阅读说明：本技术 具有优化的活性比率的fgf21化合物/glp-1r激动剂组合 (FGF21 compound/GLP-1R agonist combinations with optimized activity ratio ) 是由 K·科纳 于 2019-06-21 设计创作，主要内容包括：本发明涉及包含FGF21(成纤维细胞生长因子21)化合物和GLP-1R(胰高血糖素样肽-1受体)激动剂的组合、药物组合物和融合分子,其具有优化的GLP-1R激动剂/FGF21化合物活性比率。本发明还涉及所述组合、药物组合物和融合分子作为特定用于治疗肥胖症、体重超重、代谢综合征、糖尿病、糖尿病视网膜病变、高血糖症、血脂异常、非酒精性脂肪性肝炎(NASH)和/或动脉粥样硬化的药物的用途。(The present invention relates to combinations, pharmaceutical compositions and fusion molecules comprising an FGF21 (fibroblast growth factor 21) compound and a GLP-1R (glucagon-like peptide-1 receptor) agonist, having an optimized GLP-1R agonist/FGF 21 compound activity ratio. The invention also relates to the use of said combinations, pharmaceutical compositions and fusion molecules as a medicament in particular for the treatment of obesity, overweight, metabolic syndrome, diabetes, diabetic retinopathy, hyperglycemia, dyslipidemia, non-alcoholic steatohepatitis (NASH) and/or atherosclerosis.)

1. A combination comprising an FGF21 (fibroblast growth factor 21) compound and a GLP-1R (glucagon-like peptide-1 receptor) agonist,

wherein the FGF21 compound has FGF21 activity that is the same as or substantially the same as the FGF21 activity of native FGF21, and the FGF21 compound is an FGF21 variant, said FGF21 variant comprising at least one mutation selected from the group consisting of:

-substituting the amino acid residue at position 98 to 101 from the N-terminus of the native FGF21 from SEQ ID NO. 2 with the amino acid sequence EIRP (SEQ ID NO: 44);

-substituting the amino acid residues at positions 170 to 174 of the N-terminus of native FGF21 from SEQ ID NO 2 with the amino acid sequence TGLEAV (SEQ ID NO: 45);

-substituting the amino acid sequence TGLEAN (SEQ ID NO:46) for the amino acid residues at positions 170 to 174 from the N-terminus of the native FGF21 from SEQ ID NO: 2;

-substituting the amino acid residue at position 170 of the N-terminus of native FGF21 from SEQ ID NO:2 with amino acid N;

-substitution of the amino acid residue at position 174 of the N-terminus of native FGF21 from SEQ ID NO:2 with amino acid N;

-substitution of the amino acid residue at position 180 of the N-terminus of native FGF21 from SEQ ID No. 2 with amino acid E and one or more mutations as defined above; and

-a mutation of 1 to 10 amino acid residues which reduces the immunogenicity of the FGF21 variant compared to the native FGF21 of SEQ ID No. 2, and

wherein the GLP-1R agonist has GLP-1R agonist activity that is reduced from 1/531 to 1/9 as compared to the GLP-1R agonist activity of native GLP-1 (7-36).

2. The combination of claim 1, wherein the GLP-1R agonistic activity of the GLP-1R agonist is reduced to 1/482 to 1/9 or 1/319 to 1/9 or 1/121 to 1/9 compared to the GLP-1R agonistic activity of native GLP-1 (7-36).

3. The combination of claim 1, wherein the GLP-1R agonist has a GLP-1R agonistic activity that is reduced from 1/501 to 1/18 or 1/469 to 1/18 or 1/313 to 1/18 or 1/123 to 1/18 as compared to the GLP-1R agonistic activity of native GLP-1 (7-36).

4. The combination according to any one of claims 1 to 3, wherein the FGF21 variant has at least 80% or at least 90% or at least 95% amino acid sequence identity to the amino acid sequence of native FGF 21.

5. The combination according to any one of claims 1 to 4, wherein the FGF21 variant comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 and 64.

6. The combination according to any one of claims 1 to 5, wherein the GLP-1R agonist comprises or consists of the amino acid sequence

H-G-E-G-T-F-T-S-D-X₁₀-S-X₁₂-Q-X₁₄-X₁₅-E-E-X₁₈-V-X₂₀-X₂₁-F-I-E-W-L-X₂₇-X₂₈-X₂₉-X₃₀(SEQ ID NO:37)，

Wherein

X₁₀Is L or K;

X₁₂is K or I;

X₁₄is L or M;

X₁₅is E or D;

X₁₈is A or R;

X₂₀is R or Q;

X₂₁is L or E;

X₂₇is L, E, K or V;

X₂₈is A, N or K;

X₂₉is T or G;

X₃₀is G or R;

wherein, optionally, the amino acid sequence comprises at least one additional amino acid residue at its N-terminus; and is

Wherein, optionally, the amino acid sequence comprises a peptide extension consisting of up to 12, 11 or 10 amino acid residues at its C-terminus.

7. The combination according to any one of claims 1 to 6, wherein the GLP-1R agonist comprises or consists of an amino acid sequence selected from the group consisting of: 9, 10, 12, 14, 15, 16, 17, 19 and 20.

8. A combination according to claim 6 or 7, wherein X₁₄Is L and X₂₈Is A.

9. A pharmaceutical composition comprising an FGF21 (fibroblast growth factor 21) compound and a GLP-1R (glucagon-like peptide-1 receptor) agonist together with a pharmaceutically acceptable carrier and/or excipient,

wherein the FGF21 compound has FGF21 activity that is the same as or substantially the same as the FGF21 activity of native FGF21, and the FGF21 compound is an FGF21 variant, said FGF21 variant comprising at least one mutation selected from the group consisting of:

-substituting the amino acid residue at position 98 to 101 from the N-terminus of the native FGF21 from SEQ ID NO. 2 with the amino acid sequence EIRP (SEQ ID NO: 44);

-substituting the amino acid residues at positions 170 to 174 of the N-terminus of native FGF21 from SEQ ID NO 2 with the amino acid sequence TGLEAV (SEQ ID NO: 45);

-substituting the amino acid sequence TGLEAN (SEQ ID NO:46) for the amino acid residues at positions 170 to 174 from the N-terminus of the native FGF21 from SEQ ID NO: 2;

-substituting the amino acid residue at position 170 of the N-terminus of native FGF21 from SEQ ID NO:2 with amino acid N;

-substitution of the amino acid residue at position 174 of the N-terminus of native FGF21 from SEQ ID NO:2 with amino acid N;

-substitution of the amino acid residue at position 180 of the N-terminus of native FGF21 from SEQ ID No. 2 with amino acid E and one or more mutations as defined above; and

-a mutation of 1 to 10 amino acid residues which reduces the immunogenicity of the FGF21 variant compared to the native FGF21 of SEQ ID No. 2, and

wherein the GLP-1R agonist has GLP-1R agonist activity that is reduced from 1/531 to 1/9 as compared to the GLP-1R agonist activity of native GLP-1 (7-36).

10. A fusion molecule comprising an FGF21 (fibroblast growth factor 21) compound and a GLP-1R (glucagon-like peptide-1 receptor) agonist,

wherein the FGF21 compound has FGF21 activity that is the same as or substantially the same as the FGF21 activity of native FGF21, and the FGF21 compound is an FGF21 variant, said FGF21 variant comprising at least one mutation selected from the group consisting of:

-substituting the amino acid residue at position 98 to 101 from the N-terminus of the native FGF21 from SEQ ID NO. 2 with the amino acid sequence EIRP (SEQ ID NO: 44);

-substituting the amino acid residues at positions 170 to 174 of the N-terminus of native FGF21 from SEQ ID NO 2 with the amino acid sequence TGLEAV (SEQ ID NO: 45);

-substituting the amino acid sequence TGLEAN (SEQ ID NO:46) for the amino acid residues at positions 170 to 174 from the N-terminus of the native FGF21 from SEQ ID NO: 2;

-substituting the amino acid residue at position 170 of the N-terminus of native FGF21 from SEQ ID NO:2 with amino acid N;

-substitution of the amino acid residue at position 174 of the N-terminus of native FGF21 from SEQ ID NO:2 with amino acid N;

-substitution of the amino acid residue at position 180 of the N-terminus of native FGF21 from SEQ ID No. 2 with amino acid E and one or more mutations as defined above; and

-a mutation of 1 to 10 amino acid residues which reduces the immunogenicity of the FGF21 variant compared to the native FGF21 of SEQ ID No. 2, and

wherein the GLP-1R agonist has GLP-1R agonist activity that is reduced from 1/531 to 1/9 as compared to the GLP-1R agonist activity of native GLP-1 (7-36).

11. The fusion molecule of claim 10, wherein the fusion molecule further comprises a hybrid Fc domain comprising a combination of partial Fc regions/domains of different immunoglobulins.

12. The pharmaceutical composition according to claim 9 or the fusion molecule according to claim 10 or 11, wherein the GLP-1R agonist and/or the FGF21 compound are as defined in any one of claims 2 to 8.

13. A nucleic acid molecule encoding the fusion molecule of any one of claims 10 to 12.

14. A host cell comprising the nucleic acid molecule of claim 13.

15. A kit comprising a combination according to any one of claims 1 to 8, a pharmaceutical composition according to claim 9 or 12, a fusion molecule according to any one of claims 10 to 12, a nucleic acid molecule according to claim 13 or a host cell according to claim 14.

16. A combination according to any one of claims 1 to 8, a pharmaceutical composition according to claim 9 or 12, a fusion molecule according to any one of claims 10 to 12, a nucleic acid molecule according to claim 13 or a host cell according to claim 14 for use as a medicament.

17. The combination according to any one of claims 1 to 8, the pharmaceutical composition according to claim 9 or 12, the fusion molecule according to any one of claims 10 to 12, the nucleic acid molecule according to claim 13 or the host cell according to claim 14 for use in the treatment of a disease or disorder selected from the group consisting of: obesity, overweight, metabolic syndrome, diabetes, diabetic retinopathy, hyperglycemia, dyslipidemia, nonalcoholic steatohepatitis (NASH), and atherosclerosis.

18. Use of a combination according to any one of claims 1 to 8, a pharmaceutical composition according to claim 9 or 12, a fusion molecule according to any one of claims 10 to 12, a nucleic acid molecule according to claim 13 or a host cell according to claim 14 in the manufacture of a medicament for the treatment of a disease or disorder selected from the group consisting of: obesity, overweight, metabolic syndrome, diabetes, diabetic retinopathy, hyperglycemia, dyslipidemia, nonalcoholic steatohepatitis (NASH), and atherosclerosis.

19. A method of treating a disease or condition selected from the group consisting of: obesity, overweight, metabolic syndrome, diabetes, diabetic retinopathy, hyperglycemia, dyslipidemia, non-alcoholic steatohepatitis (NASH), and atherosclerosis, the method comprising administering to a subject in need thereof a combination according to any one of claims 1 to 8, a pharmaceutical composition according to claim 9 or 12, a fusion molecule according to any one of claims 10 to 12, a nucleic acid molecule according to claim 13, or a host cell according to claim 14.

20. The combination, pharmaceutical composition, fusion molecule, nucleic acid molecule or host cell for the use according to claim 17, the use according to claim 18 or the method according to claim 19, wherein the diabetes is type 1 diabetes or type 2 diabetes.

Technical Field

The present invention relates to combinations, pharmaceutical compositions and fusion molecules comprising an FGF21 (fibroblast growth factor 21) compound and a GLP-1R (glucagon-like peptide-1 receptor) agonist, having an optimized GLP-1R agonist/FGF 21 compound activity ratio. The invention also relates to the use of said combinations, pharmaceutical compositions and fusion molecules as medicaments, in particular for the treatment of: obesity, overweight, metabolic syndrome, diabetes, diabetic retinopathy, hyperglycemia, dyslipidemia, nonalcoholic steatohepatitis (NASH), and/or atherosclerosis.

Background

Administration of fibroblast growth factor 21(FGF21) compounds (e.g., recombinantly produced FGF21 polypeptide) results in significant reductions in body weight, blood glucose, and blood lipids, as well as improvements in insulin sensitivity, as demonstrated, for example, by Gaich et al (2013) Cell Metab 18(3): 333-. Glucagon-like peptide-1 receptor (GLP-1R) agonists are effective in reducing glucose and body weight in humans, as shown, for example, by Astrup et al (2012) Int J Obes (Lond)36(6): 843-. Combining the beneficial effects of administration of FGF21 with the hypoglycemic effects of GLP-1 receptor agonists surprisingly produces synergistic effects (see, e.g., WO 2011/089203 a1 and WO 2014/037373 a1) that provide a more comprehensive treatment of diseases/disorders such as obesity, overweight, metabolic syndrome, diabetes, diabetic retinopathy, hyperglycemia, dyslipidemia, nonalcoholic steatohepatitis (NASH), and/or atherosclerosis.

Summary of The Invention

The combination of an FGF21 compound and a GLP-1R agonist (e.g., in the form of a fusion protein) can be used, for example, to improve glycemic control in overweight to obese dyslipidemia patients with type 2 diabetes.

Notably, FGF21 and GLP-1 (as the primary GLP-1R agonist) exert their pharmacological effects at different plasma concentrations. More particularly, the FGF21 effect starts to act at higher plasma levels compared to the GLP-1 effect. Furthermore, GLP-1 is known to have adverse effects, such as nausea and vomiting, at higher levels. Taken together, this means that there is a potential risk of GLP-1 mediated adverse effects when a combination of an FGF21 compound and a GLP-1R agonist is administered (e.g., in the form of a fusion protein).

It is therefore an object of the present invention to determine an optimized GLP-1R agonist/FGF 21 compound activity ratio in order to achieve beneficial effects while avoiding potential adverse effects (e.g. nausea and vomiting). It is another object of the invention to provide corresponding combinations, pharmaceutical compositions and fusion molecules having an optimized GLP-1R agonist/FGF 21 compound activity ratio.

In one aspect, the invention relates to a combination comprising an FGF21 (fibroblast growth factor 21) compound and a GLP-1R (glucagon-like peptide-1 receptor) agonist,

wherein the FGF21 compound has FGF21 activity that is the same as or substantially the same as the FGF21 activity of native FGF21, and the FGF21 compound is an FGF21 variant, said FGF21 variant comprising at least one mutation selected from the group consisting of:

-substituting the amino acid residue at position 98 to 101 from the N-terminus of the native FGF21 from SEQ ID NO. 2 with the amino acid sequence EIRP (SEQ ID NO: 44);

-substituting the amino acid residues at positions 170 to 174 of the N-terminus of native FGF21 from SEQ ID NO 2 with the amino acid sequence TGLEAV (SEQ ID NO: 45);

-substituting the amino acid sequence TGLEAN (SEQ ID NO:46) for the amino acid residues at positions 170 to 174 from the N-terminus of the native FGF21 from SEQ ID NO: 2;

-substituting the amino acid residue at position 170 of the N-terminus of native FGF21 from SEQ ID NO:2 with amino acid N;

-substitution of the amino acid residue at position 174 of the N-terminus of native FGF21 from SEQ ID NO:2 with amino acid N;

-substitution of the amino acid residue at position 180 of the N-terminus of native FGF21 from SEQ ID No. 2 with amino acid E and one or more mutations as defined above; and

-a mutation of 1 to 10 amino acid residues which reduces the immunogenicity of the FGF21 variant compared to the native FGF21 of SEQ ID No. 2, and

wherein the GLP-1R agonist activity of the GLP-1R agonist is reduced from 1/531 to 1/9 (or 1/531.0 to 1/9.449) as compared to the GLP-1R agonist activity of native GLP-1 (7-36).

In one embodiment, the FGF21 activity is activation of the FGF21 receptor. In one embodiment, the term refers to in vitro activity. In one embodiment, activation of the FGF21 receptor is determined by measuring autophosphorylation of the FGF21 receptor when contacted with the FGF21 compound in vitro. In one embodiment, FGF21 activity is determined by using an In-Cell Western (ICW) assay, e.g., substantially as described In example 3.

In one embodiment, the GLP-1R agonistic activity is activation of the GLP-1 receptor. In one embodiment, the term refers to in vitro agonistic activity. In one embodiment, activation of the GLP-1 receptor is determined by measuring the cAMP response of a cell stably expressing the GLP-1 receptor when contacted with said agonist in vitro. In one embodiment, the activation of the GLP-1 receptor is substantially as determined in example 4.

In one embodiment, the GLP-1R agonist has GLP-1R agonistic activity that is reduced to 1/482 to 1/9 (or 1/482.396 to 1/9.449) or 1/319 to 1/9 to (or 1/319.311 to 1/9.449) or 1/121 to 1/9 (or 1/121.189 to 1/9.449) as compared to the GLP-1R agonistic activity of native GLP-1 (7-36).

In one embodiment, the GLP-1R agonist has GLP-1R agonist activity that is reduced to 1/319 to 1/9 as compared to the GLP-1R agonist activity of native GLP-1 (7-36).

In one embodiment, the GLP-1R agonist has GLP-1R agonist activity that is reduced by at most 1/9.4 or at most 1/9.45 or at most 1/9.5 as compared to the GLP-1R agonist activity of native GLP-1 (7-36).

In one embodiment, the GLP-1R agonist has GLP-1R agonist activity that is reduced by at most 1/10 compared to the GLP-1R agonist activity of native GLP-1 (7-36).

In one embodiment, the GLP-1R agonist has GLP-1R agonist activity that is reduced by at least 1/482.4 or at least 1/482.35 as compared to the GLP-1R agonist activity of native GLP-1 (7-36).

In one embodiment, the GLP-1R agonist has GLP-1R agonist activity that is reduced by at least 1/482 compared to the GLP-1R agonist activity of native GLP-1 (7-36).

In one embodiment, the GLP-1R agonist has GLP-1R agonist activity that is reduced to 1/482 to 1/10 as compared to the GLP-1R agonist activity of native GLP-1 (7-36).

In one embodiment, the GLP-1R agonist has GLP-1R agonist activity that is reduced to 1/319 to 1/10 as compared to the GLP-1R agonist activity of native GLP-1 (7-36).

In one embodiment, the GLP-1R agonist activity of the GLP-1R agonist is reduced from 1/100 to 1/90 as compared to the GLP-1R agonist activity of native GLP-1 (7-36).

In one embodiment, the GLP-1R agonist has GLP-1R agonist activity that is reduced by at most 1/18 (or at most 1/18.268) compared to the GLP-1R agonist activity of native GLP-1 (7-36).

In one embodiment, the GLP-1R agonist has GLP-1R agonistic activity that is reduced from 1/501 to 1/18 (or 1/500.686 to 1/18.268) or 1/469 to 1/18 (or 1/468.679 to 1/18.268) or 1/313 to 1/18 (or 1/313.214 to 1/18.268) or 1/123 to 1/18 (or 1/123.466 to 1/18.268) as compared to the GLP-1R agonistic activity of native GLP-1 (7-36).

In one embodiment, the GLP-1R agonist has GLP-1R agonist activity that is reduced to 1/313 to 1/18 as compared to the GLP-1R agonist activity of native GLP-1 (7-36).

In one of the above embodiments, the GLP-1R agonist has GLP-1R agonist activity that is reduced by a factor of at most 1/18.2 or at most 1/18.3 compared to the GLP-1R agonist activity of native GLP-1 (7-36).

