Factor viii compositions and methods of making and using same

Abstract

The present invention relates to compositions comprising factor VIII coagulation factors linked to extended recombinant polypeptide (XTEN), isolated nucleic acids encoding the compositions and vectors and host cells containing the same, and methods of making and using such compositions in treatment of factor VIII-related diseases, disorders, and conditions.

1 . A recombinant factor VIII fusion protein comprising a factor VIII polypeptide and at least a first extended recombinant polypeptide (XTEN), wherein said factor VIII polypeptide comprises an A1 domain including an a1 acidic spacer region, an A2 domain including an a2 acidic spacer region, an A3 domain including an a3 acidic spacer region, a C1 domain, a C2 domain and optionally all or a portion of a B domain, and wherein said first XTEN is linked to said factor VIII polypeptide at (i) the C-terminus of said factor VIII polypeptide; (ii) within the B domain of said factor VIII polypeptide if all or a portion of the B domain is present; (iii) within the A1 domain of said factor VIII polypeptide; (iv) within the A2 domain of said factor VIII polypeptide; (v) within the A3 domain of said factor VIII polypeptide; (vi) within the C1 domain of said factor VIII polypeptide; (vii) within the C2 domain of said factor VIII polypeptide; (viii) at the N-terminus of said factor VIII polypeptide, (ix) between two domains of said factor VIII polypeptide; and wherein when compared to a corresponding factor VIII protein not linked to XTEN, the fusion protein (a) retains at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 100%, 200%, 300%, 400%, or 500% of the procoagulant activity in an in vitro coagulation assay, or (b) exhibits reduced binding to an anti-factor VIII antibody in an in vitro binding assay. 
     
     
         2 . (canceled) 
     
     
         3 . The recombinant factor VIII fusion protein of  claim 1 , wherein the first XTEN is linked to said factor VIII polypeptide at an insertion site selected from Table 5, Table 6, Table 7, Table 8, or Table 9. 
     
     
         4 - 10 . (canceled) 
     
     
         11 . The recombinant factor VIII fusion protein of  claim 1 , comprising at least a second XTEN, optionally a third XTEN, optionally a fourth XTEN, optionally a fifth XTEN and optionally a sixth XTEN, wherein each of the second, third, fourth, fifth, or sixth XTEN is linked to said factor VIII polypeptide at a second, third, fourth, fifth, or sixth site selected from the group consisting of:
 (a) an insertion site from Table 5, Table 6, Table 7, Table 8, or Table 9;   (b) an insertion site within 6 amino acids of amino acid residue number 32, 220, 224, 336, 339, 390, 399, 416, 603, 1656, 1711, 1725, 1905 or 1910 with reference to full-length human factor VIII sequence as set forth in  FIG.  3   ;   (c) an insertion site between any two adjacent domains of said factor VIII polypeptide, wherein said two adjacent domains are selected from the group consisting of A1 and A2 domains, A2 and B domains, B and A3 domains, A3 and C1 domains, and C1 and C2 domains;   (d) within the B domain of said factor VIII polypeptide, wherein the second XTEN 15 linked to the C-terminal end of about amino acid residue number 741 to about 750 and to the N-terminal end of amino acid residue numbers 1635 to about 1648 with reference to full-length human factor VIII sequence as set forth in  FIG.  3   ;   (e) the C-terminus of said factor VIII polypeptide; and   (f) the N-terminus of said factor VIII polypeptide.   
     
