New paper describes multiple methods of interaction for Affimer® inhibition of Fc?R

A new paper (link is external) from the University of Leeds and colleagues at Harwell, published in Proceedings of the National Academy of Sciences (PNAS), shows how Affimer technology has been successfully applied to specifically inhibit the interaction between IgG antibodies and the Fc?RIIIa receptors on macrophages. This specific inhibition of the Fc receptors has previously proven impossible using alternative technologies due to the highly homologous nature of these receptors. With only two amino acid differences between Fc?RIIIa, found on macrophages and natural killer cells, and Fc?RIIIb, found on neutrophils, no commercial antibodies are available that specifically target Fc?RIIIa. However, Affimer binders identified in this study show total specificity for Fc?RIIIa, inhibiting the receptor and abrogating downstream effector functions of TNF? release and phagocytosis in macrophages.

Targeting these receptors has long been a clinical strategy for the treatment of many autoimmune and disease states, including rheumatoid arthritis and idiopathic thrombocytopenic purpura. Attempts to block specific signals from the Fc?R have resulted in many negative off-target reactions during clinical trials, leading to suspension of the development of such treatments. The identification of specific Affimer inhibitors offers the potential to investigate strategies to dampen macrophage-based immune responses, whilst maintaining host immunity to infections afforded by the action of neutrophils, with the possibility of developing effective treatments for the many patients of such conditions.

The team of researchers at the University of Leeds selected two Fc?RIIIa-specific Affimer binders, AfF4 and AfG3, for further analysis. Using HEK293 cell lines expressing specific Fc?R combinations they showed that both of these Affimer proteins were able to inhibit IgG binding to Fc?RIIIa expressing cell lines, but showed no significant interaction with cells expressing either of the homologous receptors, Fc?RIIIb or Fc?RIIa.

Cocrystal structures of the Affimer proteins bound to the receptor ectodomain showed that  they bind on opposite faces of the Fc?RIIIa receptor. These crystal structures together with molecular dynamics simulations revealed that AfF4 competes with IgG for binding to Fc?RIIIa via steric inhibition, with the Affimer and IgG binding regions overlapping by approximately half their interaction surfaces. The AfG3 Affimer binder by contrast interacts with Fc?RIIIa via an allosteric mode of inhibition, binding at the interdomain hinge region of the receptor, with no overlap with the IgG-binding site. Molecular dynamics simulations revealed that this is due to a narrowing of the interdomain hinge region upon Affimer binding, thus allosterically preventing Fc?RIIIa from binding to its IgG ligand.

A differentiated monocytic cell line expressing Fc?RIIIa was used to investigate the downstream effects of Affimer inhibition of the macrophage receptors. Both TNF? release and phagocytosis effector functions were shown to be reduced by the Affimer binders to levels comparable to those observed by Fab blockade of the receptor.

This study demonstrates the feasibility of generating specific Affimer inhibitors that can interrupt protein-ligand interactions, even for highly homologous proteins, and illustrates the use of Affimer binders in the study of protein function at both the molecular and cellular level, with the potential for these highly stable and reproducible proteins to target clinically relevant pathways.