Immune Netw.  2012 Feb;12(1):33-39. 10.4110/in.2012.12.1.33.

Generation of 1E8 Single Chain Fv-Fc Construct Against Human CD59

• Hong JW
• Cho WD
• Hong KP
• Kim SS
• Son SM
• Yun SJ
• Lee HC
• Yoon SS
• Song HG

• KMID: 2014654
• DOI: http://doi.org/10.4110/in.2012.12.1.33

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.

Keyword

CD59; 1E8 scFv-Fc; Complement dependent cytotoxicity (CDC)

MeSH Terms

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
Antibodies, Monoclonal
Complement System Proteins
Proteins
RNA, Messenger

Figure

• 

  Figure 1 Generation of the 1E8 scFv. (A) Amplification of the VH and VL genes from hybridoma 1E8 cells. Lane M, 100-bp DNA ladder; lane 1, VH PCR product; lane 2, VL PCR product; lane 3, VL-linker-VH PCR product. (B) Detection of the recombinant expression vector pOptiVEC-TOPO with 1E8 scFv-Fc. Lane M1, 1-kb DNA ladder; lane 4, construct digested with SfiI.

• 

  Figure 2 Nucleotide sequence and its deduced amino acid sequence of the 1E8-scFv. The shadowed part represents the linker.

• 

  Figure 3 Immunoreactivity of the anti-CD59 scFv-Fc for CCRF-CEM cells. The flow cytometry histogram demonstrates that 1E8 scFv-Fc binds to CCRF-CEM cells. (A) Negative control (FITC-labeled human IgG); (B) positive control (FITC-labeled mouse 1E8); (C) 1E8-scFv (FITC-labeled goat anti-human IgG).

• 

  Figure 4 Identification of purified recombinant 1E8 scFv-Fc on SDS-PAGE. Anti-CD59 scFv-Fc was purified using an immobilized protein A column and detected by Western blotting (A) and Coomassie blue staining (B). M, marker; LS, supernatant of the transfectant; FT, after Mabselect Sure purification; #1-3, washing buffer; #4-7, elution fraction; R, reducing conditions; NR, non-reducing conditions. Note clear bands of 120 kDa at elusion fraction (#5-7) and of 120 kDa at non-reducing and 55 kDa at reducing condition.

• 

  Figure 5 Effects of 1E8 scFv-Fc on complement-dependent cytotoxicity for CD59-expressing PC3 cells. (A) CD59 is highly expressed on PC3 cells. (B) 1E8 scFv-Fc enhances complement-dependent cytotoxicity in the presence of rabbit complement (RC) showing similar effect with mouse 1E8. negative control (IgG); mouse 1E8 (1E8). The numbers of viable cells were evaluated in a cytotoxicity assay.

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