Expression of a humanized single-chain variable fragment antibody targeting             chronic myeloid leukemia cells in Escherichia coli and its characterization.
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 Ni2+-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.
Key Laboratory of Laboratory Medical Diagnostics, Ministry of                     Education, Department of Laboratory Medicine, Chongqing Medical University,                     Chongqing 400016, P.R. China.
Xiaoying
X
Zhu
Dong
D
Wang
Shenfeng
S
Li
Qing
Q
Xiao
Kun
K
Tao
Jing
J
Hu
Weixi
W
Cao
Wenli
W
Feng
D001483:Base Sequence; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000276:immunology; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000276:immunology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification
D011994:Recombinant Proteins; D057127:Single-Chain Antibodies