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1.
J Am Soc Nephrol ; 28(12): 3616-3626, 2017 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-28827403

RESUMEN

Targeted delivery of a therapeutic agent to a site of pathology to ameliorate disease while limiting exposure at undesired tissues is an aspirational treatment scenario. Targeting diseased kidneys for pharmacologic treatment has had limited success. We designed an approach to target an extracellular matrix protein, the fibronectin extra domain A isoform (FnEDA), which is relatively restricted in distribution to sites of tissue injury. In a mouse unilateral ureteral obstruction (UUO) model of renal fibrosis, injury induced significant upregulation of FnEDA in the obstructed kidney. Using dual variable domain Ig (DVD-Ig) technology, we constructed a molecule with a moiety to target FnEDA and a second moiety to neutralize TGF-ß After systemic injection of the bispecific TGF-ß + FnEDA DVD-Ig or an FnEDA mAb, chemiluminescent detection and imaging with whole-body single-photon emission computed tomography (SPECT) revealed significantly higher levels of each molecule in the obstructed kidney than in the nonobstructed kidney, the ipsilateral kidney of sham animals, and other tissues. In comparison, a systemically administered TGF-ß mAb accumulated at lower concentrations in the obstructed kidney and exhibited a more diffuse whole-body distribution. Systemic administration of the bispecific DVD-Ig or the TGF-ß mAb (1-10 mg/kg) but not the FnEDA mAb attenuated the injury-induced collagen deposition detected by immunohistochemistry and elevation in Col1a1, FnEDA, and TIMP1 mRNA expression in the obstructed kidney. Overall, systemic delivery of a bispecific molecule targeting an extracellular matrix protein and delivering a TGF-ß mAb resulted in a relatively focal uptake in the fibrotic kidney and reduced renal fibrosis.


Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Enfermedades Renales/tratamiento farmacológico , Riñón/efectos de los fármacos , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Matriz Extracelular/metabolismo , Fibronectinas/química , Fibrosis/tratamiento farmacológico , Humanos , Hibridomas/metabolismo , Riñón/diagnóstico por imagen , Riñón/patología , Masculino , Ratones , Tomografía Computarizada de Emisión de Fotón Único , Uréter/patología
3.
Eur J Pharmacol ; 483(2-3): 195-205, 2004 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-14729107

RESUMEN

The molecular properties of the sulfonylurea receptor 2 (SUR2) subunits of K(ATP) channels expressed in urinary bladder were assessed by polymerase chain reaction (PCR). This showed that SUR2B exon 17- mRNA (72%) was predominant over the SUR2B exon 17+ splice variant (28%). The pharmacological properties of both of these isoforms stably expressed in mouse Ltk(-)cells (L-cells) with K(IR) 6.2 were determined by measuring changes in membrane potential responses evoked by K(+) channel openers using bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC(4)(3)) fluorescence. The rank order potency of a variety of structurally distinct K(+) channel openers was found to be the same in both stable cell lines and compared well with guinea pig bladder cells. The potency of these compounds in the SUR2B exon 17- cells more closely resembled the potency measured in guinea pig bladder unlike the cell line containing the SUR2B exon 17+ subtype. Analysis of the displacement of [125I]A-312110 binding with the same K(+) channel openers to the SUR2B exon 17- cells showed excellent correlation to those measured in guinea pig bladder. This study supports the notion that K(ATP) channels containing SUR2B exon 17- represent a major splice variant expressed in urinary bladder smooth muscle.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Proteínas de la Membrana/química , Canales de Potasio de Rectificación Interna/química , Canales de Potasio/genética , Receptores de Droga/genética , Vejiga Urinaria/metabolismo , Adenosina Trifosfato/genética , Adenosina Trifosfato/fisiología , Amidas/metabolismo , Amidas/farmacología , Animales , Benzofenonas/metabolismo , Benzofenonas/farmacología , ADN Recombinante/biosíntesis , ADN Recombinante/genética , Relación Dosis-Respuesta a Droga , Exones/efectos de los fármacos , Exones/fisiología , Cobayas , Humanos , Células L , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Proteínas de la Membrana/genética , Ratones , Canales de Potasio de Rectificación Interna/genética , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Receptores de Sulfonilureas , Vejiga Urinaria/efectos de los fármacos
4.
MAbs ; 5(4): 595-607, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23774760

RESUMEN

Efficient production of large quantities of therapeutic antibodies is becoming a major goal of the pharmaceutical industry. We developed a proprietary expression system using a polyprotein precursor-based approach to antibody expression in mammalian cells. In this approach, the coding regions for heavy and light chains are included within a single open reading frame (sORF) separated by an in-frame intein gene. A single mRNA and subsequent polypeptide are produced upon transient and stable transfection into HEK293 and CHO cells, respectively. Heavy and light chains are separated by the autocatalytic action of the intein and antibody processing proceeds to produce active, secreted antibody. Here, we report advances in sORF technology toward establishment of a viable manufacturing platform for therapeutic antibodies in CHO cells. Increasing expression levels and improving antibody processing by intein and signal peptide selection are discussed.


Asunto(s)
Expresión Génica , Vectores Genéticos/genética , Inteínas , Sistemas de Lectura Abierta , Anticuerpos de Cadena Única , Animales , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Humanos , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/biosíntesis , Cadenas Ligeras de Inmunoglobulina/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Anticuerpos de Cadena Única/biosíntesis , Anticuerpos de Cadena Única/genética
5.
Proc Natl Acad Sci U S A ; 104(19): 7875-80, 2007 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-17470806

RESUMEN

The pituitary adenylate cyclase-activating polypeptide (PACAP) receptor is a class II G protein-coupled receptor that contributes to many different cellular functions including neurotransmission, neuronal survival, and synaptic plasticity. The solution structure of the potent antagonist PACAP (residues 6'-38') complexed to the N-terminal extracellular (EC) domain of the human splice variant hPAC1-R-short (hPAC1-R(S)) was determined by NMR. The PACAP peptide adopts a helical conformation when bound to hPAC1-R(S) with a bend at residue A18' and makes extensive hydrophobic and electrostatic interactions along the exposed beta-sheet and interconnecting loops of the N-terminal EC domain. Mutagenesis data on both the peptide and the receptor delineate the critical interactions between the C terminus of the peptide and the C terminus of the EC domain that define the high affinity and specificity of hormone binding to hPAC1-R(S). These results present a structural basis for hPAC1-R(S) selectivity for PACAP versus the vasoactive intestinal peptide and also differentiate PACAP residues involved in binding to the N-terminal extracellular domain versus other parts of the full-length hPAC1-R(S) receptor. The structural, mutational, and binding data are consistent with a model for peptide binding in which the C terminus of the peptide hormone interacts almost exclusively with the N-terminal EC domain, whereas the central region makes contacts to both the N-terminal and other extracellular parts of the receptor, ultimately positioning the N terminus of the peptide to contact the transmembrane region and result in receptor activation.


Asunto(s)
Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/química , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/química , Secuencia de Aminoácidos , Animales , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Hormona Liberadora de Corticotropina/química , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/metabolismo , Soluciones
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