Figure S1: Characterization of binding properties and thermal stability of Fynomer C12; Figure S2: Results of binding studies with C12-pertuzumab bispecific FynomAbs; Figure S3: Additional in vitro tumour growth inhibition studies with COVA208 on more HER2 positive cancer cell lines; Figure S4: Additional data on the induction of apoptosis in NCI-N87 cells by COVA208: dose-response curve of Caspase 3/7 activity, and Tunel assay; Figure S5: Expression data (heat map) of signalling mediators in response to treatment with COVA208, pertuzumab, or trastuzumab (RPPA analysis); Supplementary Table 1 listing expression data of 167 signaling proteins (phospho- and total proteins) in response to COVA208, pertuzumab or trastuzumab treatment of NCI-N87 cells in vitro.
ARTICLE ABSTRACTUpregulation of HER2 is a hallmark of 20% to 30% of invasive breast cancers, rendering this receptor an attractive target for cancer therapy. Although HER2-targeting agents have provided substantial clinical benefit as cancer therapeutics, there is a need for the development of new agents aiming at circumventing anti-HER2 resistance. On the basis of the approved antibody pertuzumab, we have created a panel of bispecific FynomAbs, which target two epitopes on HER2. FynomAbs are fusion proteins of an antibody and a Fyn SH3–derived binding protein. One bispecific FynomAb, COVA208, was characterized in detail and showed a remarkable ability to induce rapid HER2 internalization and apoptosis in vitro. Moreover, it elicited a strong inhibition of downstream HER2 signaling by reducing HER2, HER3, and EGFR levels in vitro and in vivo. Importantly, COVA208 demonstrated superior activity in four different xenograft models as compared with the approved antibodies trastuzumab and pertuzumab. The bispecific FynomAb COVA208 has the potential to enhance the clinical efficacy and expand the scope of HER2-directed therapies, and delineates a paradigm for designing a new class of antibody-based therapeutics for other receptor targets. Mol Cancer Ther; 13(8); 2030–9. ©2014 AACR.