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

The corneal endothelium is a monolayer of metabolically active cells that lines the inner surface of the cornea. It has the important function of regulating fluid flow into the corneal stroma, thereby maintaining its clarity1,2. Because human corneal endothelial cells (hCECs) do not regenerate in vivo3,4, excessive loss of these cells due to aging, disease, or surgical trauma5 will eventually lead to corneal swelling and visual impairment6. Endothelial dysfunction is a leading cause of corneal blindness worldwide and the major indication for corneal transplantation7. While advanced surgical procedures can selectively replace the endothelial layer (e.g. Descemet’s stripping endothelial keratoplasty or DSEK), the number of patients requiring transplants far exceeds the number of available donor corneas8. To overcome the shortage of donor tissue, current research aims to induce proliferation of primary hCECs in culture9,10,11,12, or to generate hCECs from progenitor or stem cells13,14,15. Progress in this area is hampered by a lack of definitive markers to identify hCECs. Two widely used markers reported for cultured hCECs are pump-associated protein Na+/K+-ATPase16 and tight-junction protein ZO-117. In the cornea, the co-expression of these proteins indicates the presence of functional components on the corneal endothelium, but these markers are nonspecific and are expressed in other tissues including the heart18, brain19, and kidney20. Other groups have previously reported the generation of antibodies against corneal endothelial cells. Several groups used a whole-cell immunization approach to isolate mouse monoclonal antibodies against hCECs21,22,23,24, but these antibodies cross-reacted with several other human tissues. Since hCEC-specific cell surface antigens are lacking, the goal of this study was to identify antibodies that can bind to the surface of corneal endothelium and cultivated hCECs, but do not bind stromal keratocytes and fibroblasts. Several approaches could be used to identify antibodies against cell surface markers. If the surface antigen is known, then one strategy would be to express the soluble extracellular domain, which could be used to immunize animals or be used as target for antibody phage display. Other methods include whole-cell immunization or panning on whole cells using phage display. Since no specific markers have been identified for hCEC, we opted to use antibody phage display to isolate binders for these cells. Antibody phage display is a versatile technique that can be used against a wide variety of targets, including cell surface antigens25,26,27. In this study, we used a phage library based on the naive human repertoire to isolate a single-chain variable fragment (scFv) that binds selectively to the human corneal endothelium. We reformatted it to a full immunoglobulin and identified its target antigen by immunoprecipitation and mass spectrometry.