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1.
Oncogene ; 37(4): 439-449, 2018 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-28967901

RESUMEN

Toll-like receptor 5 (TLR5) is considered an attractive target for anticancer immunotherapy. TLR5 agonists, bacterial flagellin and engineered flagellin derivatives, have been shown to have potent antitumor and metastasis-suppressive effects in multiple animal models and to be safe in both animals and humans. Anticancer efficacy of TLR5 agonists stems from TLR5-dependent activation of nuclear factor-κB (NF-κB) that mediates innate and adaptive antitumor immune responses. To extend application of TLR5-targeted anticancer immunotherapy to tumors that do not naturally express TLR5, we created an adenovirus-based vector for intratumor delivery, named Mobilan that drives expression of self-activating TLR5 signaling cassette comprising of human TLR5 and a secreted derivative of Salmonella flagellin structurally analogous to a clinical stage TLR5 agonist, entolimod. Co-expression of TLR5 receptor and agonist in Mobilan-infected cells established an autocrine/paracrine TLR5 signaling loop resulting in constitutive activation of NF-κB both in vitro and in vivo. Injection of Mobilan into primary tumors of the prostate cancer-prone transgenic adenocarcinoma of the mouse prostate (TRAMP) mice resulted in a strong induction of multiple genes involved in inflammatory responses and mobilization of innate immune cells into the tumors including neutrophils and NK cells and suppressed tumor progression. Intratumoral injection of Mobilan into subcutaneously growing syngeneic prostate tumors in immunocompetent hosts improved animal survival after surgical resection of the tumors, by suppression of tumor metastasis. In addition, vaccination of mice with irradiated Mobilan-transduced prostate tumor cells protected mice against subsequent tumor challenge. These results provide proof-of-concept for Mobilan as a tool for antitumor vaccination that directs TLR5-mediated immune response toward cancer cells and does not require identification of tumor antigens.


Asunto(s)
Adenoviridae/genética , Vacunas contra el Cáncer/uso terapéutico , Vectores Genéticos/uso terapéutico , FN-kappa B/inmunología , Neoplasias de la Próstata/terapia , Receptor Toll-Like 5/metabolismo , Adyuvantes Inmunológicos/genética , Adyuvantes Inmunológicos/metabolismo , Adyuvantes Inmunológicos/uso terapéutico , Animales , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Humanos , Inmunoterapia/métodos , Inyecciones Intralesiones , Células Asesinas Naturales , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Péptidos/genética , Péptidos/inmunología , Péptidos/metabolismo , Cultivo Primario de Células , Próstata/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/mortalidad , Transducción de Señal/inmunología , Receptor Toll-Like 5/agonistas , Receptor Toll-Like 5/genética , Receptor Toll-Like 5/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto
2.
Protein Expr Purif ; 21(3): 456-61, 2001 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-11281721

RESUMEN

An approach to preparative production of polypeptides, including uneasily testable, degradable, and toxic ones, is proposed on the basis of in vitro expression systems of last generation, such as continuous-exchange cell-free and continuous-flow cell-free transcription-translation systems. The approach implies that a polypeptide of interest is synthesized as a fusion protein with the polypeptide linked to green fluorescent protein (GFP) through a cleavable spacer. The GFP moiety provides direct visualization and quantitative monitoring of the polypeptide synthesis, as well as solubility and stability of the product. The synthesis of functionally active antibacterial polypeptide cecropin P1 (31 amino acid residues) has been demonstrated.


Asunto(s)
Antibacterianos/biosíntesis , Antiinfecciosos/metabolismo , Péptidos , Secuencia de Aminoácidos , Antibacterianos/química , Antiinfecciosos/química , Secuencia de Bases , Sistema Libre de Células , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Secuencias Repetitivas de Aminoácido/genética , Secuencias Repetidas en Tándem/genética , Transcripción Genética
3.
Biochem Biophys Res Commun ; 280(3): 914-7, 2001 Jan 26.
Artículo en Inglés | MEDLINE | ID: mdl-11162611

RESUMEN

HIV-1 antigen Nef, Green Fluorescent Protein (GFP), and the fusion protein GFP-Nef ("Green Fluorescent Nef") were synthesized in bacterial cell-free system with continuous supply of substrates and continuous removal of low-molecular-weight products through a dialysis membrane during incubation, the so-called continuous-exchange cell-free (CECF) system. The identity of synthesized proteins was confirmed by electrophoretic mobility, Western blotting, and GFP fluorescence. The system produced several nanomoles (hundreds of microg) of each protein per ml of the reaction mixture. Construction of GFP-fused proteins is considered as a general strategy for visualization and monitoring of cell-free production of the proteins that have no easily testable functions.


Asunto(s)
Antígenos Virales/biosíntesis , Productos del Gen nef/biosíntesis , Productos del Gen nef/inmunología , VIH-1/inmunología , VIH-1/metabolismo , Antígenos Virales/genética , Western Blotting , Diálisis , Escherichia coli/genética , Escherichia coli/metabolismo , Fluorescencia , Productos del Gen nef/genética , Proteínas Fluorescentes Verdes , VIH-1/genética , Cinética , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana
4.
FEBS Lett ; 359(1): 89-92, 1995 Feb 06.
Artículo en Inglés | MEDLINE | ID: mdl-7851538

RESUMEN

Dihydrofolate reductase (DHFR) mRNA was inserted into Q beta phage RNA instead of its coat protein cistron. Translation of this recombinant mRNA in the Escherichia coli cell-free system resulted in the synthesis of DHFR, which was two orders of magnitude higher than that in the case of translation of the control DHFR mRNA. Additionally, it resulted in a significantly enhanced synthesis of Q beta replicase as compared with its synthesis when the original Q beta RNA was used.


Asunto(s)
Allolevivirus/genética , Vectores Genéticos , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Viral/genética , Tetrahidrofolato Deshidrogenasa/genética , Cápside/genética , Escherichia coli/genética , Técnicas de Transferencia de Gen , Genes , Proteínas Recombinantes/biosíntesis , Tetrahidrofolato Deshidrogenasa/biosíntesis
5.
FEBS Lett ; 326(1-3): 264-6, 1993 Jul 12.
Artículo en Inglés | MEDLINE | ID: mdl-8325376

RESUMEN

In order to understand the role of the 3'-terminal untranslated region (3'-UTR) of alfalfa mosaic virus (AlMV) RNA 4 in viral RNA translation we have constructed the RNA derivatives differing in the length of their 3'-terminal portions and expressed them in a wheat germ extract. The result shows that the removal of the 3'-UTR from AlMV RNA 4 causes a lagged RNA translation in the cell-free system as compared with the translation of the full length RNA 4, thus suggesting the involvement of the 3'-UTR in the translation initiation pathway.


Asunto(s)
Virus del Mosaico/genética , Biosíntesis de Proteínas , ARN Viral/química , ARN Viral/metabolismo , Sistema Libre de Células , Cinética , ARN Mensajero/química , ARN Mensajero/metabolismo , Relación Estructura-Actividad , Triticum
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