RESUMO
Previous work in our laboratory has established that the fusion of tumor-associated antigens to a truncated form of the Listeria monocytogenes virulence factor listeriolysin O (LLO) enhances the immunogenicity and antitumor efficacy of the tumor antigen when delivered by Listeria or by vaccinia. LLO contains a PEST sequence at the NH(2) terminus. These sequences, which are found in eukaryotic proteins with a short cellular half-life, target proteins for degradation in the ubiquitin-proteosome pathway. To investigate whether the enhanced immunogenicity conferred by LLO is due to the PEST sequence, we constructed new Listeria recombinants that expressed the HPV-16 E7 antigen fused to LLO, which either contained or had been deleted of this sequence. We then compared the antitumor efficacy of this set of vectors and found that Listeria expressing the fusion protein LLO-E7 or PEST-E7 were effective at regressing established macroscopic HPV-16 immortalized tumors in syngeneic mice. In contrast, Listeria recombinants expressing E7 alone or E7 fused to LLO from which the PEST sequence had been genetically removed could only slow tumor growth. Because CD8(+) T cell epitopes are generated in the ubiquitin-proteosome pathway, we also investigated the ability of the vaccines to induce E7-specific CD8(+) T cells in the spleen and to generate E7-specific tumor-infiltrating lymphocytes. A strong correlation was observed between CD8(+) T-cell induction and tumor homing and the antitumor efficacy of the Listeria-E7 vaccines. These findings suggest a strategy for the augmentation of tumor antigen-based immunotherapeutic strategies that may be broadly applicable.
Assuntos
Toxinas Bacterianas/imunologia , Vacinas Anticâncer/imunologia , Proteínas de Choque Térmico/imunologia , Listeria monocytogenes/imunologia , Proteínas Oncogênicas Virais/imunologia , Vacinas contra Papillomavirus , Sequência de Aminoácidos , Animais , Toxinas Bacterianas/genética , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/genética , Linhagem Celular Tumoral , Epitopos , Epitopos de Linfócito T/imunologia , Proteínas de Choque Térmico/genética , Proteínas Hemolisinas , Listeria monocytogenes/genética , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/terapia , Neoplasias Experimentais/virologia , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Baço/citologia , Baço/imunologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
PURPOSE: Many human tumors lose responsiveness to IFN-gamma, providing a possible mechanism for the tumor to avoid immune recognition and destruction. Here we investigate the importance of tumor responsiveness to IFN-gamma in the successful immunotherapy of TC1 tumors that were immortalized with human papillomavirus proteins E6 and E7. METHODS: To investigate the role of IFN-gamma in vivo, we constructed a variant of TC1, TC1.mugR, that is unresponsive to IFN-gamma due to overexpression of a dominant negative IFN-gamma receptor. RESULTS: Using recombinant Listeria monocytogenes that express HPV-16 E7 (Lm-LLO-E7) to stimulate an antitumor response, we demonstrate that sensitivity to IFN-gamma is required for therapeutic efficacy in that Lm-LLO-E7 induces regression of TC1 tumors but not TC1.mugR. In addition, we show that tumor sensitivity to IFN-gamma is not required for inhibition of tumor angiogenesis by Lm-LLO-E7 or for trafficking of CD4+ and CD8+ T cells to the tumor. However, it is required for penetration of lymphocytes into the tumor mass in vivo. CONCLUSIONS: Our findings identify a role for IFN-gamma in immunity to TC1 tumors and show that loss of tumor responsiveness to IFN-gamma poses a challenge to antigen-based immunotherapy.
Assuntos
Vacinas Anticâncer/uso terapêutico , Interferon gama/fisiologia , Neoplasias Experimentais/tratamento farmacológico , Infecções por Papillomavirus/tratamento farmacológico , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Vacinas Bacterianas/uso terapêutico , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Transformação Celular Viral , Modelos Animais de Doenças , Feminino , Listeria monocytogenes/genética , Listeria monocytogenes/imunologia , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Neoplasias Experimentais/genética , Neoplasias Experimentais/imunologia , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/imunologia , Papillomaviridae/genética , Proteínas E7 de Papillomavirus , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/imunologia , Receptores de Interferon/genética , Receptores de Interferon/metabolismo , Linfócitos T Citotóxicos/imunologia , Ativação Transcricional , Evasão Tumoral/imunologia , Neoplasias do Colo do Útero/virologiaRESUMO
Five overlapping fragments of rat HER-2/neu have been expressed in recombinant Listeria monocytogenes. Each fragment of HER-2/neu is secreted as a fusion protein with a truncated, nonhemolytic form of listeriolysin O (LLO). Lm-LLO-EC1, Lm-LLO-EC2, and Lm-LLO-EC3 overlap the extracellular domain of HER-2/neu, whereas Lm-LLO-IC1 and Lm-LLO-IC2 span the intracellular domain. All five strains controlled the growth of established NT-2 tumors, a rat HER-2/neu-expressing tumor line derived from a spontaneously arising mammary tumor in a FVB/N HER-2/neu-transgenic mouse. The antitumor effect of each of these vaccine constructs was abrogated by the in vivo depletion of CD8(+) T cells, although only one known epitope has been defined previously and is present in Lm-LLO-EC2. Anti-HER-2/neu CTL responses were generated by each of the rLm vaccine constructs. With the use of a panel of 3T3 cell lines expressing overlapping fragments of HER-2/neu, regions of HER-2/neu with potential CD8(+) T cell epitopes have been defined. DNA vaccines expressing either a fragment or full-length HER-2/neu were constructed in LLO-fused and non-LLO-fused forms. CTL analysis of the DNA vaccines revealed a broadening in the regions of HER-2/neu recognizable as targets when the target Ag is fused to LLO. These studies show the efficacy of L. monocytogenes-based HER-2/neu vaccines in a murine model of breast cancer and also that the immunogenicity of self-Ags can be increased by fusion to LLO and delivery by L. monocytogenes revealing subdominant epitopes.