RESUMEN
The p53 gene product is an attractive target for tumor immunotherapy. The present study aims to understand the potential of MVAp53 vaccine to induce expansion of p53-specific cytotoxic T lymphocyte ex vivo in cancer patients. The result indicated that 14 of 23 cancer patients demonstrated p53-specific IFN-γ production, degranulation, cell proliferation, and lysis of p53 overexpressed human tumor cell lines. These experiments show that MVAp53 stimulation has the potential to induce the expansion of p53-specific cytotoxic T lymphocyte from the memory T cell repertoire. The data suggest that MVAp53 vaccine is an ideal candidate for cancer immunotherapy.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Linfocitos T Citotóxicos/inmunología , Proteína p53 Supresora de Tumor/inmunología , Anciano , Anciano de 80 o más Años , Degranulación de la Célula , Línea Celular Tumoral , Proliferación Celular , Citotoxicidad Inmunológica , Femenino , Humanos , Interferón gamma/biosíntesis , Masculino , Persona de Mediana Edad , Linfocitos T Citotóxicos/citología , Vacunas Sintéticas/inmunología , Virus Vaccinia/genéticaRESUMEN
Survivin is overexpressed by 70-80% of pancreatic cancers, and is associated with resistance to chemotherapy and a poor prognosis. Gemcitabine has been a standard treatment for patients with advanced pancreatic cancer for a decade. Recent reports have demonstrated that gemcitabine treatment attenuates the tumor-suppressive environment by eliminating CD11b(+)/Gr-1(+) myeloid-derived suppressor cells (MDSCs). We hypothesize that a cancer vaccine targeting survivin can achieve enhanced efficacy when combined with gemcitabine. In this study, we tested this hypothesis using modified vaccinia Ankara (MVA) expressing full-length murine survivin. The poorly immunogenic mouse pancreas adenocarcinoma cell line, Pan02, which expresses murine survivin and is syngeneic to C57BL/6, was used for this study. Immunization with MVA-survivin resulted in a modest therapeutic antitumor effect on established Pan02 tumors. When administered with gemcitabine, MVA-survivin immunization resulted in significant tumor regression and prolonged survival. The enhanced vaccine efficacy was associated with decreased CD11b(+)/Gr-1(+) MDSCs. To analyze the survivin-specific immune response to MVA-survivin immunization, we utilized a peptide library of 15mers with 11 residues overlapping from full-length murine survivin. Splenocytes from mice immunized with MVA-survivin produced intracellular γ-interferon in response to in vitro stimulation with the overlapping peptide library. Increased survivin-specific CD8(+) T cells that specifically recognized the Pan02 tumor line were seen in mice treated with MVA-survivin and gemcitabine. These data suggest that vaccination with MVA-survivin in combination with gemcitabine represents an attractive strategy to overcome tumor-induced peripheral immune tolerance, and this effect has potential for clinical benefit in pancreatic cancer.
Asunto(s)
Adenocarcinoma/terapia , Antígenos de Neoplasias/metabolismo , Linfocitos T CD8-positivos/metabolismo , Vacunas contra el Cáncer , Proteínas Inhibidoras de la Apoptosis/metabolismo , Neoplasias Pancreáticas/terapia , Fragmentos de Péptidos/metabolismo , Proteínas Represoras/metabolismo , Virus Vaccinia/genética , Adenocarcinoma/inmunología , Adenocarcinoma/patología , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Antígeno CD11b/biosíntesis , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Línea Celular Tumoral , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Vectores Genéticos , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/inmunología , Interferón gamma/genética , Interferón gamma/metabolismo , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Células Mieloides/efectos de los fármacos , Células Mieloides/patología , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Fragmentos de Péptidos/inmunología , Biblioteca de Péptidos , Receptores de Superficie Celular/biosíntesis , Inducción de Remisión , Proteínas Represoras/genética , Proteínas Represoras/inmunología , Survivin , Vacunación , Virus Vaccinia/inmunología , GemcitabinaRESUMEN
The p53 gene product is overexpressed in approximately 50% of cancers, making it an ideal target for cancer immunotherapy. We previously demonstrated that a modified vaccinia Ankara (MVA) vaccine expressing human p53 (MVA-p53) was moderately active when given as a homologous prime/boost in a human p53 knock in (Hupki) mouse model. We needed to improve upon the inefficient homologous boosting approach, because development of neutralizing immunity to the vaccine viral vector backbone suppresses its immunogenicity. To enhance specificity, we examined the combination of 2 different vaccine vectors provided in sequence as a heterologous prime/boost. Hupki mice were evaluated as a human p53 tolerant model to explore the capacity of heterologous p53 immunization to reject human p53-expressing tumors. We employed attenuated recombinant Listeria monocytogenes expressing human p53 (LmddA-LLO-p53) in addition to MVA-p53. Heterologous p53 immunization resulted in a significant increase in p53-specific CD8 and CD4 T cells compared with homologous single vector p53 immunization. Heterologous p53 immunization induced protection against tumor growth but had only a modest effect on established tumors. To enhance the immune response we used synthetic double-strand RNA (polyinsosinic:polycytidylic acid) and unmethylated CpG-containing oligodeoxynucleotide to activate the innate immune system via Toll-like receptors. Treatment of established tumor-bearing Hupki mice with polyinsosinic:polycytidylic acid and CpG-oligodeoxynucleotide in combination with heterologous p53 immunization resulted in enhanced tumor rejection relative to treatment with either agent alone. These results suggest that heterologous prime/boost immunization and Toll-like receptor stimulation increases the efficacy of a cancer vaccine, targeting a tolerized tumor antigen.
