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

Department of Internal Medicine I, Division of Endocrinology and Metabolism, University Hospital Bergmannsheil, Ruhr-University Bochum, Bürkle de la Camp Platz 1, 44789, Bochum, Germany.
Diabetologia (Impact Factor: 6.88). 04/2010; 53(7):1384-94. DOI: 10.1007/s00125-010-1725-9
Source: PubMed

ABSTRACT 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.
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.
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.
We report a simple strategy for the generation of an SCA targeting a novel beta cell-specific epitope.


Available from: Johannes W. Dietrich, May 14, 2015
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Replenishment of beta cell mass is a key aim of novel therapeutic interventions for diabetes, and the implementation of new strategies will be aided by understanding the mechanisms employed to regulate beta cell mass under normal physiological conditions. We have recently identified a new role for the gut hormone peptide YY (PYY) and the neuropeptide Y (NPY) receptor systems in the control of beta cell survival. PYY is perhaps best known for its role in regulating appetite and body weight, but its production by islet cells, the presence of NPY receptors on islets and the demonstration that Y1 activation causes proliferation of beta cells and protects them from apoptosis, suggest a role for this peptide in modulating beta cell mass. This review introduces PYY and its potential role in glucose homeostasis, then focuses on evidence supporting the concept that PYY and NPY receptors are exciting new targets for the preservation of beta cells.
    Diabetologia 06/2014; 57(9). DOI:10.1007/s00125-014-3292-y · 6.88 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The major aim of this paper is to review the present status of the techniques for the non-invasive imaging and quantification of insulin-producing pancreatic islet β-cells. Emphasis is placed on both the expansion of prior work already considered in a prior review and novel achievements. Thus, the use of d-mannoheptulose analogs, hypoglycemic sulfonylureas and glinides, neural imaging agents, neuro-hormonal receptor ligands and nanoparticles is first dealt with. Thereafter, consideration is given on optical imaging technologies, the identification of new β-cells specific binding and target proteins, the functional imaging of islets transplanted into the eye anterior chamber and in vivo manganese-enhanced magnetic resonance imaging.
    Diabetes research and clinical practice 07/2012; 98(1):11-8. DOI:10.1016/j.diabres.2012.07.001 · 2.54 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Diabetes mellitus affects 347 million people worldwide, and over 80 % of diabetes deaths occur in low- and middle-income countries. Type 1 diabetes (T1D) is characterized by the attacks of the body's own immune system on the pancreatic β-cells. In this work, we present a new CTLA-4 Ig targeting at the surface of β-cell and prepare it from Escherichia coli aiming at clearing activated T cells around β-cells and avoiding all-round decline in systematic immunity. This fusion protein is composed of CTLA-4-Ig part and β-cell-targeting part, with properties of the therapeutic effect of CTLA-4-Ig and selective binding to β-cells. In preliminary biological activity assay, our results verified the feasibility of β-cell-targeting strategy and its activity of CTLA-4-Ig part. The fusion protein recognizes and binds specifically to CD80(+) and CD86(+) cells as well as β-cell, but not to control cells, displaying the potential to be used as a feasible and effective treatment of T1D with lessened side effect.
    Cell Biochemistry and Biophysics 11/2014; 71(2). DOI:10.1007/s12013-014-0282-0 · 2.38 Impact Factor