Discovery and Preclinical Characterization of a Novel Hepcidin Antagonist with Tunable PK/PD Properties for the Treatment of Anemia in Different Patient Populations

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 687-687 ◽  
Author(s):  
Andreas M Hohlbaum ◽  
Stefan Trentman ◽  
Hendrik Gille ◽  
Andrea Allersdorfer ◽  
Rachida Siham Belaiba ◽  
...  
Keyword(s):  
Phage Display ◽  
Half Life ◽  
Therapeutic Approach ◽  
Production Costs ◽  
Stable Cell Line ◽  
Kinetic Assay ◽  
Therapeutic Concept ◽  
Drug Candidates ◽  
High Affinity

Abstract 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.

Binding of 131I-Labeled Tissue-Type Plasminogen Activator on De-Endothelialized Lesions in Rabbits

1987 ◽  
Vol 26 (05) ◽  
pp. 224-228 ◽  
Author(s):  
Y. Isaka ◽  
H. Etani ◽  
K. Kimura ◽  
S. Yoneda ◽  
T. Kamada ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Abdominal Aorta ◽  
Half Life ◽  
Balloon Catheter ◽  
Biochemical Properties ◽  
Tissue Type ◽  
Fibrin Deposition ◽  
Tissue Type Plasminogen Activator ◽  
High Affinity ◽  
Type Plasminogen Activator

Tissue-type plasminogen activator (t-PA) which has a high affinity for fibrin in the clot, was labeled with 131I by the iodogen method, and its binding to de-endothelialized lesions in the rabbit was measured to assess the detectability of thrombi. The de-endothelialized lesion was induced in the abdominal aorta with a Fogarty 4F balloon catheter. Two hours after the de-endothelialization, 131I-labeled t-PA (125 ± 46 μCi) was injected intravenously. The initial half-life of the agent in blood (n = 12) was 2.9 ± 0.4 min. The degree of binding of 131I-labeled t-PA to the de-endothelialized lesion was evaluated at 15 min (n = 6) or at 30 min (n = 6) after injection of the agent. In spite of the retention of the biochemical properties of 131I-labeled t-PA and the presence of fibrin deposition at the de-endothelialized lesion, the binding of t-PA to the lesion was not sufficiently strong. Lesion-to-control ratios (cpm/g/cpm/g) were 1.65 ± 0.40 (at 15 min) and 1.39 ± 1.31 (at 30 min), and lesion-to-blood ratios were 1.39 ± 0.32 (at 15 min) and 1.36 ± 0.23 (at 30 min). These results suggest that radiolabeled t-PA may be inappropriate as a radiopharmaceutical for the scintigraphic detection of a pre-existing thrombotic lesion.

Characterization of a Mode of Specific Binding of Fibrin Monomer through its Amino-Terminal Domain by Macrophages and Macrophage Cell-Lines

1990 ◽  
Vol 63 (02) ◽  
pp. 193-203 ◽  
Author(s):  
John R Shainoff ◽  
Deborah J Stearns ◽  
Patricia M DiBello ◽  
Youko Hishikawa-Itoh
Keyword(s):  
Peritoneal Macrophages ◽  
Affinity Binding ◽  
Macrophage Cell ◽  
High Affinity Binding ◽  
Amino Terminal ◽  
High Affinity ◽  
Terminal Domain ◽  
Weak Binding ◽  
Amino Terminal Domain

SummaryThe studies reported here probe the existence of a receptor-mediated mode of fibrin-binding by macrophages that is associated with the chemical change underlying the fibrinogen-fibrin conversion (the release of fibrinopeptides from the amino-terminal domain) without depending on fibrin-aggregation. The question is pursued by 1) characterization of binding in relation to fibrinopeptide content of both the intact protein and the CNBr-fragment comprising the amino-terminal domain known as the NDSK of the protein, 2) tests of competition for binding sites, and 3) photo-affinity labeling of macrophage surface proteins. The binding of intact monomers of types lacking either fibrinopeptide A alone (α-fibrin) or both fibrinopeptides A and B (αβ-fibrin) by peritoneal macrophages is characterized as proceeding through both a fibrin-specific low density/high affinity (BMAX ≃ 200–800 molecules/cell, KD ≃ 10−12 M) interaction that is not duplicated with fibrinogen, and a non-specific high density/low affinity (BMAX ≥ 105 molecules/cell, KD ≥ 10−6 M) interaction equivalent to the weak binding of fibrinogen. Similar binding characteristics are displayed by monocyte/macrophage cell lines (J774A.1 and U937) as well as peritoneal macrophages towards the NDSK preparations of these proteins, except for a slightly weaker (KD ≃ 10−10 M) high-affinity binding. The high affinity binding of intact monomer is inhibitable by fibrin-NDSK, but not fibrinogen-NDSK. This binding appears principally dependent on release of fibrinopeptide-A, because a species of fibrin (β-fibrin) lacking fibrinopeptide-B alone undergoes only weak binding similar to that of fibrinogen. Synthetic Gly-Pro-Arg and Gly-His-Arg-Pro corresponding to the N-termini of to the α- and the β-chains of fibrin both inhibit the high affinity binding of the fibrin-NDSKs, and the cell-adhesion peptide Arg-Gly-Asp does not. Photoaffinity-labeling experiments indicate that polypeptides with elec-trophoretically estimated masses of 124 and 187 kDa are the principal membrane components associated with specifically bound fibrin-NDSK. The binding could not be up-regulated with either phorbol myristyl acetate, interferon gamma or ADP, but was abolished by EDTA and by lipopolysaccharide. Because of the low BMAX, it is suggested that the high-affinity mode of binding characterized here would be too limited to function by itself in scavenging much fibrin, but may act cooperatively with other, less limited modes of fibrin binding.

