Introduction: CAR T cell therapy has reshaped treatment strategies for relapsed and refractory large B cell lymphoma (rrLBCL) ; however, further breakthroughs are needed to improve long-term survival. To address the resistant mechanisms of CAR T cell therapy, we conducted scRNAseq using intratumoral T cells collected from rrLBCL patients with post-CAR T relapse and found that CAR T cells acquired a dysfunctional state in the microenvironment at relapse. In vitro serial killing assay identified reduced interleukin-2 (IL2) signaling as an acquired dysfunctional state, and in vivo PDX disseminated model supported that engineered IL2 targeting IL2Rßγ enhanced tumor cell control. Acquired dysfunction in CD8+ CAR T cells at post-CAR T progression: scRNAseq was conducted using intratumoral T cells collected from rrLBCL patients who relapsed after CAR T cell therapy (n = 7) and from patients who never received CAR T cells (n = 5). CAR-positive cells were significantly enriched in dysfunctional CD8 clusters (CD8Dys) and proliferating effector CD8 clusters (CD8Pr-Eff). TCR clonotype from T cell-rich tumors revealed that CD8Dys and CD8Pr-Eff shared major TCR clones in CAR T cells, and trajectory analysis showed the path through CD8Pr-Eff to CD8Dys, suggesting that CAR T cell clones underwent clonal expansion and progressively acquired dysfunction in the tumor microenvironment. IL2 reduction as a potential mechanism of resistance: CAR T cells generated from 9 healthy donors (HD) and 10 patient donors (PD) were cocultured with EGFP-expressing GCB LBCL cell lines at 1: 1 E: T ratio, and tumor cells were replenished every 2 days at the same input as on day 0. Monitoring EGFP intensity using Incucyte showed that PD CAR T cells exhibited less killing relative to HD CAR T cells after the 5th round of stimulation. Then, scRNAseq was conducted using day 0 HD and PD products and day 8 co-cultured T cells, and CytoSig analysis revealed significantly reduced IL2 expression in day 8 PD. IL2Rßγ agonist rescued CAR T cell dysfunction in vitro and in vivo: In the serial killing assay, supplementation with IL2Rßγ agonist pegenzileukin enhanced CAR T cell cytotoxicity even after the 5th round of stimulation in both HD and PD groups. For in vivo evaluation, a luciferase-expressing PDX cell line (PDX-CL) generated from CAR T refractory model was injected into NSG mice, and mice were treated with HD CAR T +/- pegenzileukin after tumor engraftment. All PDX-CL-bearing mice treated with CAR T cells achieved remission by bioluminescence imaging; however, mice treated without pegenzileukin relapsed early, whereas those treated with the combination showed sustained remission. T cell level in peripheral blood 10 and 19 days after CAR T injection was higher in the combination group, suggesting pegenzileukin enhanced CAR T cell expansion and persistence. Conclusion: Acquired CAR T cell dysfunction in the tumor microenvironments contributes to CAR T cell refractoriness. Pegenzileukin prevents CAR T cell dysfunction and improves tumor control in vitro and in vivo. Citation Format: Kotaro Arita, Enyu Dai, Xubin Li, Irtiza N. Shiekh, Patrick K. Reville, Jared Henderson, Ashley Wilson, Christan Dillard, Kevin Bowman, Haopeng Yang, Fuliang Chu, R Andrew. Harkins, Nathan Pate, Katie Malley, Dinesh Bangari, Estela Rojas, Jason R. Westin, Sairah Ahmed, Sattva S. Neelapu, Francisco Vega, Rui Wang, Xiangming Li, Donald Shaffer, Linghua Wang, Michael R. Green. Non-alpha IL2R agonist improves CAR T cell therapy by preventing CAR T dysfunction in relapsed/refractory large B cell lymphoma abstract. In: Proceedings of the Fifth AACR International Meeting on Advances in Malignant Lymphoma: From Discovery to Clinical Impact; 2026 Jun 24-27; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2026;7 (3Suppl): Abstract nr A002.
Arita et al. (Wed,) studied this question.