基于VEGFR2单抗的融合抗体分子设计及其激活NKG2D途径免疫监视作用的研究

NKG2D-mediated recognition of malignant cells by cytotoxic lymphocytes is enabled through the tumor-associated expression of NKG2D ligands，MIC proteins. Shedding of MIC-A is thought to constitute a major counter mechanism of tumor cells to subvert NKG2D-mediated immunosurveillance. In this project, we proposed the generation of a novel antibody fusion which consists of an anti-vascular endothelial growth factor receptor 2 (VEGFR2) antibody and one main ligand (MIC-A) of NKG2D, and postulated that the particular antibody fusion will both stimulate NKG2D and enhance antitumor activity of the parental antibody. Firstly, the heavy chain will be obtained by overlapping PCR between our patented anti-VEGFR2 antibody, linker and MIC-A gene, named H'chain. Subsequently the H' chain will be ligated with either plasmid pMH3 or pCA and recombinant plasmid pMH3-H', pCA-H'will be obtained. After co-infection to CHO cells with original pMH3-L and pCA-L, recombinant cell colonies with high production of fusion antibody will be screened and preserved. The expression will be analyzed by Western blot and ELISA, following by cultivation in serum free media. The affinity and biological activity of the fusion to VEGFR2 and NKG2D will be evaluated by flow cytometry, ELISA, etc., respectively. Also, the pathway of tumor apoptosis will be studied. Furthermore, the enhanced ADCC effect will be studied by in vitro LDH test, MTT assay. Finally, in vivo antitumor activity will be evaluated in NOD/SCID mice and the near infrared imaging of antibody targeted specific tumor.

免疫系统免疫监视作用的一个重要的机制就是肿瘤细胞表面MHC-I相关抗原分子MIC-A/B的表达，MIC-A的脱落则发生肿瘤免疫"逃逸"。本项目提出构建一种融合抗体，借助于靶向肿瘤细胞表面抗原血管内皮生长因子受体（VEGFR2）的抗体，以抗体融合的MIC-A将 NK细胞聚集，既可以重建由NKG2D激活的机体自身的肿瘤免疫监视作用，又可以增强抗体的ADCC效应。以本课题组专利抗体Mab-04为基础构建融合抗体基因并在CHO细胞表达,进行高产细胞株的无血清驯化、发酵与抗体的纯化；监测抗原/抗体作用的动力学过程，并通过MTT实验、流式细胞检测等技术进行体外与抗原VEGFR2结合相关活性和功能、抗体的抗肿瘤活性与肿瘤细胞凋亡原理进行研究。抗体MIC-A部分的功能则通过体外与NKG2D的相互作用和增强NK细胞的ADCC效应来评价。最后，应用近红外技术实时监测抗体靶向体内肿瘤与肿瘤变化的过程。

免疫系统发挥免疫监视作用的一个重要机制就是肿瘤细胞表面MHC-I相关抗原分子MICA/MICB的表达，MICA的脱落则会发生肿瘤免疫“逃逸”。本研究构建一种融合抗体mAb04-MICA，借助于靶向VEGFR2的抗体部分识别肿瘤细胞，并通过抗体融合的MICA分子募集和活化NK细胞，既可以发挥抗体的抗血管生成、抗肿瘤作用，又可以重建NKG2D途径介导的机体自身的肿瘤免疫监视作用。本文首先以mAb04基因序列为基础，通过柔性肽Gly4Ser将mAb04 的重链C末端及人MICA胞外1-3区基因连接，获得新的融合基因H’-Chain。使用HiTrap Protein A 亲和层析柱分离纯化目的蛋白，SDS-PAGE和Western blot鉴定结果说明mAb04和mAb04-MICA表达及装配正确。FCM和SPR结果揭示了mAb04和mAb04-MICA与KDR3的高亲和力，亲和力常数达到10-9 M；mAb04-MICA和NKG2D的亲和力常数为7.10×10-7 M。MTT实验结果表明mAb04-MICA能有效抑制内皮细胞、乳腺癌、胃癌及肺癌细胞的的增殖，流式凋亡和周期实验表明mAb04-MICA能够有效诱导肿瘤细胞的凋亡并将肿瘤细胞阻滞在G1期。细胞蛋白Western blot的结果显示，VEGFR2通路下游信号蛋白的磷酸化和凋亡通路抑凋亡蛋白均显著下降，而促凋亡蛋白表达量显著提高。另外，mAb04-MICA通过融合MICA，介导免疫细胞对肿瘤细胞的杀伤能力显著提高。本文接下来建立裸鼠乳腺癌、胃癌及肺癌移植瘤模型，给药后检测皮下移植瘤的肿瘤体积与瘤重，初步研究了双功能抗体mAb04-MICA的抗肿瘤活性；免疫组化测定肿瘤组织中Ki-67、VEGF、CD31的表达情况，验证了mAb04-MICA可以显著抑制肿瘤细胞增殖及新生血管生成；免疫荧光实验验证了mAb04-MICA可以显著提高肿瘤病灶部位NK细胞表达以及细胞因子IFN-γ和TNF-α的释放。综上所述，mAb04-MICA可以抗肿瘤新生血管生成、抑制肿瘤细胞增殖并激活NKG2D以发挥免疫调节作用，以达到良好的体内抗肿瘤效果。综上所述，双特异性抗体在肿瘤的治疗提供了新的方向，且MICA作为一种免疫细胞激活型受体的配体，融合单克隆抗体的设计亦可应用于其他抗体药物在抗肿瘤领域治疗的方案。