Abstract 909: Single-cell multi-omics reveals type-2 function and metabolic fitness in maintaining CAR T cell longevity associated with 8-year leukemia remission
Authors: Bai Zhiliang , Melenhorst J. Joseph , Grupp Stephan A. , Fan Rong 
Published: April 2023

Abstract
Despite high response rate in chimeric antigen receptor (CAR) T therapy for acute lymphocytic leukemia (ALL), approximately half of the patients relapse within the first year, representing an urgent question to address in the next stage of cellular immunotherapy. To interrogate the molecular determinants of ultra-long CAR T persistence, we sequenced one million pre-infusion CAR T cells from 82 pediatric ALL patients and 6 healthy donors and obtained high quality multi-omics data from ~700k single cells at the basal level or upon antigen specific stimulation. We identified that elevated type-2 functionality in both CD4+ and CD8+ subsets was associated with five patients maintaining a median B-cell aplasia duration of 8.4 years (BCA-L group), which was independently validated using both intracellular flow cytometry and multiplexed secretomic assay. Further network analysis revealed several pathways promoting the activation of the Th2 master regulator GATA3 in BCA-L cells. Through ligand-receptor interaction analysis, type-2 cytokines were found to regulate a specific cluster of Tim-3+ terminal effector cells showing overactivation of cytotoxicity, impaired immune function, elevated exhaustion signature and diminished proliferative potentiality, and CAR T cells from BCA-L patients showed reduced dysfunctional hallmarks. In addition, activated BCA-L cells maintained a significantly higher level of oxidative phosphorylation metabolism that is likely associated with their observed central memory phenotype, whereas CAR T cells from patients who developed primary resistance or relapse exhibited imbalanced functional and metabolic programs. Specifically, CAR T products from initial non-responders showed hyperactive glycolysis metabolism, and the CD19-negative relapsed patients (RL- group) had significantly attenuated gene module expression of a broad spectrum of metabolic categories, including major energy-producing metabolisms. By direct comparison with BCA-L cells, our analysis identified a strikingly impaired mTOR signaling in RL- CAR T cells, along with reduced proliferation activities and a systematic deficiency of pathways orchestrating immune response. Although the mechanism is still unclear, our hypothesis is that the impaired mTOR signaling could only endow blunted functional and metabolic response in RL- cells, thereby conferring more opportunities to leukemic cells to escape the attack. In summary, our large-scale single-cell multi-omics dataset provides valuable insights for dissecting mechanistic basis of CAR T cell longevity in mediating 8-year leukemia remission, highlighting several therapeutic strategies to edit specific CAR T functionality to ameliorate clinical outcome, particularly boosting the type-2 immunity to mitigate dysfunction thereby extending persistence.
Citation Format: Zhiliang Bai, J. Joseph Melenhorst, Stephan A. Grupp, Rong Fan. Single-cell multi-omics reveals type-2 function and metabolic fitness in maintaining CAR T cell longevity associated with 8-year leukemia remission [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 909.