Probing conformational and functional states of human hepatocyte growth factor by a panel of monoclonal antibodies

Abstract

HGF-Met signaling contributes to various biological events by controlling cell migration. Since the abnormal activation of Met receptor causes cancer progression, inhibitors such as neutralizing antibodies are regarded as promising therapeutics. HGF is secreted as a single-chain (sc) precursor and is processed by extracellular proteases to generate disulfide-bonded two-chain (tc) HGF. Although this proteolytic processing of HGF is necessary for its biological activity, exactly how the proteolysis leads to the conversion of HGF to the active form is still unclar due to the lack of structural information. In order to gain insights about this point, we generated 6 antibodies against HGF. All antibodies recognized different epitopes on the native HGF protein and showed distinct effects when tested in a cell-based HGF-Met signaling assay. They included one antibody (t1E4) that strongly blocks Met activation by tcHGF, as well as one antibody (t8E4) exclusively recognizing the active tcHGF but not inactive scHGF. Thus, a panel of anti-HGF antibodies suitable for probing the structural mechanism of HGF activation were obtained.

Fv-clasp: An Artificially Designed Small Antibody Fragment with Improved Production Compatibility, Stability, and Crystallizability

“…a6b1 integrin is a major cell surface adhesion receptor for a basement membrane component laminin, and is a critical molecule that supports growth and survival of embryonic stem cells as well as induced pluripotent stem cells during the culture (Miyazaki et al, 2012). Among 24 integrin heterodimers present in mammals (Hynes, 2002), crystal structures are available for Arg-Gly-Asp (RGD)-binding integrins (Dong et al, 2014;Nagae et al, 2012;Van Agthoven et al, 2014;Zhu et al, 2013) and for I-domain-containing integrins (Sen and Springer, 2016;Sen et al, 2013), but laminin-binding integrins that comprise a unique subgroup have escaped structural scrutiny, leading to the lack of molecular understanding of the basement membrane recognition by epithelial cells. Over the last decade, our attempts to crystallize the a6b1 ectodomain by testing more than 20 different construct designs expressed in different host cells, with and without bound Fab fragments from different anti-integrin antibodies including an anti-b1 antibody TS2/16, have all been unsuccessful.…”

Distinct Localization of Mature HGF from its Precursor Form in Developing and Repairing the Stomach

“…Because the stomach is a typical organ composed of epithelial and mesenchymal cells, and stem cells play important roles in development and regeneration [25], analyses of the localization of sc/tcHGF and its association with MET activation in the stomach may provide a better understanding of how the HGF–MET pathway participates in development, regeneration, and the stem cell behavior of endoderm-derived organs. In a recent study, we obtained several anti-HGF monoclonal antibodies that recognize different epitopes of human HGF [26], one of which selectively recognizes human tcHGF. In this study, we thus investigated the localization of tcHGF and active MET in the developing and regenerating stomach, employing human tcHGF-specific monoclonal antibody and human HGF-knock-in mice.…”

High-throughput retrieval of physical DNA for NGS-identifiable clones in phage display library

“…The clones had heterogeneous HCDR3 in the aspect of sequence length and amino acid composition. Considering the heterogeneity, the clones are expected to bind to different epitopes of hHGF, as reported before 32 .…”