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Background and Purpose Anaemia of chronic disease (ACD) has been linked to iron-restricted erythropoiesis imposed by high circulating levels of hepcidin, a 25 amino acid hepatocyte-derived peptide that controls systemic iron homeostasis. Here, we report the engineering of the human lipocalin-derived, small protein-based anticalin PRS-080 hepcidin antagonist with high affinity and selectivity. Experimental Approach Anticalin- and hepcidin-specific pharmacokinetic (PK)/pharmacodynamic modelling (PD) was used to design and select the suitable drug candidate based on t1/2 extension and duration of hepcidin suppression. The development of a novel free hepcidin assay enabled accurate analysis of bioactive hepcidin suppression and elucidation of the observed plasma iron levels after PRS-080-PEG30 administration in vivo. Key Results PRS-080 had a hepcidin-binding affinity of 0.07 nM and, after coupling to 30 kD PEG (PRS-080-PEG30), a t1/2 of 43 h in cynomolgus monkeys. Dose-dependent iron mobilization and hepcidin suppression were observed after a single i.v. dose of PRS-080-PEG30 in cynomolgus monkeys. Importantly, in these animals, suppression of free hepcidin and subsequent plasma iron elevation were sustained during repeated s.c. dosing. After repeated dosing and followed by a treatment-free interval, all iron parameters returned to pre-dose values. Conclusions and Implications In conclusion, we developed a dose-dependent and safe approach for the direct suppression of hepcidin, resulting in prolonged iron mobilization to alleviate iron-restricted erythropoiesis that can address the root cause of ACD. PRS-080-PEG30 is currently in early clinical development.

Antagonising the IL-4 receptor α (IL-4Rα) interferes with downstream IL-4/IL-13 signalling and clinically has beneficial effects in moderate to severe asthma. Anticalin® proteins are based on human lipocalins and can be engineered to bind specific ligands with high potency and selectivity. AZD1402/PRS-060, is an inhaled Anticalin protein targeting the IL-4Rα, being developed as a treatment for asthma. Potency was established using cell based assays (TF1 cell pSTAT6 activation and goblet cell differentiation). Pharmacodynamic and asthma models were developed in mice expressing human IL-4Rα, IL-4 and IL-13. AZD1402/PRS-060 was identified as a potent (KD 23 pM) and selective antagonist of IL-4Rα. AZD1402/PRS-060 and pitrakinra (hIL-4 mutein) inhibited IL-4 and IL-13 induced pSTAT6 stimulation of TF1 cells (IC50 0.1 and 8.6 nM, respectively). AZD1402/PRS-060 and dupilumab (IL-4Rα mAb) inhibited IL-4 and IL-13 induced proliferation of TF1 cells with similar potency (IC50 0.9 and 0.3 nM). In an Air Liquid Interface goblet cell differentiation assay induced by IL-13 stimulation, AZD1402/PRS-060 inhibited goblet cell metaplasia to a similar extent as AMG317 (IL-4Rα mAb) and pitrakinra. In vivo, 2 µg of AZD1402/PRS-060 (IT) inhibited IL-13 induced eotaxin expression over 24-hours. In the OVA model, inflammatory cell infiltration was reduced by 78% and TARC (CCL17) was reduced by 98% 48 h post challenge. AZD1402/PRS-060, potently inhibits IL-4 and IL-13 signalling in vitro and in vivo and based on these data and the proven benefit of targeting this receptor, AZD1402/PRS-060 has progressed into human clinical trials.

Anticalins are engineered ligand-binding proteins based on the human lipocalin scaffold. Their architecture is characterized by a rigid beta-barrel that supports four structurally hypervariable loops. Similar to antibodies, these loops form the natural ligand-binding site, usually for vitamins, hormones or secondary metabolites. Anticalins with novel specificities can be engineered by reshaping this loop region, using targeted random mutagenesis in combination with functional display and guided selection. Several drug candidates with specificities for exogenous low-molecular-weight substances, peptides and even protein targets (e.g., several disease-related cell surface receptors) have been obtained in this way. Owing to their exquisite specificity and high affinity, Anticalins are particularly attractive as antagonists for the manipulation of immune mechanisms, leading to either inhibitory or stimulatory effects. Compared with antibodies, Anticalins offer several practical advantages as they are much smaller, consist of a single polypeptide chain and can be produced easily in microbial expression systems.

