Inactivation resistant factor VIII
The present invention provides novel purified and isolated nucleic acid sequences encoding procoagulant-active FVIII proteins. The nucleic acid sequences of the present invention encode amino acid sequences corresponding to known human FVIII sequences, wherein residue Phe309 is mutated. The nucleic acid sequences of the present invention also encode amino acid sequences corresponding to known human FVIII sequences, wherein the APC cleavage sites, Arg336 and Ile562, are mutated. The nucleic acid sequences of the present invention further encode amino acid sequences corresponding to known human FVIII sequences, wherein the B-domain is deleted, the von Willebrand factor binding site is deleted, a thrombin cleavage site is mutated, an amino acid sequence spacer is inserted between the A2- and A3-domains. Methods of producing the FVIII proteins of the invention, nucleotide sequences encoding such proteins, pharmaceutical compositions containing the nucleotide sequences or proteins, as well as methods of treating patients suffering from hemophilia, are also provided.
1. A FVIII protein comprising the A1-, A2-, A3-, C1- and C2-domains of human Factor VIII, said FVIII protein having an amino acid sequence spacer between the A2- and A3-domains and a mutation at Arg740, wherein the spacer is of a sufficient length that upon thrombin activation, the FVIII protein becomes a heterodimer comprising an A1-domain and an A2-spacer-A3-C1-C2 chain, and wherein the mutation consists of a substitution of Arg at position 740 with A1a, and wherein the A2-domain remains covalently associated with the A3-, C1-, and C2-domains through the spacer.
2. A pharmaceutical composition comprising an effective amount of the protein of claim 1 in admixture with a parenterally acceptable vehicle or excipient.
3. The protein of claim 1 , wherein the amino acid sequence spacer is at least 54 amino acid residues in length.
4. The protein of claim 1 , wherein the amino acid sequence spacer consists of amino acid residues 741 to 794 of wild-type FVIII, wherein the amino acid residue at position 794 is selected from the group consisting of threonine and leucine.
5. The protein of claim 4 , wherein the amino acid residue at position 794 is threonine.