CN1187373C - Human resourced monoclone antibody of anti-blood-vessel endothelium growth factor as well as its preparing method and medicine composition - Google Patents

Human resourced monoclone antibody of anti-blood-vessel endothelium growth factor as well as its preparing method and medicine composition Download PDF

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CN1187373C
CN1187373C CNB02111093XA CN02111093A CN1187373C CN 1187373 C CN1187373 C CN 1187373C CN B02111093X A CNB02111093X A CN B02111093XA CN 02111093 A CN02111093 A CN 02111093A CN 1187373 C CN1187373 C CN 1187373C
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monoclonal antibody
variable region
chain variable
sequence
tumor
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CN1445242A (en
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李博华
钱卫珠
刘庆法
王皓
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上海中信国健药业有限公司
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Abstract

本发明提供了一种抗人VEGF单克隆抗体,轻链可变区具有SEQ ID NO:3所示的氨基酸序列,重链可变区具有SEQ ID NO:1所示的氨基酸序列。 The present invention provides an anti-human VEGF monoclonal antibody, the light chain variable region comprising SEQ ID NO: amino acid sequence of the heavy chain variable region shown in Figure 3 having the SEQ ID NO: 1 amino acid sequence. 该单克隆抗体的生物活性和宿主细胞中表达量有显著提高。 And host cells of the biological activity of the monoclonal antibody significantly increased expression. 本发明还提供了编码该抗体的DNA分子、该抗体的制备方法和含有该抗体的药物组合物。 The present invention also provides a DNA molecule encoding the antibody, method for preparing the antibodies and pharmaceutical compositions comprising the antibody.

Description

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 Humanized anti-vascular endothelial growth factor monoclonal antibody preparation method and pharmaceutical compositions

发明领域本发明涉及生物技术领域。 Field of the Invention The present invention relates to the field of biotechnology. 具体地说,本发明涉及一种新的人源化抗血管内皮生长因子(VEGF)单克隆抗体。 More specifically, the present invention relates to a novel humanized anti-growth factor (VEGF) monoclonal antibody vascular endothelium. 另外,本发明还涉及该单克隆抗体的制备方法以及含有该单克隆抗体的药物组合物。 The present invention further relates to a method for preparing the monoclonal antibody and to pharmaceutical composition containing the monoclonal antibody.

发明背景一百多年前人们就已发现,肿瘤组织较正常组织富含血管,但人们一直在争论肿瘤是由已经存在的血管提供营养,还是由新生血管提供营养,并且普遍认为这种血管反应只是一种炎症反应,并非肿瘤生长所必需。 Background of the Invention hundred years ago people had been found in tumor tissue compared with normal tissue rich in blood vessels, but people have been arguing tumor is to provide nutrition by the already existing blood vessels, or angiogenesis provided by the nutrition, and generally believe that vascular reactivity only an inflammatory reaction, is not required for tumor growth. 1971年,Folkman最早提出肿瘤生长是血管依赖性的,但当时这种观点并未为人所接受。 In 1971, Folkman first proposed the growth of tumor blood vessels is dependent, but this view was not accepted by man. 随着8年后毛细血管内皮细胞培养技术的建立,11年后第一个血管生成抑制剂的发现,13年后第一个血管生成活性蛋白的纯化等一系列工作的完成,这一观点为越来越多的证据所支持,并使这一领域成为肿瘤研究的热点及肿瘤治疗的新策略。 With the establishment of capillary endothelial cells in culture techniques 8 years, 11 years after the completion of the first discovery of angiogenesis inhibitors, angiogenesis first 13 years of activity of the protein after purification by a series of work, this view is increasing evidence supports, and this area has become a hot new strategy for cancer therapy and cancer research.

肿瘤的生长有两个明显不同的阶段,即从无血管的缓慢生长阶段转变为有血管的快速增殖阶段,血管生成使肿瘤能够获得足够的营养物质,是促成上述转变的关键环节。 Tumor growth has two distinct stages, i.e., the slow transition from the non-growing stage for rapid proliferation of the vascular vessel stage, tumor angiogenesis to obtain sufficient nutrients, is the key to facilitate the above-described transition. 如果没有血管生成,原发肿瘤的生长不会超过1~2mm3。 Without angiogenesis, the growth of the primary tumor does not exceed 1 ~ 2mm3.

肿瘤侵袭转移是肿瘤治疗失败的主要原因。 Tumor invasion and metastasis is the main cause of cancer treatment failure. 肿瘤侵袭转移是一复杂的多阶段过程,可以概括为:原发瘤增殖、肿瘤新生血管生长;瘤细胞侵袭基底膜;穿入血管或淋巴管;在循环系统中存活,形成瘤栓并转运到远隔靶器官;滞留于靶器官的微小血管中;穿出血管并形成微小转移灶;肿瘤血管形成,转移癌灶增殖。 Tumor invasion and metastasis is a complex multistage process can be summarized as: the primary tumor proliferation, tumor neovascularization; basement membrane of tumor cell invasion; penetrate the blood or lymphatic vessels; survive in the circulatory system, and transported to the tumor thrombus distant target organ; accumulated in the tiny blood vessels in the target organ; blood vessel piercing and forming micrometastases; tumor angiogenesis, proliferation of tumor foci. 可见在肿瘤发生侵袭转移的多步骤过程中,无论肿瘤转移的起始或终末阶段,血管生成均发挥着重要作用。 Visible invasive multi-step process occurs in tumor metastasis, tumor metastasis regardless of starting or end stage, both angiogenesis plays an important role.

肿瘤组织中微血管密度的测定有助于肿瘤的诊断及预后判断,如:乳腺肿瘤微血管密度与其转移性能呈正相关。 Determination of tumor microvessel density contribute to tumor diagnosis and prognosis, such as: microvessel density Metastasis positive correlation properties of breast tumors. 对于淋巴结阴性的乳腺癌患者,多因素分析表明,微血管密度相对于肿瘤的分级、肿瘤大小、雌激素受体阳性或其他标志物来说,是判断乳腺癌转移潜能的更好指标,这一结果已进一步为5年前瞻性研究所证实。 For node-negative breast cancer patients, multivariate analysis showed that microvessel density in relation to tumor grade, tumor size, estrogen receptor-positive or other markers, it is judged better indicator of breast cancer metastatic potential, this result has confirmed a five-year prospective Institute. 此外,其他多种肿瘤微血管密度亦与转移、复发、预后关系密切,如头颈鳞状细胞癌、黑色素瘤、前列腺癌、卵巢癌、膀胱癌、脑瘤、非小细胞肺癌、直肠癌、骨髓瘤等。 In addition, also various other MVD and metastasis, recurrence of, prognosis of, such as head and neck squamous cell carcinoma, melanoma, prostate cancer, ovarian cancer, bladder cancer, brain tumors, non-small cell lung cancer, colorectal cancer, myeloma Wait. 值得注意的是,新生血管的存在并不能用于良性、恶性肿瘤的鉴别,如肾上腺腺瘤属于良性肿瘤,但却是富血管的。 Notably, the presence of neovascularization can not be used to identify benign and malignant tumors, such as benign adrenal adenoma belongs, but it is rich in blood vessels. 由此可见,肿瘤的生长、转移、复发、预后与血管生成密切相关,以肿瘤的血管生成为靶点,开发血管生成抑制剂,有可能使实体瘤的治疗效果得到较大提高。 Thus, tumor growth, metastasis, recurrence, and prognosis of angiogenesis to the tumor a target for angiogenesis, the development of angiogenesis inhibitors, it is possible to make a solid tumor therapeutic effect is greatly improved.

肿瘤血管形成是肿瘤生长、转移的基础(Folkman JN Engl J Med,1971,285(21):1182)。 Tumor angiogenesis is tumor growth, metastasis base (Folkman JN Engl J Med, 1971,285 (21): 1182). 自从Weidner等(Folkman JJ Biol Chem,1992,267(16):10931)报道了肿瘤组织微血管密度(MVD)是乳腺癌患者的一个独立的预后指标以来,在许多肿瘤,包括原发性肝癌中MVD的预后价值都得到了肯定(王东,高奉浔,陈俐等,骨肉瘤微血管密度与预后的研究,中华病理学杂志,1997,26(5):266)。 Since Weidner and other (Folkman JJ Biol Chem, 1992,267 (16): 10931) since reported that tumor microvessel density (MVD) is an independent prognostic indicator in breast cancer patients, many tumors, including hepatocellular carcinoma in MVD the prognostic value have been affirmed (Wang, Feng high Xun, Chen Li, etc., density and prognosis of osteosarcoma capillaries, Chinese Journal of pathology, 1997,26 (5): 266). 肿瘤新生血管的形成是其生长、浸润、转移的重要条件。 Tumor angiogenesis is growth, invasion and metastasis important condition. 实体瘤的生长和转移是依赖于血管生成的,因此干扰和阻断肿瘤血供是有效的治疗方法。 Solid tumor growth and metastasis are dependent on angiogenesis, thus interfering with and blocking tumor blood supply is an effective treatment. 许多研究表明肿瘤内微血管密度(Intratumoral microvessel density,MVD)与骨肉瘤的转移和预后有关(王东等,同上),在多种血管生成相关因子中,血管内皮生长因子(VEGF)持续高表达,这证明VEGF是人体骨肉瘤重要的血管生成相关因子之一。 Many studies have shown that the transfer microvessel density (Intratumoral microvessel density, MVD) in the tumor and osteosarcoma and prognosis (Wang et al., Supra), in a variety of angiogenesis-related factors, vascular endothelial growth factor (VEGF) high expression, this proves that the human osteosarcoma VEGF is an important angiogenic-related factors.

