鲨鱼天然抗体IgNAR超稳定分子机制及其特异性纳米抗体(Nb/PD-L1)筛选鉴定与抗肺癌活性研究

Rich and diverse marine biological resources, not only are important sources of medicine and food for human health, but also provide rare inspiration for biomedical researches. According to the reports the sharks, are marine ancient as well as the dinosaurs at the Jurassic, and they are almost no cancer, and scientists newly discovered the IgNAR antibody with only heavy chain structure still maintained high activity and super stability in blood with urea up to 25% (more than 10 times in human blood) and high concentrations of heavy metals and other protein strong denaturant, which no doubt provides new ideas and resources for develop new anticancer drugs. We will study the causes of IgNAR ultrastability in the high concentration and strong denaturing agent (urea and mercury) and reveal the molecular mechanism of the ultrastability of the marine shark natural antibody; according to the basic framework of their active key variable region, we will construct phage nanobody library by saturation mutagenesis, screen the nanobody (Nb/PD-L1) targeting the programmed death 1 ligant 1(PD-L1), and then express, purify, test of affinity and stability and evaluate the activity by lung cancer cell and animal models in order to obtain better anti-PD-L1 antibody than currently reported in activity and stability as new drug candidates, which will provide theoretical references and innovative varieties for research and development of protein new drugs for China.

丰富多样的海洋生物资源，不仅是人类健康重要的药食来源，而且为生物医药研究提供了难得的启示。据报道与恐龙同在侏罗纪出现的海洋古老物种鲨鱼，几乎不得癌症，最新发现其仅有重链结构的IgNAR抗体在尿素高达25%（为人血的10倍以上）和高浓度重金属等蛋白强变性剂的血液中，依然保持极高活性和超级稳定性，这无疑为研发抗癌新药提供了崭新思路和资源。本研究拟经克隆表达和活性区构效解析，研究海洋鲨鱼天然抗体IgNAR在高浓度强变性剂（尿素与汞）下仍保持稳定的原因，揭示其超级稳定的分子机制；并以其起抗体活性的可变区为基础结构，利用饱和突变法构建噬菌体纳米抗体库，筛选针对程序性死亡因子1配体1(PD-L1)的纳米抗体Nb/PD-L1，进行表达纯化与亲和性和稳定性测定后，用肺癌细胞和动物模型对其活性进行评判，以期获得比目前报道的PD-L1单抗活性和稳定性更优的候选新药，为我国蛋白类新药研发提供理论借鉴和原创品种。

本研究利用生物信息学在分析超过150条鲨鱼纳米抗体的基础上优化设计了一种新型的鲨鱼 V-NAR 框架，并使用 NNK 的方法在 CDR3 区中引入突变，构建获得了库容为 1.9×109 cfu 的基于鲨鱼抗体 IgNAR 可变区（V-NAR）的合成噬菌体库。此外，从噬菌体库中筛选获得了 NbP1、NbP2、NbP3、NbP4 和 NbP5 等 5 株 Anti-PD-L1纳米抗体，这 5 株纳米抗体均与 PD-L1 特异性结合，其中NbP3、NbP4 的亲和力达到 107 M-1，并可有效阻断 PD-1 与 PD-L1 结合，同时该纳米抗体也具有良好的耐热性和耐变性剂性质，在60℃下仍可保持 50%以上的活性。因此本文所设计的新型 V-NAR 基因框架可作为鲨鱼 V-NAR 抗体库的通用骨架，为抗体库的构建提供新的理论基础；本课题构建的基于鲨鱼抗体 IgNAR 的 V-NAR 噬菌体库具有库容大，多样性良好的特征，可作为不同抗原的筛选平台，用于其它抗原的筛选，具有极高的生物医药应用价值；本研究所筛选的Anti-PD-L1 纳米抗体，具有良好的稳定性和亲和力，能有效阻断细胞表面PD-L1和PD-1de结合，具有潜在应用价值，可为新型抗肿瘤药物研发与获得提供新品种。