A pretreatment T cell signalling score identifies clinical pembrolizumab response in non-small cell lung cancer patients

Abstract Background Immune checkpoint inhibitors (ICIs) targeting the programmed death (ligand)-1 (PD-1/PD-L1) axis, like pembrolizumab, have significantly improved survival in non-small cell lung cancer (NSCLC). However, less than 50% of patients respond. Identifying early-response biomarkers is crucial to personalize therapy, thereby preventing ineffective, expensive and potentially harmful treatment. Methods We applied a novel ex vivo immunopharmacological bioassay to assess pembrolizumab-dependent T cell signalling in baseline peripheral blood mononuclear cells (PBMCs) from 64 NSCLC patients. PBMCs were stimulated with anti-CD3/CD28 with or without pembrolizumab, and phosphorylation states of PD-1-dependent T cell receptor (TCR) signalling pathways were measured by spectral flow cytometry. A composite signalling score was calculated representing the net pembrolizumab-induced phosphorylation response and patients were classified as low, optimal and high modulation responders based on this signalling score. Associations with progression-free survival and overall survival (OS) were evaluated using univariate Cox regression. Results Patients with optimal baseline pembrolizumab-induced signalling scores exhibited significantly higher signalling score outcomes than those with low modulation ( p < 0.0001) and lower than patients with excessive modulation ( p < 0.01) and had significantly longer OS (HR = 2.83, p = 0.013; and HR = 12.05, p = 0.003, respectively). Notably, conventional pharmacodynamic parameters, including half-maximal effective concentration (EC 50 ) for PD-1 receptor occupancy and maximum IL-2 production (E max ), were not associated with clinical outcomes, underscoring the unique predictive value of the phosphorylation-based signalling score. In vivo , pembrolizumab-induced T cell activation changes and TCR signalling inhibition post-treatment correlated with shorter survival (HRs = 1.33-1.95), consistent with our ex vivo findings. Conclusions We demonstrate that a pretreatment signalling score derived from ex vivo pembrolizumab-modulated T cell phosphorylation identifies clinical response in NSCLC. This functional bioassay offers a novel approach to identify patients most likely to benefit from ICI therapy, potentially enabling personalised treatment decisions before therapy initiation. Graphical abstract text Our findings reveal that pretreatment, pembrolizumab-dependent modulation of T cell phosphorylation identifies clinical response in NSCLC. Furthermore, we introduce an overall signalling score, reflecting the net phosphorylation profile, which could serve as a potential predictive biomarker to distinguish responders from non-responders, thereby supporting biomarker-driven therapeutic strategies.