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1.
J Biol Chem ; 285(27): 20607-14, 2010 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-20410301

RESUMEN

Many therapeutic antibodies act as antagonists to competitively block cellular signaling pathways. We describe here an approach for the therapeutic use of monoclonal antibodies based on context-dependent attenuation to reduce pathologically high activity while allowing homeostatic signaling in biologically important pathways. Such attenuation is achieved by modulating the kinetics of a ligand binding to its various receptors and regulatory proteins rather than by complete blockade of signaling pathways. The anti-interleukin-1beta (IL-1beta) antibody XOMA 052 is a potent inhibitor of IL-1beta activity that reduces the affinity of IL-1beta for its signaling receptor and co-receptor but not for its decoy and soluble inhibitory receptors. This mechanism shifts the effective dose response of the cytokine so that the potency of IL-1beta bound by XOMA 052 is 20-100-fold lower than that of IL-1beta in the absence of antibody in a variety of in vitro cell-based assays. We propose that by decreasing potency of IL-1beta while allowing binding to its clearance and inhibitory receptors, XOMA 052 treatment will attenuate IL-1beta activity in concert with endogenous regulatory mechanisms. Furthermore, the ability to bind the decoy receptor may reduce the potential for accumulation of antibody.target complexes. Regulatory antibodies like XOMA 052, which selectively modulate signaling pathways, may represent a new mechanistic class of therapeutic antibodies.


Asunto(s)
Anticuerpos Monoclonales/farmacología , Interleucina-1beta/fisiología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Bioingeniería , Fibroblastos/citología , Fibroblastos/fisiología , Células HeLa/efectos de los fármacos , Células HeLa/fisiología , Homeostasis/efectos de los fármacos , Homeostasis/fisiología , Humanos , Interleucina-1/fisiología , Interleucina-1beta/efectos de los fármacos , Riñón/efectos de los fármacos , Riñón/fisiología , Cinética , Ligandos , Luciferasas/genética , Pulmón/citología , Pulmón/fisiología , FN-kappa B/fisiología , Fosfoproteínas/efectos de los fármacos , Fosfoproteínas/metabolismo , Receptores de Interleucina-1/efectos de los fármacos , Receptores de Interleucina-1/fisiología , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología
2.
Obesity (Silver Spring) ; 24(8): 1687-94, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27330016

RESUMEN

OBJECTIVE: Leptin (LEP) deficiency results in major metabolic perturbations, including obesity, dyslipidemia, and diabetes. Although LEP deficiency can be treated with daily injections of a recombinant LEP, generation of an antibody activating the LEP receptor (LEPR) that has both an intrinsically long half-life and low immunogenicity could be useful in the treatment of this condition. METHODS: Phage display technology coupled with flow cytometry and cell-based in vitro assays were employed to identify an allosteric agonist of the mouse LEPR. LEP-deficient Lep(ob) /Lep(ob) mice were used to compare in vivo effects of LEP to antibody administration. To evaluate hypothalamic effects of treatment, changes in mRNA levels of neuropeptide Y and proopiomelanocortin were measured. RESULTS: XPA.80.037 is a monoclonal antibody that demonstrates allosteric agonism of the mouse LEPR. Treatment of Lep(ob) /Lep(ob) mice with XPA.80.037 markedly reduced hyperphagia and body weight, normalized blood glucose and plasma insulin levels, and corrected dyslipidemia. These metabolic alterations correlated with changes in mRNA levels of neuropeptide Y and proopiomelanocortin, suggesting that XPA.80.037 had hypothalamic effects. CONCLUSIONS: Agonist allosteric monoclonal antibodies to the LEPR can correct metabolic effects associated with LEP deficiency in vivo and thereby have the potential to treat conditions of LEP deficiency.


Asunto(s)
Glucemia/metabolismo , Leptina/metabolismo , Leptina/fisiología , Obesidad/metabolismo , Proopiomelanocortina/metabolismo , Receptores de Leptina/metabolismo , Regulación Alostérica , Animales , Peso Corporal , Diabetes Mellitus/metabolismo , Semivida , Hipotálamo/metabolismo , Resistencia a la Insulina/fisiología , Ratones , Ratones Endogámicos C57BL , Fenotipo
3.
J Immunol Methods ; 394(1-2): 10-21, 2013 Aug 30.
Artículo en Inglés | MEDLINE | ID: mdl-23624043

RESUMEN

Improper protein folding or aggregation can frequently be responsible for low expression and poor functional activity of antibody fragments secreted into the Escherichia coli periplasm. Expression issues also can affect selection of antibody candidates from phage libraries, since antibody fragments displayed on phage also are secreted into the E. coli periplasm. To improve secretion of properly folded antibody fragments into the periplasm, we have developed a novel approach that involves co-expressing the antibody fragments with the peptidyl prolyl cis-trans isomerase, FkpA, lacking its signal sequence (cytFkpA) which consequently is expressed in the E. coli cytosol. Cytoplasmic expression of cytFkpA improved secretion of functional Fab fragments into the periplasm, exceeding even the benefits from co-expressing Fab fragments with native, FkpA localized in the periplasm. In addition, panning and subsequent screening of large Fab and scFv naïve phage libraries in the presence of cytFkpA significantly increased the number of unique clones selected, as well as their functional expression levels and diversity.


