WO2010065439A1

WO2010065439A1 - Variants of fibroblast growth factor 21

Info

Publication number: WO2010065439A1
Application number: PCT/US2009/066062
Authority: WO
Inventors: David Bruce Baldwin; Craig Duane Dickinson; David Albert Driver; Radmila Micanovic
Original assignee: Eli Lilly And Company
Priority date: 2008-12-05
Filing date: 2009-11-30
Publication date: 2010-06-10

Abstract

The present invention relates to novel polypeptide variants of human fibroblast growth factor 21. The invention embodies vectors and host cells for the propagation of nucleic acid sequences encoding said polypeptides and the production thereof. Also disclosed are methods for treating type 2 diabetes or obesity.

Description

VARIANTS OF FIBROBLAST GROWTH FACTOR 21

The present invention is in the field of medicine, particularly in the field of therapeutic proteins. Specifically, the present invention relates to the identification of variants of human fibroblast growth factor 21 (FGF-21) that are useful for the treatment of type 2 diabetes and obesity.

FGF-21 is a hormone that functions as an important metabolic regulator of glucose and lipid homeostasis. FGF-21 promotes glucose uptake in adipocytes by up- regulating GLUTl expression, a mechanism distinct from that of insulin. In diabetic rodents and monkeys, human FGF-21 lowered fasting serum concentrations of glucose, and reduced fasting serum concentrations of triglycerides, insulin and glucagon. FGF-21 also lowered serum LDL cholesterol concentrations and elevated HDL cholesterol in monkeys. Furthermore, FGF-21 transgenic mice were resistant to diet induced obesity and diabetic monkeys treated with FGF-21 showed evidence of weight loss. Thus, FGF- 21 has potential utility for treatment of diabetes and obesity.

Variants of FGF-21 with improved physical/functional properties have been described previously, e.g. WO2006/028595, WO2006/065582, and WO2005/061712. However, modifying a therapeutic protein to improve a specific property i.e. pharmaceutical stability, may impact the biological activity of the protein. Additionally, a problem associated with mature wild type human FGF -21 is the relatively low potency of the molecule.

An FGF-21 variant that is more potent than mature wild type FGF-21 would have several benefits among which is that efficacy could be achieved at lower doses. Consequently, certain embodiments of the present invention have increased potency and improved pharmaceutical stability compared to mature wild type human FGF-21. The

FGF-21 variants of the present invention impart additional benefits such as a reduction in manufacturing costs and an improvement in pharmaceutical formulations due to lower concentrations necessary for administration and/or a lower injection volume utilized. Thus, the variants of human FGF-21 described herein are expected to be useful to treat type 2 diabetes and obesity. The present invention further provides a variant of human FGF-21 comprising the amino acid sequence of SEQ ID NO: 171 :

Xaai Pro He Pro Asp Ser Ser Pro Leu Leu GIn Phe GIy GIy GIn VaI Arg GIn Arg Tyr Leu Tyr Thr Asp Asp Ala GIn GIn Thr GIu Ala Xaa32 Leu GIu He Arg GIu Asp GIy Thr VaI GIy GIy Ala Ala Asp Xaa47 Ser Pro GIu Ser Leu Leu GIn Leu Lys Ala Leu Lys Pro GIy VaI He Xaa64 He Leu GIy VaI Lys Thr Ser Arg Phe Leu Cys GIn Arg Xaayg Asp GIy Ala Leu Tyr GIy Ser Leu His Phe Asp Pro GIu Xaa₉2 Cys Ser Phe Arg GIu Leu Leu Leu Xaa^oi Asp GIy Tyr Asn VaI Tyr Xaa^08 Ser GIu Ala His GIy Leu Xaa^ 15 Leu His Leu Pro GIy Asn Lys Ser Pro His Arg Xaai 27 Xaai 28 Xaai 29 Xaai 30 Arg GIy Pro Ala Arg Phe Leu Pro Leu Pro GIy Xaai 42 Pro Pro Ala Leu Xaai 47 GIu Pro Xaai 50 GIy He Leu Xaai 54 P^ro G^m P^ro P^ro Asp VaI Xaai gi Ser Ser Asp Pro Xaai gg Ser Met VaI Xaai7o Pro Ser GIn Xaaj₇4 Xaai₇₅ Ser Pro Ser Tyr XaajgQ ^Ser (^SEQ ^{ID N0: 171})_> wherein:

Xaai is H, E, F, I, or deleted; Xaa32 is H, W, F, M, or Y; Xaa47 is Q or K; Xaag4 is Q, W, F, G, L, M, N, R, S, T, or V; Xaa₇₈ is P, G, or L; Xaa₉₂ is A, H, W, G, or F;

Xaa_1Oi is E, A, K, I, Q, N, D, G, H, M, R, or W; Xaa₁₀8 is Q or Y; Xaa! ₁₅ is P or W;

Xaa^27 is D, K or R; Xaa^8 i^s P_> R_> or N; Xaa^9 is A, M, R, Q, N or G; Xaa^g is P, W, K, or R; Xaa₁₄₂ is L, P, D, T, S, I, K, M, N, R, or V; Xaa₁₄₇ is P, R, V, A, M, or S;

Xaai5o is P or F; Xaa^4 is A, W, or Q; Xaa^gi is G, E, N, F, L, K, or W; Xaa^gg is L or Y; Xaai7o is G, L, M, Q, W, or Y; Xaa^4 is G, L, Y, or F; Xaa^ is R, Q, L, or E; and Xaa 180 is A or Y; and, wherein the amino acid sequence of the variant of human FGF-21 is not mature human FGF -21 (SEQ ID NO: 1) and wherein said variant optionally has one or more substitutions from the group consisting of:

(a) the substitution of a cysteine at two, four, six, eight, of the following positions:

A26, A31, G43, G80, Ll 14, Hl 17, Ll 18, P133, or A134; (b) the substitution of S 167 with an amino acid from the group consisting of A, D, E, F, G, H, I, K, L, N, Q, R, T, V, W, or Y;

(c) the substitution of N121 with an amino acid from the group consisting of A, C, D, E, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y; (d) the substitution of L153 with an amino acid selected from the group consisting ofA, C, V, P, F, R, W, N, D, Q, E, H or M;

(e) the substitution of Sl 81 with A, D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, or

Y;

(f) the substitution of M 168 with an amino acid selected from the group consisting of I, L or Y; or

(g) the truncation at the N-terminus of one to 4 amino acids. Particular variants of SEQ ID NO: 171 are those wherein:

Xaaj is H, E, or deleted; Xaa32 is H or Y; Xaag4 is Q, L, V, or F; Xaayg is P or

G; Xaa92 is A or H; Xaai oi is E or A; Xaai Qg is Q or Y; Xaa^ 15 is P or W; Xaai 27 is D or K; Xaai 2g is P or N; Xaai 29 is A or M; Xaai 30 is P, R, or K; Xaai 42 is L or R;

Xaai 47 ^ιs P_> R_> V, or M; Xaai 54 is A or W; Xaai gi is G or E; Xaai gg is L or Y; Xaai 70 is G or Y; Xaai 74 is G, Y, F, or L; Xaai 75 is R, L, E, or Q; and, Xaai go is A or Y; and wherein said variant optionally has one or more substitutions from the group consisting of cysteine is substituted at A31 and G43; S 167 is substituted with A, R, or L; N 121 is substituted with G or A; L 153 is substituted with E or A; M 168 is substituted with I, L, or Y; S 181 is substituted with G; or, HisProIlePro is truncated from the N-terminus. Additional variants of SEQ ID NO: 171 are those wherein: Xaai is H ^or deleted; Xaa32 is H; Xaag4 is Q or L; Xaa7g is P; Xaa92 is A; Xaai 01 ^ιs E; Xaai Qg is Q; Xaai 15 is P or W; Xaai 27 is D or K; Xaai 2g is P or N; Xaai 29 i^s A; Xaai 3Q is P, R, or K; Xaai 42 is L; Xaai 47 i^s P or R; Xaai 54 is A; Xaai gi is G; Xaai 66 ^ιs L or Y; Xaa^o is G; Xaaj74 is G, F, or L; Xaai 75 is R or L; and, Xaai 80 i^s A ^or Y' ^an^ wherein said variant optionally has one or more substitutions from the group consisting of cysteine is substituted at A31 and G43; S 167 is substituted with R; N 121 is substituted with G or A; Ll 53 is substituted with E or A; M 168 is substituted with L; S 181 is substituted with G; or, HisProIlePro is truncated from the N-terminus. Additional variants of SEQ ID NO: 171 are those wherein: Xaai is H or deleted; Xaa32 is H; Xaag4 is L; Xaayg is P; Xaa92 is A; Xaa^oi is E; XaaiO8 is Q; X^aal 15 is W; Xaa^27 is K; Xaa^28 is N; Xaa^29 is A; Xaa^g is P, R, or K; Xaa^42 is L; Xaa^47 is P or R; Xaa^4 is A; Xaa^l is G; Xaa^gg is L or Y; Xaai70 is G; Xaa^4 is G, F, or L; Xaa^ is R or L; and, Xaa^gg is A or Y; and wherein said variant optionally has one or more substitutions from the group consisting of cysteine is substituted at A31 and G43; S 167 is substituted with R; N 121 is substituted with G or A; L 153 is substituted with E or A; M 168 is substituted with L; S 181 is substituted with G; or, HisProIlePro is truncated from the N-terminus. Additional variants of SEQ ID NO: 171 are those wherein: Xaa^ is H or deleted; Xaa32 is H; Xaag4 is L; Xaayg is P; Xaa92 is A; Xaa^oi is

E; XaaiO8 is Q; X^aal 15 is W; Xaa^27 is K; Xaa^28 is N; Xaa^29 is A; Xaa^o is R or K; Xaa^42 is L; Xaa^47 is P or R; Xaa^4 is A; Xaa^i is G; Xaa^gg is L or Y; Xaai 70 is G; Xaa^74 is F or L; Xaa^75 is R or L; and, Xaa^go is A or Y; and wherein said variant optionally has one or more substitutions from the group consisting of cysteine is substituted at A31 and G43; S 167 is substituted with R; N121 is substituted with G or A; L153 is substituted with E or A; M168 is substituted with L; S181 is substituted with G; or, HisProIlePro is truncated from the N-terminus.

