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Abstract 687 Rationale: Anticalins® have been developed as a new class of therapeutic proteins based on human lipocalins. We reasoned that given the diversity of compounds that these molecules naturally bind, it should be possible to engineer and identify Anticalins with specificity for small, biologically active compounds, including constrained peptides. To test this hypothesis, we asked whether it would be possible to identify an engineered lipocalin that could specifically block hepcidin, a 25 amino acid hepatic hormone. The central role of hepcidin in regulating iron homeostasis through its interaction with the only known cellular iron exporter ferroportin is supported by human genetics and preclinical data and suggests that sub-population of patients with anemia, exhibiting elevated serum levels of hepcidin, may be responsive to therapies directed at blocking hepcidin. Hepcidin-specific antagonistic Anticalins with different pharmacokinetic properties were generated to develop Anticalin drug candidates with different PK/PD relationships as different clearance rates of the Anticalin-hepcidin complex may be desirable when used as a therapeutic approach in different patient populations. Methods: State of the art phage display technology and high throughput screening were used to isolate a human lipocalin-derived Anticalin to specifically bind and antagonize hepcidin. Randomization of Anticalin binding loops and affinity-based phage display selection were used for affinity maturation and optimization of drug-like properties. Anticalins (MW ∼21 kDa) were produced in E.coli and subjected to site-directed PEGylation with different size PEG molecules. Affinity constants for hepcidin from different species were determined using ELISA-based assays and surface plasmon resonance. We then examined the ability of these Anticalin drug candidates to neutralize human hepcidin activities in cellular and in vivo assays. In addition, PK properties were determined in different animal species to predict PK properties in humans by allometric scaling. Results and Discussion: By using 2 different assay formats the lead candidate displayed high affinity (sub-nM) against human hepcidin and the extension of its plasma half-life by site directed PEGylation did not impact target binding. For example, in an SPR kinetic assay where the PEGylated Anticalin was immobilized and human hepcidin used as analyte, a dissociation constant of Kd = 50±3 pM (n=3) was determined for an Anticalin conjugated with a 40 kDa branched PEG molecule. A stable cell line expressing ferroportin fused to green fluorescent protein was established to determine blockade of hepcidin in vitro by measuring hepcidin-induced ferroportin (FPN) internalization and degradation. Hepcidin bioactivity was completely inhibited by the PEGylated Anticalin at concentrations at or above ∼40 nM (n=6), thus demonstrating the ability of the Anticalin to neutralize hepcidin's principal biological activity on the iron exporter FPN. Furthermore, the ability of PRS-080 to neutralize short-term hypoferremic effects was evaluated in mice after stimulation via a single intraperitoneal injection of 1 mg/kg synthetic human hepcidin. The PEGylated version of PRS-080, administered intravenously several hours prior to hepcidin injection, completely prevented the hypoferremic response at a dose of 95 mg/kg and showed partial prevention at 30 mg/kg (59%) and 9.5 mg/kg (23%). Tunable PK properties were demonstrated with an additional set of PEGylated molecules, tested in rats at a dose of 10 mg/kg, and exhibited terminal half-lives of 9.2, 14.2, 20.4, and 40 hours for Anticalins conjugated with 12, 20, 30 kDa linear and 40 kDa branched PEG, respectively. Conclusions: Our data describe the discovery and characterization of a hepcidin-antagonistic Anticalin for the treatment of anemia. PRS-080 displays numerous differentiating features posited to be relevant for the proposed therapeutic concept of inhibiting hepcidin-mediated hypoferremic effects, including: binding of its relatively small target with high affinity and specificity (pocket binding), favourable safety and tolerability (human scaffold, lack of immune effector cell interactions), tunable half-life and low production costs (bacterial expression). Anticalins provide a powerful novel therapeutic approach to develop antagonists against hepcidin and bioactive peptides in general. Disclosures: Hohlbaum: Pieris AG: Employment, Patents & Royalties. Trentman:Pieris AG: Employment, Patents & Royalties. Gille:Pieris AG: Employment, Patents & Royalties. Allersdorfer:Pieris AG: Employment. Belaiba:Pieris AG: Employment. Huelsmeyer:Pieris AG: Employment, Patents & Royalties. Christian:Pieris AG: Employment, Patents & Royalties. Sandal:Pieris AG: Employment. Matschiner:Pieris AG: Employment, Patents & Royalties. Jensen:Pieris AG: Employment, Patents & Royalties. Skerra:Pieris AG: Consultancy, Patents & Royalties, Research Funding. Audoly:Pieris AG: Employment.