Construction Of Human Naive Fab Library, Screening And Identification Of Phage Antibody Against C-Met

c-Met was first identified in the 1980s as an oncogene. The oncogene was found to encode a receptor tyrosine kinase, and its only known ligand is HGF/SF. The HGF receptor c-Met excessively expresses in a majority of human solid tumours and also in haematological malignancies, and links to poor clinical performance. HGF/SF and c-Met play a pivotal role in tumor initiation, progression and metastasis, which induce cell proliferation, invasiveness and prevention of apoptosis as well as angiogenesis. The comepelling evidence has strongly indicated Met as being potential target in cancer therapy. Efforts to target Met and HGF as markers with therapeutic importance in tumor cells has been studied extensively. Such strategies as receptor antisense molecules, ligand receptor antagonist NK4, c-Met kinase inhibitors SU11274 and anti-receptor or anti-ligand antibodies have been widely explored. Mouse neutralizing monoclonal antibodies against human Met and against HGF/SF have been generated, and fully human neutralizing antibodies to HGF/SF also have been reported.The development of therapeutic antibodies has been one of the hottest fields in the discovery of antineoplastic drugs for their high specificity to antigen as well as effective targeting antigen with other anti-cancer agents. However, for clinical treatment application, human anti-murine antibody (HAMA) response and other ploblems have hampered the use of murine antibodies in the human body. Therefore, the focus of therapeutic antibody development has been shifting to human antibodies. Phage antibody library now is one of the major platforms for making fully human antibodies. A diversified phage antibody library with large capacity can provide an efficient approach for preparation of human antibodies, and have the advantage of obtaining antibodies with high affinity.We report here utilized phage antibody library technique to construct a large human naive Fab phage-display library by displaying the whole set of human antibody genes on phage surface. A Fab fragment specifically against Met was successively selected from this library by using biopanning process, providing a promising candidate for the research of antineoplastic agents.Methods1. Isolated human lymphocytes from 20 normal adult bone marrow. After total RNA prepatation 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 with 25 pairs of human antibody gene primers, while the CL and CH₁ fragments were amplified from two recombinant vector templates, pComb3XTT and pComb3Xλ, respectively. Then after 2 rounds of overlap PCR, the Fab genes were assembled. The Fab genes and pComb3XSS vector were digested by Sfi I enzyme and ligated. The recombinant genes were electrotransformed into competent Ecoli. XL1-blue and a human naive Fab library then was formed.2. Selection and characterization of Anti-Met Fab fragment antibodies against c-Met were screened by biopanning with immobilized antigen. After six rounds of panning, sixty randomly selected clones were identified by phage ELISA to select specific ones with high affinity for c-Met. After analyzed by BstOI digest, the positive clone was used for soluble expression in Ecoli. Top10F' and identified by western-blot. The specifity of positive clone was characterized by ELISA.Results1. 12 pairs of height chain gene primers, 4 pairs ofκgene primers and nine pairs of X gene primers were used to amplify the V antibody fragments encoding gene repertoire. By 100 times electro-transformation, the recombinant genes were electro-transformed into competent E.coli. XL1-Blue and a human naive Fab phage library consisting of 1.2×10⁹ clones was successfully constructed.2. After 6 rounds of biopanning, the phage antibodies were 5.8×10³ folds enriched. 4 clones out of 60 were found to react specifically with the Fc-Met fusion protein by phage ELISA. BstOI fragment analysis showed these 4 binders were derived from a single clone, designated as AM2-26. Sequence analysis indicated the V genes were human variable region immunoglobulin sequences. AM2-26 was functionally expressed, which was identified by Western blot showing 2 bands between 26kD and 34kD. ELISA demonstrated that AM2-26 had a well binding ability and specificityto c-Met.ConclusionsA human Fab large naive phage antibody library was successfully constructed. And an anti-cMet antibody was obtained fom the library, which provides a promising candidate for the research of antineoplastic agents.