Simultaneous Quantification of Multiple Immune Checkpoint Interactions in Melanoma.

Background/Objectives: Immune checkpoint inhibitors (ICIs) have transformed the therapeutic landscape of advanced malignancies. However, only a subset of patients respond to treatment. Recent efforts have focused on the identification of novel biomarkers that capture the dynamic and functional state of the tumour immune microenvironment, yet their clinical translation has remained challenging. Methods: To address these limitations, we applied amplified FRET-FLIM (QF-Pro®) technology to spatially resolve and quantitatively assess functional immune checkpoint interactions directly in cells and FFPE tissue and tumour samples. Results: Using this approach, we demonstrated that PD-1/PD-L1, CTLA-4/CD80, TIGIT/CD155, and LAG-3/MHC-II interactions can be robustly quantified in routine FFPE patient samples from multiple tumour types. Furthermore, in a proof-of-concept study in a melanoma cohort treated with immune checkpoint inhibitors (ICIs) targeting PD-1 or CTLA-4, co-analysis of all four immune checkpoints suggested patterns of concomitant engagement. Notably, patients with high PD-1/PD-L1 interaction levels tended to also exhibit elevated CTLA-4/CD80 interactions. Survival analysis further showed that high LAG-3/MHC-II interaction status was associated with a trend toward improved overall survival even when corrected for tumour stage, irrespective of the specific ICI regimen administered. Conclusions: Although prospective validation in larger independent cohorts would be critical to establish clinical relevance, these findings support the exploration of LAG-3 engagement as a potential biomarker in melanoma immunotherapy, and more broadly highlight immune checkpoint interaction profiling as a promising avenue to examine in patient stratification.