A phase I clinical study of vaccination of melanoma patients with dendritic cells loaded with allogeneic apoptotic/necrotic melanoma cells. Analysis of toxicity and immune response to the vaccine and of IL-10 -1082 promoter genotype as predictor of disease progression

Abstract

Background: Sixteen melanoma patients (1 stage IIC, 8 stage III, and 7 stage IV) were treated in a Phase I study with a vaccine (DC/Apo-Nec) composed of autologous dendritic cells (DCs) loaded with a mixture of apoptotic/necrotic allogeneic melanoma cell lines (Apo-Nec), to evaluate toxicity and immune responses. Also, IL-10 1082 genotype was analyzed in an effort to predict disease progression.

Methods: PBMC were obtained after leukapheresis and DCs were generated from monocytes cultured in the presence of GM-CSF and IL-4 in serum-free medium. Immature DCs were loaded with gamma-irradiated Apo-Nec cells and injected id without adjuvant. Cohorts of four patients were given four vaccines each with 5, 10, 15, or 20 x 106 DC/Apo-Nec cell per vaccine, two weeks apart. Immune responses were measured by ELISpot and tetramer analysis. Il-10 genotype was measured by PCR and corroborated by IL-10 production by stimulated PBMC.

Results: Immature DCs efficiently phagocytosed melanoma Apo-Nec cells and matured after phagocytosis as evidenced by increased expression of CD83, CD80, CD86, HLA class I and II, and 75.2 +/- 16% reduction in Dextran-FITC endocytosis. CCR7 was also up-regulated upon Apo-Nec uptake in DCs from all patients, and accordingly DC/Apo-Nec cells were able to migrate in vitro toward MIP-3 beta. The vaccine was well tolerated in all patients. The DTH score increased significantly in all patients after the first vaccination (Mann-Whitney Test, p < 0.05). The presence of CD8+T lymphocytes specific to gp100 and Melan A/MART-1 Ags was determined by ELISpot and tetramer analysis in five HLA-A*0201 patients before and after vaccination; one patient had stable elevated levels before and after vaccination; two increased their CD8 + levels, one had stable moderate and one had negligible levels. The analysis of IL-10 promoter -1082 polymorphism in the sixteen patients showed a positive correlation between AA genotype, accompanied by lower in vitro IL-10 production by stimulated PBMC, and faster melanoma progression after lymph nodes surgery (p = 0.04). With a mean follow-up of 49.5 months post-surgery, one stage IIC patient and 7/8 stage III patients remain NED but 7/7 stage IV patients have progressed.

Conclusion: We conclude that DC/Apo-Nec vaccine is safe, well tolerated and it may induce specific immunity against melanoma Ags. Patients with a low-producing IL-10 polymorphism appear to have a worst prognosis.

Trial registration: Clinicaltrials.gov (NHI) NCT00515983.

Figures

Figure 1

Characteristics of DC/Apo-Nec vaccine. A-DCs phagocytosis. Patient #1 results are shown. Apo-Nec cells (green labeled) phagocytosis by DCs (red-labeled, left panel) was evaluated as described under Methods. DCs and Apo-Nec cells were co-cultured for 48 hs at 37°C (middle panel) or at 4°C to inhibit phagocytosis (right panel). Total DCs (red labeled population) was gated and the percentage of double positive cells (DC/Apo-Nec) was calculated. B-DC Maturation. Markers expression of iDCs, DC/Apo Nec cells and DCs + LPS from all vaccinated patients (n = 15) was evaluated by FACS using monoclonal antibodies as described under methods. Results are mean ± SD percentage of positive cells, MFI: mean fluorescence intensities. C- DC endocytosis. FITC-Dx uptake of iDCs DC/Apo-Nec cells and DC + LPS of all vaccinated patients (n = 15). Results are indicated as mean ± SD percentage of FITC-Dx positive cells after 90 min incubation and washing. *: diferences with iDCs were statistically significant (Student's t test). D-DC migration. In vitro migration towards MIP-1α and MIP-3β was measured as described under methods. iDCs, DC/Apo-Nec and DCs + LPS from patient # 9 are shown (mean ± SD, assayed in triplicate).

Figure 2

Patient#1's lymphocytes in vitro proliferation in response to autologous tumor Ags presented by DCs. Pre and post vaccination Patient#1 PBMC were incubated either alone, with Apo-Nec#1 cells prepared from patient#1 tumor cells or with DC/Apo-Nec#1 cells; (H3) dThd incorporation was measured as described under Methods. Results represent mean ± SD cpm (counts per minute) of triplicates. Positive controls incubated with PHA incorporated more than 7 × 104 cpm (not shown).

Figure 3

Detection of antigen-specific IFN-γ secreting CD8+T cells by ELISpot analysis. ELISpot assay was performed as described under Methods. Number of individual gp100 and Melan A/MART-1 specific spots/105 CD8+T cells from pre and post DC/Apo-Nec vaccination samples are shown for 5/7 HLA-A*0201 patients. Data represents mean values of triplicates. Background numbers of spots were set with non – pulsed DCs and subtracted from the number of spots.

Figure 4

Influence of Interleukin-10 -1082 promoter genotype in disease progression. A- Disease progression was calculated in months from the last surgery, reported as of December 2007. Differences between curves were significant (P = 0.04, Chi Square test). B- In vitro IL-10 secretion by patients' PBMC in response to LPS or PHA. Control PBMC (not stimulated) did not secrete IL-10. Culture supernatants were tested in triplicate by ELISA as described under methods. Lines represent median value in each case. Mann Whitney's test showed that differences between AA and AG/GG genotypes were not statistically significant. AA (PHA) vs AG/GG (PHA) P value: 0.6; AA (LPS) vs AG/GG (LPS) P value: 0.25.