Abstract 5559: Agonist-redirected checkpoint (ARC), TIM3-Fc-OX40L, for cancer immunotherapy
Authors: Fromm George , Silva Suresh de , Johannes Kellsey , Patel Arpita , Hornblower Josiah C. , Schreiber Taylor H. 
Published: August 2018

Abstract
Current attempts at 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. This is likely due to the fact that 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 fact that the ECDs are in opposite orientation. Here we report the generation of a two-sided fusion protein incorporating the ECD of TIM3 and the ECD of OX40L, adjoined by a central Fc domain.
Initial confirmation of the structural integrity of TIM3-Fc-OX40L was performed using functional ELISA assays, wherein simultaneous binding to GAL9 and OX40 was observed. A potential advantage of utilizing the intact ECD of TIM3 is the ability to bind all candidate TIM3 ligands, and the TIM3 end of the fusion protein binds both GAL9 and phosphatidylserine (PS) on the surface of human tumor cells. The OX40L end of the fusion protein binds OX40 on the surface of primary T cells. TIM3-Fc-OX40L activates NFκB signaling in cells engineered to overexpress OX40 and an NFκB-luciferase reporter in the absence of Fc receptor cross-linking. Additionally, the TIM3-Fc-OX40L ARC added to primary human PBMCs along with the super-antigen Staphylococcal enterotoxin B, induced secretion of the cytokines IL2 and TNFα. In vitro human T cell/tumor cell co-culture studies indicated that TIM3-Fc-OX40L stimulated enhanced T cell mediated killing of GAL9 positive human tumor cells as compared to OX40L-Fc controls. In vivo, TIM3-Fc-OX40L stimulated significant expansion of antigen-specific CD4 and CD8 T cells in mice adoptively transferred with OT-I/OT-II cells and vaccinated with Ova/Alum. Time-lapse high content live cell imaging has provided direct mechanistic support for TIM3-Fc-OX40L functionally tethering T cells to tumor cells and eliciting cytotoxic activity (tumor killing). Finally, the therapeutic activity of TIM3-Fc-OX40L in established murine MC38, B16.F10 and CT26 tumors was significantly superior to either TIM3 blocking antibody, OX40 agonist antibody or combination antibody therapy. Importantly, a pharmacodynamic biomarker of tumor rejection was identified by the elevation in serum cytokines post-treatment.
These data demonstrate feasibility and functional activity of a novel chimeric fusion protein platform, providing checkpoint blockade and TNF superfamily costimulation in a single molecule, which is uniquely advantageous because the construct links those two signals in the same microenvironmental context, at the time in which T cells are engaging cognate tumor antigen.
Citation Format: George Fromm, Suresh de Silva, Kellsey Johannes, Arpita Patel, Josiah C. Hornblower, Taylor H. Schreiber. Agonist-redirected checkpoint (ARC), TIM3-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 5559.