Directed evolution and modular integration of a high-affinity ICOS-L variant for potent T cell–mediated tumor elimination

BACKGROUND: Advancing cancer immunotherapy requires engineering synthetic immunomodulators that integrate precise receptor targeting, tunable activity, and compatibility with modular biologic formats. The Inducible T-cell Co-Stimulator (ICOS) is a clinically validated co-stimulatory receptor whose engagement enhances T-cell function. However, the development of ICOS-targeting biologics has been hindered by limited receptor affinity and format-dependent agonist activity. To address this, we applied a protein engineering framework to optimize the ICOS ligand (ICOS-L) as a high-affinity, modular component for precision immune modulation. RESULTS: Using yeast surface display-based directed evolution, we identified an ICOS-L variant (Y8) containing two synergistic mutations (Q51P and N57H) that improved human ICOS (hICOS) binding affinity by ~ 100-fold relative to wild-type. Structural modeli…