Monovalent bispecific IgGs cater to a distinct set of mechanisms of action but are difficult to engineer and manufacture because of complexities associated with correct heavy and light chain pairing. We have created a novel design, "DuetMab," for efficient production of these molecules. The platform uses knobs-into-holes (KIH) technology for heterodimerization of 2 distinct heavy chains and increases the efficiency of cognate heavy and light chain pairing by replacing the native disulfide bond in one of the CH1-CL interfaces with an engineered disulfide bond. Using two pairs of antibodies, cetuximab (anti-EGFR) and trastuzumab (anti-HER2), and anti-CD40 and anti-CD70 antibodies, we demonstrate that DuetMab antibodies can be produced in a highly purified and active form, and show for the first time that monovalent bispecific IgGs can concurrently bind both antigens on the same cell. This last property compensates for the loss of avidity brought about by monovalency and improves selectivity toward the target cell.
Keywords: ADCC, antibody-dependent cell-mediated cytotoxicity; Biotechnology; CDR, complementarity determining region; CH1, 2 and 3-heavy chain constant domain 1, 2 and 3; CL-, light chain constant domain; DSC-differential scanning calorimetry; E:T, ratio of effector to target cells; EGFR; EGFR, epidermal growth factor receptor; FcRn, neonatal Fc receptor; FcγR, receptor for IgG Fc; HER2; IGFR, insulin like growth factor receptor; IL-6, interleukin 6; IgG, Immunoglobulin G; PNGase, protein N-glycanase; Q1q, first component of complement 1; RAGE, receptor for advanced glycosylation; antibody engineering; bispecific antibody; cancer; disulfide; mAbs, monoclonal antibodies; multi-targeting.