Background/Purpose: Protein-protein interactions are essential for the control of cellular functions and critical for regulation of the immune system. One example is the binding of Fc regions of Immunoglobulin G to their receptors (Fcγ Receptors). High structural homology between FcγRIIIa and FcγRIIIb has led to the lack of specific agents against this important therapeutic target. We aimed to develop a novel drug development pipeline using artificial binding proteins called Adhirons both for the identification of novel therapeutics and to guide drug discovery through the identification of novel hot spots/ druggable surfaces on the receptor.

Methods: We transiently transfected vectors encoding FcγRIIIa and FcγRIIa ectodomains into HEK293T cells and the resulting secreted proteins were purified. We additionally cloned sequences encoding full-length FcγRIIIa, FcγRIIIb and FcγRIIa into expression vectors and created stably transfected HEK293 cells expressing each receptor. The FcγRIIIa ectodomain was used as a target for screening an Adhiron library using ‘phage display. High affinity binders were used in Surface Plasmon Resonance (SPR), cellular assays (IgG binding assays and immune complex-induced TNF release and phagocytosis in THP-1-derived macrophage cells) and X-ray crystallography.

Results: Here we report the identification of FcγRIIIa-specific Adhirons. SPR experiments confirmed a pool of Adhirons that bound FcγRIIIa, but not FcγRIIa. Selective blockade of IgG binding to FcγRIIIa, but not FcγRIIa or FcγRIIIb, was detected for 3 Adhirons using flow cytometry assays in HEK293 cells that ectopically expressed individual FcγRs. We also demonstrated blockade of immune-complex induced effector functions downstream of FcγRIIIa signalling in THP-1-derived macrophage cells. Co-crystal structures revealed one Adhiron bound directly to the Fc binding site whereas two others acted as allosteric inhibitors. The structural basis for the specificity of the competitive inhibitor has been defined.

Conclusion: The results suggest that Adhirons can be developed to inhibit protein-protein interactions that were previously considered to be “undruggable”. Adhirons that inhibit the activatory FcγRIIIa (expressed on macrophages) but not the highly homologous FcγRIIIb (expressed on neutrophils), have been identified. This level of specificity has not been achieved using more conventional monoclonal antibody-based technology. Furthermore, the allosteric binders highlight a novel FcγRIIIa ‘hot spot’, which could be used in in silico-based Medicinal Chemistry design tools.

« Back to 2016 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/novel-agents-for-blocking-the-interaction-of-immune-complexes-with-the-activatory-fc%ce%b3riiia-receptor/