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1.
Cancer Res ; 61(24): 8782-6, 2001 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-11751399

RESUMO

alpha-Fetoprotein (AFP) is a potential target for immunotherapy in hepatocellular carcinoma; both the murine and human T-cell repertoires can recognize AFP-derived epitopes in the context of the MHC. Protective immunity can be generated with AFP-engineered dendritic cell-based vaccines. We now report a DNA-based immunization strategy using a prime-boost approach: coadministration of plasmid DNA encoding murine AFP and murine granulocyte-macrophage colony-stimulating factor followed by boosting with an AFP-expressing nonreplicating adenoviral vector. This immunization strategy can elicit a high frequency of Th1-type AFP-specific cells leading to tumor protective immunity in mice at levels comparable with AFP-engineered dendritic cells. This cell-free mode of immunization is better suited for large-scale vaccine efforts for patients with hepatocellular carcinoma.


Assuntos
Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Carcinoma Hepatocelular/terapia , Epitopos Imunodominantes/imunologia , Neoplasias Hepáticas/terapia , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/imunologia , Adenoviridae/genética , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Epitopos de Linfócito T/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Humanos , Epitopos Imunodominantes/genética , Imunoterapia Ativa/métodos , Células Jurkat/imunologia , Células Jurkat/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Plasmídeos/genética , Linfócitos T/imunologia , Transfecção , Vacinas de DNA/genética , Vacinas de DNA/imunologia , alfa-Fetoproteínas/biossíntese
2.
Cancer Res ; 61(24): 8787-93, 2001 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-11751400

RESUMO

Genetic immunization of mice with dendritic cells (DCs) engineered to express a melanoma antigen generates antigen-specific, MHC-restricted, CD4-dependent protective immune responses. We wanted to determine the role of CD4 cells and CD40 ligation of MART-1 gene-modified DC in an animal model of immunotherapy for murine melanoma. CD4 knock-out (CD4KO) or antibody-depleted mice were immunized with DC adenovirally transduced with the MART-1 gene (AdVMART1/DC) with or without CD40 cross-linking. Tumor protection was absent in CD4-depleted mice, but protection was reestablished when the CD40 receptor was engaged using three different constructs. Transduction of DCs with vectors expressing the Th1 cytokines interleukin (IL)-2, IL-7, or IL-12 could not reproduce the CD40-mediated maturation signal in this model. CD8 T-cell depletion in CD4KO mice immunized with CD40-ligated DCs abrogated the protective response. Pooled analysis of CD40 cross-linking of AdVMART1/DC administered to wild-type C57BL/6 mice revealed an overall enhancement of antitumor immunity. However, this effect was inconsistent between replicate studies. In conclusion, maturation of AdVMART1-transduced DCs through the CD40 ligation pathway can promote a protective CD8 T-cell-mediated immunity that is independent of CD4 T-cell help.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Antígenos CD40/imunologia , Células Dendríticas/imunologia , Imunoterapia Adotiva/métodos , Melanoma Experimental/imunologia , Adenoviridae/genética , Animais , Antígenos de Neoplasias , Antígenos CD40/genética , Antígenos CD40/metabolismo , Ligante de CD40/genética , Ligante de CD40/imunologia , Linfócitos T CD8-Positivos/imunologia , Citotoxicidade Imunológica/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/fisiologia , Epitopos de Linfócito T/imunologia , Vetores Genéticos/genética , Interleucinas/biossíntese , Interleucinas/genética , Interleucinas/imunologia , Linfoma/imunologia , Linfoma/terapia , Antígeno MART-1 , Melanoma Experimental/prevenção & controle , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Transdução Genética
3.
AJR Am J Roentgenol ; 177(4): 807-12, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11566677

RESUMO

OBJECTIVE: The purpose of our study was to evaluate the sensitivity and accuracy of ferumoxides-enhanced MR imaging in comparison with surgery and intraoperative sonography. SUBJECTS AND METHODS: We prospectively evaluated 25 consecutive studies in 24 patients who underwent ferumoxides-enhanced hepatic MR imaging before surgery and intraoperative sonography. Both 1.5-T scanners (13 cases) and 0.2-T scanners (12 cases) were used. Turbo spin-echo T2-weighted sequences were performed before and after the administration of ferumoxides and the images were compared. Lesions were classified as solid or nonsolid and tabulated on standard liver maps. The liver maps from MR imaging were compared with those from surgery and intraoperative sonography. For lesions greater than 1 cm, the regions of interest were measured and contrast-to-noise ratio was calculated. RESULTS: Of 93 solid lesions found at surgery, 69 were seen on unenhanced MR imaging (sensitivity, 74.2%) and 87 were seen on ferumoxides-enhanced MR imaging (sensitivity, 93.5%) (p < 0.05). Of the seven benign lesions (five cysts, two hemangiomas) found at surgery, all were correctly identified as benign on MR imaging. Two lesions identified as solid before surgery were not found at surgery. Mean lesion contrast-to-noise ratio for the unenhanced scans was 22.9 and 34.5 (p < 0.001) for the ferumoxides-enhanced scans. Subanalysis of 1.5- and 0.2-T MR imaging revealed similar results with significant (p < 0.05) increases in sensitivity for both. The average size of the lesions missed before surgery was 0.7 cm. CONCLUSION: Turbo spin-echo T2-weighted ferumoxides-enhanced MR imaging at either 1.5 or 0.2 T has value in preoperative liver assessment.


