WO2011095604A1 - Half-life prolongation of proteins - Google Patents

Half-life prolongation of proteins Download PDF

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Publication number
WO2011095604A1
WO2011095604A1 PCT/EP2011/051681 EP2011051681W WO2011095604A1 WO 2011095604 A1 WO2011095604 A1 WO 2011095604A1 EP 2011051681 W EP2011051681 W EP 2011051681W WO 2011095604 A1 WO2011095604 A1 WO 2011095604A1
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fviii
ligand
binding site
binding
lrp
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PCT/EP2011/051681
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French (fr)
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Carola Schröder
Julia Janzon
Martina Brecelj
Christoph Kannicht
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Octapharma Biopharmaceuticals Gmbh
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Publication of WO2011095604A1 publication Critical patent/WO2011095604A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36Blood coagulation or fibrinolysis factors
    • A61K38/37Factors VIII
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082Supracellular entities, e.g. tissue, organisms
    • G01N33/5088Supracellular entities, e.g. tissue, organisms of vertebrates
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/745Assays involving non-enzymic blood coagulation factors
    • G01N2333/755Factors VIII, e.g. factor VIII C [AHF], factor VIII Ag [VWF]

Abstract

Pharmaceutical preparation comprising: - a pharmaceutical active protein having at least one LRP-binding site and/or at least one HSPG binding site and - at least one ligand binding to the LRP-binding site and/or the HSPG binding site, the at least one ligand being selected from peptides, sugars, nucleic acids and small chemical compounds, said small chemical compounds having a molecular weight of 5 KDa or less.

Description

Half-life prolongation of proteins

The present invention relates to methods for half-life prolongation of proteins.

The use of polypeptides such as proteins for therapeutic applications has expanded in recent years mainly due to advanced knowledge of the molecular biological principles underlying many diseases and the availability of improved recombinant expression and delivery systems for human polypeptides. Polypeptide therapeutics are mainly utilized in diseases where a certain natural polypeptide is defective or missing in the patient, in particular because of inherited gene defects. For example, hemophilia is a disease caused by deficiency of a certain plasma protein . Hemophiliacs suffer from hemorrhagic morbidity caused by the disturbed function of protein components of the blood coagulation cascade. Depending on the affected clotting factor two types of hemophilia can be distinguished . Both have in common the inhibited conversion of soluble fibrinogen to an insoluble fibrin-clot. They are recessive X-chromosomally- linked genetic diseases affecting mainly the male population .

Hemophilia A affects 1-2 individuals per 10.000 males. It is caused by the deficiency or absence of factor VIII, a very large glycoprotein (Mw approximately 330 kDa (1 ), which represents an important element of the blood coagulation cascade. The polypeptide sequence can be subdivided in three regions, an N-terminal region consisting of the so-called Al and A2 domains, a central B domain region and a C-terminal region composed of the A3, CI and C2 domains. In the blood, coagulation factor VIII occurs as an inactive precursor. It is bound tightly and non-covalently to von Willebrand Factor (vWF), which acts as a stabilizing carrier protein . Proteolytic cleavage of factor VIII by thrombin at three specific positions (740, 372, 1689) leads to its dissociation from vWF and releases the procoagulant function within the cascade. In its active form, factor VIII functions as a cofactor for factor IXa, thereby accelerating the proteolytic activation of factor X by several orders of magnitude. Hemophilia B occurs in about 1 of 25,000 males. It is characterized by the deficiency of the serine protease factor IX (Christmas factor). This 415 amino- acid polypeptide is synthesized in the liver as a 56 kDa glycoprotein. In order to attain its proper function a posttranslational carboxylation step is required which only occurs in the presence of vitamin K.

Treatment of both types of bleeding disorder traditionally involves infusion of human plasma-derived protein concentrates of factor VIII or factor IX. Although this method represents an efficient therapy for hemophiliacs, it carries the potential risk of transmission of various infectious agents, such as viruses causing hepatitis or AIDS, or thromboembolic factors. Alternatively, several recombinant DNA techniques for the production of clotting factors have been described. For this purpose, the corresponding cDNAs of wild type factor VIII and factor IX have been isolated and cloned into suitable expression vectors (EP-A-160457; WO-A-86/01961, U .S. Patents 4,770,999, 5,521,070 and 5,521,070).

In the case of factor VIII, recombinant expression of subunits for the production of complexes showing coagulant activity is known in the art (e.g ., from EP-A-150735, EP-A-232112, EP-A-0500734, WO-91/07490, WO- 95/13300 U .S. Patents 5,045,455 and 5,789,203). Moreover, the expression of truncated cDNA versions partially or entirely lacking the sequence coding for the highly glycosylated B domain has been described (e.g. in WO-86/06101, WO-87/04187, WO-87/07144, WO-88/00381, WO-94/29471, EP-A-251843, EP-A-253455, EP-A-254076, U .S. Patents 4,868,112 and 4,980,456, EP-A- 294910, EP-A-265778, EP-A-303540 and WO-91/09122). More recently a variety of selected point mutations have been introduced to inhibit proteolytic inactivation of factor VIII by activated protein C or to reduce the immunogenicity resulting in the formation of inhibitory antibodies by the treated patients (see e.g., U.S. Patents 5,859,204, 5,422,260 and 5,451,521, WO-97/49725, WO-99/29848). However, polypeptide therapeutics such as factor VIII are associated with many drawbacks, including short circulating half-life and proteolytic degradation. For example, the half-life of the protein factor VIII in the human body is app. 12 hours whereas in severe von Willebrand disease (vWD) patients it is app. 2 hours. Nowadays prophylactic treatment represents the state of the art treatment of hemophilia patients in developed countries. Prophylactic treatment usually results in 2 to 4 infusions per week.

There are a number of further proteins which are used for therapeutic purposes for example erythropoietin, granulocyte-colony stimulating factor (GCSF), interferons, monoclonal antibodies and the like.

In many cases, it would be helpful to increase the half-life of the therapeutic proteins to increase efficiency or reduce the amount of therapeutic proteins and/or frequency of infusions applied to patient. This would also reduce the costs of the treatment. In the prior art, the short circulating half-life of polypeptide therapeutics has been addressed by covalent attachment of a polymer to the polypeptide. For example, the attachment of polyethylene glycol (PEG), dextran, or hydroxyethyl starch (HES) has shown some improvement of the half-life of some polypeptides. However, a number of problems have been observed with the attachment of polymers. For example, the attachment of polymers can lead to decreased drug activity. Furthermore, certain reagents used for coupling polymers to a protein are insufficiently reactive and therefore require long reaction times during which protein denaturation and/or inactivation can occur. Also, incomplete or non-uniform attachment leads to a mixed population of compounds having differing properties. WO 2009/135888 discloses a complex comprising a target protein and at least one binding molecule wherein the binding molecule is bound to at least one water soluble polymer.

Because of the size of the water soluble polymer, the binding molecule has a high molecular weight.

It is the object of the invention to provide therapeutic proteins with increased half-life.

One embodiment of the invention is a pharmaceutical preparation comprising

- a pharmaceutically active protein having at least one LRP binding site and/or at least one HSPG binding site and

- at least one ligand binding to the LRP-binding site and/or the HSPG binding site,

the at least one ligand being selected from peptides, sugars, nucleic acids and small chemical compounds, said small chemical compounds having a molecular weight of 5 KDa or less.