Asunto(s)
Proteínas de Escherichia coli/fisiología , Escherichia coli/inmunología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Proteínas de la Membrana/fisiología , Isomerasa de Peptidilprolil/fisiología , Periplasma/metabolismo , Ensayo de Inmunoadsorción Enzimática , Biblioteca de Péptidos , Pliegue de Proteína
4.
J Immunol Methods ; 391(1-2): 60-71, 2013 May 31.
Artículo en Inglés | MEDLINE | ID: mdl-23454004

RESUMEN

Phage display antibody libraries have a proven track record for the discovery of therapeutic human antibodies, increasing the demand for large and diverse phage antibody libraries for the discovery of new therapeutics. We have constructed naïve antibody phage display libraries in both Fab and scFv formats, with each library having more than 250 billion clones that encompass the human antibody repertoire. These libraries show high fidelity in open reading frame and expression percentages, and their V-gene family distribution, VH-CDR3 length and amino acid usage mirror the natural diversity of human antibodies. Both the Fab and scFv libraries show robust sequence diversity in target-specific binders and differential V-gene usage for each target tested, supporting the use of libraries that utilize multiple display formats and V-gene utilization to maximize antibody-binding diversity. For each of the targets, clones with picomolar affinities were identified from at least one of the libraries and for the two targets assessed for activity, functional antibodies were identified from both libraries.


Asunto(s)
Técnicas de Visualización de Superficie Celular , Fragmentos Fab de Inmunoglobulinas/inmunología , Biblioteca de Péptidos , Receptor de Insulina/inmunología , Receptor TIE-2/inmunología , Anticuerpos de Cadena Única/inmunología , Secuencia de Aminoácidos , Animales , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Células CHO , Cricetinae , Cricetulus , Humanos , Fragmentos Fab de Inmunoglobulinas/biosíntesis , Fragmentos Fab de Inmunoglobulinas/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Sistemas de Lectura Abierta , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor de Insulina/genética , Receptor TIE-2/genética , Anticuerpos de Cadena Única/biosíntesis , Anticuerpos de Cadena Única/genética , Transfección
5.
Biochem Pharmacol ; 76(3): 340-52, 2008 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-18589401

RESUMEN

The peptide hormone gastrin is a key factor in regulation of gastric acid secretion. It has also been implicated in the development or maintenance of various types of cancer, such as pancreatic and stomach carcinoma. Inhibition of gastrin activity has potential for therapeutic use as a suppressor of acid secretion as well as an inhibitor of gastrin-responsive tumors. XPA067.06 is an affinity matured, 30 pM fully human anti-gastrin monoclonal antibody that was generated. The antibody was tested in a mouse gastric pH model to determine its effect on acid secretion. In this model, animals were treated with human gastrin, XPA067.06, and H2R or M1 receptor antagonists. Gastric fluid was collected and acid output was measured as a function of pH. XPA067.06 was shown to significantly inhibit gastrin-17-stimulated acid output for at least 48h. These results demonstrate that XPA067.06 effectively binds and neutralizes human gastrin-17 in vivo with rapid onset and prolonged duration of efficacy.


Asunto(s)
Anticuerpos Monoclonales , Afinidad de Anticuerpos/fisiología , Gastrinas/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/farmacología , Sitios de Unión de Anticuerpos , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Ácido Gástrico/metabolismo , Gastrinas/antagonistas & inhibidores , Humanos , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Cinética , Ratones , Biblioteca de Péptidos , Proteínas Recombinantes de Fusión/inmunología
6.
Anal Biochem ; 352(2): 208-21, 2006 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-16564019

RESUMEN

To explore the reliability of Biacore-based assays, 22 study participants measured the binding of prostate-specific antigen (PSA) to a monoclonal antibody (mAb). Each participant was provided with the same reagents and a detailed experimental protocol. The mAb was immobilized on the sensor chip at three different densities and a two-step assay was used to determine the kinetic and affinity parameters of the PSA/mAb complex. First, PSA was tested over a concentration range of 2.5-600 nM to obtain k(a) information. Second, to define the k(d) of this stable antigen/antibody complex accurately, the highest PSA concentration was retested with the dissociation phase of each binding cycle monitored for 1h. All participants collected data that could be analyzed to obtain kinetic parameters for the interaction. The association and the extended-dissociation data derived from the three antibody surfaces were globally fit using a simple 1:1 interaction model. The average k(a) and k(d) for the PSA/mAb interaction as calculated from the 22 analyses were (4.1+/-0.6) x 10(4) M(-1) s(-1) and (4.5+/-0.6) x 10(-5) s(-1), respectively. Overall, the experimental standard errors in the rate constants were only approximately 14%. Based on the kinetic rate constants, the affinity (K(D)) of the PSA/mAb interaction was 1.1+/-0.2 nM.


Asunto(s)
Anticuerpos Monoclonales/química , Reacciones Antígeno-Anticuerpo , Técnicas Biosensibles/métodos , Antígeno Prostático Específico/química , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/normas , Humanos , Cinética , Ligandos , Antígeno Prostático Específico/análisis , Antígeno Prostático Específico/inmunología , Unión Proteica , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Resonancia por Plasmón de Superficie/instrumentación , Resonancia por Plasmón de Superficie/métodos , Resonancia por Plasmón de Superficie/normas , Factores de Tiempo
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