The prostate cancer cell line PC3 was maintained in Dulbecco's modified Eagle's medium (DMEM) that was supplemented with 10% fetal bovine serum (FBS). Rabbit complement (Cedarlane, Burlington, ON, Canada) was reconstituted in 1 ml of ice-cold distilled water, passed through a filter with pore size of 0.22µm, and stored at -20℃ until use. The EZ-Cytox Cell Viability Assay Kit (Daeil Lab, Seoul, Korea) was used.

The PC3 cells were seeded onto culture plates and allowed to adhere for 24 h. Cells (10⁶) were incubated with saturating amounts of the purified mouse 1E8 or a control immunoglobulin for 30 min at 4℃. Thereafter, the cells were washed twice with phosphate-buffered saline (PBS), incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary antibody for 20 min at 4℃, and washed twice with PBS. The cells were fixed with 1% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA). Cells were analyzed in a FACS apparatus (Beckman Coulter, Brea, CA, USA).

PC3 cells (5×10³) in 100µl of DMEM supplemented with 10% FBS were seeded into a 96-well plate (SPL Lifesciences, Pocheon, Korea). The plates were incubated overnight at 37℃ in an atmosphere of 5% CO₂, to allow the cells to adhere to the plate. The culture supernatant was removed, and the antibody (dissolved in serum-free DMEM at a concentration of 10µg/ml), either alone or in combination with 5% rabbit complement, was added to the cells. Cells in fresh medium without antibody or complement were used as the control. The plates were incubated for 3 h at 37℃. After the incubation period, 10µl of the solution from the EZ-Cytox Cell Viability Assay kit were added to each well and incubated at 37℃ in 5% CO₂ for 4 h. The extent of the reduction to formazan within the cells was quantified by measuring the absorbance at 450 nm in an ELISA reader.

The hybridoma cell line RV1E8, which produces anti-CD59 antibody, was cultured in RPMI medium that contained 10% heat-inactivated FBS (GIBCO). The culture was incubated under at 37℃ in 5% CO₂. After the cells had grown to a density of 10⁶ cell, total RNA was purified using the Easy-BLUE RNA Extraction kit (iNtRON Biotechnology, Daejon, Korea).

The genes that encode the variable heavy and light chains of the anti-CD59 antibody were derived using multiple overlap polymerase chain reaction (PCR). The sequences of the variable segments of the heavy and light chains were amplified using: the sense primers 5'-TCCTCCTCTGGTGGCGGTGGCTCGGGCGGTGGTGGGCAGGTGCAGCTGAAG-3' (VH) and 5'-GGTTCCACTGGTGACGTGGCCCAGGCGGCCGACATCCAG ATGACT-3' (VL); and the antisense primer: 5'-AGATTTGGGCTCAGCGGCCCCACCGGCCCCTGAGGAGACGGTGAC-3' (VH) and 5'-ACCGCCACCAGAGGAGGAAGATCTAGAGGAACCACCTCTGATTTCCAACTT-3' (VL), so as to introduce SfiI restriction enzyme sites (underlined). The amplified VH and VL genes were assembled into the scFv gene using a linker. We used the VL-F and VH-R primers that contained SfiI sites in the PCR reaction (for a total of 40 cycles of 1 min at 94℃, 1 min at 60℃, and 1.5 min at 72℃), and purified the amplicons using the QIAquick gel extraction kit (Qiagen, Hilden, Germany), and confirmed by DNA sequencing (Cosmo Genetech, Seoul, Korea). The scFv DNA products were digested with SfiI, gel-purified, and then ligated into the pOptiVEC-TOPO expression vector (Dinona, Seoul, Korea), which contains the human CH2-CH3 fragment.

The day before transfection, 293T cells was cultured at 37℃ in 5% CO₂ to a density of about 5×10⁵ cells/ml in 6-well plates in DMEM medium that contained 10% heat-inactivated FBS. The cultures were 80% confluent on the day of transfection. The scFv vector was transfected into 293T cells using the Effectene transfection reagent (Qiagen). After incubation for 3 days, the culture supernatant was tested by flow cytometry.

The supernatant of the transiently transfected cells was applied to a recombinant protein column (GE Healthcare, Uppsala, Sweden) that had been previously equilibrated with binding buffer (20 mM sodium phosphate [pH 7.2], 0.15 M NaCl). The column was washed with binding buffer, and protein was eluted with elution buffer (0.1 M glycine-HCl [pH 3.0]). The eluted fractions were collected in tubes that contained neutralizing buffer (1 M Tris-HCl, [pH 8.0]), so as to adjust the pH to approximately 7. Finally, the purified samples were dialyzed against PBS. The purity of the eluted antibody fraction was analyzed by SDS-PAGE on 10% gels under reducing or non-reducing conditions. Protein bands were visualized by Coomassie blue staining. After electrophoresis, the proteins in the gel were transferred to a nitrocellulose membrane (Whatman, Dassel, Germany), which was then blocked with 5% skim milk in PBS. The membrane was incubated with a 1:5,000 dilution of HRP-conjugated goat anti-human IgG (Jackson ImmunoResearch Laboratories, Baltimore, MD, USA) for 1 h at room temperature, and then washed four times with PBST (PBS with 0.05% tween 20). Thereafter, the immunoreactive bands were visualized using an enhanced chemiluminescence detection system (ECL; Amersham Pharmacia Biotech, Uppsala, Sweden).

The VH and VL DNA fragments of mouse 1E8 were successfully amplified with the VH-F/R and VL-F/R primers, respectively. The size of the amplified VH DNA was 351 bp and that of the amplified VL DNA was 327 bp. Using VL and VH DNA, a 781bp DNA were overlapped by linking peptide inserted in the middle of VL and VH (Fig. 1A). The purified 1E8-scFv gene fragment was digested with SfiI and cloned into the pOptiVEC-TOPO transfer vector. The ligation product was transformed into competent cells and positive colonies were selected after incubation at 37℃. Plasmid DNA was extracted using a Mini-Prep kit (Qiagen). A positive clone was identified by restriction enzyme digestion (Fig. 1B). The sequencing results showed that 1E8-scFv contained the linking peptide (Fig. 2).

The constructs derived from subcloning into the pOptiVEC-TOPO vector were transfected into 293T cells using the Effectene transfection reagent. The supernatant of the transfected culture was tested for reactivity with CCRF-CEM cells by flow cytometry. The positive binding rates of the mouse 1E8 and 1E8 scFv-Fc were 97.8% and 98.8%, respectively (Fig. 3).

The recombinant antibody contained the human Fc fragment. Antibody from the supernatant was successfully purified in one step by affinity chromatography using an immobilized protein A column. The purified 1E8 scFv-Fc was analyzed by SDS-PAGE and Western blotting. SDS-PAGE disclosed discreate bands of 120 kDa at elution fraction (Fig. 4A). SDS-PAGE under reducing conditions revealed a single band of ~55 kDa. The molecular mass of the dimeric scFv-Fc under non-reducing conditions was ~120 kDa (Fig. 4B).

To examine the expression of CD59 antigens by prostate cancer cells, we assessed the binding of mouse 1E8 and 1E8 scFv-Fc on PC3 cells by FACS analysis. The mouse 1E8 and 1E8 scFv-Fc well recognized cell surface CD59 on PC3 cells (Fig. 5A). To compare the neutralizing activity of mouse 1E8 and 1E8 scFv-Fc, we tested the susceptibility of PC3 to complement-mediated damage. Approximately 20% and 25%, respectively, of the PC3 cells were sensitized by mouse 1E8 and 1E8 scFv-Fc to killing by complement (Fig. 5B).