MUTEINS OF CLOTTING FACTOR Vlll

Abstract

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 FVIII protein comprising a cysteine substitution mutation at an amino acid corresponding to a position in the amino acid sequence of SEQ ID NO:1 selected from the group of amino acid positions consisting of: 2094, 2186, 2204, 2206, 59, 239, 333, 336, 379, 481, 484, 486, 488, 489, 490, 492, 493, 495, 496, 497, 499, 500, 501, 507, 555, 562, 568, 571, 582, 1680, 1778, 1793, 1794, 1797, 1798, 1799, 1800, 1801, 1806, 1810, 1811, 1814, 1816, 1818, 1891, 2035, 2068, 2092, 2093, 2095, 2118, 2125, 2183, 2191, 2196, and 2212. 
     
     
         2 . The modified FVIII protein of  claim 1 , further comprising at least one additional substitution other than cysteine at a position corresponding to 336, 562, 1680 or 1968 in the amino acid sequence of SEQ ID NO:1 selected from the group consisting of: R336A, R562A, Y1680F and K1968A. 
     
     
         3 . The modified FVIII protein of  claim 1 , further comprising two additional substitutions other than cysteine at positions corresponding to 336 and 1680 in the amino acid sequence of SEQ ID NO:1, wherein said substitutions are R336A and Y1680F. 
     
     
         4 . (canceled) 
     
     
         5 . The modified FVIII protein of  claim 1 , wherein said FVIII protein is a single chain. 
     
     
         6 . The modified FVIII protein of  claim 1 , wherein said FVIII protein is an inactive two-chain form capable of being activated by thrombin. 
     
     
         7 . The modified FVIII protein of  claim 1 , wherein said FVIII protein lacks all or part of the B domain. 
     
     
         8 . The modified FVIII protein of  claim 7 , wherein in its single chain form said FVIII protein has the amino acid sequence of SEQ ID NO:2. 
     
     
         9 . 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 mutation. 
     
     
         10 . The conjugate of  claim 9 , wherein the conjugate further comprises a spacer. 
     
     
         11 . The conjugate of  claim 9 , wherein said covalent attachment is to the sulfur atom of the thiol group of said cysteine substitution mutation. 
     
     
         12 . The conjugate of  claim 9 , wherein said biocompatible polymer is selected from the group consisting of: polyethylene glycol (PEG), hydroxyalkyl starch (HAS), hydroxyethyl starch (HES), polysialic acid (PSA), a zwitterionic brush polymer, and a poly-phosphorylcholine branched polymer. 
     
     
         13 . The conjugate of  claim 12 , wherein said biocompatible polymer is hydroxyethyl starch (HES). 
     
     
         14 . (canceled) 
     
     
         15 . A nucleic acid encoding the modified FVIII protein of  claim 1 . 
     
     
         16 . A host cell comprising the nucleic acid of  claim 15 . 
     
     
         17 . A vector comprising the nucleic acid of  claim 15 . 
     
     
         18 . A host cell comprising the vector of  claim 17 . 
     
     
         19 . A method of producing the modified FVIII protein of  claim 1 , comprising culturing the host cell of  claim 18  under conditions where the protein is expressed and isolating said protein. 
     
     
         20 . (canceled) 
     
     
         21 . A composition comprising the conjugate of  claim 13  and a pharmaceutically acceptable excipient. 
     
     
         22 . A method of preventing or treating bleeding in a subject having a deficiency of FVIII activity comprising the step of administering to a subject having a deficiency of FVIII activity a prophylactically or therapeutically effective dose of the composition of  claim 21 . 
     
     
         23 . The method of  claim 22 , wherein said subject has Hemophilia A. 
     
     
         24 . (canceled) 
     
     
         25 . (canceled) 
     
     
         26 . (canceled) 
     
     
         27 . (canceled)