以激动性抗体靶向肿瘤坏死因子受体（TNFR）超家族的共刺激受体是癌症免疫治疗中一种有前景的方法。我们知道它们的疗效强烈依赖于FcγR的交联。在本研究中，我们利用一个基于Jurkat细胞的报道平台，分析了各个FcγR对41BB激动剂Urelumab和Utomilumab，以及CD27激动剂Varlilumab的共刺激活性的影响。我们发现，Urelumab（IgG4）可以在没有FcγR的交联的情况下激活41BB-NFκB信号传导，但FcγR（CD32A，CD32B，CD64）的存在会增强Urelumab的激动活性。人源IgG2抗体Utomilumab只有在通过CD32A和CD32B交联时才发挥激动功能。人源IgG1抗体Varlilumab在所有经过测试的FcγR上都显示出强烈的激动活性。此外，我们还分析了Urelumab，Utomilumab和Varlilumab在原代人类外周血单核细胞（PBMCs）中的共刺激效应。有趣的是，我们观察到Varlilumab在增强CD4和CD8 T细胞的细胞因子产生和增殖方面的能力非常弱。在Varlilumab存在的情况下，annexin V阳性T细胞的百分比增加，表明这种抗体介导了FcγR依赖的细胞毒性效应。总的来说，我们的数据强调了在还原化系统以及原代PBMC样本中进行研究以全面了解共刺激激动剂的活性的重要性。版权所有©2023 Leitner, Egerer, Waidhofer-Söllner, Grabmeier-Pfistershammer和Steinberger。

Targeting 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.In 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.We 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.Collectively, 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.Copyright © 2023 Leitner, Egerer, Waidhofer-Söllner, Grabmeier-Pfistershammer and Steinberger.