Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
1.
Bioconjug Chem ; 31(7): 1804-1811, 2020 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-32526138

RESUMEN

Linker design is crucial to the success of antibody-drug conjugates (ADCs). In this work, we developed a modular linker format for attaching molecular cargos to antibodies based on strand pairing between complementary oligonucleotides. We prepared antibody-oligonucleotide conjugates (AOCs) by attaching 18-mer oligonucleotides to an anti-HER2 antibody through thiol-maleimide chemistry, a method generally applicable to any immunoglobulin with interchain disulfide bridges. The hybridization of drug-bearing complementary oligonucleotides to our AOCs was rapid, stoichiometric, and sequence-specific. AOCs loaded with cytotoxic payloads were able to selectively target HER2-overexpressing cell lines such as SK-BR-3 and N87, with in vitro potencies similar to that of the marketed ADC Kadcyla (T-DM1). Our results demonstrated the potential of utilizing AOCs as a highly versatile and modular platform, where a panel of well-characterized AOCs bearing DNA, RNA, or various nucleic acid analogs, such as peptide nucleic acids, could be easily paired with any cargo of choice for a wide range of diagnostic or therapeutic applications.


Asunto(s)
Inmunoconjugados/química , Oligonucleótidos/química , Complejo Antígeno-Anticuerpo , Antineoplásicos/química , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Inmunoglobulina G/inmunología , Maitansina/química , Receptor ErbB-2/inmunología
2.
Blood ; 121(16): 3185-94, 2013 Apr 18.
Artículo en Inglés | MEDLINE | ID: mdl-23430110

RESUMEN

The functional activities of the tumor suppressor promyelocytic leukemia protein (PML) are mostly associated with its nuclear location. In the present study, we discovered an unexpected role of PML in NLRP3 inflammasome activation. In PML-deficient macrophages, the production of IL-1ß was strongly impaired. The expression of pro-IL-1ß, NLRP3, ASC, and procaspase-1 was not affected in Pml(-/-) macrophages. PML deficiency selectively reduced the processing of procaspase-1. We further showed that PML is required for the assembly of the NLRP3 inflammasome in reconstitution experiment. All PML isoforms were capable of stimulating NLRP3 inflammasome activation. In Pml(-/-) macrophages, the generation of reactive oxygen species and release of mitochondrial DNA were decreased. The involvement of PML in inflammasome activation constitutes an important activity of PML and reveals a new mechanism underlying the inflammasome activation. In addition, downregulation of PML by arsenic trioxide suppressed monosodium urate (MSU)-induced IL-1ß production, suggesting that targeting to PML could be used to treat NLRP3 inflammasome-associated diseases.


Asunto(s)
Proteínas Portadoras/inmunología , Inflamasomas/inmunología , Proteínas Nucleares/inmunología , Factores de Transcripción/inmunología , Proteínas Supresoras de Tumor/inmunología , Animales , Trióxido de Arsénico , Arsenicales/farmacología , Proteínas Portadoras/genética , Caspasa 1/inmunología , Línea Celular , Células Cultivadas , ADN Mitocondrial/inmunología , Regulación hacia Abajo/efectos de los fármacos , Eliminación de Gen , Inhibidores de Crecimiento/farmacología , Humanos , Interleucina-1beta/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR , Proteínas Nucleares/genética , Óxidos/farmacología , Proteína de la Leucemia Promielocítica , Especies Reactivas de Oxígeno/inmunología , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética
3.
Protein Sci ; 33(2): e4873, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38111376

RESUMEN

The canine anti-tumor necrosis factor-alpha (TNF-α) monoclonal antibody is a potential therapeutic option for treating canine arthritis. The current treatments for arthritis in dogs have limitations due to side effects, emphasizing the need for safer and more effective therapies. The crystal structure of canine TNF-α (cTNF-α) was successfully determined at a resolution of 1.85 Å, and the protein was shown to assemble as a trimer, with high similarity to the functional quaternary structure of human TNF-α (hTNF-α). Adalimumab (Humira), a known TNF-α inhibitor, effectively targets and neutralizes TNF-α to reduce inflammation and has been used to manage autoimmune conditions such as rheumatoid arthritis. By comparing the structure of cTNF-α with the complex structure of hTNF-α and adalimumab-Fab, the epitope of adalimumab on cTNF-α was identified. The significant structural similarities of epitopes in cTNF-α and hTNF-α indicate the potential of using adalimumab to target cTNF-α. Therefore, a canine/human chimeric antibody, Humivet-R1, was created by grafting the variable domain of adalimumab onto a canine antibody framework derived from ranevetmab. Humivet-R1 exhibits potent neutralizing ability (IC50 = 0.05 nM) and high binding affinity (EC50 = 0.416 nM) to cTNF-α, comparable to that of adalimumab for both hTNF-α and cTNF-α. These results strongly suggest that Humivet-R1 has the potential to provide effective treatment for canine arthritis with reduced side effects. Here, we propose a structure-guided antibody design for the use of a chimeric antibody to treat canine inflammatory disease. Our successful development strategy can speed up therapeutic antibody discovery for animals and has the potential to revolutionize veterinary medicine.


