FGF21 mutants comprising a mutation at position 98, 171 and/or 180

1. An isolated polypeptide comprising an amino acid sequence of SEQ ID NO:4 or 8 with one or more substitutions, wherein the sequence is at least 85% identical to the amino acid sequence of SEQ ID NO: 4 or 8 and the one or more substitutions comprise

(a) a substitution of:

(i) the leucine residue at position 98;

(ii) the proline residue at position 171; or

(iii) the alanine residue at position 180,

wherein the substitution at position 98 is L98R, L98C, L98E, L98Q, L98K or L98T; the substitution at position 171 is P171A, P171R, P171N, P171D, P171C, P171E, P171Q, P171G, P171H, P171K, P171S, P171T, P171W or P171Y; and the substitution at position 180 is A180G, A180E, A180P or A180S; and

(b) at least one substitution selected from

(i) a cysteine mutation of Table 2;

(ii) an engineered disulfide bond of Table 3;

(iii) a stability enhancing mutation from Table 4;

(iv) a proteolysis-resistant mutation from Table 5;

(v) an aggregation mutation from Table 6;

(vi) a C-terminal degradation mutation from Table 7;

(vii) a glycosylation mutation from Table 8; and

(viii) an O-glycosylation-resistant mutation selected form Table 9;

wherein the at least one substitution is not at position 98 when the polypeptide comprises L98R, L98C, L98E, L98Q, L98K or L98T;

wherein the at least one substitution is not at position 171 when the polypeptide comprises P171A, P171R, P171N, P171D, P171C, P171E, P171Q, P171G, P171H, P171K, P171S, P171T, P171W or P171Y;

wherein the at least one substitution is not at position 180 when the polypeptide comprises A180G, A180E, A180P or A180S, and

wherein said polypeptide is an FGF receptor agonist.

2. The isolated polypeptide of claim 1 , 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.

3. The isolated polypeptide of claim 2 , comprising an 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.

4. The isolated protein of claim 1 , 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.

5. The isolated protein of claim 1 , comprising:

(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.

6. The isolated protein of claim 1 , comprising:

(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.

7. The isolated polypeptide of claim 1 , comprising:

(a) G, E, P or S at position 180;

(b) G, P, K, T, A, L or Pat position 181; and

(c) A P, G, S or A at position 179.

8. The isolated polypeptide of claim 1 , comprising S167A, S167E, S167D, S167N, S167Q, S167G, S167V, S167H, S167K or S167Y.

9. The isolated polypeptide of claim 1 , wherein the mutation at position 98 is L98R, the mutation at position 171 is P171G and the mutation at position 180 is A180E.

10. The isolated polypeptide of claim 1 , further comprising

(i) an N-terminal truncation of 8 or fewer residues;

(ii) a C terminal truncation of 12 or fewer residues; or

(iii) an N-terminal truncation of 8 or fewer residues and a C terminal truncation of 12 or fewer residues.

11. The isolated polypeptide of claim 10 , wherein the polypeptide is capable of lowering blood glucose in a mammal.

12. The isolated polypeptide of claim 1 , further comprising 1 to 10 amino acid residues fused to the C-terminus of the polypeptide.

13. The isolated polypeptide of claim 12 , wherein the 1 to 10 amino acid residues are selected from the group consisting of glycine, proline and combinations thereof.

14. The isolated polypeptide of claim 1 , wherein the polypeptide is covalently linked to one or more polymers.

15. The isolated polypeptide of claim 14 , wherein the polymer is PEG.

16. A fusion polypeptide comprising the isolated polypeptide of claim 1 fused to a peptide that increases stability.

17. The fusion polypeptide of claim 16 , wherein the peptide that increases stability is an IgG constant domain or fragment thereof.

18. The fusion polypeptide of claim 17 , wherein the IgG constant domain comprises the amino acid sequence of SEQ ID NO:171 or SEQ ID NO:11.

19. The fusion polypeptide of claim 17 , wherein the polypeptide is fused to the peptide that increase stability via a linker.

20. The fusion polypeptide of claim 19 , 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).

21. A multimer comprising two or more fusion polypeptides of claim 20 .

22. A pharmaceutical composition comprising the isolated polypeptide of claim 1 and a pharmaceutically acceptable formulation agent.

23. A method for reducing triglyceride levels, improving glucose tolerance, lowering blood glucose levels, or lowering insulin levels in a subject in need thereof comprising administering to the subject the pharmaceutical composition of claim 22 .

24. The method of claim 23 , wherein the subject has diabetes.

25. The method of claim 23 , wherein the subject has obesity.