Phage display assisted discovery of a pH‐dependent anti‐α‐cobratoxin antibody from a natural variable domain library

Recycling IgG antibodies bind to their target antigen at physiological pH in the blood stream and release them upon endocytosis when pH levels drop, allowing the IgG antibodies to be recycled into circulation via FcRn‐mediated cellular pathways, while the antigens undergo lysosomal degradation. This enables recycling antibodies to achieve comparable therapeutic effect at lower doses than their non‐recycling counterparts. The development of such antibodies is typically achieved by histidine doping of their variable regions or by performing in vitro antibody selection campaigns utilizing histidine doped libraries. Both are strategies that may introduce sequence liabilities. Here, we present a methodology that employs a naïve antibody phage display library, consisting of natural variable domains, to discover antibodies that bind α‐cobratoxin from the venom of Naja kaouthia in a pH‐dependent manner. As a result, an antibody was discovered that exhibits a 7‐fold higher off‐rate at pH 5.5 than pH 7.4 in bio‐layer interferometry experiments. Interestingly, no histidine residues were found in its variable domains, and in addition, the antibody showed pH‐dependent binding to a histidine‐devoid antigen mutant. As such, the results demonstrate that pH‐dependent antigen‐antibody binding may not always be driven by histidine residues. By employing molecular dynamics simulations, different protonation states of titratable residues were found, which potentially could be responsible for the observed pH‐dependent antigen binding properties of the antibody. Finally, given the typically high diversity of naïve antibody libraries, the methodology presented here can likely be applied to discover recycling antibodies against different targets ab initio without the need for histidine doping.