Additional variants of SEQ ID NO: 171 are those wherein:

Xaai is H or deleted; Xaa32 is H, Y, or F; Xaa54 is Q, L, V, or F; Xaa7g is P;

Xaa92 is A; Xaai gi is E; Xaai Q8 is Q; Xaa^ 15 is P or W; Xaai 27 i^s D ^or ^ ^^aa128 i^s P or N; Xaa^29 is A; Xaa^o is P, R, or K; Xaa^42 is L; Xaa^47 is P, R, V, or M; Xaa^54 is A; Xaa^l is G; Xaa^66 is L or Y; Xaa^o is G; Xaa^4 is G, Y, F, or L; Xaa^75 is R, L, E, or Q; and, Xaa^go is A or Y.

Additional variants of SEQ ID NO: 171 are those wherein: Xaa^ is H or deleted; Xaa32 is H; Xaag4 is Q; Xaayg is P; Xaa92 is A; Xaa^gi is E; Xaa^08 is Q; X^aa115 is P or W; Xaa^27 is D or K; Xaa^28 *^s P or N; Xaa^29 is A; Xaai 30 is P; Xaai 42 is L; Xaai 47 is P; Xaai 54 is A; Xaai 51 is G; Xaai 55 is L or Y; Xaai 70 is G; Xaai 74 is G, Y, F, or L; Xaai 75 is R, L, E, or Q; and, Xaai gg is A or Y. The invention provides variants of human FGF-21 wherein the amino acid sequence differs from SEQ ID NO: 1 at one, two, three, four, five, six, seven, eight, nine, ten, or more of the variable positions of SEQ ID NO: 171.

The invention further provides variants of human FGF-21 wherein said variant is selected from the group consisting of L166Y/A180Y (SEQ ID NO: 35), L166Y/G174F (SEQ ID NO: 36), L166Y/G174Y (SEQ ID NO: 52), L166Y/R175L (SEQ ID NO: 97),

D127K/P130W (SEQ ID NO: 106), and Pl 15W/L166Y (SEQ ID NO: 111).

The invention further provides variants of human FGF-21 wherein said variant is selected from the group consisting of Pl 15W/L166Y/A180Y (SEQ ID NO: 33),

D127R/L166Y/G174F (SEQ ID NO: 51), D127R/P130K/L166Y (SEQ ID NO: 110), P115W/D127K/A180Y (SEQ ID NO: 80), and D127R/L166Y/A180Y (SEQ ID NO: 81); with more preferred variants selected from the group consisting of Pl 15W/L166Y/G174Y

(SEQ ID NO: 89), and D127R/P128N/L166Y (SEQ ID NO: 74).

The invention further provides variants of human FGF-21 wherein said variant is selected from the group consisting of Pl 15W/L166Y/G174Y/A180Y (SEQ ID NO: 9), P115W/D127K/P128R/L166Y (SEQ ID NO: 25), D127K/P128R/P130K/L166Y (SEQ ID

NO: 28), Q47K/P115W/P150F/R175L (SEQ ID NO: 112) and

D127R/P128N/P130R/L166Y (SEQ ID NO: 49); with more preferred variants selected from the group consisting of Pl 15W/P128R/P130K/R175L (SEQ ID NO: 5),

P115W/P128N/P130K/G174Y (SEQ ID NO: 60), P115W/P128R/P130R/L166Y (SEQ ID NO: 71), and P115W/D127K/P130R/R175Q (SEQ ID NO: 77).

The invention further provides variants of human FGF-21 wherein said variant is selected from the group consisting of Pl 15W/D127R/P128N/P130R/L166Y (SEQ ID

NO: 12), D127R/P128R/L166Y/G174L/R175L (SEQ ID NO: 86),

D127R/P128N/P130K/G174L/A180Y (SEQ ID NO: 69), Q108Y/P115W/P130R/R175E/A180Y (SEQ ID NO: 113) and D127R/P128R/P130K/L166Y/G174Y (SEQ ID NO: 161); with more preferred variants selected from the group consisting of Pl 15W/D127K/P130R/G174F/A180Y (SEQ ID NO: 42), P115W/L166Y/G174Y/R175L/A180Y (SEQ ID NO: 56), P115W/D127K/P130R/G174Y/R175L (SEQ ID NO: 90), and D127K/P128N/P130K/L166Y/R175E (SEQ ID NO: 104).

The invention further provides variants of human FGF-21 wherein said variant is selected from the group consisting of

P115W/D127R/P128R/L166Y/G174L/R175L (SEQ ID NO: 11), D127K/P128N/P130W/G174Y/R175E/A180Y (SEQ ID NO: 61), P115W/D127R/P128N/P130K/R175E/A180Y (SEQ ID NO: 67), and,

Pl 15W/D127K/P128N/P130K/G174F/R175E (SEQ ID NO: 91); with more preferred variants selected from the group consisting of P115W/D127R/P128N/P130R/G174F/R175L (SEQ ID NO: 7), and D127R/P128N/P130W/G174Y/R175Q/A180Y (SEQ ID NO: 24), The invention further provides variants of human FGF-21 wherein said variant is selected from the group consisting of

D127R/P128N/P130W/L166Y/G174Y/R175E/A180Y (SEQ ID NO: 29), D127K/P128N/P130R/L166Y/G174L/R175Q/A180Y (SEQ ID NO: 63), D127R/P128R/P130K/L166Y/G174F/R175E/A180Y (SEQ ID NO: 92), and D127K/P128N/P130R/L166Y/G174L/R175L/A180Y (SEQ ID NO: 94); with more preferred variants selected from the group consisting of P115W/D127K/P128N/L166Y/G174L/R175L/A180Y (SEQ ID NO: 82), and P115W/D127K/P128R/P130R/L166Y/G174F/R175L (SEQ ID NO: 162).

P115W/D127R/P128R/P130K/L166Y/G174L/R175Q/A180Y (SEQ ID NO: 59), and P115W/D127R/P128R/P130R/L166Y/G174L/R175Q/A180Y (SEQ ID NO: 79).

The invention further provides variants of human FGF-21 wherein said variant is selected from the group consisting of H32Y/Q64L/P115W/D127K/P128N/L166Y/G174F/R175L/A180Y (SEQ ID NO: 177), and Q64L/P115W/D127K/P128N/L166Y/S167R/G174L/R175L/A180Y (SEQ ID NO: 178).

The invention further provides variants of human FGF-21 wherein said variant is selected from the group consisting of Q64L/P115W/D127K/P128N/P147R/L166Y/S167R/G174F/R175L/A180Y (SEQ ID NO: 179).

The present invention further provides a variant of human FGF-21 comprising the amino acid sequence of SEQ ID NO: 172:

His Pro He Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln VaI Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala Xaa32 Leu Glu He Arg Glu Asp Gly Thr VaI

Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly VaI He Xaag4 He Leu Gly VaI Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu

Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn VaI Tyr Gln Ser Glu Ala His Gly Leu Xaa^ 15 Leu His Leu Pro Gly Asn Lys

Ser Pro His Arg Xaai 27 -^^aa128 Ala -^^aa130 Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Xaa^y Glu Pro Pro Gly He Leu Ala Pro Gln Pro Pro Asp VaI Gly Ser Ser Asp Pro Xaa^gg Ser Met VaI Gly Pro Ser Gln Xaai 74 Xaa^ Ser Pro Ser Tyr Xaaigo Ser (SEQ ID NO: 172), wherein: Xaa₃₂ is H, W, F, M, or Y; Xaa₆₄ is Q, W, F, G, L, M, N, R, S, T, or V; Xaai ₁₅ is P or W; Xaaj27 is D, K or R; Xaai28 ^p _> ^R _> or N; Xaa^Q is P, W, K, or R; Xaa^ is P, R, V, A, M, or S; Xaai gg is L or Y; Xaai 74 is G, L, Y, or F; Xaai 75 is R, Q, L, or E; and Xaai 80 ^ιs A ^or Y> ^and> wherein the amino acid sequence of the variant of human FGF-21 is not mature human FGF -21 (SEQ ID NO: 1) and wherein said variant optionally has one or more substitutions from the group consisting of:

(a) the substitution of a cysteine at two, four, six, or eight of the following positions: A26, A31, G43, G80, Ll 14, Hl 17, Ll 18, P133, or A134; (b) the substitution of S 167 with an amino acid from the group consisting of A, D, E, F, G, H, I, K, L, N, Q, R, T, V, W, or Y;

Y;

(g) the truncation at the N-terminus of one to 4 amino acids.

The present invention further provides a variant of human FGF-21 comprising the amino acid sequence of SEQ ID NO: 173:

His Pro He Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln VaI Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu He Arg Glu Asp Gly Thr VaI Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly VaI He Gln He Leu Gly VaI Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr

Asn VaI Tyr Gln Ser Glu Ala His Gly Leu Xaai 15 Leu His Leu Pro Gly Asn Lys Ser Pro

His Arg Xaai 27 Xaai 2g Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro

Pro Ala Leu Pro Glu Pro Pro Gly He Leu Ala Pro Gln Pro Pro Asp VaI Gly Ser Ser Asp Pro Xaai 66 Ser Met VaI Gly Pro Ser Gln Xaai 74 Xaai 75 Ser Pro Ser Tyr Ala Xaai gg Ser (SEQ ID NO: 173)

wherein:

Xaai 15 is P or W; Xaai27 is D, K or R; Xaai28 P, R, or N; Xaaigg is L or Y; Xaai 74 is G, L, Y, or F; and, Xaai 75 *^s R_> Q_> L, or E; XaaigQ is A or Y; and, wherein the amino acid sequence of the variant of human FGF-21 is not mature human FGF-21 (SEQ ID NO: 1) and wherein said variant optionally has one or more substitutions from the group consisting of: (a) the substitution of a cysteine at two, four, six, or eight of the following positions: A26, A31, G43, G80, Ll 14, Hl 17, Ll 18, P133, or A134;

(b) the substitution of S 167 with an amino acid from the group consisting of A, D, E, F, G, H, I, K, L, N, Q, R, T, V, W, or Y; (c) the substitution of N121 with an amino acid from the group consisting of A,

C, D, E, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y;

(d) the substitution of L153 with an amino acid selected from the group consisting ofA, C, V, P, F, R, W, N, D, Q, E, H or M;

(e) the substitution of Sl 81 with A, D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, or Y;

(g) the truncation at the N-terminus of one to 4 amino acids.

The invention further provides a variant of human FGF-21 wherein said variant is selected from the group consisting of

A31C/G43C/P115W/D127K/P128N/L166Y/G174L/R175L/A180Y (SEQ ID NO: 114),

A31C/G43C/P115W/D127K/P128N/L153E/L166Y/G174L/R175L/A180Y (SEQ ID NO:

129), A31C/G43C/P115W/N121G/D127K/P128N/L166Y/G174L/R175L/A180Y (SEQ ID NO:

132),

A31C/G43C/P115W/D127K/P128N/L153E/L166Y/S167R/M168L/G174L/R175L/A180

Y (SEQ ID NO: 141), and,

A31C/G43C/P115W/N121A/D127K/P128N/L153E/L166Y/S167A/M168L/G174L/R175 L/A180Y/S181G (SEQ ID NO: 147).

