Retrieving Dissociation-Resistant Antibody Mutants: An Efficient Strategy for Developing Immunoassays with Improved Sensitivities.

Previously, we generated high-affinity antibody mutants that enabled sensitive immunoassays by exploring diverse libraries of single-chain Fv fragments (scFvs) displayed on bacteriophage. To isolate rarely-occurring desirable clones, "panning" has commonly been performed but is often unsuccessful. Therefore, we previously developed a clonal array profiling (CAP) method, wherein scFv-displaying phage (scFv-Ph) clones in a library were examined individually regarding their ability to target antigens immobilized on microwells. Clones that showed strong reactivity were recovered via dissociation using an acidic treatment. The CAP successfully discovered cortisol-specific scFvs showing 17-31-fold improved Ka from libraries generated via site-directed insertions in a prototype anti-cortisol scFv (wt-scFv; Ka, 3.6 × 108 M-1), but their Ka did not exceed 1.1 × 1010 M-1. In this study, to break this possible affinity ceiling, we devised a new system employing a dissociation-independent recovery. scFv-Phs were individually reacted to target antigen (cortisol) immobilized on microwells via a linker containing a disulfide bond. Following acidic and basic treatments to eliminate scFv-Phs with "ordinary affinities," dissociation-resistant scFv-Phs remaining on the microwells were retrieved via reductive cleavage of the disulfide bonds. This system allowed for a straightforward and efficient discovery of scFv mutants with 33-56-fold increased Ka (1.2-2.0 × 1010 M-1), exceeding the previous affinity ceiling. These scFvs enabled an enzyme-linked immunosorbent assay for cortisol with 18-51-fold higher sensitivity than the assay performed using wt-scFv.

Increasing stability of antibody via antibody engineering: stability engineering on an anti-hVEGF.

Antibody stability is very important for expression, activity, specificity, and storage. This knowledge of antibody structure has made it possible for a computer-aided molecule design to be used to optimize and increase antibody stability. Many computational methods have been built based on knowledge or structure, however, a good integrated engineering system has yet to be developed that combines these methods. In the current study, we designed an integrated computer-aided engineering protocol, which included several successful methods. Mutants were designed considering factors that affected stability and multiwall filter screening was used to improve the design accuracy. Using this protocol, the thermo-stability of an anti-hVEGF antibody was significantly improved. Nearly 40% of the single-point mutants proved to be more stable than the parent antibody and most of the mutations could be stacked effectively. The T50 also improved about 7°C by combinational mutation of seven sites in the light chain and three sites in the heavy chain. Data indicate that the protocol is an effective method for optimization of antibody structure, especially for improving thermo-stability. This protocol could also be used to enhance the stability of other antibodies.