In a preferred embodiment the pharmaceutical active protein is Factor VIII, preferably human Factor VIII. Other therapeutic useful proteins having LRP binding sites include ApoE, Lipoprotein lipase, Hepatic lipase, tPA, uPA, Factor IXa, Factor VIII, Factor Villa, FactorVIIa/TFPI, MMP-13, MMP-9, Spingolipid activator protein (SAP), Pregnancy Zone Protein, a2 macroglobulin, Complement C3, PAI-1, CI inhibitor, Antithrombin III, TFPI, Heparin cofactor II, ch-Antitrypsin, APP, Thrombospondin-1, Thrombospondin-2, Pseudomonas exotoxin A, Lactoferrin, Rhinovirus, RAP, HSP-96, HIV-Tat protein.

Therapeutic proteins having HSPG binding sites include Albumin, Fibrinogen, Factor H, Fibronectin, Antithrombin III, Thrombin, Kininogen, vWF, FX, FXI, FIX, FVIII, PDGF, Platelet factor 4, VEGF, RANTES, MIP-la, bFGF, Apolipoprotein E3, Plasma protein C inhibitor. In the embodiment using Factor VIII, Factor VIII can be derived from donor plasma or it can be recombinant Factor VIII. Included are also variants of Factor VIII having mutations or the like.

A very preferred embodiment is a B domain deleted Factor VIII. FVIII as therapeutic protein used for hemophelia treatment is half-life prolonged by using small ligands binding with high affinity and high specificity to the HSPG- and/or LRP-receptor binding sites. The half-life prolonging effect is achieved by blocking a single site (for example the HSPG-site on the A2 domain) and/or by a combination of synergistically blocking several or all sites in parallel (for example synergistically blocking the HSPG- and LRPR-sites).

As ligands, peptides, sugars, small protein domains or fragments, nucleic acids (RNA, DNA) and derivatives thereof, or small chemical compounds can be used.

"Peptides" covers amino acid polymers. The term "peptides" as used in this application covers small peptides, oligopeptides, proteins.

"Sugars" are polymers of carbohydrates. The term covers saccharides of any size including oligo- and polysaccharides.

"Nucleic acids" covers polymers of nucleosides or nucleotides including oligo- and polynucleotides or -nucleosides. "Small chemical compounds" are compounds typically prepared by organic chemical synthesis and have a molecular weight of 5 kDa or less, preferably 2 kDa or 1 kDa or less. The term includes all compounds having a molecular weight of 5 kDa or less, including natural compounds from animals or plants.

The ligands can be used as single molecules co-administered with FVIII and/or two or more ligands can be bridged covalently for blocking adjacent binding sites (for example ligands blocking the HSPG-binding site and the nearby LRPR-binding site on A2 domain).

Peptide ligands can be linear or structured (for example cyclic peptides) and chemically modified (for example sulphated). Structured peptide ligands are constructed by taking into account available structural and sequence data about the targeted binding sites (for example the HSPG-binding site or the LRPR-binding site on A2 domain) as well as data about their physiological ligands (for example the LRPl-receptor, heparin), see Fig . 9 and 10. The structural 3-dimensional surface of the physiological ligands is thereby mimicked to produce a ligand with high affinity and high specificity (see Figure 10, A). The structural 3-dimensional surface of the target binding sites can be mimicked (for example the HSPG-site on A2 domain of FVIII) to design a binding site mimetic peptide which can be used for site-directed screening of ligands binding to this target site (see Figure 10, B). These ligands can be further optimized by modifications of their amino acid sequence to vary: the length of the binding region (for example including the binding regions around a known binding site), the amino acid composition by varying the overall charge (by introducing or eliminating charged amino acids), the polarity (by exchanging hydrophilic and hydrophobic amino acids) or bulkiness (by exchanging amino acids with large side chains against those with smaller side chains or vice versa).

By using elaborated and defined screening methods, also random libraries of ligands (for example phage display libraries or synthetic peptide micro arrays) can be used to identify ligands specifically binding to the targeted HSPG- and/or LRP-receptor binding sites on FVIII. These screening includes the target protein and single domains or fragments thereof (for example FVIII, A2- or A3-domains), as well as physiological ligands and their surrogates (for example heparin or Clexane), their domains or fragments thereof (for example LRP-domains), monoclonal antibodies with binding epitopes overlapping or surrounding the target binding site regions for competitions studies and constructed mimetic binding site peptides (see Figure 10, B).

In Factor VIII there are several LRP-binding sites. LRP binding sites are located in the A2 domain in residues 484-509, in the A3 domain residues 1811-1818, in the C2 domain residues 2173-2332 and in the CI domain residues 2065, 2092.

The numbering of the residues is in accordance with the numbering of SEQ ID NO. 1 (Figure 6).

A suitable HSPG binding site on Factor VIII is localized within the A2 domain residues 558-565 in accordance with SEQ ID NO. 1.

The ligand used in the pharmaceutical preparation has preferably a molecular weight below 150 KDa, preferably 100 KDa or less, 70 KDa or less, 50 KDa or less, 30 KDa or less, 10 KDa or less, 5 KDa or less, 2 KDa or less or 1 KDa or less. Suitable ligands are selected from peptides, sugars, lipids, nucleic acids and variants thereof and chemically synthesized compounds.

In preferred embodiments, the peptide is a linear, cyclic, folded or a scaffold peptide.

In some embodiments, the ligand may comprise at least one sulphate group. In preferred embodiments, the ligand does not comprise a polyalkylenglycol or derivative thereof like PEG, does not comprise a hydroxypropylmethacrylate HPMA group and its copolymers and does not comprise a starch like hydroxyalkyl starch.

A further embodiment of the invention is the method for testing a ligand's ability to bind to at least one LRP-binding site and/or at least one HSPG binding site of FVIII comprising the steps of - combining the ligand with FVIII to a FVIII-ligand complex

- measuring clearance of the FVIII-ligand complex in-vivo

wherein a reduced clearance indicates that the ligand binds to at least one LRP-binding site and/or at least one HSPG binding site of FVIII, the ligand being selected from peptides, sugars, nucleic acids and small chemical compounds, said small chemical compounds having a molecular weight of 5 KDa or less.

A further embodiment of the invention is a method for reducing clearance of FVIII comprising the step of

- combining FVIII with a ligand binding to the LRP-binding site and/or the HSPG binding site of FVIII to form a FVIII-ligand complex

- administering said FVIII-ligand complex to a patient in need of FVIII, the ligand being selected from peptides, sugars, nucleic acids and small chemical compounds, said small chemical compounds having a molecular weight of 5 KDa or less.

A further embodiment of the invention is a ligand binding with high affinity and high selectivity to or in the region of LRP and/or HSPG in order to block the mentioned sites.

Receptor-mediated clearance, proteolytic or non-proteolytic inactivation mechanisms are responsible for the "loss" of proteins from the blood stream.

This is explained in more detail for Factor VIII.

After its synthesis and secretion into the blood, 95-98% of FVIII is captured by vonWillebrandFactor, which protects FVIII from clearance and inactivation by binding tightly to its C2 domain, thus circulating in the bloodstream as a tight complex with FVIII. 2 to 5% of FVIII in the blood stream however remain vWF-unbound and are subjected to receptor-mediated clearance processes. Activation of FVIII by thrombin-cleavage leads to the loss of vWFbinding and therefore to the loss of its protecting properties. Activated FVIII (FVIIIa) is also subjected to receptor mediated clearance and proteolytic degradation, as well as non-proteolytic inactivation mechanisms. Receptor-mediated clearance of FVIII is mainly mediated by low density lipoprotein related protein receptor 1 (LRPR-1) and heparan sulphate proteoglycans (HSPGs) (for references see (2;3).

