Single-chain antibodies for highly selective targeting of positron-emission-tomography (PET)-ligands and biomolecules to pancreatic islet cells in vivo
Zusammenfassung der Projektergebnisse
A major success was the generation of a fusion protein of the single chain antibody (SCA) B1 with a NF-κB essential modifier (NEMO)-Binding Domain (NBD) peptide, thereby creating a selective inhibitor of NF-κB activation in β-cells. Firstly, it was clearly shown that our SCA B1-NBD fusion protein binds highly selective to rat β-cells in vivo. Secondly, we observed that SCA B1-mediated in vivo delivery of the NBD peptide completely blocked IL-1β + IFN-γ- and TNF-α + IFN-γ-mediated induction of NF-κB as well as islet dysfunction in culture. Finally, repeated intravenous injection of SCA B1-NBD prior to multiple low-dose administration of streptozotocin did not only induce a striking resistance to diabetes development but also preserved β-cell mass in mice. In conclusion, our data show for the first time that a SCA B1-NBD fusion peptide reliably protects β-cells against cytokines in vitro and allows protection from diabetes development in mice in vivo. This is also a proof of concept that functional domains coupled to the SCA can be selectively targeted to ß-cells without loosing the function of the domain or the SCA. It opens the perspective to selectively influence ß-cell function also with other functional domains. Despite our extensive efforts non invasive estimation of the β-cell mass by PET imaging with 124-iodine labelled SCA antibody has been a major challenge. Rapid deionisation occurred in vivo and we found impurities of the SCA-antibody preparation as possible explanation for the low stability of the iodine-124 labelling. Therefore, we optimized the purification protocol and showed by cut and count experiments consistently highly specific accumulation of the purified iodine-labelled SCA in the pancreas. These findings are very promising; however, PET imaging data with these new preparations are not yet available. Intensive efforts have been undertaken to identify the target antigens. By means of various competition assays we could rule out that the SCA-antibodies target either insulin, GAD- 65, C-peptide or IAPP. Unfortunately, all atempts to identify any target antigen of the SCAs by western blotting or classical immunoprecipitation metods were without success, suggesting that the targeted-epitopes may be structure sensitive. Therefore we plan to perform cross-linking in living cells and/or animals followed by nano-HPLC/ESI-MS/MS.
Projektbezogene Publikationen (Auswahl)
- 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. Diabetologia 53:1384-94, 2010
Ueberberg S, Ziegler D, Schechinger W, Dietrich JW, Akinturk S, Klein HH, Schneider S
- Phage Library-Screening: A powerful approach for generation of targeting-agents specific for normal pancreatic islet-cells and islet-cell carcinoma in vivo. Regul Pept 160:1-8, 2010
Ueberberg S, Schneider S
- In vitro and initial in vivo evaluation of 68Galabeled transferring receptor (Tfr) binding peptides as potential carriers for enhanced drug transport into Tfr expressing cells. Mol Imaging Biol 13:332-41, 2011
Waengler C, Nada D, Höfner G, Maschauer S, Waengler B, Schneider S, Schirrmacher E, Wanner T, Schirrmacher R, Prante O
- Protection from diabetes development by single-chain antibody-mediated delivery of a NF-B inhibitor specifically to β-cells in vivo. Am J Physiol Endocrinol Metab. 301: 83-90, 2011
Ueberberg S, Deutschbein T, Klein HH, Dietrich JW, Akinturk S, Prochnow N, Schirrmacher R, Schneider S.
