RESUMEN
D(b-/-)xbeta2 microglobulin (beta2m) null mice transgenic for a chimeric HLA-A2.1/D(b)-beta2m single chain (HHD mice) are an effective biological tool to evaluate the antitumour cytotoxic T-lymphocyte response of known major histocompatibility-restricted peptide tumour-associated antigens, and to screen for putative unknown novel peptides. We utilised HHD lymphocytes to identify immunodominant epitopes of colon carcinoma overexpressed genes. We screened with HHD-derived lymphocytes over 500 HLA-A2.1-restricted peptides derived from colon carcinoma overexpressed genes. This procedure culminated in the identification of seven immunogenic peptides, three of these were derived from the 'human 1-8D gene from interferon inducible gene' (1-8D). The 1-8D gene was shown to be overexpressed in fresh tumour samples. The three 1-8D peptides were both antigenic and immunogenic in the HHD mice. The peptides induce cytotoxic T lymphocytes that were able to kill a colon carcinoma cell line HCT/HHD, in vitro and retard its growth in vivo. One of the peptides shared by all the 1-8 gene family primed efficiently normal human cytotoxic T lymphocyte precursors. These results highlight the 1-8D gene and its homologues as putative immunodominant tumour-associated antigens of colon carcinoma.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores , Neoplasias del Colon/genética , Neoplasias del Colon/inmunología , Interferones/química , Interferones/farmacología , Proteínas de la Membrana/genética , Péptidos/farmacología , Linfocitos T Citotóxicos/inmunología , Animales , Línea Celular Tumoral , Reacciones Cruzadas , Humanos , Epítopos Inmunodominantes , Proteínas de la Membrana/inmunología , Ratones , Ratones TransgénicosRESUMEN
Bladder carcinoma is the fourth most common cancer in men and the eighth most common cancer among women. Our study is aimed to characterise tumour-associated antigen peptides of transitional cell carcinoma of the bladder (TCC). A DNA micro-array-based differential display analysis of 10 000 genes was carried out, and MAGE-A8 gene expression was detected in the tumour, and not in the normal bladder. High occurrence of MAGE-A8 expression was observed in fresh tumour samples (17 out of 23) and TCC lines (four of eight). The MAGE-A8 protein sequence was screened for HLA-A2.1-binding motifs, six potential peptides were synthesised, and peptides binding to HLA-A2.1 were assured. Immunogenicity and antigenicity of the MAGE-A8 peptides were examined in the HHD system, murine class I MHC knockout mice, transgenic for HLA-A2.1. The MAGE-A8 peptide immunogenicity was examined in three modes of vaccination, delivered intranasally with cholera toxin, injected into the tail base with complete Freund's adjuvant (CFA), or presented directly as loaded onto cell surface HLA-A2.1 molecules. Two peptides, 8.1 and 8.3, induce CTL that kills the T24 TCC line in vitro, and prime human lymphocyte response of healthy donors. These results demonstrate the potential use of the MAGE-A8 peptides for specific immunotherapy of TCC.
