Generation of 1E8 Single Chain Fv-Fc Construct Against Human CD59
Immune Network
; : 33-39, 2012.
Article
em En
| WPRIM
| ID: wpr-39026
Biblioteca responsável:
WPRO
ABSTRACT
BACKGROUND:
Therapeutic approaches using monoclonal antibodies (mAbs) against complement regulatory proteins (CRPsi.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.Palavras-chave
Texto completo:
1
Índice:
WPRIM
Assunto principal:
Neoplasias da Próstata
/
Proteínas do Sistema Complemento
/
RNA Mensageiro
/
Sequência de Bases
/
Proteínas
/
Linhagem Celular
/
Reação em Cadeia da Polimerase
/
Células Clonais
/
Citometria de Fluxo
/
Hibridomas
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Immune Network
Ano de publicação:
2012
Tipo de documento:
Article