RESUMO
Strategies using epitope-based vaccination are being considered for melanoma immunotherapy, in an attempt to overcome failure of other modalities. In the present study, we designed and produced a multiepitope polypeptide for melanoma (MEP-mel), which contains three repeats of four antigenic epitopes (gp100: 209-217 (210M); gp100: 280-288 (288V); Mart1: 26-35 (27L); tyrosinase: 368-376 (370D). The peptides were attached to each other by linkers containing sequences recognized by the proteasome, to improve protein cleavage and antigen presentation. The results show that peptide-specific T cells produced IFN-gamma when stimulated with MEP-mel-transfected dendritic cells. The presentation of peptides by MEP-mel-transfected dendritic cells was proteasome-dependent and was more long-lasting than the presentation of exogenously delivered native peptides. When dendritic cells were loaded with MEP-mel protein, weak cross presentation was induced. The production of multiepitope molecules based on several peptides linked by sequences sensitive to proteasomal cleavage represents a promising new tool for the improvement of cancer immunotherapy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Epitopos de Linfócito T/imunologia , Melanoma/imunologia , Peptídeos/imunologia , Proteínas/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Separação Celular , Células Cultivadas , DNA Complementar/genética , DNA Complementar/farmacocinética , Células Dendríticas/efeitos dos fármacos , Desenho de Fármacos , Eletroporação , Inibidores Enzimáticos/farmacologia , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/farmacologia , Escherichia/genética , Humanos , Imunoterapia/métodos , Interferon gama/metabolismo , Melanoma/genética , Peptídeos/genética , Peptídeos/farmacologia , Plasmídeos/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Proteínas/genética , Proteínas/farmacologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologiaRESUMO
Escherichia coli heat labile enterotoxin (LT) has been shown to penetrate intact skin and to activate adaptive immunity. A nontoxic mutant, nLT, and its B subunit (LTB), have been evaluated separately for their potential use as a tool for transcutaneous delivery of antigens for cancer immunotherapy. We have shown that FITC-labeled nLT is taken up by human dendritic cells (hDC) in vitro and in mouse skin, and induces maturation and activation of hDC in vitro. hDC matured with nLT enhanced nonspecific melanoma antigen uptake and presentation to autologous CD8+ T cells. In mouse in vivo studies, nLT or LTB were applied on the skin either mixed with recombinant gp100 or genetically fused with a multiepitope polypeptide (MEP). Fused LTB-MEP induced antibody production that was dependent on LTB cell binding. We conclude that LT derivatives may be useful for the transcutaneous delivery of tumor antigens for cancer immunotherapy.