Computational Design Of IgG-like Multispecific Antibodies Targeting EGFR、VEGF、PD-1 And CTLA-4

Checkpoint inhibitors against PD-1/PD-L1 and CTLA-4 kill tumor cells by releasing"brakes" on the immune system,inducing immune cell proliferation and activation.How to improve its efficiency in the treatment of NSCLC(less than 20%)has become the focus in current studies.Cetuximab is an antibody which blocks EGFR signaling pathway to kill tumors.FDA has approved cetuximab for the treatment of recurrent colon tumors.Clinical studies has not shown any significant survival benefit when treating NSCLC.At present,it is urgent to design new antibody drugs with better anti-tumor effect for the treatment of NSCLC.It was found that EGFR mutation could upregulate the expression of PD-L1 in tumor cells.PD-1/PD-L1 inhibitors combined with EGFR-targeting drugs could improve anti-tumor activity in patients with NSCLC EGFR mutation.In addition,a number of studies have shown that the combination of immune checkpoint inhibitors with VEGF antibodies can significantly enhance ant-itumor effects,as well as the combination of PD-1 antibodies and CTLA-4 antibodies.Multispecific antibodies is now becoming a hot spot of cancer immunotherapy research due to their unique multispecificity.Multispecific antibody is not exist in nature and we still need to develop more stable and effective antibody structure so that it can be mass-produced,prepared for clinical application.Objective:We aim To develop a multispecific antibody crossmab for both targeting EGFR,VEGF,PD-1 and CTLA-4.Optimizing the structure of antibody by computational design which could promote the binding of heterologous heavy chains to prepare a more stable and effective antibody drug for NSCLC.Methods1.Design multispecific antibody G6C225-9D95C4 Crossmab targeting both EGFR,VEGF,PD-1 and CTLA-4 and construct the eukaryotic expression vector of heavy chains and light chains respectively.2.The G6C225-9D95C4 Crossmab was expressed in FreeStyle 293-F Cells of mammalian expression system.The supernatant was collected and purified to obtain the target protein.The protein was identified by silver staining after SDS-PAGE.3.Using computational design to analyze the interaction between heavy chains in knobs-into-holes(KIH)by molecular dynamics simulation and modifying the amino acid residues in the corresponding sites to design better structures which were verified by silver staining after SDS-PAGE.Results:1.G6C225-9D95C4 Crossmab was designed based on knobs-into-holes(KIH)and crossover technology.And G6C225HV-knob/CMV,G6C225LV/CMV,9D95C4HV-CL-Fchole/CMV,9D95C4LV-CH1/CMV eukaryotic expression vector were successfully constructed.2.The four vectors were transiently co-transfected into FreeStyle 293-F Cells to obtain the target protein,which was identified by SDS-PAGE silver staining;Using computational optimization of KIH structure,the structure lock-key was successfully designed with high stability and binding efficiency of heavy chains in multispecific antibody.Conclusion:In this study,a multispecific antibody G6C225-9D95C4 Crossmab for simultaneously targeting EGFR,VEGF,PD-1 and CTLA-4 was successfully constructed and a look-key structure more favorable for dimerization of heterologous heavy chains was designed.Our findings will be beneficial to the improvement of the purity and stability of multispecific antibodies.