Preparation And Evaluation Of Disulfide-Stabilized Single-Chain Antibody B3(ds-scFv) Targeted SEA

The conception of superantigen(SAgs) was created by White in 1989.They are a group of protein moleculars encoded by bacterials or virus, SAgs bind as unprocessed proteins to relatively invariant regions of MHC class II antigens, which are found mainly on professional antigen presenting cells(APCs).Once bound to MHCII+ cells, SAgs can activate T lymphocytes expressing particular variable regions (Vβ) of the T cell receptor(TCR) βchain, and the number of the activated T lymphocytes is 1000-10000 folds compares to common antigens. The CTLs activated by superantigen has significant cytotoxic effect against tumor cells, therefore, as potent T lymphocytes activator, superantigen may develop to a new type of antitumor immunomolecular. SEA is the product of staphylococcus aureus, it is one of different types of superantigens, and the research on it is deeper than others recent years. On the negative side, the cytotoxic effect mediated by the T lymphocytes which is activated by SEA also has cytotoxicity against the normal cells when used to the carcinoma cells and has no effect on MHCII-carcinoma cells. Because of the reasons mentioned above, utilizing genetic engineering antibody to directed SEA to tumor tissues and decrease the negative effects is more and more necessary. In order to improve the stability of the single-chain antibody(ScFv) so as to achieve better targeted effect, a pair of interchain disulfide bond was introduced to B3ScFv and formed disulfide stable single-chain antibody(B3dsscFv) in this paper. The VH and VL fragments of the mAbB3 were ligated by overlap PCR, the PCR product was cloned to the pET22b expression vector; then the SEA fragment was inserted into the B3ds-scFv-pET22b expression vector which was digested by the same restriction enzymes. The expression plasmid was identified by restriction endonucleases digestion and tansformed into E.coli BL21(DE3) followed by IPTG inducion; the inclusion body was purified through SP-Sepharose cation exchange column after denaturing and refolding. The binding and cytotoxic ability of the purified products was examined by cell-ELISA and Non-Radioactive Cell Proliferation Assay separately. The stability assay was performed by incubating the protein sample at 37℃for a week and at different temperatures for 30min. As a result, the expression vector B3ds-scFv-SEA(D227A)-pET was constructed successfully and the expression product exists mainly in the inclusion body, amounting to 33% of the totle protein. The refolding product remains good antigen binding ability and has cytotoxic effect on HT-29 colon carcinoma cells. The stability assay showed that the resulting protein was stable at 37℃. In conclusion, this genetically engineered B3ds-scFv-SEA(D227A) fusion protein has bifunction of tumor targeting and tumor cell killing and promises to be an effective reagent for tumor targeted immunotherapy.