Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 4 de 4
Filtrar
1.
Protein Eng Des Sel ; 27(10): 419-29, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24786107

RESUMEN

Alternative scaffold molecules represent a class of proteins important to the study of protein design and mechanisms of protein-protein interactions, as well as for the development of therapeutic proteins. Here, we describe the generation of a library built upon the framework of a consensus FN3 domain sequence resulting in binding proteins we call Centyrins. This new library employs diversified positions within the C-strand, CD-loop, F-strand and FG-loop of the FN3 domain. CIS display was used to select high-affinity Centyrin variants against three targets; c-MET, murine IL-17A and rat TNFα and scanning mutagenesis studies were used to define the positions of the library most important for target binding. Contributions from both the strand and loop positions were noted, although the pattern was different for each molecule. In addition, an affinity maturation scheme is described that resulted in a significant improvement in the affinity of one selected Centyrin variant. Together, this work provides important data contributing to our understanding of potential FN3 binding interfaces and a new tool for generating high-affinity scaffold molecules.


Asunto(s)
Biblioteca de Genes , Unión Proteica , Ingeniería de Proteínas/métodos , Estructura Terciaria de Proteína , Secuencia de Aminoácidos , Animales , Interleucina-17 , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Ratas , Alineación de Secuencia , Factor de Necrosis Tumoral alfa
2.
Protein Eng Des Sel ; 25(10): 531-7, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22915597

RESUMEN

Some antibodies have a tendency to self-associate leading to precipitation at relatively low concentrations. CNTO607, a monoclonal antibody, precipitates irreversibly in phosphate-buffered saline at concentrations above 13 mg/ml. Previous mutagenesis work based on the Fab crystal structure pinpointed a three residue fragment in the heavy chain CDR-3, (99)FHW(100a), as an aggregation epitope that is anchored by two salt bridges. Biophysical characterization of variants reveals that F99 and W100a, but not H100, contribute to the intermolecular interaction. A K210T/K215T mutant designed to disrupt the charge interactions in the aggregation model yielded an antibody that does not precipitate but forms reversible aggregates. An isotype change from IgG1 to IgG4 prevents the antibody from precipitating at low concentration yet the solution viscosity is elevated. To further understand the nature of the antibody self-association, studies on the Fab fragment found high solubility but significant self- and cross-interactions remain. Dynamic light scattering data provides evidence for higher order Fab structure at increased concentrations. Our results provide direct support for the aggregation model that CNTO607 precipitation results primarily from the specific interaction of the Fab arms of neighboring antibodies followed by the development of an extensive network of antibodies inducing large-scale aggregation and precipitation.


Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Interleucina-13/inmunología , Animales , Anticuerpos Monoclonales/genética , Línea Celular , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/inmunología , Inmunoglobulina G/genética , Modelos Moleculares , Mutagénesis , Mutación , Conformación Proteica , Solubilidad
3.
Proc Natl Acad Sci U S A ; 103(11): 4005-10, 2006 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-16537476

RESUMEN

Antibody-dependent cell-mediated cytotoxicity, a key effector function for the clinical efficacy of monoclonal antibodies, is mediated primarily through a set of closely related Fcgamma receptors with both activating and inhibitory activities. By using computational design algorithms and high-throughput screening, we have engineered a series of Fc variants with optimized Fcgamma receptor affinity and specificity. The designed variants display >2 orders of magnitude enhancement of in vitro effector function, enable efficacy against cells expressing low levels of target antigen, and result in increased cytotoxicity in an in vivo preclinical model. Our engineered Fc regions offer a means for improving the next generation of therapeutic antibodies and have the potential to broaden the diversity of antigens that can be targeted for antibody-based tumor therapy.


Asunto(s)
Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Alemtuzumab , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales Humanizados , Anticuerpos Antineoplásicos/genética , Anticuerpos Antineoplásicos/metabolismo , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Citotoxicidad Celular Dependiente de Anticuerpos , Antineoplásicos/metabolismo , Linfocitos B/inmunología , Proteínas del Sistema Complemento/metabolismo , Citotoxicidad Inmunológica , Variación Genética , Humanos , Técnicas In Vitro , Depleción Linfocítica , Macaca fascicularis , Ingeniería de Proteínas , Receptores de IgG/metabolismo , Trastuzumab
4.
Science ; 301(5641): 1895-8, 2003 Sep 26.
Artículo en Inglés | MEDLINE | ID: mdl-14512626

RESUMEN

Tumor necrosis factor (TNF) is a key regulator of inflammatory responses and has been implicated in many pathological conditions. We used structure-based design to engineer variant TNF proteins that rapidly form heterotrimers with native TNF to give complexes that neither bind to nor stimulate signaling through TNF receptors. Thus, TNF is inactivated by sequestration. Dominant-negative TNFs represent a possible approach to anti-inflammatory biotherapeutics, and experiments in animal models show that the strategy can attenuate TNF-mediated pathology. Similar rational design could be used to engineer inhibitors of additional TNF superfamily cytokines as well as other multimeric ligands.


Asunto(s)
Ingeniería de Proteínas , Transducción de Señal , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/farmacología , Sustitución de Aminoácidos , Animales , Antígenos CD/metabolismo , Apoptosis , Artritis Experimental/tratamiento farmacológico , Biopolímeros , Caspasas/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Simulación por Computador , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Galactosamina/farmacología , Células HeLa , Humanos , Hígado/efectos de los fármacos , FN-kappa B/metabolismo , Mutación Puntual , Ratas , Receptores del Factor de Necrosis Tumoral/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral , Receptores Tipo II del Factor de Necrosis Tumoral , Factor de Transcripción ReIA , Transcripción Genética , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA