Factor X analog with an improved ability to be activated
This invention describes a factor Xa analog which has a substitution of a minimum of one of the amino acid between Glu226 and Arg234 and possibly Ile235, relative to the amino acid numbering according to FIG. 1 , a preparation containing the activated form of the factor X analog, and a method for the production of these molecules.
1. A Factor X analog which contains one or more modifications in SEQ ID NO:2 selected from the group consisting of:
a) Ile235 is Val or Ala;
b) Thr233 is Ser or Asn;
c) Leu232 is Phe, Arg or Ile;
d) Asn231 is Asp, Lys, Thr, or Glu;
e) Asn230 is Ser, Lys, Met, Thr, or Asp;
f) Asp229 is Phe, Thr, Ser, Pro, Leu, or Ile;
g) Gly228 is Ser, Gln, Ile, Thr, Asn, or Pro; and
h) Arg227 is Gln, Ser, His, Tyr, or Glu.
2. The Factor X analog of claim 1 , wherein the amino acid sequence of residues 227-233 is Gln227-Ser228-Phe229-Asn230-Asp231-Phe232-Thr233 (SEQ ID NO:17).
3. The Factor X analog of claim 1 , wherein the amino acid sequence of residues 227-233 is Ser227-Gln228-Thr229-Ser230-Lys231-Leu232-Thr233 (SEQ ID NO:18).
4. The Factor X analog of claim 1 , wherein the modification forms a processing site for Factor XIa or a derivative thereof.
5. A Factor X analog which
(i) contains one or more modifications in SEQ ID NO:2 selected from the group consisting of:
a) Ile235 is Val or Ala;
b) Thr233 is Ser or Asn;
c) Leu232 is Phe, Arg or Ile;
d) Asn231 is Asp, Lys, Thr, or Glu;
e) Asn230 is Ser, Lys, Met, Thr, or Asp;
f) Asp229 is Phe, Thr, Ser, Pro, Leu, or Ile;
g) Gly228 is Ser, Gln, Ile, Thr, Asn, or Pro; and
h) Arg227 is Gln, Ser, His, Tyr, or Glu; and
(ii) has a further modification occurring at Lys370 and/or within a segment extending from Arg 469 to Ser476 of SEQ ID NO:2.
6. The Factor X analog of claim 1 , wherein said modification permits an in vivo activation of the Factor X analog into native Factor Xa or a Factor Xa analog.
7. The Factor X analog of claim 1 , wherein said modification permits an in vitro activation of the Factor X analog into native Factor Xa or a Factor Xa analog.
8. The Factor X analog of claim 1 that contains an intact β-peptide.
9. The Factor X analog of claim 1 which is in the form of a double-chain molecule.
10. The Factor X analog of claim 1 having a shortened C-terminal region, wherein the C-terminal region corresponds to amino acid residues 476-487.
11. A preparation comprising the Factor X analog of claim 1 or a precursor protein thereof.
12. The preparation of claim 11 , wherein the modification forms a cleavage site for Factor XIa or a derivative thereof.
13. The preparation of claim 11 , wherein the Factor X analog is a Factor Xα analog.
14. The preparation of claim 11 , wherein the Factor X analog has a shortened C-terminal amino acid sequence, wherein the C-terminal region corresponds to amino acid residues 476-487.
15. The preparation of claim 11 , wherein the Factor X analog is a double-chain molecule.
16. The preparation of claim 11 , wherein the Factor X analog is a single-chain Factor X analog in enzymatically inactive form that is at least 80% pure; and
the preparation does not contain inactive proteolytic intermediates of Factor X/Xa analog.
17. The preparation of claim 11 , wherein the Factor X analog is a single-chain molecule.
18. The preparation of claim 11 , wherein the modification permits an in vivo activation of the Factor X analog into native Factor Xa or a Factor Xa analog.
19. The preparation of claim 11 , wherein the modification permits an in vitro activation of the Factor X analog into native Factor Xa or into a Factor Xa analog.
20. The preparation of claim 11 that is formulated as a pharmaceutical preparation.
21. A method for obtaining a preparation comprising an activated Factor X analog, the method comprising:
(a) providing the Factor X analog of claim 1 ; and
(b) activating the Factor X analog to obtain the activated Factor X analog.
22. The method of claim 21 , further comprising formulating the preparation with a physiologically acceptable matrix.
23. The method of claim 21 , further comprising combining the preparation with a blood factor or an activated form of a blood factor as an additional component.
24. The method of claim 23 , wherein the additional component comprises at least one component with Factor VIII inhibitory bypass activity.
25. The preparation of claim 11 that is formulated as a pharmaceutical compound and present as a multi-component preparation.
26. A method for preparing a pharmaceutical composition, comprising formulating the preparation of claim 11 as pharmaceutical composition.
27. The Factor X analog as set forth in claim 5 , wherein the further modification is a substitution located at the β-peptide cleavage site located between Arg469 and Gly470 of SEQ ID NO:2.
28. The Factor X analog as set forth in claim 5 , wherein the further modification is selected from a mutation, a deletion and an insertion between amino acid positions Arg469 and Ser476 of SEQ ID NO:2.
29. A Factor X analog which
(i) contains one or more modifications in SEQ ID NO:2 selected from the group consisting of:
a) Ile235 is Val or Ala;
b) Thr233 is Ser or Asn;
c) Leu232 is Phe, Arg or Ile;
d) Asn231 is Asp, Lys, Thr, or Glu;
e) Asn230 is Ser, Lys, Met, Thr, or Asp;
f) Asp229 is Phe, Thr, Ser, Pro, Leu, or Ile;
g) Gly228 is Ser, Gln, Ile, Thr, Asn, or Pro; and
h) Arg227 is Gln, Ser, His, Tyr, Glu; and
(ii) has a further modification which is a deletion of Factor X β-peptide (Gly470 to Lys488 of SEQ ID NO:2).