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

Patients with histologically proven cutaneous melanoma with verified progressive metastatic disease stage IV or unresectable stage III refractory to treatment before the start of the study and with at least one measurable target lesion were eligible for this phase I/II study. The protocol was approved by the Medical Ethics Committee of the Leiden University Medical Center and conducted in accordance with the Declaration of Helsinki. All patients gave written informed consent. Patients with brain metastases who are neurologically unstable and/or require use of dexamethasone and patients who are on continued chemo- or radiotherapy until 4 weeks before the start of IFNα injections were excluded from the study. The primary objective of this study was to investigate the feasibility and safety of adoptive T-cell transfer in combination with IFNα. Secondary endpoints include the best overall response defined as disease control (CR, PR, and SD) and evaluation of immunological parameters as predictors for response.

Patients received T cells as three consecutive infusions of on average 259 (range 38–474) million cells with a 3-week interval in combination with low-dose IFNα (3 million IU Roferon, Roche, Woerden, The Netherlands, subcutaneously daily) starting at day 7 before the first T-cell infusion and for a total of 12 weeks. Cryopreserved T cells were thawed and diluted to a total volume of 300 ml in PBS/2% human serum albumin (Albuman, Sanquin, Amsterdam, the Netherlands) and a final concentration of less than 1% DMSO. Then, the cells were administered intravenously over a time period of 30–60 min. At various time points before, during and after treatment, the tumor response was assessed by physical examination and tumor imaging (CT and MRI) and evaluated according to the Response Evaluation Criteria in Solid Tumors 1.0 (RECIST) [11] and according to the immune-related response criteria (irRC) [12]. Toxicity was scored using the CTC scale version 3.0.

Autologous melanoma cell lines were initiated and cultured using authorized Standard Operating Procedures (SOPs). Tumor tissue obtained by surgery was directly processed by mechanical dissociation and cultured in tumor medium consisting of Dulbecco’s MEM (DMEM, Lonza, Veriers, Belgium) supplemented with 8% irradiated, heat-inactivated GMP-grade pre-tested FCS, penicillin (50 U/ml), streptomycin (50 μg/ml), and l-glutamine (4 mM) (all from Lonza). A stable cell line could be established (usually within 4–12 weeks) for approximately 50% of the patients that was subsequently used as a stimulator line for the generation of tumor-reactive T-cell cultures. Before the initiation of T-cell cultures, the melanoma origin of the used autologous tumor cells was confirmed by RT–PCR [13‐20] and they were shown to express at least three of the eleven melanoma-associated antigens studied (data not shown).

Furthermore, genotyping of HLA alleles was performed in all melanoma cell lines and corresponding PBMC to confirm their origin and revealed in addition that none of the used melanoma cell lines showed complete or partial genetic loss of HLA alleles. In addition, the surface expression of HLA class I and class II molecules was assessed by flow cytometry using Alexa-648-conjugated W6/32 HLA class I A/B/C (Serotec, Dusseldorf, Germany) and FITC-conjugated Bu26 HLA-DR/DP/DQ-specific antibodies (Bio-connect, Huissen, the Netherlands). The samples were measured on a FACSCalibur and analyzed using CellQuest Software and showed that HLA class I surface expression was not downregulated on the cell lines used for the induction of T cells and that all IFNγ-stimulated melanoma cells, except Mel AB and Mel CT, displayed variable surface expression of HLA class II (data not shown).

