WO2010065439A1 - Variants de facteurde croissance 21 du fibroblaste - Google Patents

Variants de facteurde croissance 21 du fibroblaste Download PDF

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WO2010065439A1
WO2010065439A1 PCT/US2009/066062 US2009066062W WO2010065439A1 WO 2010065439 A1 WO2010065439 A1 WO 2010065439A1 US 2009066062 W US2009066062 W US 2009066062W WO 2010065439 A1 WO2010065439 A1 WO 2010065439A1
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Prior art keywords
xaa
xaai
variant
human fgf
seq
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PCT/US2009/066062
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David Bruce Baldwin
Craig Duane Dickinson
David Albert Driver
Radmila Micanovic
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Eli Lilly And Company
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factors [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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 human fibroblast growth factor 21
  • 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.
  • FGF-21 with improved physical/functional properties have been described previously, e.g. WO2006/028595, WO2006/065582, and WO2005/061712.
  • modifying a therapeutic protein to improve a specific property i.e. pharmaceutical stability may impact the biological activity of the protein.
  • a problem associated with mature wild type human FGF -21 is the relatively low potency of the molecule.
  • 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.
  • 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.
  • 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 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;
  • Xaa 78 is P, G, or L;
  • Xaa 92 is A, H, W, G, or F;
  • Xaa 1O i is E, A, K, I, Q, N, D, G, H, M, R, or W;
  • Xaa 10 8 is Q or Y;
  • Xaa! 15 is P or W;
  • Xaaj is H, E, or deleted; Xaa32 is H or Y; Xaag4 is Q, L, V, or F; Xaayg is P or
  • 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 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.
  • SEQ ID NO: 171 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 ⁇ s E; Xaai Qg is Q; Xaai 15 is P or W; Xaai 27 is D or K; 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 ⁇ s 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 Xaai
  • 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 aa l 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
  • SEQ ID NO: 171 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
  • SEQ ID NO: 171 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 ⁇ ⁇ aa 128 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 aa 115 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
  • 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),
  • 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
  • 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: 9), P115W/D127K/P128R/L166Y (SEQ ID NO: 25), D127K/P128R/P130K/L166Y (SEQ ID NO: 9), P115W/D127K/P128R/L166Y (SEQ ID NO: 25), D127K/P128R/P130K/L166Y (SEQ ID NO: 9), P115W/D127K/P128R/L166Y (SEQ ID NO: 25), D127K/P128R/P130K/L166Y (SEQ ID NO: 9), P115W/D127K/P128R/L166Y
  • 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:
  • 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
  • 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).
  • the invention further provides variants of human FGF-21 wherein said variant is selected from the group consisting of
  • 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:
  • Xaa 32 is H, W, F, M, or Y;
  • Xaa 64 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;
  • Xaa ⁇ Q is P, W, K, or R;
  • Xaa ⁇ is P, R, V, A
  • the present invention further provides a variant of human FGF-21 comprising the amino acid sequence of SEQ ID NO: 173:
  • 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;
  • the invention further provides a variant of human FGF-21 wherein said variant is selected from the group consisting of
  • the invention further provides a variant of human FGF-21 wherein said variant is selected from the group consisting of
  • 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.
  • 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
  • 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 ⁇ -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-
  • 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.
  • 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).
  • 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
  • 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.
  • 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.
  • 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.
  • 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).
  • a substitution for glutamic acid at position 30 i.e. E30
  • alanine (A) is designated as E30A.
  • a substitution for His (H) 117 with VaI (V), Trp (W), or Tyr (Y) are designated as Hl 17V, W, or Y.
  • 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, ⁇ -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.
  • 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.
  • LDL low-density lipoprotein
  • HDL high-density lipoprotein
  • 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.
