Muteins of clotting factor VIII
The present disclosure provides muteins of FVIII to which a biocompatible polymer may be attached to increase the circulatory half-life of the muteins, as well as conjugates of such muteins and biocompatible polymers.
1. A modified Factor VIII (FVIII) protein comprising amino acid substitutions at one or more positions corresponding to 336, 1680, 2094, 2186, 2204, or 2206 with reference to the amino acid sequence of SEQ ID NO:1, and wherein the substitutions are selected from the group consisting of:
a) S2094C, R336A and Y1680F;
b) S2186C, R336A and Y1680F;
c) S2204C, R336A and Y1680F;
d) S2206C, R336A and Y1680F;
e) S2094C and R336A;
f) S2186C and R336A;
g) S2204C and R336A;
h) S2206C and R336A;
i) S2094C and Y1680F;
j) S2186C and Y1680F;
k) S2204C and Y1680F;
l) S2206C and Y1680F;
m) S2094C;
n) S2186C;
o) S2204C; and
p) S2206C.
2. The modified FVIII protein of claim 1 , wherein said FVIII protein is a single chain.
3. The modified FVIII protein of claim 1 , wherein said FVIII protein is an inactive two-chain form comprising a proteolytic site of thrombin.
4. The modified FVIII protein of claim 1 , wherein said FVIII protein lacks all or part of the B domain.
5. The modified FVIII protein of claim 1 , wherein said FVIII protein is activated.
6. A conjugate comprising the modified FVIII protein of claim 1 and a biocompatible polymer covalently attached directly, or indirectly via a linker, to said cysteine substitution.
7. The conjugate of claim 6 , wherein the conjugate further comprises a spacer.
8. The conjugate of claim 6 , wherein said covalent attachment is to the sulfur atom of the thiol group of said cysteine substitution.
9. The conjugate of claim 6 , wherein said biocompatible polymer is selected from the group consisting of: polyethylene glycol (PEG), hydroxyalkyl starch (HAS), polysialic acid (PSA), a zwitterionic brush polymer, and a poly-phosphorylcholine branched polymer.
10. The conjugate of claim 9 , wherein said biocompatible polymer is hydroxyethyl starch (HES) or polyethylene glycol (PEG).
11. The conjugate of claim 10 , wherein the circulatory half-life of said modified FVIII protein is increased at least 2 times compared to unmodified FVIII protein.
12. A composition comprising the conjugate of claim 10 and a pharmaceutically acceptable excipient.