title: 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 VHH single-domain Abs (sdAbs or VHHs) are not bound by SpG and only sporadically bound by SpA. Currently, VHHs 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 VHHs. We show that SpA binding of VHHs 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 VHHs against three different targets with no adverse effects on expression yield or antigen binding. Next-generation sequencing of llama, alpaca and dromedary VHH 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 VHHs 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.
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council Canada, 100 Sussex Drive, Ottawa, Ontario, Canada, K1A 0R6.
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Henry
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council Canada, 6100 Royalmount Avenue, Montreal, Quebec, Canada, H4P 2R2.
authors.forename: Traian
authors.initials: T
authors.lastname: Sulea
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council Canada, 100 Sussex Drive, Ottawa, Ontario, Canada, K1A 0R6.
authors.forename: Henk
authors.initials: H
authors.lastname: van Faassen
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council Canada, 100 Sussex Drive, Ottawa, Ontario, Canada, K1A 0R6.
authors.forename: Greg
authors.initials: G
authors.lastname: Hussack
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council Canada, 6100 Royalmount Avenue, Montreal, Quebec, Canada, H4P 2R2.
authors.forename: Enrico O
authors.initials: EO
authors.lastname: Purisima
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council Canada, 100 Sussex Drive, Ottawa, Ontario, Canada, K1A 0R6.|School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada, N1G 2W1.
authors.forename: C Roger
authors.initials: CR
authors.lastname: MacKenzie
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council Canada, 100 Sussex Drive, Ottawa, Ontario, Canada, K1A 0R6.|School of Environmental Sciences, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada, N1G 2W1.|Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada, K1S 5B6.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Arbabi-Ghahroudi
mesh_terms: D000818:Animals; D002161:Camelids, New World / Q000276:immunology*; D015202:Protein Engineering; D061905:Single-Domain Antibodies / Q000235:genetics*; D013205:Staphylococcal Protein A / Q000235:genetics*
chemical_list: D061905:Single-Domain Antibodies; D013205:Staphylococcal Protein A