Generation of Novel Single-Chain Antibodies by Phage-Display Technology to Direct Imaging Agents Highly Selective to Pancreatic β- or α-Cells In Vivo

Abstract

OBJECTIVENoninvasive determination of pancreatic β-cell mass in vivo has been hampered by the lack of suitable β-cell–specific imaging agents. This report outlines an approach for the development of novel ligands homing selectively to islet cells in vivo.RESEARCH DESIGN AND METHODSTo generate agents specifically binding to pancreatic islets, a phage library was screened for single-chain antibodies (SCAs) on rat islets using two different approaches. 1) The library was injected into rats in vivo, and islets were isolated after a circulation time of 5 min. 2) Pancreatic islets were directly isolated, and the library was panned in the islets in vitro. Subsequently, the identified SCAs were extensively characterized in vitro and in vivo.RESULTSWe report the generation of SCAs that bind highly selective to either β- or α-cells. These SCAs are internalized by target cells, disappear rapidly from the vasculature, and exert no toxicity in vivo. Specific binding to β- or α-cells was detected in cell lines in vitro, in rats in vivo, and in human tissue in situ. Electron microscopy demonstrated binding of SCAs to the endoplasmatic reticulum and the secretory granules. Finally, in a biodistribution study the labeling intensity derived from [125I]-labeled SCAs after intravenous administration in rats strongly predicted the β-cell mass and was inversely related to the glucose excursions during an intraperitoneal glucose tolerance test.CONCLUSIONSOur data provide strong evidence that the presented SCAs are highly specific for pancreatic β-cells and enable imaging and quantification in vivo.

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

“…Next, soluble SCA B5 was radioactively labelled and its binding properties were tested in INS-1, beta TC6 and AR42J cells in vitro. These characteristics were then compared with those of the beta cell-specific SCAs identified previously [18]. Of note, in contrast to the BRASIL method, this assay was performed at 37°C (except studies for binding affinity and binding sites).…”

“…Phage-display technology may be a suitable way to overcome these limitations [15][16][17]. By applying this approach to rats in vivo or freshly isolated rat islets in vitro, we have recently reported the isolation of single-chain antibodies (SCAs) highly specific for either pancreatic beta or alpha cells [18]. However, these approaches applied collagenase-digestion for islet isolation, which might alter substantially the surface profile of the islet cells.…”

“…The controls were a phage clone without an insert (Tomlinson library I), four beta cell-specific SCAs (SCA B1-B4) and a control SCA identified previously [18].…”

“…Binding properties of SCAs in vitro SCAs (100 µg protein) were radiolabelled with iodine-125 (100 mCi/ml; Hartmann Analytics, Braunschweig, Germany) and their binding properties to cell lines (INS-1, beta TC6 and AR42J) were analysed as previously described [8,18]. Briefly, 1.5 ml tubes containing cells (10 6 ) in 200 µl medium without FCS were placed in an incubator (5% CO 2 and 37°C) for 30 min.…”

A new view of the beta cell

“…A monoclonal antibody with these characteristics (recognising an uncharacterised antigen, IC2) was generated in 1986 and was subsequently used to probe beta cells in vivo, although the analyses of specificity and correlation with beta cell mass were performed in isolated pancreases [11,12]. The fact that this antibody is a large IgM class antibody and has a long plasma half-life is likely to reduce its application in human beta cell imaging owing to a poor signal-to-noise ratio [7] Promising probes were recently generated by panning a phage display in vivo and in vitro [13]. A single-chain antibody was identified that efficiently labels beta cells in vivo, and radiolabelling demonstrated a good correlation with beta cell mass in the isolated organ.…”