Asunto(s)
Artritis Reumatoide , Factor de Necrosis Tumoral alfa , Perros , Animales , Humanos , Adalimumab/farmacología , Adalimumab/uso terapéutico , Anticuerpos Monoclonales , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/patología
4.
Food Sci Nutr ; 12(4): 2346-2363, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38628176

RESUMEN

A safety evaluation was performed of Symbiota®, which is made by a proprietary anaerobic fermentation process of soybean with multistrains of probiotics and a yeast. The battery of genotoxicity studies showed that Symbiota® has no genotoxic effects. Safety and tolerability were further assessed by acute or repeated dose 28- and 90-day rodent studies, and no alterations in clinical observations, ophthalmological examination, blood chemistry, urinalysis, or hematology were observed between the control group and the different dosing groups (1.5, 5, and 15 mL/kg/day). There were no adverse effects on specific tissues or organs in terms of weight and histopathology. Importantly, the Symbiota® treatment did not perturb hormones and other endocrine-related endpoints. Of note, the No-Observed-Adverse-Effect-Level was determined to be 15 mL/kg/day in rats. Moreover, a randomized, double-blind, placebo-controlled clinical trial was recently conducted with healthy volunteers who consumed 8 mL/day of placebo or Symbiota® for 8 weeks. Only mild adverse events were reported in both groups, and the blood chemistry and blood cell profiles were also similar between the two groups. In summary, this study concluded that the oral consumption of Symbiota® at 8 mL/day by the general population does not pose any human health concerns.

5.
Gut Microbes ; 16(1): 2380061, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-39078050

RESUMEN

Cancer immunotherapy has been regarded as a promising strategy for cancer therapy by blocking immune checkpoints and evoking immunity to fight cancer, but its efficacy seems to be heterogeneous among patients. Manipulating the gut microbiota is a potential strategy for enhancing the efficacy of immunotherapy. Here, we report that MS-20, also known as "Symbiota®", a postbiotic that comprises abundant microbial metabolites generated from a soybean-based medium fermented with multiple strains of probiotics and yeast, inhibited colon and lung cancer growth in combination with an anti-programmed cell death 1 (PD1) antibody in xenograft mouse models. Mechanistically, MS-20 remodeled the immunological tumor microenvironment by increasing effector CD8+ T cells and downregulating PD1 expression, which were mediated by the gut microbiota. Fecal microbiota transplantation (FMT) from mice receiving MS-20 treatment to recipient mice increased CD8+ T-cell infiltration into the tumor microenvironment and significantly improved antitumor activity when combined with anti-PD1 therapy. Notably, the abundance of Ruminococcus bromii, which increased following MS-20 treatment, was positively associated with a reduced tumor burden and CD8+ T-cell infiltration in vivo. Furthermore, an ex vivo study revealed that MS-20 could alter the composition of the microbiota in cancer patients, resulting in distinct metabolic pathways associated with favorable responses to immunotherapy. Overall, MS-20 could act as a promising adjuvant agent for enhancing the efficacy of immune checkpoint-mediated antitumor therapy.


Asunto(s)
Linfocitos T CD8-positivos , Microbioma Gastrointestinal , Receptor de Muerte Celular Programada 1 , Microambiente Tumoral , Animales , Ratones , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Humanos , Microambiente Tumoral/inmunología , Linfocitos T CD8-positivos/inmunología , Trasplante de Microbiota Fecal , Línea Celular Tumoral , Probióticos/administración & dosificación , Probióticos/farmacología , Inmunoterapia , Femenino , Neoplasias del Colon/inmunología , Neoplasias del Colon/terapia , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/microbiología , Inhibidores de Puntos de Control Inmunológico/farmacología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/terapia , Ratones Endogámicos BALB C , Ensayos Antitumor por Modelo de Xenoinjerto
6.
Blood ; 117(3): 960-70, 2011 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-21041719

