Abstract
FcγR activity underpins the role of antibodies in both protective immunity and auto-immunity and importantly, the therapeutic activity of many monoclonal antibody therapies. Some monoclonal anti-FcγR antibodies activate their receptors, but the properties required for cell activation are not well defined. Here we examined activation of the most widely expressed human FcγR; FcγRIIa, by two non-blocking, mAbs, 8.26 and 8.2. Crosslinking of FcγRIIa by the mAb F(ab’)2 regions alone was insufficient for activation, indicating activation also required receptor engagement by the Fc region. Similarly, when mutant receptors were inactivated in the Fc binding site, so that intact mAb was only able to engage receptors via its two Fab regions, again activation did not occur. Mutation of FcγRIIa in the epitope recognized by the agonist mAbs, completely abrogated the activity of mAb 8.26, but mAb 8.2 activity was only partially inhibited indicating differences in receptor recognition by these mAbs. FcγRIIa inactivated in the Fc binding site was next co-expressed with the FcγRIIa mutated in the epitope recognized by the Fab so that each mAb 8.26 molecule can contribute only three interactions, each with separate receptors, one via the Fc and two via the Fab regions. When the Fab and Fc binding were thus segregated onto different receptor molecules receptor activation by intact mAb did not occur. Thus, receptor activation requires mAb 8.26 Fab and Fc interaction simultaneously with the same receptor molecules. Establishing the molecular nature of FcγR engagement required for cell activation may inform the optimal design of therapeutic mAbs.
Highlights
IgG Abs, elicited by vaccination or natural infection or as therapeutic mAbs, are important mediators of human health and the activation of FcgR-expressing innate leucocytes is often fundamental to their efficacy
FcgRIIa triggers a rare thrombotic thrombocytopenia induced by anti-PF4 antibodies after vaccination for SARS CoV-2 spike vectored by a recombinant adenovirus (ChAdOx1, AstraZeneca) [23, 24]
Treatment of IIA1.6-FcgRIIa cells with agonist mAb 8.2 resulted in phosphorylation of the receptor and associated proteins with similar kinetics to that found in a conventional immunoprecipitation and Western analysis (Supplementary Figure 1)
Summary
IgG Abs, elicited by vaccination or natural infection or as therapeutic mAbs, are important mediators of human health and the activation of FcgR-expressing innate leucocytes is often fundamental to their efficacy. The activating FcgRs, including FcgRIIa, signal via an immunoreceptor tyrosine-based activation motif (ITAM) [12], with FcgRIIa and FcgRIIc containing their ITAM within their cytoplasmic domains, while other activating FcRs associate with an ITAM containing signal transduction subunit, FcRg [13, 14]. FcgRIIa has two major polymorphisms, H131, the only functional FcgR for human IgG2, and R131 which was characterized functionally by its preferential interaction with mouse IgG1 [15, 16]. FcgRIIa potently activates platelets when engaged by the Fc of anti-platelet antibodies [19, 20], including antiplatelet factor 4 (PF4) antibodies which trigger heparin induced thrombocytopenia [21, 22]. FcgRIIa triggers a rare thrombotic thrombocytopenia induced by anti-PF4 antibodies after vaccination for SARS CoV-2 spike vectored by a recombinant adenovirus (ChAdOx1, AstraZeneca) [23, 24]
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