US20150080309A1 - Compounds Suitable for Treatment of Haemophilia - Google Patents

Compounds Suitable for Treatment of Haemophilia Download PDF

Info

Publication number
US20150080309A1
US20150080309A1 US14/396,907 US201314396907A US2015080309A1 US 20150080309 A1 US20150080309 A1 US 20150080309A1 US 201314396907 A US201314396907 A US 201314396907A US 2015080309 A1 US2015080309 A1 US 2015080309A1
Authority
US
United States
Prior art keywords
fviii
seq
vwf
vwf fragment
domain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US14/396,907
Other languages
English (en)
Inventor
Gert Bolt
Ditte M. Karpf
Frederik Rode
Jesper Haaning
Kirstine Roepstorff
Lars Thim
Maj Petersen
Marianne Kjalke
Ole Hvilsted Olsen
Jens J. Hansen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novo Nordisk AS
Original Assignee
Novo Nordisk AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Nordisk AS filed Critical Novo Nordisk AS
Priority to US14/396,907 priority Critical patent/US20150080309A1/en
Publication of US20150080309A1 publication Critical patent/US20150080309A1/en
Assigned to NOVO NORDISK A/S reassignment NOVO NORDISK A/S ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: OLSEN, OLE HVILSTED, HANSEN, JENS J., KJALKE, MARIANNE, HAANING, JESPER, BOLT, GERT, KARPF, DITTE M., PETERSEN, Maj, RODE, Frederik, ROEPSTORFF, Kirstine, THIM, LARS
Priority to US15/891,425 priority patent/US20180179262A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/37—Factors VIII
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents

