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KMID : 0923620120120010033
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Immune Network
2012 Volume.12 No. 1 p.33 ~ p.39
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Generation of 1E8 Single Chain Fv-Fc Construct Against Human CD59
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Hong Jeong-Won
Cho Woon-Dong
Hong Kwon-Pyo
Kim So-Seul
Son Seung-Myoung
Yun Seok-Joong
Lee Ho-Chang
Yoon Sang-Soon
Song Hyung-Geun
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Abstract
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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.
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KEYWORD
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CD59, 1E8 scFv-Fc, Complement dependent cytotoxicity (CDC)
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