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

Area of Science:

• Immunology
• Protein Engineering
• Cancer Immunotherapy

Background:

• Advancing cancer immunotherapy necessitates synthetic immunomodulators with precise targeting and tunable activity.
• The Inducible T-cell Co-Stimulator (ICOS) receptor enhances T-cell function but faces challenges in biologic development due to limited affinity and format-dependent activity.
• Optimizing the ICOS ligand (ICOS-L) is crucial for developing high-affinity, modular components for precision immune modulation.

Purpose of the Study:

• To engineer a high-affinity ICOS ligand variant for enhanced T-cell co-stimulation.
• To investigate the functional synergy of the engineered ICOS-L with anti-PD-1 therapy.
• To explore modular fusion formats for optimizing biologic design.

Main Methods:

• Yeast surface display-based directed evolution to identify ICOS-L variants.
• Protein engineering and structural modeling to analyze mutations (Q51P, N57H) and their impact on binding.
• Functional assays measuring T-cell proliferation and cytokine secretion (IFN-γ).
• Genetic fusion of the ICOS-L variant to pembrolizumab (anti-PD-1) in various formats.

Main Results:

• An ICOS-L variant (Y8) with two mutations (Q51P, N57H) demonstrated a ~100-fold increase in binding affinity to human ICOS.
• Structural analysis revealed mutations enhance rigidity and form a salt bridge, reconfiguring the binding interface.
• Y8 induced potent T-cell proliferation and IFN-γ secretion.
• Fusion of Y8 to pembrolizumab enhanced T-cell activation and tumor cell lysis, showing synergy between PD-1 blockade and ICOS agonism.
• Light-chain conjugation (pembrolizumab-L-Y8) showed superior functional output.

Conclusions:

• Y8 is a high-affinity ICOS-L variant with robust co-stimulatory function.
• Y8 can potentiate anti-PD-1 immunotherapy through modular fusion, enhancing efficacy in cancer treatment.
• This engineering framework enables next-generation immunomodulatory biologics to overcome resistance and improve cancer immunotherapy outcomes.