Authors
Rebecca Jones, Valentina Griggio, Alfonso Piciocchi, Giorgia Mancin, Valeria Sargentini, Francesca Perutelli, Maria Chiara Montalbano, Giacomo Ortone, Riccardo Moia, Alessandro Bosi, Lydia Scarfò, Paolo Ghia, Francesco Passamonti, Gianluca Gaidano, Robin Foà, Benedetto Bruno, Candida Vitale, Marta Coscia.
Background
The therapeutic effectiveness of CAR-T cells in chronic lymphocytic leukemia (CLL) remains suboptimal, primarily due to dysfunctions in patient-derived T cells. The immunological factors affecting the functionality of CAR-T cells generated from CLL patients, as well as the impact of targeted agents used to treat CLL on T-cell fitness, has yet to be fully elucidated.
Aims
This project aims at assessing in CLL patients the impact of treatment with targeted agents (i.e. ibrutinib or venetoclax) on T cells, and on the manufacturing efficiency, the phenotype and the functional properties of the CAR-T cells generated.
Methods. Analyses were performed on 15 CLL patients enrolled in the GIMEMA CLL2020 trial (NCT04640909). Samples were collected before the start of treatment and after 6 and 12 months of therapy with ibrutinib (n=7) or venetoclax (n=8). The T-cell phenotype was evaluated by flow cytometry. Anti-CD19-4-1BB CAR-T cells were produced by lentiviral transduction, and tested for generation efficiency, immunophenotype and in vitro anti-tumor activity.
Results
CAR-T cells were successfully generated from peripheral blood mononuclear cells (PBMC) in 13 out of 15 patients (87%) at pre-treatment and at the 6-month timepoint, and in all 11 patients (100%) with available 12-month samples. Transduction efficiency remained consistent across the analyzed timepoints. Specifically, in the venetoclax-treated cohort, the mean CAR expression on T cells was 47% at pre-treatment, 41% at 6 months and 57% at 12 months, whereas in the ibrutinib-treated cohort, the corresponding values were 47%, 42% and 64%, respectively. Moreover, no significant impact of therapy was noted on the cell yield at the end of the manufacturing process in both cohorts.
Immunophenotypic analysis revealed a preferential enrichment of CD4⁺ T lymphocytes in the final CAR-T cell product compared to the starting T-cell source – a trend observed consistently across all timepoints and treatment groups. Interestingly, in the cohort of patients treated with venetoclax, CAR-T cells generated after 12 months of treatment demonstrated a more favorable distribution of the differentiation subsets – with a predominance of less mature subpopulations in both the CD4+ and CD8+ compartments – compared to the pre-treatment timepoint.
We also evaluated the expression of inhibitory immune checkpoint molecules on T cells before and after CAR-T cell manufacturing. After 6 months of treatment with targeted agents – both ibrutinib and venetoclax – we observed a significant decrease in the percentage of TIM-3⁺ CD8⁺ T cells in the pre-manufacturing T cells. Despite this positive immunomodulatory effect, TIM-3 expression was again upregulated during the generation process, across both CD4⁺ and CD8⁺ CAR-T cells and irrespective of the treatment regimen received. During treatment with targeted agents, we also observed a decreasing trend in PD-1 expression across CD4+ and CD8+ T-cell populations, which was detectable – though not significantly – in both the initial T-cell source and in the final CAR-T cell products. Notably, CAR-T cells produced after 12 months of treatment with venetoclax showed a meaningfully reduced expression of intracellular CTLA-4 compared to pre-treatment levels.
In terms of antitumor functionality, the generated CAR-T cells were able to proliferate and effectively induce target cell lysis when co-cultured with a CD19⁺ tumor target (i.e. MEC1 cell line). The magnitude of this response was dependent on the effector-to-target ratio. Despite the more favorable profile in terms of exhaustion markers expression observed after 6 and even more after 12 months of treatment with targeted agents – especially venetoclax – no significant differences were observed in the killing or proliferative ability of CAR-T cells compared to pre-treatment levels.
Conclusions
CAR-T cells can be effectively generated from CLL patients but display immunological features of dysfunction. From patients receiving 12-month venetoclax therapy we could generate CAR-T cells with a more favorable differentiation profile and partial restoration of exhaustion marker expression. Enrollment has now been completed, and assessments from an additional cohort of 8 patients, covering all timepoints, are currently underway. Conclusive results, including an evaluation of functional changes observed in CAR-T cells generated at different stages of targeted treatment, will be available at the time of data presentation.
Keywords : CAR-T cells, targeted therapies, immunomodulation
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