Heparan sulphate proteoglycans (HSPGs) are cell surface sugar structures capable of capturing proteins comprising a HSPG-binding site. They therefore enrich the local concentration of these captured proteins two-dimensionally at the cell surface, thus facilitating receptor-mediated endocytosis by cell surface receptors (see (4-6) for reference). HSPG-mediated FVIII clearance takes place in cooperation with the LRPl-receptor (LRPR-1). However, there is evidence for HSPG-mediated FVIII clearance together with other yet unidentified cell surface receptors (5).

LRPR-1 belongs to the low density lipoprotein receptor family, also comprising LDLR (low density lipoprotein receptor), VLDLR (very low density lipoprotein receptor) and megalin-receptor, which all solely play an inferior role in FVIII- clearance (4-6). LRPR-1 mainly mediates the receptor-mediated FVIII clearance either self-sufficiently or in cooperation with HSPGs (5). The effect of LRPR-1 on FVIII clearance has been extensively determined and elucidated (5;7-14) :

It has been shown in vitro that the dissociation constant for LRPR-1 and FVIII purified proteins is of high affinity (KD = 25-100 nM or 116 nM, respectively, see {8; 12).

In cell-based assays it could be demonstrated that LRPR-1 -deficient cells showed 50% less FVIII clearance, and blocking LRPR-1 with RAP (receptor associated protein, a LRPR-l-inhibitor) also resulted in a 50% reduced FVIII clearance (8;11;12). In vivo LRPR-l-knockout mice showed increased FVIII plasma levels, the half-life of intravenously administered FVIII was increased from 2.5 to 4 hours corresponding to a half-life prolongation of factor 1.5 (5;7). Furthermore, clearance studies in mice, in which LRPR-1 was inhibited by RAP showed a half-life prolongation of factor 3.5 (5). There also exist a lot of data about LRPR-l-polymorphisms in humans leading to 20% increased FVIII plasma levels (9; 10; 13; 14).

Sarafanov et al . (5) proved the effect of synergistically blocking HSPGs and LRPR-1 on FVIII clearance in mice (see Figure 2). In a first set-up they blocked HSPGs using its antagonist protamin (1 in Figure 2) resulting in a half- life prolonging effect for FVIII of factor 1.6. In a second set-up LRP-receptors were blocked by RAP (2 in Figure 2), resulting in a half-life prolonging effect of factor 3.5 for FVIII. In a third set-up both, HSPGs and LRP-receptors, were blocked synergistically, yielding a FVIII half-life prolongation of factor 5.5. Prior art has tried to block the receptor to influence receptor mediated clearance. The present invention is directed to a method for influencing receptor mediated clearance by modifying the therapeutic proteins by blocking them non-covalently via specific binding of a small ligand or covalently by attaching a small ligand . FVIII has various binding sites for proteins involved in Tenase complex formation, FVIII-clearance and proteolytic inactivation. These binding sites partially or totally overlap (for example the binding sites for LRP-receptor/HSPG and FIXa) due to their importance during different phases within the FVIII life cycle.

FVIII comprises five different so far identified binding sites for LRP-receptor and one clearly characterized binding site for HSPG, which will be described in more detail in the following.

FVIII features one clearly identified and well characterized HSPG binding site located within the A2 domain, comprising the amino acids 558-565 (see Figure 3). This site reveals high affinity for heparin and analogues (25.8 nM) used as surrogates for HSPGs (5) and overlaps with the binding region for FIXa. However, there is evidence for a second low-affinity (KD = 652 nM) HSPG-binding site elsewhere on FVIII (5).

FVIII features five characterized binding sites for LRP-receptor located within the A2, A3, CI and C2 domains (see Figure 4). Two of them are high-affinity sites: the LRP-receptor binding sites in the A2 (484-509) and in the A3 domain (1811-1818), revealing dissociation constants (for purified proteins) of 25-116 nM (8;12). Both high-affinity LRP-binding sites overlap with the binding region for FIXa involved in Tenase complex formation. The LRP-receptor binding sites within the C2 and CI domains are of low affinity and shielded by vWF, thus playing only an inferior role in receptor-mediated FVIII clearance.

Our concept for non-covalent half-life prolongation (HLP) of proteins like FVIII is based on the assumption, that blocking of certain binding sites on the surface of FVIII by small ligands (peptides, sugars, small proteins, nucleic acids, chemical compounds) will be sufficient to inhibit/decelerate receptor- mediated clearance and proteolytic inactivation mechanisms of FVIII, thus prolonging its plasma half-life.

In general the target site can be any region on the FVIII surface whose blocking by a small binding ligand results in a half-life prolonging effect of the target protein.

For this approach the A2 and A3 domains of FVIII are targeted for the following reasons:

- The A2 domain harbours a high-affinity binding site for the LRP-receptor as well as for HSPGs which are essentially involved in receptor-mediated clearance of FVIII.

- The A3 domain, in addition, features a second high-affinity binding site for the LRP-receptor.

Thus, preferred target sites for HLP of FVIII are (see Figure 5) : - the A2 LRP-receptor binding site,

- the A2 HSPG-binding site and

- the A3 LRP-receptor binding site

It has been demonstrated by Sarafanov et al ., (5) that blocking HSPGs and LRPl-receptor on cell surfaces results in effective half-life prolongation for FVIII. However, only blocking both the HSPGs and the LRPl-receptor simultaneously yielded the maximal HLP-effect (factor 5.5), whereas single inhibition of HSPGs or LRPl-receptor showed smaller FVIII-HLP (factor 1.6 for blocking HSPGs and factor 3.5 for blocking LRPl-receptors). According to the invention, site-specific blocking of the above listed target sites on FVIII by small, highly affine and specific ligands will prolong the plasma half-life of FVIII. At which combination the target sites have to be blocked and if all target sites must be blocked in parallel to yield the maximal HLP-effect needs to be determined . Ligands found binding to or around the HSPG- or LRPl-receptor binding sites on FVIII during the screening procedure can be further optimized regarding their binding affinity and/or specificity.

For FVIII the amino acid sequence is known, several structural data are available and the HSPG- and LRPl-receptor binding sites are characterized . The LRP-receptor sequence is also available, as well as structural information of LRPR-subdomains involved in direct binding of FVIII A2 and A3 domains. Additionally, various information about heparin and heparin-analogs is available.

In case of binding peptide ligands, the ligand sequence is known and the structure could be solved for example by nuclear magnetic resonance (NMR), if needed. Taking into account all the available information, the binding affinity and/or specificity can be enhanced by varying the amino acid sequence of the binding region of the ligand (changing charge, polaritiy or bulkyness of one or more amino acid positions) and/or the overall length of the ligand. Ligands can be unstructured or of structured nature, comprising a specific folding .

Unstructured, linear peptide or sugar ligands for example will adopt different flexible conformations in solution. Therefore, the binding to the target protein might be more probable and of high affinity, but maybe of low specificity. As mentioned, an optimal ligand for non-covalent half-life prolongation should bind with high affinity and specificity. It should be stable and soluble in solution, non-immunogenic and non-toxic, and should not influence the biological activity of the target protein.

Structured, folded ligands (for examples cyclic scaffold peptides, small proteins or protein domains) comprise the advantages of a stable folding and therefore a defined conformation in solution. This folding corresponds to a specific surface of this ligand, thus rendering a binding event to be specific. The ligand folding is also responsible for a better stability in solution.

As stated above a ligand for a non-covalent half-life prolongation approach should fulfill the following properties:

- The binding event should be of high affinity ("tight binding").

- The ligand should bind with high specificity to the target protein and should not cross-react with other binding partners (no "promiscuity").

The following section deals with ligand categories useful for this approach. Linear peptides of variable length and sequence are used as binding partners for non-covalent half-life prolongation of FVIII. Their conformation is flexible in solution, thus they can adopt various conformations upon a binding event. Linear peptides binding with high affinity and specificity to FVIII are identified by screening of huge random libraries.

