title: A new recombinant immunotoxin hscFv-ETA' demonstrates specific cytotoxicity against chronic myeloid leukemia cells in vitro.
abstract: Antibodies against cell surface antigens of tumor have attracted increasing attention in immunotherapy for tumor diagnosis and treatment. Recently, we constructed a new recombinant immunotoxin for possible clinical application in patients with chronic myeloid leukemia (CML). A functional humanized single chain variable fragment (hscFv) against CML patient cells was previously obtained from an anti-CML cell hybridoma derived monoclonal antibody. By insertion into the bacterial vector pWW20, the hscFv was fused with a deletion mutant of Pseudomonas exotoxin A (ETA'). Then the fusion fragment was inserted into the bacterial vector pET32a(+). After isopropyl β-d-thiogalactoside (IPTG) induction, the 6× His tagged hscFv-ETA' protein was periplasmically expressed and purified by Ni(2+)-NTA column. The characteristics of the recombinant protein were assessed by cell membrane-ELISA, flow cytometry, and toxicity assays in CML cell lines and CML patient cells. The recombinant immunotoxin showed significant toxicity toward the CML cell lines K562 and KU812 as tested by MTT and apoptosis assay. Approximately 37% of leukemia cells of CML patients were driven into apoptosis by hscFv-ETA' as measured by flow cytometric analysis. In conclusion, the hscFv-ETA' is efficacious against CML in vitro, providing the basis for a novel therapeutic strategy for the treatment of CML patients.
authors: Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics Designated by the Ministry of Education, Chongqing Medical University, 1 Yixueyuan Road, Yuzhong District, Chongqing 400016, PR China.
authors: Xiaoying
authors: X
authors: Zhu
authors: Kun
authors: K
authors: Tao
authors: Yajuan
authors: Y
authors: Li
authors: Shenfeng
authors: S
authors: Li
authors: Lijun
authors: L
authors: Zhang
authors: Dong
authors: D
authors: Wang
authors: Liang
authors: L
authors: Zhong
authors: Wenli
authors: W
authors: Feng
mesh_terms: D036002:ADP Ribose Transferases / Q000235:genetics; D017209:Apoptosis / Q000235:genetics; D001427:Bacterial Toxins / Q000235:genetics; D002469:Cell Separation; D003602:Cytotoxicity, Immunologic; D005098:Exotoxins / Q000235:genetics; D005434:Flow Cytometry; D005822:Genetic Vectors; D006801:Humans; D007167:Immunotherapy / Q000379:methods*; D000922:Immunotoxins / Q000235:genetics / Q000378:metabolism*; D020014:K562 Cells; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000276:immunology / Q000628:therapy*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics; D057127:Single-Chain Antibodies / Q000235:genetics; D037521:Virulence Factors / Q000235:genetics; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D000922:Immunotoxins; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
keywords: Chronic myeloid leukemia; Recombinant immunotoxin; Targeting therapy; hscFv