RESUMO
High-risk human papillomaviruses (HPVs) cause cervical cancer, and while there are good prophylactic vaccines on the market, these are ineffective against established infections, creating a clear need for therapeutic vaccines. The HPV E7 protein is one of the essential oncoproteins for the onset and maintenance of malignancy and is therefore an ideal therapeutic vaccine target. We fused the HPV-16 E7 protein to the Limulus polyphemus antilipopolysaccharide factor (LALF32-51 ), a small hydrophobic peptide that can penetrate cell membranes and that has immunomodulatory properties. LALF32-51 -E7 was transiently expressed in Nicotiana benthamiana, and we previously determined that it accumulated better when targeted to chloroplasts compared to being localized in the cytoplasm. Subsequently, we aimed to prove whether LALF32-51 -E7 was indeed associated with the chloroplasts by determining its subcellular localization. The LALF32-51 -E7 gene was fused to one encoding enhanced GFP to generate a LG fusion protein, and localization was determined by confocal laser scanning microscopy and transmission electron microscopy (TEM). The fluorescence observed from chloroplast-targeted LG was distinctively different from that of the cytoplasmic LG. Small spherical structures resembling protein bodies (PBs) were seen that clearly localized with the chloroplasts. Larger but less abundant PB-like structures were also seen for the cytoplasmic LG. PB-like structure formation was confirmed for both LG and LALF32-51 -E7 by TEM. LALF32-51 -E7 was indeed targeted to the chloroplasts by the chloroplast transit peptide used in this study, and it formed aggregated PB-like structures. This study could open a new avenue for the use of LALF32-51 as a PB-inducing peptide.
Assuntos
Nicotiana/metabolismo , Folhas de Planta/metabolismo , Cloroplastos/efeitos dos fármacos , Papillomavirus Humano 16/imunologia , Papillomavirus Humano 16/metabolismo , Folhas de Planta/genética , Nicotiana/genéticaRESUMO
Novel therapeutic peptides are increasingly making their way into clinical application. The cationic and amphipathic properties of certain peptides allow them to cross biological membranes in a non-disruptive way without apparent toxicity increasing drug bioavailability. By modifying the primary structure of the Limulus-derived LALF(32-51) peptide we designed a novel peptide, L-2, with antineoplastic effect and cell-penetrating capacity. Interestingly, L-2 induced cellular cytotoxicity in a variety of tumor cell lines and systemic injection into immunocompetent and nude mice bearing established solid tumor, resulted in substantial regression of the tumor mass and apoptosis. To isolate the gene transcripts specifically regulated by L-2 in tumor cells, we conducted suppressive subtractive hybridization (SSH) analysis and identified a set of genes involved in biological processes relevant to cancer biology. Our findings describe a novel peptide that modifies the gene expression of the tumor cells and exhibits antitumor effect in vivo, indicating that peptide L-2 is a potential candidate for anticancer therapy.
Assuntos
Alanina/química , Antineoplásicos/farmacologia , Neoplasias Experimentais/patologia , Biblioteca de Peptídeos , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Apoptose , Ciclo Celular , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/isolamento & purificaçãoRESUMO
Cervical cancer is a global public health problem and human papillomavirus (HPV) 16 accounts for approximately 50% of cases worldwide. Although there are several types of HPV therapeutic vaccines in clinical research, there are currently not approved for use in humans. We developed the fusion protein LALF32-51-E7 (hereafter denominated CIGB550-E7) defined by a cell-penetrating peptide linked to an E7 mutein for the treatment of HPV16-associated tumors. We have demonstrated previously the benefit on antitumor response induced by the immunization with CIGB550-E7 admixed with very small size proteoliposomes (VSSP) adjuvant compared with the adjuvant-free immunization. In this study, we obtained a similar antitumor response in mice immunized with CIGB550-E7 admixed with the new adjuvant sVSSP that does not contain any animal-derived product. Also, the immunization with the above mentioned vaccine preparation induced a cell-mediated immune response. Our results are encouraging for the future clinical trials with the vaccine candidate CIGB550-E7+sVSSP.
Assuntos
Papillomavirus Humano 16/patogenicidade , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/uso terapêutico , Adjuvantes Imunológicos , Animais , Peptídeos Penetradores de Células/química , Feminino , Papillomavirus Humano 16/imunologia , Humanos , Imunidade Celular/imunologia , Imunidade Celular/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Papillomavirus/virologia , Vacinas contra Papillomavirus/imunologia , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/prevenção & controle , Neoplasias do Colo do Útero/virologia , VacinaçãoRESUMO
High-risk human papillomaviruses (hr-HPVs) cause cervical cancer, the fourth most common cancer in women worldwide. A HPV-16 candidate therapeutic vaccine, LALF32-51-E7, was developed by fusing a modified E7 protein to a bacterial cell-penetrating peptide (LALF): this elicited both tumour protection and regression in pre-clinical immunization studies. In the current study, we investigated the potential for producing LALF32-51-E7 in a plant expression system by evaluating the effect of subcellular localization and usage of different expression vectors and gene silencing suppressors. The highest expression levels of LALF32-51-E7 were obtained by using a self-replicating plant expression vector and chloroplast targeting, which increased its accumulation by 27-fold compared to cytoplasmic localization. The production and extraction of LALF32-51-E7 was scaled-up and purification optimized by affinity chromatography. If further developed, this platform could potentially allow for the production of a more affordable therapeutic vaccine for HPV-16. This would be extremely relevant in the context of developing countries, where cervical cancer and other HPV-related malignancies are most prevalent, and where the population have limited or no access to preventative vaccines due to their typical high costs.
