COAGULATION FACTOR IX COMPOSITIONS AND METHODS OF MAKING AND USING SAME

• US 20120263703A1
• Filed: 08/02/2010
• Published: 10/18/2012
• Est. Priority Date: 08/24/2009
• Status: Active Grant

• Current Assignee: Amunix Pharmaceuticals, Inc. (Sanofi )
• Original Assignee: Amunix Operating Inc.
• Inventors: Stemmer, Willem P., To, Wayne, Silverman, Joshua, Wang, Chia-wei, Schellenberger, Volker, Geething, Nathan C., Spink, Benjamin
• Granted Patent: US 9,062,299 B2
• Time in Patent Office: Days
• US Class Current: 424/94.64
• 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 7/02 Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: A61P 7/04 Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: C07K 14/001 by chemical synthesis
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/31 fusions, other than Fc, for prolonged plasma life, e.g. albumin
• CPC Class Codes: C07K 2319/50 containing protease site
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 9/6437 Coagulation factor VIIa (3.4.21.21)
• CPC Class Codes: C12N 9/644 Coagulation factor IXa (3.4.21.22)
• CPC Class Codes: C12Y 304/21022 Coagulation factor IXa (3.4.21.22)

Abstract

The present invention relates to compositions comprising factor DC coagulation factors linked to extended recombinant polypeptide (XTEN), isolated nucleic acids encoding the compositions and vectors and host cells containing the same, and methods of making and using such compositions in treatment of coagulation factor-related diseases, disorders, and conditions.

Claims

1. An isolated factor IX polypeptide comprising an extended recombinant polypeptide (XTEN), said XTEN comprising at least 36 amino acid residues, wherein said factor DC polypeptide exhibits a terminal half-life that is longer than about 12 hours when administered to a subject.

2. The isolated factor IX polypeptide of claim 1, wherein the factor IX polypeptide exhibits at least 90% sequence identity compared to a sequence selected from Table 1 when optimally aligned.

3. The isolated factor DC polypeptide of claim 1 or 2, wherein said factor IX is linked at its C-terminus to the XTEN.

4. The isolated factor IX polypeptide of claim 1 that is linked to the XTEN via a cleavage sequence that is cleavable by a mammalian protease selected from the group consisting of factor XIa, factor XIIa, kallikrein, factor VIIa, factor IXa, factor Xa, factor IIa (thrombin), Elastase-2, MMP-12, MMP13, MMP-17 and MMP-20.

5. The isolated factor IX polypeptide of claim 4, wherein cleavage at the cleavage sequence by the mammalian protease releases said XTEN from said factor IX polypeptide.

6. The isolated factor IX polypeptide of claim 1, wherein said XTEN is characterized in that:

(a) the cumulative total of XTEN amino acid residues is greater than 200 to about 3000 amino acid residues;

(b) the sum of asparagine and glutamine residues is less than 10% of the total amino acid sequence of the XTEN;

(c) the sum of methionine and tryptophan residues is less than 2% of the total amino acid sequence of the XTEN;

(d) the XTEN sequence has a subsequence score less than 10;

(e) the XTEN sequence has greater than 90% random coil formation as determined by GOR algorithm; and

(f) the XTEN sequence has less than 2% alpha helices and 2% beta-sheets as determined by Chou-Fasman algorithm.

7. The isolated factor IX polypeptide of claim 1 exhibiting an apparent molecular weight factor of at least about 4.

8. The isolated factor IX polypeptide of claim 1, wherein said XTEN exhibits at least 90% sequence identity to a comparable length of an amino acid sequence selected from Table 4, Table 9, Table 10, Table 11, Table 12, or Table 13.

9. The isolated factor IX polypeptide of claim 1 that is configured according to formula VII:

(Gla)-(XTEN)_u-(EGF1)-(XTEN)_v-(EGF2)-(AP1)-(XTEN)_w-(AP2)-(XTEN)_x-(Pro)-(S)_y-(XTEN)_z 

 

VII

wherein independently for each occurrence,

(a) Gla is a Gla domain of factor IX;

(b) EGF1 is an EGF1 domain of factor IX;

(c) EGF2 is an EGF2 domain of factor IXI;

(d) AP1 is a portion of an activator peptide domain of factor IX;

(e) AP2 is a portion of an activator peptide domain of factor IX that includes at least a first cleavage sequence;

(f) PRO is a protease domain of factor IX;

(g) S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence;

(h) XTEN is an extended recombinant polypeptide that exhibits at least 90% sequence identity to a comparable length of an amino acid sequence selected from Table 4, Table 9, Table 10, Table 11, Table 12, or Table 13,

(i) u is either 0 or 1;

(j) v is either 0 or 1;

(k) x is either 0 or 1;

(l) y is either 0 or 1; and

(m) z is either 0 or 1, with the proviso that u+v+x+y+z≧

1.

