bioRxiv2026
Clinical studies of chimeric antigen receptor (CAR)-T therapy generate diverse molecular and clinical evidence that remains fragmented across publications, public repositories and patient-derived datasets, limiting systematic therapeutic discovery. Here we present BioPathfinder, an evidence-guided multi-agent workflow for closed-loop biomedical discovery. BioPathfinder constructs a provenance-aware knowledge resource linking publications with patient single-cell RNA sequencing (scRNA-seq) datasets and clinical metadata, and uses role-specialized large language model agents to generate, review and prioritize diverse, falsifiable and dataset-aware mechanistic hypotheses for computational and experimental validation. Applied to a curated corpus of CAR-T-treated patient studies and matched scRNA-seq datasets, BioPathfinder identified candidate mechanisms underlying CAR-T persistence, dysfunction and therapeutic resistance. The workflow prioritized the hypothesis that genes associated with an NK-like transition programme could be targeted to reduce CAR-T exhaustion and improve persistence. Analysis of patient scRNA-seq datasets showed enrichment of this programme in exhausted post-infusion CAR-T cells. Virtual perturbation prioritized transition-associated receptor genes, including KLRC1, KLRD1 and KLRG1, and expert review selected KLRC1, encoding NKG2A, for experimental validation. In vitro and in vivo chronic-stimulation models showed that NKG2A marked activated, exhaustion-associated CD8 CAR-T cells, whereas NKG2A blockade enhanced antitumour activity and persistence-associated functional readouts in vivo. BioPathfinder establishes a generalizable framework that transforms fragmented clinical single-cell evidence into experimentally validated therapeutic hypotheses, providing a scalable strategy for AI-guided biomedical discovery. HIGHLIGHTSO_LIBioPathfinder integrates fragmented CAR-T clinical studies, patient scRNA-seq datasets and metadata into a provenance-aware evidence resource for AI-guided hypothesis discovery. C_LIO_LIA multi-agent workflow generates, critiques and prioritizes diverse, falsifiable, dataset-aware mechanisms underlying CAR-T persistence, dysfunction and therapeutic resistance. C_LIO_LIBioPathfinder prioritizes KLRC1/NKG2A as a therapeutic target, and experimental validation demonstrates that NKG2A blockade enhances CAR-T persistence and antitumour function. C_LI