<fø?HPIP-A31C/G43C/Pl 15W/D127K/P128N/L166Y/G174L/R175L/A180Y (SEQ ID

NO: 149), (/esHPIP-A31C/G43C/P115W/D127K/P128N/L153E/L166Y/G174L/R175L/A180Y (SEQ ID NO: 150),

(/esHPIP-A31C/G43C/P115W/N121G/D127K/P128N/L166Y/G174L/R175L/A180Y (SEQ ID NO: 151), (/esHPIP-A31C/G43C/P115W/D127K/P128N/L153E/L166Y/S167R/M168L/G174L/ R175L/A180Y (SEQ ID NO: 152), and, desHPIP-AS lC^SC/Pl lSW/NmA/DmK/PmN/LlSSE/LlόόY/S^A/MlόδL/ G174L/R175L/A180Y/S181G (SEQ ID NO: 153).

The invention further provides variants of human FGF -21 wherein the variant is selected from the group consisting of the variants shown in Table 5; wherein preferred variants are selected from the group consisting of H32Y, A45K, Q47K, Q64L, Q76I, ElOlA, P115W, D127K, D127R, P128N, L142R, P147R, L166Y, S167R, G170Y, G174L, G174Y, R175E, R175L, and A180Y; with more preferred variants selected from the group consisting of P128R, P130K, P130R, G161E, and G174F. The invention further provides a variant of human FGF-21 wherein said variant has greater potency than the protein of SEQ ID NO: 1 as measured in the 3T3-L1 adipocyte GLUTl assay (Example 1) and the 293 β-Klotho-SRE-luc assay (Example 3).

The invention further provides a variant of human FGF-21 wherein said variant has improved pharmaceutical stability than the protein of SEQ ID NO: 1 when analyzed under simulated physiological and pharmaceutical formulation conditions (Example 6).

The invention provides a composition, preferably a pharmaceutical composition, comprising a variant of human FGF-21 of the invention and a pharmaceutically acceptable excipient.

The invention provides a method for treating type 2 diabetes and obesity comprising administering a therapeutically effective amount of a variant of human FGF-

21 of the invention to a patient in need thereof.

The invention embodies a variant of human FGF-21 according to the present invention for use as a medicament.

The invention embodies a variant of human FGF-21 according to the present invention for use in the treatment of type 2 diabetes and obesity. The invention further provides the use of a variant of human FGF-21 of the invention for the manufacture of a medicament for the treatment of type 2 diabetes and obesity.

Other embodiments of the invention are drawn to polynucleotides encoding the variants of human FGF-21, said polynucleotides are selected from the group consisting of SEQ ID NO: 154 (which encodes the amino acid sequence of SEQ ID NO: 147), SEQ ID NO: 155 (which encodes the amino acid sequence of SEQ ID NO: 42), SEQ ID NO: 156 (which encodes the amino acid sequence of SEQ ID NO: 7), SEQ ID NO: 157 (which encodes the amino acid sequence of SEQ ID NO: 59), SEQ ID NO: 158 (which encodes the amino acid sequence of SEQ ID NO: 82), and SEQ ID NO: 159 (which encodes the amino acid sequence of SEQ ID NO: 89). Another embodiment is a vector containing said polynucleotides and a host cell carrying said vector. Another embodiment is drawn to processes for producing a polypeptide by culturing host cells carrying said vector containing DNA encoding said polypeptide, expressing said polypeptide from the host cells and recovering the polypeptide from the culture media.

Brief Description of the Figures: Figure 1 is the consensus amino acid sequence (SEQ ID NO: 3) of the human FGF-21 variants of the present invention shown in Table 5 that have increased potency after a single amino acid substitution.

For purposes of the present invention, as disclosed and claimed herein, the following terms are as defined below.

Full length human wild type FGF-21 is a 208 amino acid polypeptide containing a 27 amino acid signal sequence. Mature human wild type FGF-21 comprises the full length polypeptide minus the 27 amino acid signal sequence resulting in a 181 amino acid polypeptide. Mature human wild type FGF-21 (SEQ ID NO: 1) is the polypeptide template for the variants of the present invention.

His Pro He Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln VaI Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu He Arg Glu Asp Gly Thr VaI Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly VaI He Gln He Leu Gly VaI Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn VaI Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly He Leu Ala Pro Gln Pro Pro Asp VaI Gly Ser Ser Asp Pro Leu Ser Met VaI Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser (SEQ ID NO: 1)

Thus, the amino acid positions of the variants of the present invention are determined from the 181 amino acid polypeptide of mature human wild type FGF -21 (SEQ ID NO: 1) encoded by the DNA sequence of SEQ ID NO:2.

Mutations are designated by the original amino acid, followed by the number of the amino acid position, followed by the replacement amino acid. The numerical designation of each variant is based on (SEQ ID NO: 1). For example, a substitution for glutamic acid at position 30 (i.e. E30) with alanine (A) is designated as E30A. If there are multiple substitutions possible at the same position, for example a substitution for His (H) 117 with VaI (V), Trp (W), or Tyr (Y), they are designated as Hl 17V, W, or Y. In a similar fashion, the multiple substitutions for Gln (Q) at position 47 with Lys (K), Pro (P) at position 115 with Trp (W), Pro (P) at position 150 with Phe (F) and Arg (R) at position 175 with Leu (L) is designated as Q47K/P115W/P150F/R175L. A variant of human FGF-21 is defined as human FGF-21 in which at least one amino acid of the wild type mature protein has been substituted by another amino acid and the substitution results in some modified property, structural or functional, of the wild-type mature protein.

A FGF-21 derivative is defined as a molecule having the amino acid sequence of human FGF-21 (SEQ ID NO: 1) or a FGF-21 variant but additionally having at least one chemical modification of one or more of its amino acid side groups, α-carbon atoms, terminal amino group, or terminal carboxylic acid group. For example, PEGylation of human FGF-21 or a human FGF-21 variant with polyethylene glycol results in a FGF-21 derivative. A therapeutically effective amount is the amount of an active agent necessary to impart a therapeutic benefit to a patient. For example, a therapeutically effective amount to a human patient suffering from type 2 diabetes, dyslipidimia, obesity, or metabolic syndrome is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression, physiological conditions associated with or resistance to succumbing to the afore mentioned disorders.

Type 2 diabetes is characterized by excess glucose production in spite of the availability of insulin, and circulating glucose levels remain excessively high as a result of inadequate glucose clearance. Obesity is defined as an excess of subcutaneous fat in proportion to lean body mass.

Metabolic syndrome is characterized by a group of metabolic risk factors in one person. They include: abdominal fat — in most men, a 40-inch waist or greater; high blood sugar — at least 110 milligrams per deciliter (mg/dl) after fasting; high triglycerides — at least 150 mg/dL in the bloodstream; low HDL — less than 40 mg/dl; and, blood pressure of 130/85 or higher.

Dyslipidemia can be defined as a disorder of lipoprotein metabolism, including lipoprotein overproduction or deficiency. Dyslipidemia may be manifested by elevation of the total cholesterol, low-density lipoprotein (LDL) cholesterol and the triglyceride concentrations, and a decrease in high-density lipoprotein (HDL) cholesterol concentration in the blood. Dyslipidemia comes under consideration in many situations including diabetes, a common cause of lipidemia.

The term potency or biological activity is a measurement of the relative up regulation of GLUT 1 mRNA induced by the treatment of murine 3 T3 -L 1 adipocytes with an FGF-21 variant as described in the bioassay in Example 1. This relative up regulation is compared to the wild type FGF-21 standard curve to yield a relative potency for the FGF-21 variant. Alternatively, potency is a measurement of the ability of the variants to affect luciferase expression in human 293-β-Klotho-SRE luc reporter gene assay as described in Example 3. Additionally, potency is a measurement of the ability of the variants of the present invention to lower plasma glucose levels in vivo as measured in the ob/ob mouse models as described in Examples 4 and 5.

Protein stability is defined primarily in terms of the conformational, thermodynamic and kinetic stability of a protein i.e. the protein unfolds and refolds rapidly, reversibly, and cooperatively, and maintains its biological function as a pharmaceutical composition under physiological conditions.

Variants of human FGF-21 of the present invention are summarized in Table 1. All the variants were made and compared in potency to wild type FGF-21 in the GLUTl assay (Example 1) and/or the 293 β-Klotho assay (Example 3). The potency of the variants of human FGF-21 shown in Table 1 as measured in the these assays was from about 2 fold to about 53 fold greater than the potency of wild type FGF-21. Table 1

Preferred variants of human FGF-21 shown in Table 1 and the relative fold increase (FI) of activity compared to mature human FGF-21 are: P115W/D127K/P130R/R175Q [SEQ ID NO: 77] (22.8 FI); P115W/D127K/P130R/G174F/A180Y [SEQ ID NO: 42] (53.4 FI);

Pl 15W/D127R/P128N/P130R/G174F/R175L [SEQ ID NO: 7] (35.8 FI); P115W/D127K/P128N/L166Y/G174L/R175L/A180Y [SEQ ID NO: 82] (34.4 FI); and P115W/D127R/P128R/P130K/L166Y/G174L/R175Q/A180Y [SEQ ID NO:59] (34.9 FI). Another embodiment of the present invention provides variants of human FGF-21 comprising the substitution of an amino acid residue at one to eight of the positions of SEQ ID N0:3 in combination with the substitution of a cysteine from two, four, six or eight of the positions selected from the group consisting of: A26, A31, G43, G80, Ll 14, Hl 17, Ll 18, P133, or A134, wherein said proteins have improved physical stability compared to mature wild type human FGF-21. Preferred variants of this embodiment include any protein with the substitution of an amino acid residue at one to eight of the positions of SEQ ID N0:3 in combination with a substitution selected from the group consisting of A26C/P133C (SEQ ID NO: 163); A31C/G43C (SEQ ID NO: 164); G80C/L114C (SEQ ID NO: 165); H117C/A134C (SEQ ID NO: 166); or L118C/A134C (SEQ ID NO: 167). The most preferred variant of this embodiment includes any protein with the substitution of an amino acid residue at one to eight of the positions of SEQ ID N0:3 in combination with A31C/G43C (SEQ ID NO: 164). Yet another embodiment of the present invention provides variants of human FGF-21 comprising the substitution of an amino acid residue at one to eight of the positions of SEQ ID NO: 3 in combination with the substitution of S 167 with an amino acid from the group consisting of A, D, E, F, G, H, I, K, L, N, Q, R, T, V, W, or Y, wherein said proteins have reduced capacity for O-glycosylation when expressed in yeast compared to mature wild type human FGF-21. Such O-glycosylation may introduce heterogeneity and/or new immunological determinants on a protein and may therefore be antigenic when administered to humans; may alter the pharmacokinetic properties of a protein; and/or may affect the biological activity of a protein. The most preferred variants of this embodiment include any protein with the substitution of an amino acid residue at one to eight of the positions of SEQ ID NO:3 in combination with variants from the group consisting of S167A, S167R, and S167L.

