| The Met receptor is one kind of tyrosine kinase receptors and the only known receptor for HGF/SF. Met is synthesized as a single peptide and cleaved into a disulfide-linked heterodimer made of a (Mr 45,000) and β (Mr 145,000) subunits. The Met a chain is located outside the membrane, whereas the β chain consists of an extracelluar domain, a single transmembrane domain, and a cytoplasmic moiety in which the receptor tyrosine kinase domain resides. Upon binding the HGF/SF ligand to the extracellular domain of Met, downstream cytoplasmic signal transduction pathways are activated.In vertebrates, the HGS/SF-Met signaling pair mediates a vast range of biological activities, including normal organ development during embryological processes, and tissue regeneration after damage in adult. Inappropriate expression of the receptor tyrosine kinase Met and its ligand HGF/SF is usually associated with an aggressive solid-tumor phenotype (angiogenesis, invasiveness, and metastasis) and poor clinical prognosis. Met-HGF/SF has been considered not only a marker for cancer, but also a target for therapeutic intervention, and efforts to target Met and HGF as markers with therapeutic importance in tumor cells has been studied extensively. Such strategies as receptor antisense molecules, tyrosine kinase inhibitors, ligand or/and receptor antagonist, and anti-receptor or anti-ligand antibodies have been widely explored.Mouse mAbs against human Met and against HGF/SF have been generated, and some of them showed strong affinities and blocking efficacy both in vitro and in animal models. However, clinical problems due to formation of human antimurine antibodies (HAMA) and otherpharmacodynamic effects have hampered their efficacy in the human body. In recent years, antibody phage-display technology has made it possible to overcome these limitations by enabling the generation of complete human mAbs from both immune and nonimmune sources. This technology combined with biopanning permits the selection of individual antibodies having a desired specificity.We report here the design and construction of a large human naive 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 process and its specificity was characterized in detail. Part I the Construction of Human Naive Fab Library1. The cloning of Fab encoding gene repertoireHuman lymphocytes were collected from 15 adult bone marrow samples left over from clinical diagnostic specimens. After total RNA purification and first-strand cDNA synthesis, a unique three-step PCR protocol was used to amplify the Fab antibody fragment encoding gene repertoire. The variable region gene pools were amplified from the cDNA synthesized, while the Cl and Chi fragments were amplified from two human Fab templates, pComb3XTT and pComb3XA, respectively. The variable region genes of the heavy and light chain were combined with their corresponding constant regions to develop a heavy-chain Fd and light chain repertoire by overlap extension. The Fab genes were assembled randomly by Fd fragments and light chains.2. Construction of human naive Fab libraryThe Fab genes and pComb3xss vector were digested by Sfil enzyme and ligated between each other. The recombinant genes were electro-transformed into competent E.coli. XL 1-blue. A single transformation operation can reach 2.0 X 107 tranformants by optimizing the factors, such as transformation temperature, electroporation conditions design, post-pulse treatment, and treatment of DNA samples, which may affect the transformation efficiency. By this means, a human naive Fab library with a theoretical diversity of 2.OX 109 has been constructed by 100 times electro-transformation. DNA sequencing revealed more than 86% (13/15) transformants have correct oriented full-length inserts. Part II Panning of Human Naive Fab Library and Characterization of Anti-Met Fab Fragment Generated from It 1. Biopanning of the library in search of Met specific antibodiesTo generate human antibodies against the Met epitope that would have therapeutic potential, a whole-live-cell biopanning approach was used. The library was firstly panned against SI 14 parental cell line NIH3T3 (c-Met")... |
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