Monoclonal antibodies (mAb) have transformed medicine but their ∼160 kD molecular weight and fragility make inhalation technically difficult, limiting utility in lung disease. Lipocalin-1 (LCN1) is a 17kD endogenous non-glycosylated, nest-like protein physiologically secreted at human mucosal surfaces, esp. lung and conjunctiva, and transports lipophilic molecules in a high-affinity binding pocket whose three loops resemble mAb CDR domains. Systemic mAbs targeting IL4Ra (CD123) block TH2 immunity and are promising anti-asthmatic agents, but with potential limitations of reaching the mucosal surface at high concentrations and systemic safety. We therefore used phage display to systematically randomize and affinity-mature the LCN1 binding pocket creating PRS-060, an inhalable ultra-specific “anticalin protein” that competitively and reversibly blocks hIL4Ra, inhibiting hIL-4 (0.1nM) induced TF-1 cell phospho-STAT6 with sub-nM (0.12) pEC 50 . In predictive in silico and human assays PRS-060 showed low untoward immunogenic liability, comparable to trastuzumab (Herceptin ®) , and was very stable in solution aerosol or dry powder inhalation formulations (non-aggregating, HP-SEC/RP-SEC stable). Kinetic in vivo PK/PD and allometric scaling demonstrate suitability for once daily inhalation with rapid renal excretion and no systemic accumulation. Because of limited species cross-reactivity of humanized tg mice were utilized to demonstrate in vivo activity in experimental lung inflammatory models.These data suggest PRS-060 as a potentially promising inhalable IL4Ra blocker and putative therapy for the TH2-high endotype of moderate and severe human asthma.

Autoreactive B cells are present in the lymphoid tissues of healthy individuals, but typically remain quiescent. When this homeostasis is perturbed, the formation of self-reactive antibodies can have serious pathological consequences. B cells expressing an antigen receptor specific for self-immunoglobulin-gamma (IgG) make a class of autoantibodies known as rheumatoid factor (RF). Here we show that effective activation of RF+ B cells is mediated by IgG2a-chromatin immune complexes and requires the synergistic engagement of the antigen receptor and a member of the MyD88-dependent Toll-like receptor (TLR) family. Inhibitor studies implicate TLR9. These data establish a critical link between the innate and adaptive immune systems in the development of systemic autoimmune disease and explain the preponderance of autoantibodies reactive with nucleic acid-protein particles. The unique features of this dual-engagement pathway should facilitate the development of therapies that specifically target autoreactive B cells.

Fas ligand (FasL) has been shown to mediate both apoptotic and inflammatory reactions. To rigorously assess the physiological role of different forms of the FasL molecule with regard to these two distinct processes, we isolated stably transfected lymphoma cell lines that expressed either murine wild-type FasL, membrane-only FasL, or functionally distinct forms of soluble FasL. First, the ability of these lines to induce an inflammatory response was assessed in vivo by injecting the transfectants intraperitoneally and measuring subsequent neutrophil extravasation into the peritoneal cavity. Second, lines were assessed by injecting the transfectants subcutaneously and monitoring their growth as solid tumors. Our study clearly demonstrated that the extent of inflammation induced by the transfectants directly correlated with their relative cytotoxic activities. A neutrophil response could only be elicited in mice with intact Fas death domains although Fas expression by the neutrophils was not essential. Lymphoma cells expressing the soluble FasL form corresponding to the natural cleavage product could not trigger apoptosis and did not induce a neutrophil response. In contrast to the other FasL transfectants, these cells survived as tumor transplants. However, expression of soluble FasL was not benign, but actually suppressed the inflammatory response and protected other transfectants from the effector mechanisms elicted by membrane-bound FasL.