Asunto(s)
Adenocarcinoma/inmunología , Adenocarcinoma/terapia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Oligodesoxirribonucleótidos/inmunología , ARN Bicatenario/inmunología , Proteína p53 Supresora de Tumor/inmunología , Adenocarcinoma/patología , Animales , Vacunas contra el Cáncer , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Vectores Genéticos/inmunología , Rechazo de Injerto , Humanos , Inmunización Secundaria , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/patología , Receptores Toll-Like/inmunología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND: The cellular regulatory protein p53 is overexpressed by almost 50% of all malignancies making it an attractive target for a vaccine approach to cancer. A number of immunotherapy approaches targeting p53 have been evaluated successfully in murine models, but translation of these preclinical findings to the clinic has been unsuccessful. Prior studies in our laboratory employing murine models demonstrated that a modified vaccinia virus Ankara (MVA) vaccine expressing murine p53 could stimulate p53 specific immunity. Systemic administration of the MVA vaccine was able to effect the rejection of established tumors. To better understand the immunologic mechanisms that underlie the vaccine function of human p53, we utilized a murine model in which the murine germ line copy of p53 was replaced with a modified human one. These mice, referred to as Hupki, were evaluated as a tolerant model to explore the capacity of MVA expressing human p53 to overcome tolerance and reject human p53-expressing tumors. RESULTS: MVAp53 immunization of Hupki mice resulted in the generation of p53-specific CD8(+) T cells and the rejection of a highly aggressive murine mammary carcinoma cell line 4T1(H-2d) transfected with human p53 (4T1p53). An immunologic correlate of tumor protection was evaluated utilizing an overlapping peptide library spanning the full length of human p53. This reagent was also used in combination with MVAp53 to stimulate p53-specific CD8(+) T cell responses in cancer patients. CONCLUSION: These studies demonstrate the potential of MVAp53 to overcome tolerance to p53 for cancer immunotherapy.
Asunto(s)
Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/uso terapéutico , Tolerancia Inmunológica , Inmunoterapia Activa , Neoplasias Mamarias Experimentales/terapia , Proteína p53 Supresora de Tumor/inmunología , Animales , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/farmacología , Carcinoma de Células Escamosas/inmunología , Células Cultivadas/inmunología , Citotoxicidad Inmunológica , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Neoplasias de Cabeza y Cuello/inmunología , Humanos , Interferón gamma/metabolismo , Activación de Linfocitos , Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias Mamarias Experimentales/inmunología , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/inmunología , Transfección , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The p53 gene product is overexpressed by almost 50% of cancers, making it an ideal target for cancer immunotherapy. We previously demonstrated rejection of established p53-overexpressing tumors without stimulating autoimmunity by immunization with modified vaccinia Ankara-expressing murine p53 (MVAp53). Tumor rejection was enhanced through antibody-mediated CTL-associated antigen 4 (CTLA-4) blockade. We examined the role of synthetic oligodeoxynucleotides (ODN) containing unmethylated cytosine-phosphate-guanine (CpG) motifs (CpG ODN) in enhancing MVAp53-mediated tumor rejection. CpG ODN with MVAp53 resulted in tumor rejection in BALB/c mice bearing poorly immunogenic 11A-1 murine mammary carcinomas or Meth A sarcomas and C57Bl/6 mice bearing MC-38 colon carcinomas. The effect was similar to that seen in tumor-bearing mice treated with MVAp53 along with CTLA-4 blockade. Monoclonal antibody depletion experiments demonstrated that the adjuvant effects of CpG ODN and CTLA-4 blockades were CD8 dependent. CpG ODN were partially natural killer cell dependent and ineffective in Toll-like Receptor 9(-/-) and interleukin 6(-/-) mice, whereas CTLA-4 blockade was partially CD4 dependent and functional in Toll-like Receptor 9(-/-) and interleukin 6(-/-) mice. In addition, when administered with MVAp53, both adjuvants enhanced p53-specific cytotoxicity and demonstrated an additive effect when combined. The combination of CpG ODN and CTLA-4 blockade worked synergistically to reject palpable 11A-1 and MC-38 tumors. These experiments demonstrate the potential for augmenting MVAp53-mediated antitumor immunity using CpG ODN and CTLA-4 blockade. This cell-free immunotherapy approach is a candidate for evaluation in cancer patients.