Definitions

  • the present invention relates to treatment and/or prophylaxis of haemophilia.
  • Protein replacement therapy by intravenous administration of coagulation factors is currently used for treating patients suffering from haemophilia.
  • extravascular e.g. subcutaneous (s.c.) or intradermal
  • s.c. subcutaneous
  • intradermal intravenous
  • WO10062768 it is disclosed that PEGylation of FVIII can improve the bioavailability of FVIII in connection with subcutaneous injection into mice, whereas co-formulation with VWF does not improve the bioavailability of FVIII.
  • the present invention relates to a recombinant VWF fragment comprising 1200 amino acids or less, such as e.g. the TIL′ domain or the TIL′/E′ domain (Zhou et al. Blood 2012; 120(2): 449-458).
  • the present invention furthermore relates to a pharmaceutical composition comprising: (i) a VWF fragment according to the invention and (ii) FVIII molecule (full-length/truncated B domain/conjugated).
  • the present invention furthermore relates to use thereof for treatment of haemophilia, e.g. by extravascular administration.
  • Such compounds and compositions will preferably result in a relatively high FVIII bioavailability and/or a relatively low risk of FVIII immunogenicity in connection with extravascular co-administration of FVIII.
  • VWF fragments according to the invention co-administered with FVIII molecules having a prolonged in vivo circulatory half-life have a surprisingly high bioavailability in connection with extravascular (e.g. s.c.) administration thereof.
  • the inventors of the present invention have furthermore made the surprising observation that bioavailability of FVIII molecules may be significantly improved upon extravascular co-administration with similar molar amounts of VWF fragments according to the invention.
  • high bioavailability may be achieved through extravascular co-administration of a pool of FVIII molecules, wherein the majority of said FVIII molecules are bound to VWF fragments according to the invention.
  • full length VWF does not have a positive impact on the bioavailability of FVIII.
  • VWF should be in the form of a VWF fragment that comprise the TIL′ or the TIL′/E′ domains.
  • compositions according to the present invention are thus useful for treatment and prophylaxis of haemophilia patients (in particular haemophilia A patients) with and without inhibitors, as well as for immune tolerance induction (ITI) of haemophilia patients with inhibitors.
  • ITI immune tolerance induction
  • N8-GP is a glyco-PEGylated FVIII molecule produced as described in Examples 1+2 in WO2009108806.
  • FIG. 3 FVIII activity in plasma after subcutaneous administration of 2500 U/kg
  • FIG. 9 VWF variant (764-865 SEQ ID NO 5) binding to FVIII (N8, turoctocog alfa) at 20° C.
  • the upper panel shows raw data of heat released upon each titration.
  • Lower panel shows binding isotherm obtained from integrating raw data.
  • Data analysis shows that VWF variant (SEQ ID NO 5) binds to FVIII in an exothermic reaction with a stoichiometry of 1.14, ⁇ H of -5.82 kcal/mole, ⁇ S of 9.8 cal/mol/deg and a K d of 0.33 ⁇ M.
  • “F8/N8/turoctocog alfa” is a B domain truncated FVIII molecule produced as disclosed in Example 1 in WO2009108806.
  • FIG. 10 s.c. administrated N8-GP is haemostatic effective in vivo.
  • the left panel shows blood loss in FVIIIKO mice treated s.c. with N8-GP or vehicle 24 hr before tail transection, or i.v. 5 min before tail transection.
  • N8-GP′′ is a glyco-PEGylated FVIII molecule produced as described in Examples 1+2 in WO2009108806.
  • the right panel shows clot times in whole blood from the mice ex vivo using ROTEM.
  • FIG. 11 SEC-UV (280 nm) chromatograms for FVIII, TIL′/E′/D3/A1 III, and a mixture of FVIII and TIL′/E′/D3/A1 III in 155 mM NaCl, 10 mM Calciumacetat, 10% Isopropanol at 25° C.
  • FIG. 12 SEC-UV (280 nm) chromatograms for FVIII, TIL′/E′/D3 II, and a mixture of FVIII and TIL′/E′/D3 II in 155 mM NaCl, 10 mM Calciumacetat, 10% Isopropanol at 25° C.
  • treatment refers to the medical therapy of any human or other vertebrate subject in need thereof.
  • Said subject is expected to have undergone physical examination by a medical practitioner, or a veterinary medical practitioner, who has given a tentative or definitive diagnosis which would indicate that the use of said specific treatment is beneficial to treating a disease in said human or other vertebrate.
  • the timing and purpose of said treatment may vary from one individual to another, according to the subject's health.
  • said treatment may be prophylactic, palliative, symptomatic and/or curative.
  • Compounds and pharmaceutical compositions according to the invention may be administered parenterally, such as e.g. intravenously or extravascularly (such as e.g. intradermally, intramuscularly, subcutaneously, etc).
  • Compounds and pharmaceutical compositions according to the invention may be administered prophylactically and/or therapeutically and/or on demand.
  • Extravascular administration is easier, simpler, and associated with less pain, inconvenience, and complications (and thus potentially resulting in better compliance) which is of potential benefit to all patients but of particular benefit for children and small infants.
  • Combination treatments/co-administration may be achieved in a number of different ways.
  • the two active compounds may be administered together in a single composition.
  • the two active compounds may be administered in separate compositions as part of a combined therapy.
  • the first compound may be administered before, after, or concurrently with the second compound.
  • FVIII and VWF fragment are administered extravascularly (e.g. subcutaneously) as two separate pharmaceutical compositions, they are preferably administered in close proximity in order to benefit from the improved bioavailability that can be obtained when administering these two types of compounds together (i.e.
  • the injection sites should be separated by no more than 5 cm, preferably no more than 4 cm, preferably no more than 3 cm, preferably no more than 2 cm, and most preferably no more than 1 cm).
  • the two compounds should preferably also be injected within about an hour, preferably within about 30 minutes, preferably within about 15 minutes, and most preferably within about 5 minutes.
  • Factor VIII Factor VIII (FVIII) is a large, complex glycoprotein that is primarily produced by hepatocytes. Human FVIII comprises 2351 amino acids, including a signal peptide, and contains several distinct domains as defined by homology. There are three A-domains, a unique B-domain, and two C-domains. The domain order can be listed as NH2-A1-A2-B-A3-C1-C2-COOH. The chains are connected by bivalent metal ion-bindings. The A1-A2-B chain is termed the heavy chain (HC) while the A3-C1-C2 is termed the light chain (LC).
  • HC heavy chain
  • LC light chain
  • VWF von Willebrand factor
  • Endogenous FVIII molecules circulate in vivo as a pool of molecules with B domains of various sizes, the shortest having C-terminal at position 740, i.e. at the C-terminal of A2-a2, and thus contains no B domain. These FVIII molecules with B-domains of different length all have full procoagulant activity.
  • FVIII Upon activation with thrombin, FVIII is cleaved C-terminal of A1-a1 at position 372, C-terminal of A2-a2 at position 740, and between a3 and A3 at position 1689, the latter cleavage releasing the a3 region with concomitant loss of affinity for VWF.
  • the activated FVIII molecule is termed FVIIIa.
  • the activation allows interaction of FVIIIa with phospholipid surfaces like activated platelets and activated factor IX (FIXa), i.e. the tenase complex is formed, allowing efficient activation of factor X (FX).
  • Factor VIII(a) and FVIII(a) include both FVIII and FVIIIa.
  • the term “Factor VIII” and “FVIII” may include both FVIII and FVIIIa.
  • Factor VIII” or “FVIII” as used herein refers to a human plasma glycoprotein that is a member of the intrinsic coagulation pathway and is essential to blood coagulation.
  • Wildtype(wt)/native FVIII is the human FVIII molecule derived from the full length sequence as shown in SEQ ID NO: 1 (amino acid 1-2332).
  • “FVIII(a)” includes natural allelic variants of FVIII(a) that may exist and occur from one individual to another.
  • FVIII(a) may be plasma-derived or recombinantly produced, using well known methods of production and purification.
  • the degree and location of glycosylation, tyrosine sulfation and other post-translation modifications may vary, depending on the chosen host cell and its growth conditions.
  • compositions according to the present invention may comprise native or B domain-truncated FVIII molecules wherein the remaining domains correspond closely to the sequences as set forth in amino acid numbers 1-740 and 1649-2332 of SEQ ID NO: 3.
  • mutations may be introduced.
  • Amino acid modifications such as substitutions, insertions, and deletions, may be introduced into the molecule in order to modify the binding capacity of FVIII with various other components such as low-density lipoprotein receptor-related protein (LRP) and related receptors, various other receptors, other coagulation factors, cell surfaces, introduction and/or abolishment of glycosylation sites, etc.
  • LRP low-density lipoprotein receptor-related protein
  • Other mutations that do not abolish FVIII activity may also be accommodated in the FVIII molecules herein.
  • FVIII molecules herein are capable of functioning in the coagulation cascade in a manner that is functionally similar, or equivalent, to wt/endogenous FVIII, inducing the formation of FXa via interaction with FIXa on an activated platelet and supporting the formation of a blood clot.
  • FVIII activity can be assessed in vitro using techniques well known in the art. Clot analyses, FX activation assays (often termed chromogenic assays), thrombin generation assays and whole blood thrombo-elastography are examples of such in vitro techniques.
  • FVIII molecules according to the present invention have FVIII activity that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, 100% or even more than 100% of that of native human FVIII.
  • Endogenous full length FVIII is synthesized as a single-chain precursor molecule. Prior to secretion, the precursor is cleaved into the heavy chain and the light chain.
  • Recombinant B domain-deleted or truncated FVIII can be produced by means of two different strategies. Either the heavy chain without the B-domain and the light chain are synthesized individually as two different polypeptide chains (two-chain strategy) or the B domain-deleted or truncated FVIII is synthesized as a single precursor polypeptide chain (single-chain strategy) that is cleaved into the heavy and light chains in the same way as the full-length FVIII precursor.
  • the heavy and light chain moieties are often separated by a linker.
  • the sequence of the linker is preferably derived from the FVIII B-domain.
  • amino acid 1644-1648 constitutes this recognition site.
  • the thrombin cleavage site leading to removal of the linker on activation of B domain-deleted FVIII is located in the heavy chain.
  • Deletion/truncation of the B domain is an advantage for production of FVIII. Nevertheless, parts of the B domain can be included in the linker without reducing the productivity. The negative effect of the B domain on productivity has not been attributed to any specific size or sequence of the B domain.
  • SEQ ID NO: 1 wt human FVIII (Ser750 residue shown in bold and underline) ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTL FVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHA VGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASD PLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFA VFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHR KSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLL MDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDL TDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLV
  • the B domain in FVIII spans amino acids 741-1648 of SEQ ID NO: 1.
  • the B domain is cleaved at several different sites, generating large heterogeneity in circulating plasma FVIII molecules.
  • the exact function of the heavily glycosylated B domain is unknown. What is known is that the B domain is dispensable for FVIII activity in the coagulation cascade. Recombinant FVIII is thus frequently produced in the form of B domain-deleted/truncated variants.
  • the FVIII molecule is produced by an expression vector encoding a FVIII molecule comprising a 21 amino acid residue L (linker) sequence with the following sequence: SEQ ID NO 2: SFSQNSRHP S QNPPVLKRHQR (the 0-glycan is attached to the underlined S ).
  • Alternative preferred B domain linker sequences may lack one or more of the amino acid residues set forth in SEQ ID NO 2, e.g. the C-terminal R in SEQ ID NO 2.
  • Preferred FVIII molecules are B domain deleted/truncated variants comprising an O-glycan attached to the Ser 750 residue shown in SEQ ID NO 1 optionally being conjugated to a polymeric (half life extending) moiety via this O-glycan.
  • B domain deleted FVIII molecules according to the invention having a B domain of a size from about 100 to about 400 amino acids ((preferably 150-650, more preferably 150-600, more preferably 150-550, more preferably 150-500, more preferably 150-450, more preferably 150-400, more preferably 150-350, more preferably 200-700, more preferably 200-600, more preferably 200-500, more preferably 200-400, more preferably 200-300, and most preferably about 200 to 250) have a surprisingly high bioavailability in connection with extravascular (e.g. s.c.) administration compared to e.g.
  • extravascular e.g. s.c.
  • SEQ ID NO 1 A simple and safe way of producing FVIII having improved bioavailability upon subcutaneous/intradermal administration is thus provided. It is plausible that the in vivo circulatory half-life of FVIII having B domains of 100 to about 400 amino acids may be prolonged by conjugating/fusing such variants with a half-life extending moiety.
  • An example of a FVIII molecule comprising a 226 amino acid B domain is shown in SEQ ID NO 3:
  • SEQ ID NO 3 (226 amino acid B domain variant): ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTL FVEFTDHLFNIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHA VGVSYVVKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMAS DPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLF AVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCH RKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTL LMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDD LTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLV LAPDDRSYKSQYL
  • VWF Von Willebrand Factor
  • the basic VWF monomer is a 2050 amino acid protein. Each monomer contains a number of specific domains with a specific function, including the TIL′ or TIL′/E′ domain (Zhou et al. Blood 2012; 120(2): 449-458) which binds to FVIII.
  • FVIII is bound to VWF while inactive in circulation and is released from VWF by the action of thrombin.
  • FVIII(a) not bound to VWF is rapidly cleared and/or degraded. It is shown herein, that full-length VWF does not have the ability to significantly increase bioavailability of extra-vascularly co-administered FVIII despite of its inherent FVIII protective effects.
  • the full length VWF molecule is thus a very complex protein.
  • the prepro VWF consists of 2813 amino acid residues (SEQ ID NO 22).
  • SEQ ID NO 22 The amino acid numbering is thus often based on the prepro VWF and amino acid S764 is thus the first amino acid in the mature molecule.
  • the mature molecule is believed to contain 12 Asn-linked and 10 Thr/Ser linked oligosaccharide side chains. Furthermore this molecule can form dimers, trimers etc. with multimer molecule weight of up to several million Daltons.
  • Different allelic VWF variants are found in human beings and it is thus understood that VWF fragments according to the present invention can be derived from any one of these naturally occurring variants.
  • the present invention relates to VWF fragments that are preferably easier to produce than the full length molecule.
  • VWF fragments according to the invention furthermore preferably have the ability to increase bioavailability of s.c. co-administered FVIII.
  • VWF fragments according to the present invention comprise the at least the 15 N-terminal amino acids of the TIL′ domain/subdomain (spanning amino acids 764-778 of SEQ ID NO 22) or the TIL′ domain/subdomain (spanning amino acids 764-828 of SEQ ID NO 22 or amino acids 764-829 of SEQ ID NO 22) or the TIL′/E′ domain/sub-domains (spanning amino acids 764-865 of SEQ ID NO 22) and have a size of less than 1500 amino acids, preferably less than 1400 amino acids, preferably less than 1300 amino acids, preferably less than 1200 amino acids, preferably less than 1100 amino acids, preferably less than 1000 amino acids, preferably less than 900 amino acids, preferably less than 800 amino acids, preferably less than
  • VWF fragments according to the invention preferably comprise the TIL′/E′/D3 domains (where D3 is divided into subdomains VWD3-C8-3-TIL-3-E3) spanning amino acids 764-1250 or amino acids 764-1261 or amino acids 764-1268 of SEQ ID NO 22 of SEQ ID NO 22.
  • VWF fragments according to the invention preferably comprise at least the 15 N-terminal amino acids of TIL′, TIL′ or TIL′/E′ domains (amino acids 764-778, 764-828 or amino acids 764-865 of SEQ ID NO 22).
  • VWF fragments according to the invention may furthermore contain fewer potentially antigenic regions.
  • the molecular weight of VWF fragment dimers according to the present invention may—naturally—be about twice as high as for the monomeric fragments (Dimers according to the present invention may thus comprise up to about 2400 amino acids if the monomer size is 1200 amino acids).
