Structural and Functional Characterization of a Hole–Hole\nHomodimer Variant in a “Knob-Into-Hole” Bispecific Antibody

Bispecific\nantibodies have great potential to be the next-generation\nbiotherapeutics due to their ability to simultaneously recognize two\ndifferent targets. Compared to conventional monoclonal antibodies,\nknob-into-hole bispecific antibodies face unique challenges in production\nand characterization due to the increase in variant possibilities,\nsuch as homodimerization in covalent and noncovalent forms. In this\nstudy, a storage- and pH-sensitive hydrophobic interaction chromatography\n(HIC) profile change was observed for the hole–hole homodimer,\nand the multiple HIC peaks were explored and shown to be conformational\nisomers. We combined traditional analytical methods with hydrogen/deuterium\nexchange mass spectrometry (HDX MS), native mass spectrometry, and\nnegative-staining electron microscopy to comprehensively characterize\nthe hole–hole homodimer. HDX MS revealed conformational changes\nat the resolution of a few amino acids overlapping the CH2-CH3 domain interface. Conformational heterogeneity was\nalso assessed by HDX MS isotopic distribution. The hole–hole\nhomodimer was demonstrated to adopt a more homogeneous conformational\ndistribution during storage. This conformational change is likely\ncaused by a lack of CH3 domain dimerization (due to the\nthree “hole” point mutations), resulting in a unique\nstorage- and pH-dependent conformational destabilization and refolding\nof the hole–hole homodimer Fc. Compared with the hole–hole\nhomodimer under different storage conditions, the bispecific heterodimer,\nguided by the knob-into-hole assembly, proved to be a stable conformation\nwith homogeneous distribution, confirming its high quality as a desired\ntherapeutic. Functional studies by antigen binding and neonatal Fc\nreceptor (FcRn) binding correlated very well with the structural characterization.\nComprehensive interpretation of the results has provided a better\nunderstanding of both the homodimer variant and the bispecific molecule.