Agonist Redirected Checkpoint (ARC), TIM3-Fc-OX40L, for Cancer Immunotherapy

Abstract

Abstract Combination immunotherapy with bispecific abs, linked scFv’s or T cell engagers have not shown that both checkpoint blockade and TNFR activation can be achieved with a single molecule, likely due to a loss of target avidity. Fusion proteins incorporating the extracellular domain of type I membrane proteins (eg. Enbrel) or type II membrane proteins (eg. OX40L-Fc), linked via an antibody Fc domain, are both functional despite the ECDs being in opposite orientation. We report the generation of a two-sided fusion protein (ARC) incorporating the ECD of TIM3 and the ECD of OX40L, adjoined by a central Fc domain. The TIM3 end of the ARC binds GAL9 and phosphatidylserine (PS) in vitro and in vivo, and OX40L binds OX40 on the surface of primary T cells. TIM3-Fc-OX40L activates NFκB signaling in the absence of Fc receptor cross-linking, stimulates IL2/TNFa secretion with the superantigen SEB, and increases the in vitro killing of GAL9+ tumor cells. In vivo, TIM3-Fc-OX40L stimulated expansion of antigen-specific CD4 and CD8 T cells in mice adoptively transferred with OT-I/OT-II cells (+ova/alum). Live cell imaging has provided direct mechanistic support for TIM3-Fc-OX40L 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 superior to either TIM3 blocking antibody, OX40 agonist antibody or combination antibody therapy. These data demonstrate feasibility of a novel chimeric fusion protein platform, providing checkpoint blockade and TNF superfamily costimulation in a single molecule, which is unique with this molecule compared to antibody approaches, since all TIM3 ligands can be targeted simultaneously.