Objective To construct a bispecific antibody targeting epidermal growth factor receptor(EGFR)and OX40 and evaluate the function for tumor-specific T cell activation.Methods The gene of anti-OX40/anti-EGFR bispecific antibody was cloned into eukaryotic expression vector,and then the constructed vector were transfected to 293F cells for the bispecific antibody purification.The binding activity of anti-OX40/anti-EGFR bispecific antibody with the cells expressing target proteins were detected by flow cytometry.To identify the activation of T cells mediated by anti-OX40/anti-EGFR bispecific antibody,the activation of NF-κB signal activation was evaluated by Jurkat-OX40-NF-κB-GFP reporter cells and the activation of primary T cells was detected by interleukin-2(IL-2)and interferon-γ(IFN-γ)secretion of peripheral blood mononuclear cell(PBMC).Results Anti-OX40/anti-EGFR bispecific antibody was successfully constructed and purified,and its binding ability to HEK293 cells expressing OX40 and EGFR was verified.Jurkat-OX40-NF-κB-GFP reporter cells were activated by the bispecific antibody with the crosslinking of A549 cells.Further,the anti-OX40/anti-EGFR bispecific antibody promoted the secretion of IL-2 and IFN-γ of PBMC.Conclusion Anti-OX40/anti-EGFR bispecific antibody was successfully constructed which could specifically recognize OX40 and EGFR and activate tumor specific T cells.
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