequence encoding the heavy chain variable region of the fully human anti-VEGF antibody SEQ ID NO: 5, the nucleotide sequence encoding the light chain variable region of the fully human anti-VEGF antibody is shown in SEQ ID NO: 7.
  • nucleotide of the present invention wherein the nucleotide sequence encoding the heavy chain of the fully human anti-VEGF antibody is represented by SEQ ID NO: 9, and the nucleotide sequence encoding the light chain of the fully human anti-VEGF 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 present invention also provides a host cell transformed with the above expression vector, which is a CHO-K1 cell; the present invention further provides a preparation method of the above-mentioned whole human anti-VEGF antibody, which comprises screening a phage human antibody library to obtain a high affinity full human anti-VEGF.
  • Single-chain antibody construction of a fully human anti-VEGF intact antibody molecule eukaryotic expression vector; expression of a fully human anti-VEGF intact antibody molecule in CHO cells; purification of a fully human anti-VEGF intact antibody molecule.
  • the tumor is colorectal cancer.
  • the present invention utilizes the obtained antibodies to carry out a series of experiments, and the experimental results show that: with the humanized antibody bevacizumab and the human anti-VEGF antibody 6A6 (according to Chinese Patent Application No. 02111093.X, the application date is March 20, 2002, the name of the invention Compared with the method disclosed in the method disclosed in "Humanized anti-vascular endothelial growth factor monoclonal antibody and its preparation method and pharmaceutical composition", the antibody obtained by the invention has higher antibody affinity and more strongly inhibits tumor cells. The ability to proliferate; in vivo anti-tumor experiments show that the antibodies obtained by the present invention can significantly inhibit the growth of tumors.
  • DRAWINGS the humanized antibody bevacizumab and the human anti-VEGF antibody 6A6
  • 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 ⁇ : 1 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 nucleotide sequence and amino acid sequence of the light chain constant region (( ).
  • the correct clones in this example are denoted as pGEM-T/C H and pGEM-T/C L.
  • RNA from the cells was extracted from the 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.
  • V H Back V H For
  • V L Back and VJor primers for cloning human antibody heavy chain variable region (V H ) and light chain variable region genes by reference Immunotechnology, 1998, 3 : 271 -278 .
  • the sequences of V H Back , V H For , V L Back and V L For are described in Immunotechnology, 1998, 3:271-278.
  • a phage 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 method reference
  • the specific antigen "recombinant human VEGF165 protein" (purchased from R&D) was used for panning by the recombinant Phage antibody system kit. After multiple panning of the antibody library, an anti-human VEGF single-chain antibody 11A7SCFV was obtained, and the gene sequence was obtained after sequencing.
  • SEQ ID NO: 5 and SEQ ID NO: 6 show the nucleotide and amino acid sequences V H of the heavy chain variable region 11A7 ScFv.
  • SEQ ID NO: 7 and SEQ ID NO: 8 L respectively show the nucleotide and amino acid sequences of the light chain variable region of 11A7 ScFv V.
  • the full-human antibody heavy chain gene was synthesized by overlapping PCR using the 11 A7ScFv gene and pGEM-T/C H as the template.
  • the reaction conditions were: 95 ° C for 15 minutes; 94 ° C for 50 seconds, 58 ⁇ 50 seconds, 72 ° C for 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 full humanized antibody light chain gene was synthesized by overlapping PCR using the 11A7SCFV gene and the pGEM-T/C L vector as a template.
  • the reaction conditions were: 95 ° C for 15 minutes; 94 ° 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 codon TAA and the restriction enzyme site EcoR l at the 3' end.
  • the signal peptide gene sequence is
  • the correct clones were selected and digested with Hindlll and EcoR I.
  • the full human antibody light chain fragment 11A7V L C L was purified by agarose gel electrophoresis and ligated with the plasmid pcDNA3.1/ZEO(+) (Invitrogen) vector digested with Hindlll and EcoR l to construct a human source.
  • the light chain eukaryotic expression vector pcDNA3.1/ZEO(+) (11A7V L C L ).
  • the purified antibody was dialyzed against PBS and finally quantified by ultraviolet absorption.
  • SEQ ID NO: 9 and SEQ ID NO: 10 respectively.
  • SEQ ID NO: 11 and SEQ ID NO: 12 show the light chain nucleotide sequence and amino acid sequence of the fully human antibody 11A7, respectively.
