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KMID : 1004820110120030157
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Journal of Biomedical Research
2011 Volume.12 No. 3 p.157 ~ p.164
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Generation of 1D4 Single Chain Fv-Fc
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Hong Kwon-Pyo
Moon Yoo-Ri
Ji Hyeong-Jin
Ahn Byeong-Woo
Song Hyung-Geun
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Abstract
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Immunotherapeutic approaches using agonist antibodies or fusion proteins of immunomodulatory molecules significantly inhibit tumor growth and boost cell-mediated immunity. We isolated mRNA from previously reported 1D4 hybridoma cells and amplified the variable regions of the heavy chain (VH) and light chain (VL) genes using reverse-transcriptase polymerase chain reaction. 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. VL and VH fragments were cloned into the pOptiVEC-TOPO vector containing the human CH2-CH3 fragment. Then, 293T cells were transfected with the 1D4 single-chain Fv-Fc (scFv-Fc) constructs. A549 cells were used for presentation of the 1D4 antigen. Flow cytometry was performed for analysis of the secreted 1D4 scFv-Fc constructs. The DNA sequence of 1D4 scFv-Fc was obtained. The 1D4 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 lower than that of the murine 1D4 antibody for A549 cells. A 1D4 scFv-Fc construct for immunotherapy was developed.
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KEYWORD
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Costimulatory molecules, Immunotherapy, 1D4, Single chain Fv-Fc
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