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Abstract. Human malignant melanoma is notoriously resistant to pharmacological modulation. We describe here for the first time that the synthetic retinoid CD437 has a strong dose-dependent antiproliferative effect on human melanoma cells (IC50:5 × 10-6 M) via the induction of programmed cell death, as judged by analysis of cell morphology, electron microscopical features, and DNA fragmentation. Programmed cell death was preceded by a strong activation of the AP-1 complex in CD437-treated cells as demonstrated by gel retardation and chloramphenicol transferase (CAT) assays. Northern blot analysis showed a time-dependent increase in

Cytokine gene transfer into tumor cells has been shown subsequent first clinical phase I study six patients with to mediate tumor regression and antimetastatic effects in metastatic melanoma were vaccinated with autologous, several animal models via immunomodulation. Therefore, interleukin-12 gene-modified tumor cells. Melanoma cells clinical protocols have been developed to treat cancer were expanded in vitro from surgically removed metastpatients with cytokine gene-modified tumor cells. We ases, transduced by ballistic gene transfer, irradiated and inserted the genes coding for the p35 and p40 chain of were then injected subcutaneously (s.c.) at weekly interinterleukin-12 (IL-12) in two independent eukaryotic vals. Clinically, there was no major toxicity except for mild expression vectors and transduced melanoma cells of 15 fever. All patients completed more than four s.c. vaccidifferent primary tumor cultures with both plasmids by a nations over 6 weeks and were eligible for immunological ballistic gene transfer approach. Secreted IL-12 demonevaluation. Post-vaccination, peripheral mononuclear cells strated strong bioactivity by inducing interferon-␥ release were found to contain an increased number of tumorfrom peripheral blood lymphocytes upon coculture with cell reactive proliferative as well as cytolytic cells as determculture supernatants after IL-12 gene transfer which could ined by a limiting dilution analysis in two patients. Two at least partly be blocked by IL-12-specific antisera. Further patients developed DTH reactivity against autologous enrichment of transduced tumor cells by magnetic separmelanoma cells and one had a minor clinical response. ation directly after gene transfer increased cytokine Biopsies taken from that patient's metastases revealed a secretion from a mean of 119 pg in the unsorted to 507 heavy infiltration of CD4 + and CD8 + T lymphocytes. In conpg IL-12 (24 h/10 6 cells) in the magnetically enriched cell clusion, vaccination induced immunological changes even fraction. Irradiation of these cells led to a further elevation in a group of advanced, terminally ill patients. These of secreted IL-12 (mean 987 pg). Elevated IL-12 levels changes can be interpreted as an increased antitumor were detected over 7 days after irradiation in vitro. In a immune response.

Summary Recently, cytokine gene transfer into tumour cells has been shown to mediate tumour regression in animal models via immunomodulation. Consequently, a number of clinical protocols have been developed to treat cancer patients with cytokine gene-modified tumour cells. Here, we report the results of a clinical phase trial using for the first time autologous, interleukin 7 gene-modified tumour cells for vaccination of ten patients with disseminated malignant melanoma. Melanoma cells were expanded in vitro from surgically removed metastases, transduced by a ballistic gene transfer technique and were then injected after in vitro irradiation s.c. at weekly intervals. Clinically, there was no major toxicity except for mild fever, and no major clinical response towards vaccination was observed. Eight of ten patients completed the initial three s.c. vaccinations and were eligible for immunological evaluation. Post vaccination, peripheral mononuclear cells (PBMCs) were found to contain an increased number of tumour-reactive proliferative as well as cytolytic cells, as determined by a limiting dilution analysis. In three of six patients, the frequencies of anti-melanoma cytolytic precursor cells increased between 2.6-and 28-fold. Two of these patients showed a minor clinical response. Analysis of the autologous tumour cell vaccines regarding IL-7 secretion after gene transfer, HLA class and class 11 cell surface expression, secretion of immunosuppressive mediators (TGF-,1, IL-10) and various melanoma-associated tumour antigens revealed a very diverse expression profile. In conclusion, vaccination using gene-modified autologous melanoma cells induced immunological changes in a group of advanced, terminally ill patients. These changes can be interpreted as an increased anti-tumour immune response. However, immunological modulation was most pronounced in patients in good physical condition. Therefore, patients with minimal tumour load or minimal residual disease might preferentially benefit from tumour cell vaccination in further studies. In order to evaluate the effects of the cytokine gene-modified tumour cell vaccines more precisely, an antigenically better defined vaccine is needed.