Data_Sheet_1_Impact of Human FcγR Gene Polymorphisms on IgG-Triggered Cytokine Release: Critical Importance of Cell Assay Format.docx

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 in vitro 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 FCGR2A-131H/H genotype compared to FCGR2A-131R/R in whole blood stimulated with Campath (p ≤ 0.01) and IgG1 Fc hexamer (p ≤ 0.05). Donors homozygous for both the high affinity FCGR2A-131H and FCGR3A-158V alleles mounted stronger IFN-γ responses to Campath (p ≤ 0.05) and IgG1 Fc Hexamer (p ≤ 0.05) compared to donors homozygous for the low affinity alleles. Analysis revealed significant reductions in the proportion of CD14hi monocytes, CD56dim NK cells (p ≤ 0.05) and FcγRIIIa expression (p ≤ 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 FCGR2A and FCGR3A SNPs on mAb function and the importance of using fresh whole blood assays when evaluating their association with mAb-mediated cytokine release in vitro. This knowledge can better inform on the utility of in vitro assays for the prediction of mAb therapy outcome in patients.

单克隆抗体（Monoclonal antibody, mAb）免疫治疗已彻底改变了过敏、自身免疫性疾病与癌症的治疗格局。单克隆抗体与Fcγ受体（Fc gamma receptors, FcγR）的结合相互作用，往往是其发挥疗效的关键环节。编码低亲和力Fcγ受体的基因存在单核苷酸多态性（single nucleotide polymorphisms, SNPs）与拷贝数变异，这些变异可影响免疫球蛋白G（Immunoglobulin G, IgG）Fc段与Fcγ受体的结合相互作用。基于白细胞的体外实验（in vitro assays）仍是业界用于评估单克隆抗体疗效、预测患者不良反应的标准方法之一。本研究探究了Fcγ受体遗传学特征对上述实验中免疫细胞应答的影响，并分析了实验体系的重要性。本研究采用多重连接依赖性探针扩增（Multiplex Ligation-Dependent Probe Amplification）实验，对271名健康供者进行了Fcγ受体基因分型。研究人员使用覆盖不同单克隆抗体功能类别的试剂，对供者的冻融/预培养外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）与全血样本进行刺激，以评估Fcγ受体基因型与T细胞增殖及细胞因子释放之间的关联。在使用冻融/预培养PBMCs的实验体系中，靶向T细胞的激动型单克隆抗体可诱导T细胞增殖并触发最高水平的细胞因子释放；而那些功能直接依赖Fcγ受体介导的细胞效应的单克隆抗体，其应答水平虽较低但仍可被检测到。对于单个供者而言，其应答效果在不同时间点保持一致，但采用该实验体系时，未观察到与Fcγ受体基因型存在显著关联。与之相反，在使用Campath（p ≤ 0.01）与IgG1 Fc六聚体（p ≤ 0.05）刺激的全血样本中，FCGR2A-131H/H基因型供者的干扰素γ（Interferon-γ, IFN-γ）释放水平显著高于FCGR2A-131R/R基因型供者。与携带低亲和力等位基因的纯合子供者相比，同时携带高亲和力FCGR2A-131H与FCGR3A-158V等位基因的纯合子供者，在接受Campath与IgG1 Fc六聚体刺激时，其IFN-γ应答水平更强（p ≤ 0.05）。分析结果显示，与供者匹配的全血样本相比，冻融PBMC样本中CD14高表达单核细胞、CD56低表达自然杀伤细胞（natural killer cells, NK细胞）的比例以及FcγRIIIa的表达水平均显著降低（p ≤ 0.05），这或可解释不同实验体系中Fcγ受体基因型与单克隆抗体介导的细胞因子释放之间关联的差异。本研究结果凸显了FCGR2A与FCGR3A单核苷酸多态性对单克隆抗体功能的显著影响，以及在体外评估Fcγ受体基因型与单克隆抗体介导的细胞因子释放之间关联时，采用新鲜全血实验体系的重要性。该研究结果可为利用体外实验预测患者单克隆抗体治疗结局的应用价值提供更优的参考依据。