Isolation of a recombinant antibody specific for a surface marker of the corneal endothelium by phage display

Cell surface antigens are important targets for monoclonal antibodies, but they are often difficult to work with due to their association with the cell membrane. Phage display is a versatile technique that can be applied to generate binders against difficult targets. Here we used antibody phage display to isolate a binder for a rare and specialized cell, the human corneal endothelial cell. The human corneal endothelium is a medically important cell layer; defects in this layer account for about half of all corneal transplants. Despite its importance, no specific antigens have been found to mark this cell type. By panning a phage library directly on human corneal endothelial cells, we isolated an antibody that bound to these cells and not the other types of corneal cells. Subsequently, we identified the antibody’s putative target to be CD166 by immunoprecipitation and mass spectrometry. This approach can be used to isolate antibodies against other poorly-characterized cell types, such as stem cells or cancer cells, without any prior knowledge of their discriminating markers.


Isolation and culture of stromal fibroblasts
After the DM-endothelial layer has been peeled from the stroma, an 8.5 mm stroma button was obtained by trephination. The corneal epithelial layer was carefully scraped off using a scalpel blade. Stroma buttons were washed twice in a PBS-buffered antibiotic/antimycotic solution and enzymatically digested in collagenase overnight. The following day, stromal keratocytes released from within the stroma button were briefly washed twice with PBS, seeded onto cell culture flask coated with FNC coating mixture, and cultured in F99 medium. The exposure to a serum-supplemented medium transformed the corneal stromal keratocytes into fibroblasts.
Culture medium was refreshed every two days, and confluent fibroblast cultures were passaged using TrypLE Express (Life Technologies) in a 1:5 split ratio. Stromal fibroblasts from several donor corneas were pooled for this study.

Cell based scFv and phageELISA
To monitor efficient subtraction of fibroblast specific and enrichment of hCEC specific phages, polyclonal phage libraries before and after subtraction and/or panning round were collected and tested on fibroblasts and hCEC. Cells were grown to 90% confluency in FnC-coated 96 well plates (6K per well for hCEC, 4K per well for stromal fibroblasts). Cells were fixed with 10% formaldehyde for 10min, blocked with 400 µl 3% BSA per well for 2h at RT and washed 3 times with PBS-Tween20 (0.05%) for 5min. Polyclonal phages were added in PBS and incubated at RT for 2h. KM13 helper phage was included as negative control. After washing 3 times with PBS-T for 5min, bound phages were detected by HRP conjugated anti-M13 antibody (1:5000 in 2% BSA; GE Healthcare) and the assay was developed by addition of 100 µl TMB (Promega).
Reaction was stopped by the addition of 100 µl H 2 SO 4 and reading taken at OD 450nm .
To screen for hCEC-specific monoclonal scFv, well-isolated colonies were inoculated o/n at 37°C in 2xTY, ampicillin, 1% glucose. A vector clone was used as negative control. The overnight cultures were mixed 1:6 with 2xTY, ampicillin, 1% glucose and incubated for 4-5h at 37°C. Medium was exchanged with 2xTY, ampicillin, 0.4 M sucrose, 1 mM IPTG, cultures were incubated o/n at 30°C and pelleted. 100 µl of the supernatant was tested in parallel on cultivated hCEC and fibroblast as described for the phage ELISA, except that the detection was done by a HRP conjugated c-myc specific antibody (1:10 000 in PBS: Pierce).

Immunohistochemistry
For immunohistochemistry of frozen sections, human donor cornea was rinsed in PBS twice and immersed in OCT medium and frozen at -80°C until sectioning. Eight-micron thick sections were cut using a MicromHM550 cryostat and collected on glass slides and air-dried before

Flow cytometry
Primary hCECs and fibroblasts were dissociated as described earlier by, blocked with 10% goat serum in PBS at 37°C for 15min. Cells were washed 1 time with PBS and incubated for 45 min at 4°C with 4 µg/ml scFv or IgG conjugated to DyLight™ 488 (Innova Bioscience). Conjugation of fluorescent labels was performed according to manufacturer's instructions. Cells were washed 2 times with PBS, resuspended in 0.2 ml PBS and analyzed by flow cytometry (Accuri C6, BD Bioscience). Labelled hCECs and fibroblasts were analyzed separately and gating was performed to exclude debris and doublets.

In-gel tryptic digest and mass spectrometry
The excised protein band was pre-incubated with 150 µl of washing solution containing 2.5mM ammonium bicarbonate and 50% acetonitrile (ACN) for at least 24 h at 4oC. Fresh washing solution was replaced and the gel band was incubated at room temperature for 10 min before subjecting it to reduction in buffer containing 10mM dithiothreitol DTT)/100mM ammonium bicarbonate for 1 h at 56oC. The reducing solution was aspirated from the well, followed by the addition of 55mM iodoacetic acid (IAA)/100mM ammonium bicarbonate for a period of 45 min at room temperature in order for alkylation to occur. The gel was washed with 100µl of 100mM ammonium bicarbonate followed by ACN at intervals of 10 min. The washing process was repeated and the gel was finally dried by Speedvac (Savant). Trypsin solution (10µl of 20nl/ml) was added to each well and left overnight at 37 oC for 16 h. The peptides were extracted with 50% acetonitrile and 5% formic acid and sonicated on the water-bath sonicator for 10 min. After drying down the sample using Speedvac , the pellet was reconstituted in 5.5 µl of 2% methanol, 1% formic acid for LCMS/MS (Thermos LTQ Orbitrap Elite Mass Spectrometry). The results were searched against the human database on Mascot.