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The influence of constant region polymorphisms on antibody stability and structural dynamics

Antibodies are highly effective mediators of the adaptive immune response. Their unique structural architecture makes them ideally suited for the specific targeting of disease-causing agents and the subsequent activation of potent effector functions upon binding to immune cells. These properties have led to the extensive use of antibodies in the biomedical industry as diagnostics and therapeutics. Notably, the IgG class of human antibodies (defined by the sequence of the constant region) has dominated the pool of antibody-based therapeutics, largely due to exceptional stability and efficient immune activation. The constant region genes of human IgG harbour a surprising amount of genetic variation in the form of allelic sequences encoding nonsynonymous mutations. Many allelic sequences have only been uncovered within the last five years, yet a handful of studies have already identified links between specific amino acid polymorphisms and improved receptor binding affinities that lead to enhanced activation of effector functions. This thesis furthers our understanding of the evolutionary pressures driving this genetic diversification by investigating the influence of IgG constant region diversity on antibody structural dynamics, including stability and flexibility. To analyse the structural effects in detail, I expressed a large panel of 35 trastuzumab-formatted antibodies with unique constant region allele sequences from subclasses IgG1, IgG2, and IgG3. Purified antibodies were confirmed to be of high purity and the composition of appended glycans was characterised for each variant. In this thesis, I compare the thermal stability properties of the unique IgG antibodies, measured by red edge excitation shift (REES) spectroscopy. Striking stability differences were resolved both between and within IgG subclasses, leading to new insights about the effect of specific amino acid positions on antibody stabilisation. Following the stability analysis, I extensively evaluate the limitations of REES to measure antibody stability, with a particular emphasis on tryptophan content. Using the naturally occurring mutation of arginine to tryptophan present in three of the IgG3 alleles, I generated experimental data that demonstrates the relationship between the solvation state of local tryptophan environments and the REES effect. This analysis highlights the need for matched tryptophan content for robust comparison of antibody stability using a thermodynamic model of REES behaviour. Finally, the effects of IgG3 hinge length polymorphisms on conformational flexibility are analysed. Here, I used small angle X-ray scattering to characterise the solution structures of allelic variants encoding three distinct hinge lengths. This work provides the first report of X-ray scattering data for naturally occurring IgG3 antibodies with hinge lengths of 32 and 47 amino acids. Comparison of the scattering behaviour and predicted ab initio model structures reveal more compact conformations with reduced space between the Fab domains and the Fc domain when the hinge is truncated, thereby resulting in reduced overall flexibility of the full-length structure. This study highlights dynamic differences between the IgG3 variants tested, which are likely to translate into differences in biological functions. Taken together, this data provides an important basis for future investigations into the functional roles of IgG allelic diversity. Moreover, the experimental schemes optimised throughout this thesis will enable the high-throughput screening of antibody constant region variants in the future, particularly as more genetic diversity comes to light.
Type of thesis
The University of Waikato
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