Construction of human naïve Fab library and characterization of anti-met Fab fragment generated from the library

Mol Biotechnol. 2005 Sep;31(1):41-54. doi: 10.1385/mb:31:1:041.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal* / therapeutic use
  • Antineoplastic Agents / therapeutic use
  • Blotting, Western
  • Electrophoresis, Polyacrylamide Gel
  • Hepatocyte Growth Factor
  • Humans
  • Immunoglobulin Fab Fragments / chemistry
  • Immunoglobulin Fab Fragments / genetics*
  • Immunoglobulin Fab Fragments / therapeutic use
  • Immunoprecipitation
  • Leiomyosarcoma / diagnostic imaging
  • Leiomyosarcoma / drug therapy
  • Neoplasm Invasiveness
  • Peptide Library*
  • Polymerase Chain Reaction
  • Proto-Oncogene Proteins c-met / genetics
  • Proto-Oncogene Proteins c-met / immunology*
  • Radioimmunodetection
  • Radiopharmaceuticals
  • Transplantation, Heterologous
  • Tumor Cells, Cultured


  • Antibodies, Monoclonal
  • Antineoplastic Agents
  • Immunoglobulin Fab Fragments
  • Peptide Library
  • Radiopharmaceuticals
  • Hepatocyte Growth Factor
  • Proto-Oncogene Proteins c-met