Computational Design Of Novel Bispecific Antibodies Targeting Both EGFR And PD1 And Preliminary Identify

Lung cancer is the most common malignancy in China, 2017 National Cancer Center released the latest data show that the incidence and mortality of lung cancer are ranked first, it has been quite the level of developed countries. A total of 85%cases of lung cancer are non-small cell lung cancer (NSCLC).The efficacy of conventional chemotherapy regimens for NSCLC is still limited,new treatment methods have become hot spots. Cetuximab(C225) is a chimeric monoclonal antibody that competitively binds the extracellular domain of the EGFR found on the surface of various cells, and plays a role in regulating cell growth, proliferation and differentiation. Structurally, Cetuximab, 150KDa transmembrane receptors,is composed of the variable regions of the murine EGFR monoclonal antibody and human EGFR with human IgGl heavy and K-light chain constant regions.The binding of cetuximab to the EGFR blocks phosphorylation and activation of receptor-associated kinases resulting in inhibition of cell growth, induction of apoptosis, and reduced VEGF production.It is approved for treatment of metastatic colorectal cancer and locally advanced or metastatic squamous cell cancer of the head and neck.In a variety of solid tumors,overexpression of the EGFR is ubiquitous.At present,experiments and studies in vivo have reported that Cetuximab has a role in inhibiting proliferation and promoting apoptosis in NSCLC.Clinical trials have shown that Cetuximab combined chemotherapy versus chemotherapy alone can prolong the median survival of patients, but there is no significant difference. Immune checkpoint inhibitor is a kind of new antibody medical blocking the inhibitory signal to activate the specific immune cells for killing the tumor cells ,have attracted the attention of global scholars.Programmed Death 1 and its Ligand 1 (Programmed death receptor-1,PD-1 / PD-L1) is a pair of immunosuppressive factors,promoting the formation of immune inhibitory microenvironment, inhibiting T cells functions and resuleing in tumor immune escape.In some solid tumors, such as renal cell carcinoma, malignant melanoma,NSCLC, Hodgkin’s lymphoma, head and neck squamous cell carcinoma,anti-PD1 antibody has been proven that the efficacy is superior to traditional chemotherapy.Nivolumab (Opdivo, 5C4) is an anti-PD1 antibody,blocking the binding of PD1/ PDL1 in order to restore T cell functions in the immune microenvironment and inhibition of tumor cell proliferation and diffusion.Although the efficacy of antibodies is better than conventional chemotherapy, but because the complexity of tumor development mechanism and itself heterogeneity, only through the monoclonal antibody or blocking a single signal to activate the body immune response and anti-tumor effect still be restricted.Thus, the design and optimization of specific antibodies with multifunctional and dual target structures is becoming increasingly important in the field of tumor therapy.This bispecific antibody (BsAB) can further enhance the antitumor effect by mediating cytotoxicity, mediating or blocking signal pathway, guiding functions, and so on.In this study, we used Cetuximab and Nivolumab,a EGFR antibody and a PD1 antibody, respectively as a model to optimize the EGFR / PD-1 double-targeted antibody by computational design. Under natural conditions, this bispecific antibody is absent. Our design of the antibody structure includes the structure of CrossMab and knob-into-hole. Based on this design, we constructed the eukaryotic expression vector of heavy and light chain with two kinds of antibodies by using genetic engineering technique. The expression of Cetu-PD1 CrossMab was expressed by Freestyle TM 293F cell expression system, by SDS-PAGE to identify its molecular weight and structural integrity. The results showed that the gene sequence of Cetu-PD1 CrossMab was successfully constructed and the molecular weight and structure of the expressed antibody were identified. The expression of Cetu-PD1 CrossMab was similar to that of the whole antibody. The expression of Cetu-PD1 CrossMab could be further biologically experiment.