Effect of KROS 101, a small molecule GITR ligand agonist, on T effector cells, T reg cells and intratumoral CD8 T cell cytotoxicity.

Abstract

2587 Background: A small molecule was identified that stabilizes the trimerization of the glucacorticoid-induced tumor necrosis factor receptor (GITR) ligand which then leads to the trimerization of GITR and magnified signaling of GITR. GITR signaling of T cells results in T effector cell expansion and T reg reduction. An antagonist to the GITR ligand was also indentified which stabilizes the GITR ligand dimer formation preventing trimerization. Methods: Binding of KROS 101 to the GITR ligand was assessed and the binding region of GITR ligand to KROS was determined with targeted deletion of GITR. T cell suppression studies were performed with T cell proliferation assays and T cell cytotoxicity assays with glioblastoma target cells using patient derived PBMCs. Double humanized GITR/GITRL mice bearing B16-F10-LUC2 tumors were treated with KROS 101 or controls. Tumor-infiltrating lymphocytes were analyzed by flow cytometry and tumors assessed. Results: KROS 101 agonist binds to GITRL with high affinity with a K D of 340nM by Surface Plasmon resonance. T cell suppression assay showed KROS 101 had peak proliferative induction of T effector cells at 25 uM concentration to 52% increase in proliferation, and peak proliferation induction of T effector cells with 1:1 ratio of T reg cells at 50uM concentration to 80% increase in proliferation of T effectors. KROS 101 treated T cell show enhanced effetor function and selectively target glioblastoma and cancer stem cells in vitro. KROS 101 enhances tumor immune infiltration by Increasing CD3+ T Cells, CD8+ T Cells, and M1 Macrophages While Reducing Tregs and Myeloid Cells in vivo. KROS 101 enhances cytotoxicity by increasing IFNγ and TNFα While Reducing TIGIT and TIM3 in CD4+ and CD8+ T Cells I n Vivo . Conclusions: KROS 101 is a GITR ligand agonist that increases T cell proliferation and increased cytotoxicity and reduces the T reg population more effectively than TRX 518 which is a therapeutic GITR antibody that was in clinical trial.