CHIMERA PROTEIN COMPRISING FVIII AND VWF FACTORS, AND USE THEREOF

• US 20190330311A1
• Filed: 06/23/2017
• Published: 10/31/2019
• Est. Priority Date: 06/24/2016
• Status: Active Grant

• Current Assignee: Mogam Institute For Biomedical Research
• Original Assignee: Mogam Institute For Biomedical Research
• Inventors: OH, Injae, LEE, Seung-Hoon, JO, Eui-Cheol, OH, Mee Sook, RYU, Jae Hwan, KIM, Yong Jae, KIM, So Ra, PARK, Jin-hyun
• Granted Patent: US 11,046,749 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/04 Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: C07K 14/755 Factors VIII , e.g. factor VIII C (AHF), factor VIII Ag (VWF)
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C07K 2319/50 containing protease site
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 9/64 derived from animal tissue

Abstract

A chimera protein according to the present application has a significantly increased in vivo half-life when administered because of a vWF domain coupled to FVIII, such that when used as a hemophilia A therapeutic agent, convenience for patients can be increased and medical expenses can be reduced.

Claims

1. A chimeric protein comprising:

a human Factor VIII (FVIII) comprising a light chain and a heavy chain, the human FVIII comprising a B domain which is totally deleted in its entire length or partially deleted; and

at least one vWF D′

D3 domain,

wherein the light chain and heavy chain are expressed as one polypeptide or two polypeptides, wherein;

if the light chain and heavy chain are expressed as one polypeptide, the chimeric protein comprises a linker which connects the FVIII and the vWF D′

D3 domain and has an enzyme cleavage site located therebetween, wherein the FVIII and the vWF D′

D3 domain are located in the N-terminal direction and the C-terminal direction or in the C-terminal direction and the N-terminal direction, respectively, based on the linker; and

if the light chain and heavy chain are expressed as two polypeptides, the vWF D′

D3 domain is linked to the C-terminal of the B domain contained in the heavy chain.

2. The chimeric protein of claim 1, wherein the partially deleted B domain has at least five amino acid deletions in each of the N-terminal direction and/or C-terminal direction based on the 1648 and 1649 residue positions of SEQ ID NO:

01, the deletion including the residues;

or

the B domain is partially deleted such that it includes 4 to 6 glycation sites.

3. The chimeric protein of claim 1, wherein the partially deleted B domain is represented by a sequence of amino acid residues 741 to 902 and 1654 to 1689, or amino acid residues 741 to 936, based on the sequence of SEQ ID NO:

01.

4. The chimeric protein of claim 1, which comprises two vWF D′

D3 domains, wherein the two vWF D′

D3 domains are linked by a linker with or without an enzyme cleavage site and/or form a dimer by a covalent bond there.

5. The chimeric protein of claim 1, wherein the chimeric protein further comprises at least one Fc, wherein the Fc is linked to the vWF D′

D3 domain and/or the FVIII by a linker with or without an enzyme cleavage site.

6. The chimeric protein of claim 5, wherein the chimeric protein comprises two Fcs, wherein the two Fcs are linked by a linker with or without an enzyme cleavage site and/or form a dimer by a covalent bond therebetween.

7. The chimeric protein of claim 1, wherein:

the enzyme cleavage site is a sequence which can be cleaved by thrombin, FVIIa, FXa or FXIa; and

the other sequence in the linker having the enzyme cleavage site is represented by the amino acid sequence of [G₄S]_n, wherein n is 0, or an integer between 1 and 100.

8. The chimeric protein of claim 7, wherein n is 4, 6, 8, 10, 16, 21, 22 or 58.

9. The chimeric protein of claim 7, the amino acid sequence which can be cleaved by the thrombin is DFLAEGGGVR (SEQ ID NO:

36), TTKIKPR (SEQ ID NO;

     37), or LVPRGS (SEQ ID NO;

     38);

the amino acid sequence which can be cleaved by the FVIIa is ASKPQGRIVGG (SEQ ID NO;

39);

the amino acid sequence which can be cleaved by FXa is IDGR (SEQ ID NO;

     40) or IEGR (SEQ ID NO;

     41);

the sequence which can be cleaved by FXIa is SKLTRAETVF (SEQ ID NO;

     42).

10. The chimeric protein of claim 1, wherein the two polypeptides are first and second polypeptides, wherein:

the first polypeptide comprises the heavy chain and all or a portion of the B domain of the FVIII, and the second polypeptide comprises the light chain or a portion of the B domain of the FVIII; and

the vWF D′

D3 domain is located in the N-terminal or C-terminal direction of the first polypeptide, or in the N-terminal or C-terminal direction of the second polypeptide; and

optionally, the chimeric protein further comprises at least one Fc, wherein the Fc is linked to the vWF D′

D3 domain and/or the second polypeptide.