Yet another embodiment of the present invention provides variants of human FGF-21 comprising the substitution of an amino acid residue at one to eight of the positions of SEQ ID NO: 3 in combination with the substitution of N 121 with an amino acid from the group consisting of V, L, F, G, H, W, D, S, M, Y, T, A, Q, E, C, or R wherein said variants have reduced deamidation of position 121 compared to wild type FGF-21. Preferred variants of this embodiment are N121G and N121A in combination with the substitution of an amino acid residue at one to eight of the positions of SEQ ID NO:3.

Another embodiment of the present invention provides variants of human FGF-21 comprising the substitution of an amino acid residue at one to eight of the positions of SEQ ID NO:3 in combination with the substitution of L153 with an amino acid selected from the group consisting of H, A, E, F, M, P, Q, V, or W wherein said variants have reduced susceptibility for proteolytic degradation compared to mature wild-type human FGF-21. Preferred variants of this embodiment are L153E and L153A in combination with the substitution of an amino acid residue at one to eight of the positions of SEQ ID NO:3.

Still yet another embodiment of the present invention provides variants of human FGF-21 comprising the substitution of an amino acid residue at one to eight of the positions of SEQ ID NO: 3 in combination with the substitution of S 181 with an amino acid from the group consisting of A, D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, or Y wherein said variants have reduced susceptibility for proteolytic degradation at the C- terminus compared to mature wild-type human FGF-21. A preferred variant of this embodiment is S 181 G in combination with the substitution of an amino acid residue at one to eight of the positions of SEQ ID NO:3.

Still yet another embodiment of the present invention provides variants of human FGF-21 comprising the substitution of an amino acid residue at one to eight of the positions of SEQ ID NO:3 in combination with the substitution of M168 with an amino acid selected from the group consisting of I, L or Y wherein said variants have reduced susceptibility to oxidation compared to wild-type human FGF-21. A preferred variant of this embodiment is M168L in combination with the substitution of an amino acid residue at one to eight of the positions of SEQ ID NO:3.

Yet another embodiment of the present invention provides variants of FGF-21 comprising mature human wild-type FGF -21 truncated at the N-terminus by up to 4 amino acids in combination with the substitution of an amino acid residue at one to seven of the positions of SEQ ID NO:3. The N-terminus of human wild-type FGF-21 (HisProIlePro) contains two dipeptides that could potentially be substrates to dipeptidyl peptidase IV (DPP-IV), a serine type protease involved in inactivation of neuropeptides, endocrine peptides, and cytokines. This proteolysis may result in a fragment of FGF-21 truncated at the N-terminus by up to 4 amino acids. The 4 amino acid truncated fragment of mature wild type FGF-21 (βfesHPIP-FGF-21) has been shown previously to retain biological activity (see WO2006/065582, Examples 2 and 3). It is an embodiment of this invention that any variant of FGF-21 of the present invention may have the fifesHPIP truncation with minimal affect on the potency of the variant. Preferred variants of the present invention that have a βfesHPIP truncation are selected from the group consisting of (/esHPIP-A31C/G43C/P115W/D127K/P128N/L166Y/G174L/ R175L/A180Y (SEQ ID NO: 149); desΗPJP^lC/GttC/PUSVf/DmK/PnZN/LWE/LlGβY/GπiL/ R175L/A180Y (SEQ ID NO: 150); (/esHPIP-A31C/G43C/P115W/N121G/D127K/P128N/L166Y/G174L/R175L/A180Y (SEQ ID NO: 151); desHPIP-AS lC^SC/Pl lSW/D^K/PmN/LlSSE/LlόόY/S^R/MlόδL/ G174L/R175L/A180Y (SEQ ID NO: 152); and,

(/esHPIP-A31C/G43C/P115W/N121A/D127K/P128N/L153E/L166Y/S167L/ M168L/G174L/R175L/A180Y/S181G (SEQ ID NO: 153).

The present invention provides variants of human FGF-21 that impart advantages over mature human wild-type FGF-21 that include variants that have increased potency, improved pharmaceutical stability, as well as, reduced O-glycosylation, reduced deamidation, reduced proteolytic degradation, and/or reduced susceptibility to oxidation compared to human wild-type FGF-21. Examples of variants of human FGF-21 of the present invention are shown in Table 2.

All of the variants in Table 2 were made and compared in potency to wild type FGF-21 in the 3T3-L1 adipocyte GLUTl assay (Example 1) and/or the 293 β-Klotho- SRE-luc assay (Example 3). The potency of the variants of human FGF-21 shown in Table 2 as measured in these assays was from about 2 fold to about 47 fold greater than the potency of wild type FGF-21.

Table 2

Preferred variants of human FGF-21 shown in Table 2 and the relative fold increase (FI) of activity compared to mature human FGF-21 are:

A31C/G43C/P115W/D127K/P128N/L166Y/G174L/R175L/A180Y [SEQ ID NO: 114] (37.9 FI);

A31C/G43C/P115W/D127K/P128N/L153E/L166Y/G174L/R175L/A180Y [SEQ

ID NO: 129] (37.5 FI);

A31C/G43C/P115W/N121G/D127K/P128N/L166Y/G174L/R175L/A180Y [SEQ ID NO:

132] (38.9 FI); A31C/G43C/P115W/D127K/P128N/L153E/L166Y/S167R/M168L/G174L/R175L/A180

Y [SEQ ID NO: 141] (37.5 FI); and,

A31C/G43C/P115W/N121A/D127K/P128N/L153E/L166Y/S167L/M168L/G174L/R175

L/A180Y/S181G [SEQ ID NO: 147] (34 FI).

Unexpectedly, the variants of human FGF-21 combining the multiple embodiments of the present invention shown in Table 2 retained their high potency after as many as 14 amino acid substitutions. Thus, the variants in Table 2 provide both high potency and stability over numerous parameters when compared to wild type human

FGF-21.

The variants of FGF-21 of the present invention are achieved through generating appropriate gene sequences, i.e. by arranging the appropriate nucleotide sequences and expressing these in a suitable cell line. Desired nucleotide sequences can be produced using a method such as codon based mutagenesis. Such procedures permit the production of any and all frequencies of amino acid residues at any desired codon positions within an oligonucleotide. In applying such methods, it is to be appreciated that due to the degeneracy of the genetic code, such methods as random oligonucleotide synthesis and partial degenerate oligonucleotide synthesis will incorporate redundancies for codons specifying a particular amino acid residue at a particular position, although such methods can be used to provide a master set of all possible amino acid sequences and screen these for optimal function as therapeutic proteins or for other purposes. The resulting variants of human FGF-21 of the present invention may be synthesized by recombinant organisms engineered using methods well know in the art, or alternatively, by chemical synthesis.

The polynucleotides that encode for the variants of human FGF-21 of the present invention may include the following: only the coding sequence for the variant, the coding sequence for the variant and additional coding sequence such as a functional polypeptide, or a signal or secretory sequence or a pro-protein sequence; the coding sequence for the variant and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence for the variant. Thus the term "polynucleotide encoding a variant" encompasses a polynucleotide that may include not only the coding sequence for the variant but also a polynucleotide which includes additional coding and/or non-coding sequence. It is known in the art that a polynucleotide sequence that is optimized for a specific host cell/expression system can readily be obtained from the amino acid sequence of the desired protein (see GeneArt, Toronto, Canada at geneart.com).

Examples of polynucleotides of the present invention are selected from the group consisting of SEQ ID NO: 154 (which encodes the amino acid sequence of SEQ ID NO: 147), SEQ ID NO: 155 (which encodes the amino acid sequence of SEQ ID NO: 42), SEQ ID NO: 156 (which encodes the amino acid sequence of SEQ ID NO: 7), SEQ ID NO: 157 (which encodes the amino acid sequence of SEQ ID NO: 59), SEQ ID NO: 158 (which encodes the amino acid sequence of SEQ ID NO: 82), and SEQ ID NO: 159 (which encodes the amino acid sequence of SEQ ID NO: 89). The polynucleotides of the present invention are expressed in a host cell after the sequences have been operably linked to an expression control sequence. These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers, e.g., tetracycline, neomycin, and/or dihydrofolate reductase, to permit detection of those cells transformed with the desired DNA sequences. The vectors containing the polynucleotide sequences of interest (e.g., the variants of FGF -21 and expression control sequences) are transferred into a host cell by well-known methods, which vary depending on the type of cellular host.

The variants of FGF-21 of the present invention may readily be produced in mammalian cells such as CHO, NSO, HEK293 or COS cells, in bacterial cells such as E. coli or Pseudomonas flourescence or in fungal or yeast cells. Preferably, the host cell is a fungal or yeast cell. The host cells are cultured using techniques well known in the art.

Yeast cells used for expressing the variants of the present invention include Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia august. The yeast host cells contain suitable vectors with expression control sequences, such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired. The preferred yeast host of the present invention is Pichia pastoris wherein the expression vector is integrated into the host chromosomal DNA. Various methods of protein purification may be employed and such methods are known in the art and described, for example, in Deutscher, Methods in Enzymology 182: 83-89 (1990) and Scopes, Protein Purification: Principles and Practice, 3rd Edition, Springer, NY (1994).

The pharmaceutical compositions of the FGF-21 variants of the present invention may be administered by any means known in the art that achieve the generally intended purpose to treat type 2 diabetes, dyslipidemia, obesity, or metabolic syndrome. The preferred route of administration is parenteral, defined herein as referring to modes of administration that include but are not limited to intravenous, intramuscular, intraperitoneal, subcutaneous injection and infusion. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. Typical dosage levels can be optimized using standard clinical techniques and will be dependent on the mode of administration and the condition of the patient. Generally, doses will be in the range of 1 μg/kg to 10 mg/kg; 2.5 μg/kg to 5 mg/kg; 5 μg/kg to 2.5 mg/kg; or 10 μg/kg to 500 μg/kg. Thus, an embodiment of the present invention is a method for treating type 2 diabetes or obesity in a patient in need thereof by administering a therapeutically effective amount of a variant of FGF-21 of the present invention.