     
         12 - 18 . (canceled) 
     
     
         19 . A recombinant factor VIII fusion protein comprising at least one extended recombinant polypeptide (XTEN), said fusion protein having a structure of formula IX: (A1 N )-(S) a -(XTEN) t -(S) b -(A1 C )-(A2 N )-(S) c -(XTEN) u -(S) d -(A2 C )-(B N )-(S) e -(XTEN) v -(S) f -(B C )-(A3 N )-(S) g -(XTEN) w -(S) h -(A3 C )-(C1 N )-(S) i -(XTEN) x -(S) j -(C1 C )-(C2 N )-(S) k -(XTEN) y -(S) l -(C2 C )-(S) m -(XTEN) z  IX, wherein independently for each occurrence,
 a) A1 N  is a fragment of the A1 domain of factor VIII having a sequence ranging from at least amino acid residue number 1, with reference to full-length human factor VIII sequence as set forth in  FIG.  3   , to no more than amino acid residue number 371;   b) A1 c  is a fragment of the A1 domain of factor VIII having a sequence ranging from at least amino acid residue number 2 to no more than amino acid residue number 372, with the priviso that no sequence of the A1 N  fragment is duplicated in the A1 c  is a fragment;   c) A2 N  is a fragment of the A2 domain of factor VIII having a sequence ranging from at least amino acid residue number 373 to no more than amino acid residue number 739;   d) A2 c  is a fragment of the A2 domain of factor VIII having a sequence ranging from at least amino acid residue number 374 to no more than amino acid residue number 740, with the priviso that no sequence of the A2 N  fragment is duplicated in the A2 c  is a fragment;   e) B N  is a fragment of the B domain of factor VIII having a sequence ranging from at least amino acid residue number 741 to no more than amino acid residue number 1647;   f) B c  is a fragment of the B domain of factor VIII having a sequence ranging from at least amino acid residue number 742 to no more than amino acid residue number 1648, with the priviso that no sequence of the B N  fragment is duplicated in the B c  is a fragment;   g) A3 N  is a fragment of the A3 domain of factor VIII having a sequence ranging from at least amino acid residue number 1649 to no more than amino acid residue number 2018;   h) A3 c  is a fragment of the A3 domain of factor VIII having a sequence ranging from at least amino acid residue number 1650 to no more than amino acid residue number 2019, with the priviso that no sequence of the A3 N  fragment is duplicated in the A3 c  is a fragment;   i) C1 N  is a fragment of the C1 domain of factor VIII having a sequence ranging from at least amino acid residue number 2020 to no more than amino acid residue number 2171;   j) C1 c  is a fragment of the C1 domain of factor VIII having a sequence ranging from at least amino acid residue number 2021 to no more than amino acid residue number 2172, with the priviso that no sequence of the C1 N  fragment is duplicated in the C1 c  is a fragment;   k) C2 N  is a fragment of the C2 domain of factor VIII having a sequence ranging from at least amino acid residue number 2173 to no more than amino acid residue number 2331;   l) C2 c  is a fragment of the C2 domain of factor VIII having a sequence ranging from at least amino acid residue number 2174 to no more than amino acid residue number 2332, with the priviso that no sequence of the C2 N  fragment is duplicated in the C2 c  is a fragment;   m) S is a spacer sequence having between 1 to about 50 amino acid residues that can optionally include a cleavage sequence or amino acids compatible with restrictions sites;   n) a is either 0 or 1;   o) b is either 0 or 1;   p) c is either 0 or 1;   q) d is either 0 or 1;   r) e is either 0 or 1;   s) f is either 0 or 1;   t) g is either 0 or 1;   u) h is either 0 or 1;   v) i is either 0 or 1;   w) j is either 0 or 1;   x) k is either 0 or 1;   y) l is either 0 or 1;   z) m is either 0 or 1;   aa) t is either 0 or 1;   bb) u is either 0 or 1;   cc) v is either 0 or 1;   dd) w is either 0 or 1;   ee) x is either 0 or 1;   ff) y is either 0 or 1;   gg) z is either 0 or 1 with the proviso that t+u+v+w+x+y+z≥1;   hh) each XTEN is independently an extended recombinant protein;   
       and wherein the fusion protein has at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the procoagulant activity of a corresponding factor VIII polypeptide not linked to an XTEN or wherein said fusion protein exhibits reduced binding to an anti-factor VIII antibody as compared to the corresponding factor VIII not linked to XTEN when measured by an in vitro assay which is an ELISA or Bethesda assay. 
     
