Construction And Biological Function Of Anti-VEGF-PD-1 Bispecific Recombinant Antibody

Objective: The anti-PD-1 scFv-Fc antibody,anti-VEGF scFv-Fc antibody and VEGF-PD-1 bispecific antibody were constructed and expressed by molecular biological methods,and their biological functions were researched at protein and cell levels.Methods: 1.Anti-VEGF scFv-Fc antibody plasmid and anti-PD-1 scFv-Fc antibody plasmid were contructed by digestion/connection and homologous recombination.2.293 F cells were used to express antibody,then the antibody was purified by protein A resin.3.ELISA was used to detect the affinity of anti-PD-1 scFv-Fc antibody and PD-1 antigen,anti-VEGF scFv-Fc antibody and VEGF antigen,respectively.4.ELISA was also used to detect the activation ability of PMA and calcium ionophores to Jurkat cells;Flow cytometry was used to detect the PD-1 expression level of Jurkat cells after stimulation and the binding of anti-PD-1 scFv-Fc antibody to PD-1.5.Whether the anti-PD-1 scFv-Fc antibody can reactivate Jurkat cells inhibited by PC3 prostate cancer cells was observed by detecting IL-2.6.Whether anti-VEGF scFv-Fc antibodies can inhibit the vitality of HUVEC vascular endothelial cells was observed by CCK-8.7.The protein sequence of the bispecific antibody with excellent affinity and stability was analyed through Discovery Studio software.8.The bispecific antibody had been synthesized and expressed through 293 F cells.9.The bispecific antibody sequences had been optimized and synthesized.Results: 1.The anti-PD-1 scFv-Fc antibody plasmid and the anti-VEGF scFv-Fc antibody plasmid were successfully constructed by the methods of restriction enzyme digestion and homologous recombination.2.The positive control anti-PD-1 scFv-Fc antibody and positive control anti-VEGF scFv-Fc antibody have been successfully expressed by 293 F cells and purified by protein A resin.The antibody concentration was above 100μg/ml.3.The expressed positive control anti-PD-1 scFv-Fc antibody and positive control anti-VEGF scFv-Fc antibody can bind to PD-1 protein and VEGF protein by ELISA with a strong affinity.4.PMA and calcium ionophore could smoothly activated Jurkat cells to secrete IL-2 and express PD-1.When the positive control anti-PD-1 scFv-Fc antibody reaches to 10μg/ml,the antibody is excessive and can bind to PD-1 on Jurkat cells.5.The co-culture of PC3 cells and Jurkat cells restrained the activity of Jurkat cells,and the concentration of cytokine IL-2 was clearly decreased.The effect of positive control anti-PD-1scFv-Fc antibody needs to be further confirmed on Jurkat cells activation.6.Compared with control group,the vitality of HUVEC cells significantly reduced after adding positive control anti-VEGF scFv-Fc antibody.7.The bispecific antibody structure with good affinity and stability was successfully simulated and screened out by DS software.8.The bispecific antibody was successfully expressed,but the expression level was low.9.The sequence of the bispecific antibody has been optimized and is currently being synthesized.Conclusion: In this subject,the positive control anti-PD-1 scFv-Fc antibody and positive control anti-VEGF scFv-Fc antibody can bind to the corresponding antigen.Positive control anti-VEGF scFv-Fc antibody can exert its anti-tumor biological function at the cellular level.The antineoplastic function of positive control anti-PD-1 scFv-Fc antibody is being studied and needs to be ulteriorly comfirmed.The study of anti-PD-1 antibody and anti-VEGF antibody laid the foundation for the study of the method and data of bispecific antibodies.