Abstract: 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.

Key words: OX40, epidermal growth factor receptor (EGFR), bispecific antibody (BsAb), cancer, T cell activation