Construction of human naïve Fab library and characterization of anti-met Fab fragment generated from the library.
Mol Biotechnol
; 31(1): 41-54, 2005 Sep.
Article
em En
| MEDLINE
| ID: mdl-16118414
ABSTRACT
Inappropriate expression of the receptor tyrosine kinase Met and its ligand hepatocyte growth factor (HGF)/scatter factor (SF) is usually associated with an aggressive solid tumor phenotype (angiogenesis, invasiveness, and metastasis) and poor clinical prognosis. We report here the design and construction of a large, human naïve antigen-binding fragment (Fab) phage-display library with a diversity of 2.0 x 109, which allows rapid isolation of antigen-specific human antibody fragments. A Fab fragment specifically against Met (designated hFab-Met-1) was successively selected from this library by using biopanning on Met-transfected cell line S114. The specificity of hFab-Met-1 was characterized by immunoprecipitation, Western blotting, and flow cytometry. The results demonstrate that hFab-Met-1 reacts with the extracellular domain of Met in its native conformation. Moreover, functional analysis by Madine-Darby canine kidney cell scattering and urokinase-type plasminogen activator assays demonstrated that hFab-Met-1 is not an agonist to HGF/Met signaling compared with a murine intact monoclonal antibody (MAb) Met5. To confirm that hFab-Met-1 interacts with Met-expressing tumors in vivo, I-125-labeled hFab-Met-1 was nuclear-imaged in a mouse xenograft of Met- and HGF/SF-expressing human leiomyosarcoma. Total body scintigrams were obtained between 1 and 48 h postinjection (PI). Tumor-associated activity was imaged as early as 1 h PI, and remained visible in some animals as late as 24 h PI. As expected, activity was highest in the kidneys in early images, whereas thyroid activity became predominant in later images. In conclusion, hFab-Met-1 interacts with Met both in vitro and in vivo, and is a promising candidate for clinical diagnosis and therapeutics.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fragmentos Fab das Imunoglobulinas
/
Biblioteca de Peptídeos
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Proteínas Proto-Oncogênicas c-met
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Anticorpos Monoclonais
Tipo de estudo:
Prognostic_studies
Limite:
Animals
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Humans
Idioma:
En
Revista:
Mol Biotechnol
Ano de publicação:
2005
Tipo de documento:
Article