业已证明大多数实体恶性肿瘤能分泌多种促血管生长因子,诱导血管生成,支持肿瘤生长(Folkman J,Science,1987,235(4787):442)。 Most solid malignant tumors has been shown to secrete a variety of pro-angiogenic growth factors, induce angiogenesis, supporting tumor growth (Folkman J, Science, 1987,235 (4787): 442). 肿瘤诱生的血管无论在病理、生理还是形态功能上都有着特征性,这些都为传统的化疗药物构成了“血瘤屏障”,使得肿瘤细胞得以逃逸化疗药物的杀伤(Hori K LJ Cancer Res,1991,82(1):109)。 Vascular tumors induced both in the pathology, physiological or morphological features have a characteristic, which are traditional chemotherapy drugs constitutes a "blood tumor barrier", so that the tumor cells to escape destruction (Hori K LJ Cancer Res chemotherapeutics 1991,82 (1): 109). 识别肿瘤血管生成过程与正常血管生成过程的差异,选择性攻击肿瘤血管特异性靶部位,将大大加速肿瘤治疗进程。 Identifying differences with the normal process of angiogenesis in angiogenesis process, selectively attack tumor vasculature-specific target site, will greatly speed up the process of tumor therapy.

VEGF最初是从牛垂体滤泡星状细胞培养物中纯化分离出来的强烈的内皮细胞有丝分裂原,通过与内皮细胞上的酪氨酸激酶受体flt1和KDR/flk1特异性结合,使受体自身磷酸化而激活细胞内信号传导通路,产生生物效应如诱导内皮细胞增殖、迁移,调节内皮细胞整合素及蛋白酶激活因子的表达,增加微血管通透性,促进血浆蛋白外漏,基底膜降解,导致新的基质及新生血管腔形成。 VEGF was originally isolated from bovine pituitary follicle stellate cell cultures strongly purified endothelial cell mitogen and by the tyrosine kinase receptors on the endothelial cell specific binding flt1 and KDR flk1 / the receptor itself phosphorylation activates intracellular signal transduction pathways, such as the biological effects induced endothelial cell proliferation, migration, regulating the expression of integrins and endothelial cell protease activating factor, increased microvascular permeability, promoting leakage of plasma proteins, the degradation of the basement membrane, resulting in new substrate and the cavity forming neovascularization. 在迄今发现的20多种多肽类血管生长因子中,VEGF是针对内皮细胞特异性最高,促血管生长作用最强的关键调节因子(Ferrara N.Endocr Rev,1997,18(1):4),在多种原发性恶性肿瘤组织中均呈高表达(Brown L F等Human Pathol,1995,26(1):86);新近Takahashi(Cancer Res,1995,55(18):3964)发现VEGF高表达与结肠癌转移的发生有密切联系,提示VEGF在原位肿瘤的形成和生长及转移瘤的形成中均起着举足轻重的作用。 In so far found 20 kinds of vascular endothelial growth factor polypeptide, of VEGF is an endothelial cell specific for the highest, the most potent angiogenic growth key regulator (Ferrara N.Endocr Rev, 1997,18 (1): 4), primary malignant tumors in a variety of tissues showed high expression (Brown LF et Human Pathol, 1995,26 (1): 86); newly Takahashi (Cancer Res, 1995,55 (18): 3964) found that high expression of VEGF and colon cancer metastases are closely linked, suggesting that VEGF plays an important role both in situ formation and tumor growth and metastasis in. 已经证实其它多种血管生长因子是通过直接或间接诱导、刺激VEGF表达而发挥作用的,如TGF-α、bFGF、TGF-β、TNF-α、KGF等均可上调VEGF的表达(Li J,Hampton T等,J Clin Invest,1997,100(1):18;Brown L F等,Detmar M,Claffey K,et al.Vascular permeabilityfactor/vascular endothelial growth factor:a multifunctional angiogenic cytokine.Exs,1997,79:233)。 Have demonstrated a variety of other angiogenic growth factors is directly or indirectly induce, stimulate VEGF expression functioning, such as TGF-α, bFGF, TGF-β, TNF-α, KGF etc. can upregulate the expression of VEGF (Li J, et Hampton T, J Clin Invest, 1997,100 (1): 18; Brown LF et, Detmar M, Claffey K, et al.Vascular permeabilityfactor / vascular endothelial growth factor: a multifunctional angiogenic cytokine.Exs, 1997,79: 233 ). 因此,VEGF是阻断肿瘤血供的理想靶点。 Thus, VEGF is an ideal target for tumor blood supply blocking. Kim KJ利用VEGF单抗成功地抑制了横纹肌肉瘤、恶性胶质瘤、平滑肌肉瘤在裸鼠体内的生长;Asano(Asano M等,Cancer Res,1995,55(22):5196)利用VEGF121单抗MV303不仅抑制了体外培养的人脐静脉内皮细胞生长及BALB/C鼠体内人纤维肉瘤细胞株HT-1080的生长,而且抑制了BALB/C鼠转移瘤的形成。 Kim KJ using VEGF monoclonal antibody successfully inhibited rhabdomyosarcoma, malignant glioma, leiomyosarcoma growth in nude mice; Asano (Asano M, etc., Cancer Res, 1995,55 (22): 5196) monoclonal antibody using VEGF121 MV303 not only inhibits the growth of cultured human umbilical vein endothelial cell growth and BALB / C mice human fibrosarcoma cell line HT-1080, and inhibiting the formation of BALB / C mouse metastases. 我们的实验证实VEGF多克隆抗体对骨肉瘤细胞OS-732的血管生成及肿瘤细胞的生长也有抑制作用,且VEGF多克隆抗体作用较VEGF单抗可能更全面,较小剂量时即能比较完全地阻断VEGF的作用。 Our experiments confirmed that angiogenesis and growth of osteosarcoma cells OS-732 tumor cells also inhibit VEGF polyclonal antibodies, polyclonal antibodies and VEGF VEGF monoclonal antibody may be more effective than comprehensive, when a smaller dose that is able to compare fully blocking the effects of VEGF.

血管生成过程涉及一系列形态学及生化学改变。 Angiogenic process involves a series of morphological and biochemical change. 形态学改变包括内皮细胞降解母体小静脉的基底膜、内皮细胞的定向运动、发生有丝分裂、血管腔形成、芽式生长并形成血管襻、产生新的基底膜、外膜细胞的形成等一系列步骤。 Morphological changes including endothelial cells degrade basement membrane matrix venules, directional movement of endothelial cells, mitogenesis, formation of a vascular lumen, bud growth and vascular loops are formed, the step of generating a series of new base film, or the like is formed pericytes . 通常情况下,内皮细胞处于休眠状态,是体内极度静止的细胞。 Normally, endothelial cells in a dormant state, the extreme resting cells in vivo. 内皮细胞的更新需要数百天,而骨髓细胞的更新平均只需5天,分裂速率约6×109细胞/小时。 Update endothelial cells requires hundreds of days, an average of bone marrow cell update only 5 days, the split rate of about 6 × 109 cells / hr. 血管生成中,微血管内皮细胞的增殖速度同骨髓细胞相同,其生化学机制涉及到血管生成因子与血管生成抑制因子之间的调节失衡。 Angiogenesis in microvascular endothelial cell proliferation rate of bone marrow cells with the same, which relates to the biochemical mechanism of angiogenic factors and angiogenesis inhibitory factor adjustment between imbalance.

目前已分离和纯化了20多种血管生成因子和相关因子,至少15种血管生成抑制因子。 It has been isolated and purified more than 20 related factors and angiogenic factors, at least 15 angiostatin. 血管生成因子包括血管内皮细胞生长因子(VPF/VEGF)、酸性及碱性成纤维细胞生长因子(aFGF,bFGF)、血管生成素、胎盘生长因子(PIGF)、表皮生长因子(EGF)、白介素-8、肿瘤坏死因子α(TNFα)等。 Angiogenic factors including vascular endothelial growth factor (VPF / VEGF), acidic and basic fibroblast growth factor (aFGF, bFGF), angiogenin, placental growth factor (PIGF), epidermal growth factor (EGF), interleukin - 8, tumor necrosis factor α (TNFα) and the like. 在所有的血管生成因子中,对VEGF及bFGF的研究最为深入。 In all of angiogenic factors, VEGF and bFGF in the study of the most in-depth. VEGF具有增加微血管的通透性、促进不同来源的内皮细胞分裂增殖和血管构建、促使内皮细胞的迁移等多种作用,是已知的最强的血管通透剂。 VEGF has increased microvascular permeability, promoting the proliferation of endothelial cells and vascular constructed from different sources, to promote endothelial cell migration and other role, it is the strongest known vascular permeability agent. VEGF选择性地作用于血管内皮细胞膜上的两种型酪氨酸激酶受体flt-1和KDR,通过磷酸肌醇特异性磷酯酶C使胞内IP3浓度升高而发挥作用。 VEGF selectively acts on two type of endothelial cell membrane tyrosine kinase receptors flt-1 and KDR, via phosphatidylinositol specific phospholipase C that the intracellular concentration increased IP3 play a role.

以血管生成的各个环节及其发生过程中的生化改变为靶点,研制血管生成抑制剂,控制肿瘤生长和转移,将成为肿瘤防治的一个重要途径。 Biochemical processes is changed to the target, the development of angiogenesis inhibitors, to control tumor growth and metastasis, it will be an important way to cancer prevention and all aspects of angiogenesis occurs. 总体来说,血管生成抑制剂的研究主要有如下4种策略;(1)阻断内皮细胞降解周围基质的能力;(2)直接抑制内皮细胞的功能;(3)阻断血管生成因子的合成和释放,拮抗其作用;(4)阻断内皮细胞表面整合素的作用。 Overall, studies of angiogenesis inhibitors are mainly the following four strategies; (1) blocking the endothelial cells of the surrounding matrix degradation; (2) direct inhibition of endothelial cell function; (3) blocking the synthesis of angiogenic factors and release, antagonize effect; (4) the endothelial cell surface integrin blocking effect.

目前发现的具有抗血管生成能力的因子主要有VEGF等的受体和抗VEGF单克隆抗体。 It has now been found that the ability of anti-angiogenic factors such as VEGF receptors are mainly, and anti-VEGF monoclonal antibody. 广泛的研究证明,抗VEGF单抗具有明显的抑制肿瘤血管生成的功能。 Extensive studies have shown that anti-VEGF monoclonal antibody has a significant function of suppressing tumor angiogenesis. 单克隆抗体能抑制人血管内皮细胞的增殖,在体内能抑制肿瘤新生血管形成,从而抑制肿瘤的生长和转移,其抑制率可达49%-70%。 Monoclonal antibody inhibits proliferation of human endothelial cells in vivo can inhibit tumor angiogenesis, thereby inhibiting tumor growth and metastasis, the inhibition rate of 49% -70%.

