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

Yamal Al-Ramahi,Yago Margolles,Gyorgyi Ferenc,Akos Nyerges,Víctor De Lorenzo,Csaba Pál,Lidia Cerdán,Luis Ángel Fernández

doi:10.1038/s42003-021-02702-0

Abstract

ssDNA recombineering has been exploited to hyperdiversify genomically-encoded nanobodies displayed on the surface of Escherichia coli for originating new binding properties. As a proof-of-principle a nanobody recognizing the antigen TirM from enterohaemorrhagic E. coli (EHEC) was evolved towards the otherwise not recognized TirM antigen from enteropathogenic E. coli (EPEC). To this end, E. coli cells displaying this nanobody fused to the intimin outer membrane-bound domain were subjected to multiple rounds of mutagenic oligonucleotide recombineering targeting the complementarity determining regions (CDRs) of the cognate VHH gene sequence. Binders to the EPEC-TirM were selected upon immunomagnetic capture of bacteria bearing active variants and nanobodies identified with a new ability to strongly bind the new antigen. 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.

Highlights

SsDNA recombineering has been exploited to hyperdiversify genomically-encoded nanobodies displayed on the surface of Escherichia coli for originating new binding properties
In this article we show how combination of ssDNA recombineering[16] with surface-displayed nanobodies on E. coli cells enables rapid expansion of the antigen targeting capacity of a Nb that binds the extracellular domain of the translocated intimin receptor (TirM) of enterohaemorrhagic E. coli (EHEC) strains towards the otherwise non-recognized but functionally homologous TirM domain of enteropathogenic E. coli (EPEC) strains[20,21]
The results above showcase the power of merging surface display of Nbs with genomic site-specific diversification elicited by DIvERGE technology in combination with MACSbased cyclic selection

Summary

Introduction

SsDNA recombineering has been exploited to hyperdiversify genomically-encoded nanobodies displayed on the surface of Escherichia coli for originating new binding properties. As a proof-of-principle a nanobody recognizing the antigen TirM from enterohaemorrhagic E. coli (EHEC) was evolved towards the otherwise not recognized TirM antigen from enteropathogenic E. coli (EPEC) To this end, E. coli cells displaying this nanobody fused to the intimin outer membrane-bound domain were subjected to multiple rounds of mutagenic oligonucleotide recombineering targeting the complementarity determining regions (CDRs) of the cognate VHH gene sequence. While the variable (V) domain of different antibodies adopt a very similar immunoglobulin (Ig) fold structure, their antigen-binding loops (the so-called complementarity determinant regions or CDRs) are selected out of a very large diversity pool[3] Such a process occurs upon exposure of the immune system to given antigens through an intricate molecular mechanism of clonal selection, amplification and affinity/specificity maturation[4,5]. Mutagenic oligos steps needed for more robust and versatile in vivo selection approaches (e.g. phage, bacterial or yeast display)[15]

Methods

Results

Conclusion