The variants of FGF-21 of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions. A desired formulation would be one that is a stable lyophilized product that is reconstituted with an appropriate diluent or an aqueous solution of high purity with optional pharmaceutically acceptable carriers, preservatives, excipients or stabilizers [Remington, The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA 1995].

The variants of the present invention may be combined with a pharmaceutically acceptable buffer, and the pH adjusted to provide acceptable stability, and a pH acceptable for administration. Moreover, the variants of the present invention may be placed into a container selected from the group consisting of a vial, a cartridge, a pen delivery device, a syringe, intravenous administration tubing and an intravenous administration bag, wherein the container is a unit dose container. Another aspect of the present invention provides variants of human FGF-21 for use as a medicament.

Another aspect of the invention embodies a variant of human FGF-21 according to the present invention for use in the treatment of type 2 diabetes and obesity.

Yet another aspect of the invention provides the use of variants of human FGF-21 for the manufacture of a medicament for the treatment of type 2 diabetes, dyslipidemia, obesity, or metabolic syndrome.

Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention.

Example 1

GLUTl mRNA Upregulation in Mouse 3T3-L1 Adipocytes 3T3-L1 cells are obtained from the American Type Culture Collection. Cells are cultured at 37°C, 5% CO2 in growth medium (GM) containing 10% fetal bovine serum (FBS) in Dulbecco's modified Eagle's medium. For standard adipocyte differentiation, two days after cells reach confluency (referred as day O), cells are exposed to differentiation medium (DM) containing 10% FBS, 10 μg/ml of insulin, 1 μM dexamethasone, and 500 μM isobutylmethylxanthine, for two days. Cells are then treated in post differentiation medium containing 10% fetal bovine serum and 10 μg/ml of insulin for an additional two days. Cells are maintained in post differentiation medium containing 10% fetal bovine serum for an additional nine days with media changes every two to three days.

GLUTImRNA Up regulation Assay - The medium is removed from 96 well plates of differentiated 3T3-L1 adipocytes and replaced with Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin (BSA). Transiently transfected HEK 293 cell supernatants containing wild type FGF -21 or an FGF-21 variant is added to the 3T3-L1 adipocytes so that the final concentration of FGF-21 wild type or variant is between 5OnM and 0.75nM. A two-fold dilution series of purified FGF-21 at concentrations from 5OnM to 0.75nM is included on each 96 well plate to create a standard curve. Cells are incubated at 37°C, 5% CO2 for six hours. Medium is removed from the plate and replaced with lOOμl/well Lysis/Binding Buffer. RNA is purified from the cell lysate using an RNAqueous Automated RNA purification kit (Ambion). Purified RNA is subjected to Quantitative multiplex RT-PCR, a primer-probe set specific for mouse GLUTl mRNA (Applied Biosystems, mm00441473_ml), and a primer-probe set specific for mouse hypoxanthine phosphoribosyltransferase (HPRT). Forward primer: 5 'TGGTTAAGCAGTACAGCCCCA (SEQ ID NO: 168); Reverse primer: 5 'CGAGAGGTCCTTTTCACCAGC (SEQ ID NO: 169); Probe: 5'VIC- AATGGTTAAGGTTGCAAGCT-MGBNFQ (SEQ ID NO: 170). The PCR reactions are subject to quantitative RT-PCR, PCR Cycle: 50°C 20', 95°C 15', 4Ox (94°C 45", 6O°C 45").

Relative activity of FGF-21 variants is determined as follows. The average measured threshold cycle (Ct) for GLUTl mRNA from untreated 3T3-L1 cells is subtracted from the measured GLUTl mRNA Ct from cells treated with an FGF-21 variant to yield the GLUTl mRNA change in Ct (ΔCt) for that particular variant. Similarly, the average Ct for HPRT mRNA from untreated 3T3-L1 cells is subtracted from the measured HPRT mRNA Ct from cells treated with an FGF -21 variant to yield the HPRT mRNA ΔCt. The GLUTl ΔCt minus the HPRT ΔCt yields the relative change in GLUTl mRNA Ct compared to the relatively invariant Ct of the HPRT mRNA (ΔΔCt). Because PCR amplification is exponential, 2-ΔΔCt = the relative up regulation of GLUTl mRNA induced by the treatment of 3T3-L1 adipocytes with an FGF-21 variant. This relative up regulation can be compared to the wild type FGF-21 standard curve to yield an apparent concentration for the FGF-21 variant. Variants with greater than 100% of wild type FGF-21 activity are judged to contain positive mutations. As indicated in Table 3, preferred variants of human FGF-21 of the present invention have a relative activity/potency greater than wild type human FGF-21, clearly demonstrating FGF-21 variants that have increased potency compared to mature wild type human FGF- 21.

Table 3

Example 2 Glucose Uptake in Mouse 3T3-L1 Adipocytes

3T3-L1 cells are obtained from the American Type Culture Collection. Cells are cultured in growth medium (GM) containing 10% iron-enriched fetal bovine serum in Dulbecco's modified Eagle's medium. For standard adipocyte differentiation, two days after cells reached confluency (referred as day 0), cells are exposed to differentiation medium (DM) containing 10% fetal bovine serum, 10 μg/ml of insulin, 1 μM dexamethasone, and 500 μM isobutylmethylxanthine, for 48 h. Cells then are maintained in post differentiation medium containing 10% fetal bovine serum, and 10 μg/ml of insulin.

Glucose Transport Assay— Serial dilutions of FGF21 variants are prepared in serum free medium containing 0.1% BSA. The media is aspirated from 14-30 day differentiated adipocytes. The wells are rinsed with PBS and the serial dilutions of FGF21 variants are added. The plates are incubated at 37°C for 24 hours. Hexose uptake, as assayed by the accumulation of 0.1 mM 2-deoxy-D-[14C]glucose, is measured as follows: 3T3-L1 adipocytes in 12-well plates are washed twice with Kreb's Ringer phosphate buffer (KRP) (136 mM NaCl, 4.7 mM KCl, 10 mM NaPOφ 0.9 mM CaCl2, 0.9 mM MgSOφ pH 7.4) warmed to 37 °C and containing 0.2% BSA, incubated in

Leibovitz's L- 15 medium containing 0.2% BSA for 2 h at 37°C in room air, washed twice again with KRP containing, 0.2% BSA buffer, and incubated in KRP, 0.2% BSA buffer in the absence (Me2SO only) or presence of wortmannin for 30 min at 37 °C in room air.

Insulin is then added to a final concentration of 100 nM for 15 min, and the uptake of 2- deoxy-D-[14C]glucose is measured for the last 4 min. Nonspecific uptake, measured in the presence of 10 μM cytochalasin B, is subtracted from all values. Protein concentrations are determined with the Pierce bicinchoninic acid assay. Uptake is measured routinely in triplicate or quadruplicate for each experiment.

In vitro potency is compared to the in vitro activity of mature wild type human FGF-21. The in vitro potency of variants of FGF-21 of the present invention compared to mature wild type human FGF-21 is shown in Table 4. Preferred variants of FGF-21 that have 4 or 5 amino acid substitutions have a biological potency 5.5 to 6.4 times the potency of mature wild type FGF-21. This data indicates FGF-21 variants with 4 or 5 amino acid substitutions have increased potency compared to mature wild type FGF-21.

Table 4

Example 3 Increased luciferase expression in human 293-β-Klotho-SRE luc reporter cells

Construction of 293-bKlotho-SRE luc reporter cells:

HEK-293 human embryonic kidney cells are obtained from ATCC. Cells are cultured at 37°C, 5% CO2 in growth medium (GM) containing 10% fetal bovine serum (FBS) in Dulbecco's modified Eagle's medium. Cells are cotransfected with a plasmid containing a CMV promoter driven human b-klotho expression cassette and a plasmid containing a Serum Response Element (SRE) driven luciferase expression cassette. The b-klotho expression plasmid also contains an SV40 promoter driven neomycin phosphotransferase expression cassette to confer resistance to the aminoglycoside antibiotic G418. Transfected HEK-293 cells are selected with 600ug/ml G418 to select for cells where the transfected plasmids have been integrated into the genome. Selected cells are dilution cloned and tested for an increase in luciferase production at 24 hours post addition of FGF21. The clone demonstrating the largest FGF-21 dependant increase in luciferase is chosen as the cell line used to measure relative FGF-21 variant activity. 293-bKlotho-SREluc FGF21 activity assay - 293-bKlotho-SREluc cells are rinsed and placed into CD293 suspension culture media (Invitrogen). Cells are grown in suspension overnight at 37°C, 6% CO2, 125rpm. Cells are counted, pelleted by centrifugation, and resuspended in CD293 media containing 0.1% BSA. Cells are placed in white 96 well plates at 25,000 cells per well. Transiently transfected HEK 293 cell supernatants containing wild type FGF-21 or an FGF-21 variant is added to the 293- bKlotho-SREluc cells so that the final concentration of FGF-21 wild type or variant is between 5OnM and 0.75nM. A two-fold dilution series of purified FGF-21 at concentrations from 5OnM to 0.75nM is included on each 96 well plate to create a standard curve. Cells are incubated at 37°C, 5% CO2 overnight. Cells are lysed and luciferase activity is determined by the addition of an equal volume of OneGlo luciferase substrate (Promega). Relative activity of FGF -21 variants is determined by comparing the luciferase expression to the wild type FGF -21 standard curve to yield an apparent concentration for the FGF -21 variant. Variants with greater than 100% of wild type FGF- 21 activity are judged to contain positive mutations.

FGF-21 variants that have increased biological activity after a single amino acid is changed compared to the original amino acid at that position in SEQ ID NO: 1 are shown in Table 5. All of the changed amino acid positions indicated in Table 5 were assayed in the 293-β-Klotho-SRE luc assay and had an increase in relative biological activity compared to mature wild type FGF-21. The fold increase ranged from about 1.5 to about 10.5 over the biological activity of wild type FGF-21.

Table 5

FGF-21 variants with a Single Amino Acid Substitution

* The amino acid position # is based on SEQ ID NO: 1. The variants of human FGF-21 of Table 5 are indicated by the amino acid sequence of SEQ ID NO:3.

Preferred variants of human FGF-21 of Table 5 and the relative fold increase (FI) of activity compared to mature wild type FGF-21 are selected from the group consisting of D127K (3.3 FI), P130R (4.4 FI), P130K (6.5 FI), G161E (4.6 FI), and G174F (4.8 FI).