11. The chimeric protein of claim 10, wherein the first polypeptide comprises the heavy chain and a portion of the B domain of the FVIII, and the portion of the B domain includes an amino acid sequence of amino acid residues 741 to 936 based on SEQ ID NO:

01.

12. The chimeric protein of claim 10, wherein the chimeric protein comprises two or more vWF D′

D3 domains, wherein the two or more vWF D′

D3 domains are linked by a linker with or without an enzyme cleavage site, and/or the vWF D′

D3 domains form a complex by a covalent bond between the domains.

13. The chimeric protein of claim 10, wherein the Fc or the vWF D′

D3 domain is linked by a linker with or without an enzyme cleavage site.

14. The chimeric protein of claim 1, wherein the chimeric protein which is expressed as one polypeptide has the amino acid sequence represented by SEQ ID NO:

6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, or 25, or an amino acid sequence that has at least 90% homology to said amino acid sequence.

15. The chimeric protein of claim 10, wherein the chimeric protein which is expressed as two polypeptides has the amino acid sequences represented by SEQ ID NOs:

4 and 5 (heavy chain and light chain, respectively), SEQ ID NOs;

23 and 24 (heavy chain and light chain, respectively), SEQ ID NOs;

26 and 27 (heavy chain and light chain, respectively), SEQ ID NOs;

28 and 29 (heavy chain and light chain, respectively), SEQ ID NOs;

30 and 31 (heavy chain and light chain, respectively), SEQ ID NOs;

32, and 33 (heavy chain and light chain, respectively), or SEQ ID NO;

34 and 35 (heavy chain and light chain, respectively), or the amino acid sequences that have at least 90% homology to the amino acid sequences.

16. The chimeric protein of claim 1, wherein the FVIII is conjugated to a hydrophilic polymer at one or more amino acid residues of the A and/or B domain fragments, and the conjugation position is at least one selected from the group consisting of amino acid residues 754, 781, 782, 788, 789, 825, and 897 based on the sequence of SEQ ID NO:

01 in the B domain, and/or at least one selected from the group consisting of amino acid residues 491, 495, 498 and 1806 based on the sequence of SEQ ID NO;

01 in the A domain, and the one or more residues at the conjugation position are substituted with cysteine for conjugation with the hydrophilic polymer.

17. The chimeric protein of claim 16, wherein the hydrophilic polymer is polyethylene glycol (PEG), polyethylene oxide, dextran, and the PEG is linked at the conjugation position via acryloyl, sulfone or maleimide.

18. The chimeric protein of claim 16, wherein the FVIII is conjugated to a hydrophilic polymer at one or more amino acid residues of the B domain fragment, and the conjugation position is amino acid residue 782.

19. The chimeric protein of claim 17, wherein the PEG has an average molecular weight of 20 kDa or more.

20. The chimeric protein of claim 19, wherein the PEG has an average molecular weight of 40 kDa or 60 kDa.

21. The chimeric protein of claim 1, wherein the protein has at least a half-life which is extended by at least 2-fold as compared to a wild-type FVIII protein.

22. A nucleic acid molecule encoding the chimeric protein of claim 14.

23. A nucleic acid molecule encoding the chimeric protein of claim 15.

24. The nucleic acid molecule of claim 22, which is represented by SEQ ID NOs:

48 to 64, 66, or 70.

25. The nucleic acid molecule of claim 23, wherein the nucleic acid molecule is represented by SEQ ID NOs:

46, 47, 68, 69, or 71 to 80.

26. A vector comprising the nucleic acid molecule of claim 22.

27. A cell comprising the vector of claim 26.

28. A pharmaceutical composition for the treatment of type A hemophilia comprising the chimeric protein of claim 14, a nucleic acid molecule encoding the chimeric protein of claim 14, a vector comprising the nucleic acid encoding the chimeric protein of claim 14, or a cell carrying the vector comprising the nucleic acid encoding the chimeric protein of claim 14, and a pharmacologically acceptable carrier.

29. The pharmaceutical composition of claim 28, wherein the composition has a half-life such that the composition can be administered once a day or longer, preferably once a week, or especially once a month.

30. A method for treating type A hemophilia comprising administering a therapeutically effective amount of the composition of claim 28 to a subject in need of hemophilia treatment.

31. A method of blood coagulation, comprising administering an effective amount of the composition of claim 28 to a subject in need of hemophilia treatment.

32. (canceled)