Assuntos
Meios de Contraste , Cuidados Intraoperatórios , Ferro , Neoplasias Hepáticas/diagnóstico por imagem , Neoplasias Hepáticas/patologia , Imageamento por Ressonância Magnética , Óxidos , Adulto , Idoso , Dextranos , Feminino , Óxido Ferroso-Férrico , Humanos , Neoplasias Hepáticas/cirurgia , Nanopartículas de Magnetita , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Ultrassonografia
4.
J Immunol ; 166(8): 5300-8, 2001 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-11290817

RESUMO

alpha fetoprotein (AFP)-derived peptide epitopes can be recognized by human T cells in the context of MHC class I. We determined the identity of AFP-derived peptides, presented in the context of HLA-A*0201, that could be recognized by the human (h) T cell repertoire. We screened 74 peptides and identified 3 new AFP epitopes, hAFP(137-145), hAFP(158-166), and hAFP(325-334), in addition to the previously reported hAFP(542-550.) Each possesses two anchor residues and stabilized HLA-A*0201 on T2 cells in a concentration-dependent class I binding assay. The peptides were stable for 2-4 h in an off-kinetics assay. Each peptide induced peptide-specific T cells in vitro from several normal HLA-A*0201 donors. Importantly, these hAFP peptide-specific T cells also were capable of recognizing HLA-A*0201(+)/AFP(+) tumor cells in both cytotoxicity assays and IFN-gamma enzyme-linked immunospot assays. The immunogenicity of each peptide was tested in vivo with HLA-A*0201/K(b)-transgenic mice. After immunization with each peptide emulsified in CFA, draining lymph node cells produced IFN-gamma on recognition of cells stably transfected with hAFP. Furthermore, AFP peptide-specific T cells could be identified in the spleens of mice immunized with dendritic cells transduced with an AFP-expressing adenovirus (AdVhAFP). Three of four AFP peptides could be identified by mass spectrometric analysis of surface peptides from an HLA-A*0201 human hepatocellular carcinoma (HCC) cell line. Thus, compelling immunological and physiochemical evidence is presented that at least four hAFP-derived epitopes are naturally processed and presented in the context of class I, are immunogenic, and represent potential targets for hepatocellular carcinoma immunotherapy.


Assuntos
Antígeno HLA-A2/imunologia , Ativação Linfocitária , Fragmentos de Peptídeos/imunologia , Linfócitos T Citotóxicos/imunologia , alfa-Fetoproteínas/imunologia , Adenoviridae/genética , Adenoviridae/imunologia , Alelos , Animais , Apresentação de Antígeno/genética , Linhagem Celular Transformada , Células Cultivadas , Testes Imunológicos de Citotoxicidade , Células Dendríticas/transplante , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Feminino , Adjuvante de Freund/administração & dosagem , Adjuvante de Freund/imunologia , Vetores Genéticos/administração & dosagem , Vetores Genéticos/imunologia , Antígenos H-2/genética , Antígeno HLA-A2/genética , Humanos , Células Jurkat , Células K562 , Ativação Linfocitária/genética , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , alfa-Fetoproteínas/administração & dosagem , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/metabolismo
5.
J Immunol ; 166(5): 3564-73, 2001 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-11207317

RESUMO

The discovery of human melanoma rejection Ags has allowed the rational design of immunotherapeutic strategies. One such Ag, MART-1, is expressed on >90% of human melanomas, and CTL generated against MART-1(27-35) kill most HLA A2.1(+) melanoma cells. However, variant tumor cells, which do not express MART-1, down-regulate MHC, or become resistant to apoptosis, will escape killing. Cytotoxic lymphocytes kill by two main mechanisms, the perforin/granzyme degranulation pathway and the TNF/Fas/TNF-related apoptosis-inducing ligand superfamily of apoptosis-inducing ligands. In this study, we examined whether cis-diaminedichloroplatinum (II) cisplatin (CDDP) sensitizes MART-1/HLA A2.1(+) melanoma and melanoma variant tumor cells to non-MHC-restricted, Fas ligand (FasL)-mediated killing by CTL. MART-1(27-35)-specific bulk CTL cultures were generated by pulsing normal PBL with MART-1(27-35) peptide. These CTL cultures specifically kill M202 melanoma cells (MART-1(+), HLA A2.1(+), FasR(-)), and MART-1(27-35) peptide-pulsed T2 cells (FasR(+)), but not M207 melanoma cells (MART-1(+), HLA A2.1(-), FasR(-)), FLU(58-66) peptide-pulsed T2 cells, or DU145 and PC-3 prostate cells (MART-1(-), HLA A2.1(-), FasR(+)). CDDP (0.1-10 microg/ml) sensitized non-MART-1(27-35) peptide-pulsed T2 to the CD8(+) subset of bulk MART-1-specific CTL, and killing was abolished by neutralizing anti-Fas Ab. Furthermore, CDDP up-regulated FasR expression and FasL-mediated killing of M202, and sensitized PC-3 and DU145 to killing by bulk MART-1-specific CTL cultures. These findings demonstrate that drug-mediated sensitization can potentiate FasL-mediated killing by MHC-restricted CTL cell lines, independent of MHC and MART-1 expression on tumor cells. This represents a novel approach for potentially controlling tumor cell variants found in primary heterogeneous melanoma tumor cell populations that would normally escape killing by MART-1-specific immunotherapy.


