Preclinical targeting of liver fibrosis with a 89Zr-labeled Fibrobody® directed against platelet derived growth factor receptor-ß.

PURPOSE: Hepatic fibrosis develops as a response to chronic liver injury, resulting in the formation of fibrous scars. This process is initiated and driven by collagen-producing activated myofibroblasts which reportedly express high levels of platelet derived growth factor receptor-ß (PDGFRß). We therefore regard PDGFRß as an anchor for diagnosis and therapy. The Fibrobody® SP02SP26-ABD is a biparatopic VHH-construct targeting PDGFRß. Here, we explore its potential as a theranostic vector for liver fibrosis. METHODS: Specificity, cross-species binding, and cellular uptake of SP02SP26-ABD was assessed using human, mouse and rat PDGFRß ectodomains and PDGFRß-expressing cells. Cellular uptake by PDGFRß-expressing cells was also evaluated by equipping the Fibrobody® with auristatinF and reading out in vitro cytotoxicity. The validity of PDGFRß as a marker for active fibrosis was confirmed in human liver samples and 3 mouse models of liver fibrosis (DDC, CCl4, CDA-HFD) through immunohistochemistry and RT-PCR. After radiolabeling of DFO*-SP02SP26-ABD with 89Zr, its in vivo targeting ability was assessed in healthy mice and mice with liver fibrosis by PET-CT imaging, ex vivo biodistribution and autoradiography. RESULTS: SP02SP26-ABD shows similar nanomolar affinity for human, mouse and rat PDGFRß. Cellular uptake and hence subnanomolar cytotoxic potency of auristatinF-conjugated SP02SP26-ABD was observed in PDGFRß-expressing cell lines. Immunohistochemistry of mouse and human fibrotic livers confirmed co-localization of PDGFRß with markers of active fibrosis. In all three liver fibrosis models, PET-CT imaging and biodistribution analysis of [89Zr]Zr-SP02SP26-ABD revealed increased PDGFRß-specific uptake in fibrotic livers. In the DDC model, liver uptake was 12.15 ± 0.45, 15.07 ± 0.90, 20.23 ± 1.34, and 20.93 ± 4.35%ID/g after 1,2,3 and 4 weeks of fibrogenesis, respectively, compared to 7.56 ± 0.85%ID/g in healthy mice. Autoradiography revealed preferential uptake in the fibrotic (PDGFRß-expressing) periportal areas. CONCLUSION: The anti-PDGFRß Fibrobody® SP02SP26-ABD shows selective and high-degree targeting of activated myofibroblasts in liver fibrosis, and qualifies as a vector for diagnostic and therapeutic purposes.

Combined inhibition of bile salt synthesis and intestinal uptake reduces cholestatic liver damage and colonic bile salts in mice.

Background & Aims: Intestine-restricted inhibitors of the apical sodium-dependent bile acid transporter (ASBT, or ileal bile acid transporter) are approved as treatment for several inheritable forms of cholestasis but are also associated with abdominal complaints and diarrhoea. Furthermore, blocking ASBT as a single therapeutic approach may be less effective in moderate to severe cholestasis. We hypothesised that interventions that lower hepatic bile salt synthesis in addition to intestinal bile salt uptake inhibition provide added therapeutic benefit in the treatment of cholestatic disorders. Here, we test combination therapies of intestinal ASBT inhibition together with obeticholic acid (OCA), cilofexor, and the non-tumorigenic fibroblast growth factor 15 (Fgf15)/fibroblast growth factor 19 (FGF19) analogue aldafermin in a mouse model of cholestasis. Methods: Wild-type male C57Bl6J/OlaHsd mice were fed a 0.05% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet and received daily oral gavage with 10 mg/kg OCA, 30 mg/kg cilofexor, 10 mg/kg ASBT inhibitor (Linerixibat; ASBTi), or a combination. Alternatively, wild-type male C57Bl6J/OlaHsd mice were injected with adeno-associated virus vector serotype 8 (AAV8) to express aldafermin, to repress bile salt synthesis, or to control AAV8. During a 3-week 0.05% DDC diet, mice received daily oral gavage with 10 mg/kg ASBTi or placebo control. Results: Combination therapy of OCA, cilofexor, or aldafermin with ASBTi effectively reduced faecal bile salt excretion. Compared with ASBTi monotherapy, aldafermin + ASBTi further lowered plasma bile salt levels. Cilofexor + ASBTi and aldafermin + ASBTi treatment reduced plasma alanine transaminase and aspartate transaminase levels and fibrotic liver immunohistochemistry stainings. The reduction in inflammation and fibrogenesis in mice treated with cilofexor + ASBTi or aldafermin + ASBTi was confirmed by gene expression analysis. Conclusions: Combining pharmacological intestinal bile salt uptake inhibition with repression of bile salt synthesis may form an effective treatment strategy to reduce liver injury while dampening the ASBTi-induced colonic bile salt load. Impact and Implications: Combined treatment of intestinal ASBT inhibition with repression of bile salt synthesis by farnesoid X receptor agonism (using either obeticholic acid or cilofexor) or by expression of aldafermin ameliorates liver damage in cholestatic mice. In addition, compared with ASBT inhibitor monotherapy, combination treatments lower colonic bile salt load.