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

Abstract

Bispecific antibodies have great potential to be the next-generation biotherapeutics due to their ability to simultaneously recognize two different targets. Compared to conventional monoclonal antibodies, knob-into-hole bispecific antibodies face unique challenges in production and characterization due to the increase in variant possibilities, such as homodimerization in covalent and noncovalent forms. In this study, a storage- and pH-sensitive hydrophobic interaction chromatography (HIC) profile change was observed for the hole-hole homodimer, and the multiple HIC peaks were explored and shown to be conformational isomers. We combined traditional analytical methods with hydrogen/deuterium exchange mass spectrometry (HDX MS), native mass spectrometry, and negative-staining electron microscopy to comprehensively characterize the hole-hole homodimer. HDX MS revealed conformational changes at the resolution of a few amino acids overlapping the C2-C3 domain interface. Conformational heterogeneity was also assessed by HDX MS isotopic distribution. The hole-hole homodimer was demonstrated to adopt a more homogeneous conformational distribution during storage. This conformational change is likely caused by a lack of C3 domain dimerization (due to the three "hole" point mutations), resulting in a unique storage- and pH-dependent conformational destabilization and refolding of the hole-hole homodimer Fc. Compared with the hole-hole homodimer under different storage conditions, the bispecific heterodimer, guided by the knob-into-hole assembly, proved to be a stable conformation with homogeneous distribution, confirming its high quality as a desired therapeutic. Functional studies by antigen binding and neonatal Fc receptor (FcRn) binding correlated very well with the structural characterization. Comprehensive interpretation of the results has provided a better understanding of both the homodimer variant and the bispecific molecule.

A single mutation increases heavy-chain heterodimer assembly of bispecific antibodies by inducing structural disorder in one homodimer species

“…KiH (H) was chosen as a recipient for the D84.4Q substitution over KiH "knob" (denoted K; T366W) because we found its characteristics to be the closest match to BEAT (B); both KiH (H) and BEAT (B) are the chains receiving the smaller residues, creating cavities to accommodate the larger residues introduced into KiH (K) and BEAT (A). Furthermore, some characterization data for KiH (H) homodimers had been published (13). Upon purification, we found that this antibody was not abrogated for PA binding (Fig.…”

“…The increase in Rh was less significant than for the BEAT (B) D84.4Q homodimers. Zhang et al ( 13 ) attributed the increase in Rh of the KiH (H) homodimer to a reduced compactness of the domains caused by their poor pairing capabilities. This also may explain the relatively mild reduction in FcRn and FcγR1a binding compared with the corresponding KiH heterodimer ( Fig.…”

“…10 B ). The mild increase in ANS fluorescence for KiH could be explained by the reported existence of multiple conformational isomers of varying folding compactness and varying hydrophobicity ( 13 ). Increased hydrophobicity may lead to some increase in ANS binding, which could also explain the mildly increased fluorescence emission observed for BEAT (A) homodimers, which may suffer from suboptimally paired CH3 domains.…”

“…Finally, our results shed structural insights on bsAb Hc hetero- versus homodimer assembly, and the nature of half-antibodies on which only little data could be found prior to this work ( 13 ). As in our previous studies ( 10 ), antibodies based on KiH were used as comparators.…”

Charge variant native mass spectrometry benefits mass precision and dynamic range of monoclonal antibody intact mass analysis

“…Native MS utilizes 100% aqueous solutions of low-to-moderate concentrations of volatile salt (e.g., 50 mM ammonium acetate) buffered at neutral pH. 3,4 This type of analysis proceeds commonly after removal of non-volatile salts by buffer exchange followed by nanospray infusion MS. 5–7 Native MS-based intact mass analysis is required for certain classes of biotherapeutics that require preservation of non-covalent associations of protein-protein or protein-ligand complexes. 8 Native MS also affords a fundamental benefit to intact mass analysis of all types of heterogeneous samples where proteins acquire fewer charges and yield spectra at high m/z relative to denatured conditions.…”