University of Groningen Targeted Recombinant Fusion Proteins of IFN gamma and Mimetic IFN gamma with PDGF beta R Bicyclic Peptide Inhibits Liver Fibrogenesis In Vivo Bansal,

Hepatic stellate cells (HSCs), following transdifferentiation to myofibroblasts plays a key role in liver fibrosis. Therefore, attempts to attenuate this myofibroblastic phenotype would be a promising therapeutic approach. Interferon gamma (IFNc) is a potent anti-fibrotic cytokine, but its pleiotropic receptor expression leading to severe adverse effects has limited its clinical application. Since, activated HSC express high-level of platelet derived growth factor beta receptor (PDGFbR), we investigated the potential of PDGFbR-specific targeting of IFNc and its signaling peptide that lacks IFNcR binding site (mimetic IFNc or mimIFNc) in liver fibrosis. We prepared DNA constructs expressing IFNc, mimIFNc or BiPPB (PDGFbRspecific bicyclic peptide)-IFNc, BiPPB-mimIFNc fusion proteins. Both chimeric proteins alongwith IFNc and mimIFNc were produced in E.coli. The expressed proteins were purified and analyzed for PDGFbR-specific binding and in vitro effects. Subsequently, these recombinant proteins were investigated for the liver uptake (pSTAT1a signaling pathway), for antifibrotic effects and adverse effects (platelet counts) in CCl4-induced liver fibrogenesis in mice. The purified HSC-targeted IFNc and mimIFNc fusion proteins showed PDGFbR-specific binding and significantly reduced TGFb-induced collagen-I expression in human HSC (LX2 cells), while mouse IFNc and mimIFNc did not show any effect. Conversely, mouse IFNc and BiPPB-IFNc induced activation and dose-dependent nitric oxide release in mouse macrophages (express IFNcR while lack PDGFbR), which was not observed with mimIFNc and BiPPB-mimIFNc, due to the lack of IFNcR binding sites. In vivo, targeted BiPPB-IFNc and BiPPB-mimIFNc significantly activated intrahepatic IFNc-signaling pathway compared to IFNc and mimIFNc suggesting increased liver accumulation. Furthermore, the targeted fusion proteins ameliorated liver fibrogenesis in mice by significantly reducing collagen and a-SMA expression and potentiating collagen degradation. IFNc also induced reduction in fibrogenesis but showed significant decrease in platelet counts, which was restored with targeted proteins. These results suggest that these rationally designed proteins can be further developed as novel anti-fibrotic therapeutics. Citation: Bansal R, Prakash J, De Ruiter M, Poelstra K (2014) Targeted Recombinant Fusion Proteins of IFNc and Mimetic IFNc with PDGFbR Bicyclic Peptide Inhibits Liver Fibrogenesis In Vivo. PLoS ONE 9(2): e89878. doi:10.1371/journal.pone. 0089878 Editor: Rafael Aldabe, Centro de Investigación en Medicina Aplicada (CIMA), Spain Received October 1, 2013; Accepted January 24, 2014; Published February 24, 2014 Copyright: 2014 Bansal et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Work was supported by VICI grant-in-aid from Netherlands Organization for Scientific Research (NWO; http://www.nwo.nl/) and the Technical foundation (STW; http://www.stw.nl/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: KP is scientific advisor and shareholder of BiOrion Technologies B.V. , a company dedicated to the commercialization of drug carriers, including the carrier presented here. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: R.Bansal@utwente.nl