In vitro phage display in a rat beta cell line: a simple approach for the generation of a single-chain antibody targeting a novel beta cell-specific epitope.

AIMS/HYPOTHESIS: The aim of the present study was to evaluate in vitro phage display in a beta cell line as a novel strategy for the isolation of beta cell-specific agents/biomarkers. METHODS: A single-chain antibody (SCA) library was pre-incubated with AR42J cells in order to eliminate SCAs with exocrine binding properties. It was then panned against INS-1 cells to select beta cell-targeted antibodies. RESULTS: By these means, we isolated a novel antibody, SCA B5, that binds rapidly (6.0 min) and with a 450-fold higher specificity to beta cells relative to exocrine cells. We estimated for SCA B5 a binding affinity in the low micromol/l range and 858 binding sites per beta cell. Confocal microscopy showed binding to the beta cell surface and confirmed subsequent internalisation. Moreover, staining of rat and human pancreatic tissue sections with SCA B5 suggests that the target epitope is presented in pancreatic beta cells of different origins. Infrared imaging revealed that labelling of beta cells with tracer SCA B5 is strictly dependent on beta cell mass. With competition assays we excluded insulin, glutamate decarboxylase, C-peptide and islet amyloid polypeptide as SCA B5 targets. In accordance with these predictions, SCA B5 homed in vivo highly selectively to normal beta cells and dysfunctional beta cells of diabetic rats. Moreover, accumulation of radioactively labelled SCA B5 in the pancreas was reduced by 80% after pre-injection with unlabelled SCA B5, thereby confirming the specific uptake in the pancreas. CONCLUSIONS/INTERPRETATION: We report a simple strategy for the generation of an SCA targeting a novel beta cell-specific epitope.

Phage library-screening: a powerful approach for generation of targeting-agents specific for normal pancreatic islet-cells and islet-cell carcinoma in vivo.

Phage display technology is a powerful approach for the generation of peptides and antibodies that target specific organ- or tumor-structures. By applying this approach to rats in vivo or freshly isolated rat islets in vitro, we have recently reported the successful isolation of internalizing single-chain antibodies (SCA-antibodies), which are highly specific for the endocrine-cells of a pancreatic islet (either beta- or alpha-cells) both in rodents and in humans. Moreover, others have reported peptides targeting specifically the vascular endothelium of normal or pre-malignant islets or advanced islet-cell tumors. The features of these antibodies and peptides are compatible with a potential use for in vivo delivery of molecular cargos (e.g. imaging agents and therapeutics). Therefore, this article reviews the principles of phage display, provides an overview about agents either specific for the endocrine-cells or the vascular endothelium of islets, discusses methododical key elements for the generation of these ligands and highlights remaining questions and potential future perspectives.

Synthesis and evaluation of a glibenclamide glucose-conjugate: a potential new lead compound for substituted glibenclamide derivatives as islet imaging agents.

The search for novel SUR1-ligands originates from the idea to influence the in vivo behaviour by adding new structural moieties to the glibenclamide structure while preserving its binding affinity. Important application of novel conjugates might be their use as radioactively labelled tracer probes in the non-invasive investigation of the islet mass. It is known that the imaging quality of a tracer could be improved by increasing its hydrophilicity, which leads to a reduced plasma protein binding and diminished the unspecific uptake by various organs. In this study the glucose molecule was chosen as a substitute of glibenclamide to enhance hydrophilicity. As expected glucose conjugation leads to a 12-fold increase of the hydrophilicity. In vitro evaluation showed that the conjugate binds with high affinity to SUR1. Interestingly, in vivo the hypoglycaemic action of the conjugate was of significant shorter duration compared to glibenclamide. In accordance, the conjugate was cleared much faster from the blood stream, due to a significant lower plasma protein binding. In conclusion, glycosylation proved to be a powerful tool for the development of a high affinity glibenclamide ligand with completely different pharmacodynamics. Therefore, the glucose-conjugate could be a potential lead compound for the design of substituted glibenclamide derivatives as islet imaging ligands.