RESUMO
The development of cancer immunotherapy has long been a challenge. Here, we report that prophylactic vaccination with a highly attenuated Trypanosoma cruzi strain expressing NY-ESO-1 (CL-14-NY-ESO-1) induces both effector memory and effector CD8(+) T lymphocytes that efficiently prevent tumor development. However, the therapeutic effect of such a vaccine is limited. We also demonstrate that blockade of Cytotoxic T Lymphocyte Antigen 4 (CTLA-4) during vaccination enhances the frequency of NY-ESO-1-specific effector CD8(+) T cells producing IFN-γ and promotes lymphocyte migration to the tumor infiltrate. As a result, therapy with CL-14-NY-ESO-1 together with anti-CTLA-4 is highly effective in controlling the development of an established melanoma.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Antígeno CTLA-4/imunologia , Vacinas Anticâncer/imunologia , Imunoterapia/métodos , Melanoma Experimental/terapia , Proteínas de Membrana/imunologia , Animais , Antígenos de Neoplasias/administração & dosagem , Antígenos de Neoplasias/genética , Linfócitos T CD8-Positivos/parasitologia , Antígeno CTLA-4/antagonistas & inibidores , Feminino , Humanos , Melanoma Experimental/imunologia , Melanoma Experimental/parasitologia , Proteínas de Membrana/administração & dosagem , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Trypanosoma cruzi/genética , Trypanosoma cruzi/imunologiaRESUMO
NaPi2b, a sodium-dependent phosphate transporter, is highly expressed in ovarian carcinomas and is recognized by the murine monoclonal antibody MX35. The antibody had shown excellent targeting to ovarian cancer in several early phase clinical trials but being murine the antibody's full therapeutic potential could not be explored. To overcome this impediment we developed a humanized antibody version named Rebmab200, expressed in human PER.C6® cells and cloned by limiting dilution. In order to select a clone with high therapeutic potential clones were characterized using a series of physicochemical assays, flow cytometry, real-time surface plasmon resonance, glycosylation analyses, immunohistochemistry, antibody-dependent cell-mediated cytotoxicity, complement-dependent-cytotoxicity assays and quantitative PCR. Comparative analyses of Rebmab200 and MX35 monoclonal antibodies demonstrated that the two antibodies had similar specificity for NaPi2b by flow cytometry with a panel of 30 cell lines and maintained similar kinetic parameters. Robust and high producer cell clones potentially suitable for use in manufacturing were obtained. Rebmab200 antibodies were assessed by immunohistochemistry using a large panel of tissues including human carcinomas of ovarian, lung, kidney and breast origin. An assessment of its binding towards 33 normal human organs was performed as well. Rebmab200 showed selected strong reactivity with the tested tumor types but little or no reactivity with the normal tissues tested confirming its potential for targeted therapeutics strategies. The remarkable cytotoxicity shown by Rebmab200 in OVCAR-3 cells is a significant addition to the traits of stability and productivity displayed by the top clones of Rebmab200. Antibody-dependent cell-mediated toxicity functionality was confirmed in repeated assays using cancer cell lines derived from ovary, kidney and lung as targets. To explore use of this antibody in clinical trials, GMP production of Rebmab200 has been initiated. As the next step of development, Phase I clinical trials are now planned for translation of Rebmab200 into the clinic.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/antagonistas & inibidores , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/imunologia , Especificidade de Anticorpos/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Proteínas do Sistema Complemento/imunologia , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Cinética , Camundongos , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Ligação Proteica/imunologia , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/imunologia , Ressonância de Plasmônio de SuperfícieRESUMO
Immunological adjuvants that induce T cell-mediate immunity (TCMI) with the least side effects are needed for the development of human vaccines. Glycoinositolphospholipids (GIPL) and CpGs oligodeoxynucleotides (CpG ODNs) derived from the protozoa parasite Trypanosoma cruzi induce potent pro-inflammatory reaction through activation of Toll-Like Receptor (TLR)4 and TLR9, respectively. Here, using mouse models, we tested the T. cruzi derived TLR agonists as immunological adjuvants in an antitumor vaccine. For comparison, we used well-established TLR agonists, such as the bacterial derived monophosphoryl lipid A (MPL), lipopeptide (Pam3Cys), and CpG ODN. All tested TLR agonists were comparable to induce antibody responses, whereas significant differences were noticed in their ability to elicit CD4(+) T and CD8(+) T cell responses. In particular, both GIPLs (GTH, and GY) and CpG ODNs (B344, B297 and B128) derived from T. cruzi elicited interferon-gamma (IFN-γ) production by CD4(+) T cells. On the other hand, the parasite derived CpG ODNs, but not GIPLs, elicited a potent IFN-γ response by CD8(+) T lymphocytes. The side effects were also evaluated by local pain (hypernociception). The intensity of hypernociception induced by vaccination was alleviated by administration of an analgesic drug without affecting protective immunity. Finally, the level of protective immunity against the NY-ESO-1 expressing melanoma was associated with the magnitude of both CD4(+) T and CD8(+) T cell responses elicited by a specific immunological adjuvant.
Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Imunidade Celular/imunologia , Proteínas de Membrana/imunologia , Linfócitos T/imunologia , Trypanosoma cruzi/imunologia , Adjuvantes Imunológicos , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Humanos , Melanoma/imunologia , Melanoma/metabolismo , CamundongosRESUMO
INTRODUCTION: Ovarian adenocarcinoma is frequently detected at the late stage, when therapy efficacy is limited and death occurs in up to 50% of the cases. A potential novel treatment for this disease is a monoclonal antibody that recognizes phosphate transporter sodium-dependent phosphate transporter protein 2b (NaPi2b). MATERIALS AND METHODS: To better understand the expression of this protein in different histologic types of ovarian carcinomas, we immunostained 50 tumor samples with anti-NaPi2b monoclonal antibody MX35 and, in parallel, we assessed the expression of the gene encoding NaPi2b (SCL34A2) by in silico analysis of microarray data. RESULTS: Both approaches detected higher expression of NaPi2b (SCL34A2) in ovarian carcinoma than in normal tissue. Moreover, a comprehensive analysis indicates that SCL34A2 is the only gene of the several phosphate transporters genes whose expression differentiates normal from carcinoma samples, suggesting it might exert a major role in ovarian carcinomas. Immunohistochemical and mRNA expression data have also shown that 2 histologic subtypes of ovarian carcinoma express particularly high levels of NaPi2b: serous and clear cell adenocarcinomas. Serous adenocarcinomas are the most frequent, contrasting with clear cell carcinomas, rare, and with worse prognosis. CONCLUSION: This identification of subgroups of patients expressing NaPi2b may be important in selecting cohorts who most likely should be included in future clinical trials, as a recently generated humanized version of MX35 has been developed.
Assuntos
Adenocarcinoma de Células Claras , Anticorpos Monoclonais Murinos/química , Proteínas de Neoplasias/biossíntese , Neoplasias Ovarianas , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/biossíntese , Adenocarcinoma de Células Claras/metabolismo , Adenocarcinoma de Células Claras/patologia , Idoso , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica/métodos , Pessoa de Meia-Idade , Proteínas de Neoplasias/química , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/químicaRESUMO
One of the main challenges in cancer research is the development of vaccines that induce effective and long-lived protective immunity against tumors. Significant progress has been made in identifying members of the cancer testis antigen family as potential vaccine candidates. However, an ideal form for antigen delivery that induces robust and sustainable antigen-specific T-cell responses, and in particular of CD8(+) T lymphocytes, remains to be developed. Here we report the use of a recombinant nonpathogenic clone of Trypanosoma cruzi as a vaccine vector to induce vigorous and long-term T cell-mediated immunity. The rationale for using the highly attenuated T. cruzi clone was (i) the ability of the parasite to persist in host tissues and therefore to induce a long-term antigen-specific immune response; (ii) the existence of intrinsic parasite agonists for Toll-like receptors and consequent induction of highly polarized T helper cell type 1 responses; and (iii) the parasite replication in the host cell cytoplasm, leading to direct antigen presentation through the endogenous pathway and consequent induction of antigen-specific CD8(+) T cells. Importantly, we found that parasites expressing a cancer testis antigen (NY-ESO-1) were able to elicit human antigen-specific T-cell responses in vitro and solid protection against melanoma in a mouse model. Furthermore, in a therapeutic protocol, the parasites expressing NY-ESO-1 delayed the rate of tumor development in mice. We conclude that the T. cruzi vector is highly efficient in inducing T cell-mediated immunity and protection against cancer cells. More broadly, this strategy could be used to elicit a long-term T cell-mediated immunity and used for prophylaxis or therapy of chronic infectious diseases.