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

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.

Conjugates Derived from Lapatinib Derivatives with Cancer Cell Stemness Inhibitors Effectively Reversed Drug Resistance in Triple-Negative Breast Cancer

“…Epidermal growth factor receptor (EGFR), a tyrosine kinase receptor involved in cell growth and survival, is associated with a loss of estrogen responsiveness and poor prognosis and frequently overexpressed in TNBC cases. , It has been proven that the abnormal activation of EGFR in tumor cells caused by gene mutations, overexpression, and protein overexpression can lead to uncontrolled proliferation, metastasis, invasion, and drug resistance of cancer cells, especially in TNBC. , However, therapies directed only against EGFR based on monoclonal antibodies or small-molecular inhibitors were ineffective or unsatisfactory in the treatment of TNBC . Therefore, these is an urgent need to seek new solutions based on EGFR TKIs that can enhance the therapeutic efficacy of TNBC.…”

Research Progress and Applications of Multivalent, Multispecific and Modified Nanobodies for Disease Treatment

“…Furthermore, by targeting multiple molecular targets simultaneously, multi-specific antibodies can block independent signal pathways, a condition that contributes effectively to prevent drug resistance and immune escape. For instance, targeting simultaneously EGFR and VEGFR2 by means of a bispecific construct composed by fragments of the parental IgGs cetuximab and ramucirumab was effective in inhibiting EGFR-dependent tumor growth and VEGFR2 angiogenic pathway in a mouse model of Triple Negative Breast Cancer ( 194 ), whereas a bispecific antibodies targeting TGF-β and PD-L1 showed a superior anti-tumor effect with respect to monotherapy due to enhanced anti-tumor immune response in multiple in vivo models ( 195 ).…”

Comparative Characterization of Different Molecular Formats of Bispecific Antibodies Targeting EGFR and PD-L1

“…The two plasmids of DVD-Ig format and three plasmids of KIH format anti-EGFR/PD-L1 BsAbs encoding heavy and light chains were transiently co-transfected into Expi293 cells using an Expifectamine 293 Transfection Kit (Thermo Fisher Scientific, Rockford, IL, USA) as per manufacturer’s instructions. The purification of both formats of anti-EGFR/PD-L1 BsAbs was performed by affinity chromatography using immobilized protein-A agarose resin as described previously [ 23 ]. The production yield of anti-EGFR/PD-L1 DVD-Ig and KIH formats is approximately 0.05–0.2 mg per ml from 125 mL of culture supernatant.…”

“…The standard ELISA binding assay, cell surface binding assays using flow cytometry and coimmunoprecipitation assay were performed to detect the binding of mAbs and anti-EGFR/PD-L1 BsAbs to receptors EGFR or PD-L1 as described previously [ 23 ].…”