DataSheet_1_FcγR requirements and costimulatory capacity of Urelumab, Utomilumab, and Varlilumab.docx

IntroductionTargeting costimulatory receptors of the tumor necrosis factor receptor (TNFR) superfamily with agonistic antibodies is a promising approach in cancer immuno therapy. It is known that their efficacy strongly depends on FcγR cross-linking.
MethodsIn this study, we made use of a Jurkat-based reporter platform to analyze the influence of individual FcγRs on the costimulatory activity of the 41BB agonists, Urelumab and Utomilumab, and the CD27 agonist, Varlilumab.
ResultsWe found that Urelumab (IgG4) can activate 41BB-NFκB signaling without FcγR cross-linking, but the presence of the FcγRs (CD32A, CD32B, CD64) augments the agonistic activity of Urelumab. The human IgG2 antibody Utomilumab exerts agonistic function only when crosslinked via CD32A and CD32B. The human IgG1 antibody Varlilumab showed strong agonistic activity with all FcγRs tested. In addition, we analyzed the costimulatory effects of Urelumab, Utomilumab, and Varlilumab in primary human peripheral blood mononuclear cells (PBMCs). Interestingly, we observed a very weak capacity of Varlilumab to enhance cytokine production and proliferation of CD4 and CD8 T cells. In the presence of Varlilumab the percentage of annexin V positive T cells was increased, indicating that this antibody mediated FcγR-dependent cytotoxic effects.
ConclusionCollectively, our data underscore the importance to perform studies in reductionist systems as well as in primary PBMC samples to get a comprehensive understanding of the activity of costimulation agonists.

引言：使用激动性抗体靶向肿瘤坏死因子受体（Tumor Necrosis Factor Receptor, TNFR）超家族的共刺激受体，是癌症免疫治疗中极具前景的策略。已知此类抗体的疗效高度依赖Fcγ受体（Fc gamma receptor, FcγR）交联。

方法：本研究利用基于Jurkat细胞的报告基因平台，分析了各类Fcγ受体对41BB激动剂乌瑞单抗（Urelumab）、乌托单抗（Utomilumab）以及CD27激动剂瓦利单抗（Varlilumab）共刺激活性的影响。

结果：本研究发现，乌瑞单抗（Urelumab，IgG4型）可在无Fcγ受体交联的情况下激活41BB-核因子κB（Nuclear Factor kappa B, NFκB）信号通路，但Fcγ受体（CD32A、CD32B、CD64）的存在可增强乌瑞单抗的激动活性。人源IgG2型抗体乌托单抗（Utomilumab）仅在通过CD32A与CD32B交联时方可发挥激动功能。人源IgG1型抗体瓦利单抗（Varlilumab）在与所有受试Fcγ受体结合时均表现出较强的激动活性。此外，本研究还在原代人外周血单个核细胞（Peripheral Blood Mononuclear Cell, PBMC）中分析了乌瑞单抗、乌托单抗与瓦利单抗的共刺激效应。值得注意的是，我们观察到瓦利单抗促进CD4与CD8 T细胞细胞因子产生及增殖的能力极弱。当存在瓦利单抗时，膜联蛋白V（Annexin V）阳性T细胞的比例升高，表明该抗体可介导Fcγ受体依赖性的细胞毒性效应。

结论：综上，本研究数据强调，同时在还原论模型系统与原代PBMC样本中开展研究，对于全面阐明共刺激激动剂的活性具有重要意义。