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

PLoS One

Human Health Therapeutics Portfolio, National Research Council Canada, 100 Sussex Drive, Ottawa, Ontario, Canada, K1A 0R6.

Published: August 2017

AI Article Synopsis

  • * A new mutation-based technique has been developed to enable non-SpA-binding VHHs to bind SpA, enhancing their purification potential while maintaining expression yield and binding ability.
  • * Variations in SpA binding among different camelid species may be linked to specific genetic differences, suggesting that the binding ability can be adjusted based on the source of the VHHs for improved therapeutic applications.

Article 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.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5025174PMC
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0163113PLOS

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