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CA-3008448-C - MUTATED VON WILLEBRAND FACTOR

CA3008448CCA 3008448 CCA3008448 CCA 3008448CCA-3008448-C

Abstract

The present invention provides a modified polypeptide which binds Factor VIII. The modified polypeptide comprises a sequence as shown in SEQ ID NO:3 in which the sequence comprises at least one modification at a position selected from the group consisting of L18, V42, K149, N248, S279, V320, T325, Q395 and K418 such that the modified polypeptide binds to Factor VIII with an off rate lower than a reference polypeptide comprising an unmodified SEQ ID NO:3.

Inventors

  • Arna ANDREWS
  • Con Panousis
  • Kerstin EMMRICH
  • Michael Wilson
  • Steve DOWER
  • Matthew Hardy
  • Dallas HARTMAN

Assignees

  • CSL BEHRING RECOMBINANT FACILITY AG

Dates

Publication Date
20260505
Application Date
20170106
Priority Date
20160107

Claims (14)

  1. 34 CLAIMS 1. A modified Von Willebrand Factor (VWF) polypeptide which binds to Factor VIII, wherein the modified VWF polypeptide comprises an amino acid sequence as set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:17.
  2. 2. The modified VWF polypeptide as claimed in claim 1 in which the modified VWF polypeptide binds to Factor VIII with an off rate at least 5 fold lower than a reference polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:3.
  3. 3. The modified VWF polypeptide as claimed in claim 1 in which the modified VWF polypeptide binds to Factor VIII with an off rate at least 10 fold lower than a reference polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:3.
  4. 4. The modified VWF polypeptide as claimed in claim 1 in which the modified VWF polypeptide binds to Factor VIII with a KD at least 5 fold lower than a reference polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:3.
  5. 5. The modified VWF polypeptide as claimed in claim 4 in which the modified VWF polypeptide binds to Factor VIII with an off rate at least 10 fold lower than a reference polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:3.
  6. 6. The modified VWF polypeptide as claimed in any one of claims 1 to 5 in which the modified VWF polypeptide further comprises a half-life extending moiety wherein the half-life extending moiety is: i) a heterologous amino acid sequence comprising or consisting of a polypeptide selected from the group consisting of immunoglobulin constant regions and portions thereof, transferrin and fragments thereof, the C-terminal peptide of human chorionic gonadotropin, solvated random chains with large hydrodynamic volume known as XTEN, homo-amino acid repeats (HAP), praline-alanine-serine repeats (PAS), albumin, afamin, alpha-fetoprotein, Vitamin D binding protein, and combinations thereof; or Date re~ue/Date received 2024-06-10 ii) conjugated to the modified polypeptide and is selected from the group consisting of hydroxyethyl starch (HES), polyethylene glycol (PEG), polysialic acids (PSAs), elastinlike polypeptides, heparosan polymers, hyaluronic acid and albumin binding ligands, and combinations thereof.
  7. 7. The modified VWF polypeptide as claimed in claim 6, in which the heterologous amino acid sequence comprises albumin.
  8. 8. The modified VWF polypeptide as claimed in claim 7 in which the N-terminus of the albumin is fused to the C-terminus of the modified polypeptide sequence either directly or via a spacer.
  9. 9. The modified VWF polypeptide as claimed in claim 8 in which 1 to 5 amino acids at the natural C-terminus of the modified VWF polypeptide have been deleted.
  10. 10. A complex comprising a Factor VIII molecule and the modified VWF polypeptide of any one of claims 1 to 9.
  11. 11. The modified polypeptide of any one of claims 1 to 9 or the complex of claim 10 for use in the treatment or prophylaxis of a bleeding disorder; wherein the bleeding disorder is van Willebrand's disease (VWD) or hemophilia A.
  12. 12. A pharmaceutical composition comprising the modified VWF polypeptide of any one of claims 1 to 9 or the complex of claim 10, and a pharmaceutically acceptable carrier.
  13. 13. The modified VWF polypeptide of any one of claims 1 to 9 or of the complex of claim 10 for use in the treatment of a bleeding disorder; wherein the bleeding disorder is van Willebrand's disease (VWD) or hemophilia A.
  14. 14. Use of the modified VWF polypeptide of any one of claims 1 to 9 or of the complex of claim 10 for the treatment of a bleeding disorder; wherein the bleeding disorder is van Willebrand's disease (VWD) or hemophilia A. Date re~ue/Date received 2024-06-10 36 15. Use of the modified VWF polypeptide of any one of claims 1 to 9 or of the complex of claim 10 in the preparation of a medicament for the treatment of a bleeding disorder; wherein the bleeding disorder is von Willebrand's disease (VWD) or hemophilia A. 16. A polynucleotide encoding the modified VWF polypeptide of any one of claims 1 to 9. 17. A plasmid or vector comprising the polynucleotide of claim 16. 18. The plasmid or vector of claim 17, said plasmid or vector being an expression vector. 19. A host cell comprising the polynucleotide of claim 16 or the plasmid or vector of claim 17 or 18. 20. A method of producing a polypeptide comprising a modified VWF, comprising: (i) culturing the host cells of claim 19 under conditions such that the polypeptide comprising a modified VWF is expressed; and (ii) recovering the polypeptide comprising a modified VWF from the host cells or from the culture medium. 21. A method of increasing the half-life of Factor VIII, the method comprising mixing the Factor VIII with the modified VWF polypeptide as claimed in any one of claims 1 to 9. Date re~ue/Date received 2024-06-10

