Chimeric antigen receptor (CAR) T-cell therapy that targets CD19 has shown efficacy in patients with relapsed and refractory large B-cell lymphoma (LBCL). Lymphodepleting chemotherapy (LDC) prior to cell infusion is critical to the adoptive cell therapy process, however there is relatively little prospective data dedicated to determining the optimal regimen (1). LDC can impact both short and long-term CAR T-cell efficacy by creating a favorable immune environment for in vivo product expansion and by promoting the formation of memory T-cells (2, 3). A more favorable immune environment is created through several mechanisms including the activation and recruitment of innate immune cells, increased availability of the cytokine pool, and reduction in the number of immunoregulatory T-cells (1). Despite the importance of these factors, there is significant heterogeneity among the LDC regimens reported in the literature. A recent review of 1271 publications on CAR T-cell therapy for LBCL found that fludarabine and cyclophosphamide were the most common drugs used, however, the cumulative dose varied from a total dose of 75–120 mg/m² for fludarabine and 750–1500 mg/m² for cyclophosphamide (1). The pivotal ZUMA-1 trial that led to the FDA approval of axicabtagene ciloleucel (axi-cel, Kite Pharma, Inc. Santa Monica, CA) in LBCL used fludarabine 30 mg/m² and cyclophosphamide 500 mg/m² for 3 days (4). The TRANSCEND and TRANSFORM registration studies that led to the FDA approval of lisocabtagene (liso-cel, Bristol-Myers Squibb, Bothell, WA) used fludarabine 30 mg/m² and cyclophosphamide 300 mg/m² for 3 days (5, 6). The ELIANA and JULIET registry studies led to the FDA approval of tisagenlecleucel (tisa-cel, Novartis Pharmaceuticals Corporation, East Hanover, NJ) and used fludarabine 25 mg/m² and cyclophosphamide 250 mg/m² for 3 days (7, 8). It is unclear how much the differences in outcomes between these products, if any, can be attributed to the differences in LDC dosing.

Variations in LDC exposure may influence CAR T-cell expansion and toxicity (9, 10). Higher cumulative doses of LDC have been shown to improve the depth of lymphodepletion as well as disease response after CD19 CAR T-cell therapy (10, 11). Using a fludarabine population pharmacokinetics model, it was reported that patients with low fludarabine exposure had a higher risk of relapse (9). Conversely, relatively high fludarabine exposure increased the risk of developing immune effector cell-associated neurotoxicity syndrome (ICANS). Fludarabine also has the potential to affect the hematopoietic stem cell (HSC) compartment which could play an initial role in long term myelosuppression (11). However, it is not clear if the fludarabine dose contributes to the risk for long-term cytopenias that are often seen in this patient population.

As the indications for CAR T-cell therapy have expanded rapidly, the logistical burden to providing care has also increased at a rapid pace. Due to resource constraints, many treating institutions are offering CAR T-cells as a primarily outpatient therapy to minimize hospital bed utilization. To better meet these evolving needs and to decrease the burden on patients, our center transitioned from a 3-day LDC regimen of fludarabine 30 mg/m² and cyclophosphamide 500mg/m² on days -4, -3, and -2 to a 2-day LDC regimen of fludarabine 40mg/m² and cyclophosphamide 500mg/m² on days -3 and -2 prior to CAR T-cell infusion. Here we report the outcomes of patients with non-Hodgkin lymphoma (NHL) who received this abbreviated LDC regimen prior to infusion of commercial axi-cel or tisa-cel.

A total of 92 patients received LDC prior to infusion of commercial axi-cel or tisa-cel between June 2018 and August 2023. Sixty-four patients (69.6%) received the 2-day LDC regimen and 28 (30.4%) received the 3-day regimen. Patient and disease characteristics are summarized in Table 1 . There were more lines of prior therapy in the 3-day group (median: 4 vs 3, p=0.010) and more frequent use of bridging therapies (72% vs 39%, p=0.0048) in the 2-day group ( Table 2 ). There was a numerically greater use of axi-cel (81% vs 61%) in the 2-day group that did not reach statistical significance. The most frequent indication for therapy in both cohorts was diffuse large B-cell lymphoma, not otherwise specified (DLBCL NOS) followed by transformed follicular lymphoma in the 3-day group and high-grade B-cell lymphoma in the 2-day group. The cell-of-origin differed between groups (p=0.041). The 3-day cohort included more germinal center (GC) (55% vs 75%) and the 2-day included more non-GC (33% vs 14%) B-cell phenotypes. The median follow-up time from infusion to death or last visit was 12.8 months (0.6 - 48.5) for patients receiving the 2-day regimen and 19.1 months (1.4–67.5) for the 3-day regimen.

