The tumor necrosis factor superfamily (TNFSF) member LIGHT (TNFSF14) plays an important role in regulating the activity of immune cells, especially T cells. Several studies have shown that engagement of HVEM (herpesvirus entry mediator), one of the known receptors of LIGHT, can deliver a co-stimulatory signal to support T cell activation and expansion and promote tumor clearance. The HERA technology platform developed by Apogenix generates fully human hexavalent TNFSF fusion proteins that mimic the natural receptor binding mode in order to co-stimulate T cells. HERA ligands are pure agonists whose signaling capacity does not rely on secondary Fcγ-receptor crosslinking. Here we report the in vitro and in vivo properties of a novel HERA-LIGHT construct. Similar to all HERA fusion proteins, HERA-LIGHT has been engineered as a perfect molecular mimic of the natural ligand with high clustering capacity for the cognate receptor. The core unit consists of a single chain polypeptide comprising the three minimal LIGHT-subsequences necessary for folding into a functional trivalent receptor binding domain (RBD). By fusing a silenced IgG1 Fc-domain as a dimerization scaffold to the C-terminus of the RBD we generated HERA-LIGHT, a hexavalent fusion protein. HERA-LIGHT was expressed in CHO suspension cells followed by a lab-scale purification process including AFC- and SEC-based polishing, resulting in homogenous, aggregate-free protein lots. HERA-LIGHT was proven to bind both the human and murine HVEM receptor, as determined by ELISA. Qualitative analytics revealed excellent stability following heat- and pH-stress as well as freeze-thaw cycles. Analyzing serum samples from a PK study in CD1-mice, the terminal half-life of the compound was 36.5 hours. This short half-life, relative to antibodies, allows for fast-in/fast-out dynamics essential for improving combination therapy and reducing serious side effects associated with immune system overstimulation. In order to test biological activity, T cells were isolated by magnetic sorting from human PBMCs and treated with HERA-LIGHT in vitro. Flow cytometric analysis revealed that HERA-LIGHT enhanced activation and proliferation of naïve effector T cells (Teff) following stimulation with anti-CD3 antibody, as determined by CFSE dilution. Importantly, co-stimulation with HERA-LIGHT prevented regulatory T cell (Treg)-mediated suppression of Teff proliferation. In vivo, treatment with a murine surrogate of HERA-LIGHT resulted in significant tumor growth inhibition in a pilot study using the syngeneic CT-26 colorectal cancer model.In summary, the unique hexavalent design of HERA-LIGHT mediates efficient co-stimulation of Teff even in the presence of Treg cells and independent of secondary crosslinking events. Being true agonists, all HERA molecules are unique from current antibody-based concepts rendering them attractive candidates for cancer immunotherapy.
Citation Format: Julian P. Sefrin, David M. Richards, Jaromir Sykora, Meinolf Thiemann, Christian Merz, Viola Marschall, Mauricio Redondo Müller, Karl Heinonen, Harald Fricke, Christian Gieffers, Oliver Hill. Novel hexavalent HVEM agonist HERA-LIGHT promotes T cell activation and expansion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 630.