Evaluation of Disulfide Bond Position to Enhance the Thermal Stability of a Highly Stable Single Domain Antibody

Abstract

Single domain antibodies are the small recombinant variable domains derived from camelid heavy-chain-only antibodies. They are renowned for their stability, in large part due to their ability to refold following thermal or chemical denaturation. In addition to refolding after heat denaturation, A3, a high affinity anti-Staphylococcal Enterotoxin B single domain antibody, possesses a melting temperature of ∼84°C, among the highest reported for a single domain antibody. In this work we utilized the recently described crystal structure of A3 to select locations for the insertion of a second disulfide bond and evaluated the impact that the addition of this second bond had on the melting temperature. Four double-disulfide versions of A3 were constructed and each was found to improve the melting temperature relative to the native structure without reducing affinity. Placement of the disulfide bond at a previously published position between framework regions 2 and 3 yielded the largest improvement (>6°C), suggesting this location is optimal, and seemingly provides a universal route to raise the melting temperature of single domain antibodies. This study further demonstrates that even single domain antibodies with extremely high melting points can be further stabilized by addition of disulfide bonds.

Stability of isolated antibody-antigen complexes as a predictive tool for selecting toxin neutralizing antibodies

“…The elevation of the T m of a protein can be practically useful during manufacturing and for the development of long shelf-life vaccine immunogens, 30 or for the development of field-expedient protein-based assay reagents that do not require refrigeration 31 . Incorporation of a disulfide bond at 2 sites within RTA led to 2 engineered immunogens with enhanced stability as judged by a higher apparent T m 30 .…”

Improved production of single domain antibodies with two disulfide bonds by co-expression of chaperone proteins in the Escherichia coli periplasm

“…Placement of a similar additional DSB was also shown to provide increased protease stability (Hagihara and Saerens, 2014). In earlier work other groups as well as our own examined alternate positions for placement of the additional DSB and found that many locations could accommodate the extra DSB and result in an increased melting temperature (Hagihara and Saerens, 2014;Zabetakis et al, 2014). The primary limitation to thermal stabilization by DSB addition is the loss in protein production.…”

VHH antibodies: emerging reagents for the analysis of environmental chemicals

“…In camelid VHHs, it was originally found that the long loops were constrained with a disulfide bond to form the antigen binding site [56, 57], which was considered to contribute strongly to the stability and thermal resistance of VHHs because the removal of this disulfide bond by site-directed mutagenesis resulted in a significant decrease in melting point and prevented refolding [58, 59]. Other researchers took a different approach and introduced a non-canonical disulfide bond into the hydrophobic core of llama VHHs between framework region 2 (FR2) and FR3, which proved to not only increase thermal stability at neutral pH, but also imparted resistance to proteolytic degradation and increased antibody stability at low pH [60, 61]. A recent thorough study of the relationship between number of disulfide bonds and the heat resistance of VHH concluded that while T m increased with the increase in number of disulfide bonds the half-life of VHH at 90 °C ( t 1/2 90 C ) decreased [62].…”