Constant domain polymorphisms influence monoclonal antibody stability and dynamics

Abstract

The constant regions of clinical monoclonal antibodies are derived from a select number of allotypes found in IgG subclasses. Despite a long-term acknowledgment that this diversity may impact both antibody function and developability, there is a lack of data on the stability of variants carrying these mutations. Here, we generated a panel of IgG1, IgG2, and IgG3 antibodies with 32 unique constant region alleles and performed a systematic comparison of stability using red edge excitation shift (REES). This technique exploits the fluorescent properties of tryptophan residues to measure antibody structural dynamics which predict flexibility and the propensity to unfold. Our REES measurements revealed broad stability differences between subclasses with IgG3 possessing the poorest overall stability. Further interrogation of differences between variants within each subclass enabled the high-resolution profiling of individual allotype stabilities. Crucially, these observed differences were not found to be linked to N297-linked glycan heterogeneity. Our work demonstrates diverse stabilities (and dynamics) for a range of naturally occurring constant domain alleles and the utility of REES as a method for rapid and sensitive antibody stability profiling, requiring only laboratory spectrophotometry equipment.

Keywords: Fc domain; REES; allotypes; antibody stability; constant region alleles.