综上所述,抗VEGF单克隆抗体在肿瘤治疗上显示出有巨大的应用前景。 In summary, the anti-VEGF monoclonal antibody shown to have potential application in cancer treatment.

然而,目前本领域中生产该VEGF单克隆抗体的细胞株的表达水平仅为50-100毫克/升,这远不能满足大规模生产单克隆抗体的需要。 However, those skilled in the production of the VEGF monoclonal antibody cell line expression levels of only 50-100 mg / l, which can not meet the needs of large-scale production of monoclonal antibodies. 而且,目前市售的VEGF单克隆抗体制品的活性也不高。 Furthermore, VEGF monoclonal antibody products currently on the market activity is not high. 因此,本领域中仍迫切需要提供一种活性和表达量有所改善的VEGF单克隆抗体。 Therefore, the art is still an urgent need for an active VEGF monoclonal antibodies and expression has improved.

发明目的本发明的目的是提供一种重组抗人VEGF单克隆抗体。 Purpose The purpose of the present invention is to provide a recombinant human anti-VEGF monoclonal antibody.

本发明的另一目的是提供编码上述抗VEGF单克隆抗体的DNA分子。 Another object of the present invention is to provide a DNA molecule encoding the above monoclonal anti-VEGF antibody.

本发明的另一目的是提供一种含有上述单克隆抗体的药物组合物。 Another object of the present invention is to provide a pharmaceutical composition containing the monoclonal antibody.

本发明还有一个目的是提供制备上述单克隆抗体的方法。 Another object of the present invention is to provide a process for preparing such monoclonal antibodies.

为达到上述发明目的,本发明一方面提供了一种抗人VEGF单克隆抗体,该抗体含有重链可变区和轻链可变区,其中轻链可变区具有SEQ ID NO:3所示的氨基酸序列,重链可变区具有SEQ ID NO:1所示的氨基酸序列。 To achieve the above object, an aspect of the present invention provides an anti-VEGF human monoclonal antibody comprising a heavy chain variable region and light chain variable region, wherein the light chain variable region having SEQ ID NO: 3 shown in FIG. amino acid sequence, having a heavy chain variable region SEQ ID NO: 1 amino acid sequence.

本发明另一方面提供了编码上述单克隆抗体的DNA分子。 Another aspect the present invention provides a DNA encoding the above monoclonal antibody molecule.

在一个较佳的实例中,该DNA分子含有SEQ ID NO:4所示的编码所述单抗轻链可变区的核苷酸序列,以及SEQ ID NO:2所示的编码所述单抗重链可变区的核苷酸序列。 In a preferred example, the DNA molecule comprising SEQ ID NO: 4 encoded by a nucleotide sequence of the monoclonal antibody light chain variable region, and SEQ ID NO: 2 encoding the monoclonal antibodies shown in the nucleotide sequence of the heavy chain variable region.

本发明第三方面提供了一种表达载体,该表达载体含有上述DNA序列以及与该序列操作性相连的表达调控序列。 A third aspect of the present invention provides an expression vector, the expression vector containing the DNA sequences and expression control sequences operably linked to the sequence.

本发明第四方面提供了一种宿主细胞,其中该宿主细胞被上述表达载体转化。 A fourth aspect of the present invention provides a host cell, wherein the host cell is transformed expression vector described above. 该宿主细胞优选哺乳动物细胞,更优选CHO细胞。 The host cell is preferably a mammalian cell, more preferably a CHO cell.

本发明第五方面提供了一种治疗肿瘤的药物组合物,该药物组合物含有药学上有效量的上述人单克隆抗体以及药学上可接受的载体。 A fifth aspect of the present invention provides a pharmaceutical composition for treating cancer, the pharmaceutical composition comprising a pharmaceutically effective amount of a human monoclonal antibody and a pharmaceutically acceptable carrier.

本发明第六方面提供了一种制备上述单克隆抗体的方法,该方法包括:a)提供一表达载体,该表达载体含有上述DNA编码序列以及与该序列操作性相连的表达调控序列;b)用步骤a)所述的表达载体转化宿主细胞;c)在适合所述单克隆抗体表达的条件下培养步骤b)所得的宿主细胞;和d)分离纯化获得所述单克隆抗体。 A sixth aspect of the present invention provides a method of the above monoclonal antibody preparation, the method comprising: a) providing an expression vector, the expression vector containing the DNA coding sequence and expression control sequence operably linked to the sequence; b) transformed with the expression vector of step a) of the host cell; c) culturing step b) the obtained host cell under suitable conditions for expression of the monoclonal antibody; and d) separating the purified monoclonal antibody obtained.

本发明的优点在于本发明的单克隆抗体的生理活性和在宿主细胞(如CHO细胞)中的表达量比现有的单克隆抗体有显著提高。 Advantage of the present invention is a physiologically active monoclonal antibody of the invention and expression than conventional monoclonal antibodies in host cells (e.g., CHO cells) was significantly improved. 本发明的其它目的和优点可通过下面的详细描述得知。 Other objects and advantages of the present invention may be learned by the following detailed description.

附图说明 BRIEF DESCRIPTION

图1显示了本发明所用pMG18载体的酶切图谱。 Figure 1 shows a restriction map of pMG18 vectors used in the present invention. 图中Ck表示抗体kappa轻链的恒定区基因;IgG1恒定区表示人抗体IgG1的重链恒定区基因;pA表示SV40加poly A位点。 FIG Ck constant region gene expressed antibody kappa light chain; the constant region of IgG1 heavy chain constant region gene expressed human antibodies of IgG1; SV40 pA represents a poly A addition site.

发明详述本发明涉及一种重组的抗人VEGF单克隆抗体,该抗体包含重链可变区和轻链可变区,其中轻链可变区具有SEQ ID NO:3所示的氨基酸序列,重链可变区具有SEQID NO:1所示的氨基酸序列。 DETAILED DESCRIPTION The present invention relates to a recombinant human anti-VEGF monoclonal antibody comprises a heavy chain variable region and light chain variable region, wherein the light chain variable region having SEQ ID NO: 3 amino acid sequence shown, a heavy chain variable region having SEQID NO: 1 amino acid sequence.

本文所用的术语“单克隆抗体(单抗)”指从一类基本均一的群体获得的抗体,即该群体中包含的单个抗体是相同的,除少数可能存在的天然发生的突变外。 As used herein, the term "monoclonal antibody (mAb)" refers to a class of antibodies from a substantially homogeneous population obtained, i.e., the individual antibodies comprising the population are identical, except for a few mutations that may be present naturally occurring. 单克隆抗体高特异性地针对单个抗原位点。 Monoclonal antibodies with high specificity against a single antigenic site. 而且,与常规多克隆抗体制剂(通常是具有针对不同决定簇的不同抗体)不同,各单克隆抗体是针对抗原上的单个决定簇。 Moreover, unlike conventional polyclonal antibody preparations (typically having a different antibodies against different determinants) different, each monoclonal antibody is directed against a single determinant on the antigen. 除了它们的特异性外,单克隆抗体的好处还在于它们是通过杂交瘤培养来合成的,不会被其它免疫球蛋白污染。 In addition to their specificity, the monoclonal antibodies are benefits in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins is not. 修饰语“单克隆”表示了抗体的特性,是从基本均一的抗体群中获得的,这不应被解释成需要用任何特殊方法来生产抗体。 The modifier "monoclonal" indicates the character of the antibody are obtained from a substantially homogeneous population of antibodies in, this should not be construed to require any particular method of production of the antibody.

本文所用的术语“抗体”和“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白,其由两个相同的轻链(L)和两个相同的重链(H)组成。 As used herein, the term "antibody" and "immunoglobulin" have the same structural characteristics about 150,000 daltons heterotetrameric glycoproteins, composed of two identical light (L) chains and two identical heavy chains ( H) components. 每条轻链通过一个共价二硫键与重链相连,而不同免疫球蛋白同种型的重链间的二硫键数目不同。 Each light chain by one covalent disulfide bond to a heavy chain connected to different immunoglobulins with different species of number of disulfide linkages between the heavy chains. 每条重链和轻链也有规则间隔的链内二硫键。 Each heavy and light chain also has regularly spaced intrachain disulfide bridges. 每条重链的一端有可变区(VH),其后是多个恒定区。 Each heavy chain has at one end a variable region (VH), followed by a number of constant domains. 每条轻链的一端有可变区(VL),另一端有恒定区;轻链的恒定区与重链的第一个恒定区相对,轻链的可变区与重链的可变区相对。 Each light chain has at one end a variable region (the VL), the other end of a constant region; of the light chain constant region of the heavy chain constant region is relatively variable region relative to the light chain variable region of the heavy chain . 特殊的氨基酸残基在轻链和重链的可变区之间形成界面。 Special amino acid residues form an interface between the light chain and the heavy chain variable region.

本文所用的术语“可变”表示抗体中可变区的某些部分在序列上有所不同,它形成了各种特定抗体对其特定抗原的结合和特异性。 As used herein, the term "variable" indicates that some portion of the antibody variable regions differ in sequence, it forms a variety of specific antibody binding and specificity of a particular antigen. 然而,可变性并不均匀地分布在整个抗体可变区中。 However, the variability is not evenly distributed throughout the variable domains of antibodies. 它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。 It is concentrated in the light chain and heavy chain variable regions called complementarity determining regions (CDR) fragments or super three hypervariable regions of. 可变区中较保守的部分称为构架区(FR)。 Variable region are more conserved, termed framework regions portion (FR). 天然重链和轻链的可变区中各自包含四个FR区,它们大致上呈β-折叠构型,由形成连接环的三个CDR相连,在某些情况下可形成部分β折叠结构。 The variable domains of native heavy and light chains each comprise four FR regions, β- which the substantially collapsed configuration, connected by three CDR loop of a connection, in some cases it may form part of β -sheet structure. 每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I,647-669页(1991))。 CDR in each chain by FR regions close together and form the antigen-binding site of antibodies with CDR of another chain (see Kabat et al, NIH Publ.No.91-3242, Volume I, 647-669 pages (1991)). 恒定区不直接参与抗体与抗原的结合,但是它们表现出不同的效应功能,例如参与抗体的依赖于抗体的细胞毒性。 Constant domains are not involved directly in binding an antibody to an antigen, but they exhibit different effector functions, such as participation of the antibody in antibody-dependent cytotoxicity.

