Integrated vector system for antibody display and selection
A phagemid vector, pDR-D1 (Figure 1) was used for cloning and display of antibody repertoire as described previously . The vector includes pelB signal sequences, SfiI-SfiI restriction enzyme sites for scFv cloning and sequences for myc tag between lac Z promoter and gene III. Also, hexahistidine (His6) tag is designed to be linked to myc tag after removing gene III using NotI-NotI restriction enzyme sites for soluble expression and purification of scFv in E.coli.
For rapid conversion of phage displayed scFv into Fc-fusion form, a mammalian expression vector, pDR-OriP-Fc1 (Figure 2) was used as described previously . Briefly, the vector was constructed by reengineering pcDNA3.1 (Invitrogen, Carlsbad, CA) to contain human Fcγ1 sequences and hinge region followed by scFv cloning sites as well as OriP sequences. The hybrid signal peptide sequences consisting of leader sequences of kappa chain of mouse IgG and pelB were also included upstream of SfiI-SfiI sites for direct cloning of scFv sequences from pDR-D1.
CD9 expression constructs and cell lines
Human CD9 cDNA was cloned into expression vector, pcDNA3.1/zeo(+) (Invitrogen). Human embryonic kidney-293 (HEK293) cells were transfected with the plasmid using Lipofectamine (Invitrogen). Stable transfectants were selected using 400 μg/ml of zeocine (Invitrogen) in Dubecco’s modified Eagle’s Medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Invitrogen). A stable transfectant, HEK293-CD9 was used for library panning and selection. Two human cancer cell lines were used for the characterization of CD9-specificity of selected antibodies. Human gastric cancer cell line, MKN-28 was maintained in RPMI-1640 medium (Invitrogen) supplemented with 10% FBS, and human hepatocellular carcinoma cell line, HepG2 in DMEM medium with 10% FBS.
Library construction and panning
A naïve mouse scFv library was constructed from BALB/c mice (Daehan Biolink Co., Umsong, Korea) by cloning the mouse antibody repertoire into pDR-D1 as described previously . Briefly, a mouse splenocyte suspension was prepared and subjected to total RNA extraction, cDNA synthesis, and PCR amplification of heavy and kappa chain variable regions (VH and VK). The amplified VH and VL containing BbsI site were linked by ligation, and the combined scFv repertoire was subjected to extension PCR with primers containing SfiI site. The amplified scFv inserts and pDR-D1 were digested with SfiI, and, after ligation, the final constructs were electroporated into E. coli ER2738 cells. Bacteriophages displaying the scFv repertoire were rescued by the infection of the transformed cells with VCSM13 helper phage (Stratagene, La Jolla, CA).
The rescued phage library was used for cell panning which was performed according to conventional protocols [8, 9] with slight modification. The library was pre-incubated with HEK293 cells in PBA (1% bovine serum albumin/0.02% sodium azide/PBS) at 4°C for 1 h. The subtracted phages were recovered by centrifugation and then incubated with HEK293-CD9 cells at 4°C for 1 h. The cells were washed with ice-cold PBA four times, and the bound phages on the cell surface were amplified by infecting ER2738 cells followed by helper phage super-infection. The amplified phages were then subjected to another round of panning.
Expression and purification of scFv-Fc
After third round of panning, phagemid DNA was extracted from the ER2738 cells infected with the enriched phages. The scFv inserts were cut out of the phagemid by SfiI-digestion and directly cloned into pDR-OriP-Fc1. Twenty clones were randomly selected and subjected DNA sequencing. The nucleotide sequences determined were submitted to IMGT/V-QUEST (http://imgt.cines.fr/vquest) for sequence analysis. As a negative control, scFv against Hepatitis A virus was constructed from VH and VK sequences of HA6  and also cloned into pDR-OriP-Fc1.
