Computational Design Of IgG-like Bispecific Antibodies Targeting EGFR And PD-L1

Epidermal growth factor receptor is a glycoprotein, overexpression of which is closely related to the proliferation and metastasis of lung cancer, head and neck cancer,breast cancer, prostate cancer and colorectal cancer. Cetuximab is a human mouse chimeric antibody that can specifically target human epidermal growth factor receptor and block the EGFR signaling pathway. Although cetuximab has been succeed in the treatment of colorectal cancer, compared with standard chemotherapy, the significant improvement was not observed in the treatment of non-small cell lung cancer(NSCLC).Recently, blocking PD-1/PD-L1 signaling pathway with antibody drugs has made a breakthrough progress in preclinical and clinical trials of cancer therapy. Numerous of research articles published in Science, Nature and Cell have reported that PD-1/PD-L1 blocking antibodies could modulate the tumor microenvironment, and eventually eradicate tumor cells effectively. Clinical trials have demonstrated that PD-1/PD-L1 blocking antibodies are promising therapeutic antibodies against NSCLC. FDA has approved PD-L1 antibody atezolizumab for the treatment of patients with metastatic NSCLC whose disease progressed during or following platinum-containing chemotherapy.Antibody drugs have been widely used in clinical treatment for cancer, however,due to the diversity and heterogeneity during tumorigenesis,development and recurrency,most of cancer after treatment with monoclonal antibody could ultimately develop recurrences. There is an urgent need to develop novel antibodies with potant anti-tumor efficacy.Bispecific antibody with able to recognize multiple target proteins has been demonstrated with more advantages than monoclonal antibody against cancer.Nowadays,two bispecific antibodies have been approved for cancer treatment. A large number of bispecific antibodies are evaluate in clinical trials. However,the study of bispecific antibody targeting both EGFR and the PD-L1 has not been reported. A key point in this study is design of novel bispecific antibody with potent anti-tumor efficacy against NSCLC.Objective:Computational design of IgG-like bispecific antibodies, using crossmab technology to construct bispecific antibody targeting EGFR and PD-L1,and expect to get more effective antibody in the treatment of non-small cell lung cancer.Methods:1. In this study, EGFR antibody Cetuximab and PD-L1 antibody Atezolizumab were used as parental antibody to design of novel EGFR and PD-L1 bispecific antibody with IgG architecture.2. We used FreeStyle 293-F Cells mammalian expression system to express the bispecific antibody. The purified protein was identified by Western-blot technique.3. Cell bingding of EGFR-PD-L1 CrossMab was assessed in vitro celluar assays by flow cytometry.Results:1. We designed EGFR-PD-L1 CrossMab against EGFR and PD-L1 based on the"knobs into holes" and "crossover" technology, constructed four chains as follow: C225H-knob/cmv?C225L/cmv?RG74H-CL-FChole/cmv?RG74LV-CH1/cmv.2. We constructed two heavy chain expression vectors and two light chain expression vectors which were co-transfected in FreeStyle 293F cells.EGFR-PD-L1 CrossMab protein from culture medium was purified by protein A and verified by SDS-PAGE.Conclusion:We susscessfully constructed a bispecific antibody for targeting EGFR and PD-LI,that is EGFR-PD-L1 CrossMab, and we made a preliminary identification.