Admasu, T.; Sadanala, K.; Kalyane, D.; Wang, H.; Yoon, S.; WooHyun, K.; Katiki, M.; Rudnick, J.; Ramachandran, M.; Yu, J.

Agonistic antibodies targeting the costimulatory receptor GITR have failed in clinical trials despite a strong preclinical rationale, revealing fundamental limitations of receptor-targeted approaches to TNF superfamily agonism. Here, we demonstrate that pharmacological stabilization of the trimeric structure of human GITRL represents a mechanistically distinct strategy to overcome these limitations. Small-molecule stabilization of GITRL preserves its membrane residency. It enables bidirectional signaling between antigen-presenting cells and T cells, a physiological context that receptor-targeted antibodies bypass through non-physiological clustering of GITR. In humanized GITR/GITRL double-knock-in mice bearing syngeneic tumors, GITRL stabilization expands cytotoxic CD8 T cells, selectively depletes intratumoral regulatory T cells, activates APCs, and restructures the immune spatial architecture. In patient-derived tumor explants, GITRL stabilization reactivates suppressed CD8 T cells in an APC-dependent manner. Our findings establish ligand stabilization as a mechanistically distinct therapeutic strategy and provide a framework for engaging TNF receptor superfamily costimulatory pathways by modulating their native ligands.