To the Editor: Anticalin® proteins are human lipocalins that are engineered to bind relevant targets with high affinity and specificity (1). By applying a mutation and selection process, it is possible to select Anticalin proteins that can specifically bind to very diverse protein targets in a monovalent fashion. Anticalin proteins are also suited for engaging small and compact ligands owing to their cuplike binding pocket. Compared with monoclonal antibodies, Anticalin proteins are about 8 times smaller and can be recombinantly produced in bacterial cells in large amounts. The hepatic peptide hormone hepcidin is a highly conserved molecule of only 25 amino acids with 4 disulfide bridges that plays a central role in body iron metabolism (2). Therefore, it could become a useful biomarker. Numerous hepcidin assays, by use of either mass spectrometry (MS)-based1 techniques or traditional immunochemical assays, have been described to quantify hepcidin in biological fluids (2). However, both MS and immunochemical approaches have disadvantages, including costly and sophisticated instrumentation, low throughput, need for highly specialized personnel, and difficulties in obtaining high-quality antibodies. The latter is ascribed to the small size of hepcidin, its folded nature, and its conservation throughout evolution, which complicate the generation of hepcidin antibodies for immunochemical assays such as ELISA. Thus, there is a need and opportunity for alternative approaches. We developed …

Activation of the MET oncogenic pathway has been implicated in the development of aggressive cancers that are difficult to treat with current chemotherapies. This has led to an increased interest in developing novel therapies that target the MET pathway. However, most existing drug modalities are confounded by their inability to specifically target and/or antagonize this pathway. Anticalins, a novel class of monovalent small biologics, are hypothesized to be “fit for purpose” for developing highly specific and potent antagonists of cancer pathways. Here, we describe a monovalent full MET antagonist, PRS-110, displaying efficacy in both ligand-dependent and ligand-independent cancer models. PRS-110 specifically binds to MET with high affinity and blocks hepatocyte growth factor (HGF) interaction. Phosphorylation assays show that PRS-110 efficiently inhibits HGF-mediated signaling of MET receptor and has no agonistic activity. Confocal microscopy shows that PRS-110 results in the trafficking of MET to late endosomal/lysosomal compartments in the absence of HGF. In vivo administration of PRS-110 resulted in significant, dose-dependent tumor growth inhibition in ligand-dependent (U87-MG) and ligand-independent (Caki-1) xenograft models. Analysis of MET protein levels on xenograft biopsy samples show a significant reduction in total MET following therapy with PRS-110 supporting its ligand-independent mechanism of action. Taken together, these data indicate that the MET inhibitor PRS-110 has potentially broad anticancer activity that warrants evaluation in patients. Mol Cancer Ther; 12(11); 2459–71. ©2013 AACR . This article is featured in Highlights of This Issue, [p. 2283][1] [1]: /lookup/volpage/12/2283?iss=11

Contains fulltext : 125929.pdf (Publisher’s version ) (Open Access) Mass spectrometry (MS)-based assays for the quantification of the iron regulatory hormone hepcidin are pivotal to discriminate between the bioactive 25-amino acid form that can effectively block the sole iron transporter ferroportin and other naturally occurring smaller isoforms without a known role in iron metabolism. Here we describe the design, validation and use of a novel stable hepcidin-25(+40) isotope as internal standard for quantification. Importantly, the relative large mass shift of 40 Da makes this isotope also suitable for easy-to-use medium resolution linear time-of-flight (TOF) platforms. As expected, implementation of hepcidin-25(+40) as internal standard in our weak cation exchange (WCX) TOF MS method yielded very low inter/intra run coefficients of variation. Surprisingly, however, in samples from kidney disease patients, we detected a novel peak (m/z 2673.9) with low intensity that could be identified as hepcidin-24 and had previously remained unnoticed due to peak interference with the formerly used internal standard. Using a cell-based bioassay it was shown that synthetic hepcidin-24 was, like the -22 and -20 isoforms, a significantly less potent inducer of ferroportin degradation than hepcidin-25. During prolonged storage of plasma at room temperature, we observed that a decrease in plasma hepcidin-25 was paralleled by an increase in the levels of the hepcidin-24, -22 and -20 isoforms. This provides first evidence that all determinants for the conversion of hepcidin-25 to smaller inactive isoforms are present in the circulation, which may contribute to the functional suppression of hepcidin-25, that is significantly elevated in patients with renal impairment. The present update of our hepcidin TOF MS assay together with improved insights in the source and preparation of the internal standard, and sample stability will further improve our understanding of circulating hepcidin and pave the way towards further optimization and standardization of plasma hepcidin assays.