Assuntos
Apoptose/imunologia , Citotoxicidade Imunológica , Epitopos de Linfócito T/imunologia , Melanoma/imunologia , Melanoma/patologia , Proteínas de Neoplasias/imunologia , Linfócitos T Citotóxicos/imunologia , Receptor fas/fisiologia , Adjuvantes Imunológicos/farmacologia , Animais , Antígenos de Neoplasias , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Células Cultivadas , Cisplatino/farmacologia , Testes Imunológicos de Citotoxicidade , Citotoxicidade Imunológica/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Epitopos/metabolismo , Proteína Ligante Fas , Humanos , Hibridomas , Imunização , Antígeno MART-1 , Complexo Principal de Histocompatibilidade/imunologia , Masculino , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Neoplasias/metabolismo , Perforina , Proteínas Citotóxicas Formadoras de Poros , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , RNA Mensageiro/biossíntese , Subpopulações de Linfócitos T/imunologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Células Tumorais Cultivadas , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia , Receptor fas/metabolismo
6.
Cancer Res ; 60(22): 6457-64, 2000 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-11103813

RESUMO

The cytokine interleukin-12 (IL-12) has shown potent antitumor activity in several tumor models. Recently, natural killer (NK) T cells have been proposed to mediate the antitumor effects of IL-12. In this study, the antitumor response of IL-12 was investigated in a gene therapeutic model against s.c. growing mouse hepatocellular carcinomas using an adenoviral vector expressing murine IL-12 (AdVmIL-12). An adenoviral-based system was chosen because of the ability of adenoviruses to transduce dividing and nondividing cells and because of their high transduction efficiencies. Our goals were to examine the efficacy of AdVmIL-12 in a hepatocellular carcinoma model and to investigate the mechanism of the AdVmIL-12-mediated antitumor response with specific interest in the role of NK T cells. Our studies demonstrate that intratumoral AdVmIL-12-mediated regression of s.c. hepatocellular tumors is associated with rapid antitumor responses. AdVmIL-12 treatment was associated with an immune cellular infiltrate consisting of CD4 and CD8 T lymphocytes, macrophages, NK cells, and NK T cells. Antibody ablation of CD4 and CD8 T cells and use of NK cell-defective beige mice failed to abrogate the response to AdVmIL-12. Studies in T-cell- and B-cell-deficient severe combined immunodeficient and recombinase activating gene-2-deficient mice and T-cell-, B-cell-, and NK cell-defective severe combined immunodeficient/beige mice also failed to abrogate this response. AdVmIL-12 retained potent antitumor activity in mice with specific genetic defects in immune cellular cytotoxicity (perforin knockout mice) and costimulation (CD28 knockout mice). Use of mice with specific NK T cell deficiencies, Valpha14 T-cell receptor and CD1 knockout mice, also failed to abrogate the response to AdVmIL-12. Histological and immunohistochemical studies of AdVmIL-12-treated tumors showed extensive inhibition of neovascularization and a marked decrease in factor VIII-stained endothelial cells. Our studies indicate that the antitumor response of AdVmIL-12 is independent of direct cytotoxic cellular immunity (specifically, the function of NK T cells) and suggest that the initial mechanisms of AdVmIL-12-mediated tumor regression involve inhibition of angiogenesis.


