Hinge Region Cys-to-Ser Mutation Enables Homogeneous DAR6 Anti-c-Met Biparatopic ADCs with Tolerable O-Glycosylation

Abstract Background Antibody–drug conjugates (ADCs) with homogeneous drug-to-antibody ratios (DARs) are increasingly favored for their improved physicochemical and pharmacokinetic properties; however, strategies to reliably generate homogeneous DAR6 ADCs remain limited. Methods In this study, we investigate hinge-region cysteine engineering as an approach to constrain conjugation stoichiometry, using a potent biparatopic anti–c-Met antibody as a model. Various biological and physicochemical assays were used for its characterization. Results We show that substitution of a hinge-region cysteine within a CPPC motif reduces the number of accessible conjugation sites, allowing reproducible generation of homogeneous DAR6 ADCs. During characterization of Ser- or Thr-substituted hinge variants, we observed additional mass species consistent with mucin-type O-linked glycosylation. Site-specific mass spectrometry analyses localized these modifications to a serine residue within hinge-region and identified the core structures of the O-glycans. Analysis of additional antibodies carrying analogous mutations demonstrated that hinge-associated O-glycosylation is not restricted to a single antibody scaffold. Conclusions These results demonstrate that hinge-region cysteine engineering can serve as a rational and adaptable approach for DAR6 ADC development while highlighting the need for case-by-case evaluation of associated post-translational modifications during antibody engineering.