RESUMEN
Inhibition of specific matrix metalloproteinases (MMP) is an attractive noncytotoxic approach to cancer therapy. MMP-14, a membrane-bound zinc endopeptidase, has been proposed to play a central role in tumor growth, invasion, and neovascularization. Besides cleaving matrix proteins, MMP-14 activates proMMP-2 leading to an amplification of pericellular proteolytic activity. To examine the contribution of MMP-14 to tumor growth and angiogenesis, we used DX-2400, a highly selective fully human MMP-14 inhibitory antibody discovered using phage display technology. DX-2400 blocked proMMP-2 processing on tumor and endothelial cells, inhibited angiogenesis, and slowed tumor progression and formation of metastatic lesions. The combination of potency, selectivity, and robust in vivo activity shows the potential of a selective MMP-14 inhibitor for the treatment of solid tumors.
Asunto(s)
Anticuerpos Monoclonales , Antineoplásicos/uso terapéutico , División Celular/efectos de los fármacos , Inhibidores Enzimáticos/uso terapéutico , Inhibidores de la Metaloproteinasa de la Matriz , Neovascularización Patológica/prevención & control , Animales , Anticuerpos Monoclonales Humanizados , Neoplasias de la Mama/patología , Línea Celular Tumoral , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Femenino , Genes Reporteros , Humanos , Inmunohistoquímica , Ratones , Invasividad Neoplásica/patología , Transfección , Trasplante Heterólogo , Venas Umbilicales/citología , Venas Umbilicales/efectos de los fármacosRESUMEN
Pregnancy-associated plasma protein-A (PAPP-A) is a metalloprotease that cleaves insulin-like growth factor-binding proteins (IGFBPs) to release bioactive levels of free insulin-like growth factor. Specific and potent inhibitors of PAPP-A may further elucidate the biological functions of this protease and could prove to be of therapeutic value. Phage display was used to discover fully human antibody inhibitors of PAPP-A activity towards IGFBP4 cleavage. Estimates of the inhibition constants for these antibodies were subsequently determined using a novel continuous assay of PAPP-A protease activity that uses an internally quenched synthetic peptide substrate (DX-1655). DX-1655 was hydrolyzed by PAPP-A with a K(m) of 33 muM and a k(cat) of 0.3 s(-1) (k(cat)/K(m)=9.1x10(3) M(-1) s(-1)). PAPP-A activity towards DX-1655 displays a bell-shaped pH profile, with pK(a) values of 8.2 and 10.8 and a maximum rate at approximately pH 9.5. Using this continuous assay, we measured apparent K(i) values of 1.7+/-0.2 and 7.4+/-1.5 nM for the F2 and D9 antibodies, respectively.
Asunto(s)
Anticuerpos/inmunología , Anticuerpos/farmacología , Proteína Plasmática A Asociada al Embarazo/antagonistas & inhibidores , Proteína Plasmática A Asociada al Embarazo/inmunología , Secuencia de Aminoácidos , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis/efectos de los fármacos , Cinética , Datos de Secuencia Molecular , Biblioteca de Péptidos , Proteína Plasmática A Asociada al Embarazo/química , Proteína Plasmática A Asociada al Embarazo/metabolismo , Especificidad por SustratoRESUMEN
A method was developed to rapidly identify high-affinity human antibodies from phage display library selection outputs. It combines high-throughput Fab fragment expression and purification with surface plasmon resonance (SPR) microarrays to determine kinetic constants (kon and koff) for 96 different Fab fragments in a single experiment. Fabs against human tissue kallikrein 1 (hK1, KLK1 gene product) were discovered by phage display, expressed in Escherichia coli in batches of 96, and purified using protein A PhyTip columns. Kinetic constants were obtained for 191 unique anti-hK1 Fabs using the Flexchip SPR microarray device. The highest affinity Fabs discovered had dissociation constants of less than 1 nM. The described SPR method was also used to categorize Fabs according to their ability to recognize an apparent active site epitope. The ability to rapidly determine the affinities of hundreds of antibodies significantly accelerates the discovery of high-affinity antibody leads.