Background Despite a high response rate in chimeric antigen receptor (CAR) T therapy for acute lymphocytic leukemia (ALL), ~50% of patients relapse within the first year,1 representing an urgent question to address in the next stage of cellular immunotherapy. The pioneering clinical trials conducted at Upenn/CHOP provide a unique opportunity to examine the molecular determinants of of ultra-long CAR T persistence.
Methods We performed single-cell multi-omics profiling of ~700k pre-infusion CD19-targeted 4-BB CAR T cells from 82 pediatric ALL patients and 6 healthy donors. Patient demographics were collected between Sep-2012 to July-2022. To uncover the hallmarks of CAR T longevity, we correlated the single-cell atlas with the duration of B-cell aplasia (BCA), a widely used pharmacodynamic measurement of CAR T persistence,2 and classified all the patients into 5 groups based on their BCA duration (table 1). To remove potential confounding variables between trial design, 42 patients from NCT01626495 were analyzed as the Discovery Cohort, while the other 40 patients from NCT02906371 were used as Validation Cohort.
Results Our analysis of CD19-specific stimulated CAR T cells from the Discovery Cohort revealed a prominent role of type-1 function, which was highly represented but had no discernible correlation with long-term persistence. Unexpectedly, we identified that elevated type-2 functionality was significantly associated with patients maintaining a median BCA duration of 8.4 years (BCA-L group) (figure 1A,B), and this signature was robustly presented in our Validation Cohort (figure 1C,D). Higher type-2 cytokine level of long-term persistent CAR T cells was independently validated using both flow cytometry and multiplexed secretomic assay (figure 1E,F). Through ligand-receptor interaction analysis, type-2 cytokines were found to regulate a 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 (figure 2A-G). To examine the role of type-2 function in vivo, we performed serial proteomic profiling of post-infusion sera from patients, confirming a higher level of circulating type-2 cytokines in 5-year or 8-year relapse-free responders (figure 3A-C). In a leukemic mouse model, type-2 high CAR T cell products demonstrated superior expansion and antitumor activity particularly upon leukemia rechallenge, which could be associated with their decreased exhaustion and increased memory phenotype. (figure 4A-G).
Conclusions Our findings provide key insights into the mediators of CAR T longevity and suggest a potential therapeutic strategy to maintain long-term remission by enhancing type-2 functionality in CAR T cells.
Acknowledgements We are grateful to all the participants in this study. We acknowledge the contributions of the following research cores at the University of Pennsylvania and Children’s Hospital of Philadelphia: the Translational and Correlative Studies Laboratory for providing standardized flow cytometric, quantitative PCR and cytokine multiplexing analyses, and biobanking of patient specimens on CAR T cell trials; the Clinical Cell and Vaccine Production Facility for cell processing and biobanking; the Human Immunology Core for providing healthy donor cells and analytical support; the Flow Cytometry Core for flow cytometry equipment maintenance and access to electronic sorters. We appreciate all the effort from the research and the medical teams contributed to the collection of patient samples, CAR T manufacturing, clinical administration and monitoring, and patient consent and recruitment, and the participation of the patients and families in this research. Computational data analysis was conducted with the Yale High Performance Computing clusters (HPC). The research was supported by Stand-Up-to-Cancer (SU2C) Convergence 2.0 Grant (to R.F.), 1U01CA232361 (to S.A.G), and the Packard Fellowship for Science and Engineering (to R.F.; Grant number 2012–38215). This material is based upon work supported under a collaboration by Stand Up To Cancer, a program of the Entertainment Industry Foundation and the Society for Immunotherapy of Cancer.
Trial Registration The current study is a secondary investigation using patient samples collected from existing clinical trials (ClinicalTrials.gov number, NCT01626495 and NCT02906371) for which the University of Pennsylvania Institutional Board provided insight.
Young RM, Engel NW, Uslu U, Wellhausen N, June CH. Next-Generation CAR T-cell Therapies. Cancer Discovery. 2022;12:1625–1633.
Greenbaum U, Mahadeo KM, Kebriaei P, Shpall EJ, Saini NY. Chimeric Antigen Receptor T-Cells in B-Acute Lymphoblastic Leukemia: State of the Art and Future Directions. Front Oncol. 2020;10:1594.
Ethics Approval The study obtained ethics approval from the University of Pennsylvania Institutional Board. Written informed consent for participation was obtained from patients or their guardians according to the Declaration of Helsinki. All laboratory operations were under principles of International Conference on Harmonization Guidelines for Good Clinical Practice with established Standard Operating Procedures and protocols for sample receipt, processing, freezing, and analysis. All ethical regulations were strictly followed. Healthy donor primary T lymphocytes were provided by the University of Pennsylvania Human Immunology Core. Samples are de-identified for compliance with HIPAA rules.
Experimental procedures in mouse studies were approved by the Swiss authorities (Canton of Vaud, animal protocol ID 3533) and performed in accordance with the guidelines from the CPG of EPFL.
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