Example 4 Ob/ob Mouse Model

A study in an obesity model using male ob/ob mice is done to monitor plasma glucose levels and triglyceride levels after treatment with FGF-21 or an FGF-21 variant, compared to vehicle and insulin control groups. The test groups of male ob/ob mice (7 weeks old) are injected with vehicle alone (0.9% NaCl), or FGF-21 variant (0.125 mg/kg) subcutaneously (0.1 mL, once daily) for seven days. Blood is collected by tail clip bleeding on day 7, one hour after the last compound injection and plasma glucose levels are measured using a standard protocol. The FGF-21 variant

Q47K/P115W/P150F/R175L lowers plasma glucose levels more than 2 fold greater than mature wild type human FGF -21 when compared to the vehicle control, Table 6. This data indicates that an FGF -21 variant with 4 amino acid substitutions has increased potency compared to mature wild type FGF-21. Table 6

Example 5 Glucose Lowering in Ob/ob Mouse Model Using Constant Infusion

Eight week old male ob/ob mice (C57BL/6OlaHsd-Zepob, Harlan, Indianapolis, IN) are randomized into groups (n=7) by blood glucose levels, plasma insulin levels, and body weight on Day 0. A group of lean, non-diabetic ob/m mice that are treated with vehicle and used as a control for normalization. The test compound A31C/G43C/P115W/D127K/P128N/C166Y/G174L/R175L/A180Y (SEQ ID NO: 114) is administered by constant sc infusion using Alzet minipumps model 1007 (Durect, Cupertino, CA) at the indicated dose level for 7 days. On day 7 the animals are fasted for 4 h before a blood glucose sample (80- 100 μL) is taken from the conscious mice by tail snip. Blood glucose levels are measured using a hand held glucometer (AccuCheck, Roche Diagnostics, Indianapolis, IN). Plasma insulin levels are determined using a mouse insulin assay kit (Kl 12BZC, Meso Scale Discovery, Gaithersburg, MD). At sacrifice the mice blood is collected by cardiac puncture for evaluation of protein exposure. Plasma samples are analyzed for concentrations of immunoreactive test compound SEQ ID NO: 114 using a sandwich ELISA method in 100% mouse plasma. The assay employs a monoclonal anti-FGF-21 as capture antibody and an affinity purified anti-FGF-21 polyclonal as the secondary antibody. This is then followed by an anti- rabbit IgG-HRP conjugate and detected using 3,3 ',5,5 '-Tetramethylbenzidine (TMB). The upper and lower limits of quantitation in the ELISA are 5 and 0.078 ng/mL, respectively. To obtain the ED50 for glucose lowering activity, the glucose values on day

7 are normalized to the negative control (ob/ob mice treated with vehicle) and the positive control (ob/m mice treated with vehicle) by the following equation: xl- (neg ctrl(avg)) /(pos ctrl(avg))-(neg ctrl(avg))- A four parameter logistic regression model fit is performed on the normalized blood glucose values on day 7. As shown in Table 7, the test compound of SEQ ID NO: 114 lowers blood glucose levels in a dose dependent manner. Plasma levels on Day 7 of the test compound SEQ ID NO: 114 are shown in Table 8, confirming protein exposure in a dose dependent manner. Table 7

The Test Compound is the human FGF-21 variant SEQ ID NO: 114

Table 8

"Plasma levels measured by the sandwich ELISA assay of Example 5 Example 6

Stability of FGF-21 Muteins

The stability of the FGF-21 muteins of the present invention is analyzed under simulated physiological and pharmaceutical formulation conditions and under thermal conditions.

To simulate physiological conditions, the mutein is analyzed for stability in PBS at room temperature (RT) at a target protein concentration of 10 mg/mL, pH 7.4. Solubility/physical stability of the muteins in PBS is considered satisfactory if recovery of protein following preparation resulted in >90% recovery at RT as determined by analytical size-exclusion. The muteins of the present invention indicated in Table 9 meet this criterion.

The pharmaceutical formulation of a mutein of the present invention is a preserved multi-use formulation, thus, compatibility with a common preservative is analyzed. To test for formulation compatibility, the preservative m-cresol, (3 mg/mL final concentration, a concentration usually sufficient to meet European Pharmacopia B criteria for preservative effectiveness under neutral pH conditions) is added at room temperature to a solution containing the mutein at approximately lOmg/mL in PBS, pH 7.4. Physical stability in the presence of preservative is initially accessed by determining protein recovery of the main chromatographic peak after analytical size exclusion chromatography at RT. Furthermore, the extent of aggregation as measured by DLS (dynamic light scattering) at 37°C is shown as the average diameter of particles in the presence of m-cresol after two hours, compared to wild-type human FGF-21. A larger average diameter corresponds to an increased degree protein association and/or aggregation.

High potency muteins of the present invention that are stable in PBS and compatible with preservative are designated to have enhanced or improved pharmaceutical properties as compared to wild-type FGF-21. The muteins of the present invention that have enhanced pharmaceutical stability/compatibility with the phenolic preservative , m-cresol as compared to mature human wild-type FGF-21 contain at least one engineered disulfide bond from the amino acid positions selected from the group consisting of A26C-P133C (SEQ ID NO: 163), A31C-G43C (SEQ ID NO: 164), Hl 17C- A134C (SEQ ID NO: 166), and L118C-A134C (SEQ ID NO: 167). The ability of the engineered disulfide bond to impart stability/compatibility with m-cresol as measured by average particle diameter is shown in Table 9. Table 9

*Average Particulate diameter represents a protein solution at a target cone, of 10 mg/mL, m-cresol at 3 mg/ml, after 2 hours incubation at 37 °C.

The thermal stability of variants of FGF-21 of the present invention is determined utilizing standard techniques. The protein melting point temperature (T_m) is determined by differential scanning calorimetry (DSC), with scans starting at 10°C for a reliable evaluation of baseline (see Graziano, et ah, Biochemistry 35: 13386-13392, 1996). The T_m of a protein is indicative of the thermal stability of a protein, i.e. the higher the T_m, the higher the thermal stability. Variants of the present invention that have enhanced thermal stability as compared to wild-type FGF-21 are shown in Table 10.

Table 10

*T_m is the protein melting point temperature. Example 7

PEGylation of Human FGF-21

It is well recognized in the art that PEGylation, the covalent addition of polyethylene glycol to a protein, can enhance a variety of pharmacodynamic properties of the protein (Ryan et al. Expert Opinion in Drug Delivery 2008 5(4):371-383). PEGylation is commonly done to increase the size of the protein to inhibit renal clearance, to mask the protein from proteolysis and receptor-mediated clearance, and to aid in the solubility and bioavailability of the protein. All of these qualities can contribute to a better pharmacodynamic profile for a protein drug, and could lead to lower doses or fewer injections for a patient. Different positions within the protein sequence were evaluated for their ability to tolerate the addition of a PEG moiety.

PEGylation is done using linear polymers of polyethylene glycol, ranging in size from 5-40 kDa, covalently attached to the epsilon amine of the single lysine through para- nitrophenyl chemistry (Bioconjugate Chem. 6: 150-165, 1995). The most preferred linear polymer of polyethylene glycol is the 40 kDa moiety, also indicate as 4OK PEG.

Prior to PEGylation of human FGF-21, the four endogenous lysine residues at positions 56, 59, 69, and 122 of SEQ ID NO: 1 are changed to arginine. A single lysine for PEG conjugation is placed at an amino acid position indicated by the derivative designation (i.e. K91-PEG has the lysine at position 9 lof SEQ ID NO: 1). The most preferred PEGylated derivatives of human FGF-21 are PEGylated at the lysine residue engineered at positions selected from the group consisting of K91, K120, K147, and K167.

PEGylated derivatives of FGF-21 are evaluated using the 293-β-Klotho-SRE luc reporter assay described in Example 3 or the 3T3 Ll- β-Klotho assay (as described in Micanovic et al, J. Cell. Physiol, 219: 227-234, 2009). Results of representative PEGylated derivatives of FGF-21 are shown in Table 11.

Figure 1

wherein:

R, L, E, K, H, M, Q, or W; Xaa^ is S, L, or G; Xaaj₇9 is Y, F, L, or W; Xaajgo ^{is A} _> V, E, L, M, or Y; and, wherein the amino acid sequence of the variant of human FGF-21 is not mature human FGF-21 (SEQ ID NO: 1).

Claims

We Claim:

1. A variant of human FGF -21 comprising the amino acid sequence of SEQ ID NO: 171: wherein:

Xaa! is H, E, F, or I; Xaa₃₂ is H, W, F, M, or Y; Xaa₄₇ is Q or K; Xaa₆₄ is Q, W, F, G, L, M, N, R, S, T, or V; Xaa₇g is P, G, or L; Xaa92 is A, H, W, G, or F; Xaai oi is E, A, K, I, Q, N, D, G, H, M, R, or W; Xaa₁₀₈ is Q or Y; Xa_{ai 15} is P or W; Xaa₁₂7 is D, K or R; Xaaj28 is ^p _> ^R _> or N; Xaaj29 is A, M, R, Q, N or G; Xaa^o is P, W, K, or R; Xaa₁₄₂ is L, P, D, T, S, I, K, M, N, R, or V; Xaa₁₄₇ is P, R, V, A, M, or S; Xaa₁₅₀ is P or F; Xaai 5₄ is A, W, or Q; Xaai g^ is G, E, N, F, L, K, or W; Xaai gg is L or Y; Xaai 70 is G, L, M, Q, W, or Y; Xaa^7₄ is G, L, Y, or F; Xaa^ is R, Q, L, or E; and Xaajgo is

A or Y, and, wherein the amino acid sequence of the variant of human FGF-21 is not mature human wild type FGF-21 (SEQ ID NO:1) and wherein said variant optionally has one or more substitutions from the group consisting of:

(a) the substitution of a cysteine at two, four, six, or eight of the following positions: A26, A31, G43, G80, Ll 14, Hl 17, Ll 18, P133, or A134;

(b) the substitution of S 167 with an amino acid from the group consisting of A, D, E, F, G, H, I, K, L, N, Q, R, T, V, W, or Y;

(c) the substitution of N121 with an amino acid from the group consisting of A, C, D, E, F, G, H, I, L, M, P, Q, R, S, T, V, W, or Y;

(d) the substitution of L153 with an amino acid selected from the group consisting ofA, C, V, P, F, R, W, N, D, Q, E, H or M; (e) the substitution of S 181 with A, D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, or

Y;

(g) the truncation at the N-terminus of one to 4 amino acids.

