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

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。

Optimization, construction and biological characterization of novel bispecific c-Met/PD-L1 scFv-Fc fusion protein

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. Result 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 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.