Assuntos
Antígenos CD1/imunologia , Interleucina-12/imunologia , Células Matadoras Naturais/imunologia , Neoplasias Hepáticas Experimentais/imunologia , Linfócitos T/imunologia , Adenoviridae/genética , Adenoviridae/imunologia , Animais , Antígenos CD28/imunologia , Citotoxicidade Imunológica , Modelos Animais de Doenças , Humanos , Hospedeiro Imunocomprometido/imunologia , Interleucina-12/genética , Neoplasias Hepáticas Experimentais/terapia , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Neovascularização Patológica/prevenção & controle , Perforina , Proteínas Citotóxicas Formadoras de Poros
7.
Oncologist ; 5(2): 87-98, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-10794799

RESUMO

Genetic immunization refers to treatment strategies where gene transfer methods are used to generate immune responses against cancer. Our growing knowledge of the mechanisms regulating the initiation and maintenance of cytotoxic immune responses has provided the rationale for the design of several genetic immunization strategies. Tumor cells have been gene-modified to express immune stimulatory genes and are then administered as tumor vaccines, in an attempt to overcome tumor cell ignorance by the immune system. With the description of well-characterized tumor antigens, multiple strategies have been proposed mainly aimed at optimal tumor antigen presentation by antigen-presenting cells (APC). Among APC, the dendritic cells have been recognized as the most powerful cells in this class, and have become the target for introducing tumor antigen genes to initiate antitumor immune responses. The detailed knowledge of how the immune system can be activated to specifically recognize tumor antigens, and the mechanisms involved in the control of this immune response, provide the basis for modern genetic immunization strategies for cancer treatment.


Assuntos
Antígenos de Neoplasias/imunologia , Terapia Genética/tendências , Imunoterapia/tendências , Neoplasias/imunologia , Neoplasias/terapia , Células Dendríticas/imunologia , Humanos
8.
Cancer Res ; 60(8): 2218-24, 2000 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-10786687

RESUMO

Genetic immunization with a single injection of dendritic cells (DCs) expressing a model melanoma antigen generates antigen-specific, MHC-restricted, protective immune responses. After initiating the immune response, additional vaccinations may increase the protection or conversely downregulate the immune response. Groups of mice were vaccinated several times with DCs transduced with the MART-1 gene, and the anti-tumor protection was compared with that of mice receiving a single vaccination. C3H mice had poorer protection from a syngeneic MART-1-expressing tumor challenge with multiple vaccinations. This was accompanied by lower levels of splenic CTL effectors and a shift from a type 1 to a type 2 cytokine profile. On the contrary, multiple vaccinations in C57BL/6 mice generated greater in vivo antitumor protection with no decrease in splenic CTLs and no cytokine shift. Antiadenoviral humoral or cellular immune responses did not seem to contribute to these effects. When studies were performed in Fas receptor-negative C3H.(lpr) mice, the adverse effect of multiple vaccinations disappeared, and there was no cytokine shift pattern. In conclusion, C3H mice but not C57BL/6 mice receiving multiple vaccinations with DCs expressing the MART-1 tumor antigen show decreased protection associated with deviation from a type 1 to a type 2 cytokine response attributable to a Fas-receptor mediated clearance of antigen-specific IFN-gamma-producing cells.


Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Esquemas de Imunização , Melanoma Experimental/imunologia , Receptor fas/fisiologia , Adenoviridae/genética , Adenoviridae/imunologia , Animais , Antígenos de Neoplasias/imunologia , Antígenos Virais/imunologia , Vacinas Anticâncer/administração & dosagem , Células Dendríticas/metabolismo , Células Dendríticas/transplante , Feminino , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Interferon gama/imunologia , Antígeno MART-1 , Complexo Principal de Histocompatibilidade/imunologia , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Mutação/genética , Proteínas de Neoplasias/imunologia , Linfócitos T Citotóxicos/imunologia , Células Th1/imunologia , Células Th2/imunologia , Células Tumorais Cultivadas , Vacinação , Receptor fas/genética
9.
J Immunother ; 23(1): 59-66, 2000 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10687138

RESUMO

The murine melanoma B16 expresses the murine counterpart of the human MART-1/Melan-A (MART-1) antigen, sharing a 68.6% amino acid sequence identity. In this study, mice were vaccinated with bone marrow-derived murine dendritic cells genetically modified with a replication-incompetent adenoviral vector to express the human MART-1 gene (AdVMART1). This treatment generated a protective response to a lethal tumor challenge of unmodified murine B16 melanoma cells. The response was mediated by major histocompatibility complex class I-restricted cytotoxic T lymphocytes specific for MART-1 antigen, which produced high levels of interferon-gamma when reexposed to MART-1 in vitro and lysed targets in a calcium-dependent mechanism suggestive of perforin/granzyme B lysis. MART-1 was presented by the dendritic cells used for vaccination and not by epitopes cross-presented by host antigen-presenting cells. In conclusion, dendritic cells genetically modified to express the human MART-1 antigen generate potent murine MART-1-specific protective responses to B16 melanoma.


Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Melanoma Experimental/prevenção & controle , Proteínas de Neoplasias/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas de DNA/imunologia , Animais , Antígenos de Neoplasias/genética , Reações Cruzadas , Feminino , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Interferon gama/biossíntese , Interleucina-4/biossíntese , Antígeno MART-1 , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/genética , Baço/citologia , Baço/imunologia , Vacinação
10.
Hum Gene Ther ; 11(1): 53-65, 2000 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-10646639

RESUMO

In two murine lung cancer models adenoviral interleukin 7-transduced dendritic cells (DC-AdIL-7) were administered intratumorally, resulting in complete tumor regression. Intratumoral DC-AdIL-7 therapy was as effective as DCs pulsed with specific tumor peptide antigens. Comparison with other intratumoral therapies including recombinant IL-7, AdIL-7 vector alone, unmodified DCs, IL-7-transduced fibroblasts, or DCs pulsed with tumor lysates revealed DC-AdIL-7 therapy to be superior in achieving antitumor responses and augmenting immunogenicity. Mice with complete tumor eradication as a result of either DC-AdIL-7 or AdIL-7 therapy were rechallenged with parental tumor cells 30 days or more after complete tumor eradication. All the DC-AdIL-7-treated mice completely rejected a secondary rechallenge, whereas the AdIL-7-treated mice had sustained antitumor effects in only 20-25% of the mice. DC-AdIL-7 therapy was more effective than AdIL-7 in achieving systemic antitumor responses and enhancing immunogenicity. After complete tumor eradication, those mice treated with DC-AdIL-7 evidenced significantly greater release of splenocyte GM-CSF and IFN-gamma than did controls or AdIL-7-treated mice. After intratumoral injection, gene-modified DCs trafficked from the tumor to lymph node sites and spleen. DCs were detected in nodal tissues for up to 7 days after intratumoral injection. We report that intratumoral DC-AdIL-7 leads to significant systemic immune responses and potent antitumor effects in murine lung cancer models.


Assuntos
Adenoviridae/genética , Células Dendríticas/imunologia , Interleucina-7/genética , Neoplasias Pulmonares/terapia , Animais , Feminino , Terapia Genética , Imunoterapia , Injeções Intralesionais , Interleucina-7/administração & dosagem , Neoplasias Pulmonares/imunologia , Linfonodos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Indução de Remissão , Baço/imunologia
11.
Mol Immunol ; 37(16): 943-50, 2000 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11395133

RESUMO

Human alpha-fetoprotein (AFP) is a potentially important target for the immunotherapy of hepatocellular carcinoma (HCC). AFP(542-550) (GVALQTMKQ) is one of several HLA-A2.1-restricted immunodominant AFP peptides that consistently generate AFP-specific T cell responses in human T cell cultures and in HLA-A2.1/K(b) transgenic (A2.1 tg) mice. We performed a fine specificity analysis of this nonamer to determine which amino acid side chains were critical for T cell priming and recognition. Using peptide-pulsed dendritic cells (DC) as an immunization strategy, we characterized the effects of AFP(542-550) amino acid substitutions on priming and recognition in A2.1 tg mice. Replacing the glutamine at anchor position 9 with a leucine enhanced MHC binding and AFP-specific T cell responses. Substitution of leucine at non-anchor position 4 with an alanine did not alter binding but greatly diminished T cell recognition. Computer-generated three-dimensional models provided the structural rationale for these observed effects in MHC binding and T cell responses resulted from the modifications in the AFP(542-550) sequence.


Assuntos
Antígeno HLA-A2/imunologia , Epitopos Imunodominantes/imunologia , Linfócitos T/imunologia , alfa-Fetoproteínas/imunologia , Aminoácidos/imunologia , Animais , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/terapia , Humanos , Imunoterapia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/terapia , Ativação Linfocitária , Camundongos , Modelos Moleculares , Oligopeptídeos/imunologia , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T
12.
Cancer Gene Ther ; 6(6): 523-36, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10608349

RESUMO

A murine model of dendritic cell (DC)-based genetic immunization to a defined human melanoma antigen (Ag), MART-1/Melan-A (MART-1), was developed. The MART-1 gene was stably transfected into the nonimmunogenic mouse fibrosarcoma cell line NFSA that is syngeneic in C3Hf/Sem/Kam (C3H, H-2k) mice to generate the NFSA(MART1) cell line. In vivo protection from a lethal NFSA(MART1) tumor challenge could be generated by DCs transduced with a recombinant adenovirus (AdV) vector expressing MART-1 (AdVMART1). This model has the following characteristics: (a) immunological specificity and memory, (b) comparable protection for varying transduction multiplicities of infection, cell doses, and sites of DC inoculation but, interestingly, worse protection with increasing numbers of vaccinations, (c) the ability to treat small established tumors, (d) an absolute requirement for CD8 and CD4 T cells, (e) generation of MART-1-specific splenic cytotoxic T lymphocytes, and (f) up-regulation of both T helper type 1 and T helper type 2 cytokines. Genetically engineered DCs presenting defined tumor Ags represent an attractive method to generate effective immune responses.


