Expression of a humanized single-chain variable fragment antibody targeting chronic myeloid leukemia cells in Escherichia coli and its characterization

Abstract

Abstract. Chronic myeloid leukemia (CML) is a malignant blood disease originating from hematopoietic stem cells. Drug resistance and tumor recurrence have become major problems for the treatment of this disease. Therefore, new therapeutic methods need to be developed. Antigens expressed on the surface of cancer cells are potential targets for antibody-mediated drug delivery. In our study, an anti-CML cell single-chain variable fragment (scFv) antibody has been produced and characterized because it is the first step towards the construction of a novel cancer-targeted agent for cancer diagnosis and treatment. Here, a 46 kDa antibody derivative was produced by genetic fusion of a humanized scFv antibody against a CML cell surface antigen with the 6xHis-tag, which can specifically bind to CML cells. The recombinant scFv against CML cells was expressed as a fusion protein containing the 6xHis-tag at its N-termini, and purified by Ni 2+ -NTA column chromatography. The recombinant scFv, which was soluble, was expressed and produced in bacteria, and was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot assays. Finally, its cell-binding activity and immunoactivity were demonstrated by enzyme-linked immunosorbent assay (ELISA). Furthermore, flow cytometry analysis demonstrated that this scFv specifically targeted CML cells expressing the associated antigen (47.9 and 34.4%) other than non-expressing tumor cells (1.25%) in vitro. The results presented in this study illustrate that the humanized anti-CML cell scFv antibody may function as a novel therapeutic tool for CML.