Functional Activity Evaluation Of PD-1/CTLA4 Bispecific Antibody Base On Humanized Mice

Objective:PD-1/CTLA4 bispecific antibody is the earliest developed target combination of PD-1 bispecific antibody,and several Bs Ab drugs has entered the clinical stage.However,there are few pre-clinical head-to-head comparisons and few studies on its mechanism of action.In this study,three PD-1/CTLA4 bispecific antibodies Bs Ab1,Bs Ab2,and Bs Ab3 with different structures were constructed and expressed,and their functional activities were identified through in vitro and in vivo tests,and tumor-infiltrating lymphocyte analysis was performed to explore its potential mechanism of action.The results can provide reference for the development and clinical application of bispecific antibody.Methods:(1)Bs Ab1,Bs Ab2,Bs Ab3,Bs Ab1-Ig G1、Bs Ab2-Ig G1、Bs Ab2-SI(ADCC enhanced subtype)were constructed by gene recombination technique.(2)After transfection with CHO-S cells,the antibody was purified by Protein A affinity chromatography and its molecular weight was identified by SDS-PAGE.(3)The affinity of antibody to PD-1 and CTLA4 targets was detected by SPR.(4)Luciferase reporter gene assay and flow cytometry were used to detect the blocking activity of antibody in vitro.(5)Flow cytometry was used to detect the binding activity of antibody to cell lines.(6)Establishment of MC38-h PD-L1 colon cancer model and drug efficacy test based on B-h CTLA4-h PD-1-h PDL1 humanized mice.(7)The mechanism of PD-1/CTLA4 bispecific antibody was analyzed by tumor infiltrating lymphocytes.Results:(1)Bs Ab1,Bs Ab2,Bs Ab3,Bs Ab1-Ig G1、Bs Ab2-Ig G1、Bs Ab2-SI(ADCC enhanced subtype)were successfully constructed and expressed.(2)Bs Ab1,Bs Ab2 and Bs Ab3 can bind specifically to PD-1 and CTLA-4,among which Bs Ab1 has the highest affinity with CTLA4 and PD-1.(3)Bs Ab1,Bs Ab2 and Bs Ab3 all blocked the binding of PD-1 to PD-L1 and CTLA4 to CD80/86 in vitro.(4)Bs Ab1,Bs Ab2,Bs Ab3,Bs Ab1-Ig G1 、 Bs Ab2-Ig G1 、 Bs Ab2-SI can bind to CHO-S-h CTLA4 and CHO-S-h PD-1 cell line(5)Bs Ab1,Bs Ab2 and Bs Ab3 can effectively inhibit the growth of colon cancer cells,but Bs Ab1 and Bs Ab3 do not show the pharmacodynamic activity better than the positive control Keytruda analogues,only Bs Ab2 is better than the equivalent dose of Keytruda at 4mg/kg.(6)Different antibody subtypes were studied,and it was found that Ig G1 subtype with Fc effect function had better efficacy in vivo(p<0.01).Further analysis of tumor infiltrating lymphocytes showed that Bs Ab2-Ig G1 significantly increased the percentage of cytotoxic T lymphocytes(CTL)(p<0.05).The percentage of tumor infiltrating regulatory T(Treg)cells was significantly decreased(p<0.01),and the tumor immune microenvironment was more conducive to killing tumor.However,the Fc mutant subtype Bs Ab2-SI,which enhanced the activity of ADCC,could not further improve the antitumor activity.Conclusion:Bs Ab1,Bs Ab2,and Bs Ab3 without the Fc effect can effectively block the binding of PD-1 and PD-L1,and CTLA4 and CD80/86 in vitro.Bs Ab2 had stronger activity in blocking CTLA4/CD80/86 interaction than Bs Ab1 and Bs Ab3.Bs Ab1,Bs Ab2,and Bs Ab3 all inhibited tumor growth but did not show significant pharmacodynamic activity better than the positive control Keytruda analog rams.Different antibody subtypes were studied,and it was found that the Ig G1 subtype with Fc effect function had better efficacy in vivo.Further analysis of tumor-infiltrating lymphocytes showed that the Ig G1 subtype with Fc effect function could better clear Treg in tumor-infiltrating lymphocytes.However,further enhancement of Fc mutant subtypes of ADCC did not bring better efficacy benefits.This study provides a new idea for the development of PD-1/CTLA4 bispecific antibody.