Glaucoma, the most frequent optic neuropathy, is a leading cause of blindness worldwide. Death of retinal ganglion cells (RGCs) occurs in all forms of glaucoma and accounts for the loss of vision, however the molecular mechanisms that cause RGC loss remain unclear. The pro-apoptotic molecule, Fas ligand, is a transmembrane protein that can be cleaved from the cell surface by metalloproteinases to release a soluble protein with antagonistic activity. Previous studies documented that constitutive ocular expression of FasL maintained immune privilege and prevented neoangeogenesis. We now show that FasL also plays a major role in retinal neurotoxicity. Importantly, in both TNFα triggered RGC death and a spontaneous model of glaucoma, gene-targeted mice that express only full-length FasL exhibit accelerated RGC death. By contrast, FasL-deficiency, or administration of soluble FasL, protected RGCs from cell death. These data identify membrane-bound FasL as a critical effector molecule and potential therapeutic target in glaucoma.

Biomolecular reagents that enable the specific molecular recognition of proteins play a crucial role in basic research as well as medicine. Up to now, antibodies (immunoglobulins) have been widely used for this purpose. Their predominant feature is the vast repertoire of antigen-binding sites that arise from a set of 6 hypervariable loops. However, antibodies suffer from practical disadvantages because of their complicated architecture, large size, and multiple functions. The lipocalins, on the other hand, have evolved as a protein family that primarily serves for the binding of small molecules. Here, we show that an engineered lipocalin, derived from human Lcn2, can specifically bind the T cell coreceptor CTLA-4 as a prescribed protein target with subnanomolar affinity. Crystallographic analysis reveals that its reshaped cup-like binding site, which is formed by 4 variable loops, provides perfect structural complementarity with this “antigen.” Furthermore, comparison with the crystal structure of the uncomplexed engineered lipocalin indicates a pronounced induced-fit mechanism, a phenomenon so far considered typical for antibodies. By recognizing the same epitope on CTLA-4 that interacts with the counterreceptors B7.1/B7.2 on antigen-presenting cells the engineered Lcn2 exhibits strong, cross-species antagonistic activity, as evidenced by biological effects comparable with a CTLA-4-specific antibody. With its proven stimulatory activity on T cells in vivo, the CTLA-4 blocking lipocalin offers potential for immunotherapy of cancer and infectious disease. Beyond that, lipocalins with engineered antigen-binding sites, so-called Anticalins, provide a class of small (≈180 residues), structurally simple, and robust binding proteins with applications in the life sciences in general.

