Factor viii complex with xten and von willebrand factor protein, and uses thereof

Abstract

The present invention provides a chimeric protein comprising a VWF protein comprising the D′ domain and D3 domain of VWF, one or more XTEN sequence, and a FVIII protein, wherein the VWF fragment, the XTEN sequence, or the FVIII protein are linked to or associated with each other. The chimeric protein can further comprise one or more Ig constant region or a portion thereof (e.g., an Fc region). A polypeptide chain comprising a VWF fragment of the invention binds to or is associated with a polypeptide chain comprising a FVIII protein linked to an XTEN sequence and the polypeptide chain comprising the VWF fragment can prevent or inhibit binding of endogenous VWF to the FVIII protein linked to the XTEN sequence. By preventing or inhibiting binding of endogenous VWF to the FVIII protein, which is a half-life limiting factor for FVIII, the VWF fragment can induce extension of half-life of the chimeric protein comprising a FVIII protein. The invention also includes nucleotides, vectors, host cells, methods of using the VWF fragment, or the chimeric proteins.

1 . A chimeric protein comprising (i) a von Willebrand Factor (VWF) protein comprising a D′ domain and a D3 domain of VWF, (ii) an XTEN sequence, and (iii) a FVIII protein, wherein the VWF fragment and the XTEN sequence are linked by an optional linker, wherein the VWF fragment or the XTEN sequence is linked to or associated with the FVIII protein. 
     
     
         2 . A chimeric protein comprising a formula, which comprises:
   V-X-FVIII,  (a)
     FVIII-X-V,  (b)
     V-X:FVIII,  (c)
     X-V:FVIII,  (d)
     FVIII:V-X, or  (e)
     FVIII:X-V,  (f)
   
       wherein V comprises a VWF fragment, 
       X comprises one or more XTEN sequences, 
       FVIII comprises a FVIII protein; 
       (−) is a peptide bond or one or more amino acids; and 
       (:) is a covalent bond or a non-covalent bond. 
     
     
         3 . The chimeric protein of  claim 1 , wherein the XTEN sequence is linked to the FVIII protein by a linker. 
     
     
         4 - 12 . (canceled) 
     
     
         13 . The chimeric protein of  claim 1 , further comprising a first Ig constant region or a portion thereof. 
     
     
         14 - 16 . (canceled) 
     
     
         17 . The chimeric protein of  claim 13 , wherein the chimeric protein comprises a second Ig constant region, wherein the second Ig constant region is linked to the VWF fragment by a linker. 
     
     
         18 - 19 . (canceled) 
     
     
         20 . The chimeric protein of  claim 17 , wherein the second Ig constant region or a portion thereof is associated with the first Ig constant region or a portion thereof. 
     
     
         21 . The chimeric protein of  claim 17 , wherein the second Ig constant region or a portion thereof is associated with the first Ig constant region or a portion thereof by a covalent bond. 
     
     
         22 - 67 . (canceled) 
     
     
         68 . The chimeric protein of  claim 1 , wherein the FVIII protein is linked to an XTEN sequence at the C-terminus or the N-terminus of the FVIII protein or inserted immediately downstream of one or more amino acids in the FVIII protein or any combinations thereof. 
     
     
         69 . (canceled) 
     
     
         70 . The chimeric protein of  claim 1 , wherein the FVIII protein comprises one or more domains of FVIII selected from the group consisting of an A1 domain, a1 acidic region, an A2 domain, a2 acidic region, a B domain, an A3 domain, a3 acidic region, a C1 domain, a C2 domain, one or more fragments thereof, and any combinations thereof. 
     
     
         71 - 74 . (canceled) 
     
     
         75 . The chimeric protein of  claim 1 , wherein the FVIII protein comprises a B domain or a portion thereof. 
     
     
         76 - 89 . (canceled) 
     
     
         90 . The chimeric protein of  claim 1 , wherein the amino acid sequence of the D′ domain is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 764 to 866 of SEQ ID NO: 2. 
     
     
         91 . The chimeric protein of  claim 1 , wherein the amino acid sequence of the D3 domain is at least 90%, 95%, 96%, 97%, 98%, or 99%, identical to amino acids 867 to 1240 of SEQ ID NO: 2. 
     
     
         92 - 95 . (canceled) 
     
     
         96 . The chimeric protein of  claim 91 , wherein the VWF fragment contains at least one amino acid substitution at a residue corresponding to residue 1099, residue 1142, or both residues 1099 and 1142 of SEQ ID NO: 2. 
     
     
         97 - 119 . (canceled) 
     
     
         120 . A polynucleotide or a set of polynucleotides encoding the chimeric protein of  claim 1 . 
     
     
         121 . (canceled) 
     
     
         122 . A vector comprising the polynucleotide or set of polynucleotides of  claim 120  and one or more promoter operably linked to the polynucleotide or the set of polynucleotides. 
     
     
         123 . (canceled) 
     
     
         124 . A host cell comprising the polynucleotide of or set of polynucleotides of  claim 120 . 
     
     
         125 - 126 . (canceled) 
     
     
         127 . A pharmaceutical composition comprising the chimeric protein of  claim 1 , and a pharmaceutically acceptable carrier. 
     
     
         128 - 137 . (canceled) 
     
     
         138 . A method of extending or increasing half-life of a FVIII protein in a subject, wherein the method comprises administering an effective amount of the chimeric protein of  claim 1  to a subject in need thereof, wherein the VWF fragment binds to the FVIII protein and thus extends or increases half-life of the FVIII protein. 
     
     
         139 - 142 . (canceled) 
     
     
         143 . A method of treating a bleeding disease or disorder in a subject in need thereof comprising administering an effective amount of the chimeric protein of  claim 1 , wherein the bleeding disease or disorder is selected from the group consisting of a bleeding coagulation disorder, hemarthrosis, muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma, trauma capitis, gastrointestinal bleeding, intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture, central nervous system bleeding, bleeding in the retropharyngeal space, bleeding in the retroperitoneal space, and bleeding in the iliopsoas sheath. 
     
     
         144 - 147 . (canceled) 
     
     
         148 . A method of making a chimeric protein, comprising transfecting a host cell with the polynucleotide or set of polynucleotides of  claim 120  and expressing the chimeric protein in the host cell. 
     
     
         149 - 165 . (canceled)