A Novel Antibody Engineering Strategy for Making Monovalent Bispecific Heterodimeric IgG Antibodies by Electrostatic Steering Mechanism

Abstract Producing pure and well-behaved bispecific antibodies (bsAbs) at a large scale for preclinical and clinical testing is a challenging task. Here we describe a new strategy for making monovalent bispecific heterodimeric IgG antibodies in mammalian cells. We applied electrostatic steering mechanism to engineer antibody LC-HC interface residues in such a way that each LC strongly favors its cognate HC when two different HCs and two different LCs are co-expressed in the same cell to assemble a functional bispecific antibody. We produced heterodimeric IgGs from transiently and stably transfected mammalian cells. The engineered heterodimeric IgG molecules maintain the overall IgG structure with correct LC-HC pairings; bind to two different antigens with comparable affinity when compared to their parental antibodies; and retain the functionality of parental antibodies in biological assays. In addition, the bispecific heterodimeric IgG derived from anti-HER2 and anti-EGFR antibody was shown to induce higher level of receptor internalization than the combination of two parental antibodies. Mice xenograft BxPC-3, Panc-1, and Calu-3 human tumor models showed that the heterodimeric IgGs strongly inhibited the tumor growth. The described approach can be used to generate tools from two preexistent antibodies and explore the potential of bispecific antibodies. The asymmetrically engineered Fc variants for ADCC enhancement could be embedded in monovalent bispecific heterodimeric IgG to make best-in-class therapeutic antibodies.