Description

1 MUTATED VON WILLEBRAND FACTOR .FILING OATA [0001] This application is associated with and claims priority from Australian patent application no. 2016900033 filed on 7 January 2016 . FIELD OF THE lNVENTI()N [00021 The present invention relates to polypeptides, in particular modified von Wiilebrand Factor which exhibits improved binding affinity to Fact.qr VIII. The. invention .fu1ther relates to a complex comprising the polypeptide and FVDI, to a polynucleotid~ encoding the polypeptide of the invention .and a metllqd of producing the polypeptjde. Furthermore, the.invention .concerns the therapeutic or prophylactic use of the polypeptide or complex of the invention for treating bleeding disorders, BACJ<GROUNP OF ·THE. INVENTION [0003] There are various bleeding disorders caused by deficiencies of blood coagulation factors. The mostcommon disorders are hemophilia A and B, resulting from.deficiencies of blood coagulation JactorVIU and IX, respectively. Another known bleeding diso:tder is vorl "Willebrand's disea$e. [0004] In plasma FVITI exists predominantly in a noncovalent complex with VWF and acts as a cofactor for activated factor IX in the membraae bound activated factor X g.ene:rating cottt.plex. [iJ005] Severdl attempts have been made to prolong the half-life of non-activated FVIII either by reducing its interaction with cellular receptors (WO 03/093313A2, WO 02/060951A2), by covalently attaching polymers to FVIIl" (WO 94/15625, WO 97/11957 and US 4970300), by encapsulation of FVUI {WO 99/55306). by introduction of novel metal binding sites (WO 97/03193), by c.ovalently attachio~I the A2 domain to th!.'i A.3 <,lomai.n. either by peplidic{WO 97/40145 andWO03/087355) ordi!mlfide linkage (WO 02/103024A2) or by covalently attaching the Al domain.to the A2 domain (WO2006/108590). Date Re9ue/Date Received 2023-05-16 2 [0006] Another approach to enhance the functional half-life of FVTII or VWF is by PEGylation of FVTII (WO 2007/126808, WO 2006/053299, WO 2004/075923). PEGylation ofVWF (WO 2006/071801) has also been attempted in an effort to indirectly enhance the half-life of FVTII present in plasma. Also fusion proteins of FVTII have been described (WO 2004/101740, WO2008/077616 and WO 2009/156137). [0007] VWF, which is missing, functionally defective or only available in reduced quantity in different forms of von Willebrand disease (VWD), is a multimeric adhesive glycoprotein present in plasma, which has multiple physiological functions. During primary hemostasis VWF acts as a mediator between specific receptors on the platelet surf ace and components of the extracellular matrix such as collagen. Moreover, VWF serves as a carrier and stabilizing protein for procoagulant FVIII. VWF is synthesized in endothelial cells and megakaryocytes as a 2813 amino acid precursor molecule. The amino acid sequence and the cDNA sequence of wild-type VWF are disclosed in Collins et al. 1987, Proc Natl. Acad. Sci. USA 84:4393-4397. The precursor polypeptide, pre-pro-VWF, consists of a 22-residue signal peptide, a 741- residue pro-peptide and the 2050-residue polypeptide found in plasma (Fischer et al., FEBS Lett. 351: 345-348, 1994). After cleavage of the signal peptide in the endoplasmic reticulum a C-terminal disulfide bridge is formed between two monomers of VWF. During further transport through the secretory pathway 12 N-linked and 10 O-linked carbohydrate side chains are added. Importantly, VWF dimers are multimerized via Ntenninal disulfide bridges and the propeptide of 741 amino acids is cleaved off by the enzyme PACE/furin in the late Golgi apparatus. The propeptide as well as the high-molecular-weight multimers of VWF (VWF-HMWM) are stored in the Weibel-Pallade bodies of endothelial cells or in the a-Granules of platelets. [0008] Once secreted into plasma the protease ADAMTS 13 cleaves VWF within the Al domain of VWF. Plasma VWF consists of a range of multimers ranging from single dimers of 500 kDa to multimers consisting of more than 20 dimers of a molecular weight of over 10,000 kDa. Typically VWF high molecular weight multimcrs (VWF-HMWM) have the strongest hcmostatic activity, which can be measured in ristocctin cofactor activity (VWF:RCo). The higher the ratio of VWF:RCo/VWF antigen, the higher the relative amount of high molecular weight multimcrs. 3 [0009] Defects in VWF are causal to von Willebrand disease (VWD), which is characterized by a more or less pronounced bleeding phenotype. VWD type 3 is the most severe form in which VWF is completely missing, VWD type 1 relates to a quantitative loss of VWF and its phenotype can be very mild. VWD type 2 relates to qualitative defects of VWF and can be as severe as VWD type 3. VWD type 2 has many sub forms some of them being associated with the loss or the decrease of high molecular weight multimers. Von VWD type 2a is characterized by a loss of both intermediate and large multimers. VWD type 2B is characterized by a loss of highest-molecular-weight multimers. [0010