title: Construction of human naïve Fab library and characterization of anti-met Fab fragment generated from the library.
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.
authors.affiliation: Antibody Technology Laboratory, Van Andel Research Institute, Grand Rapids, MI 49503, USA.
authors.forename: Yongjun
authors.initials: Y
authors.lastname: Jiao
authors.forename: Ping
authors.initials: P
authors.lastname: Zhao
authors.forename: Jin
authors.initials: J
authors.lastname: Zhu
authors.forename: Tessa
authors.initials: T
authors.lastname: Grabinski
authors.forename: Zhengqing
authors.initials: Z
authors.lastname: Feng
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Guan
authors.forename: R Scot
authors.initials: RS
authors.lastname: Skinner
authors.forename: Milton D
authors.initials: MD
authors.lastname: Gross
authors.forename: Rick V
authors.initials: RV
authors.lastname: Hay
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Tachibana
authors.forename: Brian
authors.initials: B
authors.lastname: Cao
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000627:therapeutic use; D000970:Antineoplastic Agents / Q000627:therapeutic use; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D017228:Hepatocyte Growth Factor; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics* / Q000627:therapeutic use; D047468:Immunoprecipitation; D007890:Leiomyosarcoma / Q000000981:diagnostic imaging / Q000188:drug therapy; D009361:Neoplasm Invasiveness; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D019859:Proto-Oncogene Proteins c-met / Q000235:genetics / Q000276:immunology*; D016719:Radioimmunodetection; D019275:Radiopharmaceuticals; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D019275:Radiopharmaceuticals; D017228:Hepatocyte Growth Factor; D019859:Proto-Oncogene Proteins c-met