NeoTCRseek: an integrated platform for high-sensitivity identification of neoantigen-specific TCR clonotypes to track clinical T-cell dynamics.

Introduction: Identifying neoantigen-specific T-cell receptor (neoTCR) clonotypes is crucial for tracking clinical T-cell dynamics in personalized cancer immunotherapy. Despite advances in experimental and computational approaches for identifying antigen-specific TCR (asTCR) clonotypes, the identification of neoTCR clonotypes remains challenging due to their low frequencies and the limited sensitivity of current methods. Methods: We introduce NeoTCRseek, an integrated platform that combines extended T-cell culture, deep TCR sequencing, and advanced TCR-clustering tools to enhance neoTCR clonotype identification. NeoTCRseek was developed using a model cytomegalovirus (CMV) antigen and subsequently validated under two neoantigen setups: a single neoantigen and a neoantigen pool representing a multi-antigen context. For each antigen setup, we used three cell sorting-based methods to detect enriched clonotypes and built a validation dataset for asTCR clonotype characterization and NeoTCRseek performance evaluation. Results: NeoTCR clonotypes exhibited a significantly higher proportion of low-frequency clonotypes (0.01%-0.1%) than CMV-specific ones (70.3% vs. 33.3%). Nonetheless, NeoTCRseek achieved a detection limit of 0.01% and high accuracy in both single- and multi-antigen contexts by integrating expanded clonotype detection with co-clustering-based TCR prediction. Compared with the benchmark, NeoTCRseek improved the mean F1 score across the two neoantigen setups from 0.21 to 0.41. Conclusion: NeoTCRseek achieves high analytical sensitivity and accuracy in neoTCR clonotype identification and supports multi-antigen analysis, providing an integrated platform for neoTCR clonotype characterization and for tracking clinical T-cell dynamics in personalized cancer immunotherapy.