Structure-guided discovery of small-molecule ICOS-ICOSL antagonists that remodel intratumoral immunity and enhance anti-PD‑1 therapy in osteosarcoma.
The inducible T‑cell co‑stimulator (ICOS)-ICOS ligand (ICOSL) axis shapes effector versus regulatory T‑cell programs in tumors, yet small‑molecule disruption of this protein-protein interaction remains underexplored. We used the ICOS-ICOSL complex to define an interfacial hotspot and applied DrugCLIP to screen approximately 5 × 108 compounds, followed by diversity clustering, developability triage, and AutoDock Vina docking. Five candidates were prioritized for experimental validation. In a TR‑FRET competition assay measuring ICOS-ICOSL complex formation, STK910592, STL167676, and OSSK‑026837 inhibited the interaction with IC50 values of 10.67, 12.59, and 14.37 μM, respectively. STK910592 and STL167676 showed favorable cellular tolerability and elicited functional immune modulation in parallel human and murine tumor-APC-T cell tri‑culture systems, increasing IL‑2, IFN‑γ, and TNF‑α secretion. In an immunocompetent orthotopic LM8 osteosarcoma model, both compounds produced dose‑dependent antitumor activity and were associated with immune remodeling, including increased CD8 infiltration, reduced FOXP3/ICOS immunoreactivity, and induction of innate‑priming and effector cytokine programs in tumor tissues. Notably, combining ICOS targeting with anti-PD-1 therapy yielded schedule‑dependent enhancement of efficacy and reduced pulmonary metastatic burden, with sequential dosing outperforming concomitant administration. These findings provide a preclinical rationale for developing ICOS-ICOSL small‑molecule antagonists and highlight temporal sequencing as an actionable parameter to optimize PD‑1-based combination immunotherapy in osteosarcoma.