Binding molecules for human factor VIII and factor VIII-like proteins
View Patent ↗It is an object of the present invention to provide novel binding molecules for factor VIII and factor VIII-like proteins. Preferred binding molecules of the present invention exhibit not only distinct characteristics for binding of the target factor VIII polypeptides but also specific and desirable characteristics for release (elution) of the target polypeptides. Especially preferred binding molecules according to the invention are short polypeptide sequences, characterized by a stable loop structure.
1. A polypeptide comprising the amino acid sequence Phe-Cys-X 1 -Val-Phe-X 2 -X 3 -X 4 -His-Cys-X 5 (SEQ ID NO: 70), wherein:
X 1 is Trp or His;
X 2 is Ala, Pro, Asn or Gln;
X 3 is Phe or Trp;
X 4 is Asp, Gln, Ser, Asn, Val, Arg or His; and
X 5 is His, Ala, Ser, Asp or Phe,
such that the polypeptide binds factor VIII.
2. The polypeptide of claim 1 , wherein the amino acid sequence is selected from the group consisting of:
(SEQ ID NO: 71)
Phe-Cys-Trp-Val-Phe-Ala-Phe-Asp-His-Cys-His;
(SEQ ID NO: 72)
Phe-Cys-Trp-Val-Phe-Pro-Phe-Gln-His-Cys-Ala;
(SEQ ID NO: 73)
Phe-Cys-Trp-Val-Phe-Asn-Phe-Ser-His-Cys-Ser;
(SEQ ID NO: 74)
Phe-Cys-Trp-Val-Phe-Pro-Phe-Asn-His-Cys-Ser;
(SEQ ID NO: 75)
Phe-Cys-Trp-Val-Phe-Asn-Trp-Val-His-Cys-Asp;
(SEQ ID NO: 76)
Phe-Cys-Trp-Val-Phe-Pro-Phe-Asn-His-Cys-Asp;
(SEQ ID NO: 77)
Phe-Cys-Trp-Val-Phe-Gln-Phe-Arg-His-Cys-His;
(SEQ ID NO: 78)
Phe-Cys-His-Val-Phe-Asn-Phe-Val-His-Cys-Ser;
and
(SEQ ID NO: 79)
Phe-Cys-Trp-Val-Phe-Pro-Phe-His-His-Cys-Phe.
3. The polypeptide of claim 1 , wherein X 1 is Trp and X 3 is Phe.
4. The polypeptide of claim 1 , wherein a terminal amino acid residue comprises a chemical modification.
5. The polypeptide of claim 4 , wherein the carboxyl terminus comprises a chemical modification.
6. The polypeptide of claim 5 , wherein the chemical modification comprises a hydrazide functional group.
7. The polypeptide of claim 4 , wherein the amino terminus comprises a chemical modification.
8. The polypeptide of claim 7 , wherein the chemical modification is an acetylation.
9. A method for purifying human factor VIII from a solution comprising:
(a) immobilizing a factor VIII binding polypeptide according to claim 4 , wherein the polypeptide is attached to a solid support;
(b) contacting a solution containing human factor VIII with the polypeptide under conditions such that factor VIII binds to the polypeptide; and
(c) separating bound factor VIII from the solution, thereby purifying human factor VIII from the solution.
10. A composition comprising a polypeptide comprising the amino acid sequence Phe-Cys-X 1 -Val-Phe-X 2 -X 3 -X 4 -His-Cys-X 5 (SEQ ID NO: 70), wherein the polypeptide can bind factor VIII and the polypeptide is attached to a solid support matrix.
11. The composition of claim 10 wherein the polypeptide is attached to the solid support matrix via a covalent or non-covalent linkage.
12. The composition of claim 11 , wherein the solid support matrix is selected from the group consisting of: sepharose, agarose and cellulose.
13. A recombinant bacteriophage expressing exogenous DNA encoding a polypeptide, the polypeptide comprising the amino acid sequence Phe-Cys-X 1 -Val-Phe-X 2 -X 3 -X 4 -His-Cys-X 5 (SEQ ID NO: 70), such that the polypeptide binds factor VIII.
14. The recombinant bacteriophage of claim 13 , wherein:
X 1 is Trp or His;
X 2 is Ala, Pro, Asn or Gln;
X 3 is Phe or Trp;
X 4 is Asp, Gln, Ser, Asn, Val, Arg or His; and
X 5 is His, Ala, Ser, Asp or Phe,
such that the polypeptide binds factor VIII.
15. The recombinant bacteriophage of claim 14 , wherein the amino acid sequence is selected from the group consisting of:
(SEQ ID NO: 71)
Phe-Cys-Trp-Val-Phe-Ala-Phe-Asp-His-Cys-His;
(SEQ ID NO: 72)
Phe-Cys-Trp-Val-Phe-Pro-Phe-Gln-His-Cys-Ala;
(SEQ ID NO: 73)
Phe-Cys-Trp-Val-Phe-Asn-Phe-Ser-His-Cys-Ser;
(SEQ ID NO: 74)
Phe-Cys-Trp-Val-Phe-Pro-Phe-Asn-His-Cys-Ser;
(SEQ ID NO: 75)
Phe-Cys-Trp-Val-Phe-Asn-Trp-Val-His-Cys-Asp;
(SEQ ID NO: 76)
Phe-Cys-Trp-Val-Phe-Pro-Phe-Asn-His-Cys-Asp;
(SEQ ID NO: 77)
Phe-Cys-Trp-Val-Phe-Gln-Phe-Arg-His-Cys-His;
(SEQ ID NO: 78)
Phe-Cys-His-Val-Phe-Asn-Phe-Val-His-Cys-Ser;
and
(SEQ ID NO: 79)
Phe-Cys-Trp-Val-Phe-Pro-Phe-His-His-Cys-Phe.
16. The recombinant bacteriophage of claim 14 , wherein X 1 is Trp and X 3 is Phe.