In one embodiment, the FGF21 variant has at least 80% or at least 90% or at least 95% amino acid sequence identity to the amino acid sequence of native FGF 21.

In one embodiment, the FGF21 variant comprises or consists of an amino acid sequence selected from the group consisting of seq id no:47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 and 64.

In one embodiment, the GLP-1R agonist comprises or consists of the amino acid sequence

H-G-E-G-T-F-T-S-D-X₁₀-S-X₁₂-Q-X₁₄-X₁₅-E-E-X₁₈-V-X₂₀-X₂₁-F-I-E-W-L-X₂₇-X₂₈-X₂₉-X₃₀(SEQ ID NO:37)，

Wherein

X₁₀Is L or K;

X₁₂is K or I;

X₁₄is L or M;

X₁₅is E or D;

X₁₈is A or R;

X₂₀is R or Q;

X₂₁is L or E;

X₂₇is L, E, K or V;

X₂₈is A, N or K;

X₂₉is T or G;

X₃₀is G or R;

wherein, optionally, the amino acid sequence comprises at least one additional amino acid residue at its N-terminus; and is

Wherein, optionally, the amino acid sequence comprises a peptide extension consisting of up to 12, 11 or 10 amino acid residues at its C-terminus.

In one embodiment, the GLP-1R agonist comprises or consists of an amino acid sequence selected from the group consisting of: 9, 10, 12, 14, 15, 16, 17, 19 and 20.

In one embodiment, X₁₄Is L and X₂₈Is A.

In one embodiment, the GLP-1R agonist comprises or consists of an amino acid sequence selected from the group consisting of: 9, 10, 12, 14, 16, 17, 19 and 20.

In another aspect, the present invention relates to a pharmaceutical composition comprising an FGF21 (fibroblast growth factor 21) compound and a GLP-1R (glucagon-like peptide-1 receptor) agonist together with a pharmaceutically acceptable carrier and/or excipient,

wherein the FGF21 compound has FGF21 activity that is the same as or substantially the same as the FGF21 activity of native FGF21, and the FGF21 compound is an FGF21 variant, said FGF21 variant comprising at least one mutation selected from the group consisting of:

-substituting the amino acid residue at position 98 to 101 from the N-terminus of the native FGF21 from SEQ ID NO. 2 with the amino acid sequence EIRP (SEQ ID NO: 44);

-substituting the amino acid residues at positions 170 to 174 of the N-terminus of native FGF21 from SEQ ID NO 2 with the amino acid sequence TGLEAV (SEQ ID NO: 45);

-substituting the amino acid sequence TGLEAN (SEQ ID NO:46) for the amino acid residues at positions 170 to 174 from the N-terminus of the native FGF21 from SEQ ID NO: 2;

-substituting the amino acid residue at position 170 of the N-terminus of native FGF21 from SEQ ID NO:2 with amino acid N;

-substitution of the amino acid residue at position 174 of the N-terminus of native FGF21 from SEQ ID NO:2 with amino acid N;

-substitution of the amino acid residue at position 180 of the N-terminus of native FGF21 from SEQ ID No. 2 with amino acid E and one or more mutations as defined above; and

-a mutation of 1 to 10 amino acid residues which reduces the immunogenicity of the FGF21 variant compared to the native FGF21 of SEQ ID No. 2, and

wherein the GLP-1R agonist has GLP-1R agonist activity that is reduced from 1/531 to 1/9 (or 1/531.0 to 1/9.449) compared to the GLP-1R agonist activity of native GLP-1 (7-36).

In yet another aspect, the invention relates to a fusion molecule comprising an FGF21 (fibroblast growth factor 21) compound and a GLP-1R (glucagon-like peptide-1 receptor) agonist,

wherein the FGF21 compound has FGF21 activity that is the same as or substantially the same as the FGF21 activity of native FGF21, and the FGF21 compound is an FGF21 variant, said FGF21 variant comprising at least one mutation selected from the group consisting of:

-substituting the amino acid residue at position 98 to 101 from the N-terminus of the native FGF21 from SEQ ID NO. 2 with the amino acid sequence EIRP (SEQ ID NO: 44);

-substituting the amino acid residues at positions 170 to 174 of the N-terminus of native FGF21 from SEQ ID NO 2 with the amino acid sequence TGLEAV (SEQ ID NO: 45);

-substituting the amino acid sequence TGLEAN (SEQ ID NO:46) for the amino acid residues at positions 170 to 174 from the N-terminus of the native FGF21 from SEQ ID NO: 2;

-substituting the amino acid residue at position 170 of the N-terminus of native FGF21 from SEQ ID NO:2 with amino acid N;

-substitution of the amino acid residue at position 174 of the N-terminus of native FGF21 from SEQ ID NO:2 with amino acid N;

-substitution of the amino acid residue at position 180 of the N-terminus of native FGF21 from SEQ ID No. 2 with amino acid E and one or more mutations as defined above; and

-a mutation of 1 to 10 amino acid residues which reduces the immunogenicity of the FGF21 variant compared to the native FGF21 of SEQ ID No. 2, and

wherein the GLP-1R agonist has GLP-1R agonist activity that is reduced from 1/531 to 1/9 (or 1/531.0 to 1/9.449) compared to the GLP-1R agonist activity of native GLP-1 (7-36).

In one embodiment, the fusion molecule further comprises a hybrid Fc domain comprising a combination of partial Fc regions/domains of different immunoglobulins.

In one embodiment, the GLP-1R agonist and/or the FGF21 compound are as defined above.

In another aspect, the invention relates to a nucleic acid molecule encoding a fusion molecule as defined above.

In another aspect, the invention relates to a host cell comprising a nucleic acid molecule as defined above.

In another aspect, the invention relates to a kit comprising a combination as defined above, a pharmaceutical composition as defined above, a fusion molecule as defined above, a nucleic acid molecule as defined above or a host cell as defined above.

In a further aspect, the invention relates to a combination as defined above, a pharmaceutical composition as defined above, a fusion molecule as defined above, a nucleic acid molecule as defined above or a host cell as defined above for use as a medicament.

In another aspect, the present invention relates to a combination as defined above, a pharmaceutical composition as defined above, a fusion molecule as defined above, a nucleic acid molecule as defined above or a host cell as defined above for use in the treatment of a disease or disorder selected from the group consisting of: obesity, overweight, metabolic syndrome, diabetes, diabetic retinopathy, hyperglycemia, dyslipidemia, nonalcoholic steatohepatitis (NASH), and atherosclerosis.

In one embodiment, the disease or disorder is diabetes. In one embodiment, the diabetes is type 1 diabetes or type 2 diabetes.

In another aspect, the invention relates to the use of a combination as defined above, a pharmaceutical composition as defined above, a fusion molecule as defined above, a nucleic acid molecule as defined above or a host cell as defined above for the manufacture of a medicament for the treatment of a disease or disorder selected from the group consisting of: obesity, overweight, metabolic syndrome, diabetes, diabetic retinopathy, hyperglycemia, dyslipidemia, nonalcoholic steatohepatitis (NASH), and atherosclerosis.

In one embodiment, the disease or disorder is diabetes. In one embodiment, the diabetes is type 1 diabetes or type 2 diabetes.

In another aspect, the invention relates to a method of treating a disease or condition selected from the group consisting of: obesity, overweight, metabolic syndrome, diabetes, diabetic retinopathy, hyperglycemia, dyslipidemia, non-alcoholic steatohepatitis (NASH), and atherosclerosis, the method comprising administering to a subject in need thereof a combination as defined above, a pharmaceutical composition as defined above, a fusion molecule as defined above, a nucleic acid molecule as defined above, or a host cell as defined above.

In one embodiment, the disease or disorder is diabetes. In one embodiment, the diabetes is type 1 diabetes or type 2 diabetes.

Drawings

Figure 1 is a graph showing EC50 for adverse effects (gastric emptying (GE) rate) and pharmacodynamics (i.e., HbA1c, triglycerides, fatty acids, non-HDL, fat mass) according to GLP-1 attenuation factor (12 month simulation):

for GLP-1 attenuating factors greater than 9.449 (rounded to 9), EC50 for GLP-1 mediated adverse effects of the gastrointestinal tract (gastric emptying; GE rate) is greater than EC50 for pharmacodynamic effects (i.e., HbA1c, fat mass, non-HDL, fatty acids, triglycerides).

The maximum distance between the maximum of pharmacodynamics (HbA1c) normalized by the extended FGF21- (lipids) and GLP-1 mediated effect (HbA1c) and the adverse effect (GE rate) was 121.189; that is, at 121.189 (which may be rounded to 121), there is a maximum distance between the maximum of the pharmacodynamic effect (HbA1c) and the adverse effect (GE rate) at the minimum distance between the GLP-1 mediated effect (HbA1c) and the mean effect mediated by FGF21 (i.e., fat mass, non-HDL, fatty acids, triglycerides) (see fig. 2);

the maximum distance between the maximum of pharmacodynamics (HbA1c) and adverse effects (GE rate) was 319.311 (roundable to 319);

the maximum distance between the mean pharmacodynamics (i.e., HbA1c, fat mass, non-HDL, fatty acids, triglycerides) and adverse effects (GE rate) was 482.396 (see FIG. 2; can be rounded to 482);

the maximum value of gastric emptying rate is 531.0;

(all: vertical lines).

Figure 2 is a graph of EC50 showing Gastric Emptying (GE) rate and average pharmacodynamic effects (i.e., HbA1c, triglycerides, fatty acids, non-HDL, fat mass) according to GLP-1 attenuation factor (12 month simulation):

the maximum distance between the mean pharmacodynamics (i.e., HbA1c, fat mass, non-HDL, fatty acids, triglycerides) and adverse effects (GE rate) was 482.396 (right vertical line; may be rounded to 482);

the maximum distance between the maximum of pharmacodynamics (HbA1c) normalized by the extended FGF21- (lipids) and GLP-1 mediated effect (HbA1c) and the adverse effect (GE rate) was 121.189 (left vertical line; can be rounded to 121). The curve "(maximum-GE ratio)/range" represents the ratio of the maximum distance between HbA1c and GE ratio relative to the minimum distance between HbA1c and the mean effect mediated by FGF21 (i.e., fat mass, non-HDL, fatty acids, triglycerides). At the minimum of the "(max-GE rate)/range" curve (i.e., at 121.189), at the minimum distance between the GLP-1 mediated effect (HbA1c) and the FGF21 mediated effect (i.e., fat mass, non-HDL, fatty acids, triglycerides), there is a maximum distance between the maximum of the pharmacodynamic effect (HbA1c) and the adverse effect (GE rate).

Figure 3 is a graph showing EC50 for adverse effects (gastric emptying (GE) rate) and pharmacodynamics (HbA1c, triglycerides, fatty acids, non-HDL, fat mass) according to GLP-1 attenuation factor (3 month simulation):

EC50 for GLP-1-mediated adverse gastrointestinal effects (gastric emptying; GE rates) greater than 18.268 (rounded to 18) for GLP-1 attenuating factors greater than EC50 for pharmacodynamic effects (i.e., HbA1c, fat mass, non-HDL, fatty acids, triglycerides);

the maximum distance between the maximum of pharmacodynamics (HbA1c) normalized by the extended FGF21- (lipids) and GLP-1 mediated effect (HbA1c) and the adverse effect (GE rate) was 123.466; that is, at 123.466 (which may be rounded to 123), there is a maximum distance between the maximum of the pharmacodynamic effect (HbA1c) and the adverse effect (GE rate) at the minimum distance between the GLP-1 mediated effect (HbA1c) and the mean effect mediated by FGF21 (i.e., fat mass, non-HDL, fatty acids, triglycerides) (see fig. 4);

the maximum distance between the maximum of the pharmacodynamics (HbA1c) and the adverse effect (GE rate) is 313.214 (rounded to 313);

the maximum distance between the mean pharmacodynamics (i.e., HbA1c, fat mass, non-HDL, fatty acids, triglycerides) and adverse effects (GE rate) was 468.679 (see FIG. 4; round to 469);

the maximum gastric emptying rate is 500.686 (rounded to 501)

(all: vertical lines).

Figure 4 is a graph of EC50 showing Gastric Emptying (GE) rate and average pharmacodynamic effects (i.e., HbA1c, triglycerides, fatty acids, non-HDL, fat mass) according to GLP-1 attenuation factor (3 month simulation):

the maximum distance between the mean pharmacodynamics (i.e., HbA1c, fat mass, non-HDL, fatty acids, triglycerides) and adverse effects (GE rate) was 468.679 (right vertical line; may be rounded to 469);

the maximum distance between the maximum of pharmacodynamics (HbA1c) normalized by the extended FGF21- (lipids) and GLP-1 mediated effect (HbA1c) and the adverse effect (GE rate) was 123.466 (left vertical line; round to 123). The curve "(maximum-GE ratio)/range" represents the ratio of the maximum distance between HbA1c and GE ratio relative to the minimum distance between HbA1c and the mean effect mediated by FGF21 (i.e., fat mass, non-HDL, fatty acids, triglycerides). At the minimum of the "(max-GE rate)/range" curve (i.e., at 123.466), at the minimum distance between the GLP-1 mediated effect (HbA1c) and the FGF21 mediated effect (i.e., fat mass, non-HDL, fatty acids, triglycerides), there is a maximum distance between the maximum of the pharmacodynamic effect (HbA1c) and the adverse effect (GE rate).

FIG. 5 shows dose response curves for (A) FGFR autophosphorylation or (B) ERK 1/2-phosphorylation In CHO cells overexpressing human FGFR1c and β -klotho as measured by In-Cell Western after stimulation with mature human FGF21(SEQ ID NO: 2).

Detailed Description

Although the invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Hereinafter, certain elements of the present invention will be described. These elements may be enumerated with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to produce other embodiments. The examples and preferred embodiments described differently should not be construed as limiting the invention to only the embodiments explicitly described. This description should be understood to support and encompass embodiments that combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Moreover, any permutation and combination of all the elements described in this application should be considered to be disclosed in the specification of this application unless the context indicates otherwise.

The term as used herein is as used herein, such as "A multilingual collaboration of biological entities" (IUPAC Recommendations) ", H.G.W.Leuenberger, B.Nagel and H.Editors, Helvetica Chimica Acta, CH-4010Basel, Switzerland, (1995).

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology and recombinant DNA techniques, which are explained in the literature of the art (Sambrook, J. et al (2001) Molecular Cloning: A Laboratory Manual, 3 rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated member, integer or step, or group of members, integers or steps, but not the exclusion of any other member, integer or step, or group of members, integers or steps, but in some embodiments the exclusion of such other member, integer or step, or group of members, integers or steps, i.e. the subject matter lies in the inclusion of a stated member, integer or step, or group of members, integers or steps. The use of the terms "a" and "an" and "the" and similar references in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Throughout the text of this specification, several documents are cited. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.) whether supra or infra is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

By using a systemic pharmacologic approach that integrates GLP-1 receptor signaling with key components of FGF21 production and action in the pathophysiology of diabetes, the inventors succeeded in determining an optimal GLP-1R agonist/FGF 21 compound activity ratio to achieve the beneficial effects of both agents (e.g., in terms of weight, lipids, glycemic control) while avoiding potential adverse effects (e.g., nausea and vomiting).

The term "combination" as used herein is intended to include the following: it is allowed to administer to the patient a combination comprising an FGF21 compound and a GLP-1R agonist either alone or in a form in which a combination product of an FGF21 compound and a GLP-1R agonist is present (e.g. in one pharmaceutical composition) or in the form of a fusion molecule/protein. When administered separately, administration may be simultaneous or sequential in either order. The amounts of FGF21 compound and GLP-1R agonist, as well as the relative timing of administration, are selected to achieve the desired combined therapeutic effect. Administration of the combination can be carried out in parallel in the following form: (1) a single pharmaceutical composition comprising all active pharmaceutical ingredients; or (2) separate pharmaceutical compositions, each comprising at least one active pharmaceutical ingredient. Alternatively, the combination may be administered separately in a sequential manner, wherein one therapeutic agent is administered first, followed by the other therapeutic agent, or vice versa. Such sequential administration may be close in time or far apart. In one embodiment, the combination is provided in the form of a kit, e.g. as defined herein.

The term "fibroblast growth factor 21" or "FGF 21" as used herein refers to any FGF21 protein known in the art, and specifically to human FGF 21. In one embodiment, human FGF21 has the amino acid sequence of SEQ ID NO. 1.

The term "FGF 21 compound" as used herein generally refers to a compound having FGF21 activity.

In one embodiment, the FGF21 compound is a peptidic compound, i.e., a peptide or a protein.

The term "peptide" as used herein refers to a polymeric form of amino acids of any length, for example, comprising two or more, or 3 or more, or 4 or more, or 6 or more, or 8 or more, or 9 or more, or 10 or more, or 13 or more, or 16 or more, or 21 or more amino acids covalently linked by peptide bonds. A peptide may, for example, consist of up to 100 amino acids. The term "polypeptide" refers to large peptides, preferably peptides having more than 100 amino acid residues. The terms "polypeptide" and "protein" are used interchangeably herein.

In one embodiment, the FGF21 compound is a native FGF21 or FGF21 variant, said FGF21 variant having at least 80% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% amino acid sequence identity to the amino acid sequence of native FGF 21.

The term "native FGF 21" as used herein refers to a naturally occurring FGF21, e.g., human wild-type FGF21 having the amino acid sequence of SEQ ID NO:1 (also referred to as "full length human wild-type FGF 21"). The term "native FGF 21" as used herein also includes mature FGF21, i.e., naturally occurring FGF21 lacking the native signal sequence (also referred to as signal peptide). In one embodiment, native FGF21 is mature human wild-type FGF21 lacking amino acids 1 through 28 of SEQ ID NO:1 (M1 through A28) and is represented by SEQ ID NO: 2.

"sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. In addition to manual means, optimal alignments of sequences can be generated for comparison by the local homology algorithm of Smith and Waterman (1981, Ads App. Math.2,482), by the local homology algorithm of Neddleman and Wunsch (1970, J.mol.biol.48,443), by the similarity search method of Pearson and Lipman (1988, Proc.Natl Acad.Sci.USA 85,2444), or by Computer programs using these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group,575Science Drive, Madison, Wis.).

FGF21 variants can be based on deletion, addition, and/or substitution of at least one amino acid residue in native FGF21 (e.g., SEQ ID NO:1 or 2).

Such deletions, additions and/or substitutions may contribute to improved stability, e.g., proteolytic stability and/or thermostability, of the variant as compared to native FGF21 (e.g., SEQ ID NO:1 or 2). This can be achieved, for example, by preventing protease cleavage at or near the substituted amino acid or by forming one or more additional disulfide bridges.

The term "amino acid" or "amino acid residue" as used herein refers to naturally occurring amino acids, non-natural amino acids that function in a similar manner as naturally occurring amino acids, amino acid analogs, and amino acid mimetics, all of which are stereoisomers if their structure allows for their D and L stereoisomeric forms. Amino acids are referred to herein by their name, by their well-known three-letter symbols, or by the one-letter symbols recommended by the IUPAC-IUB Biochemical nomenclature Commission.

