基于高活性突变MICA的融合抗体激活NKG2D途径免疫监视作用及机制研究

NKG2D-mediated recognition of malignant cells by cytotoxic lymphocytes is enabled through the tumor-associated expression of NKG2D ligands, MICA/MICB proteins. Shedding of MICA is thought to constitute a major counter mechanism of tumor cells to subvert NKG2D-mediated immunosurveillance. In previous studies, we designed and produced a novel antibody fusion protein (mAb04-MICA) based on mAb04 (an anti-VEGFR2 antibody) and MICA. The mAb04-MICA retained the anti-tumor activity, and had capacity to further strengthen the immune surveillance of NK cell activated by NKG2D pathway. However, the MICA banding to NKG2D with low affinity, and down-regulating the expression of NKG2D, resulting in limitation of the immune surveillance mediated by NKG2D. According to these findings, this program would develop a mutan MICA with high affinity and activity, and then used to anti-tumor with immunosurveillance mediated by NKG2D. Firstly, the phage display library will constructed from the gene sequence of MICA with mutantion, and clones with high affinity will be selected accordingly. After establishing cell model, the high activity clones will be further selected. Secondly, we will generat a novel antibody fusion protein (MICA-scFv) consisting of the mutant MICA and a single-chain antibody fragment targeting tumor cells (scFv).Finally, investigate the activation and mechanism of immunosurveillance mediated by NKG2D based on the mutant MCIA fusion antibody.

NKG2D通过表达在肿瘤表面的配体MICA/MICB介导淋巴细胞识别和杀伤肿瘤细胞。MICA的脱落是肿瘤逃避NKG2D介导的免疫监视作用的重要机制。在前期研究中，我们基于mAb04（一种VEGFR2单抗）设计了一种融合抗体mAb04-MICA。其在保留了抗肿瘤作用的同时可激活NKG2D途径加强免疫监视作用。然而MICA与NKG2D的亲和力低，且可下调NKG2D的表达，限制了MICA-NKG2D介导的免疫监视作用。为此，本项目拟开发一种对NKG2D同时具有高亲和力和高刺激活性的MICA突变体，通过激活NKG2D途径介导的免疫监视发挥抗肿瘤作用。以突变MICA的基因序列构建噬菌体展示文库，筛选出高亲和力的MICA突变株，继而通过构建突变MICA阳性表达细胞模型筛选出高活性的MICA突变体。与肿瘤靶向单链抗体融合后，利用细胞实验和动物模型实验研究融合抗体激活NKG2D途径的免疫监视作用及机制。

NKG2D可通过表达在肿瘤表面的配体MICA介导淋巴细胞识别和杀伤肿瘤细胞，MICA的脱落是肿瘤逃避NKG2D介导的免疫监视作用的重要机制。在前期研究中，我们基于mAb04（一种VEGFR2单抗）设计了一种融合抗体mAb04-MICA，其在保留了抗体抗肿瘤作用的同时通过激活NKG2D途径加强自然杀伤细胞的免疫监视作用。然而MICA与NKG2D的亲和力低，限制了MICA-NKG2D介导的免疫监视作用。本项目以随机突变MICA的基因序列成功构建噬菌体展示文库，其有效库容量达到4×10^13，通过5轮淘选，筛选出与NKG2D具有高亲和力的MICA突变株（mMICA），亲和力比野生型MICA提高一个数量级；mMICA与西妥昔单抗可变区构成的单链抗体（scFv）融合后，利用细胞实验和肿瘤动物模型实验验证了融合抗体的体内外抗肿瘤作用，并进一步通过ELISA、免疫组化等实验初步研究了融合抗体的作用机制。本项目为肿瘤免疫治疗提供了新的研究手段和分子工具。