RESUMEN
Fibroblast growth factor receptors (FGFRs) are transmembrane receptor tyrosine kinases that regulate multiple physiological processes. Aberrant activation of FGFR2 and FGFR3 has been linked to the pathogenesis of many tumor types, including cholangiocarcinoma and bladder cancer. Current therapies targeting the FGFR2/3 pathway exploiting small-molecule kinase inhibitors are associated with adverse events due to undesirable inhibition of FGFR1 and FGFR4. Isoform-specific FGFR2 and FGFR3 inhibitors that spare FGFR1 and FGFR4 could offer a favorable toxicity profile and improved therapeutic window to current treatments. Herein we disclose the discovery of dual FGFR2/FGFR3 inhibitors exploiting scaffold repurposing of a previously reported ALK2 tool compound. Structure-based drug design and structure-activity relationship studies were employed to identify selective and orally bioavailable inhibitors with equipotent activity toward wild-type kinases and a clinically observed gatekeeper mutant.
RESUMEN
Upregulation of the fibroblast growth factor receptor (FGFR) signaling pathway has been implicated in multiple cancer types, including cholangiocarcinoma and bladder cancer. Consequently, small molecule inhibition of FGFR has emerged as a promising therapy for patients suffering from these diseases. First-generation pan-FGFR inhibitors, while highly effective, suffer from several drawbacks. These include treatment-related hyperphosphatemia and significant loss of potency for the mutant kinases. Herein, we present the discovery and optimization of novel FGFR2/3 inhibitors that largely maintain potency for the common gatekeeper mutants and have excellent selectivity over FGFR1. A combination of meticulous structure-activity relationship (SAR) analysis, structure-based drug design, and medicinal chemistry rationale ultimately led to compound 29, a potent and selective FGFR2/3 inhibitor with excellent in vitro absorption, distribution, metabolism, excretion (ADME), and pharmacokinetics in rat. A pharmacodynamic study of a closely related compound established that maximum inhibition of downstream ERK phosphorylation could be achieved with no significant effect on serum phosphate levels relative to vehicle.
Asunto(s)
Neoplasias , Inhibidores de Proteínas Quinasas , Receptores de Factores de Crecimiento de Fibroblastos , Animales , Ratas , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/química , Transducción de Señal , Relación Estructura-Actividad , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptores de Factores de Crecimiento de Fibroblastos/química , Receptores de Factores de Crecimiento de Fibroblastos/efectos de los fármacosRESUMEN
Second generation anthrax vaccines focus on the use of recombinant protective antigen (rPA) to elicit a strong, toxin neutralizing antibody responses in immunized subjects. The main difference between the rPA vaccines compared to the current licensed vaccine, anthrax vaccine absorbed (AVA), is the rPA vaccines are highly purified preparations of only rPA. These second generation rPA vaccines strive to elicit strong immune responses with substantially fewer doses than AVA while provoking less side effects. Many of the rPA candidates have shown to be effective in pre-clinical studies, but most of the second generation molecules have stability issues which reduce their efficacy over time. These stability issues are evident even under refrigerated conditions and thus emphasis has been directed to stabilizing the rPA molecule and determining an optimized final formulation. Stabilization of vaccines for long-term storage is a major challenge in the product development life cycle. The effort required to identify suitable formulations can be slow and expensive. The ideal storage for stockpiled vaccines would allow the candidate to withstand years of storage at ambient temperatures. The Fraunhofer Center for Molecular Biotechnology is developing a plant-produced rPA vaccine candidate that shows instability when stored under refrigerated conditions in a solution, as is typical for rPA vaccines. Increased stability of our plant-produced rPA vaccine candidate was achieved in a spray dried powder formulation that could eliminate the need for conventional cold chain allowing greater confidence to stockpile vaccine for civilian and military biodefense.
Asunto(s)
Vacunas contra el Carbunco/sangre , Plantas/química , Vacunas Sintéticas/química , Animales , Carbunco/inmunología , Vacunas contra el Carbunco/inmunología , Anticuerpos Antibacterianos , Anticuerpos Neutralizantes/inmunología , Antígenos Bacterianos/inmunología , Bacillus anthracis/inmunología , Toxinas Bacterianas/inmunología , Química Farmacéutica/métodos , Estabilidad de Medicamentos , Almacenaje de Medicamentos/métodos , Inmunización/métodos , Ratones , Ratones Endogámicos BALB C , Polvos/química , Vacunas Sintéticas/inmunologíaRESUMEN
Application of tools of molecular biology and genomics is increasingly leading towards the development of recombinant protein-based biologics. As such, it is leading to an increased diversity of targets that have important health applications and require more flexible approaches for expression because of complex post-translational modifications. For example, Plasmodium parasites may have complex post-translationally modified proteins such as Pfs48/45 that do not carry N-linked glycans (Exp. Parasitol. 1998; 90, 165.) but contain potential N-linked glycosylation sites that can be aberrantly glycosylated during expression in mammalian and plant systems. Therefore, it is important to develop strategies for producing non-glycosylated forms of these targets to preserve biological activity and native conformation. In this study, we are describing in vivo deglycosylation of recombinant N-glycosylated proteins as a result of their transient co-expression with bacterial PNGase F (Peptide: N-glycosidase F). In addition, we show that the recognition of an in vivo deglycosylated plant-produced malaria vaccine candidate, Pfs48F1, by monoclonal antibodies I, III and V raised against various epitopes (I, III and V) of native Pfs48/45 of Plasmodium falciparum, was significantly stronger compared to that of the glycosylated form of plant-produced Pfs48F1. To our knowledge, neither in vivo enzymatic protein deglycosylation has been previously achieved in any eukaryotic system, including plants, nor has bacterial PNGase F been expressed in the plant system. Thus, here, we report for the first time the expression in plants of an active bacterial enzyme PNGase F and the production of recombinant proteins of interest in a non-glycosylated form.
