Site-Directed Mutagenesis of anti-CD11b Nanobody to Optimize Production

Abstract

A previous work from our lab produced nanobody-drug conjugates using 6x-Histidine affinity chromatography, followed by a sortase-mediated bioconjugation strategy. During the sortase reaction, the histidine tag is cleaved from the nanobody and is separated from unreacted nanobody using 6x-Histidine affinity chromatography again. If the nanobody contains histidine residues in its sequence, the reacted nanobody will still show affinity to the resin and yields are reduced. The reacted nanobody can be eluted using low concentration imidazole, but some of the unreacted product and sortase enzyme are then eluted too, reducing the purity of the sample. Further, the imidazole must be removed by buffer exchanging which reduces yield. We aimed to target CD11b, an integrin expressed by myeloid cells, with a nanobody but the only sequence available in literature contained two histidine residues. Using site-directed mutagenesis, we aimed to mutate both sites to four other amino acids and maintain binding affinity while eliminating histidine from the sequence. In this work, we showed that phenylalanine was the preferred mutation at both sites and binding affinity was maintained after mutagenesis. We characterized the mutated nanobodies and generated nanobody-drug conjugates as a proof-of-concept for using site-directed mutagenesis with nanobody proteins without affecting binding.