Fgf21 mutants and uses thereof

Abstract

The invention provides nucleic acid molecules encoding FGF21 mutant polypeptides, FGF21 mutant polypeptides, pharmaceutical compositions comprising FGF21 mutant polypeptides, and methods for treating metabolic disorders using such nucleic acids, polypeptides, or pharmaceutical compositions.

What is claimed is: 
     
         1 . An isolated polypeptide comprising SEQ ID NO:4 or 8, wherein the isolated polypeptide further comprises:
 (a) the substitution of any amino acid for one or more of:
 (i) the leucine residue at position 98; 
 (ii) the proline residue at position 171; 
 (iii) the alanine residue at position 180; and 
   (b) one or more substitutions selected from the mutations of Tables 2-10.   
     
     
         2 . The isolated polypeptide of  claim 1 , wherein the one or more substitutions of (b) comprises one or more mutations is selected from the group consisting of:
 (a) a cysteine mutation of Table 2;   (b) an engineered disulfide bond of Table 3;   (c) a stability enhancing mutation from Table 4;   (d) a proteolysis-resistant mutation from Table 5;   (e) an aggregation mutation from Table 6;   (f) a C-terminal degradation mutation from Table 7;   (g) a glycosylation mutation from Table 8;   (h) an O-glycosylation-resistant mutation selected form Table 9;   (i) a mutation selected from Table 10; and   (j) combinations of (a)-(i).   
     
     
         3 . The isolated polypeptide of  claim 2 , wherein the cysteine mutation of (a) comprises a cysteine at a position selected from the group consisting of: 18-31, 33, 35-50, 54, 56-62, 64-73, 75-104, 106-135, 137-140, 152-154, 163 and 167. 
     
     
         4 . The isolated polypeptide of  claim 2 , wherein the engineered disulfide bond comprises a pair of cysteine residues at one or more positions selected from the group consisting of: 19-138, 20-139, 21-33, 22-137, 22-139, 23-25, 23-28, 24-135, 25-122, 26-122, 27-123, 28-43, 28-124, 31-43, 33-21, 35-84, 41-82, 42-124, 42-126, 43-124, 50-69, 54-66, 58-62, 67-72, 67-135, 72-84, 73-93, 75-85, 75-92, 76-109, 77-79, 77-81, 80-129, 82-119, 94-110, 95-107, 100-102, 102-104, 115-117, 117-129, 117-130, 118-132, 118-134, 121-127, 123-125, 127-132, and 152-163. 
     
     
         5 . The isolated protein of  claim 2 , wherein the stability enhancing mutation comprises a D, E, R, K, H, S, T, N or Q at one or more positions selected from the group consisting of: 42, 54, 77, 81, 86, 88, 122, 125, 126, 130, 131, 139, 145, 146, 152, 154, 156, 161, 163, 170, and 172. 
     
     
         6 . The isolated protein of  claim 2 , wherein the proteolysis resistant mutation is selected from the group consisting of:
 (a) Q, I or K at position 19;   (b) H, L or F at position 20;   (c) I, F, Y or V at position 21;   (d) I, F or V at position 22,   (e) A or R at position 150;   (f) A or V at position 151;   (g) H, L, F or V at position 152;   (h) A, D, N, C, Q, E, P, or S at position 170;   (i) A, R, N, D, C, E, Q, G, H, K, S, T, W or Y at position 171;   (j) L or T at position 172; and   (k) R or E at position 173.   
     
     
         7 . The isolated protein of  claim 2 , wherein the aggregation-reducing mutation is selected from the group consisting of:
 (a) E, K or R at position 26;   (b) E K, R, Q, or T at position 45;   (c) T at position 52;   (d) C, E or S at position 58;   (e) A, E, K or R at position 60;   (f) A, C, H or R at position 78;   (g) C or T at position 86;   (h) A, E, K, R or S at position 88;   (i) C, E, K, Q, or R at position 98;   (j) C, D, E, or R at position 99;   (k) K or T at position 111;   (l) D, E, H, K, N, R or Q at position 129; and   (m) E, H, K or Y at position 134.   
     
     
         8 . The isolated polypeptide of  claim 2 , wherein the C-terminal degradation mutation is selected form the group consisting of:
 (a) G, E, P or S at position 180;   (b) G, P, K, T, A, L or P at position 181; and   (c) A P, G, S or A at position 179.   
     
     
         9 . The isolated polypeptide of  claim 2 , wherein the O-glycosylation-resistant mutation is selected from the group consisting of: S167A, S167E, S167D, S167N, S167Q, S167G, S167V, S167H, S167K and S167Y. 
     
     
         10 . The isolated polypeptide of  claim 1 , wherein
 (a) the mutation at position 98 is selected from the group consisting of L98R, L98C, L98E, L98Q, L98K and L98T;   (b) the mutation at position 171 is selected from the group consisting of P171A, P171R, P171N, P171D, P171C, P171E, P171Q, P171G, P171H, P171K, P171S, P171T, P171W and P171Y;   (c) the mutation at position 180 is selected from the group consisting of A180G, A180E, A180P and A180S.   
     
     
         11 . The isolated polypeptide of  claim 10 , wherein the mutation at position 98 is L98R, the mutation at position 171 is P171G and the mutation at position 180 is A180E. 
     
     
         12 . The isolated polypeptide of any of  claims 1 - 11 , further comprising
 (i) an N-terminal truncation of 8 or fewer residues;   (ii) a C terminal truncation of 12 or fewer residues;   (iii) an N-terminal truncation of 8 or fewer residues and a C terminal truncation of 12 or fewer residues.   
     
     
         13 . The isolated polypeptide of  claim 12 , wherein the polypeptide is capable of lowering blood glucose in a mammal. 
     
     
         14 . The isolated polypeptide of  claim 1  or  11 , wherein the polypeptide comprises an amino acid sequence that is at least 85 percent identical to the amino acid sequence of SEQ ID NO: 4 or 8, but wherein if the polypeptide comprises L98R, P171G and A180E mutations, the L98R, P171G and A180E mutations are not further modified. 
     
     
         15 . The isolated polypeptide of any of  claim 1  or  11 , further comprising 1 to 10 amino acid residues fused to the C-terminus of the polypeptide. 
     
     
         16 . The isolated polypeptide of  claim 15 , wherein the 1 to 10 amino acid residues are selected from the group consisting of glycine, proline and combinations thereof. 
     
     
         17 . The isolated polypeptide of  claim 1  or  11 , wherein the polypeptide is covalently linked to one or more polymers. 
     
     
         18 . The isolated polypeptide of  claim 17 , wherein the polymer is PEG. 
     
     
         19 . A fusion polypeptide comprising the isolated polypeptide of any of  claim 1  or  11  fused to a heterologous amino acid sequence. 
     
     
         20 . The fusion polypeptide of  claim 19 , wherein the heterologous amino acid sequence is an IgG constant domain or fragment thereof. 
     
     
         21 . The fusion polypeptide of  claim 20 , wherein the IgG constant domain comprises the amino acid sequence of SEQ ID NO:171 or SEQ ID NO:11. 
     
     
         22 . The fusion polypeptide of  claim 20 , wherein the polypeptide is fused to the heterologous amino acid sequence via a linker. 
     
     
         23 . The fusion polypeptide of  claim 22 , wherein the linker is selected from the group consisting of polyalanines, (Gly) 4  (SEQ ID NO:29), (Gly) 5  (SEQ ID NO:30), (Gly) 5 -Ser-(Gly) 3 -Ser-(Gly) 4 -Ser (SEQ ID NO:28), (Gly) 4 -Ser-(Gly) 4 -Ser-(Gly) 4 -Ser (SEQ ID NO:31), (Gly) 3 -Lys-(Gly) 4  (SEQ ID NO:32), (Gly) 3 -Asn-Gly-Ser-(Gly) 2  (SEQ ID NO:33), (Gly) 3 -Cys-(Gly) 4  (SEQ ID NO:34), Gly-Pro-Asn-Gly-Gly (SEQ ID NO:35), Gly-Ser(Gly 4 Ser) 3  (SEQ ID NO:166), (Gly 4 Ser) 4  (SEQ ID NO:167), (Gly 4 S) 2  (SEQ ID NO:168), DAAAKEAAAKDAAAREAAARDAAAK (SEQ ID NO:169), and NVDHKPSNTKVDKR (SEQ ID NO:170). 
     
     
         24 . A multimer comprising two or more fusion polypeptides of  claim 23 . 
     
     
         25 . A pharmaceutical composition comprising the isolated polypeptide of any of  claims 1 - 24  and a pharmaceutically acceptable formulation agent. 
     
     
         26 . A method for treating a metabolic disorder comprising administering to a human patient in need thereof the pharmaceutical composition of  claim 25 . 
     
     
         27 . The method of  claim 26 , wherein the metabolic disorder is diabetes. 
     
     
         28 . The method of  claim 26 , wherein the metabolic disorder is obesity. 
     
     
         29 . An isolated nucleic acid encoding the polypeptide of any of  claims 1 - 24 . 
     
     
         30 . A vector comprising the nucleic acid molecule  claim 29 . 
     
     
         31 . A host cell comprising the nucleic acid molecule of  claim 29 .