PRS-080#022 is a 20kD AnticalinTM protein linked to 30kD linear poly-ethylene-glycol that specifically binds to human hepcidin 25, thereby inhibiting its activity. PRS-080#022 is developed for the treatment of functional iron deficient anemia associated with chronic kidney disease or cancer. Elevated levels of hepcidin restrict iron availability and contribute to functional iron deficiency and anemia. Thus, antagonizing hepcidin with PRS-080#022 has the potential to improve iron availability and erythropoiesis, thereby avoiding overload with exogenous iron and reducing the administered levels of Erythropoiesis-Stimulating Agents. 48 healthy male subjects were treated in this placebo controlled, double-blind Phase I study with ascending doses of PRS-080#022 in 6 cohorts at 0.08, 0.4, 1.2, 4.0, 8.0, and 16.0 mg/kg. 6 subjects per cohort received PRS-080#022 and 2 subjects received placebo (NCT02340572). Placebo or active treatments were administered by intravenous infusion over 2 hours. Safety, tolerability, the pharmacokinetics of total and free PRS-080#022, serum hepcidin concentrations as well as parameters of iron metabolism (ferritin, serum iron, transferrin saturation, reticulocytes and hemoglobin) were investigated. PRS-080#022 was well tolerated. 39 adverse events (AE) were reported during or after treatment in 22 subjects. All such AEs were mild or moderate and no serious AE was observed. Headache was the most frequently observed AE (10 subjects). Otherwise, no association of AEs to specific organs and no apparent dose dependency or difference between placebo and active treatment were observed. Notably, no hypersensitivity or infusion reactions were noted and vital signs, body temperature and ECG were unchanged. Pharmocokinetics of total PRS-080#022 followed a two-compartment model and was consistent between dose cohorts and within subjects of each cohort (Figure 1). Maximal concentration (Cmax) and area under the time curve (AUC) increased proportionally with dose (Table 1). Cmax was reached about 1 h after the 2 h infusion period (Table 1). The terminal plasma half life (T1/2) of PRS-080#022 ranged from 71 to 81 hours among dose cohorts (Table 1). The volume of distribution was small with 49 to 65 ml/kg, consistent with a distribution mainly to the blood volume. Administration of PRS-080#022 resulted in a decrease of free hepcidin which was observed already 1 h after start of infusion. PRS-080#022 administration induced a transient increase in serum iron concentration and transferrin saturation (TSAT), with both responses exhibiting a comparable time course and at doses of 0.4 mg/kg and higher. TSAT increased to > 90% in individual subjects. Serum iron concentrations reached about 50 µmol/l in individual subjects and did not further increase with dose. Importantly, the time period at which elevated serum iron concentrations and TSAT were observed increased with dose from about 18 h at 0.4 mg/kg to about 120 h at 16 mg/kg PRS-080#022. This is reflected by an increase of the AUC of the serum iron response relative to baseline and placebo (Table 1). In contrast, ferritin levels were largely unaffected by treatment. The excellent safety profile and the confirmed activity of PRS-080#022 on iron metabolism observed in healthy subjects warrants further investigations in anemic patients. A study investigating safety, pharmacokinetics and activity on erythropoiesis in anemic end-stage chronic kidney disease patients is in preparation. aFunded by the European Community FP7 health program grant GA-No. 278408 and supported by the EUROCALIN consortium (www.eurocalin-fp7.eu) Table. Summary of pharmacokinetic and pharmacodynamic parameters PRS-080#022 dose[mg/kg] Pharmacokinetic Parameters (group means ± SD) Pharmacodynamic Parameter(group means ± SD) Cmax[µg/ml] AUC0-inf[h*µg/ml] Tmax[h] T1/2[h] Vss[ml/kg] Serum Iron AUC0-240# [h*µmol/l] 0.08 2.1±0.3 162 ± 17 2.8 ± 0.4 81.2 ± 8.7 56.2 ± 8.0 39 ± 2807 0.4 10.6 ± 1.6 761 ± 163 3.3 ± 1.6 70.5 ± 27.7 54.2 ± 9.8 1174 ± 1150 1.2 33.9 ± 4.4 2264 ±167 2.7 ± 0.8 80.0 ± 10.3 51.3 ± 4.1 958 ± 1178 4.0 120.4 ± 19.6 7491 ± 730 3.7 ± 3.1 73.1 ± 8.9 47.8 ± 5.6 1579 ± 2222 8.0 246.3 ± 56.8 15066 ± 2496 4.3 ± 2.8 79.6 ± 9.7 53.3 ± 9.3 1134 ± 2207 16.0 366.2 ± 40.9 25572 ± 4075 3.0 ± 0.6 80.2 ± 11.6 64.6 ± 14.6 3480 ± 2123 #Response as Area Under the Curve 0-240h over baseline, placebo subtracted Figure 1. Arithmetic mean plasma concentration time profiles of total PRS-080#022 Figure 1. Arithmetic mean plasma concentration time profiles of total PRS-080#022 Disclosures Moebius: Pieris Pharmaceuticals Inc.: Employment. Feuerer:Pieris Pharmaceuticals Inc.: Other: contracted clinical research. Fenzl:Pieris Pharmaceuticals Inc.: Other: contracted clinical research. van Swelm:PIERIS: Other: member of the EU FP7 Eurocalin consortium. Swinkels:PIERIS: Other: member of EU FP7 Eurocalin consortium. Hohlbaum:Pieris Pharmaceuticals Inc.: Employment.

