Abstract
In an extended 5-year follow-up of a single-arm, multicenter trial (NCT04833504) evaluating IL-7/CCL19-armored CD19 CAR-T (7×19 CAR-T) cells in 39 adults with relapsed/refractory large B-cell lymphoma (r/r LBCL)—including 41% with compromised performance status (ECOG PS 2-3)—we report sustained efficacy and safety. At a median follow-up of 63 months (range: 2-68 months), 28.2% of patients maintained ongoing responses, with 5-year overall survival (OS) and disease-specific survival rates of 43.6% (95% CI: 27.9-58.3%) and 48.7% (95% CI: 32.4-63.2%), respectively. The median OS was 31 months (95% CI: 0-78.4 months), and no late-onset severe toxicities emerged.
To investigate the impact of 7×19 CAR T-cell phenotype on long-term efficacy, we conducted single cell RNA-seq and T cell receptor (TCR)-seq analyses on thawed cryopreserved CAR T-cell products from 10 patients (4 patients with persistent complete remission (CR), 4 patients relapsed after initial response (RL) and 2 non-responders (NR)), then the cells were classified into 24 distinct clusters. Strikingly, CD8+FOXO1+ memory T (Tm) cells showing specific expression of Forkhead Box O1 (FOXO1) emerged as the most enriched population in CR patients. Quantification demonstrated that FOXO1+ Tm cells constituted 5.1% of total CAR-T cells in CR patients, representing 3.6-fold and 5.7-fold increases compared to RL and NR groups, respectively. Furthermore, our analysis revealed that CD8+FOXO1+ cells exhibited enhanced memory-associated gene signature (TCF7, CD44, and TSC1), compared to other CAR T-cell subsets. Also, this subset exhibited reduced cytotoxicity and intermediate exhaustion. In accordance, FOXO1+ Tm cells demonstrated undetectable TCR clonotypes in 90.2% of cases and reduced TRAC gene transcript levels compared to other subsets.
In order to further delineate the functional hierarchy of FOXO1+ Tm cells, we performed integrated transcriptional profiling and cellular state mapping. Unsupervised clustering revealed three phenotypically distinct subclusters with mutually exclusive expression of THEMIS, LAG3, and granzyme A (GZMA). In comparison with the other two subsets, FOXO1+THEMIS+ cells were enriched for memory/resting markers (IL7R, CD38, ITGA1) and attenuated exhaustion markers. We further reported FOXO1+THEMIS+ cells were exclusively confined to the memory branch by developmental trajectory analysis. Most importantly, FOXO1+THEMIS+ Tm cells accounted for a higher proportion in the infusion products of CR patients compared to NR patients (3.7–7.7-fold higher).
Collectively, our study provides the longest follow-up data for armored CD19 CAR-T therapies, establishes FOXO1+THEMIS+ memory T cells as biomarkers of durable remission, and validates cytokine-armored CAR-T engineering as a strategy to overcome functional attrition in aggressive B-cell lymphomas.
