全人源双特异性c-Met/PD-L1 scFv-Fc融合蛋白的优化、制备及生物学特性鉴定

Optimization,construction and biological characterization of novel bispecific c-Met/PD-L1scFv-Fc fusion protein

目的:通过优化设计、构建c-Met/PD-L1 scFv-Fc融合蛋白,探究重轻链不同组合形式对c-Met/PD-L1 scFv-Fc融合蛋白与肿瘤抗原结合的亲和性和特异性的影响。方法:使用生物信息学分析、基因工程抗体技术设计、优化及制备重轻链不同组合的双特异性c-Met/PD-L1 scFv-Fc融合蛋白CP1、CP2、PC1、PC2;生物分子相互作用分析仪BLItz分析融合蛋白对重组c-Met蛋白和PD-L1蛋白的亲和力,ELISA法分析其抗原结合特异性。结果:成功制备双特异性c-Met/PD-L1 scFv-Fc融合蛋白,融合蛋白CP1与重组c-Met蛋白和PD-L1蛋白的亲和力和抗原结合特异性均优于CP2、PC1和PC2。结论:重轻链不同组合形式会影响c-Met/PD-L1 scFv-Fc融合蛋白与c-Met和PD-L1蛋白的亲和力和抗原结合的特异性,融合蛋白CP1的亲和力和抗原结合的特异性最高,其对应的重轻链组合形式可用于c-Met/PD-L1 CAR表达载体的构建。

Objective:To optimize,screen and identify the bispecific c-Met/PD-L1 scFv fusion protein and to detect whether the different combinations of heavy chains and light chains can influence the biological activity of bispecific scFv fragments. Methods:Bioinformatics analysis,technique of gene engineered antibodies were introduced to design,optimize and construct bi-specific scFv fusion proteins. BLItz was used to analyze the affinity of those bi-specific scFv fusion proteins to c-Met and PD-L1,ELISA assay was used to detect their specific binding ability. Results:Bispecific scFv fusion proteins were produced successfully,BLItz and ELISA detection confirmed that the bispecific scFv fusion protein CP1 has higher affinity and specific binding ability. Conclusion:Different combinations of heavy and light chains can affect the affinity and specific binding activity of c-Met/PD-L1 scFv-Fc fusion proteins,CP1 has the strongest affinity and specificity for binding to c-Met and PD-L1 protein,it can be applied to the construction of c-Met/PD-L1 expression vector and subsequent research of c-Met/PD-L1 CAR-T cells.