Successful treatment of metastatic melanoma by adoptive transfer of blood-derived polyclonal tumor-specific CD4+ and CD8+ T cells in combination with low-dose interferon-alpha

Abstract

A phase I/II study was conducted to test the feasibility and safety of the adoptive transfer of tumor-reactive T cells and daily injections of interferon-alpha (IFNα) in metastatic melanoma patients with progressive disease. Autologous melanoma cell lines were established to generate tumor-specific T cells by autologous mixed lymphocyte tumor cell cultures using peripheral blood lymphocytes. Ten patients were treated with on average 259 (range 38-474) million T cells per infusion to a maximum of six infusions, and clinical response was evaluated according to the response evaluation criteria in solid tumors (RECIST). Five patients showed clinical benefit from this treatment, including one complete regression, one partial response, and three patients with stable disease. No treatment-related serious adverse events were observed, except for the appearance of necrotic-like fingertips in one patient. An IFNα-related transient leucopenia was detected in 6 patients, including all responders. One responding patient displayed vitiligo. The infused T-cell batches consisted of tumor-reactive polyclonal CD8+ and/or CD4+ T cells. Clinical reactivity correlated with the functional properties of the infused tumor-specific T cells, including their in vitro expansion rate and the secretion of mainly Th1 cytokines as opposed to Th2 cytokines. Our study shows that relatively low doses of T cells and low-dose IFNα can lead to successful treatment of metastatic melanoma and reveals a number of parameters potentially associated with this success.

Fig. 1

Clinical response. a ACT-induced regression of metastatic lesions before (upper panels) and after (lower panels) T-cell infusions. CT scans showing complete regression of two lung lesions obtained 6 month after T-cell infusion in the CR patient BO are depicted in the left two panels. Regression of a subcutaneous lesion obtained 3 months after start of the infusions in patient EN is depicted in the right panel. b Kaplan–Meier plot comparing the overall survival of the responder and non-responder patients. c IFNα treatment induced a mild leucopenia. From day 7 before infusion of the first T-cell dose, patients were treated with 3.10e6 U IFNα s.c daily for a total of 12 weeks. The absolute leukocyte count (x10e9/L) measured 1 week after the start of IFNα injections (i.e., day 0) is depicted. The values measured before treatment are shown as a black arrowhead. Absolute leukocyte counts of the responder patients are depicted as filled bars and those of the non-responders as open bars. The best overall clinical response of the individual patients is given in brackets behind the patient ID. Normal leukocyte counts range between 4 and 12 as is indicated by the light gray area. d The percentage reduction in peripheral neutrophil counts obtained after 7 days of IFNα treatment (i.e., day 0) of the non-responder (open circles) and responder (closed circles) patients correlates with their overall survival

Fig. 2

Proliferation of T cells during MLTC. T cells were produced by MLTC using autologous tumor cells as stimulator cells at day 0 and every week thereafter. The proliferation was assessed weekly after harvesting and counting of viable cell numbers using trypan blue. The proliferation rate is expressed as fold expansion of cells compared to cell number at day 0. The median and 25th percentile deviation of the weekly proliferation rate of responding (filled circle, n = 5) and non-responding (open circle, n = 5) patients are depicted

Fig. 3

Activation of infused T cells. T-cell batches were stimulated with autologous tumor cells and stained for phenotypic and activation markers as described in the Methods section. The percentage CD4+ and CD8+ T cells are given in the squares on the left of each plot. The CD154+CD4+ and CD137+CD8+ T cells (gated on CD3+ cells) are plotted, and percentages of tumor-specific activated cells (i.e., at least three times the medium control, see Fig. 1 of the on-line available supplementary materials) are given in the plots. The best overall clinical response of the individual patients is given in brackets behind the patient ID

Fig. 4

Reactivity of infused T cells. The specific reactivity of the T-cell batches was analyzed by cytotoxicity assay. Effector T cells were added in increasing ratios to radioactively ⁵¹Cr-labeled target cells (E/T) and incubated for 4 h at 37°C. Specific lysis of target cells was calculated after measuring the ⁵¹Cr release as described [22]. The percentage-specific lysis of autologous melanoma cells (black bars) and corresponding PHA blasts or EBV-LCL control cells (white bars) at different E/T ratios is depicted. The patient ID of the different T-cell batches is given in the upper right corner of the corresponding graphs. Results for the responder and non-responder patients are shown in the left and right panels, respectively

Fig. 5

Th1 and Th2 cytokine profile of T cells used for infusion. T cells were stimulated with autologous tumor cells overnight, and the supernatant was analyzed for the production of Th1 (IFNγ, IL-2 and TNFα) and Th2 (IL-10, 4 and 5) cytokines. The cytokine production after subtraction of background production after culture in medium alone is depicted for responder (black bars) and non-responder (white bars) patients, respectively. The best overall clinical response of the individual patients is given in brackets behind the patient ID. ND is not detectable