The term "naturally occurring" when used in connection with amino acids refers to the 20 conventional amino acids (i.e., alanine (a), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y)), as well as selenocysteine (selenocystein), Pyrrolysine (PYL), and pyrroline-carboxylysine (PCL).

The term "unnatural amino acid" as used herein means an amino acid that is not encoded in the genetic code of any organism or is not found in any organism. It may be, for example, a pure synthetic compound. Examples of unnatural amino acids include, but are not limited to, hydroxyproline, γ -carboxyglutamic acid, O-phosphoserine, azetidinecarboxylic acid (azetidinecarboxylic acid), 2-aminoadipic acid, 3-aminoadipic acid, β -alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, t-butylglycine, 2, 4-diaminoisobutyric acid, desmosine (desmosine), 2' -diaminopimelic acid, 2, 3-diaminopropionic acid, N-ethylglycine, N-methylglycine, N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, and N-carboxyglutamic acid, 4-hydroxyproline, isodesmine, allo-isoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthylalanine, norvaline, norleucine, ornithine, D-arginine, p-aminophenylalanine, pentylglycine, pipecolic acid (pipecolic acid) and thioproline.

The term "amino acid analog" as used herein refers to a compound having the same basic chemical structure as a naturally occurring amino acid. Amino acid analogs include natural and unnatural amino acids that are chemically blocked, reversibly or irreversibly, or that have their C-terminal carboxyl group, their N-terminal amino group, and/or their side chain functional groups chemically modified. Such analogs include, but are not limited to, methionine sulfoxide, methionine sulfone, S- (carboxymethyl) -cysteine sulfoxide, S- (carboxymethyl) -cysteine sulfone, aspartic acid- (β -methyl ester), N-ethylglycine, alanine carboxamide (alanine carboxamide), homoserine, norleucine and methionine methyl sulfonium.

The term "amino acid mimetic" as used herein refers to a chemical compound that has a structure that is different from the general chemical structure of an amino acid, but functions in a similar manner to a naturally occurring amino acid.

In some embodiments, the variant comprises at least one additional amino acid at its N-terminus. In one embodiment, the at least one additional amino acid is selected from the group consisting of naturally occurring amino acids other than proline, unnatural amino acids, amino acid analogs, and amino acid mimetics. In one embodiment, the at least one additional amino acid is selected from G, A, N and C. In particular embodiments, at least one additional amino acid is G.

Suitable FGF21 variants for use in the present invention are described in, for example, WO 2016/114633 a1, WO 2017/093465 a1, WO 2017/074117 a1, WO 2017/074123 a1 and WO 2018/088838 a1, which are incorporated herein by reference.

In one embodiment, the FGF21 compound is a FGF21 variant, said FGF21 variant comprising or consisting of an amino acid sequence selected from the group consisting of seq id no:3, 4, 5 and 6.

In one embodiment, the FGF21 compound is a FGF21 variant, said FGF21 variant comprising at least one mutation selected from the group consisting of:

-substituting the amino acid residue at position 98 to 101 from the N-terminus of the native FGF21 from SEQ ID NO. 2 with the amino acid sequence EIRP (SEQ ID NO: 44);

-substituting the amino acid residues at positions 170 to 174 of the N-terminus of native FGF21 from SEQ ID NO 2 with the amino acid sequence TGLEAV (SEQ ID NO: 45);

-substituting the amino acid sequence TGLEAN (SEQ ID NO:46) for the amino acid residues at positions 170 to 174 from the N-terminus of the native FGF21 from SEQ ID NO: 2;

-substituting the amino acid residue at position 170 of the N-terminus of native FGF21 from SEQ ID NO:2 with amino acid N;

-substitution of the amino acid residue at position 174 of the N-terminus of native FGF21 from SEQ ID NO:2 with amino acid N;

-substitution of the amino acid residue at position 180 of the N-terminus of native FGF21 from SEQ ID No. 2 with amino acid E and one or more mutations as defined above; and

-a mutation of 1 to 10 amino acid residues which reduces the immunogenicity of the FGF21 variant compared to the native FGF21 of SEQ ID No. 2.

The immunogenicity of a given FGF21 variant can be predicted by conventional methods known in the art. For example, it is possible to use, for example, iTope^TMAnd/or TCED^TMMethods to screen proteins for potential immunogenicity. Furthermore, mutations for minimizing immunogenicity can be designed by conventional methods known in the art. For example, when EpiScreen is performed^TMWhen immunogenicity is observed by analysis to assess potential immunogenicity, the amino acid sequences that induce immunogenicity can be identified by T-cell epitope mapping and designed by in silico predictionA mutant with minimized antigenicity.

In one embodiment, the FGF21 compound is a FGF21 variant, said FGF21 variant comprising or consisting of an amino acid sequence selected from the group consisting of seq id nos: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 and 64.

The FGF21 compounds comprised in the combinations, pharmaceutical compositions and fusion molecules of the invention exhibit FGF21 activity that is the same or substantially the same as the FGF21 activity of native FGF21 (e.g., SEQ ID NO: 2). In one embodiment, FGF21 activity refers to the activity of FGF21 (see below) of an FGF21 compound when not comprised in (not a component of) a fusion molecule as defined herein and/or when not further modified.

The term "substantially the same" as used herein refers to an FGF21 activity in the range of 50% to 150% or 60% to 140% or 65% to 135% of the FGF21 activity of native FGF21 (e.g., SEQ ID NO: 2).

In one embodiment, the term "FGF 21 activity" (or "FGF 21 potency") as used herein refers to the activation of FGF21 receptors (FGFRs, e.g., FGFR1 c). In one embodiment, the FGF21 receptor is a human FGF21 receptor. In one embodiment, the term refers to activity/potency in vitro. In another embodiment, the term refers to activity/potency in vivo. In one embodiment, activation of the FGF21 receptor is determined by measuring autophosphorylation of the FGF21 receptor when contacted with an FGF21 compound in vitro. In one embodiment, FGF21 activity/potency is determined by using an In-Cell Western (ICW) assay. In one embodiment, activity/potency is quantified by determining EC50 values.

The term "In-Cell Western (ICW) assay" as used herein refers to an immunocytochemical assay, more specifically a quantitative immunofluorescence assay, typically performed In a microplate (e.g., In 96-well or 384-well format). Which combines the specificity of western blotting with the reproducibility and throughput of ELISA (see, e.g., Aguilar h.n. et al (2010) PLoS ONE 5(4): e 9965). Suitable ICW assay systems are commercially available (e.g., from LI-COR Biosciences, USA). In one embodiment, anti-pFGFR and/or anti-pERK are used in the ICW assay. In one embodiment, a pFGFR ICW assay is performed. In one embodiment, the ICW assay is performed essentially as described in example 3.

In one embodiment, an FGF21 compound having the same or substantially the same FGF21 activity as the FGF21 activity of native FGF21 can be defined in terms of its EC50 value for FGF21 receptor activation. For example, an FGF21 compound having FGF21 activity in the range of 50% to 150% or 60% to 140% or 65% to 135% of the FGF21 activity of native FGF21 (e.g., SEQ ID NO:2) may also be referred to herein as an FGF21 compound that activates the FGF21 receptor with an EC50 of 2.40 to 7.20nmol/L or 2.88 to 6.72nmol or 3.12 to 6.48nmol/L, respectively, in the pFGFR ICW assay, e.g., as substantially described in example 3. In one embodiment, EC50 values are given as EC50 ± SD. In one embodiment, SD is the assay-dependent standard deviation. In one embodiment, the EC50 is 2.40 ± SD to 7.20 ± SD nmol/L or 2.88 ± SD to 6.72 ± SD nmol/L or 3.12 ± SD to 6.48 ± SD nmol/L, respectively, in the pFGFR ICW assay, e.g., as substantially described in example 3. In one embodiment, SD is 1.8 nmol/L.

According to the present invention, the FGF21 compound may be further modified, e.g., fused/conjugated to another entity/molecule, such as a polymer (e.g., PEG) or a peptide/polypeptide, such as Human Serum Albumin (HSA) or an Fc region/domain of an immunoglobulin or a variant thereof, e.g., as described further below. In one embodiment, the FGF21 activity of an FGF21 compound referred to herein is the FGF21 activity of an FGF21 compound without such further modification, also referred to herein as a "pure FGF21 compound".

The term "fused to" as used herein specifically refers to genetic fusion, e.g., by recombinant DNA techniques. The amino acid sequence of the (poly) peptide half-life extending module may be introduced at any position within the variant amino acid sequence and may, for example, be in a circular shape within the encoded protein structure, or it may be an N-terminal or C-terminal fusion.

The term "conjugated to" as used herein specifically refers to chemical and/or enzymatic conjugation that results in a stable covalent linkage between the (poly-) peptide and another molecule (e.g., a variant and half-life extending moiety). Such conjugation may occur at the N-terminus or C-terminus of the (poly-) peptide or at a specific side chain, for example, at a lysine, cysteine, tyrosine or unnatural amino acid residue.

The term "GLP-1R agonist" (abbreviated: "GLP-1 RA") as used herein generally refers to compounds that bind to and activate the GLP-1 receptor, such as GLP-1 (as a primary GLP-1R agonist).

In one embodiment, the GLP-1R agonist is a peptidic compound, i.e., a peptide or a protein. In another embodiment, the GLP-1R agonist is a small molecule, i.e., an organic compound having a molecular weight of less than 900 Da.

The GLP-1R agonist comprised in the combination, pharmaceutical composition and fusion molecule of the invention exhibits reduced GLP-1R agonistic activity as defined herein compared to native GLP-1 (7-36). The value "x" in the expression "decrease x times" as used herein may refer to a "decay factor" or a "decrease factor" herein. In one embodiment, the reduced GLP-1R agonist activity as defined herein compared to native GLP-1(7-36) is exhibited when the GLP-1R agonist is a component of the fusion molecule as defined herein.

The term "native GLP-1 (7-36)" as used herein refers to a peptide having the amino acid sequence of SEQ ID NO:7, optionally comprising an amide group at its C-terminus.

In one embodiment, the term "GLP-1R agonistic activity" (or "GLP-1R agonistic potency") as used herein refers to the activation of the GLP-1 receptor. In one embodiment, the term refers to agonistic activity/potency in vitro. In another embodiment, the term refers to agonistic activity/potency in vivo. In one embodiment, activation of the GLP-1 receptor is determined by measuring cAMP response in cells stably expressing the GLP-1 receptor when contacted with an agonist in vitro. In one embodiment, the cells are from the HEK-293 cell line. In one embodiment, the GLP-1 receptor is a human GLP-1 receptor. In one embodiment, activation of the GLP-1 receptor is determined substantially as described in example 4. In one embodiment, activity/potency is quantified by determining EC50 values.

In one embodiment, GLP-1R agonists with reduced GLP-1R agonist activity compared to the GLP-1R agonist activity of native GLP-1(7-36) may be defined in terms of their GLP-1 receptor activated EC50 value, e.g., as indicated in Table 4. For example, a GLP-1R agonist having GLP-1R agonist activity that is reduced to 1/531 to 1/9 compared to the GLP-1R agonist activity of native GLP-1(7-36) may also be referred to herein as a GLP-1R agonist that activates the GLP-1 receptor with an EC50 of 6.93 to 408.87pmol/L, and the like. In one embodiment, EC50 values are determined as described above. In one embodiment, EC50 values are given as EC50 ± SD. In one embodiment, SD is the assay-dependent standard deviation.

Suitable GLP-1R agonists with reduced GLP-1R agonist activity compared to GLP-1R agonist activity of native GLP-1(7-36) can be identified by assays described herein for determining GLP-1R agonist activity, e.g., as described in example 4 or in Xiao et al (2001) biochemistry.40(9):2860-9 or Gault et al (2013) J Biol chem.288(49):35581-91, e.g., assays for GLP-1R agonist-induced production of cytosolic cAMP, beta cell preservation (apoptosis), or glucose-stimulated insulin secretion (GSIS), etc. Suitable GLP-1R agonists as described above may be identified, for example, by: variants of known peptidic GLP-1R agonists, such as native GLP-1(7-36), are generated, for example, by random or site-directed mutagenesis or chemical synthesis (see, e.g., example 5), and their GLP-1R agonistic activity is then determined as described herein using native GLP-1(7-36) as a control. Alternatively, suitable GLP-1R agonists as described above can be identified by screening a library of small molecules for GLP-1R agonist activity using native GLP-1(7-36) as a control. These assays can be performed in the form of high throughput assays.

Variants of known peptidic GLP-1R agonists, e.g. native GLP-1(7-36), may be based on/by deletion, addition and/or substitution of at least one amino acid residue in the amino acid sequence of the known peptidic GLP-1R agonist.

In one embodiment, the variant comprises up to 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 substitutions of amino acid residues.

In one embodiment, the GLP-1R agonist is a variant of native GLP-1(7-36) comprising up to 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 substitutions of an amino acid residue in the native GLP-1(7-36) sequence. In one embodiment, the substitution is selected from the group comprising or consisting of: A8G, V16L, V16K, S18K, S18I, Y19Q, L20M, E21D, G22E, Q23E, A24R, A25V, K26R, K26Q, E27L, A30E, V33K, V33L, V33E, K34N, K34A, G35T, and R36G and/or substitutions as listed in Table 5 (see description of SEQ ID NOs: 8 to 20).

In some embodiments, the variant comprises at least one additional amino acid residue at its N-terminus. In one embodiment, the at least one additional amino acid residue is selected from the group consisting of naturally occurring amino acids other than proline, unnatural amino acids, amino acid analogs, and amino acid mimetics. In one embodiment, the at least one additional amino acid residue is selected from the group consisting of: G. a, N and C. In a particular embodiment, at least one additional amino acid residue is (single) G.

In some embodiments, the variant comprises a peptide extension at its C-terminus. A peptide extension may, for example, consist of up to 12, 11 or 10 amino acid residues. In one embodiment, the peptide extension has an amino acid sequence selected from the group consisting of: PSSGAPPPS (SEQ ID NO:38), PVSGAPPPS (SEQ ID NO:39), PSSGEPPPES (SEQ ID NO:40), PSSGEPPPE (SEQ ID NO:41), PKKQRLS (SEQ ID NO:42) and PKKIRYS (SEQ ID NO: 43).

In one embodiment, a GLP-1R agonist having reduced GLP-1R agonist activity as defined herein compared to the GLP-1R agonist activity of native GLP-1(7-36) comprises or consists of the amino acid sequence:

H-G-E-G-T-F-T-S-D-X₁₀-S-X₁₂-Q-X₁₄-X₁₅-E-E-X₁₈-V-X₂₀-X₂₁-F-I-E-W-L-X₂₇-X₂₈-X₂₉-X₃₀(SEQ ID NO:37)

wherein

X₁₀Is any amino acid, such as L or K;

X₁₂is any amino acid, such as K or I;

X₁₄is any amino acid, such as L or M;

X₁₅is any amino acid, e.g., E or D;

X₁₈is any amino acid, such as A or R;

X₂₀is any amino acid, such as R or Q;

X₂₁is any amino acid, such as L or E;

X₂₇is any amino acid, such as L, E, K or V;

X₂₈is any amino acid, such as A, N or K;

X₂₉is any amino acid, such as T or G;

X₃₀is any amino acid, such as G or R;

wherein, optionally, the amino acid sequence comprises at least one additional amino acid residue at its N-terminus; and is

Wherein, optionally, the amino acid sequence comprises a peptide extension consisting of up to 12, 11 or 10 amino acid residues at its C-terminus.

In one embodiment, X₂₇Is L, E or V, e.g., L. In one embodiment, X₂₈Is A or K, e.g., A.

In one embodiment, the at least one additional amino acid residue is selected from the group consisting of: G. a, N and C. In a particular embodiment, at least one additional amino acid residue is (single) G.

In one embodiment, the peptide extension has an amino acid sequence selected from the group consisting of: PSSGAPPPS (SEQ ID NO:38), PVSGAPPPS (SEQ ID NO:39), PSSGEPPPES (SEQ ID NO:40), PSSGEPPPE (SEQ ID NO:41), PKKQRLS (SEQ ID NO:42) and PKKIRYS (SEQ ID NO: 43).

Modifications as disclosed herein, e.g., introduction of G or X at the N-terminus₁₂Results in a modest reduction in GLP-1R agonistic activity.

In one embodiment, a GLP-1R agonist having reduced GLP-1R agonist activity as defined herein compared to the GLP-1R agonist activity of native GLP-1(7-36) comprises or consists of an amino acid sequence selected from the group consisting of: 9, 10, 12, 14, 15, 16, 17, 19 and 20.

In one embodiment, X₁₄Is L and X₂₈Is A.

In one embodiment, the GLP-1R agonist comprises or consists of an amino acid sequence selected from the group consisting of: 9, 10, 12, 14, 16, 17, 19 and 20.

According to the present invention, a GLP-1R agonist may be further modified, e.g. as described above in connection with the FGF21 compound. For example, it may be fused to an Fc region/domain of an immunoglobulin or a variant thereof, e.g., an Fc region/domain of an immunoglobulin or a variant thereof as described herein.

The pharmaceutical composition according to the invention comprises one or more carriers and/or excipients, all of which are pharmaceutically acceptable. The term "pharmaceutically acceptable" as used herein means that the material is non-toxic, preferably does not interact with the active agent of the pharmaceutical composition.

The term "carrier" refers to an organic or inorganic component, natural or synthetic in nature, in which the active components are combined to facilitate, enhance or effect the application. According to the present invention, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to a subject.

Possible carrier substances for parenteral administration are, for example, sterile water, ringer's solution, lactated ringer's solution, physiological saline, bacteriostatic saline (e.g. saline containing 0.9% benzyl alcohol), Phosphate Buffered Saline (PBS), hank's solution, polyalkylene glycols, hydrogenated naphthalenes and in particular biocompatible lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxypropylene copolymers.

The term "excipient" as used herein is intended to include all substances that may be present in a pharmaceutical composition and that are not active ingredients, such as salts, binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), fillers, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffering substances, flavoring agents, or coloring agents.

Salts that are not pharmaceutically acceptable may be used in the preparation of pharmaceutically acceptable salts and are included in the present invention. Such pharmaceutically acceptable salts include, in a non-limiting manner, those salts prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, and the like. Pharmaceutically acceptable salts may also be prepared as alkali metal or alkaline earth metal salts, such as sodium, potassium or calcium salts. Salts may be added to adjust the ionic strength or tonicity.

Suitable preservatives for use in the pharmaceutical compositions include antioxidants, citric acid, sodium citrate, benzalkonium chloride, chlorobutanol, cysteine, methionine, parabens (parabens), thimerosal, phenol, cresol and mixtures thereof.

Suitable buffering substances for pharmaceutical compositions include acetic acid in salt, citric acid in salt, boric acid in salt, phosphoric acid in salt and Tris (hydroxymethyl) aminomethane (Tris, THAM, tromethamine).

The pharmaceutical composition according to the invention is preferably sterile. The pharmaceutical compositions may be provided in unitary dosage form and may be prepared in a manner known per se. The pharmaceutical composition may, for example, be in the form of a solution or suspension.

The pharmaceutical composition may also be formulated as a stable lyophilized product for reconstitution with a suitable diluent, optionally comprising one or more excipients as defined above.

The pharmaceutical composition according to the invention may further comprise at least one other active pharmaceutical ingredient.

