CAR-T cell therapy targeting CD19 has transformed the treatment landscape for relapsed or refractory large B-cell lymphoma (R/R LBCL), demonstrating curative potential in select patients. Despite achieving high initial response rates, the durability of CAR-T therapy is often compromised by several factors. To overcome the limitations posed by the immunosuppressive tumor microenvironment. Our research team is dedicated to the development and clinical evaluation of 7×19 CAR-T cells targeting CD19, which represent a fourth-generation innovative technology integrating IL-7 and CCL19. This approach not only promotes deeper tumor infiltration by CAR-T cells but also enhances systemic anti-tumor immunity through local antigen presentation and immune cell cross-talk.

To reveal the dynamics of CAR-T cells after infusion, with a focus on the exhausted subsets, and to explore the complex interactions between CAR-T cells and other immune cell populations in patients, single-cell RNA and single-cell TCR sequencing were performed on eight patients. A total of 15 samples were sequenced, including seven samples from the CAR-T product (D00), six samples from peripheral blood mononuclear cells (PBMCs) at 7 days after infusion (D07), and two samples from either PBMCs or bone marrow at 30 days after infusion (D30). Qualified data from 141,025 single cells were obtained from all samples.

Single-cell transcriptomics and flow cytometry revealed a CAR-T trajectory from memory-like to exhausted effector phenotypes, characterized by upregulation of PDCD1, TIGIT, and TOX. In PD-L1-high (CPS≥25) TMEs, IL-7 and CCL19 engineering enhanced CAR-T cell persistence and tumor infiltration, accompanied by the upregulation of gene programs related to cytokine signaling, chemotaxis, and nucleotide metabolism, indicating improved adaptation of IL-7- and CCL19-armored CAR-T cells to suppressive TME conditions. However, expression of exhaustion markers remained elevated, and TCR clonality was restricted.

The dynamic changes of different CAR-T subpopulations were further analyzed by single-cell sequencing. The proportion of exhausted CD8 T cells increased dramatically at D07. The effector CD8 T cells also increased at D07 and D30, close to reaching significance. In addition, the expression of exhaustion-related genes, including PDCD1, CTLA4, LAG3, HAVCR2, and TIGIT, was significantly upregulated in exhausted CD8 T cells, suggesting that CAR-T is accompanied by a certain degree of functional failure. Cell interaction analysis further revealed that intermediate monocytes with high expression of CD274 (iMono-CD274) exhibited the strongest PD-L1/PD-1 interaction signal with exhausted CAR+ CD8 T cells, which was significantly different from that observed with exhausted CAR- CD8 T cells. This finding suggests that monocytes from the iMono-CD274 subpopulations in the TME can directly induce the activation of exhaustion signals by binding to PD1 on CAR-T cells through PD-L1.

This mechanism offers a novel approach to immune-blocking effects in the TME. In this phase I trial (NCT04381741), we integrated temporally guided PD-1 blockade (tislelizumab from day 30) with 7×19 CAR-T therapy in 20 patients with R/R LBCL. PD-1 blockade converted 8 of 12 partial responders (66.7%) to durable CR, elevating the cumulative CR rate to 70.0%. With a median follow-up of 21.8 months, the estimated 24-month progression-free survival (PFS) and overall survival (OS) rates were 60.0% and 71.1%, respectively. Notably, baseline PD-L1-high predicted a lower initial CR rate (10.0% vs 90.0%), but this improved to 50.0% after PD-1 blockade, achieving survival outcomes comparable to those of the PD-L1-low subgroup.

By combining PD-1 inhibitors with fourth-generation armored CAR-T cells, we not only overcame the functional impairment of traditional CAR-T therapy in a complex tumor microenvironment but also significantly delayed the exhaustion process. The introduction of IL-7 significantly enhanced the proliferation and survival of CAR-T cells, while CCL19 improved the immunosuppressive properties of TME by promoting the infiltration and migration of immune cells. In addition, PD-1 blockade further improved the efficacy of CAR-T cells by relieving the negative regulation of the PD-L1 signaling axis. In summary, the efficacy of this combined strategy in patients with R/R LBCL was significantly better than that of traditional CAR-T therapy, providing a new model for future multi-target immunotherapy.

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2025
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