A number of studies have documented a critical role for tumor-specific CD4+ cells in the augmentation of immunotherapeutic effector mechanisms. However, in the context of an extensive tumor burden, chronic stimulation of such CD4+ T cells often leads to the up-regulation of both Fas and Fas ligand, and coexpression of these molecules can potentially result in activation-induced cell death and the subsequent loss of effector activity. To evaluate the importance of T cell persistence in an experimental model of immunotherapy, we used DO11 Th1 cells from wild-type, Fas-deficient, and Fas ligand-deficient mice as effector populations specific for a model tumor Ag consisting of an OVA-derived transmembrane fusion protein. We found that the prolonged survival of Fas-deficient DO11 Th1 cells led to a more sustained tumor-specific response both in vitro and in vivo. Importantly, both Fas- and Fas ligand-deficient Th1 cells delayed tumor growth and cause regression of established tumors more effectively than wild-type Th1 cells, indicating that resistance to activation-induced cell death significantly enhances T cell effector activity.

Experimental and physiological expression of the pro-apoptotic molecule Fas-ligand can induce inflammation under certain conditions. Discussed here are the experimental situations, possible mechanisms, and pathways that mediate this response.

Fas ligand (FasL) is a potent proapoptotic type-II transmembrane protein that can cause cell death in Fas+ target populations. Despite the presumed “silent” nature of apoptotic cell death, forced expression of FasL can induce a dramatic inflammatory response. To elucidate the in vivo mechanism(s) linking FasL and inflammation, we used a membrane-bound cell-free form of FasL (mFasL-vesicle preparation (VP)). We found that i.p. injection of FasL-microvesicles led to the rapid activation and subsequent demise of Mac1high resident peritoneal macrophages. Apoptosis of Mac1high peritoneal macrophages was observed within 0.5 h of mFasL-VP injection and correlated with the detection of increased macrophage inflammatory protein (MIP)-2 levels in peritoneal lavage fluid as well as induced RNA expression of IL-1β, MIP-2, MIP-1α, and MIP-1β. In vitro culture of purified peritoneal populations identified Mac1high cells as the major cytokine/chemokine producers in response to mFasL-VP. Purified Mac1high cells exposed to FasL could restore the ability of Fas-deficient mice to mount an inflammatory response. Our data demonstrate that the FasL-mediated inflammatory response starts with the production of proinflammatory mediators by preapoptotic resident tissue macrophages and suggest a general mechanism responsible for neutrophil inflammation seen in cases of FasL-expressing allografts.

Bioactive Fas ligand (FasL)-expressing vesicles were generated (vesicle preparation, VP) from two cell lines overexpressing FasL. The effect of NOK-1 anti-FasL mAb (mouse IgG1) on the cytotoxicity of FasL VP against various targets was determined. At high concentrations (1–10 μg/ml), NOK-1 inhibited the cytotoxicity. By contrast, NOK-1 in the dose range of 1–100 ng/ml significantly enhanced cytotoxicity against the FcR+ LB27.4, M59, and LF+ targets, but not the FcR− Jurkat and K31H28 hybridoma T cell targets. The ability to enhance FasL VP-mediated cytotoxicity could be blocked by the FcR-specific mAb 2.4G2. Enhancement was also observed with FcR+ A20 B lymphoma but not with the FcR− A20 variant. Enhancement of FasL VP cytotoxicity was observed with five IgG anti-FasL mAbs, but not with an IgM anti-FasL mAb. Inhibition was observed with high doses of all mAb except the IgG anti-FasL mAb G247-4, which is specific to a segment outside the FasL binding site. Interestingly, under identical conditions but in the presence of 2.4G2, G247-4 inhibited the cytotoxicity of FasL VP. In addition, G247-4 inhibited the FasL VP-mediated killing of FcR− Jurkat. The data demonstrate that FasL-expressing bioactive vesicles display a property heretofore unknown in bioactive agents that express FasL-mediated cytotoxicity. The mechanism of the Ab-mediated, FcR-dependent enhancement of cytotoxicity of bioactive vesicles and its physiological significance are discussed.