The term "active pharmaceutical ingredient" (API) as used herein includes any pharmaceutically active chemical or biological compound and any pharmaceutically acceptable salt thereof and any mixture thereof, which provides some pharmacological effect and is used for the treatment or prevention of a disorder, e.g. a disease or disorder as defined herein. Exemplary pharmaceutically acceptable salts include (salts of) hydrochloric, sulfuric, nitric, phosphoric, hydrobromic, maleic, malic, ascorbic, citric, tartaric, pamoic, lauric, stearic, palmitic, oleic, myristic, lauryl, naphthalenesulfonic, linoleic, linolenic, and the like. The terms "active pharmaceutical ingredient", "active agent", "active ingredient", "active substance", "therapeutically active compound" and "drug" as used herein are intended to be synonymous, i.e. to have the same meaning.

According to the invention, the active pharmaceutical ingredient is optionally selected from:

-all drugs mentioned in Rote Liste 2014, such as all antidiabetics mentioned in Rote Liste 2014 chapter 12, all weight loss or appetite suppressants mentioned in Rote Liste 2014 chapter 06, all lipid lowering drugs mentioned in Rote Liste 2014 chapter 58, all antihypertensive drugs mentioned in Rote Liste 2014 chapter 17, all kidney protection drugs mentioned in Rote Liste 2014 or all diuretic drugs mentioned in Rote Liste 2014 chapter 36;

insulin and insulin derivatives, such as: insulin glargine (e.g. insulin glargine)) Insulin glargine at concentrations above 100U/mL, e.g. 270-330U/mL or 300U/mL (as disclosed in EP 2387989), insulin glulisine (e.g. insulin glargine) Insulin detemir (e.g. insulin detemir)) Insulin lispro (e.g. insulin lispro)) Insulin deglutamide (e.g. insulin deglutamide)IdegLira (NN9068)), insulin aspart, and insulin aspart formulations (e.g., insulin aspart formulations) Basal insulins and analogs (e.g., LY2605541, LY2963016, NN1436), pegylated insulin lispro (e.g., LY-275585), long-acting insulins (e.g., NN1436, Insumea (PE0139), AB-101, AB-102, Sensulin LLC), moderate-acting insulins (e.g. ) Rapid acting and short acting insulins (e.g. insulin PH20 insulin, NN1218,) Pre-mixing insulin,NN1045, insulin plusPE-0139, ACP-002 hydrogel insulin and oral, inhalable, transdermal and buccal or sublingual insulin (e.g., insulinInsulin Tegopine, TPM-02 insulin,Oral insulin, ORMD-0801, Oshadi oral insulin, NN1953, NN1954, NN1956,). Those insulin derivatives that are bonded to albumin or another protein via a bifunctional linker are also suitable;

glucagon-like peptide 1(GLP-1), GLP-1 analogues and GLP-1 receptor agonists, such as: GLP-1(7-37), GLP-1(7-36) amides, lixisenatide (e.g. peptide)) Exenatide (e.g., Exendin-4, rExendin-4, Exenatide-4, exendin-,Exenatide NexP), exenatide-LAR, liraglutide (e.g., NexP), and the like) Somaloutide, taslutide, abiglutide, dolaferin, Albugon, oxyntomodulin, geniposide (geniposide), ACP-003, CJC-1131, CJC-1134-PC, GSK-2374697, PB-1023, TTP-054, Lanlerian peptide (HM-11260C), CM-3, GLP-1Eligen, AB-201, ORMD-0901, NN9924, NN9926, NN9927, Nodexen, Viador-GLP-1, CVX-096, ZYOG-1, ZYD-1, ZP-3022, CAM-2036, DA-3091, DA-15864, ARI-2651, ARI-2255, exenatide-XTEN (VRS-859), exenatide-XTEN + glucagon-XTEN (VRS-859+ AMX-808), and polymer bound GLP-1 and GLP-1 analogs;

dual GLP-1/GIP agonists (e.g., RG-7697(MAR-701), MAR-709, BHM081, BHM089, BHM 098); dual GLP-1/glucagon receptor agonists (e.g., BHM-034, OAP-189(PF-05212389, TKS-1225), TT-401/402, ZP2929, LAPS-HMOXM25, MOD-6030);

-dual GLP-1/gastrin agonists (e.g. ZP-3022);

-gastrointestinal peptides, such as peptide YY3-36(PYY3-36) or an analogue thereof and Pancreatic Polypeptide (PP) or an analogue thereof;

-glucagon receptor agonists or antagonists, glucose-dependent insulinotropic polypeptide (GIP) receptor agonists or antagonists, ghrelin antagonists or inverse agonists, xenopus peptides and analogues thereof;

dipeptidylpeptidase-IV (DPP-4) inhibitors, such as: alogliptin (e.g. Alogliptin)) Linagliptin (e.g. linagliptin)) Saxagliptin (e.g. Saxagliptin)Komboglyze) Sitagliptin (e.g. sitagliptin) Janumet) Anegliptin, tigegliptin (e.g. anegliptin, tigegliptin)) Trelagliptin, vildagliptin (e.g. trelagliptin)) The pharmaceutical composition comprises the following components of Geogliptin, Oogliptin, Iogliptin, Doogliptin, DA-1229, MK-3102, KM-223, KRP-104, PBL-1427, penoxfloxacin hydrochloride and Ari-2243;

sodium-dependent glucose transporter 2(SGLT-2) inhibitors, such as: canagliflozin, dacemagliflozin, rigagliflozin etabonate, sjogrezin, engagliflozin, ivagliflozin, togagliflozin, ruagliflozin, EGT-0001442, LIK-066, SBM-TFC-039 and KGA-3235 (DSP-3235);

dual inhibitors of SGLT-2 and SGLT-1 (e.g., LX-4211, LIK 066);

-SGLT-1 inhibitors (e.g. LX-2761, KGA-3235) or SGLT-1 inhibitors in combination with anti-obesity drugs such as Ileal Bile Acid Transporter (IBAT) inhibitors (e.g. GSK-1614235+ GSK-2330672);

biguanides (e.g. metformin, buformin, phenformin);

thiazolidinediones (e.g. pioglitazone, rosiglitazone), glitazone analogues (e.g. lobeglitazone);

peroxisome proliferator-activated receptor (PPAR-) (alpha, gamma or alpha/gamma) agonists or modulators (e.g. saxaglitazar (e.g. SAG-glitazar)) GFT-505) or a partial PPAR γ agonist (e.g., lnt-131);

sulfonylureas (e.g. tolbutamide, glibenclamide, glimepiride, glibenclamide,Glipizide) and meglitinide (e.g., nateglinide, repaglinide, mitiglinide);

-alpha-glucosidase inhibitors (e.g. acarbose, miglitol, voglibose);

amylin and amylin analogs (e.g. pramlintide, praline, amylin, amyl,)；

G-protein coupled receptor 119(GPR119) agonists (e.g.GSK-1292263, PSN-821, MBX-2982, APD-597, ARRY-981, ZYG-19, DS-8500, HM-47000, YH-Chem 1);

GPR40 agonists (e.g. TUG-424, P-1736, P-11187, JTT-851, GW9508, CNX-011-67, AM-1638, AM-5262);

-GPR120 agonists and GPR142 agonists;

-systemic or poorly absorbing TGR5(GPBAR1 ═ G-protein coupled bile acid receptor 1) agonists (e.g. INT-777, XL-475, SB 756050);

-diabetes immunotherapeutics, such as: oral C-C chemokine receptor type 2 (CCR-2) antagonists (e.g., CCX-140, JNJ-41443532), interleukin 1 β (IL-1 β) antagonists (e.g., AC-201), or oral monoclonal antibodies (moas) (e.g., methazolamide, VVP808, PAZ-320, P-1736, PF-05175157, PF-04937319);

anti-inflammatory agents for the treatment of metabolic syndrome and diabetes, such as: nuclear factor kappa B inhibitors (e.g.)；

-adenosine monophosphate-activated protein kinase (AMPK) stimulators, such as: imeglimin (PXL-008), Debio-0930(MT-63-78), R-118;

-11-beta-hydroxysteroid dehydrogenase 1 (11-beta-HSD-1) inhibitors (e.g. LY2523199, BMS770767, RG-4929, BMS816336, AZD-8329, HSD-016, BI-135585);

activators of glucokinase (e.g. PF-04991532, TTP-399(GK1-399), GKM-001(ADV-1002401), ARRY-403(AMG-151), TAK-329, TMG-123, ZYGK 1);

inhibitors of diacylglycerol O-acyltransferase (DGAT) (e.g. pradigastat (LCQ-908)), protein tyrosine phosphatase 1 inhibitors (e.g. rodussquesmine), glucose-6-phosphatase inhibitors, fructose-1, 6-bisphosphatase inhibitors, glycogen phosphorylase inhibitors, phosphoenolpyruvate carboxykinase inhibitors, glycogen synthase kinase inhibitors, pyruvate dehydrogenase kinase inhibitors;

modulators of glucose transporter-4, somatostatin receptor 3 agonists (e.g. MK-4256);

one or more lipid lowering agents are also suitable as combination partners, for example: 3-hydroxy-3-methylglutaryl-coenzyme-A-reductase (HMG-CoA-reductase) inhibitors such as simvastatin (e.g. ) Atorvastatin (e.g.)) Rosuvastatin (e.g. rosuvastatin)) Pravastatin (e.g. pravastatin) Fluvastatin (e.g. fluvastatin) Pitavastatin (e.g. pitavastatin) Lovastatin (e.g. ) Mevastatin (e.g. mevastatin)) Lipstatin and cerivastatinFibrates (e.g. bezafibrate)Retarders), ciprofibrate (e.g. ciprofibrate) Fenofibrate (e.g. fenofibrate) Gemfibrozil (e.g. gemfibrozil)) Etofibrate, bisfibrate, clinofibrate), clofibrate, nicotinic acid and derivatives thereof (e.g., nicotinic acid, including sustained release formulations of nicotinic acid), nicotinic acid receptor 1 agonists (e.g., GSK-256073), PPAR-delta agonists, acetyl-CoA-acetyltransferase (ACAT) inhibitors (e.g., avasimibe), cholesterol absorption inhibitors (e.g., ezetimibe, clofibrate), clofibrate, nicotinic acid and derivatives thereof (e.g., nicotinic acid, including sustained release formulations of nicotinic acid), nicotinic acid receptor 1 agonists (e.g,S-556971), bile acid binding substances (e.g., cholestyramine, colesevelam), Ileal Bile Acid Transport (IBAT) inhibitors (e.g., GSK-2330672, LUM-002), microsomal triglyceride transfer protein (MTP) inhibitors (e.g., Loumetepa (AEGR-733), SLx-4090, Glan tepa), modulators of proprotein convertase subtilisin/kexin type 9 (PCSK9) (e.g., Alilizumab (REGN727/SAR236553), AMG-145, LGT-209, PF-04950615, MPSK3169A, LY3015014, ALD-306, ALN-PCS, SPC-962476, SPC5001, ISIS-394814, 1B20, 1D LGT-210, 1D LG 05, BMS-PCSK9Rx-2, SX-PCK9, RG7652), LDL receptor upregulating agents (e.g., thyroid receptor selective hormone beta agonists such as thyroid receptor agonists (Rockwell as Luo-ROVO) agonists (RCS-2115), MB07811, peritipiral (QRX-431), VIA-3196, ZYT1)), HDL-raising compounds such as: cholesteryl Ester Transfer Protein (CETP) inhibitors (e.g., Anacetrapib (MK0859), Dacetrapib, Ecetrapib, JTT-302, DRL-17822, TA-8995, R-1658, LY-2484595, DS-1442), or dual CETP/PCSK9 inhibitors (e.g., K-312), ATP-binding cassette (ABC1) modulators, lipid metabolism modulators (e.g., BMS-823778, TAP-301, DRL-21994, DRL-21995), phospholipase A2(PLA2) inhibitors (e.g., daraladil, Dacron, and the like,Varespladine, verapamil), ApoA-l enhancers (e.g., RVX-208, CER-001, MDCO-216, CSL-112), cholesterol synthesis inhibitors (e.g., ETC-1002), lipid metabolism modulators (e.g., BMS-823778, TAP-301, DRL-21994, DRL-21995) and omega-3 fatty acids and derivatives thereof (e.g., twenty-3Ethyl carbonate (AMR101),AKR-063、NKPL-66、PRC-4016、CAT-2003)；

Bromocriptine (e.g. as) Phentermine and phentermine formulations or combinations (e.g., Adipex-P, phentermine, phen,) Benzphetamine (e.g. benzphetamine)) Bupropion (e.g. bupropion)) Benzometrizine (e.g. Benzometrizine)) Bupropion and combinations (e.g.Wellbutrin) Sibutramine (e.g. Sibutramine)) Topiramate (e.g. Topiramate)) Zonisamide (e.g. zonisamide)) Tesofensine, opioid antagonists (e.g. naltrexone) (e.g. mefloxin, and mefloxin)Naltrexone + bupropionKetones)), cannabinoid receptor 1(CB1) antagonists (e.g., TM-38837), melanin-concentrating hormone (MCH-1) antagonists (e.g., BMS-830216, ALB-127158(a)), MC4 receptor agonists and partial agonists (e.g., AZD-2820, RM-493), neuropeptide Y5(NPY5) or NPY2 antagonists (e.g., virifibrate, S-234462), NPY4 agonists (e.g., PP-1420), beta-3-adrenergic receptor agonists, leptin or leptin mimetics, 5-hydroxytryptamine 2c (5HT2c) receptor agonists (e.g., rocalcin, nocillin, and,) Pramlintide/metreleptin, lipase inhibitors (such as neolistat (e.g.) Orlistat (e.g. orlistat)) Angiogenesis inhibitors (e.g., ALS-L1023), beta histidine and histamine H3 antagonists (e.g., HPP-404), AgRP (agouti-related protein) inhibitors (e.g., TTP-435), serotonin reuptake inhibitors (e.g., fluoxetine (e.g., TTP-435)), and combinations thereof) Duloxetine (e.g. duloxetine)) Dual or triple monoamine uptake inhibitors (dopamine, norepinephrine and serotonin reuptake) (e.g. sertraline)) Tesofensine), methionine aminopeptidase 2(MetAP2) inhibitors (e.g. beloranib) and antisense oligonucleotides generated against fibroblast growth factor receptor 4(FGFR4) (e.g. ISIS-FGFR4Rx) or inhibin targeting peptide-1 (e.g. ISIS-FGFR4Rx))；

Nitric oxide donor, AT1 antagonistAnti-agents or angiotensin II (AT2) receptor antagonists (such as telmisartan (e.g., TM)) Candesartan (e.g. Candesartan)) Valsartan (e.g. valsartan)) Losartan (e.g. losartan)) Eprosartan (e.g. eprosartan)) Irbesartan (e.g. irbesartan)) Olmesartan (e.g. olmesartan)) Tasosartan, azilsartan (e.g. tasosartan, azilsartan)) Dual angiotensin receptor blockers (dual ARBs), Angiotensin Converting Enzyme (ACE) inhibitors, ACE-2 activators, renin inhibitors, Endothelin Converting Enzyme (ECE) inhibitors, endothelin receptor (ET1/ETA) blockers, endothelin antagonists, diuretics, aldosterone antagonists, aldosterone synthase inhibitors, alpha-blockers, alpha-2 adrenergic receptor antagonists, beta-blockers, mixed alpha-/beta-blockers, calcium antagonists, Calcium Channel Blockers (CCBs), nasal formulations of the calcium channel blocker diltiazem (e.g., CP-404), dual mineralocorticoids/CCBs, centrally acting antihypertensive agents, neutral endopeptidase inhibitors, aminopeptidase-a inhibitors, Angiopeptin inhibitor, dual angiopeptin inhibitor (such as renal insulin residue lysozyme-ACE inhibitor or renal islet of Langerhans)An inhibitor of lysin-ECE, a dual action AT receptor-renal insulinolysin inhibitor, a dual AT1/ETA antagonist), an advanced glycation end product (AGE) catabolic agent, recombinant renalase, a blood pressure vaccine (e.g. a vaccine against RAAS (renin-angiotensin-aldosterone-system), AT 1-or AT 2-vaccine), a drug based on hypertensive pharmacogenomics (e.g. a modulator of genetic polymorphisms with anti-hypertensive responses, thrombocyte aggregation inhibitors and others) or a combination thereof is suitable.

The term "fusion molecule" generally refers to a molecule produced by: linking (particularly covalently linking) two or more different molecules (e.g., proteins and/or peptides) results in a single molecule having functional properties derived from each of the original molecules. In the case of proteins and/or peptides, the fusion molecule is referred to as a "fusion protein". Fusion molecules can be generated by genetic fusion (e.g., by recombinant DNA techniques) or by chemical and/or enzymatic conjugation. Two or more different molecules may also be linked by a suitable linker molecule, for example, a peptide linker or a non-peptidic polymer, such as polyethylene glycol (PEG).

Typically, the peptide linker is designed to provide flexibility and protease resistance. In one embodiment, the peptide linker has a length of 1 to 30, 1 to 25, or 1 to 20 amino acid residues. In one embodiment, the peptide linker comprises at least 5 amino acid residues. In one embodiment, the peptide linker is a glycine-serine rich linker, wherein at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 85% of the amino acids are glycine or serine residues, respectively. In one embodiment, the peptide linker comprises an alanine residue at its C-terminus. In another embodiment, the amino acid is selected from glycine and serine, i.e., the peptide linker consists of only glycine and serine (referred to as glycine-serine linker). In one embodiment, the peptide linker comprises or consists of the amino acid sequence of SEQ ID NO 22 or SEQ ID NO 23. The peptide linker may further comprise one or more specific protease cleavage sites.

In one embodiment, the fusion molecule is a fusion protein. In the fusion protein according to the invention, the composition of the fusion protein may be arranged in order (from N-terminus to C-terminus) A-B-C or C-B-A, wherein A is a GLP-1R agonist, B is a linker molecule, and C is an FGF21 compound.

In one embodiment, the fusion protein further comprises an Fc region/domain of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4, or IgD) or a variant thereof. In one embodiment, the variant Fc region/domain comprises up to 5, 4 or 3 mutations compared to the wild-type sequence of the Fc region/domain. In one embodiment, the mutation is selected from amino acid substitutions and deletions, e.g., N-or C-terminal deletions. In one embodiment, a variant of the Fc region/domain of IgG4 (also referred to as "IgG 4 Fc variant") comprises or consists of the amino acid sequence of SEQ ID NO: 21.

In one embodiment, the variant Fc region/domain is a hybrid Fc region/domain. Such hybrid Fc regions/domains are described, for example, in WO 2016/114633 a1, WO 2017/074117 a1, WO 2017/074123 a1 and WO 2018/088838 a1, which are incorporated herein by reference. In one embodiment, the hybrid Fc region/domain comprises a combination of partial Fc regions/domains of different immunoglobulins (e.g., IgG1, IgG2, IgG3, IgG4, or IgD). In one embodiment, the hybrid Fc region/domain comprises part of an Fc region/domain of IgG4 and IgD (also referred to as "IgG 4/IgD hybrid Fc region/domain"), preferably human IgG4 and IgD. In one embodiment, the hybrid Fc region/domain comprises a partial hinge sequence and CH2 of an IgD Fc region/domain, and CH2 and CH3 sequences of an IgG4 Fc region/domain. In one embodiment, the hybrid Fc region/domain comprises or consists of an amino acid sequence selected from the group consisting of seq id no:65, 66, 67, 68, 69 and 70 SEQ ID NOs.