脊椎动物抗体(免疫球蛋白)的“轻链”可根据其恒定区的氨基酸序列归为明显不同的两类(称为κ和λ)中的一类。 Vertebrate antibody (immunoglobulin) "light chain" may be the amino acid sequence of their constant regions into one of two distinct classes (referred to as κ and [lambda]) in. 根据其重链恒定区的氨基酸序列,免疫球蛋白可以分为不同的种类。 The amino acid sequence of the constant region of their heavy chains, immunoglobulins can be assigned to different classes. 主要有5类免疫球蛋白:IgA,IgD,IgE,IgG和IgM,其中一些还可进一步分成亚类(同种型),如IgG1,IgG2,IgG3,IgG4,IgA和IgA2。 There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. 对应于不同类免疫球蛋白的重链恒定区分别称为α、δ、ε、γ、和μ。 Correspond to the different classes of immunoglobulins heavy chain constant regions are called α, δ, ε, γ, and μ. 不同类免疫球蛋白的亚单位结构和三维构型是众所周知的。 Different classes of immunoglobulins subunit structures and three-dimensional configurations are well known.

单克隆抗体可用本领域技术人员熟知的各种方法来制得。 Monoclonal antibodies can be a variety of methods well known to those skilled be prepared. 例如,单克隆抗体可用杂交瘤方法(由Kohler等,Nature,256:495(1975)首先提出)制得,或用重组DNA方法(美国专利No.4,816,567)制得。 For example, monoclonal antibodies can hybridoma method (, Nature, 256 by Kohler et al: 495 (1975) first proposed) system, or by recombinant DNA methods (U.S. Pat. No. 4,816,567) was prepared. 单克隆抗体也可用例如Clackson等,Nature,352:624-628(1991)和Marks等,J.Mol.Biol.,222:581-597(1991)所述的技术从噬菌体抗体库中分离获得。 The monoclonal antibodies may also be used, for example, Clackson et al, Nature, 352:. 624-628 (1991) and Marks et al., J.Mol.Biol, 222: 581-597 (1991) isolated from the techniques of phage antibody library.

本发明还提供了编码本发明人源化抗VEGF单克隆抗体的DNA分子。 The present invention also provides the present invention encoding human anti-VEGF humanized monoclonal antibody of the DNA molecule. 在一个较佳的实例中,该DNA分子含有SEQ ID NO:4所示的编码所述单抗轻链可变区的核苷酸序列,以及SEQ ID NO:2所示的编码所述单抗重链可变区的核苷酸序列。 In a preferred example, the DNA molecule comprising SEQ ID NO: 4 encoded by a nucleotide sequence of the monoclonal antibody light chain variable region, and SEQ ID NO: 2 encoding the monoclonal antibodies shown in the nucleotide sequence of the heavy chain variable region.

在获得编码本发明人源化抗VEGF单克隆抗体重链可变区和轻链可变区的核苷酸序列后,通常可通过以下方法来制备本发明的单克隆抗体。 After obtaining the present invention encoding human monoclonal humanized anti-VEGF antibody heavy chain variable region and light chain variable region nucleotide sequences, monoclonal antibodies can generally be prepared by the following method of the present invention.

首先,提供含有编码本发明单克隆抗体的核苷酸序列以及与该序列操作性相连的表达调控序列的表达载体。 First, an expression vector containing a nucleotide sequence encoding the monoclonal antibodies of the present invention and the expression control sequences operably linked to the sequence. 但是,本领域普通技术人员也能预计到,将编码本发明单抗重链可变区和轻链可变区的核苷酸序列分别插入不同的表达载体中进行共表达也能获得本发明的抗VEGF单克隆抗体。 However, those of ordinary skill in the art can be expected, the nucleotide sequence encoding the monoclonal antibody heavy chain variable region and light chain variable regions of the present invention are inserted into separate expression vectors for co-expression can be obtained according to the present invention. anti-VEGF monoclonal antibody.

本文所用的术语“表达调控序列”通常指参与控制核苷酸序列表达的序列。 As used herein, the term "expression control sequence" generally refers to a control sequence involved in the expression of the nucleotide sequence. 表达调控序列包括与目标核苷酸序列操作性相连的启动子和终止信号。 Expression control sequences include promoters and termination signals connected to the target nucleotide sequence operably. 它们通常还包括核苷酸序列适当翻译所需的序列。 They also typically comprises a nucleotide sequence sequences required for proper translation. “操作性相连”是指线性DNA序列的某些部分能够影响同一线性DNA序列其他部分的活性。 "Operably linked" refers to certain portions of a linear DNA sequence can affect the activity of the same linear DNA sequence other portions. 例如,如果启动子或增强子增加了编码序列的转录,则它与编码序列是操作性相连的。 For example, if a promoter or enhancer increases the transcription of the coding sequence, the coding sequence if it is operably linked.

编码本发明单克隆抗体的DNA序列可用本领域技术人员熟知的常规手段来制得。 DNA sequences encoding the monoclonal antibodies of the present invention, conventional means well known to those skilled in the art may be used to prepare. 例如,可根据本发明公开的序列人工合成或用PCR法扩增得到编码该单克隆抗体重链可变区和轻链可变区的核苷酸序列。 For example, the present invention may be artificially synthesized sequence disclosed or amplified by PCR encoding the monoclonal antibody heavy chain variable region and light chain variable region nucleotide sequence from. 然后,用本领域熟知的各种方法通过选择合适的酶切位点将这些核苷酸序列插入合适的表达载体中,使它们分别在表达载体所携带的重链恒定区编码序列和轻链恒定区编码序列之前,并使它们在同一读框内。 Then, using a variety of methods known in the art by choosing the appropriate point of these nucleotide sequences restriction sites inserted into an appropriate expression vector, such that they are expressed in the constant region of the heavy chain and light chain constant coding sequences carried by the vector prior to the coding region sequence, and they are in the same reading frame. 本发明中所用的表达载体是本领域技术人员已知的各种市售的表达载体,例如购自Qiagen和Promega公司的表达载体,以及可购得的表达载体pMG18(《根据INCP-9质粒序列进行环境监测的工具开发》(DEVELOPMENT OF TOOLS FORENVIRONMENTAL MONITORING BASED ON INCP-9 PLASMIDS SEQUENCES),A.Greated,R.Krasowiak,M.Titok,CMThomas School of Biological Sciences,University of Birmingham,Edgbaston,Birmingham B15 2TT,UK and Faculty of Biology,Dept of Microbiology,Belarus State University Scorina Av.4,Minsk 220080 Belarus)。 Expression vectors used in the present invention are known to those skilled in various commercially available expression vector such as an expression vector was purchased from Qiagen and Promega Corporation, and a commercially available expression vector pMG18 ( "The plasmid sequences INCP-9 environmental monitoring tools to develop "(dEVELOPMENT oF tOOLS FORENVIRONMENTAL mONITORING BASED ON INCP-9 PLASMIDS SEQUENCES), A.Greated, R.Krasowiak, M.Titok, CMThomas School of Biological Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK and Faculty of Biology, Dept of Microbiology, Belarus State University Scorina Av.4, Minsk 220080 Belarus).

随后,用上述获得的表达载体转化合适的宿主细胞。 Subsequently, the above-described expression vector obtained into a suitable host cell. “宿主细胞”一般包括原核细胞和真核细胞。 "Host cell" generally includes prokaryotic cells and eukaryotic cells. 常用的原核宿主细胞的例子包括大肠杆菌、枯草杆菌等。 Examples of commonly used prokaryotic host cells include E. coli, Bacillus subtilis. 常用的真核宿主细胞包括酵母细胞,昆虫细胞、和哺乳动物细胞。 Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells. 在本发明中,优选哺乳动物细胞。 In the present invention, preferably mammalian cells. 通常用哺乳动物细胞系来作为表达真核细胞衍生多肽的宿主细胞。 Typically mammalian cell lines expressing a eukaryotic cell as a host cell-derived polypeptide. 哺乳动物细胞在培养物中的繁殖是本领域熟知的。 Propagation of mammalian cells in culture are well known in the art. 见《组织培养》,Academic Press,Kruseand Patterson编辑(1973),该文纳入本文作为参考。 See "tissue culture", Academic Press, Kruseand Patterson editor (1973), which is incorporated herein by reference. 较佳的哺乳动物细胞是许多可购得的无限增殖细胞系。 Preferred mammalian cells are commercially available in a number of immortalized cell lines. 这些无限增殖细胞系包括但不局限于,中国仓鼠卵巢(CHO)细胞、Vero细胞、海拉细胞、幼仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝细胞癌细胞(如Hep G2)和其它许多细胞系。 These immortalized cell lines include, but are not limited to, Chinese hamster ovary (CHO) cells, Vero cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2 ) and many other cell lines. 它们为蛋白质分子提供了翻译后修饰,包括正确的折叠、正确的二硫键形成以及正确位点的糖基化。 They provide post-translational modifications to protein molecules, including correct folding, correct disulfide bond formation, and sugar correct glycosylation sites. 尽管在下文实施例中,本发明仅列举了以CHO细胞作为宿主细胞的例子,但是本领域技术人员在阅读了本发明的详细描述和具体实施例可以知道,本发明也能采用上述这些细胞系。 Although embodiments Hereinafter, the present invention is only cited examples in CHO cells as a host cell, those skilled in the art upon reading the detailed description of the invention and the specific embodiments can be understood that the present invention can be employed these cell lines .

