Elarekibep (PRS-060/AZD1402), a new class of inhaled Anticalin medicine targeting IL-4Ra for type 2 endotype asthma.

BACKGROUND: Type 2 endotype asthma is driven by IL-4 and IL-13 signaling via IL-4Ra, which is highly expressed on airway epithelium, airway smooth muscle, and immunocytes in the respiratory mucosa, suggesting potential advantages of an inhalable antagonist. Lipocalin 1 (Lcn1), a 16 kDa protein abundant in human periciliary fluid, has a robust drug-like structure well suited to protein engineering, but it has never been used to make an inhaled Anticalin protein therapeutic. OBJECTIVES: We sought to reengineer Lcn1 into an inhalable IL-4Ra antagonist and assess its pharmacodynamic/kinetic profile. METHODS: Lcn1 was systematically modified by directed protein mutagenesis yielding a high-affinity, slowly dissociating, long-acting full antagonist of IL-4Ra designated PRS-060 with properties analogous to dupilumab, competitively antagonizing IL-4Ra-dependent cell proliferation, mucus induction, and eotaxin expression in vitro. Because PRS-060 displayed exquisite specificity for human IL-4Ra, with no cross-reactivity to rodents or higher primates, we created a new triple-humanized mouse model substituting human IL-4Ra, IL-4, and IL-13 at their correct syntenic murine loci to model clinical dosing. RESULTS: Inhaled PRS-060 strongly suppressed acute allergic inflammation indexes in triple-humanized mice with a duration of action longer than its bulk clearance, suggesting that it may act locally in the lung. CONCLUSION: Lcn1 can be reengineered into the Anticalin antagonist PRS-060 (elarekibep), exemplifying a new class of inhaled topical, long-acting therapeutic drugs with the potential to treat type 2 endotype asthma.

Sustained plasma hepcidin suppression and iron elevation by Anticalin-derived hepcidin antagonist in cynomolgus monkey.

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.

Functional characterization of a VEGF-A-targeting Anticalin, prototype of a novel therapeutic human protein class.

Human tear lipocalin (Tlc) was utilized as a protein scaffold to engineer an Anticalin that specifically binds and functionally blocks vascular endothelial growth factor A (VEGF-A), a pivotal inducer of physiological angiogenesis that also plays a crucial role in several neovascular diseases. Starting from a naive combinatorial library where residues that form the natural ligand-binding site of Tlc were randomized, followed by affinity maturation, the final Anticalin PRS-050 was selected to bind all major splice forms of VEGF-A with picomolar affinity. Moreover, this Anticalin cross-reacts with the murine ortholog. PRS-050 efficiently antagonizes the interaction between VEGF-A and its cellular receptors, and it inhibits VEGF-induced mitogenic signaling as well as proliferation of primary human endothelial cells with subnanomolar IC50 values. Intravitreal administration of the Anticalin suppressed VEGF-induced blood-retinal barrier breakdown in a rabbit model. To allow lasting systemic neutralization of VEGF-A in vivo, the plasma half-life of the Anticalin was extended by site-directed PEGylation. The modified Anticalin efficiently blocked VEGF-mediated vascular permeability as well as growth of tumor xenografts in nude mice, concomitantly with reduction in microvessel density. In contrast to bevacizumab, the Anticalin did not trigger platelet aggregation and thrombosis in human FcγRIIa transgenic mice, thus suggesting an improved safety profile. Since neutralization of VEGF-A activity is well known to exert beneficial effects in cancer and other neovascular diseases, including wet age-related macular degeneration, this Anticalin offers a novel potent small protein antagonist for differentiated therapeutic intervention in oncology and ophthalmology.

Multimodality multiparametric imaging of early tumor response to a novel antiangiogenic therapy based on anticalins.

Anticalins are a novel class of targeted protein therapeutics. The PEGylated Anticalin Angiocal (PRS-050-PEG40) is directed against VEGF-A. The purpose of our study was to compare the performance of diffusion weighted imaging (DWI), dynamic contrast enhanced magnetic resonance imaging (DCE)-MRI and positron emission tomography with the tracer [18F]fluorodeoxyglucose (FDG-PET) for monitoring early response to antiangiogenic therapy with PRS-050-PEG40. 31 mice were implanted subcutaneously with A673 rhabdomyosarcoma xenografts and underwent DWI, DCE-MRI and FDG-PET before and 2 days after i.p. injection of PRS-050-PEG40 (n = 13), Avastin (n = 6) or PBS (n = 12). Tumor size was measured manually with a caliper. Imaging results were correlated with histopathology. In the results, the tumor size was not significantly different in the treatment groups when compared to the control group on day 2 after therapy onset (P = 0.09). In contrast the imaging modalities DWI, DCE-MRI and FDG-PET showed significant differences between the therapeutic compared to the control group as early as 2 days after therapy onset (P<0.001). There was a strong correlation of the early changes in DWI, DCE-MRI and FDG-PET at day 2 after therapy onset and the change in tumor size at the end of therapy (r = -0.58, 0.71 and 0.67 respectively). The imaging results were confirmed by histopathology, showing early necrosis and necroptosis in the tumors. Thus multimodality multiparametric imaging was able to predict therapeutic success of PRS-050-PEG40 and Avastin as early as 2 days after onset of therapy and thus promising for monitoring early response of antiangiogenic therapy.