In one embodiment, the FGF21 compound and the GLP-1R agonist are linked by an Fc region/domain of an immunoglobulin or a variant thereof. In one embodiment, the FGF21 compound and the GLP-1R agonist are linked by a linker molecule comprising a structure selected from the group consisting of: L-Fc, Fc-L, L₁–Fc–L₂And Fc, wherein L, L₁And L₂Is as followsPeptide linker (L) as defined herein₁And L₂The same or different) and Fc is an Fc region/domain of an immunoglobulin or a variant thereof.

In one embodiment, the fusion protein comprises or consists of an amino acid sequence selected from the group consisting of seq id no:25, 26, 28, 30, 31, 32, 33, 35 and 36 SEQ ID NOs.

Further features of the fusion proteins according to the invention are described, for example, in WO 2014/037373 a1 and WO 2017/093465 a1, which are incorporated herein by reference.

According to the invention, a "nucleic acid molecule" is preferably a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). According to the invention, the nucleic acid molecule may be in the form of a single-stranded or double-stranded molecule which is linearly or covalently closed to form a loop.

The term "DNA" refers to a molecule that comprises and preferably consists entirely or essentially of deoxyribonucleotide residues. "deoxyribonucleotides" refers to nucleotides lacking a hydroxyl group at the 2' -position of the β -D-ribofuranosyl group. The term "DNA" encompasses isolated DNA, such as partially or fully purified DNA, substantially pure DNA, synthetic DNA, and recombinantly produced DNA, and includes modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides. Such changes may include the addition of non-nucleotide material, such as to one or more ends of the DNA or to the interior, for example at one or more nucleotides of the DNA. The nucleotides in the DNA molecule may also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides. These altered DNAs may be referred to as analogs or analogs of naturally occurring DNAs. The term "naturally occurring" when used in conjunction with a nucleotide refers to the bases adenine (a), cytosine (C), guanine (G), thymine (T) and uracil (U).

The term "RNA" refers to a molecule comprising and preferably consisting entirely or essentially of ribonucleotide residues. "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' -position of the β -D-ribofuranosyl group. The term "RNA" encompasses isolated RNA, such as partially or fully purified RNA, substantially pure RNA, synthetic RNA, and recombinantly produced RNA, and includes modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides. Such alterations may include the addition of non-nucleotide material, such as to one or more ends of the RNA or to the interior, for example at one or more nucleotides of the RNA. The nucleotides in the RNA molecule may also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNAs. According to the present invention, "RNA" refers to single-stranded RNA or double-stranded RNA. In one embodiment, the RNA is mRNA, e.g., in vitro transcribed RNA (ivt RNA) or synthetic RNA. The RNA can also be modified, e.g., with one or more modifications that increase the stability (e.g., half-life) of the RNA. Such modifications are known to those skilled in the art and include, for example, 5 '-cap or 5' cap analogs.

The nucleic acid molecule according to the invention may be contained in a vector. The term "vector" as used herein includes all vectors known to the skilled person, including plasmid vectors, cosmid vectors, phage vectors (such as lambda phage), viral vectors (such as adenovirus or baculovirus vectors) or artificial chromosome vectors (such as Bacterial Artificial Chromosomes (BAC), Yeast Artificial Chromosomes (YAC) or P1 Artificial Chromosomes (PAC)). The vectors include expression and cloning vectors. Expression vectors include plasmids as well as viral vectors, and typically contain the desired coding sequence and appropriate DNA sequences required for expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammalian) or in an in vitro expression system. Cloning vectors are commonly used to engineer and amplify a certain desired DNA fragment and may lack the functional sequences required for expression of the desired DNA fragment.

Alternatively, the nucleic acid molecule according to the invention may be integrated into the genome (e.g. of a host cell). Means and methods for integrating a particular nucleic acid molecule into the genome are known to those skilled in the art.

The term "cell" or "host cell" preferably relates to an intact cell, i.e. a cell which has an intact membrane and has not released its normal intracellular components (such as enzymes, organelles or genetic material). The intact cell is preferably a living cell, i.e. a living cell capable of performing its normal metabolic function. Preferably, according to the present invention, the term relates to any cell that can be transfected or transformed with an exogenous nucleic acid. Preferably, upon transfection or transformation with an exogenous nucleic acid and transfer to a recipient, the cell can express the nucleic acid in the recipient. The term "cell" includes prokaryotic cells, such as bacterial cells, as well as eukaryotic cells, such as yeast cells, fungal cells, or mammalian cells. Suitable bacterial cells include cells from: gram-negative bacterial strains, such as strains of Escherichia coli (Escherichia coli), Proteus (Proteus) and Pseudomonas (Pseudomonas), and gram-positive bacterial strains, such as strains of Bacillus (Bacillus), Streptomyces (Streptomyces), Staphylococcus (Staphylococcus), and Lactococcus (Lactococcus). Suitable fungal cells include cells from the following species: trichoderma (Trichoderma), Neurospora (Neurospora) and Aspergillus (Aspergillus). Suitable yeast cells include cells from the following species: saccharomyces (Saccharomyces) (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula (Hansenula). Suitable mammalian cells include, for example, CHO cells, BHK cells, HeLa cells, COS cells, HEK293, and the like. In one embodiment, HEK293 cells are used. However, amphibian cells, insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins may also be used. Mammalian cells are particularly preferred for adoptive transfer, such as cells from humans, mice, hamsters, pigs, goats, and primates. The cells may be derived from a wide variety of tissue types and include primary cells and cell lines, such as cells of the immune system, particularly antigen presenting cells, such as dendritic cells and T cells; stem cells, such as hematopoietic stem cells and mesenchymal stem cells; and other cell types. Antigen presenting cells are cells that display antigen in the context of a major histocompatibility complex on their surface. T cells can recognize this complex using their T Cell Receptor (TCR). A "cell" or "host cell" may be an isolated cell or part of a tissue or organism (particularly a "non-human organism").

The term "non-human organism" as used herein is intended to include non-human primates or other animals, in particular mammals, such as cows, horses, pigs, sheep, goats, dogs, cats, rabbits or rodents, e.g. mice, rats, guinea pigs and hamsters.

The term "kit of parts (abbreviation: kit)" as used herein refers to an article of manufacture comprising one or more containers and optionally a data carrier. The one or more containers may be filled with one or more of the above-mentioned medicaments (reagents). Other containers may be included in the kit, containing, for example, diluents, buffers, and other reagents. The data carrier may be a non-electronic data carrier, for example a graphical data carrier, such as an information leaflet, an information page, a bar code or an access code; or an electronic data carrier such as a Compact Disc (CD), a Digital Versatile Disc (DVD), a microchip or another semiconductor-based electronic data carrier. The access code may allow access to a database, such as an internet database, a centralized database, or a distributed database. The data carrier may contain instructions for using the agents of the invention, such as the combinations, pharmaceutical compositions and fusion molecules as described herein, as well as related agents, such as nucleic acid molecules and host cells.

The agents and compositions described herein may be administered by any conventional route, e.g., orally, pulmonarily, by inhalation, or parenterally, including by injection or infusion. In one embodiment, parenteral administration is used, e.g., intravenous, intraarterial, subcutaneous, intradermal, or intramuscular. The agents and compositions described herein can also be administered by sustained release administration.

Pharmaceutical compositions suitable for parenteral administration typically comprise a sterile aqueous or non-aqueous formulation of the active compound, which is preferably isotonic with the blood of the recipient. Examples of compatible carriers/solvents/diluents are sterile water, ringer's solution, lactated ringer's solution, physiological saline, bacteriostatic saline (e.g., saline containing 0.9% benzyl alcohol), Phosphate Buffered Saline (PBS), and hank's solution. In addition, sterile fixed oils may generally be employed as a solution or suspension medium.

The agents and compositions described herein are generally administered in a therapeutically effective amount. "therapeutically effective amount" means an amount which alone or in combination with other dosages achieves the desired therapeutic response or desired therapeutic effect, preferably without causing unacceptable side effects. In the case of treating a particular disease or a particular condition, the desired response is preferably associated with inhibiting the course of the disease. This includes slowing the disease process and, in particular, interrupting or reversing the disease process. The desired response in the treatment of a disease or condition can also be to delay the onset of the disease or condition, or to prevent the onset of the disease or condition. An effective amount of an agent or composition described herein will depend on the condition being treated, the severity of the disease, the individual parameters of the subject (including age, physiological condition, size and weight), the duration of the treatment, the type of concomitant therapy (if any), the particular route of administration, and like factors. Thus, the dosage of administration of an agent described herein may depend on a number of such parameters. In cases where the subject's response to the initial dose is insufficient, a higher dose (or an effective higher dose achieved by a different, more topical route of administration) may be used.

According to the present invention, the term "disease or disorder" refers to any pathological or unhealthy state, in particular obesity, overweight, metabolic syndrome, diabetes, diabetic retinopathy, hyperglycemia, dyslipidemia, nonalcoholic steatohepatitis (NASH) and/or atherosclerosis.

The term "obesity" refers to a medical condition in which excess body fat has accumulated to the extent that it can negatively impact health. For human (adult) subjects, obesity may be defined as a Body Mass Index (BMI) of greater than or equal toAt 30kg/m²(BMI≥30kg/m²)。

The term "overweight" refers to a medical condition in which the amount of body fat is higher than in an optimal state of health. For human (adult) subjects, obesity may be defined as a Body Mass Index (BMI) greater than or equal to 25kg/m²(e.g., 25 kg/m)²≤BMI<30kg/m²)。

BMI is a simple index of weight versus height and is commonly used to classify overweight and obesity in adults. It is defined as the weight (kilograms) of an individual divided by the height (meters) of the individual squared (kg/m)²)。

"metabolic syndrome" may be defined as the aggregation of at least three of the following medical conditions: abdominal (central) obesity (e.g., defined as waist circumference of > 94cm for European males, and > 80cm for European females, race-specific values for other ethnic groups), elevated blood pressure (e.g., 130/85mmHg or higher), elevated fasting glucose (e.g., at least 100mg/dL), high serum triglycerides (e.g., at least 150mg/dL), and low High Density Lipoprotein (HDL) levels (e.g., less than 40mg/dL for males, and less than 50mg/dL for females).

"Diabetes mellitus" (also referred to simply as "Diabetes") refers to a group of metabolic diseases characterized by high blood glucose levels due to defects in insulin production, insulin action, or both. In one embodiment, the diabetes is selected from the following: type 1 diabetes, type 2 diabetes, gestational diabetes, late adult autoimmune diabetes (LADA), juvenile onset diabetes (MODY), and other types of diabetes due to specific genetic conditions, medications, malnutrition, infection, and other diseases.

The current WHO diagnostic criteria for diabetes are as follows: fasting blood glucose is more than or equal to 7.0mmol/l (126mg/dL) or 2-h blood glucose is more than or equal to 11.1mmol/l (200 mg/dL).

"type 1 diabetes mellitus" (also known as "insulin-dependent diabetes mellitus (IDDM)" or "juvenile diabetes") is a condition characterized by high blood glucose levels caused by a complete deficiency of insulin. This occurs when the body's immune system attacks the insulin-producing beta cells in the pancreas and destroys the cells. The pancreas then produces little or no insulin. Pancreatic removal or disease can also lead to loss of insulin-producing beta cells. Type 1 diabetes accounts for 5% -10% of diabetes cases.

"type 2 diabetes" (also known as "non-insulin dependent diabetes mellitus (NIDDM)" or "adult-onset diabetes") is a condition characterized by: although insulin is available, excess glucose is produced and circulating glucose levels remain too high due to insufficient glucose clearance (insulin action). Type 2 diabetes can account for approximately 90% to 95% of all diagnosed cases of diabetes.

"gestational diabetes" is a condition in which a woman who has not previously been diagnosed with diabetes exhibits elevated blood glucose levels during pregnancy (especially during the third trimester of pregnancy). Gestational diabetes affects 3% -10% of pregnancies, depending on the population studied.

"adult delayed autoimmune diabetes (LADA)" (also referred to as "delayed type 1 diabetes") is a form of type 1 diabetes that occurs in adults, usually with a slower onset process.

"juvenile adult onset diabetes Mellitus (MODY)" refers to the hereditary form of diabetes caused by a mutation in an autosomal dominant gene that disrupts insulin production.

"diabetic retinopathy" is an ocular disease induced by metabolic disorders that occur in diabetic patients and result in the progressive loss of vision.

The term "hyperglycemia" refers to the hyperglycaemia (glucose) in the blood.

The term "dyslipidemia" refers to a disorder of lipoprotein metabolism, including either overproduction of lipoproteins ("hyperlipidemia") or deficiency ("hypolipidemia"). Dyslipidemia may be manifested as an increase in the concentration of total cholesterol, Low Density Lipoprotein (LDL) cholesterol and/or triglycerides, and/or a decrease in the concentration of High Density Lipoprotein (HDL) cholesterol in the blood.

Nonalcoholic steatohepatitis (NASH) is a liver disease characterized by the accumulation of fat (lipid droplets) and the degradation and inflammation of hepatocytes. Once it occurs, the disease is accompanied by a high risk of cirrhosis, a state in which liver function changes and may progress to hepatic insufficiency. Thereafter, NASH typically progresses to liver cancer.

"atherosclerosis" is a vascular disease characterized by the following features: lipid deposits, called plaques, are irregularly distributed in the intima of large and medium arteries, which can cause narrowing of the arterial lumen and progress to fibrosis and calcification. Lesions are usually localized and slowly and indirectly progress. Plaque rupture sometimes occurs, causing an obstruction to blood flow, causing death of tissue away from the obstruction. Blood flow limitation explains most clinical manifestations, which vary with the distribution and severity of the obstruction.

The term "drug" as used herein refers to a substance/composition for use in therapy, i.e. for treating a disease or disorder.

"treating" means administering a compound or composition or combination of compounds or compositions to the subject to prevent or eliminate a disease or disorder; preventing or slowing a disease or disorder in a subject; inhibiting or slowing the progression of a neoplastic disease or disorder in a subject; reducing the frequency or severity of symptoms and/or relapses in a subject currently suffering from or previously suffering from a disease or disorder; and/or extend (i.e., increase) the lifespan of the subject.

Specifically, the term "treating" or "treating" a disease or disorder includes curing, shortening the duration of, ameliorating, preventing, slowing or inhibiting the progression or worsening of the disease or disorder or symptoms thereof, or preventing or delaying the onset of the disease or disorder or symptoms thereof.

According to the present invention, the term "subject" means a subject for treatment, in particular a diseased subject (also referred to as "patient"), including humans, non-human primates or other animals, in particular mammals, such as cows, horses, pigs, sheep, goats, dogs, cats, rabbits or rodents, e.g. mice, rats, guinea pigs and hamsters. In one embodiment, the subject/patient is a human.

The invention will now be further described with reference to the following examples, which are intended to illustrate, but not limit the scope of the invention.

Examples

Example 1: determination of optimal GLP-1RA/FGF21 Activity ratio by systemic pharmacological modeling

An improved mechanistic understanding of the pharmacological effects of the GLP-1RA/FGF21 fusion protein in humans is used to identify the optimal GLP-1RA/FGF21 potency ratio. A mechanistic systemic pharmacological model was developed to describe the effects of GLP-1 and FGF21 on glucose, lipid and energy metabolism in humans (Cuevas-Ramos et al (2009) Curr Diabetes Rev 5(4): 216-.

The model represents the relevant pathways for GLP-1 and FGF21 action. Glycemic control (i.e., HbA1c, fasting glucose, postprandial glucose), lipid parameters (i.e., plasma triglycerides, fatty acids, cholesterol) and energy balance (i.e., body weight, food intake, energy expenditure) were captured to evaluate therapeutic response to a simulated drug therapy (e.g., GLP-1RA/FGF21 fusion protein, liraglutide, FGF21 analog LY 2405319). For LY2405319, see Kharitonenkov et al (2013) PLoS ONE 8(3): e 58575.

The model covers key aspects of glucose homeostasis controlled by the hormones insulin, glucagon and incretin (GLP-1, GIP). The primary model endpoint for glycemic control was HbA1 c. HbA1c is a clinical endpoint commonly used to estimate mean blood glucose concentration during previous months. HbA1c was estimated in the model using a linear relationship between mean blood glucose and HbA1c as reported in Nathan et al (2008) Diabetes Care 31(8): 1473-.

The model incorporates triglyceride and fatty acid metabolism at levels suitable for manipulating basal lipid metabolism, including representatives of cholesterol. HDL and non-HDL (i.e., LDL plus VLDL cholesterol) are circulating lipoproteins. The representation of lipid metabolism allows to mimic the effects of FGF21 compounds on lipids and interactions with statins. FGF21 compounds have a significant effect on lipid concentrations (Gaich et al (2013) Cell Metab 18(3): 333-3004; Fisher et al (2011) Endocrinology 152(8): 2996-3004).

Weight loss or gain in the model is measured as a change in body fat mass. There is a direct link between fat mass and body weight (Broyles et al (2011) Br J Nutr 105(8): 1272-. Food intake is based on basal and resting metabolic rates (Amirkalalii et al (2008) Indian J Med Sci 62(7): 283-. Body fat mass remains constant when energy expenditure equals caloric intake. The formulation (Gobel et al (2014) obesity (silver spring)22(10):2105-2108) was used in a model to achieve a therapeutic effect on food intake.

Food is considered to be carbohydrate (glucose equivalents), fat (fatty acid equivalents) and protein (amino acid equivalents). All nutrients enter the stomach, pass through the delayed knot, and then pass through the three-compartment gastrointestinal tract. The gastrointestinal tract design is based on work and food digestion and absorption performed by (Bastianelli et al (1996) J Anim Sci 74(8): 1873-.

Nutrients, hormones, drugs and disease conditions can cause a delay in gastric emptying. In healthy conditions, gastric emptying rates depend on the meal size, its energy density and the amount of nutrients in the stomach (Achour et al (2001) Eur J Clin Nutr 55(9): 769-. Individuals with Diabetes typically have delayed glucose absorption as seen by oral glucose tolerance tests or dietary tests (Bharucha et al (2009) Clin Endocrinol (Oxf)70(3): 415-. This delay is due to a slowing of gastric emptying. The delay between stomach and small intestine was increased in the model to account for delayed gastric emptying in diabetic subjects. Drugs and hormones (GLP-1) Can affect gastric disorientation, which reduces mechanical mixing and/or peristalsis, and this also slows gastric emptying (Jelising et al (2012) Diabetes Obes Metab 14(6): 531-.

One objective of this study was to prevent GLP-1 associated adverse effects, namely nausea and vomiting (Lean et al (2014) Int J Obes (Lond)38(5): 689-. Gastric emptying measurements provide an estimate of adverse events such as nausea and vomiting, which are associated with low gastric emptying rates. Thus, the hallmark of a gastric adverse event in the model is the sum of the gastric emptying rates.

Different virtual patients representing healthy and different disease stages of type 2 diabetic patients are implemented in the model platform. Furthermore, virtual patients covered varying degrees of obesity and dyslipidemia. Virtual patients represent variability in disease severity and pathophysiology as well as phenotypic variability observed in the clinic.