  • the VWF fragments according to the present invention comprise at least amino acids 764-828 (SEQ ID NO 4), or at least amino acids 764-865 (SEQ ID NO 5), or at least amino acids 764-1035 (SEQ ID NO 6), or at least amino acids 764-1041 (SEQ ID NO 7), or at least amino acids 764-1045 (SEQ ID NO 8), or at least amino acids 764-1128 (SEQ ID NO 9), or at least amino acids 764-1198 (SEQ ID no 10), or at least amino acids 764-1250 (SEQ ID NO 11), or at least amino acids 764-1261 (SEQ ID NO 14), or at least amino acids 764-1268 (SEQ ID NO 22).
  • the C1099 and/or the C1142 cysteines may be mutated in the VWF fragments according to the present invention. These cysteine residues are believed to be responsible for the oligomerization/dimerization of the VWF protein. VWF fragments with both cysteines intact may form dimers and homo-oligomers. Modifying both of these cysteines may lead to a product composed of monomer VWF fragments, whereas deletion of one or the other may lead to dimer VWF fragments or potentially to oligomer VWF fragments. Both of the above scenarios may lead to a simpler product purification procedure as compared to the full-length protein.
  • both of the C1099 and C1142 cysteines are kept intact which may lead to a preferentially dimeric VWF fragment. There may be a safety advantage associated with the native sequences incl. the C1099 and the C1142 cysteines.
  • VWF fragments according to the invention demonstrate improved bioavailability compared to co-formulation of FVIII with a full length VWF molecule.
  • the co-formulations according to the invention show increased bioavailability of Factor VIII when injected subcutaneously.
  • VWF fragments according to the present invention comprise the D′ domain (spanning amino acids 764-865/866 of SEQ ID NO: 22) which is thought to be the primary FVIII binding site where FVIII may dock onto D′ by electrostatic dipole-dipole like interactions.
  • VWF fragments according to the invention preferably comprise the D′ domain and/or the D3-domain (the D3 domain spans amino acids 865/866-1250/1261/1268 of SEQ ID NO: 15).
  • VWF fragments according to the invention do not to any significant degree (i.e. preferably less than 5%, more preferably less than 4%, preferably less than 3%, preferably less than 2%, more preferably less than 1%) form multimers (i.e., having more than two units, such as e.g. oligomers) because the cysteines (C1099 and C1142) essential for multimer assembly are not present or have been mutated/substituted.
  • Some VWF fragments according to the present invention do furthermore not form dimers to any significant degree in particular those wherein the C1099 and/or C1142 cysteines are not present.
  • VWF fragments forming dimers may, however, also be useful in connection with the present invention—the TIL′/E′/D3/A1 dimer has e.g. been shown to have a higher FVIII affinity than the monomer. VWF fragment dimers may furthermore be a relatively homogenous product that can be produced relatively easily.
  • VWF fragments according to the invention is that it is easier to produce such compounds on an industrial scale as a relatively homogenous product due to the low degree of multimerization and due to the fact that the compounds are smaller compounds with fewer posttranslational modifications compared to full length VWF. This means that a high expression level is easier to obtain and/or purification will be less complex due to a less complex molecule. Also, production of recombinant peptides and proteins in simple organisms such as e.g. yeast is a faster and more inexpensive production method compared to production in mammalian cell lines—some VWF fragments according to the present invention can be produced in yeast.
  • VWF fragments according to the present invention can be in the form of one single VWF fragment (such as e.g. the entire TIL′/E′/D3/A1 region spanning amino acids 764-1459 in SEQ ID NO 22) or alternatively in the form of multiple groups of sequential amino acids from VWF fused together and thus deleting intermediary fragments (such as e.g. a “fusion” of the TIL′ and the TIL′/E′ domain spanning amino acids 764-828+764-865 in SEQ ID NO 22). Another example could be amino acids 764-828+1127-1197 in SEQ ID NO 22.
  • VWF fragments according to the invention may alternatively be in the form of the repetitive elements.
  • Homologous or heterologous “spacer” sequences may be introduced between the fused VWF fragments/elements (such as e.g. a multiple fusion of TIL′/E′ domains such as e.g. TIL′/E′TIL′/E′TIL′/E′).
  • VWF fragments according to the invention may also comprise one or more amino acid alternations (e.g. substitutions, deletions, additions) in the VWF derived sequence(s).
  • Bioavailability of FVIII in connection with extravascular co-administration of FVIII and VWF fragments according to the invention may be further improved by conjugating FVIII with at least one half life extending moiety. It thus follows, that extra-vascular co-administration of VWF fragments comprising the TIL′ and/or the TIL′/E′ domains with a FVIII molecule conjugated with at least one half life extending moiety is associated with a relatively high FVIII bioavailability.
  • TIL′/E′/VWD3 I, TIL′/E′NWD3 II and TIL′/E′NWD3 III denote three versions (different lengths) of TIL′/E′NWD3.
  • SEQ ID NO 4 amino acids 764-828 (TIL′): SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCV ALERCP SEQ ID NO 5: amino acids 764-865 (TIL′/E′): SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCV ALERCPCFHQG KEYAPGETVK IGCNTCVCQDRKWNCTDHVCDA v SEQ ID NO 6: amino acids 764-1035 (TIL′/E′/VWD3 I): SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCV ALERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLK YLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKK RVTILVEGGEIE
  • This cysteine can be substituted to another amino acid, e.g. Ser: SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCV ALERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLK YLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKKRVTILVEGGEIELFDGEVNVK RPMKDETHFEWESGRYII LLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQ NNDLTSSNLQVEEDPVDFGNSWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRILT SDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCA C FCDTIAAYAHVCAQHGKVVTWRTATL CPQ SEQ ID NO 10: amino acids 764-1198 (TIL′/E′/VWD3/C
  • cysteines can be substituted to another amino acid, e.g. Ser: SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCV ALERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLK YLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKK RVTILVEGGEIELFDGEVNV KRPMKDETHFEVVESGRYII LLLGKALSWWDRHLSISVVLKQTYQEKVCGLCGNFDGIQ NNDLTSSNLQVEEDPVDFGNSWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRIL TSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCA C FCDTIAAYAHVCAQHGKVVTWRTA TLCPQSCEERNLRENGYE C EWRYNSCAPACQVTCQHPEPLA
  • cysteines can be substituted to another amino acid, e.g. Ser: SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCV ALERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLK YLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKK RVTILVEGGEIELFDGEVNV KRPMKDETHFEVVESGRYII LLLGKALSVVWDRHLSISWLKQTYQEKVCGLCGNFDGIQ NNDLTSSNLQVEEDPVDFGN SWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRIL TSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCA C FCDTIAAYAHVCAQHGKVVTWRTA TLCPQSCEERNLRENGYE C EWRYNSCAPACQVTCQHPEPLA
  • cysteines can be substituted to another amino acid, e.g. Ser: ATCSTIGMAHYLTFDGLKYLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKK RVTILVEGGEIELFDGEVNVKRPMKDETHFEVVESGRYIILLLGKALSVVWDRHLSISVVLKQT YQEKVCGLCGNFDGIQNNDLTSSNLQVEEDPVDFGNSWKVSSQCADTRKVPLDSSPATCH NNIMKQTMVDSSCRILTSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCA C FCDTIAAYAHV CAQHGKVVTWRTATLCPQSCEERNLRENGYE C EWRYNSCAPACQVTCQHPEPLACPVQC VEGCHAHCPPGKILDELLQTCVDPEDCPVCEVAGRRFASGKKVTLNPSDPEHCQICHCDVV NLTCEACQEPGGL VVPPTDA SEQ ID NO 13: amino acids from a
  • cysteines can be substituted to another amino acid, e.g. Ser: ATCSTIGMAHYLTFDGLKYLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKK RVTILVEGGEIELFDGEVNVKRPMKDETHFEVVESGRYIILLLGKALSVVWDRHLSISVVLKQT YQEKVCGLCGNFDGIQNNDLTSSNLQVEEDPVDFGNSWKVSSQCADTRKVPLDSSPATCH NNIMKQTMVDSSCRILTSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCA C FCDTIAAYAHV CAQHGKVVTWRTATLCPQSCEERNLRENGYE C EWRYNSCAPACQVTCQHPEPLACPVQC VEGCHAHCPPGKILDELLQTCVDPEDCPVCEVAGRRFASGKKVTLNPSDPEHCQICHCDVV NLTCEACQEPGGL VVPPTDAPVSPTTLYVEDIS
  • cysteines can be substituted to another amino acid, e.g. Ser: SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCV ALERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLK YLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKK RVTILVEGGEIELFDGEVNV KRPMKDETHFEVVESGRYIILLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQ NNDLTSSNLQVEEDPVDFGN SWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRIL TSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCA C FCDTIAAYAHVCAQHGKVVTWRTA TLCPQSCEERNLRENGYE C EWRYNSCAPACQVTCQHPEPLACP
  • cysteines can be substituted to another amino acid, e.g. Ser: SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCV ALERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLK YLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKK RVTILVEGGEIELFDGEVNV KRPMKDETHFEVVESGRYIILLLGKALSVVWDRHLSISWLKQTYQEKVCGLCGNFDGIQ NNDLTSSNLQVEEDPVDFGN SWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRIL TSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCA C FCDTIAAYAHVCAQHGKVVTWRTA TLCPQSCEERNLRENGYE C EWRYNSCAPACQVTCQHPEPLACPV
  • cysteines can be substituted to another amino acid, e.g. Ser: SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCV ALERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLK YLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKK RVTILVEGGEIELFDGEVNV KRPMKDETHFEVVESGRYII LLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQ NNDLTSSNLQVEEDPVDFGN SWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRIL TSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCA C FCDTIAAYAHVCAQHGKVVTWRTA TLCPQSCEERNLRENGYE C EWRYNSCAPACQVTCQHPEP
  • cysteines can be substituted to another amino acid, e.g. Ser: SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCV ALERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLK YLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKK RVTILVEGGEIELFDGEVNV KRPMKDETHFEVVESGRYIILLLGKALSVVWDRHLSISWLKQTYQEKVCGLCGNFDGIQ NNDLTSSNLQVEEDPVDFGN SWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRIL TSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTA TLCPQSCEERNLRENGYE C EWRYNSCAPACQVTCQHPEPLACPVQC
  • cysteines can be substituted to another amino acid, e.g. Ser: SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCV ALERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLK YLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKK RVTILVEGGEIELFDGEVNV KRPMKDETHFEVVESGRYII LLLGKALSVVWDRHLSISWLKQTYQEKVCGLCGNFDGIQ NNDLTSSNLQVEEDPVDFGN SWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRIL TSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCA C FCDTIAAYAHVCAQHGKVVTWRTA TLCPQSCEERNLRENGYE C EWRYNSCAPACQVTCQHPEPLA
  • cysteines can be substituted to another amino acid, e.g. Ser: SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCV ALERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLK YLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKK RVTILVEGGEIELFDGEVNV KRPMKDETHFEVVESGRYII LLLGKALSVVWDRHLSISWLKQTYQEKVCGLCGNFDGIQ NNDLTSSNLQVEEDPVDFGN SWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRIL TSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCA C FCDTIAAYAHVCAQHGKVVTWRTA TLCPQSCEERNLRENGYE C EWRYNSCAPACQVTCQHPEPLA
  • cysteines can be substituted to another amino acid, e.g. Ser: SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCV ALERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLK YLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKK RVTILVEGGEIELFDGEVNV KRPMKDETHFEVVESGRYII LLLGKALSVVWDRHLSISWLKQTYQEKVCGLCGNFDGIQ NNDLTSSNLQVEEDPVDFGN SWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRIL TSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCA C FCDTIAAYAHVCAQHGKVVTWRTA TLCPQSCEERNLRENGYE C EWRYNSCAPACQVTCQHPEPLA
  • cysteines can be substituted to another amino acid, e.g. Ser: SLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCV ALERCPCFHQGKEYAPGETVKIGCNTCVCQDRKWNCTDHVCDATCSTIGMAHYLTFDGLK YLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKK RVTILVEGGEIELFDGEVNV KRPMKDETHFEVVESGRYIILLLGKALSVVWDRHLSISWLKQTYQEKVCGLCGNFDGIQ NNDLTSSNLQVEEDPVDFGN SWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRIL TSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCA C FCDTIAAYAHVCAQHGKVVTWRTA TLCPQSCEERNLRENGYE C EWRYNSCAPACQVTCQHPEPLACPV
  • FVIII molecules/variants/derivatives/analogues The term “FVIII” as used herein, is intended to designate any FVIII molecule having FVIII activity, incl. wt FVIII, B domain deleted/truncated FVIII molecules, variants of FVIII exhibiting substantially the same or improved biological activity relative to wt FVIII and FVIII-related polypeptides, in which one or more of the amino acids of the parent peptide have been chemically modified, e.g. by protein:protein fusion, alkylation, PEGylation, HESylation, PASylation, PSAylation, acylation, ester formation or amide formation or the like (conjugated to a half-life extending moiety).
  • Half-life extending moieties/protractive groups are herein understood to refer to one or more chemical groups, e.g. a hydrophilic polymer, such as e.g. PEG and/or a polysaccharide covalently attached to FVIII via e.g. —SH, —OH, —COOH, —CONH2, —NH2, or one or more N- and/or O-glycan structures that can increase in vivo circulatory half life when conjugated to these proteins.
  • a hydrophilic polymer such as e.g. PEG and/or a polysaccharide covalently attached to FVIII via e.g. —SH, —OH, —COOH, —CONH2, —NH2, or one or more N- and/or O-glycan structures that can increase in vivo circulatory half life when conjugated to these proteins.
  • protractive groups/half-life extending moieties suitable for being conjugated to FVIII in connection with the present invention include: Biocompatible fatty acids and derivatives thereof, Hydroxy Alkyl Starch (HAS) e.g. Hydroxy Ethyl Starch (HES), Poly Ethylene Glycol (PEG), Poly (Glyx-Sery)n (HAP), Hyaluronic acid (HA), Heparosan polymers (HEP), Phosphorylcholine-based polymers (PC polymer), Fleximers, Dextran, Poly-sialic acids (PSA), an Fc domain, an Fc receptor, Transferrin, Albumin, Elastin like peptides, XTEN polymers, Albumin binding peptides, a CTP peptide, and any combination thereof.
  • HAS Hydroxy Alkyl Starch
  • HAS Hydroxy Ethyl Starch
  • PEG Poly Ethylene Glycol
  • HAP Poly (Glyx-Sery)n
  • HAP Hy
  • conjugation of FVIII with one or more half-life extending moieties generally have a better bioavailability in connection with s.c./intradermal co-administration with VWF fragments according to the invention as compared with FVIII with no half life extending moieties.
  • PEGylated FVIII molecules in connection with the present invention may have one or more polyethylene glycol (PEG) molecules attached to any part of the FVIII protein including any amino acid residue or carbohydrate moiety.
  • PEG polyethylene glycol
  • Chemical and/or enzymatic methods can be employed for conjugating PEG or other polymeric groups (half life extending moieties) to a glycan on FVIII.
  • An example of an enzymatic conjugation process is described e.g. in WO03031464.
  • the glycan may be naturally occurring or it may be inserted via e.g. insertion of an N-linked and/or O-linked glycan using methods well known in the art.
  • Cysteine-PEGylated FVIII have one or more PEG molecules conjugated to a sulfhydryl group of a cysteine present in FVIII.
  • Cysteine-acylated FVIII according to the present invention have one or more hydrophobic half-life extending moieties (e.g. fatty acids) conjugated to a sulfhydryl group of a cysteine in FVIII this cysteine residue may be introduced by genetic engineering or a part of the native amino acid sequence. It is furthermore possible to link half-life extending moieties to other amino acid residues.
  • Fusion proteins are proteins created through the in-frame joining of two or more DNA sequences which originally encoded FVIII and the fusion partner. Translation of the fusion protein DNA sequence will result in a single protein sequence which may have functional properties derived from each of the original proteins or peptides.
  • DNA sequences encoding fusion proteins may be created artificially by standard molecular biology methods such as overlapping PCR or DNA ligation and the assembly is performed excluding the stop codon in the first 5′-end DNA sequence while retaining the stop codon in the 3′end DNA sequence.
  • the resulting fusion protein DNA sequence may be inserted into an appropriate expression vector that supports the heterologous fusion protein expression in a standard host organism.
  • Fusion proteins may contain a linker or spacer peptide sequence that separates the protein or peptide parts which define the fusion protein.
  • the linker or spacer peptide sequence may facilitate the correct folding of the individual protein or peptide parts and may make it more likely for the individual protein or peptide parts to retain their individual functional properties.
  • Linker or spacer peptide sequences may be inserted into fusion protein DNA sequences during the in frame assembly of the individual DNA fragments that make up the complete fusion protein DNA sequence i.e. during overlapping PCR or DNA ligation. Examples of fusion proteins comprising FVIII and a fusion partner are shown in WO2011101284.
  • Fc fusion protein is herein meant to encompass FVIII fused to an Fc domain that can be derived from any antibody isotype.
  • An IgG Fc domain will often be preferred due to the relatively long circulatory half-life of IgG antibodies.
  • the Fc domain may furthermore be modified in order to modulate certain effector functions such as e.g. complement binding and/or binding to certain Fc receptors. Fusion of FVIII with an Fc domain, which has the capacity to bind to FcRn receptors, will generally result in a prolonged in vivo circulatory half-life.
