RESUMEN
SOT101 is a superagonist fusion protein of interleukin (IL)-15 and the IL-15 receptor α (IL-15Rα) sushi+ domain, representing a promising clinical candidate for the treatment of cancer. SOT101 among other immune cells specifically stimulates natural killer (NK) cells and memory CD8+ T cells with no significant expansion or activation of the regulatory T cell compartment. In this study, we showed that SOT101 induced expression of cytotoxic receptors NKp30, DNAM-1 and NKG2D on human NK cells. SOT101 stimulated dose-dependent proliferation and the relative expansion of both major subsets of human NK cells, CD56brightCD16- and CD56dimCD16+, and these displayed an enhanced cytotoxicity in vitro. Using human PBMCs and isolated NK cells, we showed that SOT101 added concomitantly or used for immune cell pre-stimulation potentiated clinically approved monoclonal antibodies Cetuximab, Daratumumab and Obinutuzumab in killing of tumor cells in vitro. The anti-tumor efficacy of SOT101 in combination with Daratumumab was assessed in a solid multiple myeloma xenograft in CB17 SCID mouse model testing several combination schedules of administration in the early and late therapeutic setting of established tumors in vivo. SOT101 and Daratumumab monotherapies decreased with various efficacy tumor growth in vivo in dependence on the advancement of the tumor development. The combination of both drugs showed the strongest anti-tumor efficacy. Specifically, the sequencing of both drugs did not matter in the early therapeutic setting where a complete tumor regression was observed in all animals. In the late therapeutic treatment of established tumors Daratumumab followed by SOT101 administration or a concomitant administration of both drugs showed a significant anti-tumor efficacy over the respective monotherapies. These results suggest that SOT101 might significantly augment the anti-tumor activity of therapeutic antibodies by increasing NK cell-mediated activity in patients. These results support the evaluation of SOT101 in combination with Daratumumab in clinical studies and present a rationale for an optimal clinical dosing schedule selection.
Asunto(s)
Mieloma Múltiple , Subfamilia K de Receptores Similares a Lectina de Células NK , Ratones , Animales , Humanos , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Cetuximab/metabolismo , Linfocitos T CD8-positivos/patología , Ratones SCID , Citotoxicidad Celular Dependiente de Anticuerpos , Células Asesinas Naturales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/metabolismo , Subunidad alfa del Receptor de Interleucina-15/metabolismo , Mieloma Múltiple/patologíaRESUMEN
OBJECTIVES: To improve the storage stability and reusability of various yeast strains and species by immobilization in polyvinyl alcohol (PVA) hydrogel particles. RESULTS: Debaryomyces hansenii, Pichia sorbitophila, Saccharomyces cerevisiae, Yarrowia lipolytica, and Zygosaccharomyces rouxii were immobilized in PVA particles using LentiKats technology and stored in sterile water at 4 °C. The immobilization improved the survival of all species; however, the highest storage stability was achieved for S. cerevisiae and Y. lipolytica which survived more than 1 year, in contrast to free cells that survived for only 3 months. Tests of the reusability of immobilized recombinant laccase-secreting S. cerevisiae revealed that the cells were suitable for repetitive use (55 cycles during 15 months) even after storage in water at 4 °C for 9 months. A suitable method for killing immobilized laccase-secreting cells without affecting the produced enzyme activity was also developed. CONCLUSIONS: The immobilization of yeasts in PVA hydrogel enables long-term, cheap storage with very good cell viability and productivity, thus becoming a promising approach for industrial applications.
Asunto(s)
Células Inmovilizadas , Lacasa/metabolismo , Alcohol Polivinílico/química , Preservación Biológica/métodos , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Biotecnología , Técnicas de Cultivo de Célula , Células Inmovilizadas/citología , Células Inmovilizadas/enzimología , Células Inmovilizadas/metabolismo , Equipo Reutilizado , Lacasa/química , Viabilidad Microbiana , Proteínas Recombinantes/química , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismoRESUMEN
Laccases are enzymes with a broad range of biotechnological applications and have, for example, the ability to oxidize many xenobiotics including synthetic dyes. In order to obtain an efficient laccase for the decolorization of dyes which spoil wastewater from the textile industry, genes encoding three various laccase enzymes were expressed in Saccharomyces cerevisiae. The expression of laccases from ascomycete Myceliophthora thermophila (MtL), and two basidiomycetes Trametes versicolor (TvL) and Trametes trogii (TtL) was optimized via selection of plasmids, promoters, media composition, and cultivation conditions. For the first time, the activity of the three secreted laccases was directly compared with the use of various substrates, including different dyes and a wastewater sample. A strong constitutive ADH1 promoter, minimal growth medium, optimized combination of copper and organic nitrogen source, and low cultivation temperature were shown to significantly increase the yields and relative activities of secreted laccases. Heterologous expression of three fungal laccases was successfully achieved in S. cerevisiae being the highest for MtL and the lowest for TvL. MtL, and particularly TtL, showed the decolorization capacity. This is the first report which compared decolorization of synthetic dyes and wastewater by several recombinant laccases and suggested MtL and TtL to be applicable in the ecofriendly enzymatic treatment of colored industry effluent. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:69-80, 2018.
