THROMBIN-CLEAVABLE XTEN-CONTAINING LINKER AND ITS APPLICATIONS

Abstract

The present invention provides a chimeric molecule comprising a VWF protein linked to a heterologous fragment via a VWF linker. The invention provides an effective VWF linker that can be cleaved in the presence of thrombin. The chimeric molecule may further comprise a polypeptide chain comprising a FVIII protein and a second heterologous fragment, wherein the chain comprising the VWF protein and the chain comprising the FVIII protein are associated with each other. The invention also includes nucleotides, vectors, host cells, and methods for using the chimeric proteins.

Claims

1. A chimeric molecule comprising a first polypeptide chain and a second polypeptide chain, (i) the first polypeptide chain comprises a von Willebrand factor (VWF) protein, a VWF linker, and a first Fc region; wherein the VWF linker contains the a2 region of factor VIII (FVIII), which is capable of being cleaved by thrombin; wherein the VWF protein contains a D' domain and a D3 domain, and wherein an amino acid other than cysteine ​​replaces a residue corresponding to residue 1099, residue 1142, or both residues 1099 and 1142 corresponding to full-length VWF (SEQ ID NO: 2) in the VWF protein; (ii) the second polypeptide chain comprises a factor VIII (FVIII) protein linked to a second Fc region; wherein the FVIII protein is a FVIII protein with a deletion of the B domain and wherein the first polypeptide comprises a VWF protein, a VWF linker and a first Fc region in this particular order from N-terminus to C-terminus; wherein the second polypeptide comprises a FVIII protein and a second Fc region in this particular order from N-terminus to C-terminus; wherein the VWF protein binds to the FVIII protein; and wherein the first and second Fc regions are linked by a covalent bond; and wherein the first Fc region and the second Fc region are linked to each other via one or more disulfide bonds.

2. The chimeric molecule according to claim 1, characterized in that the VWF linker contains an amino acid sequence identical to the region from Glu720 to Arg740, corresponding to full-length mature FVIII (SEQ ID NO: 16).

3. A chimeric molecule according to any one of claims 1-2, characterized in that the VWF linker has a length of at least 30 amino acids.

4. A chimeric molecule according to any one of claims 1 to 3, characterized in that the D' domain of the VWF protein contains an amino acid sequence that is at least 100% identical to amino acids 764-866 of SEQ ID NO:

2.

5. A chimeric molecule according to any one of claims 1 to 4, characterized in that the D3 domain of the VWF protein contains an amino acid sequence that is at least 99% identical to amino acids 867-1240 of SEQ ID NO:

2.

6. A chimeric molecule according to any one of claims 1 to 5, characterized in that the VWF protein contains a D' domain and a D3 domain, which contains an amino acid substitution of cysteine ​​for alanine at the position of amino acid residues 1099 and 1142, corresponding to full-length VWF (SEQ ID NO: 2).

7. A chimeric molecule according to any one of claims 1 to 6, characterized in that the VWF protein consists of a D' domain and a D3 domain, which contains an amino acid substitution of cysteine ​​for alanine at the position of amino acid residues 1099 and 1142, corresponding to full-length VWF (SEQ ID NO: 2).

8. A chimeric molecule comprising a first polypeptide chain and a second polypeptide chain, (i) the first polypeptide chain comprises, from its N-terminus to its C-terminus, a von Willebrand factor (VWF) protein, a VWF linker, and a first Fc region; (ii) the second polypeptide chain comprises a factor VIII (FVIII) protein and a second Fc region that is linked to the C-terminus of the FVIII protein; wherein the VWF protein comprises a D' domain and a D3 domain that comprises an amino acid substitution of cysteine ​​to alanine at amino acid residue positions 1099 and 1142, corresponding to full-length VWF (SEQ ID NO: 2); wherein the VWF protein binds to the FVIII protein; wherein the VWF linker comprises an a2 region of FVIII that comprises an amino acid sequence identical to the region from Glu720 to Arg740 corresponding to full-length mature FVIII (SEQ ID NO: 16), and wherein the a2 region is capable of being cleaved by thrombin; wherein the VWF linker has a length of at least 30 amino acids, and wherein the first polypeptide chain and the second polypeptide chain are linked to each other via one or more disulfide bonds between the first Fc region and the second Fc region.

9. The chimeric molecule according to claim 8, characterized in that the VWF linker contains an amino acid sequence that is at least 90% identical to SEQ ID NO:

4.

10. The chimeric molecule according to claim 9, characterized in that the VWF linker contains an amino acid sequence that is at least 95% identical to SEQ ID NO:

4.

11. The chimeric molecule of any one of claims 1 to 10, wherein the first polypeptide chain further comprises a first XTEN sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 and 78, so that the first polypeptide comprises a VWF protein, a first XTEN sequence, a VWF linker and a first Fc region in the specified order from the N-terminus to the C-terminus.

12. The chimeric molecule of any one of claims 1 to 11, wherein the second polypeptide chain further comprises a second XTEN sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 and 78, so that the second XTEN sequence is inserted into the FVIII protein.

13. A pharmaceutical composition comprising a chimeric molecule according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier.

14. Use of a composition comprising a chimeric molecule according to any one of claims 1 to 12 or a pharmaceutical composition according to claim 13 for the intravenous treatment of a blood clotting disorder in a subject in need thereof.

15. Use according to paragraph 14, where the blood clotting disorder is hemophilia A.

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