靶向抑制NRP1、KDR的双靶点新型抗体制备及其抑瘤机理研究

The occurrence, development and metastasis of malignant tumors requires adequate blood supply, and tumor angiogenesis is vital to tumor growth and metastasis for providing O2 and nutrition. VEGF is the most important angiogenesis promoting factor, tumor angiogenesis is mediated primarily through the VEGF / KDR pathway to directly enhance tumor growth. NRP1 as a non tyrosine kinase receptor of VEGF, its presence greatly enhanced the binding ability between VEGF and KDR. In tumor angiogenesis, NRP1 is an important enhancer in tumor invasion and metastasis. NRP1 is also required by Treg cells to limit anti-tumour immune responses and immune homeostasis. Lots of studies found that the use of anti-VEGF drugs for tumor treatments met drug resistance which led the failure of therapy because of the compensatory expression of other growth factors, such as NRP1, in patients or animals treated. In this study, firstly, we plan to preparate monoclonal antibodies against human NRP1. Secondly, based on the single chain antibody of KDR and reconstructed melittin which we have already generated respectively, a novel dual-to-one antibody which has the ability of inhibiting NRP1, KDR and the activity of mellitin will be generated and characterized. The novel antibody would have strong ability to block tumor angiogenesis in a dual-locking manner to block tumor angiogenesis and have the strong cytolytic activity in local tumor. The generation of the novel antibody will provide new material and good idea for anti-angiogenic tumor therapy in the future.

恶性肿瘤的的发生、发展、转移均需要充足的血液供应，肿瘤部位血管生成对于肿瘤的生长和转移提供氧气及营养至关重要。VEGF是肿瘤血管生成中起最主要作用的激活因子，肿瘤血管新生主要通过VEGF/KDR通路来直接增强肿瘤的生长。NRP1作为VEGF的非酪氨酸激酶受体，其存在大大增强了VEGF与KDR的结合能力，是肿瘤血管新生，肿瘤侵袭、转移的重要增强子，并在介导Treg细胞发挥抗肿瘤免疫、免疫自稳发面发挥着重要作用。研究发现采用抗VEGF类药物进行治疗时，机体会代偿性地高表达NRP1等因子，使得抗VEGF类的抗血管生成药物治疗失败。本研究结合临床实际，拟制备抗人NRP1单克隆抗体，结合前期工作中获得的抗人KDR单链抗体、重组蜂毒肽，制备出携带蜂毒肽的具有靶向抑制NRP1、KDR的双靶点新型抗体，以双重闭锁的方式阻断肿瘤血管生成，并发挥蜂毒肽的强大溶细胞活性，为肿瘤血管靶向治疗提供新思路、新材料。

肿瘤局部血管的生成对肿瘤发展具有着重要作用，有效遏制肿瘤局部的血管发育可促进对肿瘤的杀伤、治疗。VEGF/VEGFR信号通路是血管生成的重要通路，而人神经菌毛素1（HuNRP1）的存在有力地促进了VEGF165与其受体VEGFR2的结合，强有力地促进新生血管的生长。临床发现，采用抗VEGFR类药物进行治疗时，机体会代偿性地高表达HuNRP1等因子，使得抗VEGF/VEGFR类抗血管生成治疗失败。NRP1在肿瘤发生发展中的病理性促进作用，使其成为肿瘤治疗的一个新靶标。课题组通过构建重组HuNRP1 b结构域（HuNRP1b）蛋白制备抗HuNRP1b的单克隆抗体（McAb）。间接ELISA、间接免疫荧光（IFA）及Western-blot实验结果显示，McAb 1H4、2G7、3H4、4F1、8G9能够识别乳腺癌细胞株：MCF-7、BT474、SK-BR-3、MDA-MB-231、人白血病细胞株k562以及人脐静脉内皮细胞（HUVEC）上丰富表达的天然HuNRP1。免疫细胞化学（ICC）、免疫组织化学（IHC）结果表明本研究获得的McAbs可以特异性识别肿瘤细胞株及临床肿瘤样本中表达的HuNRP1。Transwell小室迁移、Matrigel体外成管及鸡胚尿囊膜血管生成实验及体内荷瘤实验结果均显示该McAbs具有一定程度体外抑制肿瘤细胞迁移及抑制血管生成的作用，其中McAb 1H4、8G9的作用稍强。.课题组选取稳定分泌McAb 1H4的杂交瘤细胞株，采用RT-PCR扩增其重链、轻链可变区（VH、VL），结合前期工作中获得的融合蜂毒肽的抗人KDR单链抗体（Mel-scFv(KDR），制备出携带蜂毒肽的具有靶向抑制NRP1、KDR的重组单链抗体：scFv(HuNRP1)-Mel-scFv(KDR)。Transwell实验分析表明该蛋白具有体外抑制肿瘤细胞生长的能力。.本研究制备出抗HuNRP1b McAb以及耦联蜂毒素的scFv(HuNRP1b)- Mel-scFv(KDR)双靶点重组抗体。体外实验发现McAbs以及重组双靶点抗体具有抑制肿瘤细胞迁移的能力。该特异性McAb及重组双靶点抗体的获得为进一步阐明HuNRP1的生物学功能以及靶向抑制血管生成提供重要生物材料。