     
         20 - 28 . (canceled) 
     
     
         29 . The recombinant factor VIII fusion protein of  claim 19 , wherein an XTEN is inserted immediately downstream of an amino acid which corresponds to an amino acid in mature native human factor VIII selected from the group consisting of amino acid residue numbers 32, 220, 224, 336, 339, 399, 416, 603, 1656, 1711, 1725, 1905 and 1910. 
     
     
         30 . A recombinant factor VIII fusion protein comprising: a first polypeptide comprising formula X: (A1)-a1-(A2)-a2-[B]; and a second polypeptide comprising Formula XI: a3-(A3)-(C1)-(C2);
 wherein the first polypeptide and the second polypeptide are fused or exist as a heterodimer;   wherein, a) A1 is an A1 domain of factor VIII; b) A2 is an A2 domain of factor VIII; c) [B] is a B domain of factor VIII, a fragment thereof, or is deleted; d) A3 is an A3 domain of factor VIII; e) C1 is a C1 domain of factor VIII; f) C2 is a C2 domain of factor VIII; g) a1, a2, and a3 are acidic spacer regions;   wherein the A1 domain comprises an XTEN permissive loop-1 (A1-1) region and an XTEN permissive loop-2 (A1-2) region;   wherein the A2 domain comprises an XTEN permissive loop-1 (A2-1) region and an XTEN permissive loop-2 (A2-2) region;   wherein the A3 domain comprises an XTEN permissive loop-1 (A3-1) region and an XTEN permissive loop-2 (A3-2) region;   wherein an XTEN sequence is inserted into at least one of the regions A1-1, A1-2, A2-1, A2-2, A3-1, or A3-2; and   wherein the recombinant factor VIII protein exhibits procoagulant activity.   
     
     
         31 - 73 . (canceled) 
     
     
         74 . The recombinant factor VIII fusion protein of  claim 1 , comprising one, two or three amino acid substitutions in the factor VIII sequence selected from the group consisting of residues R1645, R1648, Y1680, R1689, and any combinations thereof, numbered relative to mature human factor VIII, wherein each substitution is independently to an amino acid which is alanine, glycine, or phenylalanine. 
     
     
         75 . (canceled) 
     
     
         76 . The recombinant factor VIII fusion protein of  claim 1 , wherein the factor VIII polypeptide is linked to a least one XTEN via one or two cleavage sequences that each is cleavable by a mammalian protease selected from the group consisting of factor XIa, factor XIIa, kallikrein, factor VIIa, factor IXa, factor Xa, factor IIa (thrombin), Elastase-2, MMP-12, MMP13, MMP-17 and MMP-20, wherein cleavage at the cleavage sequence by the mammalian protease releases the factor VIII sequence from the XTEN, and wherein the released factor VIII sequence exhibits an increase in procoagulant activity compared to the uncleaved fusion protein. 
     
     
         77 - 86 . (canceled) 
     
     
         87 . A pharmaceutical composition comprising the recombinant factor VIII fusion protein of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         88 - 92 . (canceled) 
     
     
         93 . A method of treating a bleeding episode in a subject, comprising administering to said subject a clotting effective amount of the pharmaceutical composition of claim  0 , wherein the clotting effective amount of the fusion protein arrests a bleeding episode for a period that is at least two-fold, or at least three-fold longer compared to the corresponding factor VIII not linked to XTEN and administered using a comparable amount to said subject. 
     
     
         94 . (canceled) 
     
     
         95 . A recombinant factor VIII fusion protein for use in a pharmaceutical regimen for treatment of a hemophilia A subject, said regimen comprising a pharmaceutical composition comprising the recombinant factor VIII fusion protein of  claim 1 . 
     
     
         96 - 98 . (canceled) 
     
     
         99 . A recombinant factor VIII fusion protein used in the treatment of hemophilia A, comprising the recombinant factor VIII fusion protein of  claim 1 . 
     
     
         100 . A nucleic acid encoding the recombinant factor VIII fusion protein of  claim 1 , or the complement thereof. 
     
     
         101 - 107 . (canceled)