新型血管生长因子抗体药物开发及抗肾肿瘤机制研究

发布时间：2018-02-25 05:22

本文关键词： 血管内皮生长因子A 血管生成抗血管生成治疗兔单克隆抗体人源化抗体肾细胞　出处：《浙江大学》2009年博士论文　论文类型：学位论文

【摘要】： 背景: 血管生成对肿瘤的生长、转移起到关键作用。血管内皮细胞生长因子A(VEGF-A)的发现及对其在病理性血管生成中作用的研究引导了抗VEGF-A药物在治疗肿瘤和其他疾病抗血管治疗中的应用。大量研究表明,很多肿瘤中高表达VEGF-A,并与肿瘤的恶性程度及转移性相关。肾细胞癌(RCC)是一种常见的恶性肿瘤,恶性程度高的肾细胞癌只有有限的治疗方法,而且预后较差。这主要由于VHL抑癌基因失活在肾细胞癌中非常常见,从而上调了VEGF的表达。抗体具有高特异性和亲和力的靶向结合能力,是一种用于治疗的理想药物。至今为止,市场上主要的单克隆药物均为鼠源性的。兔抗体由于其更高的特异性、亲和力及可识别不同的抗原,已经广泛应用于研究和诊断领域。兔抗的这些特性是作为理想的治疗性单克隆抗体所必需的。但因为兔抗为多克隆抗体以及鼠单抗的较早开发,关于兔单克隆抗体在治疗领域的应用尚未有涉及。从转基因兔中分离出的浆细胞株作为与兔脾细胞淋巴细胞融合的滋养细胞,使兔杂交瘤及兔单克隆抗体成为可能。方法: 利用改进的兔杂交瘤技术我们制备了一组抗VEGF-A兔单克隆抗体(RabMAbs)。杂交瘤上清用直接ELISA方法和VEGF/VEGFR2配体受体阻断ELISA方法筛选。阳性杂交瘤的轻链与重链基因经克隆及表达后,重组抗体用于进一步特征性分析。抗体结合VEGF-A的特异性及亲合力用ELISA和BIAcore的方法测定。抑制VEGF诱导的VEGFR2受体磷酸化的作用由Western blotting和定量磷酸化ELISA方法检测。兔单抗的人源化是在对可变区的序列及同源性分析的引导下完成的,骨架区和互补决定区的非人源的位点均被替换。人源化抗体的体外生物活性通过抑制786-O细胞条件培养的HUVEC细胞生长来测定。抗体的体内抑瘤活性在人肾细胞癌裸鼠模型中验证。肿瘤标本的微血管密度用CD34免疫组织化学染色并定量。结果: 共筛选到15个与VEGF-A结合的中和抗体,可以阻断VEGF-A和受体VEGFR-2的相互作用及KDR/293细胞中VEGF诱导的受体水平磷酸化。这些抗体特异性和人VEGF-A结合,并不与其它VEGF亚型交叉,但其中12个抗体与鼠VEGF有交叉结合。人源化的抗体保持了其来源兔单抗的活性,并具有不同与Bevacizumab的抗原结合表位。细胞水平的实验中,该抗体可以抑制786-O条件培养基诱导的HUVEC细胞的增殖。我们在人肾细胞癌裸鼠模型中进一步验证了其抑制肿瘤生长的活性,在给予和Bevacizumab相同剂量和给药方案时,表现出更好的效果(p＜0.05,0.042);给予低剂量抗体(0.25mg/kg)时,仍然可以明显降低微血管密度(p＜0.01,0.0085)。结论: 我们研发了一个新型的人源化抗VEGF-A抗体可用于治疗肿瘤。抗体通过阻断受体水平的磷酸化,抑制血管内皮细胞的增殖,从而抑制血管生成,发挥抗肿瘤作用。本实验也为人源化兔抗应用于治疗领域提供了有利证据。
[Abstract]:Background:. Angiogenesis on the growth of tumors, Metastasis plays a key role. The discovery of vascular endothelial growth factor (VEGF) VEGF-A and its role in pathological angiogenesis have led to the application of anti- VEGF-A drugs in the treatment of tumors and other diseases. VEGF-A is highly expressed in many tumors and is related to the degree of malignancy and metastasis of the tumor. RCCs are a common malignant tumor, and there are only limited treatments for those with high malignancy. And the prognosis is poor. This is mainly due to the fact that VHL suppressor gene inactivation is very common in renal cell carcinoma, which upregulated the expression of VEGF. So far, the major monoclonal drugs on the market are murine. Rabbit antibodies are different because of their higher specificity, affinity and ability to recognize different antigens. Have been widely used in the field of research and diagnosis. These characteristics of rabbit antibodies are necessary for ideal therapeutic monoclonal antibodies. But because rabbit antibodies are polyclonal antibodies and mouse monoclonal antibodies were developed earlier, The application of rabbit monoclonal antibody in the field of treatment has not been involved. The plasma cell lines isolated from transgenic rabbits as trophoblastic cells fused with rabbit splenocyte lymphocytes make it possible for rabbit hybridoma and rabbit monoclonal antibodies to become possible. Methods:. Using the improved rabbit hybridoma technique, we have prepared a group of anti VEGF-A rabbit monoclonal antibody RabMAbs. The supernatant of the hybridoma was screened by direct ELISA method and VEGF/VEGFR2 ligand receptor blocking ELISA method. The light chain and heavy chain genes of the positive hybridoma were cloned and expressed. Recombinant antibodies were used for further characterization analysis. Specificity and affinity of antibody binding to VEGF-A were determined by ELISA and BIAcore methods. Inhibition of VEGFR2 receptor phosphorylation induced by VEGF was detected by Western blotting and quantitative phosphorylation ELISA methods. Humanization of resistance is guided by sequence and homology analysis of variable regions. The biological activity of humanized antibodies in vitro was determined by inhibiting the growth of HUVEC cells conditioned with 786-O cells. The tumor suppressor activity of antibodies in vivo was measured in nude human renal cell carcinoma. The microvessel density of tumor specimens was stained with CD34 immunohistochemical staining and quantified. Results:. Fifteen neutralizing antibodies binding to VEGF-A were screened to block the interaction between VEGF-A and receptor VEGFR-2 and phosphorylation of receptor levels induced by VEGF in KDR/293 cells. These antibodies specifically bind to human VEGF-A and do not intersect with other VEGF subtypes. However, 12 of the antibodies were cross-bound to mouse VEGF. The humanized antibody maintained the activity of rabbit monoclonal antibody derived from the antibody, and had different antigen-binding epitopes with Bevacizumab. This antibody can inhibit the proliferation of HUVEC cells induced by 786-O conditioned medium. We further demonstrated its inhibitory activity on tumor growth in nude mice model of human renal cell carcinoma. The results showed that the microvessel density was significantly decreased when the low dose antibody was given 0.25 mg / kg, and the microvessel density was less than 0.01 mg / kg, P < 0.05g / kg, P < 0.05g / kg, P < 0.05g / kg, P < 0.05g / kg, P < 0.01g / kg, respectively. Conclusion:. We have developed a new humanized anti VEGF-A antibody that can be used to treat tumors by blocking receptor level phosphorylation and inhibiting vascular endothelial cell proliferation, thereby inhibiting angiogenesis. This experiment also provides evidence for the application of humanized rabbit anti-tumor in the field of treatment.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392;R737.11

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