Abstract 5564: Agonist redirected checkpoint, VSIG8-Fc-OX40L, for cancer immunotherapy
Authors: Fromm George , Silva Suresh de , Johannes Kellsey , Patel Arpita , Hornblower Josiah , Schreiber Taylor H. 
Published: August 2018

Abstract
Combination immunotherapy with bispecific antibodies, linked scFv's or T cell engagers have not demonstrated that both checkpoint blockade and TNF receptor activation can be achieved with a single molecule, because these molecules lose target avidity when engineered to bind multiple targets with monovalent antigen binding arms. Fusion proteins incorporating the extracellular domain (ECD) of type I membrane proteins (eg. Enbrel, Orencia) or type II membrane proteins (eg. OX40L-Fc, GITRL-Fc), linked to the hinge-CH2-CH3 domain of antibodies are both functional, despite the ECDs being in opposite orientation. We report the generation of a two-sided fusion protein incorporating the ECD of VSIG8 and the ECD of OX40L, adjoined by a central Fc domain. It has been suggested that the VSIG (V-set and immunoglobulin domain containing) family of proteins play important yet diverse roles in cancer, however little is known about their MOAs or therapeutic functions.
Each of the three individual domains of VSIG8-Fc-OX40L were identified using conformation-dependent antibodies by Western blots, and binding to OX40 was demonstrated in functional ELISA assays. Although VSIG8 has been described as a putative binding partner for VISTA, no binding was detected between VSIG8-Fc-OX40L and recombinant VISTA using ELISA, Octet or a retroviral membrane display platform. Interestingly, binding was observed to VISTA positive mouse and human tumor cell lines. Membrane preparations of these cell lines were generated in order to identify the putative binding partner(s) of VSIG8, which were then re-confirmed using recombinant protein in ELISA assays. VSIG8-Fc-OX40L stimulated potent activation of NFkB signaling in reporter cells lines, and stimulated IL2 and TNFa secretion from PBMCs primed with the super-antigen SEB. Time-lapse live cell imaging demonstrated that VSIG8-Fc-OX40L led to functional tethering of primary human T cells to human tumor cells and stimulated tumor cell killing (as evidenced by cleavage of caspase 3/7) to a greater extent than antibody controls. In tumor bearing mice, VSIG8-Fc-OX40L expanded both effector CD4 and CD8 T cell subsets (without activating Tregs) in the spleen, lymph node and tumor, including a significant increase in AH1-tetramer+ cells (antigen-specific CD8+ T cells) in the isolated TIL. Finally, the therapeutic activity of VSIG8-Fc-OX40L in established murine MC38 and CT26 tumors was significantly superior to either VISTA blocking antibody, OX40 agonist antibody or combination antibody therapy.
The V-set and immunoglobulin domain containing family of proteins, including TIGIT, have potent checkpoint-like activity in cancer immunotherapy but are not well understood. These data demonstrate feasibility and functional activity of a novel chimeric fusion protein platform, which provided potent anti-tumor immunity and also facilitated the de-orphanization of the poorly understood protein, VSIG8.
Citation Format: George Fromm, Suresh de Silva, Kellsey Johannes, Arpita Patel, Josiah Hornblower, Taylor H. Schreiber. Agonist redirected checkpoint, VSIG8-Fc-OX40L, for cancer immunotherapy [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 5564.