特异抑制G蛋白偶联受体GPR52的全合成人源化纳米抗体的鉴定及验证

纳米抗体相对于化学小分子，具有更高亲和力和特异性，相比传统单抗，具有高理化稳定性、高溶解度、快速组织渗透能力、易通过血脑屏障、易于生产储存等优势。四大神经退行性疾病之一的亨廷顿病尚无有效的治疗方案，而通过纳米抗体阻断孤儿G蛋白偶联受体GPR52是种可行的治疗策略。我们拟在前期已优化GPR52表达纯化的基础上，通过本实验室已建立的高通量全合成人源化纳米抗体库，分别进行核糖体展示和噬菌体展示筛选，得到10个左右亲和力在纳摩尔级别的GPR52纳米抗体，并通过SPR，ITC和MST等方法验证其作用的动力学热力学参数，以下游cAMP为检测指标获得特异抑制GPR52的纳米抗体，在细胞模型内对抑制型纳米抗体降低变异亨廷顿蛋白效果进行验证。我们还将通过共结晶的方式，解析GPR52与纳米抗体复合物的结构，明确作用的分子机理。本项目的顺利实施，可为亨廷顿舞蹈病等神经退行性疾病的抗体治疗研究提供一种新的选择。

Compared with small molecular weight chemical compounds, nanobodies have much higher affinity and specificity. Compared with conventional antibodies, nanobodies have the advantages of high stability, high solubility, easy passing the blood-brain barrier, easy to produce, store and use. .There is no effective treatment for Huntington's disease, one of the four major neurodegenerative diseases. Blocking orphan receptor GPR52 is a possible way of treatment for Huntington's disease. We wish to obtain 10 GPR52 specific nanobodies with the affinity at the nanomole range through the ribosome display and phage display screening of high-throughput full-synthetic humanized nanobody library established in our lab. We will further verify the kinetics and thermodynamic parameters of biding to GPR52 through ELISA, SPR, ITC and other methods. We will further verify 2-3 specific inhibition nanobodies of GPR52 by downstream cAMP HTRF assay, and verify the lowing effect of mutant huntingtin proteins in the established cell model. To elucidate the molecular mechanism of action, we will also try to solve the GPR52-nanobody complexes’ structure by co-crystallization. The successful implementation of this project can provide a new option for antibody therapy research for neurodegenerative diseases such as Huntington’s disease.