Background: Activation of the c-Met oncogenic pathway has been implicated in the development of aggressive cancers which are difficult to treat with current chemotherapies. Dimerization of c-Met receptor upon binding of Hepatocyte Growth Factor (HGF) leads to the stimulation of proliferative, migratory and survival pathways implicated in tumor development. Moreover it has recently been discovered that patients who become resistant / nonresponsive to therapies such as EGFR or VEGF inhibitors often show an enhanced c-Met expression. This has led to an increased interest in developing novel therapies that target the c-Met pathway. However, most existing drug modalities are confounded by their inability to specifically target and/or antagonize this pathway. Anticalins, a novel class of small biologics, are hypothesized to be ‘fit for purpose’ for developing highly specific and potent antagonists of cancer pathways. A monovalent Anticalin c-Met antagonist displaying efficacy in both ligand-dependent and independent cancer models has been developed. Methods/Results: Here we describe the in vitro and in vivo characterisation of the Anticalin c-Met antagonist PRS-110. In protein-based binding assays PRS-110 specifically binds to c-Met with high affinity and blocks HGF interaction (IC50 3.4 ± 0.7 nM). HUVEC cell proliferation assays demonstrated that PRS-110 efficiently antagonizes HGF-mediated growth. As a monovalent antagonist PRS-110 does not induce the c-Met pathway in the absence of ligand by receptor dimerization - an unwanted activation that can occur with bivalent antibodies. In mice, rats and non-human primates, PEGylated PRS-110 displayed favourable plasma elimination half-life profiles of 41 hours, 61 hours and 72 hours (T½α) respectively, with no signs of macrotoxicity. In vivo administration of PRS-110 resulted in significant, dose-dependent tumor growth inhibition in multiple xenograft models representative of ligand-dependent and ligand-independent c-Met activation. Analysis of c-Met protein levels on xenograft biopsy samples demonstrated a significant reduction in total c-Met (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3875. doi:1538-7445.AM2012-3875

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.

Anticalins are high affinity scaffold proteins based on human lipocalins and represent a new class of biologics. Although lipocalins share limited sequence identity among each other they are all made up of a single conserved beta-barrel. The four loops protruding from the opening of the calyx contribute to the observed various binding functions of different lipocalins. The loop residues of human tear lipocalin were randomized to generate a combinatorial library and successive rounds of panning and screening were employed to generate an Anticalin with high affinity and specificity for VEGF-A, Angiocal. Angiocal was found to antagonize VEGF-A function by preventing its interaction with cognate membrane receptors. Following the initial characterization in cell-based and in vivo models as communicated previously (AACR 2008 #4077, AACR 2009 #2318) we sought to estimate the human pharmacokinetics of Angiocal by interspecies scaling. Therefore, PK profiles in mice, rats and cynomolgus monkeys were obtained and will be presented. Therapeutic antibodies such as bevacizumab are usually well tolerated while significant toxicities occur in a subset of patients, for example leading to an enhanced risk for thromboembolic events. It has been demonstrated that bevacizumab forms multimeric immune complexes in vivo which may be a cause for these effects. Furthermore, immune complex deposition in glomeruli of the kidney may cause glomerulosclerosis. With respect to thromboembolic complications of bevacizumab use, these were mirrored in human FcgammaIIa receptor transgenic mice where administration of complexes between heparin, Bevacizumab and heparin-binding isoforms of its VEGF-A target lead to platelet aggregation and thrombosis (Meyer, T. et al. J. Thromb. Haemost. 2009, 7: 171-181). Angiocal was tested in the same model and does not cause any of these adverse effects on platelets in vivo. GLP toxicology studies with Angiocal were performed in two relevant species to determine the NOAEL. In addition, we did not observe effects on inflammatory cytokines or complement factors in these studies while exaggerated pharmacodynamic effects consistent with potent VEGF neutralization were seen. Angiocal is currently undergoing phase Ib clinical testing in cancer patients to establish Anticalins as novel and safe class of therapeutic molecules. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3632. doi:10.1158/1538-7445.AM2011-3632