Linear sulphated peptides are used as HSPG-mimetics and targeted to the HSPG-site on the A2 domain on FVIII. There are some sulphated heparin- mimetic peptides known to bind to fibroblast growth factor 1 (FGFl) or vascular endothelial growth factor (VEGF), both being HSPG or heparin- binding proteins (15;16). Although this targeted approach is promising in terms of binding, the synthesis of such poly-sulphated peptides is highly complicated and time consuming, as the intermediates and products may not be stable.

Structured peptides can be any peptide showing a distinct folding or structure. These peptides can be mono-, bi-, tri- or polycyclic. Their cyclic structure can be achieved for example by introduction of cysteines and subsequent disulfide bond formation.

Structured peptides comprise the advantages of a higher stability due to their 3-dimensional structure. They have a defined surface, which renders the binding to a potential target protein much more specific than for example linear peptides would achieve. With structured peptides it is possible to mimic known binding ligands or binding surfaces (see Figure 9 and 10).

A continuation of the idea using peptides as small binding ligands is the idea to use small proteins or protein domains with a specific modified binding region for specifically binding to the targeted sites on the target protein. The function of the overall protein folding is to stabilize the specific binding region (see Figure 7).

A different substance class applicable as specific binding ligands for half-life prolongation are sugars. As an example, heparin-mimetic sulphated sugars targeted to the HSPG-binding site of FVIII can be used . In addition sugars binding to the LRP-receptor binding site on FVIII can be used as LRP-mimetic sugar ligands.

Sugars provide the advantage of low immunogenicity. However their designed synthesis can be very complex, cost and time consuming. Other ligands for the covalent or non-covalent HLP-approach can comprise any substance fitting the above mentioned ligand properties needed. These can include for example DNA or RNA-molecules (structured) (e.g . using the aptamer technology) or antibody-derived domains , any non-covalent, specific binding substance irrespective of its exact chemical nature, including peptides, DNA, RNA, sugars, oligosaccharides, or any other chemical substance.

Brief description of the figures

Figure 1 is a schematic overview of clearance and inactivation mechanisms for FVIII and FVIIIa.

Figure 2 is a schematic overview of clearance studies in mice performed by Sarafanov et al ., (5).

Figure 3 shows B-domain deleted FVIII and its binding sites for Tenase complex formation, clearance receptors and proteolytic inactivation. Capital letters indicate the domain structure; numbers give the amino acid positions of the binding sites.

Figure 4 shows an overview of binding sites for HSPG-and LRP-receptor on B- domain deleted FVIII. Capital letters give the FVIII domain structure; numbers give the amino acid positions of the corresponding binding site.

Figure 5 shows: FVIII and preferred target sites for half-life prolongation.

Figure 6 shows the full length mature FVIII protein sequence (w.o. signal peptide).

Figure 7 is a schematic representation of the non-covalent binding approach using small proteins or domains with specific, designed binding regions stabilized by the overall protein folding. Figure 8 shows the relative half-life of FVIII with bound low molecular weight heparin Clexane derived by a pharmacokinetic mouse study where wild-type mice were administered with exogenous FVIII with and without Clexane. Clexane binds to the HSPG-binding site of FVIII and therefore blocks the interaction with cell surface HSPGs partially responsible for FVIII clearance. The plasma half-life was prolonged by factor 2. All data were statistically evaluated and passed the significance test (P-values < 0.05 : P(FVIII) = 0.0414 and P(FVIII+Clexane) = 0.0363).

Figure 9 is a schematic representation of potential binding sites on a protein, their structure and possibilities to mimic these binding sites using peptides: linear, cyclic or poly-cyclic peptides; circles: single amino acids, yellow connected circles indicate cysteines bridged by sulfhydryl groups.

Figure 10 is a schematic representation of the design of ligands for half-life prolongation of FVIII (A) and construction of binding site mimetics (B). A: the ligand structures and sequence information are used to generate peptide ligands resembling and mimicking the physiological ligand (for example LRP1- receptor); B: the FVIII structures and sequence information can be used to generate binding site mimetics mimicking the FVIII HSPG- and/or LRP- receptor binding sites and can be used as screening tools for finding ligands targeted to these binding sites.

Examples

Sulphated sugars

Sulphated sugars are mimick HSPGs or heparin and are therefore likely to bind to the HSPG- or heparin binding sites of proteins. Different heparin-mimetic sugars varying in length and sulphation pattern are used to find sugar-ligands binding with high affinity and specificity to the HSPG-binding site on the A2 domain of FVIII to block the clearance interaction with cell surface HSPGs. The effect of a heparin-mimetic sugar bound to the HSPG-site on FVIII on its plasma half-life by pharmacokinetic animal study using wild-type mice is demonstrated (see Figure 8).

These were administered with exogenous recombinant FVIII with bound Clexane. Clexane is a low molecular weight (LMW) heparin (Mw = 4.5 kDa), which binds to the HSPG-binding site on the A2 domain of FVIII and therefore blocks the interaction with cell surface HSPGs partially responsible for FVIII- clearance. Binding Clexane resulted in an around 2-fold longer plasma half-life of FVIII. This clearly indicates that a small ligand (like Clexane) is able to sufficiently block an important clearance site of FVIII, thus directly resulting in a prolonged plasma half-life. The results are shown in Figure 8.

These findings are also in line with the results of Sarafanov et al . (5) who performed FVIII-clearance studies in mice and found a half-life prolonging effect for FVIII by factor 1.6 via direct blocking of the HSPGs by Protamin on cell surfaces.

Peptides a) Octet-Screening

Screening and kinetic measurements have been carried out using the Octet Red system. The principle is based on the optical measurement of the thickness of a protein layer on a biosensor tip. Streptavidin sensors are used for screening and kinetic experiments of biotinylated peptides with recombinant FVIII, recombinant FVIII domain A2. As a negative control, the C2 domain of FVIII and recombinant FIX were used and showed no binding to the ligands. The biosensor is coated with a ligand . Then the immobilized ligand is exposed to an analyte. The thickness of the resulting ligand-analyte complex/layer is observed during association and dissociation. Thereof the binding affinity between ligand and analyte is computed. Measurement took place in a 96-well plate with eight biosensors analyzing up to eight samples in parallel . The concentrations of the ligands for screening experiments have been optimized to 5 pg/rnl for peptides and antibodies. Experiments with streptavidin sensors are conducted with 10 pg/rnl biotin as quenching agent.

Screening random peptides, the following binding kinetics could be determined for SEQ ID NO 2 to 23 :

Figure imgf000019_0001
"-" indicates no detectable binding

Peptides showing binding to recombinant FVIII, but not to the A2 domain, bind through a different binding site. Phaqe-Displav

Starting from a random library, phages were selected for their ability to bind to recombinant FVIII or recombinant FVIII domains (for example A2)

An ELISA was conducted to quantify interaction between the binding ligands and FVIII or A2. The following ligands (SEQ ID NO 24 to 25) were identified :

Figure imgf000020_0001

Macroarrav-Screeninq

Intavis-(384)-peptide arrays containing FIX, LRP-II and LRP-IV sequences were used for detecting specific protein-peptide interactions on the peptide array in order to identify potential ligands for half-life prolongation of FVIII.

The peptide array was incubated with recombinant FVIII or the A2 domain.

Figure imgf000020_0002

Ligand 384-1 to ligand 384-8 (SEQ ID NO 26 to 33) show binding to both FVIII and the A2 domain . References

Furie, B. and Furie, B. C. (1988) The molecular basis of blood coagulation, Cell 53, 505-518.