RESUMEN

Interleukin-1ß (IL-1ß) is critical for inflammation and control of infection. The production of IL-1ß depends on expression of pro-IL-1ß and inflammasome component induced by inflammatory stimuli, followed by assembly of inflammasome to generate caspase-1 for cleavage of pro-IL-1ß. Here we show that tumor suppressor death-associated protein kinase (DAPK) deficiency impaired IL-1ß production in macrophages. Generation of tumor necrosis factor-α in macrophages, in contrast, was not affected by DAPK knockout. Two tiers of defects in IL-1ß generation were found in DAPK-deficient macrophages: decreased pro-IL-1ß induction by some stimuli and reduced caspase-1 activation by all inflammatory stimuli examined. With a normal NLRP3 induction in DAPK-deficient macrophages, the diminished caspase-1 generation is attributed to impaired inflammasome assembly. There is a direct binding of DAPK to NLRP3, suggesting an involvement of DAPK in inflammasome formation. We further illustrated that the formation of NLRP3 inflammasome in situ induced by inflammatory signals was impaired by DAPK deficiency. Taken together, our results identify DAPK as a molecule required for full production of IL-1ß and functional assembly of the NLRP3 inflammasome. In addition, DAPK knockout reduced uric acid crystal-triggered peritonitis, suggesting that DAPK may serve as a target in the treatment of IL-1ß-associated autoinflammatory diseases.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/deficiencia , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Adaptadoras de Señalización CARD , Proteínas Quinasas Dependientes de Calcio-Calmodulina/deficiencia , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Caspasa 1/genética , Caspasa 1/metabolismo , Línea Celular , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas Quinasas Asociadas a Muerte Celular , Células HEK293 , Humanos , Immunoblotting , Inflamación/metabolismo , Interleucina-1beta/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Proteína con Dominio Pirina 3 de la Familia NLR , Unión Proteica , Interferencia de ARN , Transfección , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo
7.
Biochem J ; 442(2): 403-12, 2012 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-22060158

RESUMEN

QC (glutaminyl cyclase) catalyses the formation of N-terminal pGlu (pyroglutamate) in peptides and proteins. pGlu formation in chemoattractants may participate in the regulation of macrophage activation and migration. However, a clear molecular mechanism for the regulation is lacking. The present study examines the role of QC-mediated pGlu formation on MCPs (monocyte chemoattractant proteins) in inflammation. We demonstrated in vitro the pGlu formation on MCPs by QC using MS. A potent QC inhibitor, PBD150, significantly reduced the N-terminal uncyclized-MCP-stimulated monocyte migration, whereas pGlu-containing MCP-induced cell migration was unaffected. QC small interfering RNA revealed a similar inhibitory effect. Lastly, we demonstrated that inhibiting QC can attenuate cell migration by lipopolysaccharide. These results strongly suggest that QC-catalysed N-terminal pGlu formation of MCPs is required for monocyte migration and provide new insights into the role of QC in the inflammation process. Our results also suggest that QC could be a drug target for some inflammatory disorders.


Asunto(s)
Aminoaciltransferasas/antagonistas & inhibidores , Movimiento Celular/fisiología , Proteínas Quimioatrayentes de Monocitos/fisiología , Aminoaciltransferasas/química , Aminoaciltransferasas/genética , Aminoaciltransferasas/fisiología , Secuencia de Bases , Movimiento Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Técnicas de Silenciamiento del Gen , Humanos , Inflamación/fisiopatología , Lipopolisacáridos/farmacología , Modelos Moleculares , Proteínas Quimioatrayentes de Monocitos/química , Monocitos/efectos de los fármacos , Monocitos/fisiología , Dominios y Motivos de Interacción de Proteínas , ARN Interferente Pequeño/genética , Células U937
8.
J Mol Biol ; 433(4): 166766, 2021 02 19.
Artículo en Inglés | MEDLINE | ID: mdl-33359099

RESUMEN

Interleukin-1ß (IL-1ß) is a potent pleiotropic cytokine playing a central role in protecting cells from microbial pathogen infection or endogenous stress. After it binds to IL-1RI and recruits IL-1 receptor accessory protein (IL-1RAcP), signaling culminates in activation of NF-κB. Many pathophysiological diseases have been attributed to the derailment of IL-1ß regulation. Several blocking reagents have been developed based on two mechanisms: blocking the binding of IL-1ß to IL-1RI or inhibiting the recruitment of IL-1RAcP to the IL-1ß initial complex. In order to simultaneously fulfill these two actions, a human anti-IL-1ß neutralizing antibody IgG26 was screened from human genetic phage-display library and furthered structure-optimized to final version, IgG26AW. IgG26AW has a sub-nanomolar binding affinity for human IL-1ß. We validated IgG26AW-neutralizing antibodies specific for IL-1ß in vivo to prevent human IL-1ß-driving IL-6 elevation in C56BL/6 mice. Mice underwent treatments with IgG26AW in A549 and MDA-MB-231 xenograft mouse cancer models have also been observed with tumor shrank and inhibition of tumor metastasis. The region where IgG26 binds to IL-1ß also overlaps with the position where IL-1RI and IL-1RAcP bind, as revealed by the 26-Fab/IL-1ß complex structure. Meanwhile, SPR experiments showed that IL-1ß bound by IgG26AW prevented the further binding of IL-1RI and IL-1RAcP, which confirmed our inference from the result of protein structure. Therefore, the inhibitory mechanism of IgG26AW is to block the assembly of the IL-1ß/IL-1RI/IL-1RAcP ternary complex which further inhibits downstream signaling. Based on its high affinity, high neutralizing potency, and novel binding epitope simultaneously occupying both IL-1RI and IL-1RAcP residues that bind to IL-1ß, IgG26AW may be a new candidate for treatments of inflammation-related diseases or for complementary treatments of cancers in which the role of IL-1ß is critical to pathogenesis.