用表达载体转化宿主细胞的方法有很多种,所用的转化程序取决于待转化的宿主。 There are many methods by transforming a host cell expression vectors, the transformation procedure used depends upon the host to be transformed. 将异源多核苷酸导入哺乳动物细胞中的方法是本领域所知的,其包括葡聚糖介导的转染、磷酸钙沉淀、Polybrene(1,5-二甲基-1,5-二氮十一亚甲基聚甲溴化物)介导转染、原生质体融合、电穿孔、脂质体介导转染以及将DNA直接显微注射到胞核中。 Methods of heterologous polynucleotides into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, Polybrene (1,5- dimethyl-1,5- polymethine nitrogen eleven methylene bromide) mediated transfection, protoplast fusion, electroporation, liposome-mediated transfection and direct microinjection of the DNA into nuclei. 在本发明中,较佳的方法是电穿孔法或脂质体介导法等。 In the present invention, the preferred method is electroporation, or liposome-mediated method or the like. 例如可采用Invitrogen公司的脂质体法试剂盒来转染诸如CHO细胞等宿主细胞。 May be employed, for example, the Invitrogen liposome kit was used to transfect host cells such as CHO cells and the like.

然后,在适合本发明单克隆抗体表达的条件下,培养转化所得的宿主细胞。 Then, under conditions suitable for expression of the monoclonal antibodies of the present invention, culturing the resulting transformed host cells. 然后用常规的免疫球蛋白纯化步骤,如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析、离子交换层析、疏水层析、分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯化手段纯化得到本发明的人源化抗VEGF单克隆抗体。 Then by conventional immunoglobulin purification procedures such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc. Those skilled in the purification conventional means well known in the art of the present invention give anti-VEGF humanized monoclonal antibody.

所得单克隆抗体可用常规手段来鉴定。 The resulting monoclonal antibodies were identified by conventional means. 单克隆抗体的结合特异性可用免疫沉淀或体外结合试验(如放射性免疫测定(RIA)或酶联免疫吸附测定(ELISA))来测定。 Binding specificity of the monoclonal antibodies can be used immunoprecipitation or in vitro (e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)) binding assay was determined. 单克隆抗体的结合亲和力例如可用Munson等,Anal.Biochem.,107:220(1980)的Scatchard分析来测定。 The binding affinity of the monoclonal antibody such as available Munson et al., Anal.Biochem, 107:. 220 (1980) determined by the Scatchard analysis.

本发明还提供了一种治疗肿瘤的药物组合物,该组合物含有药学上有效量的本发明单抗以及药学上可接受的载体。 The present invention also provides a pharmaceutical composition for treating tumors, the composition comprising a pharmaceutically effective amount of a monoclonal antibody of the present invention and a pharmaceutically acceptable carrier. 例如,本发明的药物组合物可用来治疗胃癌、肝癌。 For example, the pharmaceutical compositions of the invention may be used to treat stomach, liver. 本文所用的术语“药学上可接受的”是指当分子本体和组合物适当地给予动物或人时,它们不会产生不利的、过敏的或其它不良反应。 As used herein, the term "pharmaceutically acceptable" refers to molecular entities when administered appropriately and compositions animal or a human, they do not produce an adverse, allergic or other untoward reaction. 本文所用的“药学上可接受的载体”应当与本发明的突变蛋白相容,即能与其共混而不会在通常情况下大幅度降低药物组合物的效果。 As used herein, "pharmaceutically acceptable carrier" should be compatible with the muteins of the present invention, it can be blended therewith, i.e., without a significant reduction of the pharmaceutical composition under normal circumstances effect. 可作为药学上可接受的载体或其组分的一些物质的具体例子是糖类,如乳糖、葡萄糖和蔗糖;淀粉,如玉米淀粉和土豆淀粉;纤维素及其衍生物,如羧甲基纤维素钠、乙基纤维素和甲基纤维素;西黄蓍胶粉末;麦芽;明胶;滑石;固体润滑剂,如硬脂酸和硬脂酸镁;硫酸钙;植物油,如花生油、棉籽油、芝麻油、橄榄油、玉米油和可可油;多元醇,如丙二醇、甘油、山梨糖醇、甘露糖醇和聚乙二醇;海藻酸:乳化剂,如Tween;润湿剂,如月桂基硫酸钠;着色剂;调味剂;压片剂、稳定剂;抗氧化剂;防腐剂;无热原水;等渗盐溶液;和磷酸盐缓冲液等。 Specific examples of the material may be the pharmaceutically acceptable carriers or components thereof are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as carboxymethyl cellulose sodium, ethyl cellulose, and methyl cellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cacao oil; polyhydric alcohols such as propylene glycol, glycerine, sorbitol, mannitol and polyethylene glycol; alginic acid: emulsifying agents, such as Tween; wetting agents, such as sodium lauryl sulfate ; coloring agents; flavoring agents; compressed tablet, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; phosphate buffer and the like.

本发明的药物组合物可根据需要制成各种剂型,并可由医师根据患者种类、年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的剂量进行施用。 The pharmaceutical compositions of the invention may be made into various dosage forms as desired, and according to the type of patient, age, weight and disease condition generally, the manner of administration and other factors determined by the physician to the patient is administered a dose of the beneficial. 给药方式例如可以采用灌注和其它治疗方式。 The mode of administration may be employed, for example, perfusion and other therapeutic modalities.

下面将结合实施例进一步详细地描述本发明。 Below in conjunction with embodiments of the present invention is further described in detail. 然而应当理解,列举这些实施例只是为了起说明作用,而并不是用来限制本发明。 However, it should be understood that these examples are mentioned for illustrative purposes, and are not intended to limit the present invention.

实施例1从抗体库中筛选VEGF的抗体可变区基因1)人源抗体库的构建按照Marks等人J.Mol.Biol.,222,581-597;Hoogenboom和Winte,J.Mol.Biol.,227,381-388;Haidaris CG等,J Immunol Methods.2001 Nov 1;257(1-2):185-202;Griffiths,AD等EMBO J.,13,3245-3260(1994);Nissim,A.等EMBO J.,13,692-698(1994)中描述的方法构建人源抗体库。 Example 1 Construction of Embodiment antibody library screened VEGF antibody variable region gene 1) human antibody library according to Marks et al., J.Mol.Biol, 222,581-597;. Hoogenboom and Winte, J.Mol.Biol. , 227,381-388; Haidaris CG like, J Immunol Methods.2001 Nov 1; 257 (1-2): 185-202; Griffiths, AD et EMBO J., 13,3245-3260 (1994); Nissim, A et al. EMBO J., 13,692-698 method (1994) Construction of humanized antibody library.

2)筛选:将复苏的抗体库菌株1毫升加入新鲜LB培养基14毫升,于50毫升三角瓶中37℃培养16小时。 2) Screening: The strain recovery antibody library was added 1 ml of fresh LB medium 14 ml, 50 ml Erlenmeyer flask and cultured at 37 ℃ 16 hours.

12000rpm高速离心10分钟,转移上清至一个无菌的50毫升离心管中,保存备用。 12000rpm 10 minutes high speed centrifugation, the supernatant was transferred to a 50 ml sterile centrifuge tube, stored for use. 其滴度应在2×1011以上。 Titers should be 2 × 1011 or more. 以纯化的VEGF蛋白(购自深圳晶美公司)为抗原,包被25毫升细胞培养瓶。 Purified VEGF protein (commercially available from Shenzhen Crystal Corp.) antigen, the cell-coated 25 ml flasks. 在包被后的细胞瓶中加入不少于3×1010噬菌体颗粒,37℃温育1小时。 Cell packets are added after the bottle is not less than 3 × 1010 phage particles and incubated for 37 ℃ 1 hour. 然后,倒掉瓶中的液体,用10毫升加入了1%Tween-20的PBS洗涤培养瓶10次。 Then, the bottle is drained of liquid, was added with 10 ml of PBS 1% Tween-20 flasks were washed 10 times. 在培养瓶中加入1毫升对数期的TG1细胞,37℃温育震荡培养16小时。 1 ml of the culture flask log phase TG1 cells, 37 ℃ incubated shaking culture for 16 hours.

重复上段所述步骤共4次。 Repeat steps four times the segment.

将上述获得的细胞稀释至100000细胞/毫升,然后在加入0.1%氨苄青霉素的1.5%琼脂平板上进行培养以获得单克隆。 The above-obtained cells were diluted to 100,000 cells / ml, then cultured to obtain a monoclonal ampicillin and 0.1% on 1.5% agar plates. 取上述平板上的克隆在96孔深孔板上进行培养,每孔一个克隆,共作960个克隆(10块96孔板)。 Colonies on plates taken in the above-described 96-well deep well plates the culture, a clone per well, 960 clones (10 96) complex. 将上述深孔板在96孔板离心机上5000RPM离心20分钟,将上清转移到新的无菌深孔板,封口后保藏于4度备用。 In the above-described 96-well deep well plate 5000RPM centrifuged for 20 minutes on a centrifuge, the supernatant was transferred to new sterile deep-well plates, after the sealing of the backup 4 deposited.

取96孔板10块,每孔中加入VEGF(10微克/毫升)10微升常规包被后,分别加入上述保存的上清10微升,37℃温育1小时后用含有1%Tween-20的PBS洗涤20次。 Take 96 10, of VEGF added to each well (10 [mu] g / ml) 10 microliters of a conventional After coating the saved supernatant were added to the 10 [mu] l, 37 [deg.] C After 1 hour incubation containing 1% Tween- 20 of the 20 washes PBS. 加入1微升HRP标记的羊抗M13单抗,37℃温育30分钟后用1%Tween-20的PBS洗涤10次。 Add 1 l of HRP-labeled goat anti-M13 mAb, for 30 minutes incubation at 37 ℃ washed with PBS 1% Tween-20 by 10 times.

加入含有0.025%DAB显色剂的PBS 200微升和1微升1%的H2O2,37℃温育显色20分钟后在读板机上读取595纳米处的光吸收。 Add PBS containing 0.025% DAB color-developing agent and 1 l 200 l 1% H2O2,37 ℃ incubated for 20 minutes the color reading light absorption at 595 nanometers on a plate reader. 根据光吸收读数确定显色反应强的孔,这些孔相对应的克隆即为亲和力较强的抗体可变区克隆。 Strong light absorption readings determined in accordance with the color reaction holes, these holes corresponding to the clones is the strong affinity of the antibody variable region clone.