Lenting, P. J., Christophe, O. D., and Gueguen, P. (2008) The disappearing act of factor VIII, Haemophilia.

Lillicrap, D. (2008) Extending half-life in coagulation factors: where do we stand?, Thromb. Res. 122 Suppl 4, S2-S8.

Spijkers, P. P., Denis, C. V., Blom, A. M ., and Lenting, P. J. (2008) Cellular uptake of C4b-binding protein is mediated by heparan sulfate proteoglycans and CD91/LDL receptor-related protein, Eur. J Immunol.

38, 809-817.

Sarafanov, A. G., Ananyeva, N. M ., Shima, M ., and Saenko, E. L. (2001) Cell surface heparan sulfate proteoglycans participate in factor VIII catabolism mediated by low density lipoprotein receptor- related protein, J Biol. Chem. 276, 11970-11979.

Mikhailenko, I., Kounnas, M . Z., and Strickland, D. K. (1995) Low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor mediates the cellular internalization and degradation of thrombospondin. A process facilitated by cell-surface proteoglycans, J Biol. Chem. 270, 9543-9549.

Bovenschen, N ., Herz, J., Grimbergen, J. M ., Lenting, P. J., Havekes, L. M ., Mertens, K., and van Vlijmen, B. J. (2003) Elevated plasma factor VIII in a mouse model of low-density lipoprotein receptor-related protein deficiency, Blood 101 , 3933-3939.

Lenting, P. J., van Mourik, J. A., and Mertens, K. (1998) The life cycle of coagulation factor VIII in view of its structure and function, Blood 92, 3983-3996.

Marchetti, G., Lunghi, B., Legnani, C, Cini, M ., Pinotti, M., Mascoli, F., and Bernard, F. (2006) Contribution of low density lipoprotein receptor- related protein genotypes to coagulation factor VIII levels in thrombotic women, Haematologica 91 , 1261-1263.

Morange, P. E., Tregouet, D. A., Frere, C, Saut, N., Pellegrina, L., Alessi, M . C, Visvikis, S., Tiret, L., and Juhan-Vague, I. (2005) Biological and genetic factors influencing plasma factor VIII levels in a healthy family population : results from the Stanislas cohort, Br. J Haematol. 128, 91-99. Neels, J. G., Bovenschen, N ., van Zonneveld, A. J., and Lenting, P. J. (2000) Interaction between factor VIII and LDL receptor-related protein. Modulation of coagulation?, Trends Cardiovasc Med. 10, 8-14.

Saenko, E. L., Yakhyaev, A. V., Mikhailenko, I., Strickland, D. K., and Sarafanov, A. G. (1999) Role of the low density lipoprotein-related protein receptor in mediation of factor VIII catabolism, J Biol. Chem. 274, 37685-37692.

Vormittag, R., Bencur, P., Ay, C, Tengler, T., Vukovich, T., Quehenberger, P., Mannhalter, C, and Pabinger, I. (2007) Low-density lipoprotein receptor-related protein 1 polymorphism 663 C > T affects clotting factor VIII activity and increases the risk of venous thromboembolism, J Thromb. Haemost. 5, 497-502. 14. Cunningham, N., Laffan, M. A., Manning, R. A., and O'Donnell, J. S.

(2005) Low-density lipoprotein receptor-related protein polymorphisms in patients with elevated factor VIII coagulant activity and venous thrombosis, Blood Coagul. Fibrinolysis 16, 465-468.

15. Maynard, H. D. and Hubbell, J. A. (2005) Discovery of a sulfated tetrapeptide that binds to vascular endothelial growth factor, Acta Biomater. 1, 451-459.

16. Kim, S. H. and Kiick, K. L. (2007) Heparin-mimetic sulfated peptides with modulated affinities for heparin-binding peptides and growth factors, Peptides 28, 2125-2136.

17. Timmerman, P., Beld, J., Puijk, W. C, and Meloen, R. H. (2005) Rapid and quantitative cyclization of multiple peptide loops onto synthetic scaffolds for structural mimicry of protein surfaces, Chembiochem. 6, 821-824.

18. Timmerman, P., Puijk, W. C, and Meloen, R. H. (2007) Functional reconstruction and synthetic mimicry of a conformational epitope using CLIPS technology, J Mol. Recognit. 20, 283-299.

19. Dolmer, K., Huang, W., and Gettins, P. G. (1998) Characterization of the calcium site in two complement-like domains from the low-density lipoprotein receptor-related protein (LRP) and comparison with a repeat from the low-density lipoprotein receptor, Biochemistry 37, 17016-17023.

20. Jensen, G. A., Andersen, O. M., Bonvin, A. M ., Bjerrum-Bohr, I., Etzerodt, M ., Thogersen, H . C, O'Shea, C, Poulsen, F. M ., and Kragelund, B. B.

(2006) Binding site structure of one LRP-RAP complex: implications for a common ligand-receptor binding motif, J Mol. Biol. 362, 700-716.

21. Simonovic, M ., Dolmer, K., Huang, W., Strickland, D. K., Volz, K., and Gettins, P. G. (2001) Calcium coordination and pH dependence of the calcium affinity of ligand-binding repeat CR7 from the LRP. Comparison with related domains from the LRP and the LDL receptor, Biochemistry 40, 15127-15134.

22. Huang, W., Dolmer, K., and Gettins, P. G. (1999) NMR solution structure of complement-like repeat CR8 from the low density lipoprotein receptor- related protein, J Biol. Chem. 274, 14130-14136.

23. Sarafanov, A. G., Makogonenko, E. M ., Pechik, I. V., Radtke, K. P., Khrenov, A. V., Ananyeva, N. M., Strickland, D. K., and Saenko, E. L.

(2006) Identification of coagulation factor VIII A2 domain residues forming the binding epitope for low-density lipoprotein receptor-related protein, Biochemistry 45, 1829-1840.

24. Sarafanov, A. G., Makogonenko, E. M ., Andersen, O. M ., Mikhailenko, I.

A., Ananyeva, N. M., Khrenov, A. V., Shima, M., Strickland, D. K., and

Saenko, E. L. (2007) Localization of the low-density lipoprotein receptor- related protein regions involved in binding to the A2 domain of coagulation factor VIII, Thromb. Haemost. 98, 1170-1181.

