Generation of 1E8 Single Chain Fv-Fc Construct Against Human CD59.
- Author:
Jeong Won HONG
1
;
Woon Dong CHO
;
Kwon Pyo HONG
;
So Seul KIM
;
Seung Myoung SON
;
Seok Joong YUN
;
Ho Chang LEE
;
Sang Soon YOON
;
Hyung Geun SONG
Author Information
- Publication Type:Original Article
- Keywords: CD59; 1E8 scFv-Fc; Complement dependent cytotoxicity (CDC)
- MeSH: Animals; Antibodies, Monoclonal; Base Sequence; Cell Line; Centers for Disease Control and Prevention (U.S.); Clone Cells; Complement System Proteins; Flow Cytometry; Humans; Hybridomas; Light; Mice; Polymerase Chain Reaction; Prostatic Neoplasms; Proteins; RNA, Messenger
- From:Immune Network 2012;12(1):33-39
- CountryRepublic of Korea
- Language:English
- Abstract: BACKGROUND: Therapeutic approaches using monoclonal antibodies (mAbs) against complement regulatory proteins (CRPs:i.e.,CD46,CD55 and CD59) have been reported for adjuvant cancer therapy. In this study, we generated a recombinant 1E8 single-chain anti-CD59 antibody (scFv-Fc) and tested anti-cancer effect.by using complement dependent cytotoxicity (CDC). METHODS: We isolated mRNA from 1E8 hybridoma cells and amplified the variable regions of the heavy chain (VH) and light chain (VL) genes using reverse-transcriptase polymerase chain reaction (RT-PCR). Using a linker, the amplified sequences for the heavy and light chains were each connected to the sequence for a single polypeptide chain that was designed to be expressed. The VL and VH fragments were cloned into the pOptiVEC-TOPO vector that contained the human CH2-CH3 fragment. Then, 293T cells were transfected with the 1E8 single-chain Fv-Fc (scFv-Fc) constructs. CD59 expression was evaluated in the prostate cancer cell lines using flow cytometry. The enhancement of CDC effect by mouse 1E8 and 1E8 scFv-Fc were evaluated using a cytotoxicity assay. RESULTS: The scFv-Fc constructs were expressed by the transfected 293T cells and secreted into the culture medium. The immunoreactivity of the secreted scFv-Fc construct was similar to that of the mouse 1E8 for CCRF-CEM cells. The molecular masses of 1E8 scFv-Fc were about 120 kDa and 55 kDa under reducing and non-reducing conditions, respectively. The DNA sequence of 1E8 scFv-Fc was obtained and presented. CD59 was highly expressed by the prostate cancer cell line. The recombinant 1E8 scFv-Fc mAb revealed significantly enhanced CDC effect similar with mouse 1E8 for prostate cancer cells. CONCLUSION: A 1E8 scFv-Fc construct for adjuvant cancer therapy was developed.