  • Human antibody 6A6 Prepared according to the method disclosed in the invented name "Humanized anti-vascular endothelial growth factor monoclonal antibody, preparation method and pharmaceutical composition thereof" of Chinese Patent Application No. 02111093.X, dated February 30, 2002
  • VEGF165 product of R&D was covalently bound to the CM5 biosensor chip (GE Healthcare) by amino acid, and fully human antibody 11A7, Bevacizumab, human antibody 6A6 (according to Chinese Patent Application No. 02111093.X Application Date 2002) The method disclosed in the method of "humanized anti-vascular endothelial growth factor monoclonal antibody, its preparation method and pharmaceutical composition" was prepared on February 30.
  • a negative control antibody (commercially available from Rituximab) (with PBS/0.05% TWEEN-20 (ICI Americas) (detergent)), formulated in different concentrations (2 times diluted than concentration), with 50 ⁇ 1/ ⁇ The flow rate is passed through the chip. After each examination, the residual antibody was eluted from the immobilized ligand by washing with a 5 ⁇ l 50 mM aqueous hydrochloric acid solution at a flow rate of 3 ⁇ l to ⁇ . Binding curves were analyzed by nonlinear regression using BIAevalution software (T100 evalution version 2.0, GE Healthcare). The results are shown in Table 1.
  • the KD value of the fully human antibody 11A7 was significantly lower than that of Bevacizumab and human antibody 6A6, indicating that the affinity of the fully human antibody 11A7 to VEGF is higher than that of Bevacizumab and human antibody 6A6.
  • the affinity test results are shown in Table 1. .
  • TdR (18.5 kBq/well) was incubated for 7 h in a 37 °C incubator.
  • the cell harvester collected cells in glass fiber. On the filter, the measurement was carried out using a [3H] liquid scintillation counter. As shown in Figure 1.
  • LM3 cells human liver cancer cells, from the Institute of Liver Cancer, Fudan University Medical College, Shanghai, China
  • VEGF human liver cancer cells
  • Center subcutaneously on the right flank
  • VEGF on the day of tumor inoculation
  • the antibody 25 mg/kg and the unrelated control protein Rituximab were subcutaneously injected every other day for 4 weeks. After 6 weeks, the length and width of the tumor were measured every 3 days, and the tumor volume was calculated. The results are shown in Fig. 2.

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Description

一种全人源抗 VEGF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
肿瘤尤其是恶性肿瘤是当今世界严重危害人类健康的疾病，在各种疾病所 致死亡中高居第二位。 而且近年来， 其发病率呈明显上升趋势。 恶性肿瘤治疗 效果差，晚期转移率高，预后多不佳。 目前临床上所采用的常规治疗方法如放、 化疗和手术治疗虽然在很大程度上緩解了病痛，延长了生存时间，但这些方法 均存在 4艮大的局限性， 其疗效难以进一步提高。
肿瘤的生长有两个明显不同的阶段，即从无血管的緩慢生长期转为有血管 的快速增殖期，血管的生成使肿瘤能获得足够的营养而完成血管切换期，如果 没有血管的生成， 原发肿瘤的生长不超过 l-2mm, 转移则无法实现。 血管内 皮生长因子 (VEGF)是一类能促进内皮细胞分裂增殖、 促进新生血管的形成、 提高血管通透性的生长因子， 它与细胞表面的血管内皮生长因子受体结合，通 过激活酪氨酸激酶信号转导途径发挥功能。 在肿瘤组织中，肿瘤细胞、 肿瘤侵 入的巨噬细胞和肥大细胞能分泌高水平的 VEGF,以旁分泌的形式刺激肿瘤血 管内皮细胞,促进内皮细胞增殖、迁移,诱导血管形成，促进肿瘤持续生长，并提高 血管通透性,引起周围组织纤维蛋白沉着，促进单核细胞、 成纤维细胞内皮细胞 侵润,有利于肿瘤基质形成和肿瘤细胞进入新生血管,促进肿瘤转移， 因而抑制 肿瘤血管生成被认为是当前最具有前途的肿瘤治疗方法之一。