10. The isolated factor DC polypeptide of claim 9, wherein y=1 and S comprises a cleavage sequence that is cleavable by a mammalian protease selected from the group consisting of FIXa, FXIIa, kallikrein, FVIIa, FIXa, FXa, FIIa (thrombin), Elastase-2, MMP-12, MMP13, MMP-17 and MMP-20.

11. The isolated factor IX polypeptide of claim 1 comprising more than one XTEN.

12. The isolated factor IX polypeptide of claim 1, wherein said XTEN is incorporated between any two adjacent domains contained in said factor IX, wherein said two adjacent domains are selected from the group consisting of Gla, EGF1, EGF2, AP, and peptidase S1 (Pro).

13. The isolated factor IX polypeptide of claim 1, characterized in that:

(i) it has a longer terminal half-life when administered to a mammal compared to the corresponding factor IX that lacks the XTEN when administered to a mammal at a comparable molar dose;

(ii) when a smaller molar amount of the factor IX polypeptide is administered to a mammal in comparison to the corresponding factor IX that lacks the XTEN administered to a mammal under an otherwise equivalent dose regimen, the factor IX polypeptide achieves a comparable area under the curve (AUC) as the corresponding factor IX that lacks the XTEN;

(iii) when a smaller molar amount of the factor IX polypeptide is administered to a mammal in comparison to the corresponding factor IX that lacks the XTEN administered to a mammal under an otherwise equivalent dose regimen, the factor IX polypeptide achieves a comparable therapeutic effect as the corresponding factor IX that lacks the XTEN;

(iv) when the factor IX polypeptide is administered to a mammal less frequently in comparison to the corresponding factor IX that lacks the XTEN administered to a mammal using an otherwise equivalent molar amount, the factor IX polypeptide achieves a comparable area under the curve (AUC) as the corresponding factor IX that lacks the XTEN;

(v) when the factor IX polypeptide is administered to a mammal less frequently in comparison to the corresponding factor IX that lacks the XTEN administered to a mammal using an otherwise equivalent molar amount, the factor IX polypeptide achieves a comparable therapeutic effect as the corresponding factor IX that lacks the XTEN;

(vi) when an accumulatively smaller molar amount of the factor IX polypeptide is administered to a mammal in comparison to the corresponding factor IX that lacks the XTEN administered to a mammal under an otherwise equivalent dose period, the factor IX polypeptide achieves comparable area under the curve (AUC) as the corresponding factor IX that lacks the XTEN;

or

(vii) when an accumulatively smaller molar amount of the factor IX polypeptide is administered to a mammal in comparison to the corresponding factor IX that lacks the XTEN administered to a mammal under an otherwise equivalent dose period, the factor IX polypeptide achieves comparable therapeutic effect as the corresponding factor IX that lacks the XTEN.

14. A method of treating coagulopathy in a subject, comprising administering to said subject a composition comprising a therapeutically effective amount of the factor IX polypeptide of any of claim 1.

15. The method of claim 14, wherein said coagulopathy is hemophilia B.

16. A method of treating a bleeding episode in a subject comprising administering to said subject a composition comprising a therapeutically effective amount of the factor IX polypeptide of claim 1.

17. A method of treating a subject deficient in a clotting protein, comprising:

administering to said subject a composition comprising a therapeutically effective amount of the factor IX of any of claims 1-13.

18. The method of claim 17, wherein the clotting protein substitutes wild-type factor VII, factor IX, or factor XI.

19. The isolated factor IX polypeptide of claim 1, wherein said XTEN comprises at least 100 amino acid residues.

20. An isolated factor IX polypeptide comprising an extended recombinant polypeptide (XTEN), wherein the XTEN in characterized in that:

(a) the XTEN comprises at least 36 amino acid residues;

(b) the sum of glycine (G), aspartate (D), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues contained in the URP, constitutes at least 80% of the total amino acids of the XTEN;

(c) the XTEN sequence is substantially non-repetitive such that (i) the XTEN sequence contains no three contiguous amino acids that are identical unless the amino acids are serine, or (ii) at least about 80% of the XTEN sequence consists of non-overlapping sequence motifs, each of the sequence motifs comprising about 9 to about 14 amino acid residues, wherein any two contiguous amino acid residues does not occur more than twice in each of the sequence motifs;

(d) the XTEN sequence lacks a predicted T-cell epitope when analyzed by TEPITOPE algorithm, wherein the TEPITOPE algorithm prediction for epitopes within the XTEN sequence is based on a score of −

9;

(e) the XTEN sequence has greater than 90% random coil formation as determined by GOR algorithm; and

(f) the XTEN sequence has less than 2% alpha helices and 2% beta-sheets as determined by Chou-Fasman algorithm.