Background: Autologous chimeric antigen receptor (CAR) T-cell therapy is standard of care for patients with relapsed or refractory Large B cell Lymphoma (R/R LBCL). Each patient's CAR T product is unique, and the patient-specific determinants of CAR T-cell quality are poorly understood. CD39 is an ectonucleosidase that participates in the conversion of ATP and ADP and is upregulated in cancer. On T-cells, CD39 is expressed on CD4+ Tregs and on exhausted CD8+ T-cells that are prone to apoptosis.
Methods: This is a retrospective single-center cohort study (N=72) of patients who received CD19 CAR T-cell therapy for LBCL (axi-cel n=52; tisa-cel n=20). Leukapheresis material was obtained from n=51 patients and flow cytometry was performed to phenotype CD4+ and CD8+ stem-central memory, memory, and effector T-cell subsets (using CCR7 and CD45RO), and for immune checkpoint expression (PD-1, LAG3, TIGIT, and CD39). 10x multiome (ATAC and RNA) single cell sequencing (scRNA-seq) was performed on n=8 leukapheresis products for deeper characterization. Serum metabolomics was performed on paired samples from n=21 patients. 10X Genomics Chromium scRNAseq was also performed on n=57 CAR T-cell infusion products, including n=36 with paired leukapheresis material previously phenotyped by flow cytometry. In vitro CAR T-cells were manufactured from starting patient T-cells and analyzed for CAR T-cell phenotype and function.
Results: In the leukapheresis material of patients that did not achieve long-term remission after CAR T-cell therapy, and in those with high tumor burden, we found higher numbers of CD4+CD39+ and CD8+CD39+ T-cells ( Figs. 1 and 2). 10X multiome sequencing confirmed that CD39+ T-cells exhibit characteristics of exhaustion. Recent platinum-based chemotherapy did not affect CD39+ T-cell levels. In n=21 patients with paired serum analyzed by metabolomics, high CD8+CD39+ T-cells associated with reduced metabolites in the inosine-hypoxanthine pathway, which are downstream of the ectonucleotidase function of CD39 (inosine; P=0.006; hypoxanthine P=0.07). Increased numbers of CD39+ T-cells in the starting leukapheresis material translated into manufactured CAR T-cells having less favorable product characteristics, including higher CD39 expression and fewer memory cells in both paired patient samples and in experiments of in vitro CAR T-cell manufacturing. ScRNA-seq of the infused CAR T-cell product revealed differential gene signatures, both globally and in association with response, between axi-cel and tisa-cel CAR T-cell products, highlighting the profound product-specific differences. Nonetheless, both products were adversely affected by high CD39+ T-cells in the leukapheresis material used for manufacture.
Conclusions: CD39+ T-cells, found in patients with high lymphoma tumor burden and an unfavourable immunometabolic environment, associate with poor CAR T-cell quality and adverse patient outcomes in R/R LBCL. Strategies are needed to improve patient T-cell quality prior to leukapheresis and/or improve CAR T-cell manufacturing from patients with high levels of exhausted CD39+ T-cells.
Figure 1: Progression-free survival (PFS) stratified by leukapheresis CD8+CD39+ T-cells in the leukapheresis product. Blue - below cohort median percentage of CD8+T-cells that are CD39+ (low); Red - above median percentage of CD8+T-cells that are CD39+ (high). P-value by Log-Rank test. Overall survival (not shown) P-value 0.01.
Figure 2: Proportion of CD8+CD39+ T-cells found in patients with low or high baseline metabolic tumor volume (MTV) based on a previously established cutoff (Dean et al. Blood Adv. 2020). MTV was measured on the pre-CAR T-cell PET/CT. P-value by T-test.
MDJ, XY, and RMA are co-first authors with equal contribution.
Disclosures
Jain:Myeloid Therapeutics: Consultancy, Honoraria; Kite/Gilead: Consultancy, Honoraria, Research Funding; Incyte: Research Funding; Loxo@Lilly: Research Funding. Abraham Miranda:Moffitt Cancer Center: Current Employment. Liu:BioLineRx: Membership on an entity's Board of Directors or advisory committees. Shah:Incyte, Jazz Pharmaceuticals, Kite/Gilead, SERVIER: Research Funding; Pharmacyclics/Janssen, Spectrum/Acrotech, BeiGene, Gilead Sciences: Honoraria; Moffitt Cancer Center: Current Employment; DSMC, Pepromene Bio: Membership on an entity's Board of Directors or advisory committees; Celgene, Novartis, Pfizer, Janssen, Seattle Genetics, AstraZeneca, Stemline Therapeutics, Kite/Gilead: Other: Travel, Accommodations, Expenses; Takeda, AstraZeneca, Adaptive Biotechnologies, BMS/Celgene, Novartis, Pfizer, Amgen, Precision Biosciences, Kite/Gilead, Jazz Pharmaceuticals, Century Therapeutics, Deciphera, Autolus Therapeutics, Lilly, Pepromene: Consultancy. Chavez:Karyopharm: Membership on an entity's Board of Directors or advisory committees; Genmab: Honoraria; Epizyme: Speakers Bureau; Cellectar: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Beigene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astra Zeneca: Research Funding; ADC Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive: Research Funding; Kite/Gilead: Membership on an entity's Board of Directors or advisory committees; Lilly: Honoraria; Merck: Research Funding; Morphosys: Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees. Saini:Panbela Theraputics: Research Funding; GSK: Research Funding. Hanash:Abbott: Honoraria; Dynex: Research Funding; Cosmos Wisdom: Research Funding; BMS: Honoraria. Davila:Syncopation Life Sciences: Consultancy; Synthekine: Consultancy; Legend Biotech: Consultancy; Precision Biosciences: Other: Ownership interest (stock, stock options in a publicly owned company); Kite Pharma Inc.: Other: Teaching and Speaking; Caribou Biosciences: Consultancy; Capstan: Other: Advisor or review panel participant; CRISPR (CRSP): Patents & Royalties: Intellectual property rights (Royalties or patent sales); Bellicum Pharmaceuticals, Inc.: Other: Advisor or review panel participant; Ownership interest (stock, stock options in a publicly owned company); Atara Biotherapeutics: Consultancy; Adaptive Biotechnologies: Other: Ownership interest (stock, stock options in a publicly owned company); Adicet: Consultancy. Locke:Aptitude Health: Other: Travel Support; GammaDelta Therapeutics: Consultancy; EcoR1: Consultancy; Cellular Medicine Group: Consultancy; Daiichi Sankyo: Consultancy; ASH: Other: Travel Support; Society for Immunotherapy of Cancer: Other; National Cancer Institute: Other; Caribou: Consultancy; Imedex: Other; Leukemia and Lymphoma Society: Other; Clinical Care Options Oncology: Other; BioPharma Communications CARE Education: Other: Institutional; Emerging Therapy Solutions: Consultancy, Other; Calibr: Consultancy; Pfizer: Membership on an entity's Board of Directors or advisory committees; Legend Biotech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sana: Consultancy, Membership on an entity's Board of Directors or advisory committees; CERo Therapeutics: Other: (Institutional); Cowen: Consultancy; Umoja: Consultancy, Membership on an entity's Board of Directors or advisory committees; Wugen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Iovance: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Institutional; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Allogene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Institutional ; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Institutional , Research Funding; Kite, a Gilead Company: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Institutional , Research Funding; Bristol Myers Squibb/ Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Institutional , Research Funding; Gerson Lehrman Group (GLG): Consultancy; A2 Biotherapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel support.
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