While LDC is critical for effective CAR T-cell therapy, prospective studies to maximize both efficacy and efficiency of this regimen are limited. For patients receiving commercial therapy with axi-cel or tisa-cel for non-Hodgkin’s lymphoma, a common LDC regimen is a 3-day regimen of fludarabine and cyclophosphamide per the licensing trials. As the use of CAR T-cell therapy has expanded rapidly, treatment centers must maximize resource utilization. Many institutions are meeting the rising demand for CAR T-cell infusion by moving parts of the treatment into the outpatient setting. It has been shown that outpatient administration of CAR T-cell therapy increases quality of life and has similar safety and efficacy to inpatient administration (16–18). Outpatient CAR T-cell therapy can also improve cost savings by 2-to-4-fold (19). One area which can be maximized further is the potential consolidation of the LDC regimen. Our center utilized a similar 3-day fludarabine and cyclophosphamide regimen. To maximize efficiency and facilitate moving therapy to the outpatient setting, a shift was made to a 2-day regimen that would maintain fludarabine AUC. Here we demonstrate that there were no significant differences in outcomes or safety between the regimens.

The primary limitation of this study is the inability to control for numerous competing factors which may impact CAR T-cell associated outcomes aside from lymphodepleting chemotherapy. There were notable differences in characteristics between the cohorts. The 2-day cohort included more patients with a non-GCB histology, which likely influenced the more frequent use of bridging therapy. There were also differences in bridging therapies between the groups which reflects an evolution in practice patterns and available therapy options given the limited use of polatuzumab in the earlier, 3-day LDC group. The reported effects of bridging therapy in the available literature have been mixed and may be confounded by high-risk patients preferentially receiving bridging therapy (20, 21). Despite having more aggressive histology and more bridging therapy in the 2-day cohort there were no differences in overall response, complete response, survival rates, or toxicities between these regimens. The 2-day cohort also had fewer median prior lines of therapy (3 vs 4) and a greater proportion of patients receiving axi-cel, reflecting the interval approval of axi-cel as second-line therapy. Although they have not been prospectively compared, differences in response rates and toxicity have been reported between the products. The small patient subgroups limit extensive comparison, however, there were no appreciable differences in efficacy or toxicity outcomes between the 2-day and 3-day LDC regimens for either product. This also suggests that further studies evaluating tisa-cel and axi-cel with this regimen may be beneficial.

Since fludarabine exposure has been related to response, we evaluated this using the same published fludarabine population pharmacokinetics model and did not observe a difference in the optimal median predicted fludarabine AUC in the 2-day cohort compared to the 3-day cohort (9, 13). While the median fludarabine AUC was in the optimal reported range for both groups, there were only 25% of patients that fell into this range. The percentage above or below this range was also similar between the cohorts. Ideally, this suggests that future regimens would be designed to prospectively adjust fludarabine doses per patient to potentially achieve better outcomes. The response rates and safety evaluations are similar to those reported in pivotal trials as well as in real world reports for axi-cel and tisa-cel.

This study highlights the need for dedicated prospective studies to determine an optimal regimen and minimize patient exposure to unnecessary chemotherapy. Experience has also been reported for bendamustine as an alternative fludarabine-sparing regimen that may incur less toxicities (22, 23). Within the limitations of a single center retrospective study, it appears that fludarabine 40mg/m² and cyclophosphamide 500 mg/m² on days -3 and -2 prior to CAR T-cell infusion is safe and efficacious while conserving resources.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving humans were approved by Institutional Review Boards of the University of Michigan Medical Campus. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required from the participants or the participants’ legal guardians/next of kin in accordance with the national legislation and institutional requirements.

DF: Conceptualization, Data curation, Formal analysis, Writing – original draft, Writing – review & editing. MaG: Conceptualization, Formal analysis, Investigation, Methodology, Project administration, Writing – original draft, Writing – review & editing. SK: Data curation, Formal analysis, Investigation, Writing – review & editing. DM: Data curation, Formal analysis, Investigation, Writing – review & editing. GS: Data curation, Formal analysis, Investigation, Writing – review & editing. TF: Data curation, Formal analysis, Investigation, Writing – review & editing. AH: Data curation, Formal analysis, Investigation, Writing – original draft. HM: Data curation, Formal analysis, Investigation, Writing – original draft. AO: Data curation, Formal analysis, Investigation, Writing – original draft. HD’A: Data curation, Formal analysis, Investigation, Writing – original draft. SA: Writing – review & editing. SC: Writing – review & editing. JMac: Writing – review & editing. AP: Writing – review & editing. MR: Writing – review & editing. MT: Writing – review & editing. JMag: Writing – review & editing. MoG: Supervision, Writing – original draft, Writing – review & editing.