Comparison of non-myeloablative lymphodepleting preconditioning regimens in patients undergoing adoptive T cell therapy

Abstract

Background: Adoptive cell therapy with T cells genetically engineered to express a chimeric antigen receptor (CAR-T) or tumor-infiltrating T lymphocytes (TIL) demonstrates impressive clinical results in patients with cancer. Lymphodepleting preconditioning prior to cell infusion is an integral part of all adoptive T cell therapies. However, to date, there is no standardization and no data comparing different non-myeloablative (NMA) regimens.

Methods: In this study, we compared NMA therapies with different doses of cyclophosphamide or total body irradiation (TBI) in combination with fludarabine and evaluated bone marrow suppression and recovery, cytokine serum levels, clinical response and adverse events.

Results: We demonstrate that a cumulative dose of 120 mg/kg cyclophosphamide and 125 mg/m² fludarabine (120Cy/125Flu) and 60Cy/125Flu preconditioning were equally efficient in achieving deep lymphopenia and neutropenia in patients with metastatic melanoma, whereas absolute lymphocyte counts (ALCs) and absolute neutrophil counts were significantly higher following 200 cGyTBI/75Flu-induced NMA. Thrombocytopenia was most profound in 120Cy/125Flu patients. 30Cy/75Flu-induced preconditioning in patients with acute lymphoblastic leukemia resulted in a minor ALC decrease, had no impact on platelet counts and did not yield deep neutropenia. Following cell infusion, 120Cy/125Flu patients with objective tumor response had significantly higher ALC and significant lower inflammatory indexes, such as neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR). Receiver-operating characteristics curve analysis 7 days after cell infusion was performed to determine the cut-offs, which distinguish between responding and non-responding patients in the 120Cy/125Flu cohort. NLR≤1.79 and PLR≤32.7 were associated with clinical response and overall survival. Cytokine serum levels did not associate with clinical response in patients with TIL. Patients in the 120Cy/125Flu cohort developed significantly more acute NMA-related adverse events, including thrombocytopenia, febrile neutropenia and cardiotoxicity, and stayed significantly longer in hospital compared with the 60Cy/125Flu and TBI/75Flu cohorts.

Conclusions: Bone marrow depletion and recovery were equally affected by 120Cy/125Flu and 60Cy/125Flu preconditioning; however, toxicity and consequently duration of hospitalization were significantly lower in the 60Cy/125Flu cohort. Patients in the 30Cy/75Flu and TBI/75Flu groups rarely developed NMA-induced adverse events; however, both regimens were not efficient in achieving deep bone marrow suppression. Among the regimens, 60Cy/125Flu preconditioning seems to achieve maximum effect with minimum toxicity.

Trial registration: ClinicalTrials.gov NCT00287131 NCT03166397 NCT02772198.

Keywords: adoptive; chimeric antigen; clinical trials; immunotherapy; lymphocytes; phase II as topic; receptors; tumor-infiltrating.

Figure 1

Cell counts following non-myeloablative lymphodepletion. (A) Absolute neutrophil count (ANC), (B) absolute lymphocyte count (ALC) and (C) absolute platelet count (APC). (D–F) Comparison of ALC between (D) the 120Cy/175Flu and 60Cy/175Flu cohorts, (E) the 120Cy/175Flu and TBI/75Flu cohorts and (F) the 60Cy/125Flu and TBI/75Flu cohorts. Day 0, day of cell infusion. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. Cy, cyclophosphamide; Flu, fludarabine; TBI, total body irradiation.

Figure 2

Absolute cell counts and inflammatory ratios of responders and non-responders in the 120Cy/125Flu cohort. (A) Absolute neutrophil count (ANC), (B) absolute lymphocyte count (ALC) and (C) absolute platelet count (APC), (D) platelet-to-neutrophil ratio (PLR), (E) neutrophil-to-lymphocyte ratio (NLR) and (F) systemic immune-inflammatory index (SII) of objective responders (ORs) and non-responders (NRs). Data are shown as mean±SEM. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. Cy, cyclophosphamide (Cy); Flu, fludarabine.

Figure 3

Kaplan-Meier curves for assessment of median overall survival. Kaplan-Meier curves for the inflammatory indexes (A) NLR and (B) PLR according to cut-off values received by ROC curve analysis. Survival analysis was performed by the log-rank test. ROC, Receiver operating characteristics; NLR, neutrophil-to-lymphocyte ratio; PLR, platelet-to-lymphocyte ratio;.

Figure 4

Cytokine serum levels on the day of cell infusion. (A) IL-7, (B) IL-15 and (C) IL-21. (D–F) Comparison of cytokine serum levels between objective responders (ORs) and non-responders (NRs) in the 120Cy/125Flu cohort. Data are shown as mean±SEM analyzed using a non-parametric two-tailed Student’s t-test. *p<0.05 and **p<0.01. Cy, cyclophosphamide; Flu, fludarabine; IL, interleukin; TBI, total body irradiation.