Asunto(s)
Anticuerpos Bloqueadores/química , Anticuerpos Monoclonales/química , Proteína Accesoria del Receptor de Interleucina-1/química , Interleucina-1beta/química , Modelos Moleculares , Conformación Proteica , Receptores Tipo I de Interleucina-1/química , Animales , Antineoplásicos Inmunológicos/química , Antineoplásicos Inmunológicos/farmacología , Sitios de Unión , Línea Celular Tumoral , Mapeo Epitopo/métodos , Epítopos/inmunología , Humanos , Inmunoglobulina G/química , Proteína Accesoria del Receptor de Interleucina-1/metabolismo , Interleucina-1beta/metabolismo , Ratones , Modelos Biológicos , FN-kappa B/metabolismo , Biblioteca de Péptidos , Unión Proteica/efectos de los fármacos , Receptores Tipo I de Interleucina-1/metabolismo , Transducción de Señal , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de Xenoinjerto
9.
Sci Rep ; 5: 12411, 2015 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-26202883

RESUMEN

Humoral immunity against diverse pathogens is rapidly elicited from natural antibody repertoires of limited complexity. But the organizing principles underlying the antibody repertoires that facilitate this immunity are not well-understood. We used HER2 as a model immunogen and reverse-engineered murine antibody response through constructing an artificial antibody library encoded with rudimentary sequence and structural characteristics learned from high throughput sequencing of antibody variable domains. Antibodies selected in vitro from the phage-displayed synthetic antibody library bound to the model immunogen with high affinity and specificities, which reproduced the specificities of natural antibody responses. We conclude that natural antibody structural repertoires are shaped to allow functional antibodies to be encoded efficiently, within the complexity limit of an individual antibody repertoire, to bind to diverse protein antigens with high specificity and affinity. Phage-displayed synthetic antibody libraries, in conjunction with high-throughput sequencing, can thus be designed to replicate natural antibody responses and to generate novel antibodies against diverse antigens.


Asunto(s)
Reacciones Antígeno-Anticuerpo/inmunología , Inmunidad Innata/inmunología , Receptor ErbB-2/química , Receptor ErbB-2/inmunología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Humanos , Ratones , Datos de Secuencia Molecular , Unión Proteica , Relación Estructura-Actividad
10.
Mol Pharmacol ; 66(5): 1317-24, 2004 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-15496512

RESUMEN

GTP cyclohydrolase I (GCH) is the rate-controlling enzyme in the production of tetrahydrobiopterin (BH4) that is essential for the synthesis of nitric oxide and catecholamines including dopamine and serotonin. Therefore, the regulation of GCH expression is important in determining the catecholamine levels in the brain under pathophysiological conditions. During the study of human disease dopa-responsive dystonia, we found that coactivator RNF4 is involved in the GCH gene expression. Through serial deletion and mutagenesis studies of the GCH promoter, we defined the RNF4-responsive element on GCH proximal promoter as a CCAAT box. RNF4 did not possess specific DNA binding activity toward this CCAAT box, which suggests that RNF4 may be a coactivator of the CCAAT boxbinding protein nuclear factor Y (NF-Y). Cotransfection of a dominant-negative mutant of NF-Y resulted in a significant reduction in RNF4-mediated CCAAT box activation. In addition, overexpression of RNF4 could not activate the CCAAT box in Drosophila melanogaster SL2 cells, which are devoid of endogenous NF-Y, whereas overexpression of RNF4 and NF-Y could. Furthermore, immunoprecipitation experiments revealed the physical association between RNF4 and the NF-Y complex. These data indicate that RNF4 imposes functional importance on GCH promoter.


Asunto(s)
Factor de Unión a CCAAT/fisiología , GTP Ciclohidrolasa/genética , Proteínas Nucleares/fisiología , Regiones Promotoras Genéticas , Factores de Transcripción/fisiología , Secuencia de Bases , Factor de Unión a CCAAT/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/fisiología , Células Cultivadas , GTP Ciclohidrolasa/metabolismo , Expresión Génica/fisiología , Humanos , Inmunoprecipitación , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Factores de Transcripción/metabolismo , Activación Transcripcional
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA