Coagulation Factor VII polypeptides

Abstract

The present invention relates to novel coagulation Factor VII polypeptides, polynucleotide constructs encoding such polypeptides, as well as vectors and host cells comprising and expressing the polynucleotide, pharmaceutical compositions, uses and methods of treatment.

1 . A Factor VII polypeptide with a Tissue Factor binding affinity lower than recombinant wild type human Factor VIIa and substantially the same activity or increased activity compared to recombinant wild type human Factor VIIa, wherein said Factor VII polypeptide is selected from the group consisting of: 
 i) a Factor VII polypeptide comprising one or more amino acid substitutions at positions selected from the group consisting of: K18X 1 , R36X 2 , S43X 3 , K62X 4 , Q64X 5 , L65X 6 , I69X 7 , F71X 8 , L73X 9 , P74X 10 , E77X 11 , G78X 12 , R79X 13 , K85X 14 , Q88X 15 , N93X 16 , F275X 17 , R277X 18 , M306X 19 , T307X 20 , Q308X 21 , D309X 22 , or R379X 23  corresponding to amino acids at positions of SEQ ID NO: 1, wherein    X 1  is E, D, A, or F;    X 2  is E, D, K, A, F, or N;    X 3  is E, D, K, R, A, or F;    X 4  is D, A, or F;    X 5  is E, D, K, R, A, F, or N;    X 6  is E, D, K, R, A, or F;    X 7  is C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, or Y;    X 8  is A, G, H, I, K, L, M, P, R, S, T, V, or W;    X 9  is A, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W, or Y;    X 10  is A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, or Y;    X 11  is A, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;    X 12  is A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;    X 13  is A, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y;    X 14  is A, D, E, F, G, I, L, M, P, S, T, V, W, or Y;    X 15  is A, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y;    X 16  is A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, or Y;    X 17  is A, D, E, G, I, K, L, M, N, P, Q, R, S, T, V, W, or Y;    X 18  is A, D, E, F, G, H, I, K, L, M, P, Q, S, T, V, W, or Y;    X 19  is A, E, F, G, H, I, K, L, P, Q, R, S, T, V, W, or Y;    X 20  is A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y;    X 21  is A, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y;    X 22  is A, F, G, H, I, K, L, M, N, P, Q, R, V, W, or Y; and    X 23  is A, D, E, F, G, H, I, K, L, M, P, Q, S, T, V, W, or Y;    and/or    ii) a Factor VII polypeptide in which one or more amino acids corresponding to amino acids at positions selected from K18, R36, K62, Q64, L65, I69, F71, L73, P74, E77, G78, R79, K85, Q88, N93, F275, R277, M306, Q308, D309, or R379 of SEQ ID NO: 1 have been substituted with a cysteine amino acid residue, and wherein the cysteine amino acid residue may optionally be conjugated with a chemical group that increases the molecular weight of the Factor VII polypeptide;    and/or    iii) a Factor VII polypeptide comprising one or more N-glycosylation site N-Xaa-S/T introduced by amino acid substitutions corresponding to amino acids starting at positions selected from R36, Q64, I69, F71, P74, E77, G78, Q88, N93, F275, M306, T307, or D309 of SEQ ID NO: 1, wherein Xaa is any amino acid except P.    
     
     
         2 . The Factor VII polypeptide according to  claim 1 , further comprising one or more amino acid substitutions selected from the group consisting of L305V, L305V/M306D/D309S, L3051, L305T, F374P, V158T/M298Q, V158D/E296V/M298Q, K337A, M298Q, V158D/M298Q, L305V/K337A, V158D/E296V/M298Q/L305V, V158D/E296V/M298Q/K337A, V158D/E296V/M298Q/L305V/K337A, K157A, E296V, E296V/M298Q, V158D/E296V, V158D/M298K, and S336G, L305V/K337A, L305V/V158D, L305V/E296V, L305V/M298Q, L305V/V158T, L305V/K337A/V158T, L305V/K337A/M298Q, L305V/K337A/E296V, L305V/K337A/V158D, L305V/V158D/M298Q, L305V/V158D/E296V, L305V/V158T/M298Q, L305V/V158T/E296V, L305V/E296V/M298Q, L305V/V158D/E296V/M298Q, L305V/V158T/E296V/M298Q, L305V/V158T/K337A/M298Q, L305V/V158T/E296V/K337A, L305V/V158D/K337A/M298Q, L305V/V158D/E296V/K337A, L305V/V158D/E296V/M298Q/K337A, L305V/V158T/E296V/M298Q/K337A, S314E/K316H, S314E/K316Q, S314E/L305V, S314E/K337A, S314E/V158D, S314E/E296V, S314E/M298Q, S314E/V158T, K316H/L305V, K316H/K337A, K316H/V158D, K316H/E296V, K316H/M298Q, K316H/V158T, K316Q/L305V, K316Q/K337A, K316Q/V158D, K316Q/E296V, K316Q/M298Q, K316Q/V158T, S314E/L305V/K337A, S314E/L305V/V158D, S314E/L305V/E296V, S314E/L305V/M298Q, S314E/L305V/V158T, S314E/L305V/K337A/V158T, S314E/L305V/K337A/M298Q, S314E/L305V/K337A/E296V, S314E/L305V/K337A/V158D, S314E/L305V/V158D/M298Q, S314E/L305V/V158D/E296V, S314E/L305V/V158T/M298Q, S314E/L305V/V158T/E296V, S314E/L305V/E296V/M298Q, S314E/L305V/V158D/E296V/M298Q, S314E/L305V/V158T/E296V/M298Q, S314E/L305V/V158T/K337A/M298Q, S314E/L305V/V158T/E296V/K337A, S314E/L305V/V158D/K337A/M298Q, S314E/L305V/V158D/E296V/K337A, S314E/L305V/V158D/E296V/M298Q/K337A, S314E/L305V/V158T/E296V/M298Q/K337A, K316H/L305V/K337A, K316H/L305V/V158D, K316H/L305V/E296V, K316H/L305V/M298Q, K316H/L305V/V158T, K316H/L305V/K337A/V158T, K316H/L305V/K337A/M298Q, K316H/L305V/K337A/E296V, K316H/L305V/K337A/V158D, K316H/L305V/V158D/M298Q, K316H/L305V/V158D/E296V, K316H/L305V/V158T/M298Q, K316H/L305V/V158T/E296V, K316H/L305V/E296V/M298Q, K316H/L305V/V158D/E296V/M298Q, K316H/L305V/V158T/E296V/M298Q, K316H/L305V/V158T/K337A/M298Q, K316H/L305V/V158T/E296V/K337A, K316H/L305V/V158D/K337A/M298Q, K316H/L305V/V158D/E296V/K337A, K316H/L305V/V158D/E296V/M298Q/K337A, K316H/L305V/V158T/E296V/M298Q/K337A, K316Q/L305V/K337A, K316Q/L305V/V158D, K316Q/L305V/E296V, K316Q/L305V/M298Q, K316Q/L305V/V158T, K316Q/L305V/K337AN/158T, K316Q/L305V/K337A/M298Q, K316Q/L305V/K337A/E296V, K316Q/L305V/K337A/V158D, K316Q/L305V/V158D/M298Q, K316Q/L305V/V158D/E296V, K316Q/L305V/V158T/M298Q, K316Q/L305V/V158T/E296V, K316Q/L305V/E296V/M298Q, K316Q/L305V/V158D/E296V/M298Q, K316Q/L305V/V158T/E296V/M298Q, K316Q/L305V/V158T/K337A/M298Q, K316Q/L305V/V158T/E296V/K337A, K316Q/L305V/V158D/K337A/M298Q, K316Q/L305V/V158D/E296V/K337A, K316Q/L305V/V158D/E296V/M298Q/K337A, K316Q/L305V/V158T/E296V/M298Q/K337A, F374Y/K337A, F374Y/V158D, F374Y/E296V, F374Y/M298Q, F374Y/V158T, F374Y/S314E, F374Y/L305V, F374Y/L305V/K337A, F374Y/L305V/V158D, F374Y/L305V/E296V, F374Y/L305V/M298Q, F374Y/L305V/V158T, F374Y/L305V/S314E, F374Y/K337A/S314E, F374Y/K337A/V158T, F374Y/K337A/M298Q, F374Y/K337A/E296V, F374Y/K337A/V158D, F374Y/V158D/S314E, F374Y/V158D/M298Q, F374Y/V158D/E296V, F374Y/V158T/S314E, F374Y/V158T/M298Q, F374Y/V158T/E296V, F374Y/E296V/S314E, F374Y/S314E/M298Q, F374Y/E296V/M298Q, F374Y/L305V/K337A/V158D, F374Y/L305V/K337A/E296V, F374Y/L305V/K337A/M298Q, F374Y/L305V/K337A/V158T, F374Y/L305V/K337A/S314E, F374Y/L305V/V158D/E296V, F374Y/L305V/V158D/M298Q, F374Y/L305V/V158D/S314E, F374Y/L305V/E296V/M298Q, F374Y/L305V/E296V/V158T, F374Y/L305V/E296V/S314E, F374Y/L305V/M298Q/V158T, F374Y/L305V/M298Q/S314E, F374Y/L305V/V158T/S314E, F374Y/K337A/S314E/V158T, F374Y/K337A/S314E/M298Q, F374Y/K337A/S314E/E296V, F374Y/K337A/S314E/V158D, F374Y/K337A/V158T/M298Q, F374Y/K337A/V158T/E296V, F374Y/K337A/M298Q/E296V, F374Y/K337A/M298Q/V158D, F374Y/K337A/E296V/V158D, F374Y/V158D/S314E/M298Q, F374Y/V158D/S314E/E296V, F374Y/V158D/M298Q/E296V, F374Y/V158T/S314E/E296V, F374Y/V158T/S314E/M298Q, F374Y/V158T/M298Q/E296V, F374Y/E296V/S314E/M298Q, F374Y/L305V/M298Q/K337A/S314E, F374Y/L305V/E296V/K337A/S314E, F374Y/E296V/M298Q/K337A/S314E, F374Y/L305V/E296V/M298Q/K337A, F374Y/L305V/E296V/M298Q/S314E, F374Y/V158D/E296V/M298Q/K337A, F374Y/V158D/E296V/M298Q/S314E, F374Y/L305V/V158D/K337A/S314E, F374Y/V158D/M298Q/K337A/S314E, F374Y/V158D/E296V/K337A/S314E, F374Y/L305V/V158D/E296V/M298Q, F374Y/L305V/V158D/M298Q/K337A, F374Y/L305V/V158D/E296V/K337A, F374Y/L305V/V158D/M298Q/S314E, F374Y/L305V/V158D/E296V/S314E, F374Y/V158T/E296V/M298Q/K337A, F374Y/V158T/E296V/M298Q/S314E, F374Y/L305V/V158T/K337A/S314E, F374Y/V158T/M298Q/K337A/S314E, F374Y/V158T/E296V/K337A/S314E, F374Y/L305V/V158T/E296V/M298Q, F374Y/L305V/V158T/M298Q/K337A, F374Y/L305V/V158T/E296V/K337A, F374Y/L305V/V158T/M298Q/S314E, F374Y/L305V/V158T/E296V/S314E, F374Y/E296V/M298Q/K337A/V158T/S314E, F374Y/V158D/E296V/M298Q/K337A/S314E, F374Y/L305V/V158D/E296V/M298Q/S314E, F374Y/L305V/E296V/M298Q/V158T/S314E, F374Y/L305V/E296V/M298Q/K337A/V158T, F374Y/L305V/E296V/K337A/V158T/S314E, F374Y/L305V/M298Q/K337A/V158T/S314E, F374Y/L305V/V158D/E296V/M298Q/K337A, F374Y/L305V/V158D/E296V/K337A/S314E, F374Y/L305V/V158D/M298Q/K337A/S314E, F374Y/L305V/E296V/M298Q/K337A/V158T/S314E, F374Y/L305V/V158D/E296V/M298Q/K337A/S314E, S52A, S60A; R152E, S344A, P11Q/K33E, T106N, K143N/N145T, V253N, R290N/A292T, G291N, R315N/V317T; 
 K143N/N145T/R315N/V317T; one or more substitutions, additions or deletions in the amino acid sequence from 233Thr to 240Asn; and one or more substitutions, additions or deletions in the amino acid sequence from 304Arg to 329Cys.    
     