The resulting scFv-Fc expression plasmid was introduced into 293E cells (CRL-10852, ATCC) using Lipofectamine. The transfected cells were grown in DMEM containing 10% FBS and subsequently, the media were changed to serum-free media, and the culture supernatant was harvested every 3 day. The culture supernatant was confirmed for scFv-Fc expression by western blot and used for flow cytometric screening of specific binders. For purification of scFv-Fc, the culture supernatant was subjected to affinity chromatography on a Protein G-agarose column (Merck Millipore, Darmstadt, Germany) as described previously .
For the screening of scFv-Fcs binding to HEK293-CD9, flow cytometric analysis was performed using the culture supernatant. HEK293 and HEK293-CD9 cells were grown to 70–80% confluence and harvested by trypsinization. About 1 × 106 cells were washed with ice-cold PBS and then blocked with PBA for 10 min at 4 °C. 100 μL of the culture supernatant was added to the cells and incubated for 1–2 h at 4 °C. After washing with PBA, fluorescein isothiocyanate (FITC)-conjugated rabbit anti-human IgG (Fc-fragment specific, Pierce, Rockford, IL) was added to the cells for the detection of bound scFv-Fcs. After 30 min incubation, the cells were washed twice with PBA and resuspended in PBS containing propodium iodide (PI). PI negative cells were gated and analyzed for scFv-Fc binding by using FACSCalibur (BD Bioscience, San Jose, CA) and Cell Quest software (BD Bioscience).
Staining of cancer cell lines with purified scFc-Fv was also performed on MKN-28 and HepG2 cells. Cells grown to 70–80% confluence were harvested and stained as described above. For comparison, a monoclonal antibody (mAb) against CD9, clone MM2/57 (Merck millipore) was used as positive control, and its binding to the cells was detected using FITC-conjugated goat anti-mouse IgG (Pierce).
Immunoprecipitation and immunoblotting
The binding specificity of scFv-Fc to CD9 was tested by immunoprecipitation and immunoblotting. HEK293-CD9 cells grown to 70-80% confluence were lysed at 4 °C in a lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 5 mM sodium fluoride, 1 mM ethylenediaminetetraaceticacid (EDTA), 1 mM ß-glycerophosphate, Xpert Protease Inhibitor Cocktail solution (GenDEPOT, Barker, Tx)). After removing insoluble fraction by centrifugation, the cell lysate was depleted with protein G-agarose for 30 min at 4 °C. The depleted cell lysate was then incubated with scFv-Fc or MM2/57 for 3 h at 4 °C. The immune complexes were captured on protein G-agarose and washed three times with the lysis buffer. After treatment with non-reducing protein loading buffer, the immunoprecipitates were resolved by 14% SDS-PAGE and transferred to PVDF membranes. The membrane was blocked with 4% skim milk in PBS and incubated with MM2/57. After washing with 0.01% Tween20 in PBS, the bound antibody was detected with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Fc specific, Jackson ImmunoResearch Laboratories, West Grove, PA).
Immunofluorescence confocal microscopy
Confocal laser scanning microscopy was performed on MKN-28 and HepG2 cells to visualize cell surface binding of scFv-Fcs. The glass coverslips were coated with 0.1% gelatin overnight, washed once with PBS and dried overnight. The cells were seeded on the gelatin-coated coverslips at 2 × 105 cells/slip and allowed to adhere for 48 h at 37 °C. After washing with ice cold PBS twice, the cells were incubated with scFv-Fc or MM2/57 for 2 h at room temperature. Unbound antibody was removed by washing with ice-cold PBS, and the cells were fixed with 4% formalin solution at room temperature for 15 min. After three times washing with ice-cold PBS, bound scFv-Fc and MM2/57 were stained with FITC-conjugated rabbit anti-human IgG and FITC-conjugated goat anti-mouse IgG1, respectively. Finally, coverslips were mounted by applying Vectashield solution (Vector laboratories, Burlingame, CA) and, the stained cells were observed with a confocal laser scanning microscope (Carl zeiss 510 META, Carl Zeiss, Oberkochen, Germany).