Asunto(s)
Bacterias/enzimología , Biotecnología/métodos , Nicotiana/metabolismo , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Proteínas Recombinantes/biosíntesis , Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/inmunología , Toxinas Bacterianas/inmunología , Western Blotting , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Glicosilación , Espectrometría de Masas , Mapeo Peptídico , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/aislamiento & purificación , Plantas Modificadas Genéticamente , Plasmodium falciparum/metabolismo , Polisacáridos/metabolismo , Unión Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , SolubilidadRESUMEN
BACKGROUND: Influenza virus is a globally important respiratory pathogen that causes a high degree of annual morbidity and mortality. Significant antigenic drift results in emergence of new, potentially pandemic, virus variants. The best prophylactic option for controlling emerging virus strains is to manufacture and administer pandemic vaccines in sufficient quantities and to do so in a timely manner without impacting the regular seasonal influenza vaccine capacity. Current, egg-based, influenza vaccine production is well established and provides an effective product, but has limited capacity and speed. OBJECTIVES: To satisfy the additional global demand for emerging influenza vaccines, high-performance cost-effective technologies need to be developed. Plants have a potential as an economic and efficient large-scale production platform for vaccine antigens. METHODS: In this study, a plant virus-based transient expression system was used to produce hemagglutinin (HA) proteins from the three vaccine strains used during the 2008-2009 influenza season, A/Brisbane/59/07 (H1N1), A/Brisbane/10/07 (H3N2), and B/Florida/4/06, as well as from the recently emerged novel H1N1 influenza A virus, A/California/04/09. RESULTS: The recombinant plant-based HA proteins were engineered and produced in Nicotiana benthamiana plants within 2 months of obtaining the genetic sequences specific to each virus strain. These antigens expressed at the rate of 400-1300 mg/kg of fresh leaf tissue, with >70% solubility. Immunization of mice with these HA antigens induced serum anti-HA IgG and hemagglutination inhibition antibody responses at the levels considered protective against these virus infections. CONCLUSIONS: These results demonstrate the feasibility of our transient plant expression system for the rapid production of influenza vaccine antigens.
Asunto(s)
Antígenos Virales/genética , Expresión Génica , Vacunas contra la Influenza/genética , Gripe Humana/inmunología , Nicotiana/genética , Animales , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H3N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/prevención & control , Gripe Humana/virología , Ratones , Ratones Endogámicos BALB C , Orthomyxoviridae/genética , Orthomyxoviridae/inmunología , Nicotiana/metabolismoRESUMEN
The global spread of highly pathogenic avian influenza virus (H5N1 subtype) has promoted efforts to develop human vaccines against potential pandemic outbreaks. However, current platforms for influenza vaccine production are cumbersome, limited in scalability and often require the handling of live infectious virus. We describe the production of hemagglutinin from the A/Indonesia/05/05 strain of H5N1 influenza virus by transient expression in plants, and demonstrate the immunogenicity and protective efficacy of the vaccine candidate in animal models. Immunization of mice and ferrets with plant-derived hemagglutinin elicited serum hemagglutinin-inhibiting antibodies and protected the ferrets against challenge infection with a homologous virus. This demonstrates that plant-derived H5 HA is immunogenic in mice and ferrets, and can induce protective immunity against infection with highly pathogenic avian influenza virus. Plants could therefore be suitable as a platform for the rapid, large-scale production of influenza vaccines in the face of a pandemic.