  • a modified IgG Fc domain of a fusion protein according to the invention comprises one or more of the following mutations that will result in decreased affinity to certain Fc receptors (L234A, L235E, and G237A) and in reduced C1q-mediated complement fixation (A330S and P331S), respectively.
  • the Fc domain may be an IgG4 Fc domain, preferably comprising the S241P/S228P mutation.
  • Bioavailability describes the percentage of compound absorbed to the blood after extravascular is calculated as the Area under the concentration time curves after extravascular dosing of the compound. This is calculated from the Area under the concentration curves of FVIII after s.c. administration divided by the dose, relatively to the area under the concentrations curve divided by the dose of the same FVIII compound, dosed i.v.
  • the bioavailability of FVIII molecules is at least 3%, preferably at least 5%, preferably at least 6%, preferably at least 7%, preferably at least 8%, preferably at least 9%, preferably at least 10%, preferably at least 11%, preferably at least 12%, preferably at least 13%, preferably at least 14%, preferably at least 15%, preferably at least 16%, preferably at least 17%, preferably at least 18%, preferably at least 19%, preferably at least 20%, preferably at least 21%, preferably at least 22%, preferably at least 23%, preferably at least 24%, preferably at least 25%, preferably at least 26%, preferably at least 27%, preferably at least 28%, preferably at least 29%, preferably at least 30%, preferably at least 31%, preferably at least 32%, preferably at least 33%, preferably at least 34%,
  • Bioavailability can be measured as described herein.
  • the FVIII bioavailability (FVIII antigen and/or activity) of formulations according to the invention will be high enough to exert prophylactic effects under conditions with normal activity when such formulations are administered extravascularly (e.g. subcutaneously or intra-dermally) e.g. once or twice a day or once, twice or three times a week.
  • FVIII dosages are comparable with those used in connection with I.V. administration of FVIII, preferably twice as high, and more preferably three times as high, more preferably four times as high, more preferably about 10 times as high, more preferably about 15 times as high, more preferably about 20 times as high, and most preferably about 25 times as high.
  • Safety and cost considerations may be considered in connection with dosage determinations.
  • Saturation of FVIII with VWF fragments saturation of FVIII with VWF fragment/the relative amount of FVIII bound to or in complex with VWF/the amount of FVIII bound to VWF divided by the total amount of FVIII. This calculation is based on the KD value of the binding between FVIII and the protein. For FVIII binding to VWF fragments, the measured KI values are used as KD.
  • compositions comprising VWF fragments and preferably also FVIII.
  • one object of the invention is to provide a pharmaceutical composition comprising a FVIII molecule present in a concentration from 40 IU/ml to 25,000 IU/ml, and wherein said composition has a pH from 2.0 to 10.0.
  • the FVIII molecules are co-administered together with VWF fragments.
  • Pharmaceutical compositions according to the invention may thus comprise FVIII in a concentration of from 40 IU/ml to 25,000 IU/ml, such as e.g.
  • compositions according to the invention may further comprise one or more pharmaceutically acceptable excipients such as e.g. a buffer system, a preservative, a tonicity agent, a chelating agent, a stabilizer, or a surfactant, as well as various combinations thereof.
  • a buffer system e.g. a preservative, a tonicity agent, a chelating agent, a stabilizer, or a surfactant, as well as various combinations thereof.
  • preservatives, isotonic agents, chelating agents, stabilizers and surfactants in pharmaceutical compositions is well-known to the skilled person. Reference may be made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.
  • the pharmaceutical composition is an aqueous composition.
  • aqueous composition is typically a solution or a suspension, but may also include colloids, dispersions, emulsions, and multi-phase materials.
  • aqueous composition is defined as a composition comprising at least 50% w/w water.
  • aqueous solution is defined as a solution comprising at least 50% w/w water
  • aqueous suspension is defined as a suspension comprising at least 50% w/w water.
  • the pharmaceutical composition is a freeze-dried composition, to which the physician or the patient adds solvents and/or diluents prior to use.
  • the pharmaceutical composition comprises an aqueous solution of such an antibody, and a buffer, wherein the antibody is present in a concentration from 1 mg/ml or above, and wherein said composition has a pH from about 2.0 to about 10.0.
  • compositions according to the present invention are preferably suitable for extravascular administration (e.g. s.c. or intradermal administration) in prophylactic/therapeutic treatment of blood clotting diseases.
  • preferred ratios of FVIII and VWF/VWF fragment include FVIII/VWF ratios (molar ratios) from 0.5:1 to 1:50, such as e.g. 1:1 to 1:50, such as e.g. 1:1 to 1:25, such as e.g. 1:1 to 1:20, or 1:1 to 1:15, or 1:1 to 1:10, or 1:1 to 1:7,5, or 1:7 to 1:8, or 1:6 to 1:8, or 1:6 to 1:9, or 1:5 to 1:10.
  • Preferred ratios thus include: 1:1, 1:2, 1:3, 1:4, 1:5, 1:5,5; 1:6; 1:6,5, 1:7; 1:7,1; 1:7,2; 1:7,3; 1:7,4; 1:7,5; 1:7,6; 1:7,7; 1:7,8; 1:7,9, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, and 1:50.
  • Preferred ratios include: 0.5:1; 0.6:1; 0.7:1; 0.8:1; 0.9:1; 1:1; 1.1:1; 1.2:1; 1.3:3; 1.4:1, and 1.5:1.
  • a molar ratio close to 1:1 generally has the advantage of minimizing the required amount of active substance.
  • the optimal ratio between FVIII and VWF fragment in a co-formulation mixture may be determined by calculating the amount of bound FVIII:VWF at certain protein concentrations based on the binding affinity to the VWF variant for the FVIII species in question.
  • the binding affinity can be determined e.g. by ELISA, SPR or by ITC.
  • Haemophilia Haemophilia/hemophilia/blood clotting diseases is a group of hereditary genetic disorders that impair the body's ability to control blood clotting or coagulation (“bleeding disorders”), which is used to stop bleeding when a blood vessel is broken.
  • Haemophilia A clotting factor VIII deficiency
  • Haemophilia encompasses von Willebrand disease.
  • a pharmaceutical formulation according to the invention wherein the concentration of FVIII is at least about 100, 150, 200, 250, 300, 350, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000, or 30,000 IU/ml.
  • FVIII KO mice exon 16 knock-out in a mixed background of C57B1/6 and SV129, bred at Taconic M&B (B6.129S4-F8tm1Kaz/J) with an approximate weight of 22 g were dosed subcutaneously in the flank with 10000 IU/kg FVIII or FVIII/VWF, 6 mice with each test compound.
  • Blood was sampled at 1, 3, 7, 17, 24, 30, 48, 72 and 96 h post administration.
  • the mice were anaesthetized by Isoflurane/O 2 /N 2 O prior to blood sampling via the retroorbital plexus. Three samples were taken from each mouse.
  • Blood 45 ⁇ l was stabilised with 5 ⁇ l of sodium-citrate (0.13 M) and added 200 ⁇ l FVIII coatest SP buffer (50 mM TRIS-HCl, 1% BSA, Ciprofloxacin 10 mg/L, pH 7.3). After centrifugation at 4000 g for 5 minutes at room temperature, the supernatants were immediately frozen on dry ice before storage at ⁇ 80° C. prior to analysis.
  • FIG. 1 The circulating profiles of FVIII activity are shown graphically in FIG. 1 , the circulating concentrations of FVIII antigen are shown in FIG. 2 .
  • FVIII KO mice exon 16 knock-out in a mixed background of C57Bl/6 and SV129, bred at Taconic M&B (B6.12954-F8tm1Kaz/J) with an approximate weight of 22 g were dosed subcutaneously in the flank with 2500 IU/kg FVIII or FVIII/VWF, 6 mice with each test compound.
  • mice were anaesthetized by Isoflurane/O 2 /N 2 O prior to blood sampling via the retroorbital plexus. Three samples were taken from each mouse. 45 ⁇ l of blood was stabilised with 5 ⁇ l of sodium-citrate (0.13 M) and added 200 ⁇ l FVIII coatest SP buffer (50 mM TRIS-HCl, 1% BSA, Ciprofloxacin 10 mg/L, pH 7.3). After centrifugation at 4000 g for 5 minutes at room temperature, the samples were immediately frozen on dry ice before storage at ⁇ 80° C. prior to analysis.
  • FVIII coatest SP buffer 50 mM TRIS-HCl, 1% BSA, Ciprofloxacin 10 mg/L, pH 7.3
  • the circulating profiles of FVIII activity are shown graphically in FIG. 3 , the circulating concentrations of FVIII antigen are shown in FIG. 4 .
  • mice Before tail transection, the mice are anaesthetised with isoflurane and placed on a heating pad
  • the tails are placed in pre-heated saline at 37° C. for 10 min
  • I.v. controls are injected 5 min, 24 or 48 hr prior to injury
  • the tail is transected 4 mm from the tip
  • Parallel animals are used for blood sampling and subsequent analysis of their clotting parameters (ex vivo efficacy).
  • the prophylactic effect of the co-formulation is determined from comparing the blood loss during the 30 min study period at a certain time after s.c. administration (5 min until 168 hr) to that of 1, a vehicle control and 2, an i.v. control group with FVIII or glycoPEGylated FVIII.
  • FIG. 10 shows that glycoPEGylated FVIII are haemostatic effective 24 hr after s.c. administration of 2500 U/kg as shown by reduction of blood loss and shortening of clot time ex vivo. Similar effect is seen for FVIII co-formulated with a VWF fragment.
  • Bioavailability of co-compositions of FVIII and VWF/VWF fragments according to the invention can be determined from evaluations of the effect on bioavailability in PK experiments as those described in examples 1 and 2 as well as evaluations of the prophylactic effect as described in example 3.
  • the bioavailability of a FVIII compound co-formulated with a concentration of VWF fragment that enables the majority of FVIII to be bound to a VWF fragment compound in the injection composition can be determined from the concentration of FVIII compound in the composition and from experiments evaluating the binding affinity of the VWF fragment compound to the FVIII compound such as e.g. surface plasmon resonance experiments.
  • Dose titration can be carried out as disclosed in examples 1-3. Briefly, plasma concentration of FVIII will be evaluated after s.c. administration of doses of 70, 100, 150, 280, 500, 1000 and 2500 IU/kg (FVIII units) alone or together with a VWF fragment in FVIII k/o mice.
  • FVIII to VWF fragment For PK evaluation, doses of 280, 500, 1000 or 2500 IU/kg FVIII compound will be co-formulated with VWF fragments at a molar ratio of 1:1, 1:1.5, 1:2, 1:3, 1:4, 1:5, 1:7.7 or up to 1:100 (FVIII to VWF fragment) and plasma concentration of FVIII evaluated in FVIII k/o mice after s.c. administration.
  • the maximum molar surplus of VWF fragment to FVIII will be determined from binding affinities of the fragment to the FVIII compound in question; the highest molar surplus used will be the one that should result in at least 99% of the FVIII used bound to a VWF fragment.
  • the candidate compositions from the PK experiments will be evaluated in efficacy models, such as the tail bleeding described in example 3.
  • the immuno-modulatory effect of VWF co-formulated with a FVIII compound is evaluated in comparison to wild type FVIII and FVIII compounds alone.
  • the relative immunogenicity is evaluated from the titer of FVIII binding antibodies and the determination of the level of neutralizing antibodies (inhibitors) at certain time points after administration.
  • the assay for detection of FVIII binding antibodies is a radioimmunoassay (RIA). Briefly, anti-FVIII antibodies from a sample bind to radioactive 125 I-labelled rFVIII. Immunoglobulin and immune complexes bind to protein G-sepharose and is precipitated by centrifugation. The radioactivity in the precipitate is measured and this is proportional to the amount of anti-FVIII antibodies in the sample. The result is expressed in per cent of the total amount of added radioactivity. i.e. as % bound/total (% B/T).
  • Samples positive for anti-FVIII antibodies are analysed for the presence of FVIII neutralizing antibodies using a chromogenic assay. Briefly, samples are incubated with 1 IU/ml FVIII for 1 hr. The remaining FVIII activity is determined by addition of FIX, FX, thrombin, CaCl 2 and phospholipids. After incubation the amount of generated FXa is determined by addition of the chromogenic substrate S-2760 and the change in optical density (OD) is measured. The OD change is proportional to FVIII activity in the samples, and is compared to samples containing a known amount of FVIII and no inhibitors.
  • a chromogenic assay Briefly, samples are incubated with 1 IU/ml FVIII for 1 hr. The remaining FVIII activity is determined by addition of FIX, FX, thrombin, CaCl 2 and phospholipids. After incubation the amount of generated FXa is determined by addition of the chromogenic substrate S
  • the % remaining activity of the test sample is calculated compared to the reference samples without inhibitors/anti-FVIII antibodies added. Furthermore, the presence of anti-VWF antibodies is measured by ELISA using monoclonal or polyclonal anti-human VWF antibodies which does not cross react with murine VWF. If a strong anti-VWF response is detected, this can be expected to interfere with the binding of VWF to FVIII and the in vivo analysis is repeated using murine VWF fragments.
  • the appearance of anti-drug antibodies is evaluated after repeated (e.g. once weekly for 4 weeks or once daily for three weeks) s.c. administration of the compounds in nave mice, in FVIII k/o mice as well as in mice tolerized to human FVIII.
  • the readout is the ratio of animals with positive titres at certain time points after the first and/or the last administration (e.g. 1, 2, 3, 4, 5, 6, 7 or 8 weeks).
  • FVIII k/o mice are injected weekly e.g. with 1000 IU/kg FVIII alone or in combination with VWF in a molar ratio ensuring that at least e.g. 87% of FVIII is bound to VWF.
  • the FVIII dose is lower and based upon the bioavailability of the FVIII-VWF complex.
  • Mice tolerized to hFVIII are injected weekly for e.g. eight weeks s.c. with e.g. 1000 IU/kg FVIII with or without VWF and in some experiments including additional challenge with complete Freund's adjuvant (CFA) for the first injection followed by weekly challenges by incomplete Freund's adjuvant (IFA).
  • CFA complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • Relative immunogenicity of VWF versus VWF fragments and of wild type FVIII versus a FVIII compound co-formulated with VWF is furthermore evaluated in vitro in a human CD4+ T-cell assay. This is done using peripheral blood mononuclear cells (PBMCs) depleted of CD8+ T-cells. FVIII is added to the cell culture e.g. for eight days. T-cell proliferation is evaluated during the course of the assay by pulsing for e.g. 18 h with 3 H-thymidine in sub-samples from the cultures and subsequently measuring 3 H-thymidine incorporation. Interleukin 2 production is measured at the end of the assay using an ELISPOT IL-2 kit e.g. from R&D Systems, following the manufacturer's instructions. The data obtained in the assays are converted to a “stimulation index” describing the ratio between compound-stimulated versus un-stimulated cells.
  • PBMCs peripheral blood mononu
  • the HLA-binding capacity of VWF has been evaluated using in silico analysis of HLA-binding properties. Strong binding to a sequence in a modified VWF may indicate novel T-cell epitopes, although the in silico analysis tool is predicting epitopes that may not be processed by the naturally occurring proteases. In order to predict if the Cys->Ser mutation will induce a risk of induced immunogenicity in the VWF-mutants, the VWF protein sequences are applied to an in silico peptide/HLA-II binding prediction software.
  • the peptide/HLA-II binding prediction software is based on two different algorithms, NetMHCIIpan 2.1 (NetMHCIIpan-2.0—Improved pan-specific HLA-DR predictions using a novel concurrent alignment and weight optimization training procedure. Nielsen M, Lundegaard C, Justesen S, Lund O, and Buus S. Immunome Res. 2010 Nov. 13; 6(1):9) performing pan-specific HLA-DR predictions—and NetMHCII 2.0 (NN-align—A neural network-based alignment algorithm for MHC class II peptide binding prediction. Nielsen M and Lund O. BMC Bioinformatics. 2009 Sep. 18; 10:296) performing HLA-DP/DQ predictions.
  • NetMHCIIpan 2.1 NetMHCIIpan-2.0—Improved pan-specific HLA-DR predictions using a novel concurrent alignment and weight optimization training procedure. Nielsen M, Lundegaard C, Justesen S, Lund O, and Buus S. Immunome Res. 2010 Nov. 13; 6(1):9) performing pan
  • Twenty-three amino acid long peptides with the point of mutation in position 12 are used as input to the algorithms.
  • the optimal processed peptide is assumed to be a 15′mer peptide with a nine amino acid core peptide binding to the HLA-II.
  • the output is 15 amino acid long peptides with 9 amino acid long core peptides (in contact with HLA-II) and the predicted binding affinities in nanomolar.
  • the predicted binding affinities of the VWF mutant peptides are in the same range as the binding affinities of the wild type sequences (data not shown) and because the peptides are predicted to bind with relatively poor affinity to the HLA-II molecules, the risk of inducing novel CD4+ T-cell epitopes is considered to be very low.
  • the in silico peptide/HLA-II binding predictions are based on experimental peptide/HLA-II binding data where it is very challenging to test cysteine-rich peptides (due to the nature of the peptides).
