In vitro priming with adenovirus/gp100 antigen-transduced dendritic cells reveals the epitope specificity of HLA-A*0201-restricted CD8+ T cells in patients with melanoma

Replication-deficient recombinant adenovirus (Ad) encoding human gp100 or MART-1 melanoma Ag was used to transduce human dendritic cells (DC) ex vivo as a model system for cancer vaccine therapy, A second generation E1/E4 region deleted Ad which harbors the CMV immediate-early promoter/enhancer and a unique E4-ORF6/pIX chimeric gene was employed as the backbone vector, We demonstrate that human monocyte-derived DC are permissive to Ad infection at multiplicity of infection between 100 and 500 and occurs independent of the coxsackie Ad receptor. Fluorescent-labeled Ad was used to assess the kinetics and distribution of viral vector within DC. Ad-transduced DC show peak transgene expression at 24-48 h and expression remains delectable for at least 7 days. DC transduced with replication-deficient Ad do not exhibit any unusual phenotypic characteristics or cytopathic effects, DC transduced with Ad2/gp100v2 can elicit tumor-specific CTL in vitro from patients bearing gp100(+) metastatic melanoma, Using a panel of gp100-derived synthetic peptides, we show that Ad2/gp100v2-transduced DC elicit Ag-specific CTL that recognize only the G209 and G280 epitopes, both of which display relatively short half-lives (similar to 7-8 h) on the surface bf HLA-A*0201(+) cells, Thus, patients with metastatic melanoma are not tolerant to gp100 Ag based on the detection of CD8(+) T cells specific for multiple HLA-A*0201-restricted, gp100-derived epitopes.