贝伐单抗（ Avastin, bevacizumab )是抗人 VEGF的人源化抗体（ Cancer Res 1997;57:4593-9 ), 它于 2004年被美国食品药品管理局批准为转移性结直肠癌 的一线治疗药物。 但 Bevacizumab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 选获得了一株全人 源抗 VEGF抗体 11A7。
更具体地， 本发明提供一种全人源抗 VEGF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； 本发明所述的全人源抗 VEGF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；
本发明还提供一种分离的核苷酸， 其编码上述的全人源抗 VEGF抗体； 本发明所述的核苷酸， 其中编码全人源抗 VEGF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 VEGF抗体轻链可变区的核苷酸 序列为 SEQ ID NO:7所示；
本发明所述的核苷酸， 其中编码全人源抗 VEGF抗体重链的核苷酸序列 为 SEQ ID NO:9所示， 编码全人源抗 VEGF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 (+);
本发明还提供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 VEGF 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 VEGF单链抗体； 全人源抗 VEGF完 整抗体分子真核表达载体的构建； 全人源抗 VEGF完整抗体分子在 CHO细胞 中的表达； 全人源抗 VEGF完整抗体分子的纯化。 其中肿瘤为结直肠癌。
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 bevacizumab和人源抗 VEGF抗体 6A6 (按照中国专利申请号 02111093.X, 申 请日 2002年 3月 20日，发明名称为《人源化抗血管内皮生长因子单克隆抗体 及其制法和药物组合物》中公开的方法制备得到）相比， 本发明得到的抗体具 有更高的抗体亲和力， 更强地抑制肿瘤细胞增殖的能力； 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 , 细胞增殖实验结果； 图 2， 肿瘤生长曲线图。 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（ Cell,1980，22: 197-207 )和文献（Nucleic Acids Research, 1982, 10: 4071-4079 )报道的序 列分别设计引物采用 RT-PCR反应扩增抗体重链和轻链恒定区基因。 PCR产物 经琼脂糖凝胶电泳纯化回收并克隆到 pGEM-T载体（ Promega公司产品） 中， 测序验证后确认获得了正确的克隆。 SEQ ID ΝΟ:1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 (( )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/CL。
(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 基因的 VHBack, VHFor, VLBack和 VJor引 物 。 VHBack , VHFor ， VLBack 和 VLFor 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 序歹1 J atg gcc cag ccg gcc atg gcc, 在 VHFor引物 ό 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，耑力口上序歹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 'J atgcggccgCo ( 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）构建噬菌体单链抗体库， 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 "重组人 VEGF165蛋白"（购自 R&D公司）。 经多次淘选抗体库后获得了一株 抗人 VEGF单链抗体 11A7SCFV,测序后获得其基因序列。 SEQ ID NO:5和 SEQ ID NO:6分别显示了 11A7 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 11A7 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 11 A7ScFv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， 58Ό50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 11A7VHCH, 与用 Hindlll和 EcoR l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 (+)
Figure imgf000005_0001
以 11A7SCFV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； 94Ό 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 l。 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 11A7VLCL, 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(+) (11A7VLCL)。