Tumor-reactive T cells were cultured using a mixed lymphocyte tumor cell culture (MLTC) as described [21]. Briefly, PBMC were isolated from heparinized venous blood and cryopreserved until use. Approximately 50 million PBMC were thawed, washed, counted and resuspended (1–2 × 10e⁶ cells per ml) in T-cell medium; Iscoves MDM (Lonza) supplemented with 8% heat-inactivated pooled human serum (Sanquin Bloodbank, Dordrecht, The Netherlands), penicillin (50 U/ml), streptomycin (50 μg/ml), and l-glutamine (4 mM) (all GMP-grade from Lonza) and plated 1 ml per well in a 24-well culture plate. Next, autologous tumor cells were lethally irradiated (100 Gy) and resuspended (1 × 10e⁵ cells per ml) in T-cell medium. One milliliter of this tumor cell suspension was added per well containing PBMC. Human recombinant IL-4 (5 ng/ml, Cellgenix, Sieversen, Germany and Gentaur, Brussels Belgium) was added at day 0. Starting from day 2, low-dose (150 IU/ml) IL-2 (Aldesleukin, Novartis, Arnhem, The Netherlands) was added. Culture medium containing IL-2 was refreshed every 2–3 days. The T cells were cultured for a total of 4 weeks and stimulated weekly with irradiated tumor cells as described above. After 4 weeks of culture, the T-cell batches were evaluated for sterility, phenotype, and tumor reactivity before they were released for infusion. Phenotypic analysis was performed using a FACSCalibur and CellQuest software (BD Pharmingen, San Jose, CA, USA). T cells were released when they consist of more than 80% of T cells (CD3+CD4+ and CD3+CD8+) or NK cells (CD3-CD56+) (All conjugated-mAb were from BD Pharmingen except CD8-APC from DAKO, Heverlee, Belgium). In addition, the frequency of Treg CD4+CD25+FoxP3+ T cells was evaluated in the T-cell batches using CD4-APC and CD25-FITC mAb (both from BD Pharmingen) in combination with the human Treg/FoxP3-PE staining kit (eBiosciences, Vienna, Austria). Tumor reactivity was assessed by a ⁵¹Cr-cytotoxicity assay [22], and in order to be released for infusion, T cells were required to specifically lyse autologous tumor cells but not autologous EBV-LCL or PHA blasts (difference >10% at an E/T ratio of 30). Alternatively, tumor reactivity was assessed by IFNγ ELISA (Sanquin, Amsterdam, The Netherlands) where T-cell batches were required to secrete >200 pg/ml IFNγ in response to autologous tumor cells and at least twice the amount induced by the negative controls, i.e., autologous EBV-LCL or PHA blasts [23].

To examine T-cell reactivity, 0.5–1 × 10e⁶ T cells were seeded in a 24-well plate in T-cell medium and stimulated with 0.5–1 × 10e⁶ autologous melanoma cells. After 1 h, Brefeldin-A (10 μg/ml) was added and stimulation was continued overnight. The next day T cells were harvested, and part of the cells were stained with a mix of antibodies to CD154-PECy5, CD137-APC, CD4-PECy7, CD8-APCCy7, IFNγ-FITC, and IL-2-PE (all from BD Pharmingen) and CD3-Pacific Blue (DAKO) see [24]. Non-stimulated T cells or T cells stimulated with PHA (5 μg/ml) served as controls. Responses were considered positive when the percentage of tumor-stimulated CD154+ and/or CD137+ T cells was at least three times the medium control (see Fig 1. of the on-line available supplementary materials).

Polyclonality of the T-cell batches was evaluated by the anti-IFNγ-FITC and IL-2-PE Ab by either one of the 8 different mixes of FITC- and/or PE-conjugated anti-TCRVβ mAb (BETAmark, Beckman Coulter) as previously described [25]. The different T-cell clones were operationally defined as the percentage of activated tumor-specific CD4+ or CD8+ T cells expressing the same TCRVβ-chain, within the population of tumor-specifically activated CD154+ and/or CD137+ T cells, respectively. All samples were measured on a calibrated LSRII and analyzed using DIVA software (BD). A TCRVβ was considered dominant (>10%), subdominant (3–10%), or minor (<3%) based on the percentage of tumor-specific cells using the same TCRVβ [25].

In parallel, T cells were stimulated with autologous melanoma cell lines in the absence of Brefeldin-A, and supernatant was harvested after 24 h in order to analyze the cytokine secretion using the human Th1/Th2 cytometric bead array (BD Pharmingen). T cells stimulated with PHA (5 μg/ml), medium alone, and autologous EBV-LCL or PHA blasts were included as controls. Antigen-specific cytokine production was defined by a cytokine concentration above the cut-off value (IFNγ 50 pg/ml; other cytokines 10 pg/ml) and >2 × the concentration of the medium control [26].

The overall survival was analyzed using Kaplan–Meier and SPSS version 2.0. The Cox regression could not performed due to the low patient number, and therefore no confidence interval and hazard ratio could be provided. Post hoc statistical analyses were performed to determine potential correlates with clinical responsiveness using the non-parametric Mann–Whitney Test or Wilcoxon Signed Ranks Test. All p values are two-sided and are considered significant when they are less than 0.05.