  • potency is a measurement of the ability of the variants to affect luciferase expression in human 293- ⁇ -Klotho-SRE luc reporter gene assay as described in Example 3.
  • 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 ⁇ -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);
  • 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.
  • 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.
  • 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.
  • 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 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 ( ⁇ fesHPIP-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 ⁇ fesHPIP truncation are selected from the group consisting of (/esHPIP-A31C/G43C/P115W/D127K/P128N/L166Y/G174L/ R175L/A180Y (SEQ ID NO: 149); des ⁇ PJP ⁇ lC/GttC/PUSVf/DmK/PnZN/LWE/LlG ⁇ Y/G ⁇ iL/ 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/Ll ⁇ Y/S ⁇ R/Ml ⁇ L/ G174L/R175L/A180Y
  • 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.
  • variants of human FGF-21 of the present invention are shown in Table 2.
  • 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.
  • the variants in Table 2 provide both high potency and stability over numerous parameters when compared to wild type human
  • 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.
  • 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.
  • 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).
  • 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).
  • 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:
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ g/ml of insulin, 1 ⁇ M dexamethasone, and 500 ⁇ M isobutylmethylxanthine, for two days.
  • GM growth medium
  • FBS fetal bovine serum
  • Dulbecco's modified Eagle's medium Dulbecco's modified Eagle's medium.
  • DM differentiation medium
  • Cells are then treated in post differentiation medium containing 10% fetal bovine serum and 10 ⁇ g/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.
  • BSA bovine serum albumin
  • 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).
  • 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 ( ⁇ Ct) for that particular variant.
  • Ct measured threshold cycle
  • 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 ⁇ Ct.
  • the GLUTl ⁇ Ct minus the HPRT ⁇ Ct yields the relative change in GLUTl mRNA Ct compared to the relatively invariant Ct of the HPRT mRNA ( ⁇ Ct).
  • 2- ⁇ Ct 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.
  • 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.
  • 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 ⁇ g/ml of insulin, 1 ⁇ M dexamethasone, and 500 ⁇ M isobutylmethylxanthine, for 48 h. Cells then are maintained in post differentiation medium containing 10% fetal bovine serum, and 10 ⁇ g/ml of insulin.
  • GM growth medium
  • DM differentiation medium
  • 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 NaPO ⁇ 0.9 mM CaCl2, 0.9 mM MgSO ⁇ pH 7.4) warmed to 37 °C and containing 0.2% BSA, incubated in KRP) (136 mM NaCl, 4.7 mM KCl, 10 mM NaPO ⁇ 0.9 mM CaCl2, 0.9 mM MgSO ⁇ 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 ⁇ M 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.
  • Example 3 Increased luciferase expression in human 293- ⁇ -Klotho-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.
  • GM growth medium
  • FBS fetal bovine serum
  • SRE Serum Response Element
  • 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.
  • FGF -21 variants are 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- ⁇ -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.
  • 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).
  • 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 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.
  • a blood glucose sample 80- 100 ⁇ L 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).
  • 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.
  • the Test Compound is the human FGF-21 variant SEQ ID NO: 114
  • the stability of the FGF-21 muteins of the present invention is analyzed under simulated physiological and pharmaceutical formulation conditions and under thermal 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.
  • 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.
  • 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.
  • 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.
  • 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- ⁇ -Klotho-SRE luc reporter assay described in Example 3 or the 3T3 Ll- ⁇ -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.
  • Xaa ⁇ is S, L, or G
  • Xaaj 7 9 is Y, F, L, or W
  • Xaajgo is A > V, E, L, M, or Y
  • amino acid sequence of the variant of human FGF-21 is not mature human FGF-21 (SEQ ID NO: 1).

Abstract

La présente invention concerne à de nouveaux variants de polypeptides du facteur de croissance 21 du fibroblaste. L’invention concerne des vecteurs et des cellules hôtes pour la propagation de séquences d’acide nucléique codant pour lesdits polypeptides et leur production. L’invention concerne également des procédés pour le traitement du diabète de type 2 et de l’obésité.
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