2. The variant of human FGF -21 of Claim 1 wherein Xaa^ is H, E or deleted; Xaa32 is H or Y; Xaag4 is Q, L, V, or F; Xaayg is P or G; Xaa92 is A or H; Xaa^gi is E or A; Xaai o8 is Q or Y; Xaai 15 is P or W; Xaai 27 i^s D or K; Xaai 2g is P or N; Xaai 29 is A or M; Xaai 30 is P, R, or K; Xaai 42 is L or R; Xaai 47 i^s P_> R_> V, or M; Xaai 54 is A or W; Xaa^l is G or E; Xaa^gg is L or Y; Xaa^g is G or Y; Xaa^74 is G, Y, F, or L; Xaa^ 75 is R_> L, E, or Q; and, Xaa^gQ is A or Y.

3. The variant of human FGF-21 of Claim 2 wherein Xaa^ is H or deleted; Xaa32 is H; Xaag4 is Q or L; Xaa7g is P; Xaa92 is A; Xaa^oi is E; Xaa^08 is Q; X^aal 15 is P ^or W; Xaa^27 is D or K; Xaa^28 ^ιs P ^or N; Xaa^29 is A; Xaa^o is P, R,or K; Xaa^42 is L; Xaa^47 is P or R; Xaa^4 is A; Xaa^i is G; Xaa^66 is L or Y; Xaa^o is G; Xaa^74 is G, F, or L; Xaa^75 is R or L; and, Xaa^go is A or Y.

4. The variant of human FGF-21 of Claim 3 wherein Xaag4 is L; Xaa^ 15 is W; Xaa^27 is K; Xaa^28 is N; Xaa^o is P, R,or K; Xaa^47 is P or R; Xaa^66 is L or Y;

Xaa^74 is G, F, or L; Xaa^75 is R or L; and, Xaa^go is A or Y.

5. The variant of human FGF-21 of Claim 4 wherein Xaa^o is R or K; Xaa^47 is P or R; Xaai 55 is L or Y; Xaai 74 is F or L; Xaai 75 is R or L; and, Xaai gg is A or Y.

6. The variant of human FGF-21 of Claim 5 wherein Xaa^o is R; Xaa^47 is P;

Xaa^66 ^ιs L or Y; Xaa^74 is F or L; Xaa^5 is R or L; and, Xaa^go is A or Y.

7. The variant of human FGF-21 of Claim 6 wherein Xaa^66 is Y; Xaa^74 is L; Xaai 75 i^s R ^or L; and, Xaa^gg is A or Y.

8. The variant of human FGF-21 of Claim 7 wherein Xaa^ is L; and, Xaa^gg is Y.

9. The variant of human FGF-21 of Claim 3 wherein Xaag4 is L; Xaa^ 15 is W; Xaa^27 is K; Xaa^28 is N; Xaa^g is P or K; Xaa^47 is R; Xaa^gg is Y; Xaa^74 is G, F, or L; Xaa^75 is R or L; and, Xaa^gg is A or Y.

10. The variant of human FGF-21 of Claim 9 wherein Xaai 30 is P; Xaai 74 is F;

Xaai 75 is L; and, Xaai gg is Y-

11. The variant of human FGF-21 of Claim 9 wherein Xaai 30 is K; Xaai 74 is L; Xaai 75 is L; and, Xaai go is Y-

12. The variant of human FGF-21 of Claim 9 wherein Xaai 30 is P; Xaai 74 is L; Xaai 75 is L; ^and, Xaai gg is Y-

13. The variant of human FGF-21 of Claim 4 wherein Xaai 39 is P or R; Xaai 47 is P or R; Xaa^gg is L or Y; Xaa^74 is F or L; Xaa^ is R or L; and, Xaa^gg is A or Y.

14. The variant of human FGF-21 of Claim 13 wherein Xaai 39 is P; Xaai 47 is P; Xaaigg is Y; Xaa^74 is F or L; Xaa^ is R or L; and, Xaa^gg is A or Y.

15. The variant of human FGF-21 of Claim 14 wherein Xaa^74 is F; Xaa^ is L; and, Xaai go i^s Y-

16. The variant of human FGF-21 of Claim 14 wherein Xaaj74 is L; Xaa^ is L; and, Xaa^ go is Y.

17. The variant of human FGF-21 of Claim 2 wherein Xaa^ is H or deleted; Xaa32 is Y; Xaag4 is Q or L; Xaa7g is P or G; Xaa92 is H; Xaa^Ql is A; Xaa^Qg is Q; Xaan5 is P or W; Xaa^27 is D or K; Xaai2g is P or N; Xaa^29 is M; Xaa^o is P, R, or K; Xaa^42 is R; Xaa^47 is R; Xaa^4 is W; Xaaigi is G; Xaaigg is L or Y; Xaa^o is Y; Xaai 74 is G, F, or L; Xaai 75 is R, L, or Q; and, Xaai go is A or Y.

18. The variant of human FGF-21 of Claim 17 wherein Xaa54 is Q; Xaa7g is P;

Xaai 15 i^s W; Xaa^7 is K; Xaa^8 is N; Xaa^g is P; Xaaigg is Y; Xaai74 is F; Xaai 75 is L; and, Xaai go is Y-

19. The variant of human FGF-21 of Claim 17 wherein Xaag4 is L; Xaayg is P; Xaai 15 i^s W; Xaai27 is D; Xaai2g is N; Xaa^g is R; Xaaigg is Y; Xaai74 is L;

Xaai 75 is Q; ^and, X^aa180 ^ιs Y-

20. The variant of human FGF-21 of Claim 17 wherein Xaag4 is L; Xaa7g is G; Xaai 15 i^s W; Xaai27 is K; Xaai28 is P; Xaa^g is K; Xaaigg ^ιs Y; Xaai74 is G; Xaai 75 is L; and, Xaai go is Y-

21. The variant of human FGF-21 of Claim 17 wherein Xaag4 is L; Xaa7g is P; Xaai 15 i^s W; Xaai27 is K; Xaai28 is N; Xaai3o is P; Xaaigg ^ιs Y; Xaai74 is F; Xaai 75 is L; and, Xaai go ^ιs Y-

22. The variant of human FGF-21 of Claim 1 wherein Xaai i^s H, E or deleted;

Xaa32 is H or Y; Xaag4 is Q, L, V, or F; Xaa7g is P or G; Xaa92 is A or H; Xaaioi is E or A; XaaiQg is Q or Y; Xaai 15 i^s P ^or W; Xaai27 is D, R, or K; Xaai28 is P, R, or N; Xaai29 is A; Xaai3o is P, R, W, or K; Xaai42 is L or R; Xaai47 is P or R; Xaai54 is A; Xaaigi is G; Xaaigg is L or Y; Xaai70 is G; Xaai74 is G, Y, F, or L; Xaai75 is R, L, E, or Q; and, Xaai go is A or Y.

23. The variant of human FGF-21 of Claim 22 wherein Xaai is H or deleted; Xaa32 is H; Xaag4 is Q or L; Xaayg is P; Xaa92 is A; Xaa^oi is E or A; Xaaigg is Q; Xaai 15 is P or W; Xaai27 is D, R, or K; Xaai28 is P, R, or N; Xaai3o is P, R, W, or K; Xaai 42 is L or R; Xaai 47 is P or R; Xaaigg is L or Y; Xaai 74 is G, Y, F, or L; Xaai 75 is R, L, E, or Q; and, Xaai gg is A or Y.

24. The variant of human FGF-21 of Claim 23 wherein Xaag4 is Q; Xaaioi is E; Xaai 15 i^s P; X^aa127 i^s ^ ^^aa128 i^s N; Xaa^g is W; Xaai42 is L; Xaai47 is P; Xaaigg is Y; Xaa^74 is Y; Xaa^ is E; and, Xaaigg ^ιs Y-

25. The variant of human FGF-21 of Claim 23 wherein Xaag4 is Q; Xaaioi is E;

Xaai 15 ^ιs P; X^aa127 ^ιs ^ Xaaj28 ^ιs N; X^aa130 ^ιs ^ X^aal42 is L; Xaai47 is P; Xaaigg is L; Xaai74 is Y; Xaai75 is Q; and, Xaaigg is Y-

26. The variant of human FGF-21 of Claim 23 wherein Xaag4 is Q; Xaai_Qi is E;

Xaai 15 is W; Xaai27 is R; Xaai28 is R; Xaa^g is R; Xaai42 is L; Xaai47 is P ; Xaaigg is Y; Xaai 74 is L; Xaai 75 is Q; ^and, X^aa180 ^ιs Y-

27. The variant of human FGF-21 of Claim 23 wherein Xaag4 is Q; Xaaioi is E; Xaai 15 is W; Xaai27 is K; Xaai28 is N; Xaai3o is P; Xaai42 is L; Xaai47 is P; Xaaigg is Y; Xaai 74 is L; Xaai 75 is L; and, Xaai go is Y-

28. The variant of human FGF-21 of Claim 22 wherein Xaai is H or deleted; Xaa32 is H; Xaag4 is Q; Xaa7g is P; Xaa92 is A; Xaaioi is E; XaaiQ8 is Q; Xaai 15 is W; Xaai27 is D; Xaai28 is P; X^aa130 is R; X^aa142 is L or R; Xaaigg is L or Y; Xaai74 is G, Y, F, or L; Xaai 75 is R_> L, E, or Q; and, Xaai go is A or Y.

29. The variant of human FGF-21 of Claim 28 wherein Xaai42 is L; Xaaigg is L; Xaai74 is G; Xaai75 is E; and, XaaigQ is Y.

30. The variant of human FGF-21 of Claim 28 wherein Xaa^gg ^1S Y; Xaa^74 is F; Xaa^75 is L; and, Xaa^gg is Y.

31. The variant of human FGF-21 of Claim 22 wherein Xaa^42 is R; Xaa^gg is

Y; Xaai 74 is L; Xaai 75 is L; and, Xaai gg is Y-

32. The variant of human FGF-21 of Claim 22 wherein Xaa^ is H or deleted; Xaa32 is Y; Xaa54 is L; Xaa7g is P; Xaa92 is A; Xaai gi is E; Xaai Q8 is Q; Xaa^ 15 is W; Xaai 27 is K; Xaai 28 is N; Xaai 30 is K; Xaai 42 is L; Xaai 47 is P; Xaai 55 is L or Y; Xaa^74 is G, Y, F, or L; Xaa^5 is R, L, E, or Q; and, Xaa^go is A or Y.

33. The variant of human FGF-21 of Claim 32 wherein Xaai gg is Y; Xaai 74 is F; Xaai 75 ^ιs L; and, Xaa^gg is Y.

34. The variant of human FGF-21 of Claim 32 wherein Xaa^gg is Y; Xaa^74 is

L; Xaa^75 is Q; and, Xaa^go is Y.