Claims

Claims
Pharmaceutical preparation comprising
- a pharmaceutically active protein having at least one LRP-binding site and/or at least one HSPG binding site and
- at least one ligand binding to the LRP-binding site and/or the HSPG binding site, the at least one ligand being selected from peptides, sugars, nucleic acids and small chemical compounds, said small chemical compounds having a molecular weight of 5 KDa or less.
Pharmaceutical preparation according to claim 1 wherein the pharmaceutically active protein is Factor VIII, preferably human FVIII.
Pharmaceutical preparation according to claim 2 wherein FVIII is recombinant FVIII.
Pharmaceutical preparation according to at least one of claims 2 to 3, wherein FVIII is B domain deleted FVIII.
Pharmaceutical preparation according to at least one of claims 2 to 4, wherein the FVIII LRP binding site is localized within the A2 domain residues 484-509 and/or the A3 domain residues 1811-1818 C2 domain residues 2173-2332 and/or CI domain residues 2065, 2092 in accordance with SEQ ID NO. 1.
Pharmaceutical preparation according to at least one of claims 2 to 5, wherein the FVIII HSPG binding site is localized within the A2 domain residues 558-565 in accordance with SEQ ID NO. 1.
Pharmaceutical preparation according to at least one of claims 1 to 6, wherein the ligand has a molecular weight below 5 kDa.
8. Pharmaceutical preparation according to at least one of claims 1 to 7, wherein the ligand is selected from peptides, sugars, lipids, nucleic acids and variants thereof and chemically synthesized compounds.
9. Pharmaceutical preparation according to claim 8, wherein the peptide is a linear, cyclic, folded or a scaffold peptide.
10. Pharmaceutical preparation according to at least one of claims 1 to 9, wherein the ligand comprises at least one sulphate group.
11. Pharmaceutical preparation of claims 1 to 10, wherein the ligand is selected from peptides comprising SEQ ID No. 2 to 33.
12. A pharmaceutical preparation comprising
- a pharmaceutically active protein having at least one LRP-binding site and/or at least one HSPG binding site and
- at least one molecule binding to the LRP-binding site and/or the HSPG binding site, the at least one molecule
comprising two or more ligands covalently bridged for blocking adjacent binding sites.
13. The pharmaceutical preparation of claim 12 wherein one of the binding sites is a HSPG-binding sites and the other binding site is a LRP binding site.
14. A method for testing a ligand's ability to bind to at least one LRP binding site and/or at least one HSPG binding site of FVIII comprising the steps of
- combining the ligand with FVIII to a FVIII-ligand complex
- measuring clearance of the FVIII-ligand complex in-vivo
wherein a reduced clearance indicates that the ligand binds to at least one LRP-binding site and/or at least one HSPG binding site of FVIII, , the ligand being selected from peptides, sugars, nucleic acids and small chemical compounds, said small chemical compounds having a molecular weight of 5 KDa or less.
15. A method for reducing clearance of FVIII comprising the step of
- combining FVIII with at least one ligand binding to the LRP-binding site or the HSPG binding site of FVIII to form a FVIII-ligand complex
- administering said FVIII-ligand complex to a patient in need of FVIII, , the ligand being selected from peptides, sugars, nucleic acids and small chemical compounds, said small chemical compounds having a molecular weight of 5 KDa or less.
16. A ligand selected from peptides having one of the SEQ. ID. No 2 to 33.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013057171A1 (en) * 2011-10-18 2013-04-25 Csl Behring Gmbh Combined use of a sulfated glycosaminoglycan and a hyaluronidase for improving the bioavailability of factor viii
WO2013057167A1 (en) * 2011-10-18 2013-04-25 Csl Behring Gmbh Use of sulfated glycosaminoglycans for improving the bioavailability of factor viii

Citations (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0150735A2 (en) 1984-01-12 1985-08-07 Chiron Corporation Protein composition exhibiting coagulation activity and method for the preparation thereof
EP0160457A1 (en) 1984-04-20 1985-11-06 Genentech, Inc. Human factor VIII, Compositions containing it, methods and materials for use in it production
WO1986001961A1 (en) 1984-09-14 1986-03-27 Sony Corporation Device for controlling beam current of a tv camera device
WO1986006101A1 (en) 1985-04-12 1986-10-23 Genetics Institute, Inc. Novel procoagulant proteins
WO1987004187A1 (en) 1986-01-03 1987-07-16 Genetics Institute, Inc. METHOD FOR PRODUCING FACTOR VIII:c-TYPE PROTEINS
EP0232112A2 (en) 1986-01-27 1987-08-12 Chiron Corporation Recombinant protein complex having human factor VIII:C activity, its production and use
WO1987007144A1 (en) 1986-05-29 1987-12-03 Genetics Institute, Inc. Novel procoagulant proteins
EP0251843A1 (en) 1986-06-06 1988-01-07 Transgene S.A. Process for the preparation of factor VIII from mammalian cells
WO1988000381A1 (en) 1986-07-08 1988-01-14 Palle Pedersen Locking device
EP0253455A1 (en) 1986-07-18 1988-01-20 Gist-Brocades N.V. Method for the preparation of proteins with factor VIII activity by microbial host cells; expression vectors, host cells, antibodies
EP0254076A1 (en) 1986-07-11 1988-01-27 Miles Inc. Improved recombinant protein production
EP0265778A1 (en) 1986-10-15 1988-05-04 RORER INTERNATIONAL (OVERSEAS) INC. (a Delaware corporation) Factor VIII-C analogs
US4770999A (en) 1985-04-22 1988-09-13 Genetics Institute, Inc. High yield production of active Factor IX
EP0294910A1 (en) 1987-06-12 1988-12-14 Immuno Ag Novel proteins with factor VIII activity, process for their preparation using genetically engineered cells and pharmaceutical compositions containing them
EP0303540A1 (en) 1987-08-11 1989-02-15 Transgene S.A. Factor VIII analog, process for its preparation and composition containing it
US4980456A (en) 1987-04-06 1990-12-25 Scripps Clinic And Research Foundation Recombinant factor VIIIC derived fragments
WO1991007490A1 (en) 1989-11-17 1991-05-30 Novo Nordisk A/S Protein complexes having factor viii:c activity and production thereof
WO1991009122A1 (en) 1989-12-15 1991-06-27 Kabivitrum Ab A recombinant human factor viii derivative
US5045455A (en) 1984-01-12 1991-09-03 Chiron Corporation Factor VIII:C cDNA cloning and expression
WO1994029471A1 (en) 1993-06-10 1994-12-22 Genetic Therapy, Inc. Adenoviral vectors for treatment of hemophilia
WO1995013300A1 (en) 1993-11-12 1995-05-18 Novo Nordisk A/S New factor viii polypeptides
US5422260A (en) 1986-05-29 1995-06-06 Genetics Institute, Inc. -Legal Affairs Human factor VIII:c muteins
US5521070A (en) 1988-11-09 1996-05-28 Transgene S.A. DNA sequence coding for human factor IX or a similar protein, expression vector, transformed cells, method for preparing factor IX and corresponding products obtained
WO1997049725A1 (en) 1996-06-26 1997-12-31 Emory University Modified factor viii
US5789203A (en) 1986-01-27 1998-08-04 Chiron Corporation Protein complexes having factor VIII:C activity and production thereof
WO1999029848A1 (en) 1997-12-05 1999-06-17 The Immune Response Corporation Novel vectors and genes exhibiting increased expression
EP1454632A1 (en) * 2003-02-07 2004-09-08 Aventis Behring GmbH, Intellectual Property/Legal Pharmaceutical preparation for the treatment of blood-clotting disorders containing factor VIII derived peptides
WO2006103298A2 (en) * 2005-04-01 2006-10-05 Novo Nordisk Health Care Ag Blood coagulation fviii analogues
US20080219983A1 (en) * 2002-04-29 2008-09-11 Trimester Cushion Company Antagonists of Factor VIII Interaction with Low-Density Lipoprotein Receptor Related Protein
WO2009135888A2 (en) 2008-05-06 2009-11-12 Octapharma Ag Complex

