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.

HER2, a member of the Erythroblastosis Protein B/Human Epidermal Growth Factor Receptor (ErbB/HER) family of receptor tyrosine kinase, is overexpressed in 20~30% of human breast cancers. Trastuzumab, a HER2-targeted therapeutic monoclonal antibody, was developed to interfere with the homodimerization of HER2 in HER2-overexpressing breast cancer cells, which attenuates HER2-mediated signaling. Trastuzumab binds to the domain IV of the HER2 extracellular domain and does not directly block the dimerization interface of HER2-HER2 molecules. The three-dimensional structures of the tyrosine kinase domains of ErbB/HER family receptors show asymmetrical packing of the two monomers with distinct conformations. One monomer functions as an activator, whereas the other acts as a receiver. Once activated, the receiver monomer phosphorylates the activator or other proteins. Interestingly, in our previous work, we found that the binding of trastuzumab induced phosphorylation of HER2 with the phosphorylation pattern of HER2 that is different from that mediated by epidermal growth factor (EGF) in human epidermal growth factor receptor 2 (HER2)-positive breast cancer. Binding of trastuzumab to HER2 promoted an allosteric effect of HER2, in both tyrosine kinase domain and ectodomain of HER2 although details of allosteric regulation were missing. In this study, we utilized molecular dynamics (MD) simulations to model the allosteric consequences of trastuzumab binding to HER2 homodimers and heterodimers, along with the apo forms as controls. We focused on the conformational changes of HER2 in its monomeric and dimeric forms. The data indicated the apparent dual role of trastuzumab as an antagonist and an agonist. The molecular details of the simulation provide an atomic level description and molecular insight into the action of HER2-targeted antibody therapeutics.

Bispecific T-cell-engaging antibodies are a growing class of therapeutics with numerous molecules being tested in clinical trials and, currently, seven of them have received market approval. They are structurally complex and function as adaptors to redirect the cytotoxicity of T cells to kill tumor cells. T-cell-engaging bispecific antibodies can be generally divided into two categories: IgG/IgG-like and non-IgG-like formats. Different formats may have different intrinsic potencies and physiochemical properties, and comprehensive studies are needed to gain a better understanding of how the differences in formats impact on structural and functional characteristics. In this study, we designed and generated bispecific T-cell-engaging antibodies with IgG-like (DVD-Ig) and non-IgG (BiTE) formats. Both target the same pair of antigens (EGFR and CD3) to minimize the possible influence of targets on functional characterization. We performed a side-by-side comparison to assess differences in the physiochemical and biological properties of these two bispecific T-cell-engaging antibodies using a variety of breast and ovarian cancer cell-based functional assays to delineate the structural–functional relationships and anti-tumor activities/potency. We found that the Fc portion of T-cell-engaging bispecific antibodies can significantly impact antigen binding activity, potency, and stability in addition to eliciting different mechanisms of action that contribute the killing of cancer cells.

We generated two IgG1-like bispecific antibodies (BsAbs) with different molecular formats, symmetrical DVD-Ig and asymmetrical knob-in-hole (KIH), targeting the same antigens, EGFR and PD-L1 (designated as anti-EGFR/PD-L1). We performed the physiochemical and biological characterization of these two formats of anti-EGFR/PD-L1 BsAbs and compared some key quality attributes and biological activities of these two formats of BsAbs. Physiochemical binding characterization data demonstrated that both formats bound EGFR and PD-L1. However, the binding affinity of the KIH format was weaker than the DVD-Ig format in Biacore binding assays. In contrast, both DVD-Ig and KIH BsAbs had similar ELISA and cell surface binding activities, comparable to mAbs. Triple-negative breast cancer (TNBC) cells and a xenograft model were used to test the potency of BsAbs and other biological activities. Results showed that anti-EGFR/PD-L1 BsAbs exhibited in vitro and in vivo antitumor proliferation activity, but there was a difference in the potencies of the respective BsAb formats (DVD-Ig and KIH) when different cells or assays were used. This study provides evidence that the potency of the BsAbs targeting the same antigens can be affected by the respective molecular features, and selection of appropriate cell lines and assays is critically important for the assay development and potency testing of BsAbs.