A Rational Engineering Strategy for Designing Protein A-Binding Camelid Single-Domain Antibodies

Abstract

Staphylococcal protein A ( SpA) and streptococcal protein G ( SpG) affinity chromatography are the gold standards for purifying monoclonal antibodies ( mAbs) in therapeutic applications. However, camelid V _HH single-domain Abs ( sdAbs or V _H Hs) are not bound by SpG and only sporadically bound by SpA. Currently, V _HHs require affinity tag-based purification, which limits their therapeutic potential and adds considerable complexity and cost to their production. Here we describe a simple and rapid mutagenesis-based approach designed to confer SpA binding upon a priori non-SpA-binding V _HHs. We show that SpA binding of V _HHs is determined primarily by the same set of residues as in human mAbs, albeit with an unexpected degree of tolerance to substitutions at certain core and non-core positions and some limited dependence on at least one residue outside the SpA interface, and that SpA binding could be successfully introduced into five V _HHs against three different targets with no adverse effects on expression yield or antigen binding. Next-generation sequencing of llama, alpaca and dromedary V _HH repertoires suggested that species differences in SpA binding may result from frequency variation in specific deleterious polymorphisms, especially Ile57. Thus, the SpA binding phenotype of camelid V _HHs can be easily modulated to take advantage of tag-less purification techniques, although the frequency with which this is required may depend on the source species.