通过上述过程共筛选出415个阳性克隆,根据读数确定其中5个亲和力最强的克隆。 By the above procedure were screened 415 positive clones, of which five strongest affinity clones determined based on readings. 将这5个克隆分别接种在100毫升LB培养基中,在37℃260rpm条件下震荡培养9小时后加入终浓度为1mM的异丙基硫代半乳糖苷(IPTG)诱导培养10小时。 After these five clones were inoculated in 100 ml LB medium, shake culture at 37 ℃ 260rpm conditions of a final concentration of 9 hours galactoside (IPTG) induced by isopropylthio-culture 10 hours of 1mM. 然后用Pharmacia的“重组人scFv纯化系统”参照厂家说明书分离纯化重组scFv抗体蛋白。 Then specification Purification of recombinant scFv antibody protein using a Pharmacia "recombinant human scFv Purification System" reference manufacturers. 抽提纯化的重组scFv蛋白质用于亲和力研究。 Affinity Study of extraction and purification for recombinant proteins scFv. 亲和力研究证明,在这415个阳性克隆中有3个具有较高的亲和力,它们分别命名为5H4、9D2和3G6。 Affinity studies have shown, there are three high affinities in this 415 positive clones, which were designated as 5H4,9D2 and 3G6. 其中,3G6即具有最高的表达量,又具有最高的亲和力,将此克隆将用在后续的研究。 Wherein, i.e., 3G6 has the highest expression levels, but also has the highest affinity for the clone to be used in subsequent studies.

3)抗体可变区编码序列向表达载体的克隆将上述3G6克隆的菌株在100毫升LB培养基中扩增,用Promega公司的质粒DNA抽提纯化试剂盒纯化质粒DNA。 3) the antibody variable region coding sequence of strain 3G6 clone described above in 100 ml of LB medium to amplify the expression cloning vector, plasmid DNA was purified using the Promega plasmid DNA extraction and purification kit.

用XbaI和NheI酶切上述质粒DNA后在1.5%琼脂糖凝胶电泳上分离酶切片段,取350bp左右的带进行胶回收,所得片段即为重链可变区编码序列。 Above plasmid DNA was digested with XbaI and NheI were separated on 1.5% agarose gel electrophoresis fragment, taken approximately 350bp gel band recovered, the resulting fragment is the heavy chain variable region encoding sequences.

用HindIII和Bsi WI酶切上述质粒DNA后在1.5%琼脂糖凝胶电泳上分离酶切片段,取320bp左右的带进行胶回收,所得片段即为轻链可变区编码序列。 Above plasmid DNA was digested with HindIII and Bsi WI separated on 1.5% agarose gel electrophoresis fragment, taken approximately 320bp gel band recovered, the resulting fragment is the light chain variable region encoding sequences.

然后首先将上述重链可变区编码序列插入到表达载体pMG18-3K(参见DEVELOPMENT OF TOOLS FOR ENVIRONMENTAL MONITORING BASED ONINCP-9 PLASMIDS SEQUENCES.A.Greated,R.Krasowiak,M.Titok,CMThomasSchool of Biological Sciences,University of Birmingham,Edgbaston,Birmingham B152TT,UK and Faculty of Biology,Dept of Microbiology,Belarus State University ScorinaAv.4,Minsk 220080 Belarus)的XbaI/NheI位点中,再用HindIII和Bsi WI将上述抗体轻链可变区编码序列插入到插入有重链可变区编码序列的pMG18-3K的HindIII/BsiWI位点中,构建成人源化抗VEGF抗体基因的表达载体。 The above-described first and then the heavy chain variable region encoding sequence into the expression vector pMG18-3K (see DEVELOPMENT OF TOOLS FOR ENVIRONMENTAL MONITORING BASED ONINCP-9 PLASMIDS SEQUENCES.A.Greated, R.Krasowiak, M.Titok, CMThomasSchool of Biological Sciences, University of Birmingham, Edgbaston, Birmingham B152TT, UK and Faculty of Biology, Dept of Microbiology, Belarus State University ScorinaAv.4, Minsk 220080 Belarus) the XbaI / NheI sites, HindIII and Bsi WI and then the above-described antibody light chain variable region encoding sequences inserted into the inserted pMG18-3K heavy chain variable region coding sequence of HindIII / BsiWI sites, construct an expression vector of the anti-VEGF humanized antibody gene.

4)CHO细胞的转染与重组克隆的筛选上述步骤3)中构建的带有抗体基因的表达载体在大肠杆菌DH5a菌株接种于100毫升LB培养基中进行扩增,用Qiagen公司的超纯质粒DNA纯化试剂盒(UltrapurePlasmid DNA Purification Kit)抽提纯化质粒DNA。 4) Screening the above step CHO cell transfected with the recombinant expression vector cloned 3) constructed with the antibody genes were amplified in E. coli DH5a strain was inoculated into 100 ml LB medium, with a Qiagen plasmid ultrapure DNA purification kit (UltrapurePlasmid DNA purification kit) extraction and purification of plasmid DNA. 将上述纯化的质粒DNA采用Invitrogen公司的脂质体法试剂盒转染CHO细胞,操作参照厂家的说明书进行。 The above-described purified plasmid DNA using the Invitrogen liposome kit transfected CHO cells, the operation with reference to the manufacturer's instructions.

转化的CHO细胞在选择培养基上进行连续9周的选择,最后在96孔板上进行极度稀释培养,连续进行3次,进行单克隆化。 Transformed CHO cells selected for 9 consecutive weeks on selective medium, and finally cultured extreme dilution in 96 well plates, 3 consecutive, monocloned.

挑出的单克隆细胞系在RPMI 1641培养基上进行培养,对上清进行Western印迹实验,根据染色反应判断表达强度,挑选出10个表达较强的克隆作为候选细胞株(见表1)。 Picked monoclonal cell lines were cultured in RPMI 1641 medium, Western blot of supernatant experiment, expression of the dyeing reaction is determined, 10 selected clones expressing strong as candidate cell lines (see Table 1).

5)单克隆抗体的纯化上述单抗的纯化采用Protein A(Pharmacia公司)亲和层析柱从细胞培养上清中直接分离纯化,并用SDA-PAGE电泳证明,所得产物纯度大于90%。 5) Purification of monoclonal antibody described above was purified using monoclonal antibody Protein A (Pharmacia Corporation) affinity chromatography separation and purification of culture supernatants from the cells, and demonstrated by SDA-PAGE electrophoresis, the resulting product purity greater than 90%. 以上亲和层析的产物再次经过分子筛层析,获得了纯度>98%的样品。 Affinity chromatography of the above product was again through the molecular sieve chromatography, is obtained with a purity of> 98% of the sample. 将这些样品用于以下的进一步分析与研究。 These samples will be used in the following further analysis and research.

实施例2抗体基因在CHO细胞中表达强度的研究将上述筛选得到的高表达候选克隆培养于10cm的组织培养皿中,采用ELISA法测量抗体的表达量:羊抗人IgG(Fc)包被于ELISA板,4℃过夜,经2%BSA于37℃封闭2小时,加入待测的培养上清和标准品(人IgG1),37℃孵育2小时,加入HRP-羊抗人IgG(κ)进行结合反应,37℃孵育1小时,加入TMB于37℃作用10分钟,最后用H2SO4终止反应,测A450值。 Example 2 Strength of antibody gene expression studies embodiment the above screening candidate obtained high expression clones were cultured in 10cm tissue culture dishes, the expression amount of the antibody measured by ELISA in CHO cells: goat anti-human IgG (Fc) were coated in ELISA plates, 4 ℃ overnight, blocked for 2 hours at 37 [deg.] C over 2% BSA, culture supernatants to be tested was added and standards (human an IgGl), incubated for 2 h 37 ℃, added HRP- goat anti-human IgG (κ) binding The reaction, 37 [deg.] C for 1 hour, at 37 ℃ at TMB was added for 10 minutes and finally with H2SO4 to terminate the reaction, measured A450 values. 测得上述10个候选克隆的表达量如下:表1抗体基因在CHO细胞中表达强度细胞株编号 3E9 5D7 4C6 5H8 6A6 3B4 4D8 4F6 5G7 6G2表达量(ug/毫升) 116.7 128.9 366.7 343.2 408.6 372.1 332.1 176.4 231.4 146.9从上表可以看出,编号为4C6、5H8、6A6和3B4的细胞株的表达水平具有很高的表达水平(340-410毫克/毫升),已经远远超出了国内外单抗类的表达水平(50~100mg/毫升)。 Expression level measured with the 10 candidate clones as follows: Table 1 Antibody gene expression of cell line No. 3E9 5D7 4C6 5H8 6A6 3B4 4D8 4F6 5G7 6G2 expression level (ug / ml) 116.7 128.9 366.7 343.2 408.6 372.1 332.1 176.4 in CHO cells 231.4 146.9 as can be seen from the table, number of cell lines the expression level 4C6,5H8,6A6 and 3B4 has a high expression level (340-410 mg / mL), far beyond the mAbs abroad The expression level (50 ~ 100mg / mL).

实施例3单克隆抗体生理活性和亲和力研究1)生理功能研究由于至今只发现血管内皮细胞对VEGF有特异的增殖反应,本试验根据Gospodarowicz D等的方法(PNAS,1989,86:7311)检测抗VEGF单抗的生物学活性。 Example 3 physiologically active monoclonal antibodies and affinity studies 1) Research Since the physiological function has found only on endothelial cells. VEGF-specific proliferative responses, in accordance with this test method Gospodarowicz D, etc. (PNAS, 1989,86: 7311) Detection of anti VEGF monoclonal antibody biological activity.

具体方法如下:取96孔细胞培养板一块,每孔中加入0.5毫升含有1×104牛肾上腺血管内皮细胞(可从中国科学院上海细胞生物学研究所细胞库获得)培养液和200IU的VEGF(购自深圳晶美公司),在第4天单独加入同样量的VEGF以持续刺激牛肾上腺内皮细胞增殖。 Specific methods are as follows: Take a 96-well cell culture plates, each well containing 0.5 ml of 1 × 104 bovine endothelial cells (available from the Shanghai Institute of Cell Biology, CAS Cell Bank) culture medium and VEGF 200IU (available since Shenzhen crystal Corp.), was added on day 4 the same amount of VEGF alone to continue to stimulate the proliferation of bovine endothelial cells.

