EFFECT OF FRAMEWORK AND COMPLEMENTARITY DETERMINING REGION CHARGE ON VH DOMAIN SOLUBILITY
Sultana, Sharmin 1987-
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The use of VH domains in diagnostic and therapeutic applications circumvents many of the issues associated with antibodies. Nevertheless, VH domains still have limitations such as a tendency to aggregate. They usually contain hydrophobic residues within their complementarity determining regions (CDRs) that facilitate binding to target antigens but also mediate VH domain aggregation, which is a great concern for therapeutic applications. This thesis focuses on the engineering of the VH framework and CDR1 to prevent VH aggregation. We used two strategies to increase the stability and solubility of the VH domain. First, we used a previously discovered autonomous human VH-B1a as a starting point and increased its net charge by introducing charge mutations in the framework region. We observed that our designed +VH domain showed increased thermostability, but the reversible folding ability and solubility were lower compared to near-neutrally charged VH-B1a. Second, we mutated CDR1 of VH-B1a and +VH domains by introducing a series of positively and negatively charged residues, and assessed the effect of net framework charge and CDR1 charge on improving VH domain thermostability and solubility. From the analysis of CDR1-mutated VH-B1a and +VH domains, we noticed a position specific effect of a single lysine or two lysines in CDR1. Moreover, +VH can overcome the position specific effect of single lysine mutation, and the positional effect of double lysine mutation was better tolerated in +VH domain compared to VH-B1a. Significant aggregation resistance of VH domain was noticed in the presence of three negative (DDD) or positive (KKK) charge in the CDR1 of VH-B1a and +VH, respectively. Furthermore, VH-B1a was better at tolerating negative charge in CDR1, whereas +VH was better at tolerating either positive or negative charge. Our findings demonstrate that in addition to the net framework charge and CDR1 charge, a positional effect of CDR1 mutation affects thermostability and aggregation resistance. Our results demonstrate that the +VH domain will be a good starting template for the construction of phage displayed libraries that will be enriched in highly thermostable and aggregation resistant VH variants.
DegreeMaster of Science (M.Sc.)
CommitteeLee, Jeremy; Cygler, Miroslaw; Napper, Scott; Phenix, Christopher
Copyright DateNovember 2017
Complementarity Determining Regions (CDRs), aggregation, thermostability, reversible folding ability