强啡肽单抗融合蛋白的克隆、表达及功能分析

G蛋白偶联受体(GPCR)长期以来是最重要的药物靶点家族,小分子药物层出不穷。然而受研发难度的限制,针对GPCR的抗体或大分子类药物屈指可数。我们利用选择性靶向κ阿片受体(KOR)且无法激活下游信号的单克隆抗体连接强啡肽(Dynorphin)基因、HEK 293F系统进行表达,纯化获得KOR强啡肽单抗融合蛋白,我们将此融合蛋白命名为APF(antibody-peptide fusion)。结果显示,获得的单抗融合蛋白二级结构未显著改变,保持了Dynorphin活性,可激活KOR相关下游蛋白(Gi)活性,调动β-arrestin信号。结果证明,基因层面实现抗体药物改构的可行性,该法可指导新一代抗体偶联药物的改造,为以GPCR为靶点的大分子药物开发提供了新的空间。

The cloning,expression and functional analysis of Dynorphin-monoclonal antibody fusion protein

G protein-coupled receptors(GPCRs) present the most valuable targets for decades, the majority of which are small molecules or peptides, though. Development of functional biologics targeting on GPCRs is technically difficult yielding limited success. Here, we genetically fused a peptide ligand Dynorphin to a nonfunctional monoclonal antibody targeting on κ opioid receptor(KOR) and obtained a fusion protein hereafter we named as antibody-peptide fusion(APF) by HEK293 F expression system. The results showed that without alteration of the antibody secondary structure and conformation, the APF acquired the agonist activity from Dynorphin, such as activating the KOR related downstream protein(Gi) signaling, and recruiting the β-arrestin signal. Together, we proved that it is feasible to modify non-functional antibody on gene level and convert it to a fusion protein with desired function. This method can accelerate the pace of biologics development for GPCRs and open up new opportunity for next-generation GPCR therapeutics.