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

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.