     
         3 . The Factor VII polypeptide according to  claim 1 , wherein the dissociation constant K d  of said Factor VII polypeptide is higher than 5 nM,  
     
     
         4 . The Factor VII polypeptide according to  claim 3 , wherein the dissociation constant K d  of said Factor VII polypeptide is higher than 20 nM.  
     
     
         5 . The Factor VII polypeptide according to  claim 4 , wherein the dissociation constant K d  of said Factor VII polypeptide is dissociation constant K d  of said Factor VII polypeptide is higher than 100 nM.  
     
     
         6 . The Factor VII polypeptide according to  claim 5 , wherein the dissociation constant K d  of said Factor VII polypeptide is dissociation constant K d  of said Factor VII polypeptide is higher than 1 μM.  
     
     
         7 . A composition comprising a Factor VII polypeptide according to  claim 1 .  
     
     
         8 . A pharmaceutical composition comprising a Factor VII polypeptide according to  claim 1  and a pharmaceutically acceptable carrier or diluent.  
     
     
         9 . A polynucleotide construct encoding a Factor VII polypeptide according to  claim 1 .  
     
     
         10 . The polynucleotide construct according to  claim 9 , wherein said construct is a vector.  
     
     
         11 . A eukaryotic host cell comprising the polynucleotide construct according to  claim 9 .  
     
     
         12 . The eukaryotic host cell according to  claim 11 , wherein said cell is of mammalian origin.  
     
     
         13 . The eucaryotic host cell according to  claim 12 , wherein said cell is selected from the group consisting of CHO cells, BHK cells or HEK cells.  
     
     
         14 . A method for producing the Factor VII polypeptide, the method comprising (i) cultivating a eukaryotic host cell according to  claim 11  in an appropriate growth medium under conditions allowing protein synthesis from said polynucleotide construct and (ii) recovering said Factor VII polypeptide from the culture medium.  
     
     
         15 . A method for the treatment of bleeding episodes or bleeding disorders in a subject or for the enhancement of the normal haemostatic system, the method comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a Factor VII polypeptide as defined in  claim 1.