Several therapies were implemented in the model, i.e., GLP-1RA/FGF21 fusion protein, liraglutide, FGF21 analog LY2405319, metformin, atorvastatin, sitagliptin, human insulin. These therapies can be turned on or off in the simulation. It was assumed that the virtual patients had a background of metformin and atorvastatin when administered the GLP-1RA/FGF21 fusion protein.

A virtual GLP-1RA/FGF21 fusion protein was implemented in the model. The fusion protein contains both FGF21 and GLP-1 agonist activity and has the same effect as both FGF21 and GLP-1 receptor agonists. The pharmacokinetic profile of the virtual fusion protein is assumed to be similar to that of dolauda peptide (Geiser et al (2016) Clin Pharmacokinet 55(5): 625-34).

The model is validated by comparison to a plurality of data sets. The simulation results are qualitatively consistent with relevant data and knowledge, e.g., Hellerstein et al (1997) J Clin Invest 100(5) 1305-; muscellli et al (2008) Diabetes 57(5): 1340-1348. The model is matched to relevant quantitative test data, e.g., Ashtner et al (2006) Diabetes Care 29(12): 2632-; dalla Man, Caumo et al (2005) Am J Physiol Endocrinol Metab 289(5) E909-914; dalla Man et al (2005) Diabetes 54(11): 3265-; Fiallo-Scharer (2005) J Clin Endocrinol Metab 90(6) 3387-; hahn et al (2011) the or Biol Med Model 8: 12; herman et al (2005) Clin Pharmacol Ther 78(6) 675-688; herman et al (2006) J Clin Pharmacol 46(8): 876-; hojlund et al (2001) Am J Physiol Endocrinol Metab 280(1) E50-58; monauni et al (2000) Diabetes 49(6) 926-935; nauck et al (2009) Diabetes Care 32(1) 84-90; nauck et al (1993) J Clin Invest 91(1): 301-307; nauck et al (2004) Regul Pept 122(3): 209-217; tzamaloukas et al (1989) West J Med 150(4): 415-; sikaris (2009) J Diabetes Sci Technol 3(3): 429-); vicini and Cobelli (2001) Am J Physiol Endocrinol Metab 280(1) E179-186; vollmer et al (2008) Diabetes 57(3) 678-687.

Existing therapies are implemented in models for direct comparison, including FGF21 analogs and GLP-1 receptor agonists. The effect of FGF21 analogs was verified using clinical data, e.g., Gaich et al 2013. The GLP-1 receptor agonist liraglutide is a direct competitor against the target and is implemented in comparison to a variety of clinical data, e.g., Jacobsen et al (2009) Br J Clin Pharmacol 68(6): 898-; elbrond et al (2002) Diabetes Care 25(8), 1398-1404; chang et al (2003) Diabetes52(7): 1786-; kolterman et al (2003) J Clin Endocrinol Metab 88(7): 3082-; degn et al (2004) Diabetes53(5): 1187-1194; kolterman et al (2005) Am J Health Syst Pharm 62(2): 173-181; vilsball et al (2008) Diabet Med 25(2): 152-; rose 374(9683) 39-47 of Buse et al (2009); jelising et al (2012) Diabetes Obes Metab 14(6): 531-538; hermansen et al (2013) Diabetes Obes Metab 15(11): 1040-1048; suzuki et al (2013) Intern Med 52(10): 1029-1034; van Can et al (2013) Int J Obes (Lond)38(6): 784-93); zinman et al (2009) Diabetes Care 32(7) 1224-1230; Russell-Jones et al (2009) Diabetologia 52(10): 2046-2055; pratley et al (2011) Int J Clin practice 65(4) 397-; nauck et al (2013) Diabetes Obes Metab 15(3) 204-212; flint et al (2011) Adv Ther 28(3) 213-226; 650-; astrup et al (2012) Int J Obes (Lond)36(6): 843-854.

The model platform allows for the modeling of the beneficial and adverse effects of the virtual GLP-1RA/FGF21 fusion protein at varying activity ratios. Effective FGF 21-mediated EC50 values were set to constant values from Gaich et al (2013) Cell Metab 18(3): 333-340. Potent GLP-1 mediated EC50 values were reduced by a factor of 2 to 600 increments of 1 relative to endogenous GLP-1 (Table 1).

Table 1: the GLP-1R agonist/FGF 21 fusion protein pharmacodynamics (EC50 values).

^*Relative to endogenous GLP-1

^**FGF21 EC50 values were set based on the assumption of half-maximal effect by Gaich et al (2013) Cell Metab 18(3): 333-340.

For each virtual fusion protein, the exposure-response relationship was modeled for the relevant pharmacodynamic endpoints (i.e., HbA1c, triglycerides, fatty acids, non-HDL cholesterol, and fat mass). Gastric emptying rate was used as a marker for GLP-1 mediated adverse events. Simulating a virtual patient's 52-week treatment with GLP-1RA/FGF21 fusion protein for a wide dose range for obese dyslipidemia type 2 diabetes. After 52 weeks of treatment, all relevant pharmacodynamic endpoints are expected to reach steady state. For each endpoint, the half maximal effective concentration (EC50 value) was determined from the exposure-response curve. Especially for the major GLP-1 mediated endpoint HbA1c and gastric emptying rate, EC50 values varied with activity ratio. Figure 1 depicts EC50 values as a function of GLP-1 attenuation factor. An increased GLP-1 attenuation factor indicates a decreased GLP-1R agonistic activity.

This procedure allows identification of relevant activity ratios for which adverse effects act at higher plasma levels compared to pharmacodynamic effects. For GLP-1 attenuating factors greater than 9, EC50 for GLP-1 mediated gastrointestinal adverse effects is greater than EC50 for pharmacodynamic effects. Thus, the gastropathic effects act at plasma levels higher than the pharmacodynamic effects. It is possible to find a dose that provides all the desired pharmacodynamic effects while avoiding GLP-1 mediated gastrointestinal adverse effects. Therefore, an activity ratio below 1:10 is not relevant.

The maximum EC50 value for gastric emptying rate was reached at attenuation factor 531. The maximum distance between the adverse effect and the mean pharmacodynamic effect is reached at the decay factor 482 (figure 2). Therefore, activity ratios above 1:482 are not relevant. The maximum distance between the maximum of pharmacodynamics (HbA1c) and adverse effects was 319. The maximum distance between the maximum of pharmacodynamics (HbA1c) normalized by the extended FGF21- (lipid) and GLP-1 mediated effects (HbA1c) and adverse effects was 121.

The GLP-1RA/FGF21 fusion protein with a predicted potency ratio of 1:10-1:482 is most beneficial in improving lipid profile, body weight and glucose metabolism, and may not cause serious adverse events based on gastric emptying response. A lower potency ratio may not be a good candidate based on its predicted strong inhibition of gastric emptying and the likelihood of adverse events. A higher efficiency ratio may not be sufficiently effective and thus competitive.

Furthermore, a 12-week treatment of a virtual patient with common obese dyslipidemia type 2 diabetes with a GLP-1RA/FGF21 fusion protein was simulated for a wide dose range, as the primary GLP-1 mediated parameter HbA1c clinically reached steady state after 12 weeks of treatment.

Figure 3 depicts EC50 values dependent on a 12-week simulated GLP-1 attenuation factor. For GLP-1 attenuating factors greater than 18, EC50 for GLP-1 mediated gastrointestinal adverse effects is greater than EC50 for pharmacodynamic effects. The maximum EC50 value for gastric emptying rate was reached at attenuation factor 501. The maximum distance between the adverse effect and the mean pharmacodynamic effect was reached at attenuation factor 469 (figure 4). The maximum distance between the maximum of pharmacodynamics (HbA1c) and adverse effects was 313. The maximum distance between the maximum of pharmacodynamics (HbA1c) normalized by the extended FGF21- (lipid) and GLP-1 mediated effects (HbA1c) and adverse effects was 123.

The efficacy and potential of GLP-1RA/FGF21 fusion proteins with different ratios of activity against adverse events was investigated with the aid of the systemic pharmacological approach. Fusion proteins with a speculatively calculated ideal potency ratio were identified that were predicted to be beneficial in improving lipid profile, body weight and glycemic control, while possibly not causing serious adverse GLP-1 RA-related effects based on gastric emptying responses. Thus, compounds predicted to have the selected model informing potency ratio provide good potency versus risk profiles.

Example 2: expression of GLP1RA-FGF21 fusion protein in HEK293 cells

The FGF21 protein of SEQ ID NO. 2 was fused directly to GLP1RA or a linker sequence was inserted between the GLP1RA and FGF21 sequences. In all constructs, the FGF21 construct was fused C-terminally to the GLP1RA sequence. If a linker is inserted, GLP1RA is fused N-terminally to the linker sequence and FGF21 is C-terminallyThe termini are fused to a linker sequence. The DNA sequence of the GLP1RA-FGF21 fusion protein was N-terminally fused to the IL2 signal sequence, followed by a histidine-rich sequence (His-tag) and a Tev cleavage site. The GLP1RA-FGF21 fusion protein was produced by transient transfection of HEK293 cells. The signal sequence is required for secretion of the desired fusion protein into the culture medium. The desired fusion protein was purified from the culture supernatant using immobilized metal ion affinity chromatography (IMAC). After elution from the IMAC column, the N-terminal His-tag can be cleaved by addition of Tev protease. For construct screening purposes, the His-tag was cleaved by adding Tev protease directly to the incubation medium for GLP1RA activity assay. The incubation time before starting the assay was 10-60 minutes to ensure complete lysis of the His tag. Constructs with GLP1RA activity in the desired range were produced on a larger scale. The GLP1RA-Fc-FGF21 fusion protein was produced by transient transfection in HEK293 cells. The desired fusion protein was purified from the culture supernatant using IMAC and a complete His-tag purification resin (Roche). After cleavage of the His tag, the cleavage reaction solution was passed through an IMAC column (cOmplexite) for a second time^TMHis-tag purification resin (Roche)), collecting (no His-tag) flow-through fractions. The fusion protein was further purified using a gel filtration column with phosphate buffered saline (PBS, Gibco) as running buffer. Fractions containing the desired fusion protein were collected, pooled, concentrated and stored at-80 ℃ until further use.

Example 3: in vitro cell assay for the efficacy of human FGF21 receptor in CHO cells (intracellular Western)

The In-Cell Western (ICW) assay with specificity and high sensitivity was used to measure the In vitro efficacy of mature human FGF21(SEQ ID NO:2) or FGF21 variants. ICW assays are immunocytochemical assays, usually performed in microplate format. FGF21 receptor autophosphorylation assay (Aguilar h.n. et al (2010) PLoS ONE 5(4): e9965) was performed using CHO Flp-ln cells (Invitrogen, Darmstadt, Germany) stably expressing human FGFR1c (═ FGF receptor 1c isoform) and human β -klotho (klb) using In-Cell Western. To determine the level of autophosphorylation or downstream activation of the receptor for MAP kinase ERK1/2, 2X10⁴Individual cellPerwell were seeded into 96-well plates and allowed to grow for 48 hours. Cells were serum-starved for 3-4 hours with Ham's F-12 nutrient mixture of serum-free medium with GlutaMAX (Gibco, Darmstadt, Germany). The cells were then treated with increasing concentrations of mature human FGF21(SEQ ID NO:2) for 5 minutes at 37 ℃. After incubation, the medium was discarded and the cells were fixed in 3.7% freshly prepared paraformaldehyde for 20 minutes. Cells were permeabilized for 20 minutes with 0.1% Triton-X-100 in PBS. Blocking was performed with Odyssey blocking buffer (LICOR, Bad Homburg, Germany) for 2 hours at room temperature. Primary antibodies (anti pGFR Tyr653/654(New England Biolabs, Frankfurt, Germany) or anti pERK phosphate p44/42MAP kinase Thr202/Tyr204 (cell signaling)) were added and incubated overnight at 4 ℃. After incubation of the primary antibody, cells were washed with PBS plus 0.1% Tween 20. The secondary mouse 800CW antibody (LICOR, Bad Homburg, Germany) was incubated at room temperature for 1 hour. Subsequently, the cells were washed again with PBS plus 0.1% Tween20 and the infrared dye signal was quantified with an Odyssey imager (LICOR, Bad Homburg, Germany). The results were normalized by quantifying the DNA with the TO-PRO3 dye (Invitrogen, Karlsruhe, Germany). Data were obtained in Arbitrary Units (AU) and EC50 values were obtained from dose response curves and summarized in table 2. Figure 5 shows the results from ICW using CHO cells overexpressing human FGFR1c plus KLB.

Table 2: EC50 values of mature human FGF21(SEQ ID NO:2) measured by ICW pGFFR or ICW PERK in CHO cells overexpressing human FGFR1c and KLB.

Example 4: in vitro cellular assay for human GLP-1 receptor efficacy

Agonism of a compound at the human glucagon-like peptide-1 (GLP-1) receptor was determined by a functional assay measuring cAMP response in HEK-293 cell lines stably expressing the human GLP-1 receptor.

The cAMP content of the cells was determined based on HTRF (homogeneous time resolved fluorescence) using a kit from Cisbio corp (catalog No. 62AM4 PEC). For preparation, cells were assigned to T175 culture flasks and grown overnight in media (DMEM/10% FBS) to near confluence. The medium was then removed and the cells were washed with calcium and magnesium deficient PBS prior to protease treatment with Accutase (Sigma-Aldrich catalog No. a 6964). The detached cells were washed and resuspended in assay buffer (1 XHBSS; 20mM HEPES, 0.1% BSA,2mM IBMX) and cell density was determined. It was then diluted to 4x10⁵Individual cells/mL and 25 μ L aliquots were dispensed into wells of a 96-well plate. For measurement, 25 μ Ι _ of test compound in assay buffer was added to the wells, followed by incubation at room temperature for 30 minutes. After addition of HTRF reagent diluted in lysis buffer (kit components), the plates were incubated for 1 hour before measuring the fluorescence ratio at 665/620 nm. By determining the concentration (EC) that causes 50% activation of the maximal response₅₀) To quantify the in vitro potency of agonists. The results are summarized in Table 3.

Table 3: EC50 values for GLP-1 receptor agonists (SEQ ID NOS: 7 and 24-36) measured by detecting a cAMP response in a HEK-293 cell line stably expressing the human GLP-1 receptor. A corresponding ratio of GLP-1R agonistic activity is also shown (native GLP-1(7-36)/GLP-1R agonist). The ratio X means that GLP-1R agonistic activity is reduced by 1/X compared to GLP-1R agonistic activity of native GLP-1 (7-36).

Table 4: selected ratios of GLP-1R agonist activity (native GLP-1(7-36)/GLP-1R agonist) and corresponding calculated EC50 values (based on the results obtained above). The ratio X means that GLP-1R agonistic activity is reduced by 1/X compared to GLP-1R agonistic activity of native GLP-1 (7-36).

Example 5: synthesis of peptide Compounds

Fusion proteins are produced by recombinant methods (see example 2), whereas isolated peptidic GLP-1R agonists are chemically synthesized.

More specifically, the peptides were synthesized by the following synthetic procedure:

0.3g of oven-dried Rink amide MBHA resin (0.66mmol/g) was placed in a polyethylene container equipped with a polypropylene filter. The resin was swollen in DCM (15ml) for 1 h and in DMF (15ml) for 1 h. The Fmoc group on the resin was deprotected by treating it twice (5 min and 15 min) with 20% (v/v) piperidine/DMF solution. The resin was washed with DMF/DCM/DMF (6: 6:6 times each). Removal of Fmoc from the solid support was confirmed using Kaiser test (quantitative method). The C-terminal Fmoc-amino acid in dry DMF (corresponding to a 5 equivalent excess of resin loading) was added to the deprotected resin and the coupling of the next Fmoc-amino acid was initiated with DIC and HOBT in 5 equivalent excess in DMF. The concentration of each reactant in the reaction mixture was about 0.4M. The mixture was rotated on a rotator at room temperature for 2 hours. The resin was filtered and washed with DMF/DCM/DMF (6: 6:6 times each). The Kaiser test on an aliquot of peptide resin was negative (no color on the resin) after the coupling was complete. After the first amino acid attachment, unreacted amino groups in the resin (if present) were capped/blocked using acetic anhydride/pyridine/DCM (1:8:8) for 20 min to avoid any deletion of the sequence. After capping/blocking, the resin was washed with DCM/DMF/DCM/DMF (6/6/6/6 times each). The Fmoc group on the C-terminal amino acid attached to the peptidyl resin was deprotected by treating it twice (5 min and 15 min) with 20% (v/v) piperidine/DMF solution. The resin was washed with DMF/DCM/DMF (6: 6:6 times each). After Fmoc deprotection was complete, a Kaiser test on aliquots of the peptide resin was positive.

The remaining amino acids in the target sequence on Rink amide MBHA resin were coupled in sequence using the Fmoc AA/DIC/HOBt method using a 5-equivalent excess corresponding to the resin loading in DMF. The concentration of each reactant in the reaction mixture was about 0.4M. The mixture was rotated on a rotator at room temperature for 2 hours. The resin was filtered and washed with DMF/DCM/DMF (6: 6:6 times each). After each coupling step and Fmoc deprotection step, a Kaiser test was performed to confirm completion of the reaction.

After completion of the linear sequence, the epsilon amino group of the lysine used as branch or modification point was deprotected by using 2.5% hydrazine hydrate (hydrazine hydrate) in DMF over 15 min x2 and washed with DMF/DCM/DMF (6: 6:6 times each). The gamma carboxy terminus of glutamic acid was attached to the epsilon amino group of Lys in DMF using Fmoc-Glu (OH) -OtBu and DIC/HOBt methods (5 equivalent excess relative to resin loading). The mixture was rotated on a rotator at room temperature for 2 hours. The resin was filtered and washed with DMF/DCM/DMF (6X 30ml each). The Fmoc group on glutamic acid was deprotected by treatment with 20% (v/v) piperidine/DMF solution twice for 5 min and 15 min (25ml each). The resin was washed with DMF/DCM/DMF (6: 6:6 times each). After Fmoc deprotection was complete, a Kaiser test on aliquots of the peptide resin was positive.

If the side chain branch also contains another gamma-glutamic acid, a second Fmoc-Glu (OH) -OtBu is used to attach to the free amino group of gamma-glutamic acid using the DIC/HOBt method (5 equivalent excess relative to resin loading) in DMF. The mixture was rotated on a rotator at room temperature for 2 hours. The resin was filtered and washed with DMF/DCM/DMF (6X 30ml each). The Fmoc group on gamma-glutamic acid was deprotected by treating it twice with 20% (v/v) piperidine/DMF solution for 5 min and 15 min (25 mL). The resin was washed with DMF/DCM/DMF (6: 6:6 times each). After Fmoc deprotection was complete, a Kaiser test on aliquots of the peptide resin was positive.

