Factor ix fusion proteins and methods of making and using same

Abstract

The present invention provides Factor IX (FIX) fusion proteins comprising at least one heterologous moiety, such as an XTEN. The present invention further discloses methods of making and using the FIX fusion proteins.

1 . A Factor IX (FIX) fusion protein comprising a FIX polypeptide and an XTEN that is inserted within the FIX polypeptide, wherein the FIX fusion protein comprises the FIX polypeptide amino acid sequence of SEQ ID NO: 2 with the XTEN inserted at an insertion site in the activation peptide of SEQ ID NO: 2 (amino acids 146-180), and wherein the FIX fusion protein exhibits procoagulant activity. 
     
     
         2 . The FIX fusion protein of  claim 1 , wherein the insertion site corresponds to an amino acid selected from the group consisting of amino acid 149 of SEQ ID NO: 2, amino acid 162 of SEQ ID NO: 2, amino acid 166 of SEQ ID NO: 2, amino acid 174 of SEQ ID NO: 2 and any combination thereof. 
     
     
         3 . The FIX fusion protein of  claim 1 , wherein the XTEN comprises at least about 5 amino acids, at least about 6 amino acids, at least about 12 amino acids, at least about 36 amino acids, at least about 42 amino acids, at least about 72 amino acids, at least about 144 amino acids, or at least about 288 amino acids. 
     
     
         4 . The FIX fusion protein of  claim 1 , which further comprises a second XTEN. 
     
     
         5 . The FIX fusion protein of  claim 4 , wherein the XTEN is inserted within the FIX polypeptide at an insertion site corresponding to amino acid 166 of SEQ ID NO: 2, and wherein the second XTEN is fused to the C-terminus of the FIX polypeptide. 
     
     
         6 . The FIX fusion protein of  claim 1 , further comprising a first Fc domain. 
     
     
         7 . The FIX fusion protein of  claim 6 , further comprising a second Fc domain. 
     
     
         8 . The FIX fusion protein of  claim 7 , which comprises two polypeptide chains, wherein
 (i) the first polypeptide chain comprises the first Fc domain fused to the N-terminus or C-terminus of the FIX polypeptide comprising the XTEN, and   (ii) the second polypeptide chain comprises the second Fc domain,   wherein the first Fc domain and the second Fc domain are associated by a covalent bond.   
     
     
         9 . The FIX fusion protein of  claim 1 , wherein the FIX polypeptide is a R338L FIX variant. 
     
     
         10 . An isolated polynucleotide comprising a sequence encoding the FIX fusion protein of  claim 1 . 
     
     
         11 . An expression vector comprising the polynucleotide of  claim 10 . 
     
     
         12 . A host cell comprising the vector of  claim 11 . 
     
     
         13 . A pharmaceutical composition comprising the FIX fusion protein of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         14 . A method of preventing, treating, ameliorating, or managing a clotting disease or condition in a patient in need thereof, comprising administering to the patient an effective amount of the FIX fusion protein of  claim 1 . 
     
     
         15 . A Factor IX (FIX) fusion protein comprising a first polypeptide chain and a polypeptide second chain, wherein:
 a. the first polypeptide chain comprises:
 i. a FIX polypeptide and an XTEN that is inserted within the FIX polypeptide, wherein the FIX fusion protein comprises the FIX polypeptide amino acid sequence of SEQ ID NO: 2 with the XTEN inserted at an insertion site in the activation peptide (amino acids 146-180) of SEQ ID NO: 2, and wherein the at least one XTEN comprises an amino acid sequence having at least about 72 amino acids; and 
 ii. a first Fc domain, wherein the first Fc domain is fused to the FIX polypeptide comprising the XTEN; and 
   b. the second polypeptide chain comprises a second Fc domain, wherein the first Fc domain and the second Fc domain are associated by a covalent bond; and wherein the FIX fusion protein exhibits procoagulant activity.