Abstract

Recycling antibodies can bind to their target antigen at neutral pH in the blood stream and release them upon endocytosis when pH levels drop, allowing the antibodies to be recycled into circulation via FcRn-mediated pathway, while the antigens undergo lysosomal degradation. This enables recycling antibodies to achieve the same therapeutic effect at lower doses than their non-recyclable counterparts. The development of such antibodies is typically achieved by histidine doping of the variable regions of specific antibodies or by performing in vitro antibody selection campaigns utilizing histidine doped libraries. While often successful, these strategies may introduce sequence liabilities, as they often involve mutations that may render the resultant antibodies to be non-natural. Here, we present a methodology that employs a naive antibody phage display library, consisting of natural variable domains, to discover antibodies that bind -cobratoxin from the venom of Naja kaouthia in a pH-dependent manner. Upon screening of the discovered antibodies with immunoassays and bio-layer interferometry, a pH-dependent antibody was discovered that exhibits an 8-fold higher dissociation rate at pH 5.5 than 7.4. Interestingly, the variable domains of the pH-dependent antibody were found to be entirely devoid of histidines, demonstrating that pH-dependency may not always be driven by this amino acid. Further, given the high diversity available in a naive antibody library, the methodology presented here can likely be applied to discover pH-dependent antibodies against different targets ab initio without the need of histidine doping. For broader audienceHere, we present the discovery of an -cobratoxin targeting pH-dependent antibody, with a variable region devoid of histidines, from a naive antibody library with natural variable domains. Our findings suggest that the commonly taken approach of histidine doping to find pH-dependent antibodies may not always be required, and thus offer an alternative strategy for the discovery of pH-dependent antibodies.