AUTHOR=Hussain Khiyam , Hargreaves Chantal E. , Rowley Tania F. , Sopp Joshua M. , Latham Kate V. , Bhatta Pallavi , Sherington John , Cutler Rona M. , Humphreys David P. , Glennie Martin J. , Strefford Jonathan C. , Cragg Mark S. 

TITLE=Impact of Human FcγR Gene Polymorphisms on IgG-Triggered Cytokine Release: Critical Importance of Cell Assay Format

JOURNAL=Frontiers in Immunology

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2019.00390

DOI=10.3389/fimmu.2019.00390

ISSN=1664-3224

ABSTRACT=<p>Monoclonal antibody (mAb) immunotherapy has transformed the treatment of allergy, autoimmunity, and cancer. The interaction of mAb with Fc gamma receptors (FcγR) is often critical for efficacy. The genes encoding the low-affinity FcγR have single nucleotide polymorphisms (SNPs) and copy number variation that can impact IgG Fc:FcγR interactions. Leukocyte-based <italic>in vitro</italic> assays remain one of the industry standards for determining mAb efficacy and predicting adverse responses in patients. Here we addressed the impact of FcγR genetics on immune cell responses in these assays and investigated the importance of assay format. FcγR genotyping of 271 healthy donors was performed using a Multiplex Ligation-Dependent Probe Amplification assay. Freeze-thawed/pre-cultured peripheral blood mononuclear cells (PBMCs) and whole blood samples from donors were stimulated with reagents spanning different mAb functional classes to evaluate the association of FcγR genotypes with T-cell proliferation and cytokine release. Using freeze-thawed/pre-cultured PBMCs, agonistic T-cell-targeting mAb induced T-cell proliferation and the highest levels of cytokine release, with lower but measurable responses from mAb which directly require FcγR-mediated cellular effects for function. Effects were consistent for individual donors over time, however, no significant associations with FcγR genotypes were observed using this assay format. In contrast, significantly elevated IFN-γ release was associated with the <italic>FCGR2A</italic>-131H/H genotype compared to <italic>FCGR2A</italic>-131R/R in whole blood stimulated with Campath (<italic>p</italic> ≤ 0.01) and IgG1 Fc hexamer (<italic>p</italic> ≤ 0.05). Donors homozygous for both the high affinity <italic>FCGR2A</italic>-131H and <italic>FCGR3A</italic>-158V alleles mounted stronger IFN-γ responses to Campath (<italic>p</italic> ≤ 0.05) and IgG1 Fc Hexamer (<italic>p</italic> ≤ 0.05) compared to donors homozygous for the low affinity alleles. Analysis revealed significant reductions in the proportion of CD14<sup>hi</sup> monocytes, CD56<sup>dim</sup> NK cells (<italic>p</italic> ≤ 0.05) and FcγRIIIa expression (<italic>p</italic> ≤ 0.05), in donor-matched freeze-thawed PBMC compared to whole blood samples, likely explaining the difference in association between FcγR genotype and mAb-mediated cytokine release in the different assay formats. These findings highlight the significant impact of <italic>FCGR2A</italic> and <italic>FCGR3A</italic> SNPs on mAb function and the importance of using fresh whole blood assays when evaluating their association with mAb-mediated cytokine release <italic>in vitro</italic>. This knowledge can better inform on the utility of <italic>in vitro</italic> assays for the prediction of mAb therapy outcome in patients.</p>