A heterodimeric Fc-based bispecific antibody simultaneously targeting VEGFR-2 and Met exhibits potent antitumor activity.

Heterodimeric Fc designed by engineering the CH3 homodimeric interface of immunoglobulin G1 serves as an attractive scaffold for the generation of bispecific antibodies (bsAb) due to the favorable properties of the Fc region. In this study, we describe a heterodimeric Fc generated by substituting the conserved electrostatic interactions at the CH3 core interface with asymmetric hydrophobic interactions and introducing asymmetric, long-range electrostatic interactions at the rim of the CH3 interface. Coexpression of Fc proteins carrying the combined CH3 variant pairs in HEK293F cells produced the heterodimer, which was purified with more than 90% yield, and retained wild-type Fc biophysical properties. The heterodimeric Fc was exploited to generate a bsAb simultaneously targeting both the Met receptor tyrosine kinase and the VEGF receptor 2 (VEGFR-2), with two respective antigen-specific, single-chain variable fragments (scFv) into the N-terminus. The Met × VEGFR-2 bsAb bound concurrently to the two target antigens, efficiently inhibited the downstream signaling and tube formation stimulated by the two receptors in human endothelial cells, and exhibited more potent antitumor efficacy in MKN45 human gastric cancer xenograft models than both the parent monospecific antibody alone. Collectively, based on the newly designed heterodimeric Fc-based bsAb, our results provide the therapeutic potential of bsAb targeting both Met and VEGFR-2 simultaneously for the treatment of human cancers.