待测样品和阳性对照储存液浓度均为500微克/毫升。 Test sample and the positive control stock solution concentration is 500 g / ml. 阳性对照为Genentech公司的RhuFab V2。 The positive control was Genentech's RhuFab V2. 用含有0.2%明胶的PBS稀释液系列稀释待测样品和阳性对照后,每孔中加入0.5毫升待测样品和阳性对照。 Diluted test sample and the positive control were diluted with PBS containing 0.2% gelatin series, each well 0.5 ml of the test sample and positive control. 阴性对照孔加入0.5毫升细胞培养液。 Negative control wells 0.5 ml of cell culture medium. 每个处理设3次重复。 Each treatment was repeated 3 times. 在37℃ 5%CO2培养箱中培养4~5天。 Incubated at 37 ℃ 5% CO2 incubator for 4 to 5 days.

取经过上述处理的细胞0.2毫升,加入1/10体积浓度为5毫克/毫升的MTT溶液,37℃培养30分钟后在酶标仪上读取570nm处的光吸收。 Taking cells processed as described above 0.2 ml, 1/10 volume of a 5 mg / ml MTT solution was incubated for 30 minutes at a reading light absorption 570nm on a microplate reader 37 ℃. 参考波长为630nm。 Reference wavelength of 630nm.

试验结果显示在下表2中。 Test results are shown in Table 2 below.

表2VEGF生物活性分析 Table 2VEGF analysis of biological activity

从表2可以看出,本发明的人源抗人VEGF单克隆抗体具有很高的生物活性,比阳性对照RhuFab V2高出约8倍。 As can be seen from Table 2, the present invention is the source of human anti-VEGF monoclonal antibody having a high biological activity than the positive control RhuFab V2 approximately 8 times higher.

B)小鼠肝癌灌注治疗效果研究大鼠移植性肝癌模型的建立将皮下荷瘤大鼠断颈处死后,局部消毒,取皮下瘤块,拨去纤维组织,挑选无出血、无坏死肿瘤组织,用利刀片切成0.2mm3大小的小块备用。 After B) Mouse liver perfusion treatment Establishment transplanted carcinoma model will be sacrificed by cervical dislocation rat subcutaneous tumor-bearing rats, local disinfection, Subcutaneous tumor mass, fibrous tissue to dial, the selection of no bleeding, no necrotic tumor tissue, 0.2mm3 size of the cut pieces of spare blades with Li.

另取Wistar大鼠,以戊巴比妥钠按40毫克/千克腹腔注射麻醉,取仰卧位,四肢固定于手术板上,手术区用75%酒精消毒后,在腹正中切开皮肤10毫米左右,用显微组织镊子将肝包膜戳一小口,将肿瘤块沿隧道嵌入。 Another Wistar rats with sodium pentobarbital at 40 mg / kg i.p. supine position, limb fixed on the operating plate, the operation area after disinfection with 75% alcohol, in the ventral midline skin incision approximately 10 mm with forceps microstructure liver capsule poke a small mouth, embedded tumor mass along the tunnel. 移植完毕后逐层缝合腹壁,饲养待用,整个过程在无菌条件下进行。 After completion of transplantation sutured abdominal wall, raising stand, the whole process is conducted under sterile conditions.

治疗方法动脉插管参照Lindell等(转引自周梅芳,蒙坚:1999,肝动脉插管化疗与lak/il-2联合治疗晚期肝癌的护理,护理经验,V18(6))的方法,肝脏接种瘤块后第7天,大鼠腹腔麻醉后固定,再次剖腹(开口约2~3厘米),暴露肝脏,测量肿瘤表面最大径(a)和最小径(b),分离胃十二指肠动脉、肝动脉及肝固有动脉,结扎胃十二指肠动脉的远端。 Treatment of arterial reference Lindell, etc. (Quoted from Zhoumei Fang, Meng Jian: 1999, hepatic arterial chemotherapy and lak / il-2 in patients with advanced liver cancer nursing, nursing experience, V18 (6)) method, liver inoculation after the tumor mass on day 7, rats were anesthetized and the abdominal cavity is fixed, re-laparotomy (opening of about 2 to 3 cm), liver exposure, measuring tumor surface maximum diameter (a) and the minimum diameter (B), separating the gastroduodenal artery , hepatic artery and hepatic artery inherent distal ligation of the gastroduodenal artery. 以银夹暂时阻断肝总动脉,于手术显微镜下在胃十二指肠动脉上切一小口,由此将外径约0.3mm的特制导管上行插入肝固有动脉。 In temporary occlusion silver clip hepatic artery, to the surgical microscope cut a small hole on the gastroduodenal artery, whereby the outer diameter of about 0.3mm of the uplink special catheter inserted into the hepatic artery. 待回血后,按以下分组及剂量(表3)分别灌注盐水和本发明的上述单抗制剂。 After the return of blood grouping and the following dose (Table 3) were perfused with brine and the above monoclonal antibody preparation of the present invention. 灌注后拔管、结扎胃十二指肠动脉近端,放开肝总动脉上的银夹,逐层缝合切口后擦净伤口、外涂金霉素软膏,分笼恒温、自动供水供食饲养。 Perfusion extubation, gastroduodenal artery ligated proximal release silver clip on hepatic artery, the wound sutured incision wipe, coated chlortetracycline ointment, a thermostat, cage, automatic water feeding served .

肿瘤生长率测定每组部分大鼠于治疗后第7天处死,再次测量肿瘤的最大径(a)和最小径(b),按公式计算肿瘤体积:V=ab2/2,再根据治疗前后的肿瘤体积计算肿瘤生长率(GR)∶GR=(治疗后的肿瘤体积/治疗前的肿瘤体积)。 Tumor growth in rats for determination of the maximum diameter of each part on day 7 after treatment were sacrificed, tumors were measured again (a) and the minimum diameter (B), tumor volume was calculated according to the formula: V = ab2 / 2, then according to the before and after treatment tumor volume was calculated tumor growth rate (GR) :GR = (tumor volume of tumor volume / pre-treatment post-treatment).

5组大鼠灌注前后的肿瘤体积及肿瘤生长率方差分析结果见表3。 The results are shown in Table 3 tumor volume and tumor growth rates before and after the variance of 5 rats reperfusion. 治疗前各组肿瘤体积之间均无显著性差异,肝动脉灌注生理盐水后(阴性对照),所有大鼠的肿瘤均明显增大,平均肿瘤体积显著大于灌注前。 There was no significant difference between tumor volume of each group before and after treatment of hepatic arterial infusion of saline (negative control), all tumors were significantly increased in rats, the mean tumor volume was significantly greater than the previous infusion. 灌注抗VEGF单克隆抗体后,肿瘤体积较治疗前亦明显增大,但肿瘤生长率低于对照组。 After infusion of anti-VEGF monoclonal antibody, the tumor volume was also significantly increased compared with that before the treatment, but the rate of tumor growth than the control group. 这说明,灌注抗VEGF单克隆抗体可以明显抑制肝肿瘤的生长。 This shows that the infusion of anti-VEGF monoclonal antibody can inhibit the growth of liver tumors.

表3大鼠动脉灌注抗VEGF单抗后肝肿瘤体积及肿瘤增长率( x±s,n=10) Table 3 rat artery after liver perfusion anti-VEGF monoclonal antibody in tumor volume and tumor growth (x ± s, n = 10)

c)胃静脉灌注治疗大鼠胃癌的研究用上述类似方法对大鼠胃癌模型进行研究,结果显示在表4中。 Therapeutic rat gastric c) stomach perfused rats with gastric cancer research by the above methods analogous results are shown in Table 4.

表4大鼠动脉灌注抗VEGF单克隆抗体治疗前后肿瘤体积及平均肿瘤生长率( x±s,N=12) Table tumor volume and mean tumor growth rate of anti-VEGF monoclonal antibody perfusion before and after treatment 4 Artery (x ± s, N = 12)

从表4可以看出,该灌注该单克隆抗体可以明显抑制胃癌的生长,效果比对肝癌还要好。 As can be seen from Table 4, the infusion of the monoclonal antibodies can inhibit the growth of stomach cancer, liver cancer effect is even better than.

d)亲和力鉴定亲和力测定采用Scatchard分析法(Munson等人,1980,Anal.BioChem.,107:220)进行。 d) Identification of affinity was determined by Scatchard analysis the affinity (of Munson et al., 1980, Anal.BioChem, 107:. 220) for. 结果表明,4C6、5H8、6A6和3B4四种单克隆抗体的亲和力分别为6.8×10-9、3.21×10-8、7.1×10-9和6.64×10-6。 The results showed that, 4C6,5H8,6A6 four kinds of monoclonal antibodies and 3B4 were affinity of 6.8 × 10-9,3.21 × 10-8,7.1 × 10-9 and 6.64 × 10-6. 其中4C6和6A6两株单克隆抗体的亲和力均已达到0.1nM的水平。 4C6 and 6A6 in which two monoclonal antibodies have reached the level of affinity of 0.1nM. 与阳性对照相比(见表2),6A6比阳性对照的明显高。 Compared to the positive control (see Table 2), 6A6 was significantly higher than that of the positive control. 这很可能是由于阳性对照的分子结构为Fab(抗原结合片段),而本发明的6A6是具有完整分子结构的单克隆抗体。 This is probably due to the molecular structure of the positive control Fab (antigen binding fragment), and the present invention is a monoclonal antibody 6A6 having a complete molecular structure.

实施例4.VEGF单克隆抗体基因的DNA测序根据系谱,对上述细胞株6A6的抗VEGF单克隆抗体基因进行DNA测序。 Example 4.VEGF monoclonal antibody DNA sequencing of genes according pedigree embodiment, the above cell lines to anti-VEGF monoclonal antibody 6A6 gene DNA sequencing. 结果显示在序列表中,其中SEQ ID NO:2和SEQ ID NO:4分别是本发明获得的高活性高表达VEGF单克隆抗体的重链可变区和轻链可变区的DNA编码序列;SEQ ID NO:1和SEQ ID NO:3分别是根据上述DNA编码序列推测的重链可变区和轻链可变区的氨基酸序列。 The results are shown in the sequence listing, wherein SEQ ID NO: 2 and SEQ ID NO: 4 of the present invention are highly active to obtain a high expression of the DNA sequence encoding the heavy chain variable region and light chain variable regions of the VEGF monoclonal antibody; SEQ ID NO: 1 and SEQ ID NO: 3 are an amino acid sequence encoded by the DNA sequence of putative heavy chain variable region and light chain variable region according to.