Assuntos
Antígenos de Neoplasias/imunologia , Células Dendríticas/imunologia , Melanoma Experimental/terapia , Animais , Antígenos de Neoplasias/genética , Modelos Animais de Doenças , Feminino , Terapia Genética , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Antígeno MART-1 , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C3H , Proteínas de Neoplasias/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
Cancer Res ; 59(17): 4369-74, 1999 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-10485485

RESUMO

An E1B gene-attenuated adenovirus (dl1520) has been proposed to have a selective cytolytic activity in cancer cells with a mutation or deletion in the p53 tumor suppressor gene (p53-null), a defect present in almost half of human hepatocellular carcinomas (HCCs). In this study, the in vitro and in vivo antitumor activity of dl1520 was investigated focusing on two human HCC cell lines, a p53-wild type (p53-wt) cell line and a p53-null cell line. dl1520 was tested for in vitro cytopathic effects and viral replication in the human HCC cell lines Hep3B (p53-null) and HepG2 (p53-wt). The in vivo antitumor effects of dl1520 were investigated in tumors grown s.c. in a severe combined immunodeficient mouse model. In addition, the combination of dl1520 infection with systemic chemotherapy was assessed in these tumor xenografts. At low multiplicities of infection, dl1520 had an apparent p53-dependent in vitro viral growth in HCC cell lines. At higher multiplicities of infection, dl1520 viral replication was independent of the p53 status of the target cells. In vivo, dl1520 significantly retarded the growth of the p53-null Hep3B xenografts, an effect augmented by the addition of cisplatin. However, complete tumor regressions were rare, and most tumors eventually grew progressively. dl1520 had no effect on the in vivo growth of the p53-wt HepG2 cells, with or without cisplatin treatment. The E1B-deleted adenoviral vector dl1520 has an apparent p53-dependent effect in HCC cell lines. However, this effect is lost at higher viral doses and only induces partial tumor regressions without tumor cures in a human HCC xenograft model.


Assuntos
Adenoviridae/fisiologia , Proteínas E1B de Adenovirus/fisiologia , Carcinoma Hepatocelular/terapia , Genes p53/fisiologia , Neoplasias Hepáticas/terapia , Replicação Viral , Animais , Humanos , Masculino , Camundongos , Camundongos SCID , Transplante de Neoplasias , Transplante Heterólogo , Células Tumorais Cultivadas
14.
Cancer Res ; 59(13): 3064-7, 1999 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-10397245

RESUMO

The majority of human hepatocellular carcinomas overexpress alpha-fetoprotein (AFP). Two genetic immunization strategies were used to determine whether AFP could serve as a target for T-cell immune responses. Dendritic cells engineered to express AFP produced potent T-cell responses in mice, as evidenced by the generation of AFP-specific CTLs, cytokine-producing T cells, and protective immunity. AFP plasmid-based immunization generated less potent responses. These novel observations demonstrate that this oncofetal antigen can serve as an effective tumor rejection antigen. This provides a rational, gene therapy-based strategy for this disease, which is responsible for the largest number of cancer-related deaths worldwide.


Assuntos
Células Dendríticas/imunologia , Terapia Genética , Imunoterapia , Neoplasias Hepáticas Experimentais/terapia , Neoplasias Hepáticas/terapia , Linfócitos T/imunologia , alfa-Fetoproteínas/genética , Animais , Citotoxicidade Imunológica , Feminino , Vetores Genéticos , Humanos , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas Experimentais/imunologia , Linfoma/imunologia , Complexo Principal de Histocompatibilidade , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/imunologia , Transcrição Gênica , alfa-Fetoproteínas/imunologia
15.
Cancer Res ; 59(13): 3134-42, 1999 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-10397256

RESUMO

Alpha-fetoprotein (AFP) is often derepressed in human hepatocellular carcinoma. Peptide fragments of AFP presented in the context of major histocompatibility molecules could serve as potential recognition targets by CD8 T cells, provided these lymphocytes were not clonally deleted in ontogeny. We therefore wished to determine whether the human T-cell repertoire could recognize AFP-derived peptide epitopes in the context of a common class I allele, HLA-A2.1. Dendritic cells genetically engineered to express AFP were capable of generating AFP-specific T-cell responses in autologous human lymphocyte cultures and in HLA-A2.1/Kb transgenic mice. These T cells recognize a 9-mer peptide derived from the AFP protein hAFP(542-550) (GVALQTMKQ). Identified as a potential A2.1-restricted peptide epitope from a computer analysis of the AFP sequence, hAFP(542-550) proved to have low binding affinity to A2.1, but slow off-kinetics. AFP-specific CTL- and IFN-gamma-producing cells recognize hAFP(542-550)-pulsed targets. Conversely, hAFP(542-550) peptide-generated T cells from both human lymphocyte cultures and A2.1/Kb transgenic mice recognized AFP-transfected targets in both cytotoxicity assays and cytokine release assays. These lines of evidence clearly demonstrate that AFP-reactive clones have not been deleted from the human T-cell repertoire and identify one immunodominant A2.1-restricted epitope. These findings also clearly establish AFP as a potential target for T-cell-based immunotherapy.


