Abstract A83: Hexavalent agonists targeting co-stimulatory receptors of the tumor necrosis factor superfamily
Authors: Gieffers Christian , Merz Christian , Richards David , Redondo Mauricio , Marschall Viola , Sykora Jaromir , Thiemann Meinolf , Fricke Harald , Hill Oliver 
Published: March 2017

Tumor necrosis factor receptor superfamily (TNFRSF) proteins are widely expressed by immune and tumor cells highlighting their importance in multiple locations and phases of the anti-tumor immune response. Whereas agonistic stimulation of TRAIL-Receptor-1 or -2 can directly induce apoptosis in tumor cells, signaling through many TNFRSF members, such as CD40, CD27, OX40, HVEM, GITR and 4-1BB, results in co-stimulation of immune cells. Therefore, agonistic stimulation of certain members of the TNFRSF is considered to have a positive impact on immune-based therapeutic concepts in clinical oncology. Apogenix has developed a proprietary technology platform for the construction of novel hexavalent TNFRSF agonists (HERA) for the treatment of cancer. HERA fusion proteins are based on a perfect molecular mimic of the TNFSF cytokine structure. The HERA core unit consists of one single polypeptide chain comprising the three receptor-binding domain-forming subsequences (TNFSF-protomers). These single-chain TNFSF receptor-binding domains (scTNFSF-RBD) preserve the structural organization of the trimeric natural TNFSF cytokine and can be utilized to engineer fully human fusion-proteins in a modular manner. Fusing an IgG1 Fc-domain as a dimerization scaffold to the C-terminus of a scTNFSF-RBD creates a hexavalent agonist from two trivalent scTNFSF-RBDs. As a result of this molecular design, each molecule is capable of clustering six receptors in a spatially well-defined manner in close proximity to each other. Therefore, signaling following treatment with the Apogenix HERA scTNFSF-RBD-Fc fusion proteins in vivo is independent of secondary clustering through Fc-γ receptors that is required for many agonistic anti-TNFRSF antibodies (e.g., anti-TRAIL-R2 or anti-CD40). The described HERA engineering concept has been successfully translated to TRAIL, CD40L, LIGHT and CD27L resulting in hexavalent agonists suitable for further development. CD27-Ligand is a potent co-stimulatory molecule that drives T-cell activation and survival through interaction with its receptor (CD27). Here we show in vitro and in vivo data for APG1293 (scCD27L-RBD-Fc), a hexavalent CD27 agonist. APG1293 was expressed in CHO suspension cells followed by a lab-scale purification process including affinity chromatography and SEC-based polishing, resulting in homogenous aggregate-free protein lots. The purified protein binds its respective target-receptor with high affinity. In vivo stability/PK studies have been performed in addition to in vitro experiments with primary human and mouse lymphoid and myeloid cell populations. Specifically, APG1293 was able to bind CD27 expressed on primary human CD4+ and CD8+ T cells and importantly, binding significantly increased T-cell expansion following activation. In vivo the efficacy of APG1293 was evaluated in the colorectal syngeneic murine tumor models MC38-CEA and CT26. In both models APG1293 treatment resulted in a dose dependent tumor growth inhibition. In summary, the data on the hexavalent APG1293 indicate a potent immune cell driven anti tumor efficacy. Therefore, APG1293 could be applied as a single agent or in combination with check-point inhibitors.

Citation Format: Christian Gieffers, Christian Merz, David Richards, Mauricio Redondo, Viola Marschall, Jaromir Sykora, Meinolf Thiemann, Harald Fricke, Oliver Hill. Hexavalent agonists targeting co-stimulatory receptors of the tumor necrosis factor superfamily. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A83.