Covalent Complexes Between Low Molecular Weight Heparin Fragments and Antithrombin III – Inhibition Kinetics and Turnover Parameters

1983 ◽  
Vol 49 (02) ◽  
pp. 109-115 ◽  
Author(s):  
M Hoylaerts ◽  
E Holmer ◽  
M de Mol ◽  
D Collen
Keyword(s):  
Molecular Weight ◽  
Half Life ◽  
Low Molecular Weight Heparin ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Life Time ◽  
Low Molecular Weight ◽  
High Affinity ◽  
Plasma Half Life ◽  
Covalent Complex

SummaryTwo high affinity heparin fragments (A/r 4,300 and M, 3,200) were covalently coupled to antithrombin III (J. Biol. Chem. 1982; 257: 3401-3408) with an apparent 1:1 stoichiometry and a 30-35% yield.The purified covalent complexes inhibited factor Xa with second order rate constants very similar to those obtained for antithrombin III saturated with these heparin fragments and to that obtained for the covalent complex between antithrombin III and native high affinity heparin.The disappearance rates from plasma in rabbits of both low molecular weight heparin fragments and their complexes could adequately be represented by two-compartment mammillary models. The plasma half-life (t'/j) of both low Afr-heparin fragments was approximately 2.4 hr. Covalent coupling of the fragments to antithrombin III increased this half-life about 3.5 fold (t1/2 ≃ 7.7 hr), approaching that of free antithrombin III (t1/2 ≃ 11 ± 0.4 hr) and resulting in a 30fold longer life time of factor Xa inhibitory activity in plasma as compared to that of free intact heparin (t1/2 ≃ 0.25 ± 0.04 hr).

Combination of ribosome and phage display for fast selection of high affinity VHH antibody fragments

2019 ◽  
pp. 27-33
Author(s):  
YuE Kravchenko ◽  
SV Ivanov ◽  
DS Kravchenko ◽  
EI Frolova ◽  
SP Chumakov
Keyword(s):  
Phage Display ◽  
Selection Procedure ◽  
Free System ◽  
Phage Displays ◽  
High Affinity ◽  
Binding Domains ◽  
A Cell ◽  
Vhh Antibodies ◽  
Efficiency Of Selection ◽  
Selection Of

Selection of antibodies using phage display involves the preliminary cloning of the repertoire of sequences encoding antigen-binding domains into phagemid, which is considered the bottleneck of the method, limiting the resulting diversity of libraries and leading to the loss of poorly represented variants before the start of the selection procedure. Selection in cell-free conditions using a ribosomal display is devoid from this drawback, however is highly sensitive to PCR artifacts and the RNase contamination. The aim of the study was to test the efficiency of a combination of both methods, including pre-selection in a cell-free system to enrich the source library, followed by cloning and final selection using phage display. This approach may eliminate the shortcomings of each method and increase the efficiency of selection. For selection, alpaca VHH antibody sequences suitable for building an immune library were used due to the lack of VL domains. Analysis of immune libraries from the genes of the VH3, VHH3 and VH4 families showed that the VHH antibodies share in the VH3 and VH4 gene groups is insignificant, and selection from the combined library is less effective than from the VHH3 family of sequences. We found that the combination of ribosomal and phage displays leads to a higher enrichment of high-affinity fragments and avoids the loss of the original diversity during cloning. The combined method allowed us to obtain a greater number of different high-affinity sequences, and all the tested VHH fragments were able to specifically recognize the target, including the total protein extracts of cell cultures.

Acid-Sensing Ion Channels

2020 ◽  
Author(s):  
Stefan Gründer
Keyword(s):  
Nervous System ◽  
Ion Channels ◽  
Excitatory Synapses ◽  
Detailed Characterization ◽  
High Affinity ◽  
Acid Sensing Ion Channels ◽  
Long Time ◽  
Pathological Pain

Acid-sensing ion channels (ASICs) are proton-gated Na+ channels. Being almost ubiquitously present in neurons of the vertebrate nervous system, their precise function remained obscure for a long time. Various animal toxins that bind to ASICs with high affinity and specificity have been tremendously helpful in uncovering the role of ASICs. We now know that they contribute to synaptic transmission at excitatory synapses as well as to sensing metabolic acidosis and nociception. Moreover, detailed characterization of mouse models uncovered an unanticipated role of ASICs in disorders of the nervous system like stroke, multiple sclerosis, and pathological pain. This review provides an overview on the expression, structure, and pharmacology of ASICs plus a summary of what is known and what is still unknown about their physiological functions and their roles in diseases.

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