Recombinant factor viii having enhanced stability following mutation at the a1-c2 domain interface

Abstract

The invention relates to a recombinant factor VIII that includes one or more mutations at an interface of A1 and C2 domains of recombinant factor VIII. The one or more mutations include substitution of one or more amino acid residues with either a cysteine or an amino acid residue having a higher hydrophobicity. This results in enhanced stability of factor VIII. Methods for making the recombinant factor VIII, pharmaceutical compositions containing the recombinant factor VIII, and use of the recombinant factor VIII for treating hemophilia A are also disclosed.

What is claimed: 
     
         1 . A recombinant factor VIII comprising substitution of two or more amino acid residues with a Cysteine residue at an interface of A1 and C2 domains of the recombinant factor VIII to afford a disulfide bond between the A1 and C2 domains, and one or more of the following:
 (i) a substitution of one or more amino acid residues with an amino acid residue having a higher hydrophobicity at a C2 domain interface of the A1 domain;   (ii) a substitution of one or more amino acid residues with an amino acid residue having a higher hydrophobicity at an A1 domain interface of the C2 domain; and   (iii) a substitution of one or more charged amino acid residues with a hydrophobic amino acid residue at an interface of A1 and A2 domains or an interface of A2 and A3 domains of the recombinant factor VIII.   
     
     
         2 . The recombinant factor VIII according to  claim 1 , wherein the A1 domain having the Cysteine substitution comprises the sequence SXXX (SEQ ID NO: 20) wherein X at the third position is Cysteine, and the C2 domain having the Cysteine substitution comprises the sequence PPXX (SEQ ID NO: 23) wherein X at the fourth position is Cysteine. 
     
     
         3 . The recombinant factor VIII according to  claim 1 , wherein the two or more amino acid residues substituted with a Cysteine residue comprise an Arg→Cys substitution at a position corresponding to residue 121 of SEQ ID NO: 2 and an Leu→Cys substitution at a position corresponding to residue 2302 of SEQ ID NO: 2. 
     
     
         4 . The recombinant factor VIII according to  claim 1 , wherein the substitution of one or more amino acid residues with an amino acid residue having a higher hydrophobicity at a C2 domain interface of the A1 domain is present. 
     
     
         5 . The recombinant factor VIII according to  claim 4 , wherein the C2 domain interface of the A1 domain comprises the amino acid sequence of:
 KXS (SEQ ID NO: 19), where the substitution is at the second position and X is Valine, Isoleucine, or Leucine; or   TYXW (SEQ ID NO: 21), where the substitution is at the third position and X is Leucine, Isoleucine, or Valine.   
     
     
         6 . The recombinant factor VIII according to  claim 4 , wherein the substitution of one or more amino acid residues with an amino acid residue having a higher hydrophobicity at the C2 domain interface of the A1 domain comprises an Ala→Ile substitution at a position corresponding to residue 108 of SEQ ID NO: 2. 
     
     
         7 . The recombinant factor VIII according to  claim 1 , wherein the substitution of one or more amino acid residues with an amino acid residue having a higher hydrophobicity at an A1 domain interface of the C2 domain is present. 
     
     
         8 . The recombinant factor VIII according to  claim 1 , wherein the substitution of one or more charged amino acid residues with a hydrophobic amino acid residue at an interface of A1 and A2 domains or an interface of A2 and A3 domains of the recombinant factor VIII is present. 
     
     
         9 . The recombinant factor VIII according to  claim 8 , wherein the charged amino acid residue is either Glu or Asp, and the hydrophobic amino acid substitution is one of Ala, Val, Ile, Leu, Met, Phe, or Trp. 
     
     
         10 . The recombinant factor VIII according to  claim 8  wherein the substitution of one or more charged amino acid residues with a hydrophobic amino acid residue at the interface of A1 and A2 domains or the interface of A2 and A3 domains of the recombinant factor VIII comprises substitution of a Glu287 residue of wild type factor VIII, substitution of an Asp302 residue of wild type factor VIII, substitution of an Asp519 residue of wild type factor VIII, substitution of a Glu665 residue of wild type factor VIII, substitution of a Glu1984 residue of wildtype factor VIII, or a combination thereof. 
     
     
         11 . The recombinant factor VIII according to  claim 8 , wherein the substitution of one or more charged amino acid residues with a hydrophobic amino acid residue at an interface of A1 and A2 domains or an interface of A2 and A3 domains of the recombinant factor VIII comprises:
 (i) an Asp→Ala substitution at a position corresponding to residue 302 of SEQ ID NO: 2;   (ii) a Glu→Ala substitution at a position corresponding to residue 287 of SEQ ID NO: 2;   (iii) a Glu→Ala or Glu→Val substitution at a position corresponding to residue 665 of SEQ ID NO: 2;   (iv) an Asp→Ala or Asp→Val substitution at a position corresponding to residue 519 of SEQ ID NO: 2;   (v) a Glu→Ala or Glu→Val substitution at a position corresponding to residue 1984 of SEQ ID NO: 2; or   (vi) combinations of any two or more of the substitutions (i)-(v).   
     
     
         12 . The recombinant factor VIII according to  claim 1 , wherein the recombinant factor VIII further comprises one or more of (i) factor IXa and/or factor X binding domains modified to enhance the affinity of the recombinant factor VIII for one or both of factor IXa and factor X; (ii) modified sites that enhance secretion in culture; (iii) modified serum protein binding sites that enhance the circulating half-life thereof; (iv) at least one glycosylation recognition sequence that is effective in decreasing antigenicity and/or immunogenicity thereof; (v) a modified A1 domain calcium-binding site that improves specific activity of the recombinant factor VIIIa; and (vi) a modified activated protein C-cleavage site. 
     
     
         13 . A pharmaceutical composition comprising the recombinant factor VIII according to  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         14 . A method of treating an animal for hemophilia A, said method comprising:
 administering to an animal exhibiting hemophilia A an effective amount of the recombinant factor VIII of  claim 1 , whereby the animal exhibits effective blood clotting following vascular injury.   
     
     
         15 . An isolated nucleic acid molecule encoding the recombinant factor VIII according to  claim 1 . 
     
     
         16 . The isolated nucleic acid molecule according to  claim 15 , wherein the nucleic acid is DNA. 
     
     
         17 . A recombinant expression system comprising a DNA molecule of  claim 16 . 
     
     
         18 . A recombinant host cell comprising the nucleic acid molecule according to  claim 15 . 
     
     
         19 . An implantable device comprising a plurality of the recombinant host cells according to  claim 18  contained within the device. 
     
     
         20 . A method of treating an animal for hemophilia A, said method comprising:
 administering to an animal exhibiting hemophilia A a recombinant host cell of  claim 18 , whereby the recombinant host cell expresses the recombinant factor VIII and the animal exhibits effective blood clotting following vascular injury.