Patent Citations (34)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5045455A (en) 1984-01-12 1991-09-03 Chiron Corporation Factor VIII:C cDNA cloning and expression
EP0150735A2 (en) 1984-01-12 1985-08-07 Chiron Corporation Protein composition exhibiting coagulation activity and method for the preparation thereof
EP0160457A1 (en) 1984-04-20 1985-11-06 Genentech, Inc. Human factor VIII, Compositions containing it, methods and materials for use in it production
WO1986001961A1 (en) 1984-09-14 1986-03-27 Sony Corporation Device for controlling beam current of a tv camera device
US4868112A (en) 1985-04-12 1989-09-19 Genetics Institute, Inc. Novel procoagulant proteins
WO1986006101A1 (en) 1985-04-12 1986-10-23 Genetics Institute, Inc. Novel procoagulant proteins
US4770999A (en) 1985-04-22 1988-09-13 Genetics Institute, Inc. High yield production of active Factor IX
WO1987004187A1 (en) 1986-01-03 1987-07-16 Genetics Institute, Inc. METHOD FOR PRODUCING FACTOR VIII:c-TYPE PROTEINS
EP0232112A2 (en) 1986-01-27 1987-08-12 Chiron Corporation Recombinant protein complex having human factor VIII:C activity, its production and use
US5789203A (en) 1986-01-27 1998-08-04 Chiron Corporation Protein complexes having factor VIII:C activity and production thereof
WO1987007144A1 (en) 1986-05-29 1987-12-03 Genetics Institute, Inc. Novel procoagulant proteins
US5422260A (en) 1986-05-29 1995-06-06 Genetics Institute, Inc. -Legal Affairs Human factor VIII:c muteins
US5451521A (en) 1986-05-29 1995-09-19 Genetics Institute, Inc. Procoagulant proteins
EP0251843A1 (en) 1986-06-06 1988-01-07 Transgene S.A. Process for the preparation of factor VIII from mammalian cells
WO1988000381A1 (en) 1986-07-08 1988-01-14 Palle Pedersen Locking device
EP0254076A1 (en) 1986-07-11 1988-01-27 Miles Inc. Improved recombinant protein production
EP0253455A1 (en) 1986-07-18 1988-01-20 Gist-Brocades N.V. Method for the preparation of proteins with factor VIII activity by microbial host cells; expression vectors, host cells, antibodies
EP0265778A1 (en) 1986-10-15 1988-05-04 RORER INTERNATIONAL (OVERSEAS) INC. (a Delaware corporation) Factor VIII-C analogs
US4980456A (en) 1987-04-06 1990-12-25 Scripps Clinic And Research Foundation Recombinant factor VIIIC derived fragments
EP0294910A1 (en) 1987-06-12 1988-12-14 Immuno Ag Novel proteins with factor VIII activity, process for their preparation using genetically engineered cells and pharmaceutical compositions containing them
EP0303540A1 (en) 1987-08-11 1989-02-15 Transgene S.A. Factor VIII analog, process for its preparation and composition containing it
US5521070A (en) 1988-11-09 1996-05-28 Transgene S.A. DNA sequence coding for human factor IX or a similar protein, expression vector, transformed cells, method for preparing factor IX and corresponding products obtained
EP0500734A1 (en) 1989-11-17 1992-09-02 Chiron Corporation Protein complexes having factor viii:c activity and production thereof
WO1991007490A1 (en) 1989-11-17 1991-05-30 Novo Nordisk A/S Protein complexes having factor viii:c activity and production thereof
WO1991009122A1 (en) 1989-12-15 1991-06-27 Kabivitrum Ab A recombinant human factor viii derivative
US5859204A (en) 1992-04-07 1999-01-12 Emory University Modified factor VIII
WO1994029471A1 (en) 1993-06-10 1994-12-22 Genetic Therapy, Inc. Adenoviral vectors for treatment of hemophilia
WO1995013300A1 (en) 1993-11-12 1995-05-18 Novo Nordisk A/S New factor viii polypeptides
WO1997049725A1 (en) 1996-06-26 1997-12-31 Emory University Modified factor viii
WO1999029848A1 (en) 1997-12-05 1999-06-17 The Immune Response Corporation Novel vectors and genes exhibiting increased expression
US20080219983A1 (en) * 2002-04-29 2008-09-11 Trimester Cushion Company Antagonists of Factor VIII Interaction with Low-Density Lipoprotein Receptor Related Protein
EP1454632A1 (en) * 2003-02-07 2004-09-08 Aventis Behring GmbH, Intellectual Property/Legal Pharmaceutical preparation for the treatment of blood-clotting disorders containing factor VIII derived peptides
WO2006103298A2 (en) * 2005-04-01 2006-10-05 Novo Nordisk Health Care Ag Blood coagulation fviii analogues
WO2009135888A2 (en) 2008-05-06 2009-11-12 Octapharma Ag Complex

Non-Patent Citations (32)

