靶向PD-1/PD-L1的非天然核酸适配体的体外筛选及其在肿瘤免疫治疗中的应用探索

Tumor cells escape immunological surveillance by interacting with immune checkpoint proteins and inhibiting T cell activation. One of the key inhibitory immune signaling pathways involves the interaction of programmed death-1 (PD-1) protein and its ligand (PD-L1). PD-1:PD-L1 blockade using anti-PD-1 monoclonal antibodies such as nivolumab (Opdivo, Bristol-Myers Squibb) and pembrolizumab (Keytruda, Merck) has proven to be an effective strategy to overcome immune evasion in patients with various cancers such as melanoma and non-small cell lung cancer. However, monoclonal antibody therapy inherently possesses some disadvantages including high manufacturing costs, long production cycle and immunogenicity with repeated administration. Aptamers are a new class of nucleic acid molecules capable of recognizing and binding a wide variety of targets with high affinity and specificity. Aptamers are identified rapidly by an in vitro selection and amplification process, and can be synthesized in a large scale by solid-phase synthesis. I hereby propose to isolate TNA, which is a type of unnatural nucleic acids, aptamers with affinity towards protein targets PD-1 and PD-L1. Compared with DNA and RNA, these TNA molecules contain unnatural backbones and exhibit intrinsic resistance to nuclease-mediated degradation. Importantly, these aptamers 1) exhibit strong affinity and high specificity, which is comparable to antibodies, towards their targets; 2) possess superior stability than natural nucleic acid counterparts in biological environment; 3) lack immunogenicity which often causes immune-related adverse events. Those aptamers with the ability to interfere with or block the PD-1:PD-L1 interaction would be studied further for their anti-tumor effects. Therefore, it is believed that the unnatural nucleic acid aptamers targeting PD-1/PD-L1 would have a promising prospect in future cancer immunotherapy.

肿瘤细胞可通过结合免疫检查点蛋白，抑制T细胞激活，从而逃避免疫杀伤。程序性死亡蛋白-1（PD-1）与配体PD-L1之间的相互作用是其中一条关键的免疫抑制信号通路。基于单克隆抗体技术的PD-1抑制剂已在多种肿瘤适应症中取得了良好的治疗效果，但是目前的抗体药物仍然存在成本高、周期长、易引发免疫相关不良反应等问题。核酸适配体是一类具有识别功能的单链寡核苷酸分子，其序列可以通过体外筛选技术结合高通量测序快速鉴定获得，并可以通过固相合成实现大规模生产。本项目计划利用体外筛选技术分离靶向PD-1/PD-L1信号通路的非天然核酸TNA适配体，并评估此类适配体分子作为PD-1/PD-L1抑制剂对肿瘤生长的抑制作用。该类适配体不但具有可与抗体媲美的高亲和力和特异性，而且拥有比天然核酸更好的生物稳定性，同时在体内不具免疫原性。因此，靶向PD-1/PD-L1的非天然核酸适配体在肿瘤免疫治疗中拥有广阔的应用前景。

免疫检查点疗法是近年发展迅速的肿瘤治疗新策略，靶向PD-1/PD-L1信号通路的单克隆抗体类药物已展现了良好的临床效果。抗体类药物存在生产成本高、易引发免疫相关不良反应等缺陷。本项目发展了基于非天然核酸适体的免疫检查点抑制剂，并展现了在肿瘤免疫治疗方向的应用潜力。本项目首先化学合成了苏糖核酸的核苷酸单体分子，并在适宜的聚合酶的催化下产生了随机序列的苏糖核酸文库，利用体外筛选技术鉴定了结合PD-L1靶蛋白的苏糖核酸适体；这些苏糖核酸适体结合PD-L1蛋白的亲和力在100-1000 nM范围内，其中N5和S42序列能够有效抑制PD-1/PD-L1之间的相互作用；最后，在荷瘤小鼠模型上，苏糖核酸适体N5展现了良好的抗肿瘤效果。本项目为肿瘤的免疫治疗提供了基于非天然核酸适体的新型免疫检查点抑制剂。