The objective is to generate, by molecular cloning, a library of human F(ab) fragments in a bacteriophage cloning/expression system and then compare the immunoglobulin usage in this in vitro derived repertoire with the antibodies obtained using classical hybridoma technology. Major findings to demonstrate that the cloning technique, PCR primers (developed in this laboratory) and cloning/expression vectors (obtained from Dr. Lerner's laboratory at Scripps Research Institute), can be used to generate a human F(ab) in E. Coli. We have acquired two different human hybridoma cell lines (which had been developed in LOM). The F(abs) from both of these cell lines have been successfully cloned and expressed, but whether they bind to their respective antigens has not yet been determined. A model system for a large combinatorial library is being developed using PBLs from a patient suffering with rheumatoid arthritis. This source of blood alleviates the need to immunize the patient before obtaining a blood sample. A screening system has been developed in the laboratory which consists of purified human Fc fragment covalently linked to the enzyme alkaline phosphatase. To date, a light chain recombinant library (kappa chain specific) and two heavy chain libraries (one gamma and one mu) have been cloned into a lambda phage prokaryotic expression vector. The light chain library (1c2-kappa) contains approximately 50% recombinant phage. The heavy chain libraries (hc2-gamma, hc2-mu) contain between 25 to 50% recombinant phage.