Assuntos
Citotoxicidade Imunológica , Epitopos/farmacologia , Antígeno HLA-A2/imunologia , Linfócitos T/imunologia , alfa-Fetoproteínas/imunologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Células Cultivadas , Células Dendríticas/imunologia , Epitopos/química , Antígeno HLA-A2/genética , Humanos , Camundongos , Camundongos Transgênicos , Proteínas Recombinantes/imunologia , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Transfecção , alfa-Fetoproteínas/química , alfa-Fetoproteínas/farmacologia
16.
Anticancer Res ; 19(2A): 1165-70, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10368670

RESUMO

Peptides extracted from tumor cells after mild acid treatment can function as antigenic epitopes when presented by cultured dendritic cells. Peptides were extracted from four tumors syngeneic to C3H mice, three weakly immunogenic tumors (FSA, MCAK, HCA) and one non-immunogenic tumor (NFSA). Dendritic cells pulsed with peptides extracted from the three weakly immunogenic tumors partially protect mice from a tumor challenge with the parental cell line. This protection was evident by a slower rate of tumor appearance and a slower tumor growth curve when compared to control, non-immunized mice. However, vaccination of mice with dendritic cells pulsed with peptides derived from the non-immunogenic cell line NFSA did not elicit a protective response. Neither the route of immunization, the number of immunizations, nor the amount of peptides significantly affected the antitumor protection. Dendritic cells genetically engineered to produce IL-2 did not increase the protective effect of peptide-pulsed dendritic cells. These results suggest that only a partial protection against immunogenic tumors can be achieved when dendritic cells pulsed with acid-eluted tumor peptides are used as antitumor vaccination.


Assuntos
Células Dendríticas/imunologia , Proteínas de Neoplasias/imunologia , Neoplasias Experimentais/terapia , Ácidos , Animais , Feminino , Imunização , Interleucina-2/biossíntese , Camundongos , Camundongos Endogâmicos C3H , Neoplasias Experimentais/imunologia
17.
Cancer Res ; 58(23): 5305-9, 1998 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-9850054

RESUMO

Both CD8+ and CD4+ T cells have demonstrated roles in antitumor immune response in many animal tumor systems. In many human tumor systems, although abundant literature exists on the evidence of tumor antigen-specific CD8+ CTL response, only limited information is available on tumor antigen-specific CD4+ T-cell response. Using the MART-1/Melan-A (MART-1) antigen system as a prototype human tumor-associated antigen (TAA)- and dendritic cell (DC)-based MART-1 antigen presentation system (i.e., DCs transduced with an adenoviral vector-based construct carrying the MART-1 gene), we explored, in vitro, the feasibility of generating both CD8+ and CD4+ T-cell responses in the same individual. Here, we show that autologous DCs from both HLA-A2-positive melanoma patients and normal healthy individuals that are transduced with an adenoviral vector containing the MART-1 antigen are capable of inducing both MART-1-specific CD8+ and CD4+ T cells in in vitro coculture. After several rounds of stimulation, both the CD4+ and CD8+ T cells synthesized IFN-gamma when they were specifically stimulated. The CD8+ T cells generated in such cocultures also recognized the MART-1(27-35) peptide, AAGIGILTV, in 4-h cytotoxicity assays. These observations, therefore, suggest that Th1-type responses can be generated, in vitro, by stimulation with DCs that are genetically modified to express a TAA. Although the outcome of this type of genetically engineered DC-based stimulation may vary from system to system, this type of in vitro antigen presentation may be very useful in more comprehensive analyses of CD4+ T-cell response to defined TAAs, and such genetically engineered autologous DCs might be better candidates to serve as surrogate cancer vaccines.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Proteínas de Neoplasias/imunologia , Antígenos de Neoplasias , Comunicação Celular/fisiologia , Células Dendríticas/metabolismo , Células Dendríticas/fisiologia , Epitopos/imunologia , Epitopos de Linfócito T/imunologia , Humanos , Ativação Linfocitária/fisiologia , Antígeno MART-1 , Melanoma/imunologia , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Transdução Genética
18.
J Immunol ; 161(10): 5607-13, 1998 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-9820539

RESUMO

Dendritic cells (DC) are potent stimulators of primary T cell responses. In this study, we demonstrate that DC, genetically engineered to express the MART-1/Melan-A (MART-1) tumor-associated Ag, express MART-1 mRNA and protein, correctly process and present the HLA-A2.1-restricted immunodominant MART-1 peptide (MART-1(27-35)), and serve as potent stimulators of MART-1-specific CTL in vitro. A replication-defective E1-deleted adenovirus (AdV) was constructed that expresses MART-1 (AdVMART1). Transduced DC produce full length MART-1 mRNA as well as MART-1 protein. AdVMART1 does not significantly down-regulate cell surface class I expression despite having an intact E3 region. Transduction of an HLA-A2-positive/MART-1-negative cell line with AdVMART1 renders these cells sensitive to lysis by CTL specific for the MART-1(27-35) immunodominant peptide. In addition, DC transduced with AdVMART1 stimulated MART-1(27-35)-specific tumor-infiltrating lymphocytes to synthesize IFN-gamma. Finally, AdVMART1-transduced DC were able to generate MART-1(27-35) peptide-specific, class I-restricted CTL in PBL cultures from normal donors. This study supports the use of tumor Ag-engineered DC in genetic immunotherapy.


