A Novel Bispecific Antibody Targeting EGFR and VEGFR2 Is Effective against Triple Negative Breast Cancer via Multiple Mechanisms of Action

Simple Summary Triple-negative breast cancer (TNBC) accounts for approximately 10–20% of all diagnosed breast cancers and is often associated with a poor prognosis. There is therefore an urgent need to develop novel and targeted therapeutic approaches against TNBC. Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2) are prominent therapeutic protein targets that are frequently overexpressed in TNBC. In this investigation, we developed a novel bispecific antibody (BsAb) targeting EGFR and VEGFR2 (designated as anti-EGFR/VEGFR2 BsAb) and investigate its anti-tumor activity using TNBC cellular and xenograft mouse models. Data from these studies indicate that anti-EGFR/VEGFR2 BsAb elicited more comprehensive anti-tumor activity via multiple mechanisms of action, including direct inhibition of EGFR and VEGFR2 in TNBC cells, and disruption of autocrine and paracrine pathways in TNBC and endothelial cells, compared to the individual parental mAbs. Our data suggest that this novel BsAb warrants further investigation as a targeted antibody therapeutic to treat TNBC. Abstract Both EGFR and VEGFR2 frequently overexpress in TNBC and cooperate with each other in autocrine and paracrine manner to enhance tumor growth and angiogenesis. Therapeutic mAbs targeting EGFR (cetuximab) and VEGFR2 (ramucirumab) are approved by FDA for numerous cancer indications, but none of them are approved to treat breast cancers. TNBC cells secrete VEGF-A, which mediates angiogenesis on endothelial cells in a paracrine fashion, as well as promotes cancer cell growth in autocrine manner. To disrupt autocrine/paracrine loop in TNBC models in addition to mediating anti-EGFR tumor growth signaling and anti-VEGFR2 angiogenic pathway, we generated a BsAb co-targeting EGFR and VEGFR2 (designated as anti-EGFR/VEGFR2 BsAb), using publicly available sequences in which cetuximab IgG backbone is connected to the single chain variable fragment (scFv) of ramucirumab via a glycine linker. Physiochemical characterization data shows that anti-EGFR/VEGFR2 BsAb binds to both EGFR and VEGFR2 in a similar binding affinity comparable to parental antibodies. Anti-EGFR/VEGFR2 BsAb demonstrates in vitro and in vivo anti-tumor activity in TNBC models. Mechanistically, anti-EGFR/VEGFR2 BsAb not only directly inhibits both EGFR and VEGFR2 in TNBC cells but also disrupts autocrine mechanism in TNBC xenograft mouse model. Furthermore, anti-EGFR/VEGFR2 BsAb inhibits ligand-induced activation of VEGFR2 and blocks paracrine pathway mediated by VEGF secreted from TNBC cells in endothelial cells. Collectively, our novel findings demonstrate that anti-EGFR/VEGFR2 BsAb inhibits tumor growth via multiple mechanisms of action and warrants further investigation as a targeted antibody therapeutic for the treatment of TNBC.