Asunto(s)
Hemaglutininas Virales/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Plantas Modificadas Genéticamente , Animales , Anticuerpos Antivirales/sangre , Peso Corporal , Hurones , Pruebas de Inhibición de Hemaglutinación , Hemaglutininas Virales/genética , Hemaglutininas Virales/aislamiento & purificación , Humanos , Subtipo H5N1 del Virus de la Influenza A/genética , Vacunas contra la Influenza/genética , Gripe Humana/prevención & control , Masculino , Ratones , Ratones Endogámicos BALB C , Análisis de Supervivencia , Viremia/prevención & controlRESUMEN
Influenza is a globally important respiratory pathogen that causes a high degree of morbidity and mortality annually. Although current vaccines are effective against virus infection, new strategies need to be developed to satisfy the global demand for an influenza vaccine. To address this point, we have engineered and produced the full-length hemagglutinin (HA) protein from the A/Wyoming/03/03 (H3N2) strain of influenza in plants. The antigenicity of this plant-produced HA was confirmed by ELISA and single-radial immunodiffusion (SRID) assays. Immunization of mice with plant-produced HA resulted in HA-specific humoral (IgG1, IgG2a and IgG2b) and cellular (IFNgamma and IL-5) immune responses. In addition, significant serum hemagglutination inhibition (HI) and virus neutralizing (VN) antibody titers were obtained with an antigen dose as low as 5mug. These results demonstrate that plant-produced HA protein is antigenic and can induce immune responses in mice that correlate with protection.
Asunto(s)
Hemaglutininas/inmunología , Vacunas contra la Influenza/inmunología , Plantas/genética , Animales , Anticuerpos Antivirales/biosíntesis , Western Blotting , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática , Pruebas de Inhibición de Hemaglutinación , Hemaglutininas/biosíntesis , Inmunodifusión , Vacunas contra la Influenza/biosíntesis , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Plantas/metabolismo , NicotianaRESUMEN
BACKGROUND: Influenza A viruses are of major concern for public health, causing worldwide epidemics associated with high morbidity and mortality. Vaccines are critical for protection against influenza, but given the recent emergence of new strains with pandemic potential, and some limitations of the current production systems, there is a need for new approaches for vaccine development. OBJECTIVE: To demonstrate the immunogenicity and protective efficacy of plant-produced influenza antigens. Method We engineered, using influenza A/Wyoming/3/03 (H3N2) as a model virus, the stem and globular domains of hemagglutinin (HA) produced in plants as fusions to a carrier protein and used purified antigens with and without adjuvant for ferret immunization. RESULTS: These plant-produced antigens were highly immunogenic and conferred complete protection against infection in the ferret challenge model. The addition of plant-produced neuraminidase was shown to enhance the immune response in ferrets. CONCLUSIONS: Plants can be used as a production vehicle for vaccine development against influenza. Domains of HA can generate protective immune responses in ferrets.
Asunto(s)
Hemaglutininas Virales/inmunología , Subtipo H3N2 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/prevención & control , Animales , Anticuerpos Antivirales/sangre , Temperatura Corporal , Peso Corporal , Ensayo de Inmunoadsorción Enzimática , Femenino , Hurones , Pruebas de Inhibición de Hemaglutinación , Hemaglutininas Virales/genética , Humanos , Subtipo H3N2 del Virus de la Influenza A/genética , Vacunas contra la Influenza/genética , Plantas Modificadas Genéticamente , Índice de Severidad de la Enfermedad , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Esparcimiento de VirusRESUMEN
The current approved vaccine against anthrax is based on protective antigen (PA) of Bacillus anthracis, requires six injections over an 18-month period and has a known history of side effects. Therefore, there is significant effort towards developing an improved vaccine against B. anthracis. Here we separately engineered and expressed domain 4 of PA (PAD4) and domain 1 of lethal factor (LFD1) as fusions to lichenase (LicKM), a thermostable enzyme from Clostridium thermocellum, and transiently expressed these fusions in Nicotiana benthamiana. Plant-produced antigens were combined and immunogenicity was evaluated in mice. All animals that received the experimental vaccine developed high antibody titers that were predominantly IgG1 and were able to neutralize the effects of LeTx in vitro.
Asunto(s)
Vacunas contra el Carbunco/inmunología , Carbunco/prevención & control , Bacillus anthracis/inmunología , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/inmunología , Animales , Carbunco/inmunología , Carbunco/patología , Vacunas contra el Carbunco/administración & dosificación , Vacunas contra el Carbunco/química , Vacunas contra el Carbunco/genética , Toxinas Bacterianas/biosíntesis , Toxinas Bacterianas/genética , Toxinas Bacterianas/inmunología , Inmunización , Ratones , Proteínas Recombinantes/inmunología , Nicotiana/genética , Nicotiana/metabolismo , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/químicaRESUMEN
Historically, most vaccines have been based on killed or live-attenuated infectious agents. Although very successful at immunizing populations against disease, both approaches raise safety concerns and often have limited production capacity. This has resulted in increased emphasis on the development of subunit vaccines. Several recombinant systems have been considered for subunit vaccine manufacture, including plants, which offer advantages both in cost and in scale of production. We have developed a plant expression system utilizing a 'launch vector', which combines the advantageous features of standard agrobacterial binary plasmids and plant viral vectors, to achieve high-level target antigen expression in plants. As an additional feature, to aid in target expression, stability and purification, we have engineered a thermostable carrier molecule to which antigens are fused. We have applied this launch vector/carrier system to engineer and express target antigens from various pathogens, including, influenza A/Vietnam/04 (H5N1) virus.