Asunto(s)
Antígenos de Neoplasias/genética , Carcinoma de Células Transicionales/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/genética , Oligopéptidos/genética , Neoplasias de la Vejiga Urinaria/genética , Animales , Antígenos de Neoplasias/inmunología , Carcinoma de Células Transicionales/patología , Toxina del Cólera/administración & dosificación , Citotoxicidad Inmunológica , Adyuvante de Freund , Perfilación de la Expresión Génica , Antígeno HLA-A2/inmunología , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas de Neoplasias/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos , Oligopéptidos/farmacología , Linfocitos T/inmunología , Neoplasias de la Vejiga Urinaria/patología , Vacunación , Microglobulina beta-2/genética , Microglobulina beta-2/fisiologíaRESUMEN
Peptide vaccination of homozygous mice against syngeneic tumors using single major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocyte (CTL) epitopes elicits effective immune responses against metastatic growth. So far, single-peptide vaccination of patients against their autologous tumors seems to elicit less satisfactory results. In this study, the authors tried to determine whether effective anti-metastatic immunity requires the presentation of peptides restricted by the two parental class I major histocompatibility complex alleles in heterozygous hosts. The immune response against the H-2b-derived 3LL Lewis lung carcinoma was evaluated in heterozygous recombinant congenic F1 mice (Kk x K(b)) and (Kd x K(b)). Vaccination of such heterozygous animals with dendritic cells expressing the two parental H-2K alleles, pulsed with total tumor extract, elicited a potent anti-metastatic response. A comparable response was obtained after vaccination with tumor cells genetically modified to express the two class I alleles. In contrast, vaccination of the heterozygous mice with dendritic cells expressing only one of the parental F1 H-2K alleles or with tumors expressing only one H-2K allele failed to elicit effective immunity against tumor metastasis in recombinant congenic F1 mice. It appears, therefore, that to achieve effective anti-metastatic immunotherapy in heterozygous organisms, presentation of cytotoxic T lymphocyte epitopes restricted by the two parental class I alleles is required.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/uso terapéutico , Células Dendríticas/inmunología , Epítopos/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Neoplasias Pulmonares/terapia , Péptidos/inmunología , Linfocitos T Citotóxicos/inmunología , Alelos , Animales , Pruebas Inmunológicas de Citotoxicidad , Citometría de Flujo , Genes MHC Clase I , Heterocigoto , Antígenos de Histocompatibilidad Clase I/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Ratones , Ratones Congénicos , Metástasis de la Neoplasia/prevención & control , Células Tumorales CultivadasRESUMEN
The MUC1 protein was found to be up-regulated in a spectrum of malignant tumors. T-cell responses to the MUC1 extracellular tandem repeat array (TRA) were observed in murine models as well as in breast-carcinoma patients. In the present study, we evaluated the anti-tumor potential of HLA-A2.1-motif-selected peptides from non-TRA domains of the molecule. Peptide immunogenicity was examined in the Db-/- x beta2 microglobulin (beta2m) null mice transgenic for a modified HLA-A2.1/Db-beta2 microglobulin single chain (HHD mice). Our results show the existence of 3 novel HLA-A2.1-restricted MUC1-derived cytotoxic T-lymphocyte (CTL) epitopes. These peptides are processed and presented by the HHD-transfected breast-tumor cell line MDA-MB-157. Moreover, CTL induced by these 3 peptides show higher lysis of target cells pulsed with breast-carcinoma-derived peptides than of targets pulsed with normal breast-tissue-derived peptides. These data suggest an important role for non-TRA MUC1-derived peptides as inducers of a MHC-restricted CTL reaction to a breast-carcinoma cell line and patient-derived tumor extracts.
Asunto(s)
Neoplasias de la Mama/inmunología , Vacunas contra el Cáncer/inmunología , Epítopos de Linfocito T/inmunología , Antígeno HLA-A2/inmunología , Mucina-1/inmunología , Fragmentos de Péptidos/inmunología , Linfocitos T Citotóxicos/inmunología , Microglobulina beta-2/inmunología , Animales , Presentación de Antígeno , Neoplasias de la Mama/metabolismo , Vacunas contra el Cáncer/química , Vacunas contra el Cáncer/genética , Citotoxicidad Inmunológica , Epítopos de Linfocito T/química , Femenino , Fluorescencia , Regulación Neoplásica de la Expresión Génica , Antígenos H-2/genética , Antígenos H-2/inmunología , Antígeno de Histocompatibilidad H-2D , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mucina-1/química , Mucina-1/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Péptidos/inmunología , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Células Tumorales Cultivadas , Microglobulina beta-2/química , Microglobulina beta-2/genéticaRESUMEN