Assuntos
Papillomavirus Humano 16/química , Nicotiana/genética , Proteínas E7 de Papillomavirus/biossíntese , Vacinas contra Papillomavirus/biossíntese , Peptídeos/metabolismo , Proteínas Recombinantes/biossíntese , Agrobacterium/genética , Agrobacterium/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Expressão Gênica , Inativação Gênica/imunologia , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Papillomavirus Humano 16/imunologia , Humanos , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/imunologia , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/prevenção & controle , Infecções por Papillomavirus/virologia , Vacinas contra Papillomavirus/genética , Vacinas contra Papillomavirus/imunologia , Peptídeos/genética , Peptídeos/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Nicotiana/metabolismo , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/prevenção & controle , Neoplasias do Colo do Útero/virologiaRESUMO
One important goal of cancer immunotherapy is to prevent and treat tumor metastasis. We have previously reported the significant antitumor effect induced by the immunization with our human papillomavirus therapeutic protein-based vaccine (LALF32-51-E7) without adjuvant and admixed with clinically relevant adjuvants in the subcutaneous TC-1 tumor challenge model. In the present study, we evaluated the efficacy of the above mentioned vaccine formulations in controlling the hematogenous spread of TC-1 tumor cells using a more tumourigenic clone named TC-1* and other intravenous injection site less stressful than the tail vein. We generated a lung metastasis model by injecting TC-1* cells into the retro-orbital venous sinus and this is the first study describing it. Also, this is the first study that demonstrates the efficacy of the immunization with LALF32-51-E7 without adjuvant and admixed with VSSP or Al(OH)3 in controlling metastatic tumors increasing the survival of the mice. Our TC-1 lung metastasis model can be used to test the efficacy of other immunotherapeutic strategies based on E6/E7 antigens.
Assuntos
Imunoterapia , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Proteínas E7 de Papillomavirus/imunologia , Vacinas contra Papillomavirus/uso terapêutico , Neoplasias do Colo do Útero/terapia , Animais , Feminino , Vetores Genéticos , Humanos , Neoplasias Pulmonares/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteolipídeos , Linfócitos T Citotóxicos , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/patologiaRESUMO
Protein Kinase (casein kinase 2, CK2) is a serine-threonine kinase that is frequently dysregulated in many human tumors. Therefore we hypothesized that peptides capable of binding to the CK2 acidic domain may exhibit potential anticancer properties. By screening a random cyclic peptide phage display library, we have identified a novel peptide, P15, that abrogated CK2 phosphorylation by blocking the substrate in vitro. To verify its potential antineoplastic effect, P15 was fused to the cell-penetrating peptide derived from the HIV-Tat protein. Interestingly, P15-Tat induced apoptosis as evidenced by rapid caspase activation and cellular cytotoxicity in a variety of tumor cell lines. Furthermore, direct injection of P15-Tat into C57BL6 mice bearing day 7-established solid tumors, resulted in substantial regression of the tumor mass. Our findings describe a new proapoptotic cyclic peptide that blocks the CK2 phosphorylation and exhibits antitumor effect in vivo, indicating that the P15 peptide may potentially be used clinically to treat solid tumors or as an adjuvant for cancer therapy.
Assuntos
Apoptose/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Caseína Quinase II , Linhagem Celular Tumoral , Produtos do Gene tat/genética , Produtos do Gene tat/farmacocinética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Biblioteca de Peptídeos , Peptídeos Cíclicos/genética , Peptídeos Cíclicos/farmacocinética , Fosforilação/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
A fusion protein comprising a cell penetrating and immunostimulatory peptide corresponding to residues 32 to 51 of the Limulus polyphemus protein linked to human papillomavirus (HPV)-16 E7 antigen (LALF32-51-E7) was expressed in E. coli BL21 (DE3) cells. The recombinant protein in E. coli accounted for approximately 18% of the total cellular protein and purified with a single affinity chromatographic step. Yields of approximately 38 mg purified LALF32-51-E7 per liter of induced culture was obtained with an overall 52% recovery and constitutes a promising setting for the future production and scaling-up. Purified protein was characterized as soluble aggregates with molecular weight larger than 670 kDa, which is considered an important property to increase the immunogenicity of an antigen preparation. The recombinant fusion protein LALF32-51-E7 will be a promising vaccine candidate for the treatment of HPV-16 related malignancies.