尽管本发明描述了具体的例子,但是有一点对于本领域技术人员来说是明显的,即在不脱离本发明的精神和范围的前提下可对本发明作各种变化和改动。 Although the present invention is described with specific examples, but one thing for the skilled person will be apparent, i.e., without departing from the spirit and scope of the present invention can make various changes and modifications of the present invention. 因此,所附权利要求覆盖了所有这些在本发明范围内的变动。 Accordingly, the appended claims cover all such variations within the scope of the claimed invention.

序列表<110>上海中信国健药业有限公司<120>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<130>020773<160>4<170>PatentIn version 3.0<210>1<211>122<212>PRT<213>智人(Homo sapiens)<400>1Glu Val Gln Leu Leu Glu Ser Gly Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr20 25 30Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile35 40 45Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe50 55 60Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr65 70 75 80Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Ala Arg Pro Ser Ile Tyr Tyr Gly Ser Asn His SEQUENCE LISTING & lt; 110 & gt; Shanghai CITIC Pharmaceutical Co., Ltd. & lt; 120 & gt; humanized anti-growth factor monoclonal antibody preparation method and pharmaceutical compositions & lt vascular endothelium; 130 & gt; 020773 & lt; 160 & gt; 4 & lt; 170 & gt; PatentIn version 3.0 & lt; 210 & gt; 1 & lt; 211 & gt; 122 & lt; 212 & gt; PRT & lt; 213 & gt; Homo sapiens (Homo sapiens) & lt; 400 & gt; 1Glu Val Gln Leu Leu Glu Ser Gly Ala Glu Leu Val Lys Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr20 25 30Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile35 40 45Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe50 55 60Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr65 70 75 80Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Ala Arg Pro Ser Ile Tyr Tyr Gly Ser Asn His Trp Tyr Phe Asp Val100 105 110Trp Gly Ala Gly Thr Ser Val Thr Val Ser115 120<210>2<211>366<212>DNA<213>智人(Homo sapiens)<400>2gaggtgcagc tgctggagtc cggcgccgag ctggtgaagc ccggcgcctc cgtgaagctg 60 Trp Tyr Phe Asp Val100 105 110Trp Gly Ala Gly Thr Ser Val Thr Val Ser115 120 & lt; 210 & gt; 2 & lt; 211 & gt; 366 & lt; 212 & gt; DNA & lt; 213 & gt; Homo sapiens (Homo sapiens) & lt; 400 & gt; 2gaggtgcagc tgctggagtc cggcgccgag ctggtgaagc ccggcgcctc cgtgaagctg 60

tcctgcaccg cctccggctt caacatcaag gacacctaca tgcactgggt gaagcagcgc 120cccgagcagg gcctggagtg gatcggccgc atcgaccccg ccaacggcaa caccaagtac 180gaccccaagt tccagggcaa ggccaccatc accgccgaca cctcctccaa caccgcctac 240ctgcagctgt cctccctgac ctccgaggac accgccgtgt actactgcgc ccgcccctcc 300atctactacg gctccaacca ctggtacttc gacgtgtggg gcgccggcac ctccgtgacc 360gtgtcc 366<210>3<211>111<212>PRT<213>智人(Homo sapiens)<400>3Asp Ile Val Leu Thr Gln Phe Pro Ala Ser Leu Ser Val Phe Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Tyr20 25 30Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro35 40 45Arg Leu Leu Ile Tyr Leu Val Ser Ash Leu Glu Phe Gly Val Pro Ala50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr L tcctgcaccg cctccggctt caacatcaag gacacctaca tgcactgggt gaagcagcgc 120cccgagcagg gcctggagtg gatcggccgc atcgaccccg ccaacggcaa caccaagtac 180gaccccaagt tccagggcaa ggccaccatc accgccgaca cctcctccaa caccgcctac 240ctgcagctgt cctccctgac ctccgaggac accgccgtgt actactgcgc ccgcccctcc 300atctactacg gctccaacca ctggtacttc gacgtgtggg gcgccggcac ctccgtgacc 360gtgtcc 366 & lt; 210 & gt; 3 & lt; 211 & gt; 111 & lt; 212 & gt; PRT & lt; 213 & gt; Homo sapiens ( Homo sapiens) & lt; 400 & gt; 3Asp Ile Val Leu Thr Gln Phe Pro Ala Ser Leu Ser Val Phe Leu Gly1 5 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Tyr20 25 30Gly Tyr Ser Tyr Met His Trp Asn gln gln Lys Pro Gly gln Pro Pro35 40 45Arg Leu Leu Ile Tyr Leu Val Ser Ash Leu Glu Phe Gly Val Pro Ala50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr L eu Asn Ile His65 70 75 80Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg85 90 95Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys100 105 110<210>4<211>333<212>DNA<213>智人(Homo sapiens)<400>4gacatcgtgc tgacccagtt ccccgcctcc ctgtccgtgt tcctgggcca gcgcgccacc 60atctcctgcc gcgcctccca gtccgtgtcc acctacggct actcctacat gcactggaac 120 eu Asn Ile His65 70 75 80Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg85 90 95Glu Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys100 105 110 & lt; 210 & gt; 4 & lt; 211 & gt; 333 & lt; 212 & gt ; DNA & lt; 213 & gt; Homo sapiens (Homo sapiens) & lt; 400 & gt; 4gacatcgtgc tgacccagtt ccccgcctcc ctgtccgtgt tcctgggcca gcgcgccacc 60atctcctgcc gcgcctccca gtccgtgtcc acctacggct actcctacat gcactggaac 120

cagcagaagc ccggccagcc cccccgcctg ctgatctacc tggtgtccaa cctggagttc 180ggcgtgcccg cccgcttctc cggctccggc tccggcaccg acttcaccct gaacatccac 240cccgtggagg aggaggacgc cgccacctac tactgccagc acatccgcga gctgccctac 300accttcggcg gcggcaccaa gctggagatc aag 333 cagcagaagc ccggccagcc cccccgcctg ctgatctacc tggtgtccaa cctggagttc 180ggcgtgcccg cccgcttctc cggctccggc tccggcaccg acttcaccct gaacatccac 240cccgtggagg aggaggacgc cgccacctac tactgccagc acatccgcga gctgccctac 300accttcggcg gcggcaccaa gctggagatc aag 333

Claims (13)

1.一种抗人血管内皮生长因子单克隆抗体,它包含重链可变区和轻链可变区,其特征在于,轻链可变区具有SEQ ID NO:3所示的氨基酸序列,重链可变区具有SEQID NO:1所示的氨基酸序列。 An anti-human VEGF monoclonal antibody, comprising a heavy chain variable region and light chain variable region, wherein the light chain variable region comprising SEQ ID NO: 3 the amino acid sequence shown by weight chain variable region having SEQID NO: 1 amino acid sequence.
2.一种DNA分子,其特征在于,它编码SEQ ID NO:3所示的氨基酸序列。 A DNA molecule, characterized in that, which encodes SEQ ID NO: 3 amino acid sequence.
3.根据权利要求2所述的DNA分子,其特征在于,该DNA分子含有SEQ ID NO:4所示的核苷酸序列。 3. The DNA molecule of claim 2, wherein the DNA molecule comprises SEQ ID NO: 4 nucleotide sequence.
4.一种DNA分子,其特征在于,它编码SEQ ID NO:1所示的氨基酸序列。 4. A DNA molecule, characterized in that, which encodes SEQ ID NO: 1 amino acid sequence.
5.根据权利要求4所述的DNA分子,其特征在于,该DNA分子含有SEQ IDNO:2所示的核苷酸序列。 5. The DNA molecule according to claim 4, wherein the DNA molecule comprises SEQ IDNO: 2 nucleotide sequence.
6.一种表达载体,其特征在于,它含有权利要求2和4所述的DNA序列以及与所述序列操作性相连的表达调控序列。 6. An expression vector, characterized in that it comprises a DNA sequence as claimed in claim 2 and 4, and the expression control sequences operably linked to the sequence.
7.一种宿主细胞,其特征在于,它被权利要求6所述的表达载体转化。 A host cell, characterized in that it is an expression vector according to claim 6 conversion.
8.根据权利要求7所述的宿主细胞,其特征在于,所述宿主细胞是哺乳动物细胞。 8. The host cell of claim 7, wherein said host cell is a mammalian cell.
9.根据权利要求8所述的宿主细胞,其特征在于,所述宿主细胞是CHO细胞。 9. The host cell of claim 8, wherein said host cell is a CHO cell.
10.一种治疗肿瘤的药物组合物,其特征在于,它含有药学上有效量的权利要求1所述的人单克隆抗体以及药学上可接受的载体。 10. A pharmaceutical composition for treating tumors, characterized in that it comprises a pharmaceutically effective amount of a claimed in claim 1, the human monoclonal antibody and a pharmaceutically acceptable carrier.
11.权利要求1所述的人单克隆抗体在制备治疗肿瘤的药物中的应用。 Manufacture of a medicament for treating tumors in a human the monoclonal antibody of claim 11.
12.根据权利要求11所述的应用,其中所述肿瘤选自胃癌和肝癌。 12. The use according to claim 11, wherein the tumor is selected from the stomach and liver cancer.
13.一种制备权利要求1所述的单克隆抗体的方法,其特征在于,该方法包括:a)提供一表达载体,该表达载体含有权利要求2和4所述的DNA序列以及与该序列操作性相连的表达调控序列;b)用步骤a)所述的表达载体转化宿主细胞;c)在适合所述单克隆抗体表达的条件下培养步骤b)所得的宿主细胞;和d)分离纯化获得所述单克隆抗体。 Monoclonal antibodies 13. A preparation according to claim 1, characterized in that, the method comprising: a) providing an expression vector, the expression vector comprising the DNA sequence of claim 2 and 4, and the sequence expression control sequences operably linked; expression vector b) of step a) transforming said host cell; c) culturing step b) the obtained host cell under suitable conditions for expression of the monoclonal antibody; and d) purification obtaining said monoclonal antibody.
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CN102002104A (en) 2009-08-28 2011-04-06 宜康公司 Anti-VEGF monoclonal antibody and medicinal composition containing same
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