Reagents For scFv and IgG purification and characterization: CHO-S-SFM II media and G418 (Invitrogen, Carlsbad, CA, USA), 1 L spinner flask (Corning, Corning, NY, USA), CellgroTM Stirrers (Barnstead, Dubuque, IA, USA), HisTrapTMHP, HiTrap protein A HP and HiTrap desalting columns (GE Healthcare, Piscataway, NJ, USA). For FACS: streptavidin-phycoerythrin (SA-PE) (Invitrogen/BioSource, Camarillo, CA, USA), PE-conjugated anti-human Fc-specific mAb (Jackson ImmunoResearch, West Grove, PA, USA), murine anti-ICAM-1 mAb (clone BBIG-l1) (R&D System, Minneapolis, MN, USA), and biotin-labeled rabbit anti-fd bacteriophage (Sigma-Aldrich, St. Louis, MO, USA). For immunoprecipitation: mammalian protease inhibitor cocktails (Sigma-Aldrich), NP40 and protein A agarose beads (Pierce, Rockford, IL, USA). For cell invasion assay: Matrigel Basement Membrane Mix (BD Biosciences, San Jose, CA, USA), Transwell (CorningCostar, Corning, NY, USA), and Diff-Quik (Dade Behring Inc, Newark, DE, USA).
LNCaP was purchased from American Type Culture Collection (ATCC) and C4-2B from DIANON/Urocor (Oklahoma City, OK, USA). The CHO-ICAM-1 line, a stably transfected CHO cell line expressing the full-length human ICAM-1 protein, was purchased from the ATCC. The CHO DG44 cell line (dhfr-) was purchased from Invitrogen. The BPH-1 line was originally obtained from Dr. Gerald Cunha [22
] at UCSF and maintained in the lab.
Selection of C4-2B-specific phage antibodies
For counter-selection, a naïve library containing 500 million members was incubated with LNCaP cells at 4°C for 8 h, centrifuged to collect the supernatants, and the counter-selected library was further incubated with C4-2B cells at 37°C for 2 h. The cells were then washed three times with ice-cold RPMI media containing 0.5% fetal bovine serum (FBS), once with 100 mM glycine containing 150 mM NaCl, pH 2.8, and once with phosphate-buffered saline (PBS), and lysed with 300 µl 100 mM triethylamine at RT for 5 min, neutralized with 150 µl 1 M Tris–HCl, and used to infect exponentially growing TG1 bacteria. The infected TG1 were plated on LB-agar plates containing tetracycline (50 µg/ml) and incubated at 37°C overnight. Colonies on the plate were scraped off and used to inoculate 2× YT media containing tetracycline (50 µg/ml) at 37°C for 12 h, the supernatants were collected by centrifugation and phage prepared by polyethylene glycol precipitation as described [16
]. The polyclonal phage antibodies were used as the input for the next round of selection using the same counter-selection then selection procedure described above.
Analysis of binding phage antibodies
FACS was used to screen for monoclonal phage antibodies that bind specifically to C4-2B but not LNCaP. Phage-infected bacteria grown on agar plates were picked into 96-well U bottom culture plates in 2× YT media containing tetracycline (50 µg/ml) and grown overnight at 37°C. After centrifugation, an identical amount of supernatants (50 µl per well per plate) containing phage particles were transferred into two V-bottom plates, and incubated with LNCaP and C4-2B cells, respectively, at RT for 1 h. Following washing with PBS, bound phage were detected by biotin-labeled anti-fd antibodies followed by streptavidin-phycoerythrin (SA-PE), using FACS LSRII (BD Biosciences, San Jose, CA, USA) as described [17
Recombinant human IgG1
The development of recombinant IgG1 from scFv has been described previously [23
]. We have previously constructed a mammalian expression vector that produces recombinant fully human M10A12 IgG1 using genetic information of the M10A12 scFv [23
]. CHO DG44 cells stably transfected with the M10A12 IgG1-expressing plasmid were grown in CHO-S-SFM II containing G418 in 1 L spinner flasks on CellgroTM
Stirrers as described [23
]. Supernatants were collected and purified on a HiTrap Protein A column using an AKTAprime (GE Healthcare).
Identification of the target antigen as ICAM-1/CD54/rhinovirus receptor
We used the M10A12 IgG1 to immunoprecipitate the target antigen from C4-2B lysates, and analyzed the product by tandem mass spectrometry. Ten million tumor cells were harvested, washed, and pelleted in an Eppendorf microcentrifuge tube. Cell pellets were resuspended in cell lysis buffer (1% NP-40 in PBS, pH 7.2, with mammalian protease inhibitor cocktails added at v
1:200 per manufacturer’s instruction) and incubated on ice for 1 h. Following centrifugation in an Eppendorf 5417R at 10,000×g
for 15 min, the supernatants were collected, pre-cleared by incubating with protein A beads on ice for 4 h, centrifuged at 10,000×g
at 4°C for 15 min to remove the beads, divided into two parts, and further incubated with the M10A12 IgG1 and a control non-binding human IgG1 respectively on ice for 4 h. Immunoprecipitation products of both the M10A12 IgG1 and the control IgG1 were analyzed on a gradient SDS-PAGE gel (4–20%, Invitrogen), stained with coomassie blue and bands unique to the M10A12 IgG immunoprecipitation products were excised, digested with trypsin, and analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Peptides were separated by reverse phase chromatography using an Ultimate HPLC (Dionex) and then analyzed on-line using a QSTAR Pulsar Mass Spectrometer (MDS Sciex/Applied Biosystems). Raw data was converted to peaklists using the Mascot dll in Analyst (version 1.6b16), then searched using Batch-Tag in Protein Prospector (version 5.0) [24
] against mammalian proteins in the SwissProt Database (downloaded June 2008: 52,897 entries searched), allowing a precursor mass accuracy tolerance of 50 ppm and a fragment mass tolerance of 0.1 Da. Acceptance criteria was a peptide expectation value of less than 0.05. To confirm the identification, CHO (control) and CHO cells stably transfected with the full-length human ICAM-1 gene were incubated with the M10A12 IgG1 at RT for 30 min, washed three times with PBS/0.5% FBS, further incubated with PE-conjugated anti-human Fc antibodies, and then analyzed by FACS. As a further control for background staining, the experiment was repeated using a recombinant anti-botulinum toxin human IgG1, CR-2, which does not bind to prostate cancer cells.
Cell invasion assay
MatriGel basement membrane was used as the matrix for the cell invasion assay. About 2.5
cells were mixed with 50 µg/ml IgGs at 37°C for 1 h. In the meantime, MatriGel was dissolved in RPMI media at 4°C and placed on the top chamber (insert) at 37°C to solidify. The cells were placed on top of the MatriGel layer and incubated for 48 h. Cells remaining in the top layer of the chamber were removed. After Diff-Quik staining, viable cells that migrated to the lower layer of the chamber were counted under an inverted microscope (Nikon, Japan). The experiments were performed in triplicate and the data were analyzed using a student t
test. A p
value of less than 0.05 was used as indication of a significant difference.