  • cysteine-rich peptides are underrepresented in data sets used to train the different prediction algorithms. Therefore, the peptide/HLA-II binding predictions of these cysteine-rich VWF peptides are uncertain and should be analysed further using other immunogenicity prediction platforms (etc. in vitro peptide/HLA-II binding assays or ex vivo T-cell assays).
  • FVIII KO mice exon 16 knock-out in a mixed background of C57Bl/6 and SV129, bred at Taconic M&B (B6.129S4-F8tm1Kaz/J) with an approximate weight of 22 g were dosed subcutaneously in the flank with 10000 IU/kg FVIII or FVIII/VWF, 6 mice with each test compound.
  • mice were anaesthetized by Isoflurane/O 2 /N 2 O prior to blood sampling via the retroorbital plexus. Three samples were taken from each mouse. 45 ⁇ l of blood was stabilised with 5 ⁇ l of sodium-citrate (0.13 M) and added 200 ⁇ l FVIII coatest SP buffer (50 mM TRIS-HCl, 1% BSA, Ciprofloxacin 10 mg/L, pH 7.3). After centrifugation at 4000 g for 5 minutes at room temperature, the supernatants were immediately frozen on dry ice before storage at ⁇ 80° C. prior to analysis.
  • FVIII coatest SP buffer 50 mM TRIS-HCl, 1% BSA, Ciprofloxacin 10 mg/L, pH 7.3
  • the circulating profiles of FVIII activity are shown graphically in FIG. 5 and antigen levels are shown in FIG. 6 .
  • FVIII KO mice exon 16 knock-out in a mixed background of C57Bl/6 and SV129, bred at Taconic M&B (B6.129S4-F8tm1Kaz/J) with an approximate weight of 22 g were dosed subcutaneously in the flank with 10000 IU/kg FVIII or FVIII/VWF, 6 mice with each test compound.
  • mice were anaesthetized by Isoflurane/O 2 /N 2 O prior to blood sampling via the retro-orbital plexus. Three samples were taken from each mouse. 45 ⁇ l of blood was stabilised with 5 ⁇ l of sodium-citrate (0.13 M) and added 200 ⁇ l FVIII coatest SP buffer (50 mM TRIS-HCl, 1% BSA, Ciprofloxacin 10 mg/L, pH 7.3). After centrifugation at 4000 g for 5 minutes at room temperature, the supernatants were immediately frozen on dry ice before storage at ⁇ 80° C. prior to analysis.
  • FVIII coatest SP buffer 50 mM TRIS-HCl, 1% BSA, Ciprofloxacin 10 mg/L, pH 7.3
  • F8-500 Plasmid with insert encoding the F8-500 FVIII molecule (F8-500 equals turoctocog alfa/N8 encoding sequence) was used for production of FVIII.
  • the F8-500 vector encodes the FVIII heavy chain without the B domain (amino acids 1-740), a 21 amino acid linker (SFSQNSRHPSQNPPVLKRHQR SEQ ID NO 2), and the FVIII light chain (amino acids 1649-2332 of full-length wild-type human FVIII).
  • the sequence of the 21 amino acid linker is derived from the FVIII B domain and consists of amino acids 741-750 and 1638-1648 of full length wild-type human FVIII. Fragments of FVIII cDNA were amplified from full length FVIII cDNA and inserted into F8-500 coding plasmid giving rise to DNA constructs encoding the BDD FVIII.
  • F8-500D-HIS-C2-linked-(GGGS)6-hFc(IgG1), F8-500D-HIS-C2-linked-(GGGS)6-mFc(IgG2A), and F8-500D-HIS-C2-linked-(GGGS)6-albumin were established as described in the following.
  • the internal BamHI site (aa 604-606) in F8-500 coding DNA was eliminated by site-directed mutagenesis and DNA encoding the flexible (GGGS) 6 linker was inserted 3′ to the coding region.
  • a new BamHI site was introduced in the 3′ end of the linker-coding DNA in order to ease cloning of C-terminal fusion partners between BamHI and Notl sites.
  • a construct encoding F8-500-C2-linked-(GGGS)6 was generated.
  • DNA encoding human Fc (IgG1), mouse Fc (IgG2a), and human serum albumin was amplified.
  • the PCR products were inserted between the BamHI and Not I sites of the F8-500-C2-linked-(GGGS)6 coding vector giving rise to constructs encoding F8-500-C2-linked-(GGGS)6-hFc(IgG1), F8-500-C2-linked-(GGGS)6-mFc(IgG2A), and F8-500-C2-linked-(GGGS)6-albumin.
  • a SphI/ClaI restriction fragment from the latter constructs were transferred to a F8-500D-His coding constructs in order to generate F8-500D-HIS-C2-linked-(GGGS)6-hFc(IgG1)-, F8-500D-HIS-C2-linked-(GGGS)6-mFc(IgG2A)-, and F8-500D-HIS-C2-linked-(GGGS)6-albumin coding constructs.
  • DNA constructs consisting of the mammalian expression vector pTT5 with insert encoding BDD FVIII were utilized.
  • the vector pTSV7 is utilized. This vector encodes dihydrofolate reductase allowing selection of transfected cells with the dihydrofolate reductase system.
  • a SpeI/AgeI restriction fragment from a pTT5-derived vector encoding F8-500D-His was transferred to a pTSV7-derived vector encoding F8-500 leading to construct #1917 consisting of pTSV7 with insert encoding F8-500D-His.
  • HKB11 cells at a density of 0.9-1.1 ⁇ 10 6 were transfected with a complex of plasmid (0.7 mg/l or 1.0 mg/l) and the transfection agent, 293Fectin (Invitrogen) (1.0 ml/l or 1.4 ml/l).
  • the transfection complex was prepared by diluting the plasmid and the transfection separately, mixing the two solutions, and incubating the mixture at room temperature for 20 minutes.
  • the complex mixture was added to the cell suspension and the suspension was incubated in shaker incubator for 4 or 5 days at 36.5° C. or 37° C. and at 5% or 8% CO 2 .
  • Cell culture harvests were analysed by chromogenic FVIII assay as described in Example 14 and/or filtered through a 0.22 ⁇ m membrane filter and utilized for purification of FVIII as described in Example 13.
  • Serum-free adapted CHO-DUKX-B11 cells were transfected with the expression plasmid construct #1917 described in Example 10 and encoding the FVIII F8-500D-His.
  • Transfected cells were selected with the dihydrofolate reductase system and cloned by limiting dilution. Clones were screened for FVIII production by ELISA and chromogenic activity assay. The clone GedT019A was selected for upscaling. The cells were transferred to a bioreactor. The F8-500D-His protein was purified from cell culture harvests as described in Example 13
  • a column was packed with the resin VIIISelect (GE Healthcare), with the dimensions 1.6 cm in diameter and 4 cm in bed height giving 8 mL, and was equilibrated with 20 mM
  • the culture filtrate prepared as described in Example 3 was applied to the column, and the column was subsequently washed with first equilibration buffer and then 20 mM Imidazole+10 mM CaCl 2 +0.01% Tween80+1.5M NaCl, pH7.3.
  • the bound FVIII was eluted isocratic at 90 cm/h with 20 mM Imidazole+10 mM CaCl 2 +0.01% Tween80+1M Ammoniumacetate+6.5M Propylenglycol, pH7.3.
  • the fractions containing FVIII were pooled and diluted 1:10 with 20 mM Imidazole+10 mM CaCl 2 +0.01% Tween80, pH7.3 and applied to a column packed with F25-Sepharose (Thim et al., Haemophilia, 2009).
  • the column dimension was 1.6 cm in diameter and 2 cm in bed height giving 4 mL in column volume.
  • the column was equilibrated at 180 cm/h with 20 mM Imidazole+10 mM CaCl 2 +0.01% Tween80+150 mM NaCl+1M Glycerol, pH7.3 prior to application.
  • the fractions containing FVIII were pooled and diluted 1:15 with 20 mM Imidazole+10 mM CaCl 2 +0.01% Tween80, pH7.3, except FVIII-variants with deletions of the a3 domain which were diluted 1:45 in the same buffer.
  • the diluted pool was applied to a column packed with Poros 50HQ (PerSeptive Biosystem), with the column dimensions 0.5 cm in diameter and 5 cm in bed height giving 1 mL in column volume.
  • the column was equilibrated at 300 cm/h with 20 mM Imidazole+10 mM CaCl 2 +0.01% Tween80+50 mM NaCl+1M Glycerol, pH7.3 prior to application.
  • the column was washed with equilibration buffer before the elution using a linear gradient over 5 column volumes from equilibration buffer to 20 mM Imidazole+10 mM CaCl 2 +0.01% Tween80+1M NaCl+1M Glycerol, pH7.3.
  • the fractions containing FVIII were pooled and the pool was stored at ⁇ 80° until use.b
  • the FVIII molecules with HIS-tag were purified essentially as described above, however the second purification step (F25-sepharose) was exchanged to Chelating Sepharose FF (GE Healtcare) charged with 2 column volumes of 1M NiSO 4 .
  • the column dimension was 0.5 cm in diameter and 5 cm bed height giving 1 mL column volume.
  • the column was equilibrated with 30 mM Imidazole+10 mM CaCl 2 +0.01% Tween80+1.5M NaCl, pH7.3 at 180 cm/h prior to application.
  • the FVIII activity (FVIII:C) of the rFVIII compound was evaluated in a chromogenic FVIII assay using Coatest SP reagents (Chromogenix) as follows: rFVIII samples and a FVIII standard (Coagulation reference, Technoclone) were diluted in Coatest assay buffer (50 mM Tris, 150 mM NaCl, 1% BSA, pH 7.3, with preservative). Fifty ⁇ l of samples, standards, and buffer negative control were added to 96-well microtiter plates (Spectraplates MB, Perkin Elmer). All samples were tested diluted 1:100, 1:400, 1:1600, and 1:6400.
  • the factor IXa/factor X reagent, the phospholipid reagent and CaCl 2 from the Coatest SP kit were mixed 5:1:3 (vol:vol:vol) and 75 ⁇ l of this added to the wells. After 15 min incubation at room temperature, 50 ⁇ l of the factor Xa substrate S-2765/thrombin inhibitor I-2581 mix was added and the reactions were incubated 5 min at room temperature before 25 ⁇ l 1 M citric acid, pH 3, was added. The absorbance at 405 nm was measured on an Envision microtiter plate reader (Perkin Elmer) with absorbance at 620 nm used as reference wavelength. The value for the negative control was subtracted from all samples and a calibration curve prepared by linear regression of the absorbance values plotted vs. FVIII concentration. The yields of the present FVIII relative to that of the F8-500 protein are shown in Table 1.
  • the FVIII activity (FVIII:C) of the rFVIII compound was evaluated in a chromogenic FVIII assay using Coatest SP reagents (Chromogenix) as follows: rFVIII samples and a FVIII standard (e.g. purified wild-type rFVIII calibrated against the 7th international FVIII standard from NIBSC) were diluted in Coatest assay buffer (50 mM Tris, 150 mM NaCl, 1% BSA, pH 7.3, with preservative). Fifty ⁇ l of samples, standards, and buffer negative control were added to 96-well microtiter plates (Nunc) in duplicates.
  • the factor IXa/factor X reagent, the phospholipid reagent and CaCl 2 from the Coatest SP kit were mixed 5:1:3 (vol:vol:vol) and 75 ⁇ l of this added to the wells. After 15 min incubation at room temperature 50 ⁇ l of the factor Xa substrate S-2765/thrombin inhibitor I-2581 mix was added and the reactions incubated 10 min at room temperature before 25 ⁇ l 1 M citric acid, pH 3, was added. The absorbance at 415 nm was measured on a Spectramax microtiter plate reader (Molecular Devices) with absorbance at 620 nm used as reference wavelength.
  • the value for the negative control was subtracted from all samples and a calibration curve prepared by linear regression of the absorbance values plotted vs. FVIII concentration.
  • the specific activity was calculated by dividing the activity of the samples with the protein concentration determined by HPLC.
  • the concentration of the sample was determined by integrating the area under the peak in the chromatogram corresponding to the light chain and compare with the area of the same peak in a parallel analysis of a wild-type rFVIII, where the concentration was determined by amino acid analyses. The results are shown in Table 1.
  • FVIII activity (FVIII:C) of the rFVIII compounds was further evaluated in a one-stage FVIII clot assay as follows: rFVIII samples and a FVIII standard (e.g. purified wild-type rFVIII calibrated against the 7th international FVIII standard from NIBSC) were diluted in HBS/BSA buffer (20 mM hepes, 150 mM NaCl, pH 7.4 with 1% BSA) to approximately 10 U/ml followed by 10-fold dilution in FVIII-deficient plasma containing VWF (Dade Behring or Siemens). The samples were subsequently diluted in HBS/BSA buffer.
  • a FVIII standard e.g. purified wild-type rFVIII calibrated against the 7th international FVIII standard from NIBSC
  • the APTT clot time was measured on an ACL300R or an ACL9000 instrument (Instrumentation Laboratory) using the single factor program.
  • FVIII-deficient plasma with VWF Dade Behring or Siemens
  • SynthASil, (HemosILTM, Instrumentation Laboratory) was used as assay plasma and SynthASil, (HemosILTM, Instrumentation Laboratory) as aPTT reagent.
  • the diluted sample or standard is mixed with FVIII-deficient plasma, aPTT reagents at 37° C. Calcium chloride is assed and time until clot formation is determined by turbidity.
  • the FVIII activity in the sample is calculated based on a standard curve of the clot formation times of the dilutions of the FVIII standard. The results are shown in table 1.
  • DNA fragments encoding the VWF signal peptide, followed by different C-terminally truncated versions, the VWF D′ domain and the VWF D3 domain, an Ala-Leu-Ala spacer and a HPC4 tag were generated by polymerase chain reaction (PCR) using plasmid pLC095 as template (Plasmid pLLC095 is described in Example 26.
  • the primer JP1000 was used as forward primer in all PCR reactions in combination with the reverse primers JP1001-JP1008 shown in Table 2.
  • PCR products were digested with HindIII and NheI and were subsequently cloned into a HindIII and NheI digested pJSV164 vector using Rapid DNA Ligation kit (Roche Diagnostics GmbH, Mannheim, Germany).
  • pJSV164 is a pTT5 based expression vector (Yves Durocher, CNRC, Montreal, Canada) containing a CD33 signal peptide and a HPC4 tag.
  • the resulting eight plasmids were named as shown in Table 3.
  • the amino acid sequences of the generated proteins are outlined in SEQ ID NO 4, 5, 6, 7, 8, 11 and 16.
  • the three PCR fragments VWF(864-1250)-HPC4, VWF(764-1128)-HPC4 and VWF(764-1198)-HPC4 were 5685/5610/5817 by in size respectively.
  • the PCR fragments were DpnI treated to remove methylated template DNA.
  • the PCR fragments were subsequently purified from gel and were self-ligated by LIC using the In-Fusion HD Cloning Kit (Clontech, Mountain View, Calif., USA) to generate circular DNA fragments and subsequently transformed into Top10 cells (Life Technologies, Carlsbad, Calif., USA).
  • the resulting three plasmids were named as shown in Table 5.
  • the amino acid sequences of the generated proteins are outlined in SEQ ID NOs 12, 9, and 10.
  • Human embryonic kidney 293 6E suspension cells at a density of 0.9-1.1 ⁇ 10 6 cells/ml were transfected with a complex of VWF fragment coding plasmid (0.7 mg/l or 1.0 mg/l) and the transfection agent 293Fectin (Invitrogen) (1.0 ml/l or 1.4 ml/l).
  • the transfection complex was prepared by diluting the plasmid and the transfection separately, mixing the two solutions, and incubating the mixture at room temperature for 20 minutes.
  • the complex mixture was added to the cell suspension and the suspension was incubated in shaker incubator for 5 days at 36.5° C. or 37° C. and at 5% or 8% CO 2 .
  • Cell culture harvests were filtered through a 0.22 ⁇ m membrane filter and utilized for purification of VWF fragment as described in Example 22.
  • a dimeric form of the VWF fragments is wanted. This can be accomplished in several ways:
  • the cDNA encoding the desired VWF fragment is including the presequence of VWF e.g the D1D2 sequence of VWF (amino acid residues 23-763 of SEQ ID NO 22). This will, during processing in the golgi apparatus align two monomers of a given VWF fragment in a configuration allowing a dimeric molecule to be formed with two disulphide bonds in which Cys1099 in monomer 1 is connected to a Cys1099 in monomer 2 and Cys1142 in monomer 1 is connected to Cys1142 in monomer 2.
  • dimer formation Another method to accomplish dimer formation is to avoid the inclusion of the presequence (amino acid residues 23-763 of SEQ ID NO 22) and simply let a recombinant VWF fragment with Cys in position 1099 and 1142 form a dimeric molecule. This can in principle result in a series of different dimers e.g.:
  • Yet another method to accomplish dimer formation may be toto replace one of the cysteine residues 1099 or 1142 with other amino acid residues (e.g. Serine, Arginine).
  • the molecule may form a dimer by establishment of a disulphide bond between Cys1142 in monomer 1 with Cys1142 in monomer 2.
  • Cys1142 is replaced with a non-Cysteine residue, the molecule may form a dimer by establishment of a disulphide bond between Cys1099 in monomer 1 with Cys1099 in monomer 2.
  • the dimeric forms mentioned above may be constructed either with or without the D1D2 presequence of VWF (amino acid residues 23-763 of SEQ ID NO 22).
  • the different monomeric and dimeric forms will have different properties with regards to their binding to FVIII, their ease of production and their effect on bioavailability of FVIII when injected subcutaneously as a co-formulation.
  • the purified protein is characterised by 1) SDS-gel electrophoreses, 2) analytical HPLC and 3) amino acid sequence analysis.
  • the anti-VWF antibody recognise amino acid residue number 764-865 of VWF (SEQ ID NO 5).
  • the purified VWF fragment is characterised by 1) SDS-gel electrophoreses, 2) analytical HPLC and 3) amino acid sequence analysis.
  • All protein samples are dialyzed in 50 mM Hepes pH 7.4, 150 mM NaCl, 10 mM CaCl 2 buffer.
  • Each iTC experiment involves filling the iTC cell with FVIII (approximately 250 ⁇ L) and the syringe with VWF variants (approximately 40 ⁇ L). Temperature is set as required and the protein sample is allowed to equilibrate under given experimental conditions (approximately 10 minutes). Typically 17-20 injections (of 2-2.5 ⁇ L) of VWF variants into cell, containing FVIII, are performed. The first injection is always of 0.2 ⁇ L and is discarded from the final data analysis in order to account for diffusion during equilibration step.
  • Stirring speed is set between 700-1000 rpm.
  • Filter period for data collection is 5 sec with a high feedback mode setting. Each titration is spaced by 120 sec. Appropriate control experiments are performed.
  • Raw data is processed to set baseline and integrated to obtain a final isotherm.
  • This binding isotherm is fit to a single-site model to yield K d , stoichiometry (n), and ⁇ S values to complete characterization of VWF variant binding to FVIII.
