Ph.D. (Doctor of Philosophy)
Department of Chemistry and Biochemistry
Antibodies are widely applied as affinity reagents across a variety of fields, due to their high affinity and specificity. While high affinity across a range of conditions is useful for many applications, the ability to modulate affinity with environmental conditions (e.g., pH) opens new opportunities for antibody applications. This dissertation focuses on exploring the impact of an antibody’s interface size on engineered pH dependence, as well as exploring engineered pH-dependence through perturbing oligomerization equilibria that are linked to target binding. An anti-maltose binding protein (MBP) Fab 7O antibody fragment was used as a large protein-protein interface model to explore a library-based introduction of histidine residues within the antigen binding interface. After rounds of phage display selection, the Fab’s antigen binding interface incorporated five to six histidine residues, which was roughly twice the number observed in a smaller antibody interface, previously studied. Surprisingly, while the histidine-containing Fab variants retained near wild-type binding affinity at the permissive pH, they displayed only modest pH dependence, despite the significant number of histidine residues present in the antigen binding site. Such variants may originate due to complexities of the selection strategy or may result from the significant number of acidic residues present within the MBP epitope. To explore pH-dependent engineering via linked oligomerization equilibria, two different models were used, the anti-MBP Fab 7O VHVL interface and the anti-caffeine VHH homodimer interface. A phage display approach introducing histidine protonation triggers within the anti-MBP Fab 7O VHVL interface resulted in the selection of two prominent clones HC W47H/LC Q38H and LC A34H/Q89H, which is notable considering the conserved nature of the VHVL framework. The anti-MBP Fab variant HC W47H/LC Q38H displayed marginal pH dependence, although it possessed a calorimetric signature suggesting changes in protonation. The lack of an appreciable pH response may stem from an asymmetric energetic dominance of the VH domain towards MBP recognition. In a second model, anti-caffeine VHH variants containing aspartic acid residues within the homodimer interface were examined to determine the driving force of the observed neutral pH switch. With an increase in pH from 4.0 to 7.5, anti-caffeine VHH variants S35D and T52D displayed decreases in binding affinity of ~340-fold and ~20-fold, and estimated ΔpKa values of 1.4 and 0.7, respectively. Double mutant S35D/T52D experienced an 11-fold drop in binding affinity with a ΔpKa of 0.7, suggesting lack of simple additivity. Overall, these studies reveal that pH dependence can be engineered in a direct manner using histidine and anti-MBP Fab 7O model system, and in an indirect manner using aspartic acid residues and the anti-caffeine VHH homodimer model system. Although the indirect pH switch was not successful using anti-MBP Fab 7O and the VHVL interface, a pH-switch can still be engineered using an alternate model system with an even distribution of heavy and light chain binding residues.
Laughlin, Tosha, "Engineering pH-Dependent Antibody interactions Through introduction of Linked Protonation Equibria Within The Antigen Binding interface Or Linked Oligomeric interfaces" (2023). Graduate Research Theses & Dissertations. 7159.
Northern Illinois University
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