RESUMO
The ultimate success of cancer vaccination is dependent upon the generation of tumor-specific CTLs. In this study, we designed and evaluated a novel fusion protein comprising a cell penetrating and immunostimulatory peptide corresponding to residues 32-51 of the Limulus polyphemus protein (LALF(32-51)) linked to human papillomavirus (HPV) 16 E7 antigen (LALF(32-51)-E7). We demonstrated that LALF(32-51) penetrates the cell membrane and delivers E7 into cells. In a preclinical model of HPV16-induced cervical carcinoma we showed that vaccination with adjuvant-free LALF(32-51)-E7 fusion protein significantly improves the presentation of E7-derived peptides to T-cells in vitro and induces suppression of tumor growth.
Assuntos
Carcinoma/prevenção & controle , Caranguejos Ferradura/imunologia , Papillomavirus Humano 16/imunologia , Proteínas E7 de Papillomavirus/imunologia , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/imunologia , Neoplasias do Colo do Útero/prevenção & controle , Animais , Carcinoma/imunologia , Modelos Animais de Doenças , Feminino , Caranguejos Ferradura/genética , Papillomavirus Humano 16/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas E7 de Papillomavirus/administração & dosagem , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/imunologia , Vacinas contra Papillomavirus/administração & dosagem , Vacinas contra Papillomavirus/genética , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/imunologia , Neoplasias do Colo do Útero/imunologiaRESUMO
Objective. CIGB-228 is a novel therapeutic vaccine consisting of HLA-restricted HPV16 E7 epitope adjuvated with VSSP. This trial was designed to evaluate the toxicity, safety, immunogenicity, HPV clearance, and lesion regression. Methods. Seven women were entered. All were HLA-A2 positive, had biopsy-proven high-grade CIN, histologically positive for HPV16, and beared persistent postbiopsy lesions visible by digital colposcopy. HLA-A2 women with biopsy-proven high-grade CIN, HPV16-positive, and beared persistent postbiopsy lesions visible by digital colposcopy were vaccinated. One weekly injections of CIGB-228 vaccine was given for four weeks. Then, loop electrosurgical excision procedure (LEEP) of the transformation zone was performed. Study subjects were followed for 1 year after LEEP. Results. No toxicity beyond grade 1 was observed during and after the four vaccinations. Five of seven women had complete and partial regression. Cellular immune response was seen in all patients. HPV was cleared in three of the patients with complete response. Conclusion. CIGB-228 vaccination was well tolerated and capable to induce IFNγ-associated T-cell response in women with high-grade CIN. In several patients, lesion regression and HPV clearance were observed.
RESUMO
Passive immunotherapy against soluble pro-angiogenic factors and/or their receptors in endothelial cells has become a promising approach in cancer therapeutics. There is also experimental evidence indicating that an active immunotherapy strategy directed towards these target molecules could also be effective. In this paper we show that it is possible to reduce tumor growth or increase the survival of tumor-bearing C57Bl/6 mice when animals are vaccinated with the human vascular endothelial growth factor (VEGF) isoform 121 gene (hVEGF(121)), and later challenged with melanoma or lung carcinoma tumor cells. Immunization was done with 10 microg DNA doses of the hVEGF121 gene, which is highly homologous to its mouse counterpart, administered on a weekly basis using a plasmid bearing 5 CpG bacterial motifs. Histopathology analyses of tumors of hVEGF(121) immunized animals showed a decrease in tumor cell density around vessels and in mitotic figures, as well as an increase in apoptotic tumor cells. A statistically significant cell cytotoxic response was found when spleen cells of immunized mice were co-cultured in vitro with mouse tumor VEGF-producing cells. Vaccination with an hVEGF121 gene mutated to make it deficient for VEGF receptor binding, produced similar in vitro and in vivo results, and significantly reduced the number of spontaneous metastases produced by the mouse Lewis lung carcinoma. Our results indicate that human VEGF DNA can be employed for anti-angiogenic active immunotherapy in mice, and that direct cell cytotoxicity is a contributor mechanism to the overall anti-tumor effects seen in immunized animals.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Vacinas Anticâncer/uso terapêutico , Carcinoma Pulmonar de Lewis/prevenção & controle , Melanoma Experimental/prevenção & controle , Vacinas de DNA/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/uso terapêutico , Inibidores da Angiogênese/imunologia , Animais , Vacinas Anticâncer/imunologia , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Carcinoma Pulmonar de Lewis/secundário , Linhagem Celular Tumoral , Feminino , Técnicas de Transferência de Genes , Terapia Genética/métodos , Humanos , Imuno-Histoquímica , Interferon gama/imunologia , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/secundário , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/fisiopatologia , Neovascularização Patológica/prevenção & controle , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Vacinas de DNA/imunologiaRESUMO
Peptide-based vaccines aimed at the induction of effective T-cell responses against established tumors have not been successful in clinic and require the use of new adjuvants. One of those is a new adjuvant in which gangliosides are incorporated into the outer membrane protein complex of Neisseria meningitidis to form very small size proteoliposomes (VSSP). In a preclinical model of human papillomavirus HPV16-induced cervical cancer we show that vaccination with HPV 16 E7 derived minimal CTL peptide and VSSP protects mice against tumor challenge, induces regression of established tumors and produces E7-specific CD8+ T-cell responses.