  • An example binding isotherm is shown in FIG. 9 . These data are being used for determining the optimal concentrations of the FVIII and the VWF fragment in co-formulations intended for subcutaneous administrations.
  • FVIII KO mice exon 16 knock-out in a mixed background of C57Bl/6 and SV129, bred at Taconic M&B (B6.129S4-F8tm1Kaz/J) with an approximate weight of 22 g were dosed subcutaneously in the flank with FVIII in combination with various proteins, 6-9 mice with each test compound.
  • the dose volume was 5 ml/kg or 0.25 ml/kg if indicated in table 7.
  • mice were anaesthetized by Isoflurane/O 2 /N 2 O prior to blood sampling via the retroorbital plexus.
  • 45 ⁇ l of blood was stabilised with 5 ⁇ l of sodium-citrate (0.13 M) and added 200 ⁇ l FVIII Coatest SP buffer (50 mM TRIS-HCl, 1% BSA, Ciprofloxacin 10 mg/L, pH 7.3). After centrifugation at 4000 g for 5 minutes at room temperature, the supernatants were immediately frozen on dry ice before storage at ⁇ 80° C. prior to analysis.
  • the s.c. FVIII bioavailabilities of the test compounds are shown in table 7 below and in FIGS. 7 and 8 .
  • the column labelled “FVIII Saturation” denotes the calculated fraction of FVIII that is binding the co-formulated protein at the concentrations used in the experiment.
  • the column labelled “F %” denotes the bioavailability of FVIII obtained in the experiment.
  • the s.c. bioavailability of FVIII co-formulated with a VWF fragment appear to depend on the saturation of the FVIII VWF binding sites in the co-formulation rather than on the VWF fragment length.
  • the dimer form of the 764-1464 dosed in a lower volume of 0.25 ml/kg resulted in a FVIII bioavailability of 8.4%.
  • Fragments shorter than 764-1250 which do not contain the entire D3 region, bind FVIII with a higher 1050 (K i ) than longer fragments.
  • K i 1050
  • 1 to 1 molar formulation of FVIII and VWF fragments shorter than 764-1250 displayed lower FVIII bioavailabilities, i.e. less than 4%.
  • the s.c. FVIII bioavailability-improving effect of VWF fragments according to the invention may thus be obtained by saturation of the FVIII VWF binding sites with VWF-fragment.
  • Short VWF fragments with relatively low FVIII binding affinity should thus be used in higher ratios compared to longer VWF fragments with better binding FVIII binding properties in order to obtain a high degree of bioavailability.
  • FVIII derived from the full-length sequence displayed the same degree of bioavailability as FVIII with a truncated B domain (turoctocog alfa/N8) when co-formulated with the 764-1464 VWF fragment. This indicates that high FVIII bioavailability is not dependent on co-formulation with turoctocog alfa/N8 but is dependent on presence of the VWF fragment.
  • Serum albumin did not improve the s.c. bioavailability of FVIII (turoctocog alfa/N8). Thus, presence of additional protein in a FVIII formulation does not appear to increase the s.c. bioavailability of FVIII—unless this protein is a VWF fragment according to the present invention.
  • VWF dose was not critical for FVIII s.c. bioavailability as seen for molar ratios between 1:1 and 1:7.7 of FVIII:VWF fragment.
  • the critical factor for achieving a high FVIII bioavailability thus appear to be a high degree of FVIII saturation (binding) with VWF fragment. All compositions in these experiments comprising a calculated saturation of N8 of at least 86.8% thus resulted in similar bioavailabilities.
  • VWF fragments according to the invention may thus protect FVIII at the s.c. injection site.
  • FVIII with a 226 amino acid (aa) B domain displayed a higher s.c. FVIII bioavailability than turoctocog alfa/N8.
  • bioavailability of this FVIII with a 226 aa B-domain was comparable to turoctocog alfa/N8 in connection with s.c. co-administration with the VWF-fragment 764-1464 (TIL′/E′/D3/A1) monomer.
  • FVIIIK1804C-HEP157 displayed a bioavailability of 50% dosed in co-administration with the VWF-fragment 764-1464 (TIL′/E′/D3/A1) monomer and a bioavailability of 27% dosed alone.
  • PSA40Kd-O-Glycan-N8 displayed a bioavailability of 8.8% dosed in co-administration with the VWF-fragment 764-1464 (TIL′/E′/D3/A1) monomer and 6.11% dosed alone. It may thus be speculated that conjugation of FVIII molecules with Heparosan polymers and/or Polysialic acid polymers either protects FVIII against breakdown/uptake in the sub cutis or enhances s.c. absorption. Heparosan appear to be more effective than Sialic acid polymers in enhancing the s.c. bioavailability. Both FVIII variants displayed higher bioavailability's when dosed together with VWF fragment.
  • N8-GP and FVIIIK1804C-HEP157+764-1464 (TIL′/E′/D3/A1) monomer and dimer, resulted in the highest bioavailability obtained.
  • Bioavailability of N8-GP may thus be increased by increasing the dose or the concentration in the co-formulation.
  • Dose volume was 5 ml/kg in all dosing's, thus the N8-GP concentration in the dosing solution was 2 times higher in the 20000 IU/kg dosing than in the 10000 IU/kg dosing. This resulted in 28% and 19% bioavailability respectively.
  • the 764-1464 dimer VWF fragment does not contain any mutations.
  • the 764-1464 dimer VWF fragment binds stronger to Turoctocog alfa and N8-GP (table 6) but result in a similar bioavailability of FVIII as the monomer version of the fragment.
  • substituting Cys1099 and/or Cys1142 in the VWF fragments according to the invention does not influence the bioavailability of FVIII.
  • the binding affinity of VWF fragments to N8-GP does not influence the effect on bioavailability of N8-GP as long as more than 80% of the FVIII molecules are in complex with VWF fragment in co-formulation.
  • the dimer version of VWF fragment 764-1464 improves the bioavailability, the maximum molecular weight of a desired VWF fragment may be equal to or larger than 158.8 KDa.
  • the antibody 4F30 (further characterised in WO2012035050), which bind to Cl and inhibits cellular uptake of FVIII, did not improve the bioavailability of N8-GP.
  • 2000 IU/ml N8-GP was co-formulated with 1 mg/ml of 4F30 which means that 99.6% of FVIII was bound to the mAb also after in vivo dilution assuming a K d of 0.6 nM, an in vivo dilution of 20 ⁇ , a molecular weight for FVIII (turoctocog alfa/N8) of 170000 g/mol, a specific activity of 10000 IU/mg for turoctocog alfa/N8, and a molecular weight for 4F30 of 150000 g/mol.
  • the PEGylated FVIII with K2092A+F2093A mutations displayed decreased uptake in cells but the mutations did not improve the bioavailability compared to N8-GP. Inhibition of cellular FVIII uptake does thus not appear to be the mechanism by which co-formulated VWF fragments result in increased s.c. bioavailability of FVIII.
  • Test compounds were formulated in 18 mg/ml NaCl, 3 mg/ml saccharose, 1.5 mg/ml L-histidine, 0.1 mg/ml polysorbate 80, 0.25 mg/ml CaCl 2 , pH ⁇ 7.3.
  • mice Female New Zealand white rabbits weighing approximately 2-3 kg were used for the study. The animals were allowed free access to feed and water. The rabbits were dosed subcutaneously over the thigh with FVIII in combination with various proteins, 4-5 rabbits with each test compound. The dose volume was 0.2 ml/kg or 1 ml/kg.
  • FVIII antigen analysis using two FVIII light chain antibodies (4F45 and 4F11) in a FVIII LOCI assay (Luminescence oxygen channelling immunoassay).
  • the s.c. bioavailability in rabbits of N8-GP and N8-GP co-formulated with VWF fragment TIL′/E′/D3/A1 dosed in a dosing volume of 0.2 ml/kg was 40 and 59%, respectively.
  • the bioavailability of N8-GP+VWF dosed in a dosing volume of 1 ml/kg was 34%.
  • the bioavailability of N8-GP may thus be influenced either by the species or by the differences in dosing volumes (5 ml/kg in mice and 0.2 ml/kg or 1 ml/kg in rabbits).
  • 0.2 ml/kg is closest to a dosing volume relevant for humans.
  • FVIII (turoctocog alfa/N8) dosed together with VWF fragment TIL′/E′/D3/A1 displayed a similar bioavailability in rabbits compared to mice despite the higher dosing concentration.
  • Plasmid #796 consisting of the pZEMHygro vector with insert consisting of wild-type human VWF cDNA was utilized as the starting point for generating DNA constructs for the expression of truncated human VWF proteins.
  • DNA encoding the VWF signal peptide, followed by the VWF TIL′E′ domain, the VWF D3 domain, the VWF A1 domain, and a HPC4 tag was generated by polymerase chain reaction (PCR) using plasmid #796 as template, forward primer oLLC089 VWF forward, and reverse primer oLLC092 VWF A1 HPC4 reverse. These primers contain a Nhe I and a Not I restriction site, respectively. The resulting PCR product was inserted into the pCR2.1-TOPO vector (Invitrogen).
  • the VWF(TIL′/E′/D3/A1)-HPC4 coding DNA was excised with the Nhe I and a Not I restriction enzymes and inserted into pZEM219b digested with the same restriction enzymes.
  • the pLLC089 construct was established consisting of pZEM219b with insert encoding VWF(TIL′/E′/D3/A1)-HPC4.
  • Nucleotide substitutions leading to the amino acid replacements C1099/1142S in the VWF VWF(TIL′/E′/D3/A1)-HPC4 protein encoded by pLLC089 were introduced by site-directed mutagenesis of pLCC089 using the QuikChange XL Site-directed Mutagenesis kit (Stratagene) and the oLLC101-f, oLLC102-r, oLLC103-f, and oLLC104-r mutagenesis primers.
  • the site.directed mutagenesis gave rise to the pLLC095 vector consisting of pZEM219b with insert encoding VWF (TIL′/E′/D3/A1)C1099/1142S-HPC4.
  • Baby hamster kidney (BHK) cells grown in Dulbecco's modified Eagle's medium with 10% fetal calf serum were transfected with pLL095 using Genejuice transfection reagent (Merck).
  • a pool of transfected cells was generated by selection with 1.5 ⁇ M methotrexate giving rise to a non-clonal BHK cell line producing VWF (TIL′/E′/D3/A1)C1099/1142S-HPC4.
  • the cells were seeded in a biofermentor and the VWF (TIL′/E′/D3/A1)C1099/1142S-HPC4 protein was purified from the cell culture supernatant as described in Example 22.
  • CHO-DUKX-B11 suspension cells grown in suspension were transfected with pLLC095 by electroporation.
  • a pool of transfected cells was generated by adaptation to growth in medium without nucleosides. Subsequently, the pool was adapted to growth in the presence of 100 mM methotrexate giving rise to the VWF (TIL′/E′/D3/A1)C1099/1142S-HPC4 producing non-clonal CHO-DUKX-B11 cell line MBML001.
  • the cells were seeded in a biofermentor and the VWF (TIL′/E′/D3/A1)C1099/1142S-HPC4 protein was purified from the cell culture supernatant as described in Example 22.
  • U87 MG cells are obtained from ATCC (HTB-14). The cells are cultured in fibronectin-coated 24-well plates for 48 hours in EMEM supplemented with 10% heat inactivated FCS at 37° C.
  • buffer A 10 mM HEPES, 150 mM NaCl, 4 KCl, 11 mM Glucose, pH 7.4
  • buffer B buffer A supplemented with 5 mM CaCl 2 and 1 mg/ml BSA
  • Radioactively labelled FVIII ( 125 I-FVIII, final concentration 1 nM) is incubated alone or premixed with different concentrations of pdVWF (American Diagnostica, final concentration 0.001 nM 50 nM based on monomer content) or TIL′/E′/D3/A1 (final concentration 0.25 nM-500 nM or 1000 nM) 10 min prior to addition to the U87 MG cells and incubated with the cells 1 hour at 37° C. to allow binding and internalization. Cells are subsequently washed three times with ice-cold buffer B.
  • pdVWF American Diagnostica, final concentration 0.001 nM 50 nM based on monomer content
  • TIL′/E′/D3/A1 final concentration 0.25 nM-500 nM or 1000 nM
  • Dendritic cells and macrophages are differentiated from monocytes isolated from buffy coats by magnetic separation using magnetic anti-CD14-beads (Miltenyi Biotec) and a MACS column (Miltenyi Biotec) according to the manufactures instructions.
  • Monocytes 0.5 ⁇ 10 6 cells/nil
  • IMDM media GIBCO
  • FBS fetal bovine serum
  • penicillin/streptomycin 3.3 ng/ml M-CSF
  • R&D Systems 3.3 ng/ml M-CSF
  • the monocytes can alternatively be differentiated into dendritic cells by stimulating with 40 ng/ml GM-CSF (R&D Systems) and 40 ng/ml IL-4 for five days.
  • Dendritic cells are washed in buffer B and transferred to low binding Nunc tubes with 0.5 ⁇ 10 6 cells/tube.
  • Fluorescently labelled FVIII e.g. Oregon-Green FVIII (e.g. 30 and 100 nM) are added and incubated 1 hour at 37° C.
  • Cells are washed once and analysed by flow cytometry using a LRS Fortessa instrument (BD). The macrophages are after six days culturing washed with PBS and incubated 10-20 min at 4° C.
  • Macrophages (7 ⁇ 10 5 /well) are seeded on fibronectin-coated 96-well glass bottom tissue culture plates (Perkin Elmer ViewPlate Black). 24 hours post seeding the cells are washed once with buffer B before addition of 30 nM fluorescently-labelled FVIII (e.g. OregonGreen-FVIII) alone or in the presence of increasing concentrations (15-240 nM) of pdVWF (American Diagnostica) or TIL′/E′/D3/A1. Macrophages are incubated for 1 hour at 37° C.
  • FVIII fluorescently-labelled FVIII
  • Dead or apoptotic cells are excluded from the analysis based on nuclei fragmentation and/or excessive binding of FVIII to the plasma membrane.
  • the integrated fluorescent intensity of the vesicular FVIII signal is calculated and plotted against time.
  • IC50 values for inhibition of FVIII binding and internalization in U87 MG cells and macrophages are shown in table 10. Both pdVWF and TIL′/E′/D3/A1 are able to inhibit FVIII cell binding/uptake in both cell types providing sufficient high concentrations are used.
  • the data may indicate that a reduced immune response can be achieved upon co-formulation of FVIII with a VWF fragment.
  • FVIII deficient, FVIII-KO mice 12-16 weeks old, male and females are divided into 3 groups of 12 animals. In each group, eight animals are subjected to tail bleeding and 4 animals are used in parallel for ex vivo efficacy testing using ROTEM analysis.
  • GlycoPEGylated FVIII or vehicle is dosed s.c. 24 hr prior to tail transection.
  • the s.c injection is performed in the neck and the i.v. injection in a lateral tail vein.
  • the dose volume is 5 ml/kg.
  • GlycoPEGylated FVIII is prepared in buffer (10 mM L-Histidine, 8.8 mM Sucrose, 0.01% Polysorbate 80, 308 mM NaCl, 1.7 mM CaCl 2 (dihydrate), 0.01% Polysorbate 80 0.1 mg/ml, pH 6.9) to a concentration of 40 and 500 U/ml and stored at ⁇ 80° C. until use.
  • mice Before tail transection, the mice are anaesthetised with isoflurane and placed on a heating pad. The tails are placed in pre-heated saline at 37° C. for 10 min. The tail is transected 4 mm from the tip.
  • Parallel animals are used for blood sampling and subsequent analysis of their clotting parameters (ex vivo efficacy).
  • a blood sample is taken from the peri-orbital plexus with 20 ⁇ L capillary tubes without additive.
  • the blood sample is diluted 1:10 in 0.13M sodium citrate and carefully mixed and stored at rum temperature for immediate thromboelastography by ROTEM.
  • the blood sample is re-calcified by adding 7 ⁇ L CaCl 2 to a mini curvet (StarTEM). Thereafter, 105 ⁇ L of blood is added to the mini curvet and mixed. The analysis is performed until the maximum amplitude is reached.
  • the prophylactic effect of s.c. administered FVIII is determined by comparing the blood loss during the 30 min study period at 24 hr after s.c. administration to that of 1) a vehicle control group and 2) an i.v. control group with glycoPEGylated FVIII.
  • the blood loss in the group dosed s.c. with glycoPEGylated FVIII is comparable to the blood loss in the group dosed i.v. ( FIG. 10 , left panel).
  • the blood loss data are supported by the ex vivo efficacy parallel study of the examined clotting parameters, e.g. clot time ( FIG. 10 , right panel).
  • subcutaneously administered FVIII appear to be hemostatically active based on the PK profile and the results from the ex vivo activity. Therefore, subcutaneously administered FVIII co-formulated with a VWF fragment is also believed to be hemostatically active as can be predicted from its pharmacokinetic profile.
  • Test compounds are prepared in 10 mM L-Histidine (1.55 mg/ml), 8.8 mM Sucrose (3.0 mg/ml), 308 mM NaCl (18 mg/ml), 1.7 mM CaCl2 dihydrate (0.25 mg/ml), 0.01% Polysorbate 80 (0.1 mg/ml), pH 7.3.
  • mice Experiments are performed using groups of F8 knockout (FVIII k/o) mice (129/C57BL/6 or C57BL/6, exon 16 disrupted). Animals are included in experiments when 12-18 weeks old at which time they are weighing roughly 18-25 grams. Twelve to 15 animals are included per group.
  • F8 knockout mice FVIII k/o mice
  • Test compounds are administered subcutaneously (or intravenously for controls) using a dose volume of maximally 10 ml/kg (or 5 ml/kg for controls).
  • a tail vein transection (TVT) bleeding model is conducted with the mice under full isoflurane anaesthesia. Briefly, following anaesthesia the bleeding challenge comprises a template-guided transection of a lateral tail vein at a tail diameter of 2.7 mm. The tail is immersed in saline at 37° C. allowing visual recording of the bleeding for 60 min, where after the blood is isolated and the blood loss determined by measuring the haemoglobin concentration as described in “Example 3”. When feasible and justified, blood is sampled for assessment of FVIII activity (FVIII:C) in plasma as described above.
  • FVIII activity FVIII activity
  • FVIII or FVIII co-formulated with VWF fragments are injected subcutaneously at defined time point(s) prior to TVT.
  • Vehicle and intravenous control/treatment groups are included for no effect and maximal effect, respectively.
  • FVIII or FVIII/VWF is injected s.c. to identify prolonged effect, i.e. improved bleeding phenotype after treatment.
  • TVT is performed at several time points, e.g. 24, 48, 72, 96, after dosing.
  • FVIII or FVIII/VWF fragment is dosed s.c. once daily for several days.
  • TVT is performed at different time points to assess any improvement in the bleeding phenotype.
  • Data processing and analyses Data are physically recorded throughout the experiment. Hereafter, data are aggregated for analysis using MS Excel (Microsoft, WA, USA) before being analysed in GraphPad Prism version 5 (GraphPad Software, Inc, CA, USA).
