A bispecific nanobody targeting the dimerization interface of epidermal growth factor receptor: Evidence for tumor suppressive actions in vitro and in vivo

• Published in: Biochemical and biophysical research communications 2021-04, Vol.548, p.78-83
• Main Authors: Xu, Zhimin, Qiu, Chuangnan, Wen, Biyan, Wang, Shuang, Zhu, Linfeng, Zhao, Lin, Li, Huangjin
• Format: Article
• Language: English
• Subjects: Anti-Tumor; Bispecific nanobody; Dimer interface; EGFR; Heterodimerization
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2021.02.059

Targeting the dimer interface for the epidermal growth factor receptor (EGFR) that is highly conserved in the structure and directly involved in dimerization may solve the resistance problem that plagues anti-EGFR therapy. Heavy chain single domain antibodies have promising prospects as therapeutic antibodies. A bispecific nanobody was constructed based on previously screened humanized nanobodies that target the β-loop at the EGFR dimer interface, an anti-FcγRIIIa (CD16) of natural killer cells (NK) nanobodies and anti-human serum albumin (HSA) nanobodies. The target gene was effectively expressed and secreted while controlled by promoter GAP in Pichia pastoris X33, and the expressed product was purified with a cation exchange and nickel chelation chromatography. The bispecific nanobody specifically bound to the surfaces of EGFR-overexpressed human epidermal carcinoma A431 cells and effectively inhibited tumor cell growth both in vitro and in vivo. In the A431 cell nude mouse xenograft model, the growth inhibition effect from the bispecific nanobody was significantly increased with the assistance of peripheral blood mononuclear cells (PBMCs), which was consistent with the results obtained in vitro, suggesting that there was an antibody-dependent cell-mediated cytotoxicity (ADCC) effect. In addition, the intraperitoneal administration of bispecific nanobodies effectively reached tumor tissues in the shoulder dorsal region, but in significantly less distributed quantities than EGFR Dimer Nb77. To conclude, a bispecific nanobody targeting the EGFR dimer interface with ADCC effect was successfully constructed. •The first reported bispecific nanobody that targets the EGFR dimer interface.•A bispecific nanobody was designed to resolve the drug resistance issue for anti-EGFR therapy.•An antibody can be efficiently produced in Pichia pastoris.•The bispecific nanobody exhibited growth inhibition and ADCC effects.