ssDNA recombineering boosts in vivo evolution of nanobodies displayed on bacterial surfaces

SUMMARYIn vivo evolution of antibodies facilitates emergence of novel target specificities from pre-existing clones. In this work we show how mutagenic ssDNA recombineering of camel-derived nanobodies encoded in a bacterial genome enables clonal hyper-diversification and the rise of new properties. As a proof-of-principle we used a nanobody recognizing the antigen TirM from enterohaemorrhagic E. coli (EHEC) and evolved it towards the otherwise not recognized TirM antigen from enteropathogenic E. coli (EPEC). To this end, E. coli cells displaying on their surface this nanobody fused to the intimin outer membrane anchor domain were subjected to multiple rounds of mutagenic ssDNA recombineering targeted to the CDR1, CDR2 and CDR3 regions of its genomically encoded VHH sequence. Binders to the new antigen (EPEC TirM) were then selected upon immunomagnetic capture of bacteria bearing the corresponding nanobody variants. As a result, several modified nanobodies were identified which maintained recognition of EHEC TirM but acquired the ability to bind the new antigen with high affinity (Kd ~20 nM). The results highlight the power of combining evolutionary properties of bacteria in vivo with oligonucleotide synthesis in vitro for the sake of focusing diversification to specific segments of a gene (or protein thereof) of interest. Our experimental workflow empowers the evolution of nanobodies displayed on the surface of bacterial cells for a large number of potential applications in medical and industrial biotechnology.