  • FVIII or FVIII/VWF are injected subcutaneously before assessing ex vivo effect, before inducing a bleeding challenge, or as a means to treat or prevent spontaneous bleeds.
  • Test compounds are prepared in 10 mM L-Histidine (1.55 mg/ml), 8.8 mM Sucrose (3.0 mg/ml), 308 mM NaCl (18 mg/ml), 1.7 mM CaCl2 dihydrate (0.25 mg/ml), 0.01% Polysorbate 80 (0.1 mg/ml), pH 7.3.
  • Test compounds are administered subcutaneously (or intravenously for controls) using a dose volume of maximally 10 ml/kg (or 5 ml/kg for controls).
  • Dog effect model In dogs with haemophilia A the effect is assessed ex vivo using surrogate markers, e.g. thrombelastography as previously described (Knudsen et al, 2011; Haemophilia, 17, 962-970), or in vivo, e.g. using a standardized bleeding challenge monitored by acoustic force radiation force impulse (ARFI) ultrasound as described (Scola et al, 2011; Ultrasound in Med. & Biol., 37(12), 2126-2132). Capacity allowing, test compound are administered to treat spontaneously bleeding dogs. Effect is monitored by assessing the resolution of clinical manifestation in comparison with historic data on i.v. treatment.
  • surrogate markers e.g. thrombelastography as previously described (Knudsen et al, 2011; Haemophilia, 17, 962-970)
  • ARFI acoustic force radiation force impulse
  • Rat effect model In rats with haemophilia A the effect is assessed ex vivo using surrogate markers, e.g. thrombelastography as described above for mice and dogs, or in vivo, e.g. using a standardized bleeding challenge as described for mice. Capacity allowing, test compound are administered to treat spontaneously bleeding rats. Effect is monitored by assessing the resolution of clinical manifestation in comparison with historic data on i.v. treatment.
  • surrogate markers e.g. thrombelastography as described above for mice and dogs
  • in vivo e.g. using a standardized bleeding challenge as described for mice.
  • Capacity allowing, test compound are administered to treat spontaneously bleeding rats. Effect is monitored by assessing the resolution of clinical manifestation in comparison with historic data on i.v. treatment.
  • a nucleotide substitution leading to the amino acid replacement 51142C in the VWF(764-1250)-C1099/1142S-ALA-HPC4 protein encoded by pJSV348 described in Example 17 was introduced by PCR-based site-directed mutagenesis using the VWF 1099C S and VWF 1099C AS primers (Table P). This gave rise to the pGB237 vector consisting of pTT5 with insert encoding VWF(764-1250)-C1099S-ALA-HPC4 (SEQ ID NO 11). The cysteine at position 1142 allows dimerization of the protein as described in Example 20.
  • the S1099C amino acid replacement was introduced in the VWF(764-1128)-C1099S-HPC4 protein encoded by pJSV406 described in Example 18, giving rise to the pGB249 vector consisting of pTT5 with insert encoding VWF(764-1128)-HPC4 (SEQ ID NO 9).
  • the cysteine at position 1099 allows dimerization of the protein as described in Example 20.
  • cDNA encoding amino acid 1-1250 of human VWF was amplified by PCR using plasmid #796 (described in Example 26) as template, forward primer JP1000 VWF-HindIII S (Table 2), and reverse primer JP1006 VWF764-1250 (Table 2).
  • Primer JP1006 VWF764-1250 contains a Nhe I site.
  • the resulting PCR product was inserted into the pCR4BLUNT-TOPO vector (Invitrogen) downstream of Pme I restriction site.
  • the vWF(1-1250) coding DNA was excised with the Pme I and a Nhe I restriction enzymes and inserted into pJSV164 described in Example 17 generating the pGB242 vector consisting of pTT5 with insert encoding vWF(1-1250)-ALA-HPC4.
  • the cysteines at position 1099 and 1142 allow dimerization of the protein as described in Example 20, and proteolytic removal of the presequence will generate vWF(764-1250)-ALA-HPC4 (SEQ ID NO 11).
  • DNA sequences of pJSV348 (described in Example 17) and construct #796 (described in Example 26) were inverse amplified by PCR using overlapping primers.
  • the pJSV348 sequence was amplified using primer 2764pJSV348 and 1202pJSV348R (Table P), while the construct #796 sequence was amplified using primer 221#796F and 3537#796R (Table P).
  • pJSV348 The amplification products from pJSV348 (recipient) and construct #796 (donor) were excised from an agarose gel and joined by ligation independent cloning (LIC) using the In-Fusion HD Cloning Kit (Clontech) to generate circular DNA and subsequently transformed into Stellar competent cells (Clontech).
  • the resulting expression vector, named pGB252 consists of PTT5 with insert encoding VWF(1-1128)-ALA-HPC4.
  • the cystein at position 1099 allows dimerization of the protein as described in Example 20, and proteolytic removal of the presequence will generate vWF(764-1128)-ALA-HPC4 (SEQ ID NO 9).
  • amplification using pJSV348 (described in Example 17) as template with the primers 2764pJSV348 and 1202pJSV348R (Table P) and amplification using #796 (described in Example 26) as template with the primers 221#796F and 3747#796R (Table P) generated pJSV348 (recipient) and construct #796 (donor) amplification products that were also excised from an agarose gel and joined by ligation independent cloning (LIC) using the In-Fusion HD Cloning Kit (Clontech) to generate circular DNA and subsequently transformed into Stellar competent cells (Clontech).
  • LIC ligation independent cloning
  • the resulting expression vector named pGB253 consists of PTT5 with insert encoding VWF(1-1198)-ALA-HPC4.
  • the cysteines at position 1099 and 1142 allow dimerization of the protein as described in Example 20, and proteolytic removal of the presequence will generate vWF(764-1198)-ALA-HPC4 (SEQ ID NO 10).
  • DNA sequences of pJSV348 (described in Example 17) and construct #796 (described in Example 26) were inverse amplified by PCR using overlapping primers.
  • the pJSV348 sequence was amplified using primer 2764pJSV348 and 2420pJSV348R (Table 11), while the construct #796 sequence was amplified using primer 3666#796F and 5203#796R (Table P).
  • pGB250 consists of PTT5 with insert encoding VWF(764-1873)-C1099/1142C-ALA-HPC4 (SEQ ID NO 20).
  • Human VWF cDNA sequences amplified from construct #796 (described in Example 26) were combined generating the pLLC122 vector consisting of pZEM219b with insert encoding vWF (1-1464)-HPC4.
  • the cysteines at position 1099 and 1142 allow dimerization of the protein as described in Example 20, and proteolytic removal of the presequence will generate vWF(764-1464)-HPC4 (SEQ ID NO 19).
  • Human monocyte-derived dendritic cells were prepared as described in example 28. Expression of the dendritic cell markers CD209 and CD86 were controlled by flow cytometry using a LRS Fortessa instrument (BD). Fluorescent labelled FVIII (Oregon green—FVIII, 30 nM final concentration) was premixed with different concentrations of plasma-derived VWF or VWF fragments before incubating 1 h at 37° C. with dendritic cells. Live/Dead cell kit (Invitrogen # L10119, APC-Cy7) was used for gating on live dendritic cells, and FVIII uptake within this cell population was quantified. Data was normalized for each individual experiment.
  • FVIII Fluorescent labelled FVIII
  • the signal in samples without VWF was defined as 100% FVIII uptake, and the signal in the sample with the highest concentration of plasma-derived VWF (240 nM based on monomer content) was defined as 0%.
  • Values from 3-5 experiments were combined and 1050 values calculated using non-linear regression in Prism software (log(inhibitor) vs. response—Variable slope (four parameters)). The resulting 1050 values are shown in table 12.
  • the data show that all tested VWF fragments were able to inhibit FVIII uptake by the dendritic cells provided sufficiently high concentrations are used.
  • FVIII uptake by antigen-presenting cells is the initial step in presenting FVIII to the immune system the data suggests that co-formulation of FVIII with sufficiently high concentration of VWF fragment may have a potential in reducing immunogenicity of FVIII.
  • the objective is to evaluate the potential of pharmaceutical compositions to treat haemophilia A patients with inhibitors against FVIII.
  • FVIII alone or co-formulated with VWF-fragments subcutaneously to nave FVIII-KO mice or FVIII-KO mice where inhibitors are induced by repeated subcutaneous or intravenous administrations of FVIII prior to treatment with the compositions, or by injecting a polyclonal or monoclonal anti-FVIII antibody.
  • the effect of the treatments is evaluated in anaesthetized mice after transection of a lateral tail vein. The tail is placed in pre-warmed saline at 37° C. and the bleeding is observed for 60 minutes. The blood loss during the experiment is a measure of the effect of the composition.
  • Murine VWF fragment TIL′/E′/D3/A1 1.829 nmol/ml, 0.015 mg/ml
  • test compound was formulated in 20 mM imidazol 150 mM NaCl, 0.02% Tween 80, 1.1M Glycerol, 10 mM CaCl2, pH 7.3
  • Blood was sampled pre-dose and at 0.08, 0.33, 0.5, 1, 2, 4, 7, 18 and 24 h post administration in a sparse sample design with 2 mice sampled per time point.
  • the mice were anaesthetized by Isoflurane/O2/N2O prior to blood sampling via the retroorbital plexus. Three samples were taken from each mouse.
  • Blood (45 ⁇ l) was stabilised with 5 ⁇ l of sodium-citrate (0.13 M) and added 200 ⁇ l FVIII coatest SP buffer (50 mM TRIS-HCl, 1% BSA, Ciprofloxacin 10 mg/L, pH 7.3). After centrifugation at 4000 g for 5 minutes at room temperature, the supernatants were immediately frozen on dry ice before storage at ⁇ 80° C. prior to analysis.
  • HX-MS technology exploits that hydrogen exchange (HX) of a protein can readily be followed by mass spectrometry (MS).
  • MS mass spectrometry
  • incorporation of a deuterium atom at a given site in a protein will give rise to an increase in mass of 1 Da.
  • This mass increase can be monitored as a function of time by mass spectrometry in quenched samples of the exchange reaction.
  • the deuterium labelling information can be sub-localized to regions in the protein by pepsin digestion under quench conditions and following the mass increase of the resulting peptides.
  • HX-MS One use of HX-MS is to probe for sites involved in molecular interactions by identifying regions of reduced hydrogen exchange upon protein-protein complex formation. Usually, binding interfaces will be revealed by marked reductions in hydrogen exchange due to steric exclusion of solvent. Protein-protein complex formation may be detected by HX-MS simply by measuring the total amount of deuterium incorporated in either protein members in the presence and absence of the respective binding partner as a function of time.
  • the HX-MS technique uses the native components, i.e., protein and antibody or Fab fragment, and is performed in solution. Thus HX-MS provides the possibility for mimicking the in vivo conditions (for a recent review on the HX-MS technology, see Wales and Engen, Mass Spectrom. Rev. 25, 158 (2006)).
  • D′D3A1 (SEQ ID NO 19; Cys1099Ser; Cys1142Ser) Batch 0129-0000-0170-6B; 2304 (SEQ ID NO 5) Batch 0129-0000-2304-1B; 2307 (SEQ ID NO 8) Batch 0129-0000-2307-1B; 2308 (SEQ ID NO 11) Batch 0129-0000-2308 2B.
  • the HX experiments were performed on a nanoACQUITY UPLC System with HDX Technology (Waters Inc.) coupled to a Synapt G2 mass spectrometer (Waters Inc.).
  • the Waters HDX system contained a Leap robot (H/D-x PAL; Waters Inc.) operated by the LeapShell software (Leap Technologies Inc/Waters Inc.), which performed initiation of the deuterium exchange reaction, reaction time control, quench reaction, injection onto the UPLC system and digestion time control.
  • the Leap robot was equipped with two temperature controlled stacks maintained at 20° C. for buffer storage and HX reactions and maintained at 2° C. for storage of protein and quench solution, respectively.
  • the Waters HDX system furthermore contained a temperature controlled chamber holding the pre- and analytical columns, and the LC tubing and switching valves at 1° C.
  • a separately temperature controlled chamber holds the pepsin column at 25° C.
  • 100 ⁇ L quenched sample containing 100 pmol hIL-21 was loaded and passed over a Poroszyme® Immobilized Pepsin Cartridge (2.1 ⁇ 30 mm (Applied Biosystems)) placed at 25° C. using a isocratic flow rate of 100 ⁇ L/min (0.1% formic acid:CH 3 CN 95:5).
  • the resulting peptides were trapped and desalted on a VanGuard pre-column BEH C18 1.7 ⁇ m (2.1 ⁇ 5 mm (Waters Inc.)). Subsequently, the valves were switched to place the pre-column in-line with the analytical column, UPLC-BEH C18 1.7 ⁇ m (1 ⁇ 100 mm (Waters Inc.)), and the peptides separated using a 8 min gradient of 8-45% B delivered at 120 ⁇ l/min from the nanoAQUITY UPLC system (Waters Inc.). The mobile phases consisted of A: 0.1% formic acid and B: 0.1% formic acid in CH 3 CN.
  • HX Amide hydrogen/deuterium exchange
  • the HX time-course of 191 peptides, covering 83% of the primary sequence of FVIII were monitored in the absence or presence of the vWF fragments 2304 or 2307 for i.e., 10, 20, 30, 40, 60, 120, and 240 sec.
  • the vWF fragments 2304 and 2307 both induce identical alterations in the exchange profile of FVIII and will be described together here.
  • the observed exchange pattern in the time points (i.e., 10, 20, 30, 40, 60, 120, and 240 sec) in the presence or absence of 2304/2307 can be divided into different groups: One group of peptides display an exchange pattern that is unaffected by the binding of 2304/2307. In contrast, another group of peptides in FVIII show protection from exchange upon 2304/2307 binding.
  • the regions displaying protection upon 2304/2307 binding encompass peptides covering residues 1855-1875, 1857-1875, 2062-2070, 2125-2147, 2125-2148, 2127-2147, 2275-2291, 2275-2302, 2275-2305, 2292-2305, and 2293-2312 (Table 14).
  • the regions that display reduced deuterium incorporation can be narrowed to residues 1862-1875, 2062-2070, 2125-2147, and 2285-2299.
  • vWF fragments D′D3A1 and 2308 both induce identical alterations in the exchange profile of FVIII and will be described together here.
  • the regions displaying protection upon D′D3A1 or 2308 binding encompass peptides covering residues 1669-1680, 1738-1765, 1743-1765, 1856-1869, 1870-1874, 2061-2074, 2063-2074, 2123-2146, and 2260-2280 (Table 15).
  • the regions that display reduced deuterium incorporation can be narrowed to residues 1671-1680, 1745-1754, 1858-1874, 2063-2074, 2125-2146, 2262-2280.
  • the region displaying exchange protection upon FVIII binding encompass the peptide covering residues 768-778 (Table 16).
  • the identified regions of FVIII showing protection upon binding to vWF fragments D′D3A1, 2308, 2304, or 2307 are structurally situated at remote distances when mapping on to the crystal structure PDB: 2R7E. This makes it highly unlikely that they can all be assigned to protection induced by binding interface between FVIII and the vWF fragments D′D3A1, 2308, 2304, or 2307.
  • the HX-MS analysis is unable to distinguish between exchange protection induced by binding interface with exchange protections induced by rapid conformational changes.
  • domains C1 and C2 are essential for the membrane binding affinity of FVIII. It can be speculated that conformational changes of these part of FVIII will reduce the membrane binding ability of FVIII.
  • the conformational position of domains C1 and C2 of FVIII complex bound to the vWF fragments might be unfavourable for membrane binding affinity of FVIII.
  • the fragments in complex with FVIII will shield for the membrane binding affinity of FVIII as it has been established for the membrane binding characteristics of FVIII complex bound to endogenous vWF.
  • a reduced membrane binding affinity of FVIII complex bound to the vWF fragments in comparison to free FVIII would lead to a reduced binding of FVIII to cell membranes of the immune system, e.g. antigen presenting cells. This could decrease presentation of FVIII-derived peptides on MHC class II and it can therefore be speculated that FVIII complex bound to vWF fragments will be less immunogenic than free FVIII.
  • Sequence Domain 2304 2307 L1855-E1875 A3 EX EX V1857-E1875 A3 EX EX W2062-W2070 A3 EX EX V2125-R2147 C1 EX EX V2125-Y2148 C1 EX EX F2127-R2147 C1 EX EX F2275-T2291 C2 EX EX F2275-L2302 C2 EX EX F2275-Y2305 C2 EX EX EX P2292-Y2305 C2 EX EX V2293-S2312 C2 EX EX EX EX: exchange protection of FVIII residues upon 2304 or 2307 binding indicating interaction region (40 sec incubation in D2O, >0.4 Da).
  • FVIII (N8, Turoctocog alfa, SEQ ID NO 2) Batch 0155-0000-0004-37A; TIL′/E′/D3/A1 III (SEQ ID NO 19; Cys1099Ser; Cys1142Ser) Batch 0129-0000-0170-6B; TIL′/E′/D3 II (SEQ ID 14; Cys1099Ser; Cys1142Ser) Batch 0129-0000-2309-1B.
  • Size-exclusion chromatography was performed on a Waters Biosuite, 4.6 ⁇ 300 mm column using a flow rate of 0.3 ml/min and a running buffer of 155 mM NaCl, 10 mM Calciumacetat, 10% Isopropanol at 25° C. The absorbance of the effluent was monitored by a UV detector at 280 nm. SEC-UV characterization were performed of FVIII, TIL′/E′/D3/A1 III, TIL′/E′/D3 II, and 1:2 complexes of FVIII—TIL′/E′/D3/A1 III and of FVIII—TIL′/E′/D3 II. Samples of FVIII 10 ⁇ M, TIL′/E′/D3/A1 III 20 ⁇ M, TIL′/E′/D3 II 20 ⁇ M, and in complex were prepared and 15 ⁇ L were loaded on to the column.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Hematology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Toxicology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Dermatology (AREA)
  • Diabetes (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicinal Preparation (AREA)
US14/396,907 2012-04-24 2013-03-13 Compounds Suitable for Treatment of Haemophilia Abandoned US20150080309A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US14/396,907 US20150080309A1 (en) 2012-04-24 2013-03-13 Compounds Suitable for Treatment of Haemophilia
US15/891,425 US20180179262A1 (en) 2012-04-24 2018-02-08 Compounds Suitable for Treatment of Haemophilia

