A phase I study of nonmyeloablative chemotherapy and adoptive transfer of autologous tumor antigen-specific T lymphocytes in patients with metastatic melanoma

Abstract

This report describes a phase I clinical trial using nonmyeloablative, lympho-depleting chemotherapy in combination with adoptive immunotherapy in patients with metastatic melanoma. The chemotherapy-conditioning schedule that induced transient lymphopenia consisted of cyclophosphamide (30 or 60 mg/kg per day for 2 days) followed by fludarabine (25 mg/m(2) per day for 5 days). Immunotherapy for all patients consisted of in vitro expanded, tumor-reactive, autologous T-cell clones selected for high avidity recognition of melanoma antigens. Cohorts of three to six patients each received either no interleukin (IL)-2, low-dose IL-2 (72,000 IU/kg intravenously three times a day to a maximum of 15 doses), or high-dose IL-2 (720,000 IU/kg intravenously three times a day for a maximum of 12 doses). The toxicities associated with this treatment were transient and included neutropenia and thrombocytopenia that resolved in all patients. High dose intravenous IL-2 was better tolerated by patients after chemotherapy than during previous immunotherapy cycles without chemotherapy. No patient exhibited an objective clinical response to treatment, although five patients demonstrated mixed responses or transient shrinkage of metastatic deposits. This study established a nonmyeloablative-conditioning regimen that could be safely administered in conjunction with adoptive T-cell transfer and IL-2 in patients with metastatic melanoma.

FIG. 1

Patients who underwent treatment including nonmyelodepleting chemotherapy exhibited profound but transient lymphopenia, neutropenia, and thrombocytopenia. Average absolute lymphocyte counts (ALC), absolute neutrophil counts (ANC), and platelet counts from all patients in each cohort are plotted as a function of time. All patients received chemotherapy consisting of cyclophosphamide once per day on days –7 and –6 (Cohort 1, 30 mg/Kg; Cohorts 2, 3, and 4, 60 mg/Kg) and fludarabine once per day on days –5 through –1 (25 mg/m²). T-cell clones and peptide vaccinations were administered on day 0. Patients in Cohorts 3 and 4 received IL-2 starting on day 1. A dashed line indicates the lower limit of normal values. Immunosuppression was evident for about 5 days in all cohorts, and then counts recovered to safe levels within a few days. After day 7, values were not obtained for each patient everyday, and “average” counts may not include patients who were discharged as their counts normalized.

FIG. 2

Recovery of lymphocyte subsets after treatment (absolute cell counts per mm³). Flow cytometric analysis of lymphocyte subsets was performed on peripheral blood samples within the indicated time ranges. All data from patients who received 60 mg/Kg cyclophosphamide (Cohorts 2, 3, and 4) was included. No patient is represented more than once in a single time range. Pre: values assayed before the start of chemotherapy. A dashed line represents the lower limit of normal values. B cells (CD20+), and NK cells (CD16+ or CD56+ and CD3−) and CD8+ T cells recovered to pre-ablation levels within a few months. CD4+ cell counts remained low for the duration of the study, resulting in low values for total T cells (CD3+), and a depressed CD4+/CD8+ cell ratio.