Final cleavage of the peptide from the resin:

the peptidyl resin synthesized by the manual synthesis was washed with DCM (6x10ml), MeOH (6x10ml) and ether (6x10ml) and dried overnight in a vacuum desiccator. Cleavage of the peptide from the solid support is achieved by: the peptide-resin was treated with the reagent mix (80% TFA/5% thioanisole/5% phenol/2.5% EDT/2.5% DMS/5% DCM) for 3 hours at room temperature. The cleavage mixture was collected by filtration and the resin was washed with TFA (2ml) and DCM (2X5 ml). The excess TFA and DCM was concentrated to a small volume under nitrogen and a small amount of DCM (5-10ml) was added to the residue and evaporated under nitrogen. The process was repeated 3-4 times to remove most of the volatile impurities. The residue was cooled to 0 ℃ and anhydrous ether was added to precipitate the peptide. The precipitated peptide was centrifuged and the ether in the supernatant was removed, and fresh ether was added to the peptide and centrifuged again. The crude sample was purified by preparative HPLC and lyophilized. The identity of the peptide was confirmed by LCMS.

Table 5: sequence listing

Sequence listing

<110> Sainuo Fei (Sanofi)

<120> FGF21 compound/GLP-1R agonist combination with optimized activity ratio

<130> 589-264 PCT2

<150> EP 18 305 784.3

<151> 2018, 6 months and 21 days

<160> 70

<170> PatentIn version 3.5

<210> 1

<211> 209

<212> PRT

<213> human (Homo sapiens)

<400> 1

Met Asp Ser Asp Glu Thr Gly Phe Glu His Ser Gly Leu Trp Val Ser

1 5 10 15

Val Leu Ala Gly Leu Leu Leu Gly Ala Cys Gln Ala His Pro Ile Pro

20 25 30

Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr

35 40 45

Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg

50 55 60

Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu

65 70 75 80

Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val

85 90 95

Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly

100 105 110

Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu

115 120 125

Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu

130 135 140

His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly

145 150 155 160

Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Pro Pro Glu

165 170 175

Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp

180 185 190

Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala

195 200 205

Ser

<210> 2

<211> 181

<212> PRT

<213> human (Homo sapiens)

<400> 2

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Pro Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser

165 170 175

Pro Ser Tyr Ala Ser

180

<210> 3

<211> 181

<212> PRT

<213> Artificial sequence

<220>

<223> FGF21(His29-Ser209) A59C,G71C

<400> 3

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Cys His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Cys Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Pro Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser

165 170 175

Pro Ser Tyr Ala Ser

180

<210> 4

<211> 181

<212> PRT

<213> Artificial sequence

<220>

<223> FGF21(His29-Ser209) Q55C,N149C,G198Y

<400> 4

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Cys Gln Thr Glu Ala His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Pro Gly Cys Lys Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Pro Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Ser Met Val Tyr Pro Ser Gln Gly Arg Ser

165 170 175

Pro Ser Tyr Ala Ser

180

<210> 5

<211> 180

<212> PRT

<213> Artificial sequence

<220>

<223> FGF21(His29-Ser209) Q55C,P147C,delP199

<400> 5

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Cys Gln Thr Glu Ala His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Cys Gly Asn Lys Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Pro Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Ser Met Val Gly Ser Gln Gly Arg Ser Pro

165 170 175

Ser Tyr Ala Ser

180

<210> 6

<211> 180

<212> PRT

<213> Artificial sequence

<220>

<223> FGF21(His29-Ser209) Q55C,N149C,delP199

<400> 6

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Cys Gln Thr Glu Ala His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Pro Gly Cys Lys Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Pro Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Ser Met Val Gly Ser Gln Gly Arg Ser Pro

165 170 175

Ser Tyr Ala Ser

180

<210> 7

<211> 30

<212> PRT

<213> human (Homo sapiens)

<400> 7

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg

20 25 30

<210> 8

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> GLP1(7-36)

A8G,V16L,S18K,Y19Q,L20M,G22E,Q23E,A25V,K26R,E27L,A30E,V33K,K34N,R

36G,insPSSGAPPPS

<400> 8

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu

1 5 10 15

Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 9

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> GLP1(7-36)

A8G,V16L,S18K,Y19Q,G22E,Q23E,A25V,K26Q,E27L,A30E,V33L,K34A,G35T,R

36G,insPSSGAPPPS

<400> 9

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu

1 5 10 15

Glu Ala Val Gln Leu Phe Ile Glu Trp Leu Leu Ala Thr Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 10

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> GLP1(7-36)

A8G,V16L,S18I,Y19Q,E21D,G22E,Q23E,A25V,K26R,E27L,A30E,V33L,K34A,G

35T,R36G,insPVSGAPPPS

<400> 10

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Ile Gln Leu Asp Glu

1 5 10 15

Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Leu Ala Thr Gly Pro Val

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 11

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> GLP1(7-36)

A8G,V16L,S18I,Y19Q,E21D,G22E,Q23E,A25V,K26R,E27L,A30E,V33E,K34A,G

35T,R36G,insPVSGAPPPS

<400> 11

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Ile Gln Leu Asp Glu

1 5 10 15

Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Glu Ala Thr Gly Pro Val

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 12

<211> 40

<212> PRT

<213> Artificial sequence

<220>

<223> GLP1(7-36)

insG,A8G,V16L,S18I,Y19Q,G22E,Q23E,A25V,K26R,E27L,A30E,V33L,K34A,R

36G,insPSSGAPPPS

<400> 12

Gly His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Ile Gln Leu Glu

1 5 10 15

Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Leu Ala Gly Gly Pro

20 25 30

Ser Ser Gly Ala Pro Pro Pro Ser

35 40

<210> 13

<211> 40

<212> PRT

<213> Artificial sequence

<220>

<223> GLP1(7-36)

insG,A8G,V16L,S18I,Y19Q,G22E,Q23E,A25V,K26R,E27L,A30E,V33L,K34A,G

35T,R36G,insPSSGAPPPS

<400> 13

Gly His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Ile Gln Leu Glu

1 5 10 15

Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Leu Ala Thr Gly Pro

20 25 30

Ser Ser Gly Ala Pro Pro Pro Ser

35 40

<210> 14

<211> 40

<212> PRT

<213> Artificial sequence

<220>

<223> GLP1(7-36)

A8G,V16L,S18K,Y19Q,E21D,G22E,Q23E,A25V,K26Q,E27L,A30E,V33L,K34A,G

35T,R36G,insPSSGEPPPES

<400> 14

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu

1 5 10 15

Glu Ala Val Gln Leu Phe Ile Glu Trp Leu Leu Ala Thr Gly Pro Ser

20 25 30

Ser Gly Glu Pro Pro Pro Glu Ser

35 40

<210> 15

<211> 30

<212> PRT

<213> Artificial sequence

<220>

<223> GLP1(7-36)

A8G,V16L,S18K,Y19Q,L20M,E21D,G22E,Q23E,A25V,K26R,E27L,A30E,V33K,K

34N,R36G

<400> 15

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu

1 5 10 15

Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly

20 25 30

<210> 16

<211> 30

<212> PRT

<213> Artificial sequence

<220>

<223> GLP1(7-36)

A8G,V16L,S18K,Y19Q,G22E,Q23E,A25V,K26Q,E27L,A30E,V33L,K34A,G35T,R

36G

<400> 16

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu

1 5 10 15

Glu Ala Val Gln Leu Phe Ile Glu Trp Leu Leu Ala Thr Gly

20 25 30

<210> 17

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> GLP1(7-36)

A8G,V16L,S18K,Y19Q,G22E,Q23E,A25V,K26Q,E27L,A30E,V33L,K34A,G35T,R

36G,insPSSGEPPPE

<400> 17

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu

1 5 10 15

Glu Ala Val Gln Leu Phe Ile Glu Trp Leu Leu Ala Thr Gly Pro Ser

20 25 30

Ser Gly Glu Pro Pro Pro Glu

35

<210> 18

<211> 40

<212> PRT

<213> Artificial sequence

<220>

<223> GLP1(7-36)

insG,A8G,V16L,S18K,Y19Q,G22E,Q23E,A24R,A25V,K26Q,A30E,insPSSGAPPP

S

<400> 18

Gly His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu

1 5 10 15

Glu Glu Arg Val Gln Glu Phe Ile Glu Trp Leu Val Lys Gly Arg Pro

20 25 30

Ser Ser Gly Ala Pro Pro Pro Ser

35 40

<210> 19

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> GLP1(7-36)

A8G,V16L,S18K,Y19Q,G22E,Q23E,A25V,K26Q,E27L,A30E,V33E,K34A,G35T,R

36G,insPSSGAPPPS

<400> 19

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu

1 5 10 15

Glu Ala Val Gln Leu Phe Ile Glu Trp Leu Glu Ala Thr Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser

35

<210> 20

<211> 38

<212> PRT

<213> Artificial sequence

<220>

<223> GLP1(7-36)

insG,A8G,V16L,S18I,Y19Q,G22E,Q23E,A25V,K26R,E27L,A30E,V33L,K34A,R

36G,insPKKQRLS

<400> 20

Gly His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Ile Gln Leu Glu

1 5 10 15

Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Leu Ala Gly Gly Pro

20 25 30

Lys Lys Gln Arg Leu Ser

35

<210> 21

<211> 228

<212> PRT

<213> Artificial sequence

<220>

<223> IgG4 Fc variant, IGHG4_ human (Glu99-Gly326)

<400> 21

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly

225

<210> 22

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> (G7S) (G4S) (G4S) A Joint (19GS)

<400> 22

Gly Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

1 5 10 15

Gly Ser Ala

<210> 23

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> (G3S) (GS) A Joint (7GS)

<400> 23

Gly Gly Gly Ser Gly Ser Ala

1 5

<210> 24

<211> 474

<212> PRT

<213> Artificial sequence

<220>

<223> GLP1(7-36)

A8G,V16L,S18K,Y19Q,L20M,G22E,Q23E,A25V,K26R,E27L,A30E,V33K,K34N,R

36G, insPSSGAPPPS- [19GS ] -IgG 4 Fc-variant [7GS ] \u

FGF21(His29-Ser209)_A59C,G71C

<400> 24

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu

1 5 10 15

Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Gly Gly Gly Ser Gly

35 40 45

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro

50 55 60

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val

65 70 75 80

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

85 90 95

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

100 105 110

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

115 120 125

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

130 135 140

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

145 150 155 160

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

165 170 175

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

180 185 190

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

195 200 205

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

210 215 220

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

225 230 235 240

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

245 250 255

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

260 265 270

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly

275 280 285

Gly Ser Gly Ser Ala His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln

290 295 300

Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln

305 310 315 320

Gln Thr Glu Cys His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Cys

325 330 335

Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys

340 345 350

Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys

355 360 365

Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu

370 375 380

Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr

385 390 395 400

Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser

405 410 415

Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu

420 425 430

Pro Gly Leu Pro Pro Ala Pro Pro Glu Pro Pro Gly Ile Leu Ala Pro

435 440 445

Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro

450 455 460

Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser

465 470

<210> 25

<211> 474

<212> PRT

<213> Artificial sequence

<220>

<223> GLP1(7-36)

A8G,V16L,S18K,Y19Q,G22E,Q23E,A25V,K26Q,E27L,A30E,V33L,K34A,G35T,R

36G,insPSSGAPPPS_[19GS] _IgG4 Fc

Variant- [7GS ] -FGF21 (His29-Ser209) Q55C, N149C, G198Y

<400> 25

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu

1 5 10 15

Glu Ala Val Gln Leu Phe Ile Glu Trp Leu Leu Ala Thr Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Gly Gly Gly Ser Gly

35 40 45

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro

50 55 60

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val

65 70 75 80

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

85 90 95

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

100 105 110

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

115 120 125

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

130 135 140

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

145 150 155 160

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

165 170 175

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

180 185 190

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

195 200 205

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

210 215 220

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

225 230 235 240

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

245 250 255

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

260 265 270

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly

275 280 285

Gly Ser Gly Ser Ala His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln

290 295 300

Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Cys

305 310 315 320

Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly

325 330 335

Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys

340 345 350

Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys

355 360 365

Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu

370 375 380

Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr

385 390 395 400

Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Cys Lys Ser

405 410 415

Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu

420 425 430

Pro Gly Leu Pro Pro Ala Pro Pro Glu Pro Pro Gly Ile Leu Ala Pro

435 440 445

Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met Val Tyr Pro

450 455 460

Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser

465 470

<210> 26

<211> 473

<212> PRT

<213> Artificial sequence

<220>

<223> GLP1(7-36)

A8G,V16L,S18I,Y19Q,E21D,G22E,Q23E,A25V,K26R,E27L,A30E,V33L,K34A,G

35T, R36G, insPVSGAPPPS- [19GS ] -IgG 4 Fc-variant

[7GS]_FGF21(His29-Ser209)Q55C,P147C,delP199

<400> 26

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Ile Gln Leu Asp Glu

1 5 10 15

Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Leu Ala Thr Gly Pro Val

20 25 30

Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Gly Gly Gly Ser Gly

35 40 45

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro

50 55 60

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val

65 70 75 80

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

85 90 95

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

100 105 110

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

115 120 125

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

130 135 140

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

145 150 155 160

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

165 170 175

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

180 185 190

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

195 200 205

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

210 215 220

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

225 230 235 240

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

245 250 255

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

260 265 270

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly

275 280 285

Gly Ser Gly Ser Ala His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln

290 295 300

Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Cys

305 310 315 320

Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly

325 330 335

Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys

340 345 350

Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys

355 360 365

Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu

370 375 380

Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr

385 390 395 400

Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Cys Gly Asn Lys Ser

405 410 415

Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu

420 425 430

Pro Gly Leu Pro Pro Ala Pro Pro Glu Pro Pro Gly Ile Leu Ala Pro

435 440 445

Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met Val Gly Ser

450 455 460

Gln Gly Arg Ser Pro Ser Tyr Ala Ser

465 470

<210> 27

<211> 473

<212> PRT

<213> Artificial sequence

<220>

<223> GLP1(7-36)

A8G,V16L,S18I,Y19Q,E21D,G22E,Q23E,A25V,K26R,E27L,A30E,V33E,K34A,G

35T, R36G, insPVSGAPPPS- [19GS ] -IgG 4 Fc-variant

[7GS]_FGF21(His29-Ser209)Q55C,N149C,delP199

<400> 27

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Ile Gln Leu Asp Glu

1 5 10 15

Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Glu Ala Thr Gly Pro Val

20 25 30

Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Gly Gly Gly Ser Gly

35 40 45

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro

50 55 60

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val

65 70 75 80

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

85 90 95

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

100 105 110

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

115 120 125

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

130 135 140

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

145 150 155 160

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

165 170 175

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

180 185 190

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

195 200 205

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

210 215 220

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

225 230 235 240

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

245 250 255

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

260 265 270

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly

275 280 285

Gly Ser Gly Ser Ala His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln

290 295 300

Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Cys

305 310 315 320

Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly

325 330 335

Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys

340 345 350

Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys

355 360 365

Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu

370 375 380

Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr

385 390 395 400

Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Cys Lys Ser

405 410 415

Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu

420 425 430

Pro Gly Leu Pro Pro Ala Pro Pro Glu Pro Pro Gly Ile Leu Ala Pro

435 440 445

Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met Val Gly Ser

450 455 460

Gln Gly Arg Ser Pro Ser Tyr Ala Ser

465 470

<210> 28

<211> 474

<212> PRT

<213> Artificial sequence

<220>

<223> GLP1(7-36)

insG,A8G,V16L,S18I,Y19Q,G22E,Q23E,A25V,K26R,E27L,A30E,V33L,K34A,R

36G, insPSSGAPPPS- [19GS ] -IgG 4 Fc-variant

[7GS]_FGF21(His29-Ser209)Q55C,N149C,delP199

<400> 28

Gly His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Ile Gln Leu Glu

1 5 10 15

Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Leu Ala Gly Gly Pro

20 25 30

Ser Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Gly Gly Gly Ser

35 40 45

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly

50 55 60

Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser

65 70 75 80

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

85 90 95

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

100 105 110

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

115 120 125

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

130 135 140

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

145 150 155 160

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

165 170 175

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

180 185 190

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

195 200 205

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

210 215 220

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

225 230 235 240

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser

245 250 255

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

260 265 270

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly

275 280 285

Gly Gly Ser Gly Ser Ala His Pro Ile Pro Asp Ser Ser Pro Leu Leu

290 295 300

Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala

305 310 315 320

Cys Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly

325 330 335

Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu

340 345 350

Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu

355 360 365

Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro

370 375 380

Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val

385 390 395 400

Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Cys Lys

405 410 415

Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro

420 425 430

Leu Pro Gly Leu Pro Pro Ala Pro Pro Glu Pro Pro Gly Ile Leu Ala

435 440 445

Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met Val Gly

450 455 460

Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser

465 470

<210> 29

<211> 474

<212> PRT

<213> Artificial sequence

<220>

<223> GLP1(7-36)

insG,A8G,V16L,S18I,Y19Q,G22E,Q23E,A25V,K26R,E27L,A30E,V33L,K34A,G

35T, R36G, insPSSGAPPPS- [19GS ] -Ig 4G Fc-variant

[7GS]_FGF21(His29-Ser209)Q55C,P147C,delP199

<400> 29

Gly His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Ile Gln Leu Glu

1 5 10 15

Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Leu Ala Thr Gly Pro

20 25 30

Ser Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Gly Gly Gly Ser

35 40 45

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly

50 55 60

Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser

65 70 75 80

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

85 90 95

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

100 105 110

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

115 120 125

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

130 135 140

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

145 150 155 160

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

165 170 175

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

180 185 190

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

195 200 205

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

210 215 220

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

225 230 235 240

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser

245 250 255

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

260 265 270

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly

275 280 285

Gly Gly Ser Gly Ser Ala His Pro Ile Pro Asp Ser Ser Pro Leu Leu

290 295 300

Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala

305 310 315 320

Cys Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly

325 330 335

Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu

340 345 350

Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu

355 360 365

Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro

370 375 380

Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val

385 390 395 400

Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Cys Gly Asn Lys

405 410 415

Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro

420 425 430

Leu Pro Gly Leu Pro Pro Ala Pro Pro Glu Pro Pro Gly Ile Leu Ala

435 440 445

Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met Val Gly

450 455 460

Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser

465 470

<210> 30

<211> 475

<212> PRT

<213> Artificial sequence

<220>

<223> GLP1(7-36)

A8G,V16L,S18K,Y19Q,E21D,G22E,Q23E,A25V,K26Q,E27L,A30E,V33L,K34A,G

35T, R36G, insPSSGEPPPES- [19GS ] -IgG 4 Fc-variant

[7GS]_FGF21(His29-Ser209)Q55C,N149C,G198Y

<400> 30

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu

1 5 10 15

Glu Ala Val Gln Leu Phe Ile Glu Trp Leu Leu Ala Thr Gly Pro Ser

20 25 30

Ser Gly Glu Pro Pro Pro Glu Ser Gly Gly Gly Gly Gly Gly Gly Ser

35 40 45

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly

50 55 60

Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser

65 70 75 80

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

85 90 95

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

100 105 110

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

115 120 125

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

130 135 140

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

145 150 155 160

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

165 170 175

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

180 185 190

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

195 200 205

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

210 215 220

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

225 230 235 240

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser

245 250 255

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

260 265 270

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly

275 280 285

Gly Gly Ser Gly Ser Ala His Pro Ile Pro Asp Ser Ser Pro Leu Leu

290 295 300

Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala

305 310 315 320

Cys Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly

325 330 335

Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu

340 345 350

Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu

355 360 365

Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro

370 375 380

Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val

385 390 395 400

Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Cys Lys

405 410 415

Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro

420 425 430

Leu Pro Gly Leu Pro Pro Ala Pro Pro Glu Pro Pro Gly Ile Leu Ala

435 440 445

Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met Val Tyr

450 455 460

Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser

465 470 475

<210> 31

<211> 465

<212> PRT

<213> Artificial sequence

<220>

<223> GLP1(7-36)