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
EP12165301.8 2012-04-24
EP12165301 2012-04-24
US201261641434P 2012-05-02 2012-05-02
EP13150576 2013-01-09
EP13150576.0 2013-01-09
US201361752612P 2013-01-15 2013-01-15
US14/396,907 US20150080309A1 (en) 2012-04-24 2013-03-13 Compounds Suitable for Treatment of Haemophilia
PCT/EP2013/055106 WO2013083858A1 (en) 2012-04-24 2013-03-13 Compounds suitable for treatment of haemophilia

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2013/055106 A-371-Of-International WO2013083858A1 (en) 2012-04-24 2013-03-13 Compounds suitable for treatment of haemophilia

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US15/146,313 Continuation US20160318991A1 (en) 2012-04-24 2016-05-04 Compounds Suitable for Treatment of Haemophilia

Publications (1)

Publication Number Publication Date
US20150080309A1 true US20150080309A1 (en) 2015-03-19

Family

ID=48573612

Family Applications (4)

Application Number Title Priority Date Filing Date
US14/396,907 Abandoned US20150080309A1 (en) 2012-04-24 2013-03-13 Compounds Suitable for Treatment of Haemophilia
US13/916,262 Expired - Fee Related US9125890B2 (en) 2012-04-24 2013-06-12 Compounds suitable for treatment of haemophilia
US15/146,313 Abandoned US20160318991A1 (en) 2012-04-24 2016-05-04 Compounds Suitable for Treatment of Haemophilia
US15/891,425 Abandoned US20180179262A1 (en) 2012-04-24 2018-02-08 Compounds Suitable for Treatment of Haemophilia

Family Applications After (3)

Application Number Title Priority Date Filing Date
US13/916,262 Expired - Fee Related US9125890B2 (en) 2012-04-24 2013-06-12 Compounds suitable for treatment of haemophilia
US15/146,313 Abandoned US20160318991A1 (en) 2012-04-24 2016-05-04 Compounds Suitable for Treatment of Haemophilia
US15/891,425 Abandoned US20180179262A1 (en) 2012-04-24 2018-02-08 Compounds Suitable for Treatment of Haemophilia

Country Status (5)

Country Link
US (4) US20150080309A1 (zh)
EP (1) EP2841451A1 (zh)
JP (2) JP2015515482A (zh)
CN (1) CN104411716B (zh)
WO (1) WO2013083858A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150224203A1 (en) * 2014-02-12 2015-08-13 Novo Nordisk A/S Factor VIII Conjugates

Families Citing this family (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2291523B1 (en) 2008-06-24 2014-12-17 CSL Behring GmbH Factor viii, von willebrand factor or complexes thereof with prolonged in vivo half-life
AU2010290131C1 (en) 2009-08-24 2015-12-03 Amunix Operating Inc. Coagulation factor VII compositions and methods of making and using same
ES2771208T3 (es) 2012-02-15 2020-07-06 Bioverativ Therapeutics Inc Composiciones de factor VIII y métodos de preparación y uso de las mismas
DK2822577T3 (en) 2012-02-15 2019-04-01 Bioverativ Therapeutics Inc RECOMBINANT FACTOR VIII PROTEINS
DK2796145T3 (da) 2013-04-22 2018-01-29 Csl Ltd Et kovalent kompleks af von Willebrand-faktor og faktor VIII linket af en disulfidbro
SG10201913738YA (en) * 2013-06-28 2020-03-30 Bioverativ Therapeutics Inc Thrombin cleavable linker with xten and its uses thereof
WO2015023891A2 (en) 2013-08-14 2015-02-19 Biogen Idec Ma Inc. Factor viii-xten fusions and uses thereof
DK3091997T5 (da) 2014-01-10 2024-10-14 Bioverativ Therapeutics Inc Kimære faktor viii-proteiner og anvendelser deraf
KR20170010895A (ko) * 2014-06-06 2017-02-01 옥타파마 아게 인자 ⅷ 및 폰 빌레브란트 인자 펩티드를 포함하는 조제
US10626164B2 (en) 2014-07-25 2020-04-21 Csl Limited Purification of VWF
ES2788870T3 (es) * 2014-08-04 2020-10-23 Csl Ltd Formulación de factor VIII
KR20170125941A (ko) 2015-03-06 2017-11-15 체에스엘 베링 리컴비넌트 퍼실리티 아게 개선된 반감기를 갖는 변형된 폰 빌레브란트 인자
DK3298036T3 (da) 2015-05-22 2022-06-07 CSL Behring Lengnau AG Fremgangsmåder til forberedelse af modificeret von Willebrand faktor
CA2986626A1 (en) * 2015-05-22 2016-12-01 Csl Behring Recombinant Facility Ag Truncated von willebrand factor polypeptides for treating hemophilia
WO2017024060A1 (en) 2015-08-03 2017-02-09 Biogen Ma Inc. Factor ix fusion proteins and methods of making and using same
RU2018128582A (ru) 2016-01-07 2020-02-11 Цсл Беринг Ленгнау Аг Мутированный укороченный фактор фон виллебранда
EP3538133B1 (en) 2016-11-11 2021-02-17 CSL Behring Lengnau AG Truncated von willebrand factor polypeptides for treating hemophilia
CA3043397A1 (en) 2016-11-11 2018-05-17 CSL Behring Lengnau AG Truncated von willebrand factor polypeptides for extravascular administration in the treatment or prophylaxis of a blood coagulation disorder
PL3641800T3 (pl) * 2017-06-22 2024-03-18 CSL Behring Lengnau AG Modulacja immunogenności fviii przez skrócony vwf
WO2019094521A1 (en) 2017-11-07 2019-05-16 Rani Therapeutics, Llc Clotting factor preparations for delivery into tissue of the intestinal tract using a swallowable drug delivery device
NZ769677A (en) 2018-05-18 2024-07-05 Bioverativ Therapeutics Inc Methods of treating hemophilia a
US10654911B1 (en) 2019-04-02 2020-05-19 Beijing Neoletix Biological Technology Co., Ltd. Vector co-expressing truncated von Willebrand factor and factor VIII
AU2020300820A1 (en) 2019-07-04 2022-03-03 CSL Behring Lengnau AG A truncated von willebrand factor (vWF) for increasing the in vitro stability of coagulation factor VIII
WO2021094344A1 (en) 2019-11-11 2021-05-20 CSL Behring Lengnau AG Polypeptides for inducing tolerance to factor viii
WO2024124136A1 (en) * 2022-12-09 2024-06-13 Vega Therapeutics, Inc. Von willebrand disease animal models

Family Cites Families (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2651437A1 (fr) 1989-09-05 1991-03-08 Lille Transfusion Sanguine Procede de preparation de concentre du complexe facteur viii-facteur von willebrand de la coagulation sanguine a partir de plasma total.
IL113010A (en) 1994-03-31 1999-10-28 Pharmacia & Upjohn Ab Pharmaceutical formulation comprising factor viii with an activity of at least 500iu/ml and an enhancer for improved subcutaneous intramuscular or intradermal administration
SE9503380D0 (sv) 1995-09-29 1995-09-29 Pharmacia Ab Protein derivatives
US6005077A (en) 1995-11-10 1999-12-21 Immuno Aktiengesellschaft Use of von willebrand factor and pharmaceutical formulation
AT403765B (de) 1996-04-12 1998-05-25 Immuno Ag Verfahren zur herstellung einer präparation enthaltend einen hochgereinigten komplex
FR2748028B1 (fr) * 1996-04-30 1998-08-14 Lab Francais Du Fractionnement Peptides derives du facteur von willebrand et leur utilisation comme anticoagulant
US7033994B2 (en) 1999-12-24 2006-04-25 Juridical Foundation The Chemo-Sero-Therapeutic Research Institute Medicinal compositions for preventing and treating hemorrhagic diseases associating thrombopathy
EP1148063A1 (de) 2000-04-18 2001-10-24 Octapharma AG Haemostatisch aktives vWF enthaltendes Präparat und Verfahren zu seiner Herstellung
DK1578771T3 (da) 2001-10-10 2013-06-10 Novo Nordisk As Remodellering og glycokonjugering af peptider
DE10246125A1 (de) 2002-10-01 2004-04-15 Aventis Behring Gmbh Konzentrat eines Faktor VIII:C-haltigen von-Willebrand-Faktors und das dazu gehörige Verfahren
PT1558643E (pt) * 2002-11-09 2009-08-24 Immunocore Ltd Apresentação de um receptor das células t
WO2005012354A1 (en) 2003-07-31 2005-02-10 Zlb Behring Gmbh Method for extending the half-life of fviii
ES2229931B1 (es) 2003-10-03 2006-01-16 Grifols, S.A. Composicion liquida bilogicamente estable de fviii, de fvw o del complejo fviii/fvw humanos.
FR2861395B1 (fr) 2003-10-23 2006-02-17 Lab Francais Du Fractionnement Facteur viii viralement securise a faible teneur en multimeres superieurs
EP1935429A1 (en) 2006-12-22 2008-06-25 CSL Behring GmbH Synergistic therapeutic use of prothrombin complex concentrates with FVIII concentrates
CN101678119B (zh) 2006-12-27 2014-02-12 尼克塔治疗公司 具有可释放的键合的血管性血友病因子-和因子ⅷ-聚合物轭合物
EP2486936A1 (en) 2007-06-13 2012-08-15 CSL Behring GmbH A composition comprising VWF and FVIII preparations for use in treating of bleeding disorders
FR2920429B1 (fr) * 2007-08-30 2012-10-05 Lfb Biotechnologies Procede de purification du facteur viii et du facteur von willebrand
CA2704234A1 (en) 2007-11-09 2009-05-14 Baxter International Inc. Modified recombinant factor viii and von willebrand factor and methods of use
ES2298096B1 (es) 2008-01-08 2009-01-01 Grifols, S.A. Procedimiento para la obtencion de un concentrado de factor von willebrand o del complejo de factor viii/factor von willebrand y utilizacionde los mismos.
CN103497247A (zh) * 2008-02-27 2014-01-08 诺沃—诺迪斯克有限公司 缀合的因子viii分子
KR100981092B1 (ko) * 2008-02-29 2010-09-08 고려대학교 산학협력단 제8 혈액응고 인자와 폰 빌리 블란트 인자 변이체의 재조합발현 벡터 시스템
EP2291523B1 (en) 2008-06-24 2014-12-17 CSL Behring GmbH Factor viii, von willebrand factor or complexes thereof with prolonged in vivo half-life
DE102008032361A1 (de) 2008-07-10 2010-01-21 Csl Behring Gmbh Der Einsatz von Faktor VIII und vWF bzw. vWF-enthaltenden Konzentraten zur Therapie der durch Thrombocyten-Inhibitoren induzierte Koagulopathie
CN102202684A (zh) 2008-11-03 2011-09-28 拜耳医药保健有限公司 用于治疗血友病的方法
CN102648212B (zh) * 2009-11-13 2014-12-03 基立福疗法公司 包含冯维勒布兰德因子(vWF)的制剂及其相关制备方法、试剂盒和用途
WO2011101284A1 (en) * 2010-02-16 2011-08-25 Novo Nordisk A/S Factor viii fusion protein
AU2011303916A1 (en) 2010-09-15 2013-03-21 Novo Nordisk A/S Factor VIII variants having a decreased cellular uptake

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
The sequence listing for WO2011060242, accessed on 12/2/2015. *
UniProt Protein Database, Factor VIII, Protein Accession P00451, accessed on 12/2/2015. *
UniProt Protein Database, Von Willebrand Factor, Protein Accession P04275, published online January 2011. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150224203A1 (en) * 2014-02-12 2015-08-13 Novo Nordisk A/S Factor VIII Conjugates

Also Published As

Publication number Publication date
US20160318991A1 (en) 2016-11-03
US9125890B2 (en) 2015-09-08
CN104411716B (zh) 2018-09-07
EP2841451A1 (en) 2015-03-04
US20180179262A1 (en) 2018-06-28
JP2018104459A (ja) 2018-07-05
WO2013083858A1 (en) 2013-06-13
WO2013083858A9 (en) 2013-08-01
CN104411716A (zh) 2015-03-11
JP2015515482A (ja) 2015-05-28
US20140018297A1 (en) 2014-01-16

Similar Documents

Publication Publication Date Title
US20180179262A1 (en) Compounds Suitable for Treatment of Haemophilia
US20160120954A1 (en) Pharmaceutical Composition Suitable for Treatment of Haemophilia
US20160207977A1 (en) Compounds Suitable for Treatment of Haemophilia
JP6527918B2 (ja) 第ｖｉｉｉ因子組成物、ならびに組成物の作製方法および用途
US10906960B2 (en) Factor VIII variants having a decreased cellular uptake
Stennicke et al. A novel B-domain O-glycoPEGylated FVIII (N8-GP) demonstrates full efficacy and prolonged effect in hemophilic mice models
US20220213200A1 (en) Anti-gpiib/iiia antibodies or uses thereof
CN114391024A (zh) 具有增加的半衰期的因子viii蛋白
DK2616486T3 (en) FACTOR VIII VARIATIONS WITH DISABLED CELLULAR ADMISSION
AU2013204897A1 (en) Factor VIII variants having a decreased cellular uptake

Legal Events

Date Code Title Description
AS Assignment

Owner name: NOVO NORDISK A/S, DENMARK

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:BOLT, GERT;KARPF, DITTE M.;RODE, FREDERIK;AND OTHERS;SIGNING DATES FROM 20150210 TO 20150326;REEL/FRAME:035406/0927

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION