Publication Date


Document Type


First Advisor

Horn, James R.

Degree Name

Ph.D. (Doctor of Philosophy)

Legacy Department

Department of Chemistry and Biochemistry


Engineered antibodies are frequently used in life science applications, such as therapeutics and clinical diagnostics. These customized antibodies typically possess enhanced binding affinity and/or specificity, yet these interactions are only one-way binding events that cannot be easily controlled without extreme changes in conditions, such as pH, denaturant, and/or temperature that likely irreversible damage the antibody and antigen. Here, we explore the design of proton-linked binding events in the context of both antibody/hapten (i.e., a low molecular weight ligand target) and antibody/antigen complexes. Specifically, this work looks to overcome the challenges of engineering pH-dependent small ligand/protein interactions, as well as develop a genetic mechanism to create pH-dependent Fab/antigen interactions, through engineering proton-linked binding within inter-domain interfaces that are required for target recognition, thus indirectly control antigen interaction in a pH-dependent manner.Our model system for an antibody/hapten interaction was an anti-caffeine VHH that requires dimerization to bind its target, caffeine. This the VHH homodimer complex with caffeine is structurally analogous to the heterodimer formed between the various heavy (VH) and light (VL) domains found in conventional antibodies. Individual or pairwise histidine residues were introduced in the anti-caffeine VHH homodimer interface (Y108H, F49H, and Y108H/F49H) to generate a pH-dependent interaction. Due to the symmetrical interface, introduction of a single histidine residue within the anti-caffeine VHH interface results in two interface histidine residues. The observed binding affinity of each variant was determined using ITC. Notably, all three variants displayed significant pH dependence in their interaction with caffeine. Based on the pH dependence of the observed binding constant (Kb) the individual protonation constant values (pKa) were estimated. To explore whether linked protonation events can be introduced within the VL/VH heterodimer of conventional antibody systems to generate pH-dependent antibodies, an anti-maltose binding protein Fab was used as a model system. Using a combinatorial histidine scanning library approach, histidine residues were sampled throughout the interface of the two variable domains of the anti-MBP Fab antibody. Two clones were selected from phage ELISA assays and further characterized to determine their contribution to a pH-dependent binding response. Clone #3 possessed a single histidine within the VL domain and clone #7 possessed three histidines within the VL domain. Clone #3 displayed eight times reduced binding affinity at pH 4.0 compared with pH 7.4. On the other hand, clone #7 displayed thirty times reduced binding affinity at pH 4.0. While the extent of each contributions is not fully understood, it is notable that the incorporation of multiple histidine residues within the heterodimer domain interface did not result in an energetic penalty to binding at the permissive pH (7.4), while generating increased pH-sensitive binding at pH 4.0, as compared to the wild-type Fab. The introduction of histidine residues within interdomain interfaces allowed the engineering of pH dependent anti-caffeine VHH and anti-MBP Fab 7O antibodies where binding affinity decreased with a decrease in pH. Here, we explore the design of “reverse” pH control, where binding affinity decreases with an increase in pH, through the introduction of acidic residues in the anti-caffeine VHH dimer interface. Single aspartic acid or glutamic acid residues were introduced within the anti-caffeine VHH dimer interface to probe the possibility of generating “reverse” pH-sensitive antibodies. Nine positions were subjected to mutagenesis, introducing an aspartic or glutamic acid. ITC analysis of the binding affinity revealed that six of the nine variants (T52D, S35D, V106D, Y61D, Y108E, and Y61E) displayed the hypothesized “reverse” pH-dependent binding profile. While variants showed a rage of energetic penalties to binding at pH 4.0, along with a range of pH sensitivities when raising the pH, the anti-caffeine VHH S35D variant possessed significant reverse pH dependency, possessing a 165-fold decrease in binding affinity between pH 4.0 and 7.4, with a KD similar to wild type at pH 4.0.


142 pages




Northern Illinois University

Rights Statement

In Copyright

Rights Statement 2

NIU theses are protected by copyright. They may be viewed from Huskie Commons for any purpose, but reproduction or distribution in any format is prohibited without the written permission of the authors.

Media Type