First-in-human phase I study of PRS-050 (Angiocal), an Anticalin targeting and antagonizing VEGF-A, in patients with advanced solid tumors.

BACKGROUND: To report the nonrandomized first-in-human phase I trial of PRS-050, a novel, rationally engineered Anticalin based on human tear lipocalin that targets and antagonizes vascular endothelial growth factor A (VEGF-A). METHODS: Patients with advanced solid tumors received PRS-050 at 0.1 mg/kg to 10 mg/kg by IV in successive dosing cohorts according to the 3+3 escalation scheme. The primary end point was safety. RESULTS: Twenty-six patients were enrolled; 25 were evaluable. Two patients experienced dose-limiting toxicity, comprising grade (G) 3 hypertension and G3 pyrexia, respectively. The maximum tolerated dose was not reached. Most commonly reported treatment-emergent adverse events (AEs) included chills (52%; G3, 4%), fatigue (52%; G3, 4%), hypertension (44%; G3, 16%), and nausea (40%, all G1/2). No anti-PRS-050 antibodies following multiple administration of the drug were detected. PRS-050 showed dose-proportional pharmacokinetics (PK), with a terminal half-life of approximately 6 days. Free VEGF-A was detectable at baseline in 9/25 patients, becoming rapidly undetectable after PRS-050 infusion for up to 3 weeks. VEGF-A/PRS-050 complex was detectable for up to 3 weeks at all dose levels, including in patients without detectable baseline-free VEGF-A. We also detected a significant reduction in circulating matrix metalloproteinase 2, suggesting this end point could be a pharmacodynamic (PD) marker of the drug's activity. CONCLUSIONS: PRS-050, a novel Anticalin with high affinity for VEGF-A, was well-tolerated when administered at the highest dose tested, 10 mg/kg. Based on target engagement and PK/PD data, the recommended phase II dose is 5 mg/kg every 2 weeks administered as a 120-minute infusion. TRIAL REGISTRATION: ClinicalTrials.gov NCT01141257 http://clinicaltrials.gov/ct2/show/NCT01141257.

Improved mass spectrometry assay for plasma hepcidin: detection and characterization of a novel hepcidin isoform.

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.

A highly potent and specific MET therapeutic protein antagonist with both ligand-dependent and ligand-independent activity.

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.

Identification of proangiogenic genes and pathways by high-throughput functional genomics: TBK1 and the IRF3 pathway.

A genome-wide phenotype screen was used to identify factors and pathways that induce proliferation of human umbilical vein endothelial cells (HUVEC). HUVEC proliferation is a recognized marker for factors that modulate vascularization. Screening "hits" included known proangiogenic factors, such as VEGF, FGF1, and FGF2 and additional factors for which a direct association with angiogenesis was not previously described. These include the kinase TBK1 as well as Toll-like receptor adaptor molecule and IFN regulatory factor 3. All three proteins belong to one signaling pathway that mediates induction of gene expression, including a mixture of secreted factors, which, in concert, mediate proliferative activity toward endothelial cells. TBK1 as the "trigger" of this pathway is induced under hypoxic conditions and expressed at significant levels in many solid tumors. This pattern of expression and the decreased expression of angiogenic factors in cultured cells upon RNA-interference-mediated ablation suggests that TBK1 is important for vascularization and subsequent tumor growth and a target for cancer therapy.

Development of secreted proteins as biotherapeutic agents.

As one of the most important classes of proteins, secreted factors account for about one-tenth of the human genome, 3000 - 4000 in total, including factors of signalling pathways, blood coagulation and immune defence, as well as digestive enzymes and components of the extracellular matrix. Secreted proteins are a rich source of new therapeutics and drug targets, and are currently the focus of major drug discovery programmes throughout the industry. Many of the most important novel drugs developed in biotechnology have resulted from the application of secreted proteins as therapeutics. Secreted proteins often circulate throughout the body and, therefore, have access to most organs and tissues. Because of that, many of the factors are themselves therapeutic agents. This paper gives an overview on the features and functions of human secreted proteins and peptides, as well as strategies by which to discover additional therapeutic proteins from the human 'secretome'. Furthermore, a variety of examples are provided for the therapeutic use of recombinant secreted proteins as 'biologicals', including features and applications of recombinant antibodies, erythropoietin, insulin, interferon, plasminogen activators, growth hormone and colony-stimulating factors.