Vaccination of melanoma patients using dendritic cells loaded with an allogeneic tumor cell lysate

Abstract

The aim of the present phase I/II study was to evaluate the safety, immune responses and clinical activity of a vaccine based on autologous dendritic cells (DC) loaded with an allogeneic tumor cell lysate in advanced melanoma patients. DC derived from monocytes were generated in serum-free medium containing GM-CSF and IL-13 according to Good Manufacturing Practices. Fifteen patients with metastatic melanoma (stage III or IV) received four subcutaneous, intradermal, and intranodal vaccinations of both DC loaded with tumor cell lysate and DC loaded with hepatitis B surface protein (HBs) and/or tetanus toxoid (TT). No grade 3 or 4 adverse events related to the vaccination were observed. Enhanced immunity to the allogeneic tumor cell lysate and to TAA-derived peptides were documented, as well as immune responses to HBs/TT antigens. Four out of nine patients who received the full treatment survived for more than 20 months. Two patients showed signs of clinical response and received 3 additional doses of vaccine: one patient showed regression of in-transit metastases leading to complete remission. Eighteen months later, the patient was still free of disease. The second patient experienced stabilization of lung metastases for approximately 10 months. Overall, our results show that vaccination with DC loaded with an allogeneic melanoma cell lysate was feasible in large-scale and well-tolerated in this group of advanced melanoma patients. Immune responses to tumor-related antigens documented in some treated patients support further investigations to optimize the vaccine formulation.

Fig. 1

Expression of HLA-DR and CD80 by DC prepared from melanoma patients. Expression of HLA-DR and CD80 molecules in DC cell suspensions are shown for each patient preparation. The percentage of positive cells is given after gating on large (FSC/SSC) alive cells. Coefficient of variations between DC preparations were 20 and 18% for HLA-DR and CD80 molecules, respectively

Fig. 2

Clinical evolution of patient 02 with a complete response after treatment with lysate-loaded DC. a Pictures of patient 02 were taken before treatment (day 0) with lysate-loaded DC and at different time points posttreatment (months 7, 11, and 24). The patient received two cycles of vaccinations (days 0, 22, 57, 85, and months 8, 10, and 12). The site of eczema presented by this patient at the inclusion is indicated with an arrow. The correspondent site is indicated in the picture taken after vaccination (month 7), showing the modification of the skin reaction. Complete response was diagnosed at this point by biopsy and histopathological analysis. The picture taken at month 11 shows the exacerbation of the eczema reaction following the sixth immunization. The last picture was taken 12 months after beginning of the treatment with lysate-loaded DC. b Histological examination (original magnification ×100) of the biopsy of a cutaneous metastasis in this patient 02 two months after the fourth vaccination showed the presence of melanophages (arrows) which were interpreted as signs of tumor regression. c The cutaneous biopsy performed after the sixth vaccination demonstrated a lymphocytic infiltrate and spongiosis (original magnification ×40, arrowheads) confirming the delayed immune response (eczema)

Fig. 3

Immune responses to HBs- and TT-proteins in vaccinated patients. a, b PBMC were tested in proliferation assay for reactivity against (a) TT- or (b) HBs-proteins at baseline and after 1, 2, 3, or 4 vaccinations. Results are expressed as stimulation index (SI = cpm with antigen/cpm without antigen). Results from patients with pre-existing reactivity (open symbols) and those without reactivity at baseline (filled symbols) are shown. Patients with increased reactivity during treatment are presented on the left side of graphs (a) and (b). c, d Fresh PBMC were cultured with autologous DC and TT (c) or HBs (d) proteins and IFN-γ production was measured by ELISpot. Results are expressed as IFN-γ spot forming cell (SFC) among 4×10⁵ PBMC. Patient 07 could not be evaluated by ELISpot because of a high reactivity against autologous DC at baseline

Fig. 4

Immune responses to lysate-loaded DC detected by IFN-γ ELISpot. Fresh PBMC were cultured overnight with autologous unloaded DC or DC loaded with M17 lysate. Results are expressed as IFN-γ spot forming cells after subtraction of the background among 4×10⁵ PBMC collected before (white bars) and during treatment (gray bars)

Fig. 5

Immune responses to TAA derived peptides detected by IFN-γ ELISpot in HLA-A2⁺ patients. Fresh PBMC were cultured overnight with autologous DC and TAA-derived peptides (10 μg/ml). Results are expressed as IFN-γ spot forming cells among 4×10⁵ PBMC collected before (white bars) and during treatment (gray bars)