Preparation And Anticancer Activity Of ScFvs And Immunotoxin ScFv-Gz B Against CD33

Background:Leukemia is a kind of malignant cloning disease caused by hematopoietic stem cell. According to the report, the incidence of leukemia is the sixth place in various tumors in our country. The main therapeutic approaches of leukemia are bone-marrow transplantation and chemotherapy at present, which often lead to recurrence and side effects. Immunotherapy is becoming a new method after bone-marrow transplantation and chemotherapy, since it has lower side effects and stronger specificity. It often achieves good clinical outcomes if combined with conventional chemotherapy and transplantation. With the progress of humanized antibody technology, antibody drugs have made breakthroughs in clinical treatment of cancer and are becoming the hot spot in global bio-pharmaceutical field.Objective:Our research laboratory had successfully identified several scFvs against CD33 extracellular domain (CD33-ECD) from a phage-displayed library. In order to achieve better anti-cancer effects, single-chain immunotoxins (sc-ITs) composed of scFv with human granular enzyme B(Gz B)were constructed and expressed for evaluation of anticancer efficiency.Methods:The scFv antibody genes were amplified by PCR using the corresponding primers, and inserted into the expression vector PET30a(+). The expression plasmid was transformed into the E. coli BL21, the scFvs were then expressed and detected. Through designing primers and linkers, DNA fragments encoding scFv and Gz-B were fused together. The recombinant scFvs and sc-ITs were expressed in E. coli, and further in a yeast expression system (P. pastoris) to achieve higher activity. The cytotoxicity was analysed using the CCK-8 method.Results:Three corresponding expression vectors of scFv and sc-IT were constructured successfully. The recombinant proteins were expressed in E. coli and P. pastoris, and purified by affinity chromatography. The bioactivity of the recombinant scFv and scIT was determined By ELISA and Western blotting. Our results indicated that the recombinant scFvs were able to bind to the antigen-CD33 strongly. The studies in cytotoxicity assay have shown that scFv-1 could kill CD33 positive leukemia cells.Conclusion:The three recombinant scFvs and their corresponding sc-ITs were expressed, purified and renatured successfully. The recombinant proteins have shown the CD33-binding ability and efficiency in killing CD33-positive leukemia cells in vitro.