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

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(H)H single-domain Abs (sdAbs or V(H)Hs) are not bound by SpG and only sporadically bound by SpA. Currently, V(H)Hs 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(H)Hs. We show that SpA binding of V(H)Hs 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(H)Hs against three different targets with no adverse effects on expression yield or antigen binding. Next-generation sequencing of llama, alpaca and dromedary V(H)H 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(H)Hs 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.