RESUMO
PURPOSE: Autologous melanoma cells display a broad variety of tumor antigens and were used for treatment of American Joint Committee on Cancer stages III and IV melanoma as an adjuvant or active therapy. Survival data and immune response were evaluated in vaccinated patients. EXPERIMENTAL DESIGN: Forty-seven patients received 2,4-dinitrophenyl-conjugated autologous melanoma vaccine as an adjuvant (23 patients) or therapy (24 patients). CD4 and CD8 T-cell response in blood sampled before vaccination and after five or eight vaccine doses was evaluated against melanoma cells and autologous peripheral blood mononuclear cells using IFNgamma enzyme-linked immunospot. Serum levels of antilivin, an inhibitor of apoptosis, and anti-gp100 IgG were determined. RESULTS: The immunologic effect of the vaccine differed between the two groups of patients. In the adjuvant group, there was a significant increase in CD8 melanoma-reactive T cells (P = 0.035) after vaccination and an increase in antimelanoma CD4 T cells correlating with improved overall survival (P = 0.04). In the therapeutic group, there was no objective tumor regression; antimelanoma T-cell reactivity increased by a small amount, stayed the same, or in some cases decreased. In all patients, a significant increase was noted in CD4 T-cell reactivity against autologous peripheral blood mononuclear cells (P = 0.02), which did not affect survival. Increased antilivin IgG was associated with improved survival. Expression of MHC class II on melanoma cells was vital for the immunogenicity of the vaccine. CONCLUSION: Autologous melanoma cell vaccine is capable of inducing effective antimelanoma CD4 T-cell activity associated with improved survival. Patients with active metastatic disease generally displayed reduced immune response and gained little from active immunization.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Melanoma/terapia , Neoplasias Cutâneas/terapia , 2,4-Dinitrofenol/imunologia , Proteínas Adaptadoras de Transdução de Sinal/sangue , Autoimunidade/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer/uso terapêutico , Linhagem Celular Tumoral , Humanos , Imunidade Celular , Imunidade Humoral , Imunoterapia , Proteínas Inibidoras de Apoptose/sangue , Interferon gama/imunologia , Interferon gama/metabolismo , Estimativa de Kaplan-Meier , Melanoma/imunologia , Melanoma/mortalidade , Glicoproteínas de Membrana/sangue , Proteínas de Neoplasias/sangue , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/mortalidade , Antígeno gp100 de MelanomaRESUMO
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.
Assuntos
Adjuvantes Imunológicos/uso terapêutico , Toxinas Bacterianas/uso terapêutico , Vacinas Anticâncer/uso terapêutico , Enterotoxinas/uso terapêutico , Proteínas de Escherichia coli/uso terapêutico , Imunoterapia , Neoplasias/terapia , Sequência de Aminoácidos , Animais , Formação de Anticorpos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Portadores de Fármacos , Citometria de Fluxo , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Humanos , Interleucina-12/biossíntese , Melanoma/imunologia , Camundongos , Dados de Sequência Molecular , Monócitos/imunologia , Peroxidase/metabolismo , Pele/patologiaRESUMO
Native gp100, a glycoprotein highly expressed in the majority of melanomas, contains several immunogenic peptides that are recognized by cytotoxic lymphocytes (CTLs) in the context of major histocompatibility complex (MHC) class I molecules. The objective of this study was to evaluate the ability of dendritic cells (DCs) from melanoma patients to take up gp100 protein and stimulate specific autologous CTL. The gp100 used in this study was a recombinant molecule with diminished hydrophobicity, HR-gp100, produced in Escherichia coli bacteria and in Pichia pastoris yeast. Stimulation of CD8+ T cells from melanoma patients with HR-gp100-loaded DC was visualized by confocal microscopy using stained target cells, and was quantitatively measured by the production of IFN-gamma using an ELISPOT assay. The results showed that HR-gp100 protein, produced either in bacteria or in yeast, when loaded on DC from melanoma patients, stimulated autologous CD8+ lymphocytes. By direct visualization, these lymphocytes were found in close contact with dead melanoma cells, and to contain membrane material transferred from stained melanoma cells; in cultures containing control lymphocytes stimulated with unloaded DC, no melanoma cell killing was observed. In ELISPOT assays, increased number of IFN-gamma-producing CD8+ T lymphocytes from patients, but not from healthy controls, were measured upon stimulation with HR-gp100-loaded DC. HR-gp100 could represent a useful tool to load DC with multiple immunogenic epitopes/antigen-derived epitopes for the immunotherapy of melanoma.