CTLs specific for tumor antigens play a major role in immunity against cancer. Improved binding affinity of putative TAA peptides could enhance the in vivo immunogenicity of these self-altered self- tumor antigens. We examined here the efficacy of tumor vaccines composed of an altered peptide ligand of MUT-1, designated MUT-D, which exhibited significantly higher class-I allele K(b) binding affinity than its native counterpart MUT-1. The peptide was loaded on antigen presenting cells composed of the C57BL/6-syngeneic fibroblast cell line BLK.CL4. These cells were treated with proteasome inhibitor in order to shut off the degradation of proteins and the subsequent loading of endogenous peptides onto MHC class-I molecules, thus allowing for the pulsing of these cells with the modified peptide MUT-D. Proteasome-inhibited and modified peptide-loaded fibroblasts induced a peptide-specific CTL that significantly delayed primary tumor progression and protected the pre-immunized mice against the development of lung metastasis following the surgical removal of the primary tumor. Genetic modification of the fibroblasts to express the immunostimulatory cytokine IL-2 did not improve the APC function of the modified cells, nor did it result in augmentation of the potency of the vaccine. Our results suggest that the proteasome-inhibited fibroblasts pulsed with modified, high binder tumor-associated antigen peptide are good antigen-presenting cells and represent an effective form of tumor vaccine.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Fibroblastos/inmunología , Neoplasias Pulmonares/terapia , Oligopéptidos/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Antígenos de Neoplasias/inmunología , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Epítopos de Linfocito T/inmunología , Fibroblastos/enzimología , Inmunoterapia , Interleucina-2/biosíntesis , Interleucina-2/genética , Leupeptinas/farmacología , Neoplasias Pulmonares/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Complejos Multienzimáticos/metabolismo , Metástasis de la Neoplasia/prevención & control , Trasplante de Neoplasias , Complejo de la Endopetidasa ProteasomalRESUMEN
CTL induction by immunization with synthetic peptide epitopes has been shown to inhibit tumor growth and its metastatic spread. Ex vivo pulsing of peptides on MHC class I-bearing cells such as RMA-S cells or professional APCs elicits an effective CTL response. Since the stability of the MHC-peptide complex is strongly correlated with the overall immunogenecity, we compared the effect of immunization with low affinity, high affinity, and irreversibly bound MHC peptides in the context of immunotherapy of metastasis. MUT1, a tumor-associated antigen peptide that was isolated from 3LL Lewis lung carcinoma, is a low H-2Kb binder. MUT1 was modified into a high binder by changing positions 3, 5, and 8 to the favorable anchor residues. In addition, we introduced a photo-active chemical moiety, which can bind irreversibly to MHC upon illumination. These peptides, loaded onto RMA-S, were used to immunize mice against the 3LL tumor. Vaccination via the covalent conjugation of the low binder peptide was found to increase the CTL response measured against MUT1 loaded cells and against H-2Kb transfected D122 cells relative to the native MUT1 peptide. However, the photo cross-linking of the high affinity peptide to the MHC did not significantly improve the induction of specific CTL. The level of CTL activity was elevated to the same extent by either cross-linking the peptide to the MHC or by modifying it into a high-binder peptide. The protective capacity of all the peptide-based vaccines against D122 metastatic spread to the lungs was found to be comparable. These results indicate that augmentation of the affinity of a TAA peptide to the RMA-S surface MHC molecules, by conversion to a high-affinity mimotope or by photo-conjugation, can significantly enhance the immune response. There seems to be, however, a ceiling beyond which increase in the peptide-binding affinity does not lead to a corresponding enhancement of the overall immunogenicity of the peptide.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Antígenos H-2/inmunología , Péptidos/inmunología , Vacunas Sintéticas/inmunología , Animales , Vacunas contra el Cáncer/química , Trasplante de Células , Epítopos/inmunología , Ratones , Ratones Endogámicos C57BL , Imitación Molecular , Péptidos/química , Linfocitos T/inmunología , Linfocitos T/trasplante , Linfocitos T Citotóxicos/inmunología , Células Tumorales Cultivadas , Vacunas Sintéticas/químicaRESUMEN
Epitope spreading is a process whereby epitopes distinct from and non-cross-reactive with an inducing epitope become targets of an evolving immune response. This phenomenon has been associated most notably with the progression of naturally occurring or experimentally induced chronic autoimmune diseases. We have investigated the potential occurrence of epitope spreading in the context of antitumor cytotoxic T cell (CTL) responses using chicken ovalbumin (OVA) as a model antigen. Our results indicate that following rejection of OVA-expressing EG.7 tumor cells effectuated by a CTL response which is induced against the MHC class I-restricted immunodominant epitope OVA257-264, there occurs intramolecular diversification of the CTL response to two additional OVA-derived epitopes, OVA176-183 and OVA55-62, as well as intermolecular spreading to other endogenous tumor-derived determinants. It seems that CTL-mediated tumor cell destruction in vivo favors cross-presentation of additional epitopes with the consequent activation of additional tumor-reactive lymphocytes. The process of epitope spreading in that context has obvious important implications for the design of antigen-specific antitumor immunotherapies.