FcγR requirements and costimulatory capacity of Urelumab, Utomilumab, and Varlilumab

Abstract

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.

Plant‐derived Durvalumab variants show efficient PD‐1/PD‐L1 blockade and therapeutically favourable FcR binding

“…In addition, Fc N ‐glycosylation has been shown to affect the affinity of IgG for all FcγR classes (CD64, CD32 and CD16) and to C1q (Hayes et al ., 2014 ). We used a flow cytometry‐based assay to test the binding of our plant‐derived DL variants to cells expressing high levels of different human FcγRs—CD16A, the natural V176 high‐affinity variant of CD16A (C16V), CD32A, CD32B and CD64 (Leitner et al ., 2023 ). Cells not expressing human FcγR were used as a control.…”

“…To evaluate the binding of DL variants to human FcγRs, we used a set of BW5147 lines stably expressing high levels of human FcγR—CD16A (FcγRIIIA), the natural V176 high‐affinity variant of CD16A (FcγRIIIA 176 V), CD32A (FcγRIIA), CD32B (FcγRIIB) and CD64 (FcγRI) (Leitner et al ., 2023 ). BW5147 cells not expressing human FcγR were used as a control.…”

Transcriptional reprogramming via signaling domains of CD2, CD28, and 4-1BB

“…Specifically, we found that engagement of 4-1BB and CD27 by their natural ligands also induced potent upregulation of NF-kB and AP-1 reporter gene expression and a modest downregulation of NFAT activity. 31 We also performed some experiments with chimera harboring mICOS ectodomains lacking the dimerization motif, but due to a poor expression of these chimeric receptors their costimulatory capacity was weak. Furthermore, downregulation of NFAT activation via 4-1BB and CD27 signaling, which also occurs upon engagement of 4-1BB and CD27 with their natural ligands, was not observed with mICOS chimera lacking the dimerization motif.…”

Inhibitory CARs fail to protect from immediate T cell cytotoxicity

“…Flow cytometry analysis was performed using constant cell volumes, flow rates, and acquisition time for all samples to detect target cell loss as described recently. 85 Cytotoxicity was measured as percentage of Annexin V-positive target cells of all target cells.…”