Assuntos
Adenoviridae/genética , Antígenos de Neoplasias/imunologia , Células Dendríticas/imunologia , Epitopos de Linfócito T/imunologia , Melanoma/imunologia , Proteínas de Neoplasias/imunologia , Linfócitos T Citotóxicos/imunologia , Adenoviridae/imunologia , Antígenos de Neoplasias/genética , Células Clonais , Citotoxicidade Imunológica , Células Dendríticas/metabolismo , Epitopos/imunologia , Epitopos de Linfócito T/genética , Engenharia Genética , Antígenos de Histocompatibilidade Classe I/biossíntese , Humanos , Linfócitos do Interstício Tumoral/imunologia , Antígeno MART-1 , Proteínas de Neoplasias/genética , Subpopulações de Linfócitos T/imunologia
19.
Anticancer Res ; 18(3A): 1357-60, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-9673340

RESUMO

BACKGROUND: E-selectin expression is very low in normal adult blood vessels, but is significantly elevated in newly formed tumor capillaries. We hypothesized that a viral vector which has transcriptional specificity for the tumor vasculature may be a tool for angiogenesis-targeted gene therapy. We therefore designed an adenoviral vector which would only be expressed in cells that transcribe the E-selectin gene. MATERIALS AND METHODS: The E-selectin promoter was inserted into an adenoviral vector driven by the luciferase reporter gene. The resulting AdV-Esel-Luc vector was then used to transduce endothelial cells as well as other cell types, and luciferase activity measured with a luminometric assay. RESULTS: Exposure of transduced endothelial cells to TNF-alpha (tumor necrosis factor-alpha), a known inducer of the E-selectin promoter, generated a 30-fold increase in luciferase expression compared to untreated cells (p = 0.01). Endothelial cells cultured in tumor conditioned media as an in vitro recreation of the tumor environment resulted in even higher induction of luciferase (p = 0.0001). Furthermore, many non-endothelial cell lines expressed minimal levels of luciferase when transduced with the AdV-Esel-Luc vector under identical conditions. CONCLUSIONS: We conclude that the E-selectin promoter can be used in order to confer transcriptional specificity to an adenoviral vector. This transcriptional specificity may in the future enable us to deliver the specific expression of therapeutic genes to the tumor vasculature.


Assuntos
Selectina E/biossíntese , Selectina E/genética , Endotélio Vascular/metabolismo , Regiões Promotoras Genéticas , Adenoviridae , Adulto , Neoplasias da Mama , Células Cultivadas , Neoplasias do Colo , Feminino , Genes Reporter , Vetores Genéticos , Humanos , Luciferases/biossíntese , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Recombinantes de Fusão/biossíntese , Transfecção , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologia , Veias Umbilicais
20.
Cancer ; 82(9): 1704-8, 1998 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-9576292

RESUMO

BACKGROUND: The sentinel lymph node is defined as the first lymph node to receive drainage from a primary tumor. Based on this concept, the authors set out to evaluate whether the status of the sentinel lymph node can accurately predict whether breast tumor cells have metastasized to the axillary lymphatic basin. METHODS: Radiolabeled dextran was injected into the site of the breast tumor. In the operating room, a portable gamma detector probe was used to identify the exact location of the sentinel lymph node(s). After identifying and excising the radioactive sentinel lymph node specimens, a routine axillary lymph node dissection was performed. All lymph nodes were then subjected to hematoxylin and eosin (H & E) staining, and the sentinel lymph nodes were subjected to additional cytokeratin immunohistochemistry. RESULTS: Of the 41 patients who participated in the study, 18 had tumor metastasis to their axillary lymph nodes. In all 18 of these cases, the sentinel lymph node(s) contained cancer detected by either H & E staining or cytokeratin immunohistochemistry. CONCLUSIONS: The status of the sentinel lymph node(s) appears to predict accurately whether breast tumor cells have metastasized to the axillary lymphatic basin. This new, minimally invasive technique for staging breast carcinoma should be further validated in a large, multi-institutional clinical trial.


Assuntos
Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/patologia , Dextranos , Linfonodos/diagnóstico por imagem , Linfonodos/patologia , Compostos de Organotecnécio , Compostos Radiofarmacêuticos , Feminino , Humanos , Linfonodos/cirurgia , Metástase Linfática , Estadiamento de Neoplasias , Cintilografia
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