35. The variant of human FGF-21 of Claim 32 wherein Xaa^gg is L; Xaa^74 is F; Xaa^75 is Q; and, Xaa^go is Y.

36. The variant of human FGF-21 of Claim 32 wherein Xaa^gg is L; Xaa^74 is L; Xaa^75 is L; and, Xaa^go is Y.

37. The variant of human FGF-21 of Claim 32 wherein Xaa^gg is Y; Xaa^74 is

G; Xaai 75 i^s Q; ^and, X^aa180 i^s Y-

38. The variant of human FGF-21 of Claim 32 wherein Xaa^gg is Y; Xaa^74 is F; Xaai 75 i^s ^ ^and, X^aa180 i^s A-

39. The variant of human FGF -21 of Claim 22 wherein Xaa^ is H or deleted; Xaa32 is Y; Xaag4 is L; Xaayg is G; Xaa92 is H; Xaa^oi is A; Xaa^08 is Q; Xaa^ 15 is W; Xaa^27 is R; Xaa^28 i^s N; Xaa^g is W; Xaa^42 is R; Xaa^47 is R; Xaa^gg is L or Y; Xaa^74 is G, Y, F, or L; Xaa^ is R, L, E, or Q; and, Xaa^go is A or Y.

40. The variant of human FGF-21 of Claim 39 wherein Xaa^gg ^ιs L; Xaa^74 is G; Xaai 75 is R; and, Xaai gg is Y-

41. The variant of human FGF-21 of Claim 39 wherein Xaa^gg is Y; Xaa^74 is

G; Xaa^75 is R; and, Xaa^go is Y.

42. The variant of human FGF-21 of Claim 39 wherein Xaai 55 is Y; Xaai 74 is

F; Xaai 75 i^s ^ ^and, X^aa180 i^s Y-

43. The variant of human FGF-21 of Claim 39 wherein Xaa^gg is Y; Xaa^4 is

G; Xaai 75 i^s Q; ^and, X^aa180 i^s Y-

44. The variant of human FGF-21 of Claim 39 wherein Xaa^gg is Y; Xaai74 is L; Xaa^75 is L; and, Xaa^g_Q is A.

45. The variant of human FGF-21 of Claim 22 wherein Xaa^ is H or deleted; Xaa32 is Y; Xaag4 is L; Xaa7g is P; Xaa92 is A; Xaa^oi is E; Xaa^o8 ^ιs Q; ^^aal 15 ^ιs W; Xaa^27 is D, R, or K; Xaai 28 is P, R, or N; Xaa^g is P, R, W, or K; Xaai42 is L; Xaa^47 is P; Xaa^gg is Y; Xaai 74 is F; Xaa^ is E; and, Xaa^gø is Y.

46. The variant of human FGF-21 of Claim 45 wherein Xaai27 is R; Xaai28 ^ιs R; and, Xaai 3o is K.

47. The variant of human FGF -21 of Claim 45 wherein Xaa^27 is K; Xaa^28 is N; and, Xaa^g is R.

48. The variant of human FGF -21 of Claim 45 wherein Xaa^27 is R; Xaa^28 is R; and, Xaa 130 is R.

49. The variant of human FGF-21 of Claim 45 wherein Xaai 27 is R; Xaai 28 i^s N; and, Xaai 30 is W.

50. The variant of human FGF-21 of Claim 45 wherein Xaai 27 i^s K; Xaai 28 i^s

R; and, Xaa \ 30 is W.

51. The variant of human FGF-21 of any one of Claims 1-50 wherein cysteine is substituted at A31 and G43; N121 is substituted with G or A; L 153 is substituted with E or A; S 167 is substituted with A, R, or L or is S; M 168 is substituted with I, L, or Y; S 181 is substituted with G; and, HisProIlePro is truncated from the N-terminus.

52. The variant of human FGF-21 of Claim 51 wherein N121 is substituted with G; L 153 is substituted with E; S 167 is substituted with A; and M 168 is substituted with Y.

53. The variant of human FGF-21 of Claim 51 wherein N121 is substituted with A; L 153 is substituted with A; S 167 is substituted with L; and M 168 is substituted with I.

54. The variant of human FGF-21 of Claim 51 wherein N121 is substituted with

A; L 153 is substituted with E; S 167 is substituted with R; and, M 168 is substituted with L.

55. The variant of human FGF-21 of Claim 51 wherein N121 is substituted with G; L153 is substituted with A; S167 is substituted with L; and, M168 is substituted with Y.

56. The variant of human FGF-21 of Claim 51 wherein N121 is substituted with

A; L 153 is substituted with E; S 167 is substituted with L; and, M 168 is substituted with Y.

57. The variant of human FGF-21 of Claim 51 wherein N121 is substituted with G; L 153 is substituted with E; S 167 is S; and, M 168 is substituted Y.

58. The variant of human FGF-21 of Claim 51 wherein N121 is substituted with A; L 153 is substituted with E; S 167 is S; and, M 168 is substituted with I.

59. The variant of human FGF-21 of Claim 51 wherein N121 is substituted with

G; L 153 is substituted with A; S 167 is S; and, M 168 is substituted with L.

60. The variant of human FGF-21 of Claim 1 selected from the group consisting of P115W, P128N, L166Y, G174F, and G174Y.

61. The variant of human FGF-21 of Claim 1 selected from the group consisting of L166Y/A180Y (SEQ ID NO: 35), L166Y/G174F (SEQ ID NO: 36), L166Y/G174Y (SEQ ID NO: 52), L166Y/R175L (SEQ ID NO: 97), D127K/P130W (SEQ ID NO: 106), and P115W/L166Y (SEQ ID NO: 111).

62. The variant of human FGF-21 of Claim 1 selected from the group consisting of P115W/L166Y/G174Y (SEQ ID NO: 89), and D127R/P128N/L166Y (SEQ ID NO: 74).

63. The variant of human FGF-21 of Claim 1 selected from the group consisting of P115W/P128R/P130K/R175L (SEQ ID NO: 5), P115W/P128N/P130K/G174Y (SEQ ID NO: 60), P115W/P128R/P130R/L166Y (SEQ ID NO: 71), and P115W/D127K/P130R/R175Q (SEQ ID NO: 77).

64. The variant of human FGF-21 of Claim 1 selected from the group consisting of P115W/D127K/P130R/G174F/A180Y (SEQ ID NO: 42),

Pl 15W/L166Y/G174Y/R175L/A180Y (SEQ ID NO: 56), P115W/D127K/P130R/G174Y/R175L (SEQ ID NO: 90), and D127K/P128N/P130K/L166Y/R175E (SEQ ID NO: 104).

65. The variant of human FGF-21 of Claim 1 selected from the group consisting of P115W/D127R/P128N/P130R/G174F/R175L (SEQ ID NO: 7), and D127R/P128N/P130W/G174Y/R175Q/A180Y (SEQ ID NO: 24),

66. The variant of human FGF-21 of Claim 1 selected from the group consisting of P115W/D127K/P128N/L166Y/G174L/R175L/A180Y (SEQ ID NO: 82), and P115W/D127K/P128R/P130R/L166Y/G174F/R175L (SEQ ID NO: 162).

67. The variant of human FGF-21 of Claim 1 selected from the group consisting of P115W/D127R/P128R/P130K/L166Y/G174L/R175Q/A180Y (SEQ ID NO: 59), and P115W/D127R/P128R/P130R/L166Y/G174L/R175Q/A180Y (SEQ ID NO: 79).

68. Variants of human FGF-21 of Claim 1 selected from the group consisting of A31C/G43C/P115W/D127K/P128N/L166Y/G174L/R175L/A180Y (SEQ ID NO: 114), A31C/G43C/P115W/D127K/P128N/L153E/L166Y/G174L/R175L/A180Y (SEQ ID NO: 129),

A31C/G43C/P115W/N121G/D127K/P128N/L166Y/G174L/R175L/A180Y (SEQ ID NO: 132), A31C/G43C/P115W/D127K/P128N/L153E/L166Y/S167R/M168L/G174L/R175L/A180 Y (SEQ ID NO: 141), and,

69. Variants of human FGF-21 of Claim 10 wherein said variant is truncated at the N-terminus of SEQ ID NO: 1 and wherein said variant is selected from the group consisting of <fø>HPIP-31C/G43C/P115W/D127K/P128N/L166Y/G174L/R175L/A180Y (SEQ ID NO: 149), desHFIF- A31C/G43C/P115W/D127K/P128N/L153E/L166Y/G174L/R175L/A180Y (SEQ ID NO:

150), desHPIP-ASlC^SC/Pl lSW/NmG/DmK/PmN/LlόόY/GmL/RπSL/AlδOY

(SEQ ID NO: 151), desHPIP-ASlC^SC/Pl lSW/D^K/PmN/LlSSE/LlόόY/S^R/MlόδL/GmL/ R175L/A180Y (SEQ ID NO: 152), and, desHPIP-ASlC^SC/Pl lSW/NmA/DmK/PmN/LlSSE/LlόόY/S^L/MlόδL/

G174L/R175L/A180Y/S181G (SEQ ID NO: 153).

70. The variant of human FGF-21 of any one of Claims 1 to 69 wherein said variant has greater potency than the protein of SEQ ID NO: 1 as measured in the 3T3-L1 adipocyte GLUTl assay (Example 1) and the 293 β-Klotho-SRE-luc assay (Example 3).

71. The variant of human FGF-21 of any one of Claims 1 to 70 wherein said variant has greater stability than the protein of SEQ ID NO: 1 when analyzed under simulated physiological and pharmaceutical formulation conditions (Example 6).

72. A pharmaceutical composition comprising a variant of human FGF-21 of any one of claims 1-71 and a pharmaceutically acceptable excipient.

73. A method for treating type 2 diabetes or obesity comprising administering a variant of human FGF-21 of any one of claims 1-71 to a patient in need thereof.

74. A variant of human FGF-21 of any one of claims 1-71 for use as a medicament.

75. A variant of human FGF-21 of any one of claims 1-71 for use in the treatment of type 2 diabetes or obesity.

76. An isolated polynucleotide that encodes the amino acid sequence of any one of claims 1-71.

77. A recombinant expression vector comprising a polynucleotide of claim 76.

78. A host cell transfected with the expression vector of claim 77.

79. A process for producing a variant of human FGF-21 of any one of claims 1-71 comprising cultivating the host cell of Claim 78 and recovering the polypeptide encoded by said polynucleotide from the culture media.

80. A derivative of human FGF-21 of any one of the variants of claims 1-71 wherein said derivative is PEGylated at the lysine residue engineered at one of the amino acid positions selected from the group consisting of K91, K120, K147, and K167.

81. The derivative of claim 80 wherein said derivative is SEQ ID NO: 160.