Abstract 1204: A construction platform for hexavalent agonists targeting receptors of the tumor necrosis factor superfamily: Where death meets co-stimulation

Tumor necrosis factor receptor superfamily (TNFRSF) proteins are widely expressed by immune and tumor cells. Their importance in many locations and phases of the anti-tumor immune response is now broadly appreciated and several TNFR agonists are currently in preclinical and clinical development. Importantly, signaling through many TNFRSF members, such as CD40, CD27, OX40, 4-1BB, HVEM and GITR, is potentially associated with an enhanced anti tumor response via co-stimulation of immune cells. Apogenix has established a development platform for a novel class of TNFRSF-agonists for the treatment of cancer. Unlike their natural homotrimeric counterparts, the Apogenix recombinant TNFSF proteins consist of one single polypeptide chain composed of three receptor-binding domain-forming protomers. These single-chain TNFSF receptor-binding domains (scTNFSF-RBD) are mimics of the three-dimensional organization of the natural TNFSF-cytokine and can be used to engineer fully human fusion-proteins from a modular toolbox. For example, fusing an IgG1 Fc-domain to the C-terminus of a scTNFSF-RBD creates a hexavalent agonist as the Fc-domain acts as a dimerization scaffold for two trivalent scTNFSF-RBDs. As a result of this molecular design, each drug molecule is capable of clustering six receptors in a spatially well-defined manner. Consequently, TNFSF receptor signaling following treatment with the Apogenix scTNFSF-RBD-Fc in vivo is independent of secondary clustering through Fc-γ receptors that is required for many anti-TNFRSF agonistic antibodies (e.g., anti-TRAILR2 or -CD40). Following up the scTRAIL-RBD-Fc prototype, this engineering concept has now been successfully translated to CD40L and CD27L resulting in hexavalent agonists suitable for further development. Expression of the drug candidates in CHO suspension cells followed by an AFC and SEC-based lab-scale purification process resulted in homogenous aggregate-free protein lots. The purified proteins bind their respective target-receptors 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, it was shown that scCD27L-RBD-Fc was able to bind CD27 expressed on primary human CD4+ and CD8+ T cells. Importantly, binding significantly increased T cell expansion following activation. Treatment with scCD40L-RBD-Fc induced differentiation of B cells and enhanced primary human monocyte differentiation into DCs or M1 macrophages. Encouraged by the promising results obtained with TRAIL, CD40L, and CD27L, Apogenix is currently expanding the TNFRSF-agonist pipeline to target additional cell populations, locations and phases of the immune response in order to develop novel therapies to treat cancer and other conditions. Citation Format: Christian Merz, Christian Gieffers, Michael Kluge, David M. Richards, Tim Schnyder, Jaromir Sykora, Meinolf Thiemann, Harald Fricke, Oliver Hill. A construction platform for hexavalent agonists targeting receptors of the tumor necrosis factor superfamily: Where death meets co-stimulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1204.