Asunto(s)
Colorantes/química , Lacasa/química , Saccharomyces cerevisiae/enzimología , Aguas Residuales/química , Medios de Cultivo/química , Lacasa/genética , Nitrógeno/química , Oxidación-Reducción , Saccharomyces cerevisiae/genética , Industria Textil , Trametes/química , Trametes/enzimologíaRESUMEN
Laccases are multi-copper oxidoreductases which catalyze the oxidation of a wide range of substrates during the simultaneous reduction of oxygen to water. These enzymes, originally found in fungi, plants, and other natural sources, have many industrial and biotechnological applications. They are used in the food, textile, pulp, and paper industries, as well as for bioremediation purposes. Although natural hosts can provide relatively high levels of active laccases after production optimization, heterologous expression can bring, moreover, engineered enzymes with desired properties, such as different substrate specificity or improved stability. Hence, diverse hosts suitable for laccase production are reviewed here, while the greatest emphasis is placed on yeasts which are commonly used for industrial production of various proteins. Different approaches to optimize the laccase expression and activity are also discussed in detail here.
Asunto(s)
Lacasa/genética , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/genética , Levaduras/genética , Animales , Bacterias/genética , Hongos/genética , Insectos/citología , Insectos/genética , Lacasa/metabolismo , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
The bacterial adenylate cyclase two-hybrid (BACTH) system has been widely used to characterize protein-protein interactions in the prokaryotic world. This system relies on the interaction-mediated reconstitution of adenylate cyclase activity in Escherichia coli by bringing together two complementary fragments of the catalytic domain of the adenylate cyclase toxin of Bordetella pertussis. A limiting factor in performing large-scale two-hybrid interaction screens with full-length open reading frames (ORFs) is the need to clone each ORF individually into the plasmids used to express the hybrid proteins. The Gateway(®) (GW) cloning system (Life Technologies, Grand Island, NY, USA) partially circumvents this limitation, and we describe here modifications to the BACTH system for compatibility with this recombineering technology. We validated and tested the functionality of the BACTH Gateway (BACTHGW ) system using several models of protein-protein interactions, focusing particularly on those involved in bacterial cell division. We further modified the BACTH plasmids to incorporate a transmembrane (TM) segment downstream of the cyclase fragments to permit analysis of extracytoplasmic protein interactions. This approach was also useful to identify putative TM segments and to experimentally validate bioinformatically identified TM domains. The BACTHGW system will prove a useful addition to the study of protein-protein interactions.
Asunto(s)
Adenilil Ciclasas/genética , Proteínas Bacterianas/genética , Vectores Genéticos/genética , Técnicas del Sistema de Dos Híbridos , Adenilil Ciclasas/metabolismo , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Citoplasma/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Orden Génico , Plásmidos/genética , Unión Proteica , Mapeo de Interacción de Proteínas , Reproducibilidad de los ResultadosRESUMEN
The aim of this work was to construct transgenic plants with increased capabilities to degrade organic pollutants, such as polychlorinated biphenyls. The environmentally important gene of bacterial dioxygenase, the bphC gene, was chosen to clone into a plant of Nicotiana tabacum. The chosen bphC gene encodes 2,3-dihydroxybiphenyl-1,2-dioxygenase, which cleaves the aromatic ring of dihydroxybiphenyl, and we cloned it in fusion with the gene for ß-glucuronidase (GUS), luciferase (LUC) or with a histidine tail. Several genetic constructs were designed and prepared and the possible expression of desired proteins in tobacco plants was studied by transient expression. We used genetic constructs successfully expressing dioxygenase's genes we used for preparation of transgenic tobacco plants by agrobacterial infection. The presence of transgenic DNA , mRNA and protein was determined in parental and the first filial generation of transgenic plants with the bphC gene. Properties of prepared transgenic plants will be further studied.
Asunto(s)
Burkholderiaceae/enzimología , Dioxigenasas/genética , Dioxigenasas/metabolismo , Nicotiana/genética , Plantas Modificadas Genéticamente/genética , Bifenilos Policlorados/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biodegradación Ambiental , Burkholderiaceae/genética , Clonación Molecular , Glucuronidasa/genética , Glucuronidasa/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Luciferasas/genética , Luciferasas/metabolismo , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Nicotiana/enzimología , Nicotiana/metabolismoRESUMEN
Varied therapeutic peptides and proteins represent a rapidly growing part of marketed drugs and have an undisputed place alongside other established therapies. Nevertheless, such biodrugs have several drawbacks that hinder their therapeutic application. These are undesirable physicochemical properties, such as variable solubility, low bioavailability and limited stability. These issues can be overcome by addition of stabilizing agents and directed injectable administration, which can however result in low patient compliance. Hence, there is a drive in the biotechnology industry to produce needle-free and more user-friendly drugs, and this has led to the growth of nano-enabled drug delivery systems in the last decade. As discussed here, nanobiotechnology is becoming a commercially feasible and promising opportunity for oral, pulmonary and transdermal administration routes.