HERA-CD27L, a true CD27 agonist, is a hexavalent CD27 ligand that enhances T cell activation and induces potent anti-tumor immunity

Agonistic stimulation of TNFRSF members like CD27 is a promising strategy to boost anti-tumor responses. Although antibodies are effective inhibitors of signaling, they have shown minimal agonistic activity due to their limited binding domains, flexibility and the toxicity mediated by Fc/FcγR interactions. TNFRSF signaling is a structurally well-defined event that takes place during cell contact. The trimeric-trivalent TNFSF-receptor binding domain (TNFSF-RBD) on the conducting cell and the resulting multi-trimer-based receptor clustering on the receiving cell are essential for signaling. In contrast to antibodies, HERA-CD27L mimics the natural ligand and induces potent activity. In order to understand the activity of HERA-CD27L, human T cells were stimulated in the presence of HERA-CD27L, the trimeric CD27L or a clinical benchmark anti-CD27 antibody. In all assays, treatment with the hexavalent HERA-CD27L significantly boosted T cell activation, proliferation and differentiation. Furthermore, the hexavalent molecule was always superior to the trimeric CD27L and bivalent antibody. In fact, treatment with the anti-CD27 antibody resulted in significantly weaker proliferation compared to anti-CD3 antibody alone. To understand early events, we tested CD27 signaling using a reporter cell assay. Treatment with HERA-CD27L and CD27L resulted in high and intermediate, respectively, reporter activity. In contrast, the anti-CD27 antibody failed to show any signaling activity across a wide range of concentrations. Since most T cells express CD27, there is potential for non-specific T cell activation. This was examined by comparing OVA-specific and non-specific T cells in the same environment using the CD8+ “OT-I” T cell adoptive transfer mouse model. Following a single dose of HERA-CD27L, serial blood samples showed a significant and HERA-CD27L dose-dependent clonal expansion of OT-I T cells. OT-I T cells expressed high levels of activation markers, while the endogenous T cells failed to show any response. The potent single-agent anti-tumor efficacy of the hexavalent HERA-CD27L was demonstrated in two different mouse models. With CT26wt, HERA-CD27L also showed superior activity compared to anti-PD-1 antibody. Furthermore, combination of HERA-CD27L and anti-PD-1 antibody showed additive effects. Finally, early treatment with HERA-CD27L significantly increased overall survival, from 19 to 41 days, and tumor-free animals still alive at the end of the study were protected from tumor re-challenge. Various strategies have been proposed for targeting CD27 for cancer therapy. As we have shown here, the hexavalent HERA-CD27L has superior activity compared to bivalent antibodies. Altogether, HERA-CD27L shows single-agent anti-tumor efficacy, is well tolerated by multiple relevant species and the lead candidate is currently ready for GMP cell line development. Citation Format: Julian P. Sefrin, David M. Richards, Katharina Billian-Frey, Karl Heinonen, Viola Marschall, Christian Merz, Mauricio Redondo Muller, Matthias Schroder, Jaromir Sykora, Meinolf Thiemann, Harald Fricke, Christian Gieffers, Oliver Hill. HERA-CD27L, a true CD27 agonist, is a hexavalent CD27 ligand that enhances T cell activation and induces potent anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4845.