Humanization Of Selenium-Containing Single-Chain Abzyme

Reactive oxygen species(ROS) are products of the normal metabolic activities of aerobic living organism and are produced in response to various stimuli. Under normal conditions, there is a balance between the production of ROS and their destruction. In certain pathogenic states the production of ROS is enhanced and the excess ROS damage biomacromolecules such as RNA, DNA, protein, sugared and lipids, therefore this results in ROS-mediated diseases. ROS-related diseases include reperfusion injury, inflammatory process, aged-related disease, neuronal apoptosis, cancer and cataract. In order to scavenge ROS, the living organism has several lines of defense system, including enzymatic and non-enzymatic action. The enzymatic antioxidant system consists of glutathione peroxidase (GPX), catalase (CAT) and superoxide dismutase (SOD). The non-enzymatic antioxidant system includes vitamine E, ascorbate, glutathione (GSH) and uric acid. GPX plays an important role in antioxidant defense system of enzymatic action. However, due to the shortcomings of native GPX (solution instability, poor availability, short half-lives and proteolytic digestion), mimics with high GPX efficiency were prepared by many biochemists. One of the well-known GPX mimics is Ebselen (2-phenyl-l,2- benziososelenazol-3 (2H)-one) . Although this intresting molecule is undergoing phase III clinical trial in Japan as antioxidant, it has some drawbacks such as its low GPX activity and its insolubility in water. We have successfully generated several mouse catalytic antibodies, and expressed mouse single-chain variable region antibodies (ScFv's) in Escherichia coli (E.coli)by protein engineering and found high GPX efficiencies by chemical modified serine into selenocysteine (Sec). Although some antibodies from murine sources have good affinity and showed excellent results in animal models, the problem with human antimurine (HAMA) antibodies must be resolved. In 2001, the full human antibodies were selected from a semi-synthetic antibody library and the GPX efficiency of the catalytic antibody was found to be 80 U/umol, upon Sec was introduced. But this efficiency was almost as much as that of Se- y IgGIn this study, we described the construction of a library displayed human antibody fragments that can bind the specific substrate-glutathione (GSH). Our strategy was to construct a single-chain variable fragments (ScFv) library by reforming a mouse fragment variant region into a phagemid vector pFUW-80. The constructed library was a chimeric one that contained both conservative fragments of mouse ScFv and incorporated the human homologous fragments. Two designed haptens were synthesized GSH-S-DNP-Me and GSH-S-DNP-Bu and were coupled to BSA by glutaradehyde. The selected ScFv's were identified by ELISA and were cloned into the expression vector pPelB. The expressed protein was purified, identified and the catalytic group, Sec was incorporated into the protein by chemical mutation. The GPX activity of the single-chain antibody was 880 U/umol, which is 11-fold that of first reported human glutathione peroxidase mimic. The selenium-containing human single-chain abzyme with GPX activity shows great prospects in medicine.1. Construction of ScFv antibody libraryThe heavy chain (VH) and light chain (VL) of the reshaping antibody of variable fragment were designed respectively. By homology sequence analysis from protein data bank (PDB), homology replacements of the VH and VL fragments regions were chosen. There is a need to select a correct pair of variable regions to achieve the right specificity. Key residues from the mouse single-chain antibody (heavy chain CDR3 sequences and sometimes also CDR2) are incorporated into human repertoires in order to preserve the desired binding function. By computerstimulation, the conservative residues, which are relevant to the structure of the non-human antibodies, as well as CDRs, were retained. The VH genes were ligated by the synthesized VH fragments, while the VL genes were amplified by over-lap PCR with the primers o...