于 3.5cm组织培养 JDL中接种 3 x 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 10 g(质粒 pcDNA3.1(+)(l lA7VHCH)4 g， 质粒 pcDNA3.1/ZEO(+) (l lA7VLCL)6 g)和 20μ1 Lipofectamine2000 Reagenti 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 600 g/ml G418 ( Invitrogen公司产品）和 250 g/ml Zeocin ( Invitrogen公司产品）的 DMEM 培养基 选抗性克隆。 取细胞培养上清用 ELISA检测薛选高表达克隆： 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, 加入 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 11A7。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 11A7的重链核苷酸序列和氨基酸序 列。 SEQ ID NO:ll和 SEQ ID NO: 12分别显示了全人源抗体 11A7的轻链核苷 酸序列和氨基酸序列。
实验例
人源抗体 6A6: 按照中国专利申请号 02111093.X申请日 2002年 2月 30 日发明名称《人源化抗血管内皮生长因子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
实验例 1.VEGF抗体亲和力测定
运用 Biacore T100(Biacore AB, Uppsala, Sweden)检测 VEGF抗体的亲和力 常数。 将 VEGF165(R&D公司产品)通过氨基共价结合与 CM5生物传感芯片 (GE Healthcare)上， 将全人源抗体 11A7， Bevacizumab, 人源抗体 6A6 (按照 中国专利申请号 02111093.X申请日 2002年 2月 30日发明名称 《人源化抗血 管内皮生长因子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） 和阴性对照抗体 （ Rituximab 市售产品） （以 PBS/0.05%TWEEN-20(ICI Americas) (去污剂）配）溶液， 配成不同的浓度 (2倍比浓度稀释)， 以 50μ1/ιηίη 的流速通过芯片。 每次检查之后， 用 5μ1 50mM盐酸水溶液以 3μ1/ιηίη的流速 洗涤， 从而将残留的抗体从固定化的配体上洗脱下来。 用 BIAevalution软件 ( T100 evalution 2.0版， GE Healthcare公司）， 通过非线性回归法分析结合曲 线。 结果如表 1所示， 全人源抗体 11A7的 KD值显著低于 Bevacizumab和人 源抗体 6A6， 说明全人源抗体 11A7对 VEGF的亲和力高于 Bevacizumab和人 源抗体 6A6， 亲和力实验结果见表 1。
表 1 亲和力实验结果
抗体 Kon ( M-'S-'/lO4 ) Koff ( lO4^1 ) KD ( nM )
11A7 8.24 0.71 0.86
Bevacizumab 5.12 1.03 2.01
6A6 4.19 0.95 2.27
Rituximab ND ND ND 实验例 2.VEGF抗体抑制 HUVEC细胞增殖实验
实验步糠： 取生长状态良好的 HUVEC细胞（ Cascade Biologies ), 调整细 胞浓度为 2.5xl04/ml, 接种于 96孔细胞培养板， 200μ1/孔， 于 37°C、 5% C02 孵箱中培养 24 h后换无血清培养基， 继续培养 72 h， 加入不同浓度梯度的 VEGF抗体,以抗 CD20抗体 Rituximab为阴性对照， 每个浓度取 3个平行孔， 37°C孵育 1 h, 加入终浓度为 25 ng/ml的 VEGF165 ( R&D )继续培养 24 h后， 加入 10μ1[3 Η]. TdR(18.5 kBq/孔)， 37°C孵箱中孵育 7 h, 细胞收集器收集细胞 于玻璃纤维滤膜上， 釆用 [3H]液闪计数仪进行测定。 如图 1所示。
结果表明：阴性对照抗体（Rituximab )不能有效抑制 VEGF引起的 HUVEC 细胞增殖，而全人源抗体 11A7, Bevacizumab,人源抗体 6A6 (均可有效抑制 VEGF引起的 HUVEC细胞增殖。全人源抗体 11A7抑制 VEGF引起的 HUVEC 细胞增殖的活性明显强于 Bevacizumab和人源抗体 6A6 (尸<0.05， t检验， 抗 体浓度范围为 0.4-3.2 nM )o
实验例 2.VEGF抗体体内抑制肿瘤生长实验
实验步骤： 为检测 VEGF抗体体内抑瘤活性,首先用 LM3细胞 (人肝癌细 胞， 来自复旦大学医学院肝癌研究所， 中国上海），接种于到雌性重症免疫缺 陷小鼠（第二军医大学实验动物中心）右胁侧皮下,接种肿瘤当天给予各 VEGF 抗体 25mg/kg及无关对照蛋白 Rituximab,然后隔天皮下注射持续 4周.6周后， 每 3天测量肿瘤的长宽，计算肿瘤体积，结果如图 2所示。
结果表明： 阴性对照抗体（Rituximab ) 不能有效抑制肿瘤的生长,而全人 源抗体 11A7, Bevacizumab，人源抗体 6A6 (均可有效抑制重症免疫缺陷小鼠接 种肿瘤的生长， 且从第 18天起， 全人源抗体 11A7对肿瘤生长的抑制作用较 Bevacizumab和人源抗体 6A6强， 有显著差异（ P<0.05， Mann- Whitney检验, 当观察时间大于 35天以后）。

Claims

权利要求书
1. 一种全人源抗 VEGF单克隆抗体， 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 VEGF 单克隆抗体， 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. 编码权利要求 1所述的全人源抗 VEGF单克隆抗体的核苷酸， 其具有 如 SEQ ID NO:5所示的重链可变区的核苷酸序列， 以及如 SEQ ID NO:7所示 的轻链可变区的核苷酸序列。
4. 权利要求 3所述的核苷酸， 其具有如 SEQ ID NO:9所示的重链核苷酸 序列 , 以及如 SEQ ID NO: 11所示的轻链核苷^ 列。
5. 含有权利要求 3或 4所述核苷酸的表达载体， 为 pcDNA3.1/ZEO(+)或 pcDNA3.1 (+)。
6. 含有权利要求 5所述的表达载体的宿主细胞， 为 CHO-K1 细胞。
7. 权利要求 1或 2所述的全人源抗 VEGF单克隆抗体的制备方法， 包括 从噬菌体人源抗体库筛选获得高亲和力全人源抗 VEGF 单链抗体、 全人源抗 VEGF 完整抗体分子真核表达载体的构建、 全人源抗 VEGF 完整抗体分子在 CHO细胞中的表达和全人源抗 VEGF完整抗体分子的纯化四个步骤。
8. 权利要求 1或 2所述的全人源抗 VEGF单克隆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， 其中所述肿瘤为结直肠癌。
PCT/CN2010/000513 2010-02-25 2010-04-16 一种全人源抗vegf单克隆抗体、其制备方法及用途 WO2011103702A1 (zh)

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