Assuntos
Anticorpos/química , Imunoconjugados/química , Neoplasias , Preparações Farmacêuticas/química , Animais , Anticorpos/imunologia , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Feminino , Fibronectinas/química , Fibronectinas/imunologia , Imunocompetência , Imunoconjugados/uso terapêutico , Camundongos , Neoplasias/tratamento farmacológicoRESUMO
The in vivo perfusion of rodent models of disease with biotin derivatives and the subsequent comparative proteomic analysis of healthy and diseased tissues represent a promising methodology for the identification of vascular accessible biomarkers. A novel, triply charged biotinylation reagent, NHS-ß-Ala-(L-Asp)(3)-biotin, was synthesized and validated in terms of its applicability for in vivo protein biotinylation. Compared to sulfo-NHS-LC-biotin, NHS-ß-Ala-(L-Asp)(3)-biotin exhibited a reduced membrane permeability and a preferential labeling of proteins localized in compartments readily accessible in vivo from the vasculature.
Assuntos
Biotina/análogos & derivados , Biotinilação/métodos , Proteínas/análise , Animais , Biotina/análise , Biotina/química , Permeabilidade da Membrana Celular , Rim/química , Fígado/química , Camundongos , Succinimidas/químicaRESUMO
Antibody fragments can recognize their cognate antigen with high affinity and can be produced at high yields, but generally display rapid blood clearance profiles. For pharmaceutical applications, the serum half-life of antibody fragments is often extended by chemical modification with polymers or by genetic fusion to albumin or albumin-binding polypeptides. Here, we report that the site-specific chemical modification of a C-terminal cysteine residue in scFv antibody fragments with a small organic molecule capable of high-affinity binding to serum albumin substantially extends serum half-life in rodents. The strategy was implemented using the antibody fragment F8, specific to the alternatively spliced EDA domain of fibronectin, a tumor-associated antigen. The unmodified and chemically modified scFv-F8 antibody fragments were studied by biodistribution analysis in tumor-bearing mice, exhibiting a dramatic increase in tumor uptake for the albumin-binding antibody derivative. The data presented in this paper indicate that the chemical modification of the antibody fragment with the 2-(3-maleimidopropanamido)-6-(4-(4-iodophenyl)butanamido)hexanoate albumin-binding moiety may represent a general strategy for the extension of the serum half-life of antibody fragments and for the improvement of their in vivo targeting performance.
Assuntos
Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/metabolismo , Albumina Sérica/química , Animais , Afinidade de Anticorpos , Clonagem Molecular , Feminino , Meia-Vida , Fragmentos de Imunoglobulinas/isolamento & purificação , Fragmentos de Imunoglobulinas/farmacologia , Camundongos , Camundongos Endogâmicos , Neoplasias Experimentais , Distribuição TecidualRESUMO
We describe the synthesis and characterization of two acetazolamide derivatives containing either a charged fluorophore or an albumin-binding moiety, which restrict binding to carbonic anhydrase IX and XII present on tumor cells. In vivo studies showed the preferentially targeting of tumor cells by the fluorescent acetazolamide derivative and the ability of the albumin-binding acetazolamide derivative to cause tumor retardation in a SK-RC-52 xenograft model of cancer.