* Cited by examiner, † Cited by third party
Title
ANANYEVA N M ET AL: "Catabolism of the coagulation factor VIII: can we prolong lifetime of f VIII in circulation?", TRENDS IN CARDIOVASCULAR MEDICINE, ELSEVIER SCIENCE, NEW YORK, NY, US LNKD- DOI:10.1016/S1050-1738(01)00124-4, vol. 11, no. 6, 1 August 2001 (2001-08-01), pages 251 - 257, XP002320945, ISSN: 1050-1738 *
BOVENSCHEN, N.; HERZ, J.; GRIMBERGEN, J. M.; LENTING, P. J.; HAVEKES, L. M.; MERTENS, K.; VAN VLIJMEN, B. J.: "Elevated plasma factor VIII in a mouse model of low-density lipoprotein receptor-related protein deficiency", BLOOD, vol. 101, 2003, pages 3933 - 3939
CUNNINGHAM, N.; LAFFAN, M. A.; MANNING, R. A.; O'DONNELL, J. S.: "Low-density lipoprotein receptor-related protein polymorphisms in patients with elevated factor VIII coagulant activity and venous thrombosis", BLOOD COAGUL. FIBRINOLYSIS, vol. 16, 2005, pages 465 - 468, XP009141760
DATABASE BIOSIS, [Online] 16 November 2003 (2003-11-16), MERTENS KOEN ET AL: "The endocytic receptors megalin and low-density lipoprotein receptor-related protein share binding to coagulation factor VIII", XP002589830, retrieved from BIOSIS Database accession no. PREV200400172501 *
DOLMER, K.; HUANG, W.; GETTINS, P. G.: "Characterization of the calcium site in two complement-like domains from the low-density lipoprotein receptor-related protein (LRP) and comparison with a repeat from the low-density lipoprotein receptor", BIOCHEMISTRY, vol. 37, 1998, pages 17016 - 17023
FAY P J ET AL: "Mutating factor VIII: lessons from structure to function", BLOOD REVIEWS, CHURCHILL LIVINGSTONE LNKD- DOI:10.1016/J.BLRE.2004.02.003, vol. 19, no. 1, 1 January 2005 (2005-01-01), pages 15 - 27, XP004661269, ISSN: 0268-960X *
FURIE, B.; FURIE, B. C.: "The molecular basis of blood coagulation", CELL, vol. 53, 1988, pages 505 - 518, XP023908863, DOI: doi:10.1016/0092-8674(88)90567-3
HUANG, W.; DOLMER, K.; GETTINS, P. G.: "NMR solution structure of complement-like repeat CR8 from the low density lipoprotein receptor-related protein", J BIOL. CHEM., vol. 274, 1999, pages 14130 - 14136
JENSEN, G. A.; ANDERSEN, O. M.; BONVIN, A. M; BJERRUM-BOHR, I.; ETZERODT, M.; THOGERSEN, H. C.; O'SHEA, C.; POULSEN, F. M.; KRAGEL: "Binding site structure of one LRP-RAP complex: implications for a common ligand-receptor binding motif", J MOL. BIOL., vol. 362, 2006, pages 700 - 716, XP024951404, DOI: doi:10.1016/j.jmb.2006.07.013
KIM, S. H.; KIICK, K. L.: "Heparin-mimetic sulfated peptides with modulated affinities for heparin-binding peptides and growth factors", PEPTIDES, vol. 28, 2007, pages 2125 - 2136, XP022309379, DOI: doi:10.1016/j.peptides.2007.07.002
LENTING P J ET AL: "THE LIGHT CHAIN OF FACTOR VIII COMPRISES A BINDING SITE FOR LOW DENSITY LIPOPRETEIN RECEPTOR-RELATED PROTEIN", JOURNAL OF BIOLOGICAL CHEMISTRY, THE AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, INC., BALTIMORE, MD, US, vol. 274, no. 34, 1 August 1999 (1999-08-01), pages 23743 - 23749, XP000882874, ISSN: 0021-9258, DOI: DOI:10.1074/JBC.274.34.23734 *
LENTING, P. J.; CHRISTOPHE, O. D.; GUEGUEN, P.: "The disappearing act of factor VIII", HAEMOPHILIA, 2008
LENTING, P. J.; VAN MOURIK, J. A.; MERTENS, K.: "The life cycle of coagulation factor VIII in view of its structure and function", BLOOD, vol. 92, 1998, pages 3983 - 3996, XP002333863
LILLICRAP, D.: "Extending half-life in coagulation factors: where do we stand?", THROMB. RES., vol. 122, no. 4, 2008, pages S2 - S8, XP025627027, DOI: doi:10.1016/S0049-3848(08)70027-6
MARCHETTI, G.; LUNGHI, B.; LEGNANI, C.; CINI, M.; PINOTTI, M.; MASCOLI, F.; BERNARD, F.: "Contribution of low density lipoprotein receptor-related protein genotypes to coagulation factor VIII levels in thrombotic women", HAEMATOLOGICA, vol. 91, 2006, pages 1261 - 1263
MAYNARD, H. D.; HUBBELL, J. A.: "Discovery of a sulfated tetrapeptide that binds to vascular endothelial growth factor", ACTA BIOMATER., vol. 1, 2005, pages 451 - 459, XP027820209
MEIJER ET AL: "Functional duplication of ligand-binding domains within low-density lipoprotein receptor-related protein for interaction with receptor associated protein, alpha2-macroglobulin, factor IXa and factor VIII", BIOCHIMICA ET BIOPHYSICA ACTA (BBA) - PROTEINS & PROTEOMICS, ELSEVIER, vol. 1774, no. 6, 5 June 2007 (2007-06-05), pages 714 - 722, XP022104838, ISSN: 1570-9639 *
MIKHAILENKO, I.; KOUNNAS, M. Z.; STRICKLAND, D. K.: "Low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor mediates the cellular internalization and degradation of thrombospondin. A process facilitated by cell-surface proteoglycans", J BIOL. CHEM., vol. 270, 1995, pages 9543 - 9549
MORANGE, P. E.; TREGOUET, D. A.; FRERE, C.; SAUT, N.; PELLEGRINA, L.; ALESSI, M. C.; VISVIKIS, S.; TIRET, L; JUHAN-VAGUE, I.: "Biological and genetic factors influencing plasma factor VIII levels in a healthy family population: results from the Stanislas cohort", BR. J HAEMATOL., vol. 128, 2005, pages 91 - 99
NEELS, J. G.; BOVENSCHEN, N.; VAN ZONNEVELD, A. J.; LENTING, P. J.: "Interaction between factor VIII and LDL receptor-related protein. Modulation of coagulation?", TRENDS CARDIOVASC MED., vol. 10, 2000, pages 8 - 14, XP002264412, DOI: doi:10.1016/S1050-1738(00)00036-0
SAENKO E L ET AL: "Role of the low density lipoprotein-related protein receptor in mediation of factor VIII catabolism", JOURNAL OF BIOLOGICAL CHEMISTRY, THE AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, INC., BALTIMORE, MD, US, vol. 274, no. 53, 31 December 1999 (1999-12-31), pages 37685 - 37692, XP002152276, ISSN: 0021-9258, DOI: DOI:10.1074/JBC.274.53.37685 *
SAENKO, E. L.; YAKHYAEV, A. V.; MIKHAILENKO, I.; STRICKLAND, D. K.; SARAFANOV, A. G.: "Role of the low density lipoprotein-related protein receptor in mediation of factor VIII catabolism", J BIOL. CHEM., vol. 274, 1999, pages 37685 - 37692, XP002264411, DOI: doi:10.1074/jbc.274.53.37685
SARAFANOV A G ET AL: "Cell surface heparan sulfate proteoglycans participate in factor VIII catabolism mediated by low density lipoprotein receptor-related protein", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, INC, US LNKD- DOI:10.1074/JBC.M008046200, vol. 276, no. 15, 13 April 2001 (2001-04-13), pages 11970 - 11979, XP002233161, ISSN: 0021-9258 *
SARAFANOV, A. G.; ANANYEVA, N. M.; SHIMA, M.; SAENKO, E. L.: "Cell surface heparan sulfate proteoglycans participate in factor VIII catabolism mediated by low density lipoprotein receptor-related protein", J BIOL. CHEM., vol. 276, 2001, pages 11970 - 11979, XP002233161, DOI: doi:10.1074/jbc.M008046200
SARAFANOV, A. G.; MAKOGONENKO, E. M.; ANDERSEN, O. M.; MIKHAILENKO, I. A.; ANANYEVA, N. M.; KHRENOV, A. V.; SHIMA, M.; STRICKLAND,: "Localization of the low-density lipoprotein receptor-related protein regions involved in binding to the A2 domain of coagulation factor VIII", THROMB. HAEMOST., vol. 98, 2007, pages 1170 - 1181
SARAFANOV, A. G.; MAKOGONENKO, E. M; PECHIK, I. V.; RADTKE, K. P.; KHRENOV, A. V.; ANANYEVA, N. M.; STRICKLAND, D. K.; SAENKO, E.: "Identification of coagulation factor VIII A2 domain residues forming the binding epitope for low-density lipoprotein receptor-related protein", BIOCHEMISTRY, vol. 45, 2006, pages 1829 - 1840
SCHWARZ HANS PETER ET AL: "Involvement of low-density lipoprotein receptor-related protein (LRP) in the clearance of factor VIII in von Willebrand factor-deficient mice", BLOOD, vol. 95, no. 5, 1 March 2000 (2000-03-01), pages 1703 - 1708, XP002589828, ISSN: 0006-4971 *
SIMONOVIC, M.; DOLMER, K.; HUANG, W.; STRICKLAND, D. K.; VOLZ, K.; GETTINS, P. G.: "Calcium coordination and pH dependence of the calcium affinity of ligand-binding repeat CR7 from the LRP. Comparison with related domains from the LRP and the LDL recepto", BIOCHEMISTRY, vol. 40, 2001, pages 15127 - 15134
SPIJKERS, P. P.; DENIS, C. V.; BLOM, A. M.; LENTING, P. J.: "Cellular uptake of C4b-binding protein is mediated by heparan sulfate proteoglycans and CD91/LDL receptor-related protein", EUR. J IMMUNOL., vol. 38, 2008, pages 809 - 817
TIMMERMAN, P.; BELD, J.; PUIJK, W. C.; MELOEN, R. H.: "Rapid and quantitative cyclization of multiple peptide loops onto synthetic scaffolds for structural mimicry of protein surfaces", CHEMBIOCHEM, vol. 6, 2005, pages 821 - 824, XP002378236, DOI: doi:10.1002/cbic.200400374
TIMMERMAN, P.; PUIJK, W. C.; MELOEN, R. H.: "Functional reconstruction and synthetic mimicry of a conformational epitope using CLIPS technology", J MOL. RECOGNIT., vol. 20, 2007, pages 283 - 299, XP055012496, DOI: doi:10.1002/jmr.846
VORMITTAG, R.; BENCUR, P.; AY, C.; TENGLER, T.; VUKOVICH, T.; QUEHENBERGER, P.; MANNHALTER, C.; PABINGER, I.: "Low-density lipoprotein receptor-related protein 1 polymorphism 663 C > T affects clotting factor VIII activity and increases the risk of venous thromboembolism", J THROMB. HAEMOST., vol. 5, 2007, pages 497 - 502

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