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

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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 2.0×109, 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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Authors and Affiliations

  1. Antibody Technology Laboratory, Van Andel Research Institute, Grand Rapids, MI

    Yongjun Jiao, Ping Zhao, Jin Zhu, Tessa Grabinski & Brian Cao

  2. Molecular Oncology Laboratory, Van Andel Research Institute, Grand Rapids, MI

    Rick V. Hay

  3. Institute of Molecular Biology, Nanjing Medical University, Nanjing, Jiangsu, China

    Yongjun Jiao, Jin Zhu & Xiaohong Guan

  4. Department of Pathology, Nanjing Medical University, Nanjing, Jiangsu, China

    Zhengqing Feng

  5. Department of Veterans Affairs Healthcare System, Ann Arbor, MI

    R. Scot Skinner & Milton D. Gross

  6. Department of Infectious Diseases, Tokai University School of Medicine, Kanagawa, Japan

    Hiroshi Tachibana

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  1. Yongjun Jiao
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  2. Ping Zhao
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  3. Jin Zhu
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Correspondence to Brian Cao.

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These two authors contributed equally to this work.

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Jiao, Y., Zhao, P., Zhu, J. et al. Construction of human naïve fab library and characterization of anti-met fab fragment generated from the library. Mol Biotechnol 31, 41–54 (2005). https://doi.org/10.1385/MB:31:1:041

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