Lymphodepletion (LD) is a critical component of chimeric antigen receptor T-cell (CAR T-cell) therapy, maximizing the engraftment, efficacy and long-term survival of infused cells [1]. Although these drugs provided a therapeutical breakthrough, notably in patients with non-Hodgkin lymphoma (NHL), less than half of them achieve a durable response and the prognosis of post CAR T-cell relapse is very poor [2, 3]. Mechanisms contributing to CAR T-cells treatment failure include disease-related characteristics (total metabolic tumor volume [TMTV], lactate dehydrogenase [LDH] and C-reactive protein levels, performance status and targeted antigen loss), as well as CAR T-cell-related factors (poor T-cell fitness, T-cell exhaustion, lack of persistence and type of costimulatory molecules in the CAR construct) [1].

LD is administered to create a favorable microenvironment for CAR T-cell proliferation by eliminating immunosuppressive cells, particularly regulatory T-cells and myeloid-derived suppressor cells [4, 5] and improving the availability of such homeostatic cytokines as IL-7 and IL-15, crucial for CAR T-cell expansion and survival, associated with treatment efficacy [1, 6].

LD regimens use the combination of fludarabine (Flu) and cyclophosphamide (Cy) at varying doses, depending on the type of approved drug, respectively Flu 25 mg/m² and Cy 250 mg/m² (Flu25/Cy250) for tisagenlecleucel (tisa-cel), Flu30/Cy300 for lisocabtagene maraleucel (liso-cel) and Flu30/Cy500 for axicabtagene ciloleucel (axi-cel) and brexucabtagene autoleucel (brexu-cel). Axi-cel has provided better disease control compared to tisa-cel in cohorts of patients with relapsed and/or refractory (R/R) large B-cell lymphoma (LBCL) [7, 8], possibly owing to the different LD regimen, raising the question of whether a more intensive LD before tisa-cel infusion could lead to greater benefit.

This issue was addressed here, where the outcome of four patients treated with tisa-cel after a modified LD of Flu30/Cy500 for three days (mLD) is reported and compared to that of tisa-cel recipients who received standard Flu25/Cy250 LD (sLD) in 4 centers.

The study and all methods were performed under institutional review board-approved protocols and complied with Foundation for the Accreditation of Cellular Therapy (FACT)-JACIE criteria under the responsibility of Saint Louis Hospital in accordance with the Declaration of Helsinki. All patients signed informed consent with approval of ethic committee (CPP SUD-EST I; number 2019-77) and agreed for data re-use (RNIPH - MR004). They were in third line or more and had been selected based on severe NHL criteria of bulky disease and/or extranodal sites including central nervous system (CNS) involvement [9]. Clinical data were collected via institutional electronic medical records, retrospectively from 101 patients with R/R LBCL treated in third line or more with tisa-cel after sLD between January 2020 and December 2023. They segregated into two cohorts respectively treated i) in the same center as the patients who received mLD (Paris St-Louis Hospital; control cohort, n = 60), and ii)in 3 other French centers (Lyon, Rennes, Toulouse; validation cohort, n = 41).

Peripheral blood CAR T-cell kinetics was assessed in flow cytometry [10] in flow cytometry, on days 4, 7, 10, 14, 21 and 28 (±2) post-infusion.

Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) were managed according to the American Society for Transplantation and Cellular Therapy recommendations [11].

Baseline disease burden was evaluated based on TMTV. Response was assessed and graded according to 2014 Lugano criteria [12].

The primary endpoint was to assess the impact of mLD on tisa-cel clinical efficacy. Overall response rate (ORR) was defined as the percentage of patients who achieved a partial (PR) or complete response (CR). Progression-free survival (PFS) was defined as the time from CAR T-cell infusion until relapse, progression or death from any cause. Overall survival (OS) was defined as the time from CAR T-cell infusion until death of any cause. Duration of response (DOR) was defined as the time from CR or PR to relapse, progression or death from any cause. The secondary endpoint was to assess the effect of mLD versus sLD on CAR T-cell expansion and persistence.

Descriptive statistics included median and interquartile range (IQR) for continuous variables, number and percentages for categorical variables. Survivals were compared using log-rank test. Statistical analyses used R software version 4.2.2.

The median age of the 4 patients was 70 years and 3 were male. At the time of treatment decision, one presented bulky disease (TMTV 4500 mL), and two had stage IV disease with CNS involvement. All received bridging therapy consisting of conventional immunochemotherapy (rituximab, ifosfamide and etoposide, n = 3; rituximab, methotrexate and aracytine, n = 1). At the time of tisa-cel infusion, 2 patients presented progressive disease as assessed by PET/CT scan.

Patient characteristics in the three cohorts are summarized in Supplementary Table S1 showing good balance between the three groups except for more CNS involvement in the four patients with mLD, and significantly higher IPI in the control compared to validation cohorts (Chi² p = 0.004).

The primary end-point of mLD better efficacy in the four patients compared to the control and validation cohort was fulfilled, as shown in Table 1. Indeed the four patients who received mLD all reached CR and none has relapsed although one died of secondary cancer. Results are poorer in the comparative cohorts with high level of relapse, more than 40% of the patients deceased at one year and median DOR of about 6 months.

Table 1 Impact of LD protocol.
Full size table

The secondary objective of a positive impact of the stronger mLD on CAR T-cell kinetics was also reached (Table 1). Indeed, mLD improved the speed of CAR T- cell expansion with a peripheral blood peak at 6.5 days (2 on day 6, 2 on day 7) compared to medians of 9 and 8 days in the control and validation cohorts. Similarly, peripheral CAR T-cells detected on peak day were higher in numbers per microliter and as proportion of CD3+ T-cells.

Toxicity related to mLD was evaluated in the four patients. CRS was observed in all of them, at a median time of 1 day, with a median duration of 4 days (3–7), of grade 3 in only one. ICANS occurred at a median time of 3 days and for a duration of 5-8 days in three patients (grade 3 n = 1) and resolved. Toxicity treatment included tocilizumab, anakinra, and siltuximab, each used in one patient, while all received dexamethasone. There was no grade 4 or 5 toxicity. Two patients required admission in intensive care unit respectively for 12 days (concomitant grade 3 CRS and grade 3 ICANS) and 2 days (grade 2 CRS). No tumor lysis syndrome was observed. One patient developed colitis due to Clostridium difficile. Grade 4 neutropenia occurred in all four patients and resolved by month 1 in 3 patients. All also had late cytopenias, anemia and thrombocytopenia, during the first year. Toxicities are detailed in Supplementary Table S2.

Although definitive conclusions cannot be drawn from such a small number of patients, observations reported here are likely attributable to mLD, since other parameters, such as LDH and TMTV values were within normal limits. Importantly, although the strong CAR T-cell expansion was associated with significant toxicity, all adverse events were manageable, and the survival rate of these high-risk patients is remarkable.

LD intensity therefore appears as a major modifiable factor to improve CAR T-cell expansion, thereby improving treatment outcomes. Pharmacokinetic-directed dosing based on weight, renal function, and drug monitoring could be helpful to enhance treatment efficacy while mitigating toxicity and should be prospectively evaluated.