A8G,V16L,S18K,Y19Q,L20M,E21D,G22E,Q23E,A25V,K26R,E27L,A30E,V33K,K

34N, R36G- [19GS ] -. IgG4 Fc _ variant

[7GS]_FGF21(His29-Ser209)Q55C,N149C,G198Y

<400> 31

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu

1 5 10 15

Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Gly Gly

20 25 30

Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

35 40 45

Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu

50 55 60

Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

65 70 75 80

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

85 90 95

Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

100 105 110

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

115 120 125

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

130 135 140

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

145 150 155 160

Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

165 170 175

Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn

180 185 190

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

195 200 205

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

210 215 220

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg

225 230 235 240

Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys

245 250 255

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

260 265 270

Ser Leu Ser Leu Gly Gly Gly Gly Ser Gly Ser Ala His Pro Ile Pro

275 280 285

Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr

290 295 300

Leu Tyr Thr Asp Asp Ala Cys Gln Thr Glu Ala His Leu Glu Ile Arg

305 310 315 320

Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu

325 330 335

Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val

340 345 350

Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly

355 360 365

Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu

370 375 380

Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu

385 390 395 400

His Leu Pro Gly Cys Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly

405 410 415

Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Pro Pro Glu

420 425 430

Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp

435 440 445

Pro Leu Ser Met Val Tyr Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala

450 455 460

Ser

465

<210> 32

<211> 465

<212> PRT

<213> Artificial sequence

<220>

<223> GLP1(7-36)

A8G,V16L,S18K,Y19Q,G22E,Q23E,A25V,K26Q,E27L,A30E,V33L,K34A,G35T,R

36G _ [19GS ] _ IgG4 Fc _ variant [7GS ]

FGF21(His29-Ser209)Q55C,N149C,G198Y

<400> 32

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu

1 5 10 15

Glu Ala Val Gln Leu Phe Ile Glu Trp Leu Leu Ala Thr Gly Gly Gly

20 25 30

Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

35 40 45

Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu

50 55 60

Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

65 70 75 80

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

85 90 95

Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

100 105 110

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

115 120 125

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

130 135 140

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

145 150 155 160

Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

165 170 175

Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn

180 185 190

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

195 200 205

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

210 215 220

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg

225 230 235 240

Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys

245 250 255

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

260 265 270

Ser Leu Ser Leu Gly Gly Gly Gly Ser Gly Ser Ala His Pro Ile Pro

275 280 285

Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr

290 295 300

Leu Tyr Thr Asp Asp Ala Cys Gln Thr Glu Ala His Leu Glu Ile Arg

305 310 315 320

Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu

325 330 335

Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val

340 345 350

Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly

355 360 365

Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu

370 375 380

Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu

385 390 395 400

His Leu Pro Gly Cys Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly

405 410 415

Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Pro Pro Glu

420 425 430

Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp

435 440 445

Pro Leu Ser Met Val Tyr Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala

450 455 460

Ser

465

<210> 33

<211> 474

<212> PRT

<213> Artificial sequence

<220>

<223> GLP1(7-36)

A8G,V16L,S18K,Y19Q,G22E,Q23E,A25V,K26Q,E27L,A30E,V33L,K34A,G35T,R

36G, insPSSGEPPPE- [19GS ] _ IgG4 Fc _ variant

[7GS]_FGF21(His29-Ser209)Q55C,N149C,G198Y

<400> 33

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu

1 5 10 15

Glu Ala Val Gln Leu Phe Ile Glu Trp Leu Leu Ala Thr Gly Pro Ser

20 25 30

Ser Gly Glu Pro Pro Pro Glu Gly Gly Gly Gly Gly Gly Gly Ser Gly

35 40 45

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro

50 55 60

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val

65 70 75 80

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

85 90 95

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

100 105 110

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

115 120 125

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

130 135 140

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

145 150 155 160

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

165 170 175

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

180 185 190

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

195 200 205

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

210 215 220

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

225 230 235 240

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

245 250 255

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

260 265 270

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly

275 280 285

Gly Ser Gly Ser Ala His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln

290 295 300

Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Cys

305 310 315 320

Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly

325 330 335

Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys

340 345 350

Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys

355 360 365

Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu

370 375 380

Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr

385 390 395 400

Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Cys Lys Ser

405 410 415

Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu

420 425 430

Pro Gly Leu Pro Pro Ala Pro Pro Glu Pro Pro Gly Ile Leu Ala Pro

435 440 445

Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met Val Tyr Pro

450 455 460

Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser

465 470

<210> 34

<211> 474

<212> PRT

<213> Artificial sequence

<220>

<223> GLP1(7-36)

insG,A8G,V16L,S18K,Y19Q,G22E,Q23E,A24R,A25V,K26Q,A30E,insPSSGAPPP

S _ [19GS ] _ IgG4 Fc _ variant

[7GS]_FGF21(His29-Ser209)Q55C,N149C,delP199

<400> 34

Gly His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu

1 5 10 15

Glu Glu Arg Val Gln Glu Phe Ile Glu Trp Leu Val Lys Gly Arg Pro

20 25 30

Ser Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Gly Gly Gly Ser

35 40 45

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly

50 55 60

Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser

65 70 75 80

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

85 90 95

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

100 105 110

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

115 120 125

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

130 135 140

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

145 150 155 160

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

165 170 175

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

180 185 190

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

195 200 205

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

210 215 220

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

225 230 235 240

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser

245 250 255

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

260 265 270

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly

275 280 285

Gly Gly Ser Gly Ser Ala His Pro Ile Pro Asp Ser Ser Pro Leu Leu

290 295 300

Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala

305 310 315 320

Cys Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly

325 330 335

Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu

340 345 350

Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu

355 360 365

Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro

370 375 380

Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val

385 390 395 400

Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Cys Lys

405 410 415

Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro

420 425 430

Leu Pro Gly Leu Pro Pro Ala Pro Pro Glu Pro Pro Gly Ile Leu Ala

435 440 445

Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met Val Gly

450 455 460

Ser Gln Gly Arg Ser Pro Ser Tyr Ala Ser

465 470

<210> 35

<211> 473

<212> PRT

<213> Artificial sequence

<220>

<223> GLP1(7-36)

A8G,V16L,S18K,Y19Q,G22E,Q23E,A25V,K26Q,E27L,A30E,V33E,K34A,G35T,R

36G, insPSSGAPPPS- [19GS ] -IgG 4 Fc-variant

[7GS]_FGF21(His29-Ser209)Q55C,N149C,delP199

<400> 35

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Leu Glu Glu

1 5 10 15

Glu Ala Val Gln Leu Phe Ile Glu Trp Leu Glu Ala Thr Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser Gly Gly Gly Gly Gly Gly Gly Ser Gly

35 40 45

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro

50 55 60

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val

65 70 75 80

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

85 90 95

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

100 105 110

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

115 120 125

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

130 135 140

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

145 150 155 160

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

165 170 175

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

180 185 190

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

195 200 205

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

210 215 220

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

225 230 235 240

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

245 250 255

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

260 265 270

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly

275 280 285

Gly Ser Gly Ser Ala His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln

290 295 300

Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Cys

305 310 315 320

Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly

325 330 335

Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys

340 345 350

Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys

355 360 365

Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu

370 375 380

Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr

385 390 395 400

Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Cys Lys Ser

405 410 415

Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu

420 425 430

Pro Gly Leu Pro Pro Ala Pro Pro Glu Pro Pro Gly Ile Leu Ala Pro

435 440 445

Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met Val Gly Ser

450 455 460

Gln Gly Arg Ser Pro Ser Tyr Ala Ser

465 470

<210> 36

<211> 472

<212> PRT

<213> Artificial sequence

<220>

<223> GLP1(7-36)

insG,A8G,V16L,S18I,Y19Q,G22E,Q23E,A25V,K26R,E27L,A30E,V33L,K34A,R

36G, insPKKQRLS- [19GS ] _ IgG4 Fc _ variant

[7GS]_FGF21(His29-Ser209)Q55C,N149C,delP199

<400> 36

Gly His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Ile Gln Leu Glu

1 5 10 15

Glu Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Leu Ala Gly Gly Pro

20 25 30

Lys Lys Gln Arg Leu Ser Gly Gly Gly Gly Gly Gly Gly Ser Gly Gly

35 40 45

Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro Pro

50 55 60

Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe

65 70 75 80

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

85 90 95

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

100 105 110

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

115 120 125

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

130 135 140

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

145 150 155 160

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

165 170 175

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

180 185 190

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

195 200 205

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

210 215 220

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

225 230 235 240

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

245 250 255

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

260 265 270

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly

275 280 285

Ser Gly Ser Ala His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe

290 295 300

Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Cys Gln

305 310 315 320

Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala

325 330 335

Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro

340 345 350

Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln

355 360 365

Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala

370 375 380

Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln

385 390 395 400

Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Cys Lys Ser Pro

405 410 415

His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro

420 425 430

Gly Leu Pro Pro Ala Pro Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln

435 440 445

Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met Val Gly Ser Gln

450 455 460

Gly Arg Ser Pro Ser Tyr Ala Ser

465 470

<210> 37

<211> 30

<212> PRT

<213> Artificial sequence

<220>

<223> GLP-1RA, Universal sequences

<220>

<221> misc _ feature

<222> (10)..(10)

<223> Xaa is any amino acid, e.g., L or K

<220>

<221> misc _ feature

<222> (12)..(12)

<223> Xaa is any amino acid, e.g., K or I

<220>

<221> misc _ feature

<222> (14)..(14)

<223> Xaa is any amino acid, e.g., L or M

<220>

<221> misc _ feature

<222> (15)..(15)

<223> Xaa is any amino acid, e.g., E or D

<220>

<221> misc _ feature

<222> (18)..(18)

<223> Xaa is any amino acid, e.g., A or R

<220>

<221> misc _ feature

<222> (20)..(20)

<223> Xaa is any amino acid, e.g., R or Q

<220>

<221> misc _ feature

<222> (21)..(21)

<223> Xaa is any amino acid, e.g., L or E

<220>

<221> misc _ feature

<222> (27)..(27)

<223> Xaa is any amino acid, e.g., L, E, K or V

<220>

<221> misc _ feature

<222> (28)..(28)

<223> Xaa is any amino acid, e.g., A, N or K

<220>

<221> misc _ feature

<222> (29)..(29)

<223> Xaa is any amino acid, e.g., T or G

<220>

<221> misc _ feature

<222> (30)..(30)

<223> Xaa is any amino acid, e.g., G or R

<400> 37

His Gly Glu Gly Thr Phe Thr Ser Asp Xaa Ser Xaa Gln Xaa Xaa Glu

1 5 10 15

Glu Xaa Val Xaa Xaa Phe Ile Glu Trp Leu Xaa Xaa Xaa Xaa

20 25 30

<210> 38

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> C-terminal peptide extension I

<400> 38

Pro Ser Ser Gly Ala Pro Pro Pro Ser

1 5

<210> 39

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> C-terminal peptide extension II

<400> 39

Pro Val Ser Gly Ala Pro Pro Pro Ser

1 5

<210> 40

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> C-terminal peptide extension III

<400> 40

Pro Ser Ser Gly Glu Pro Pro Pro Glu Ser

1 5 10

<210> 41

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> C-terminal peptide extension IV

<400> 41

Pro Ser Ser Gly Glu Pro Pro Pro Glu

1 5

<210> 42

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C-terminal peptide extension V

<400> 42

Pro Lys Lys Gln Arg Leu Ser

1 5

<210> 43

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> C-terminal peptide extension VI

<400> 43

Pro Lys Lys Ile Arg Tyr Ser

1 5

<210> 44

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<400> 44

Glu Ile Arg Pro

1

<210> 45

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<400> 45

Thr Gly Leu Glu Ala Val

1 5

<210> 46

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> peptide

<400> 46

Thr Gly Leu Glu Ala Asn

1 5

<210> 47

<211> 181

<212> PRT

<213> Artificial sequence

<220>

<223> FGF21 variants

<400> 47

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser

165 170 175

Pro Ser Tyr Ala Ser

180

<210> 48

<211> 182

<212> PRT

<213> Artificial sequence

<220>

<223> FGF21 variants

<400> 48

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Ser Met Val Thr Gly Leu Glu Ala Val Arg

165 170 175

Ser Pro Ser Tyr Ala Ser

180

<210> 49

<211> 182

<212> PRT

<213> Artificial sequence

<220>

<223> FGF21 variants

<400> 49

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Ser Met Val Thr Gly Leu Glu Ala Asn Arg

165 170 175

Ser Pro Ser Tyr Ala Ser

180

<210> 50

<211> 181

<212> PRT

<213> Artificial sequence

<220>

<223> FGF21 variants

<400> 50

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Ser Met Val Asn Pro Ser Gln Gly Arg Ser

165 170 175

Pro Ser Tyr Ala Ser

180

<210> 51

<211> 181

<212> PRT

<213> Artificial sequence

<220>

<223> FGF21 variants

<400> 51

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Asn Arg Ser

165 170 175

Pro Ser Tyr Ala Ser

180

<210> 52

<211> 182

<212> PRT

<213> Artificial sequence

<220>

<223> FGF21 variants

<400> 52

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Ser Met Val Thr Gly Leu Glu Ala Val Arg

165 170 175

Ser Pro Ser Tyr Ala Ser

180

<210> 53

<211> 182

<212> PRT

<213> Artificial sequence

<220>

<223> FGF21 variants

<400> 53

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Ser Met Val Thr Gly Leu Glu Ala Asn Arg

165 170 175

Ser Pro Ser Tyr Ala Ser

180

<210> 54

<211> 181

<212> PRT

<213> Artificial sequence

<220>

<223> FGF21 variants

<400> 54

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Ser Met Val Asn Pro Ser Gln Gly Arg Ser

165 170 175

Pro Ser Tyr Ala Ser

180

<210> 55

<211> 181

<212> PRT

<213> Artificial sequence

<220>

<223> FGF21 variants

<400> 55

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Asn Arg Ser

165 170 175

Pro Ser Tyr Ala Ser

180

<210> 56

<211> 181

<212> PRT

<213> Artificial sequence

<220>

<223> FGF21 variants

<400> 56

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser

165 170 175

Pro Ser Tyr Glu Ser

180

<210> 57

<211> 182

<212> PRT

<213> Artificial sequence

<220>

<223> FGF21 variants

<400> 57

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Ser Met Val Thr Gly Leu Glu Ala Val Arg

165 170 175

Ser Pro Ser Tyr Glu Ser

180

<210> 58

<211> 182

<212> PRT

<213> Artificial sequence

<220>

<223> FGF21 variants

<400> 58

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Ser Met Val Thr Gly Leu Glu Ala Asn Arg

165 170 175

Ser Pro Ser Tyr Glu Ser

180

<210> 59

<211> 181

<212> PRT

<213> Artificial sequence

<220>

<223> FGF21 variants

<400> 59

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Ser Met Val Asn Pro Ser Gln Gly Arg Ser

165 170 175

Pro Ser Tyr Glu Ser

180

<210> 60

<211> 181

<212> PRT

<213> Artificial sequence

<220>

<223> FGF21 variants

<400> 60

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Asn Arg Ser

165 170 175

Pro Ser Tyr Glu Ser

180

<210> 61

<211> 182

<212> PRT

<213> Artificial sequence

<220>

<223> FGF21 variants

<400> 61

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Ser Met Val Thr Gly Leu Glu Ala Val Arg

165 170 175

Ser Pro Ser Tyr Glu Ser

180

<210> 62

<211> 182

<212> PRT

<213> Artificial sequence

<220>

<223> FGF21 variants

<400> 62

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Ser Met Val Thr Gly Leu Glu Ala Asn Arg

165 170 175

Ser Pro Ser Tyr Glu Ser

180

<210> 63

<211> 181

<212> PRT

<213> Artificial sequence

<220>

<223> FGF21 variants

<400> 63

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Ser Met Val Asn Pro Ser Gln Gly Arg Ser

165 170 175

Pro Ser Tyr Glu Ser

180

<210> 64

<211> 181

<212> PRT

<213> Artificial sequence

<220>

<223> FGF21 variants

<400> 64

His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Asn Arg Ser

165 170 175

Pro Ser Tyr Glu Ser

180

<210> 65

<211> 223

<212> PRT

<213> Artificial sequence

<220>

<223> hybrid Fc variants

<400> 65

Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln Pro Leu Gly Val

1 5 10 15

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

20 25 30

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

35 40 45

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

50 55 60

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

65 70 75 80

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

85 90 95

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

100 105 110

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

115 120 125

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

130 135 140

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

145 150 155 160

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

165 170 175

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

180 185 190

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

195 200 205

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

210 215 220

<210> 66

<211> 245

<212> PRT

<213> Artificial sequence

<220>

<223> hybrid Fc variants

<400> 66

Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys

1 5 10 15

Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His

20 25 30

Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

35 40 45

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

50 55 60

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

65 70 75 80

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

85 90 95

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

100 105 110

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

115 120 125

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

130 135 140

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

145 150 155 160

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

165 170 175

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

180 185 190

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

195 200 205

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

210 215 220

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

225 230 235 240

Leu Ser Leu Gly Lys

245

<210> 67

<211> 233

<212> PRT

<213> Artificial sequence

<220>

<223> hybrid Fc variants

<400> 67

Glu Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu

1 5 10 15

Cys Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys

20 25 30

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

35 40 45

Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

65 70 75 80

Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

85 90 95

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

100 105 110

Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

115 120 125

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met

130 135 140

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

145 150 155 160

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

165 170 175

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

180 185 190

Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val

195 200 205

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

210 215 220

Lys Ser Leu Ser Leu Ser Leu Gly Lys

225 230

<210> 68

<211> 255

<212> PRT

<213> Artificial sequence

<220>

<223> hybrid Fc variants

<400> 68

Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg Asn Thr Gly Arg Gly

1 5 10 15

Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys Glu Glu Gln Glu Glu Arg

20 25 30

Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln Pro Leu Gly Val

35 40 45

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

50 55 60

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

65 70 75 80

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

85 90 95

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

100 105 110

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

115 120 125

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

130 135 140

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

145 150 155 160

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

165 170 175

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

180 185 190

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

195 200 205

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

210 215 220

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

225 230 235 240

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

245 250 255

<210> 69

<211> 264

<212> PRT

<213> Artificial sequence

<220>

<223> hybrid Fc variants

<400> 69

Ala Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro

1 5 10 15

Ala Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu

20 25 30

Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys

35 40 45

Pro Ser His Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro

50 55 60

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

65 70 75 80

Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val

85 90 95

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

100 105 110

Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

115 120 125

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly

130 135 140

Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

145 150 155 160

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr

165 170 175

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

180 185 190

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

195 200 205

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

210 215 220

Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe

225 230 235 240

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

245 250 255

Ser Leu Ser Leu Ser Leu Gly Lys

260

<210> 70

<211> 253

<212> PRT

<213> Artificial sequence

<220>

<223> hybrid Fc variants

<400> 70

Glu Thr Lys Thr Pro Glu Cys Pro Ser His Thr Gln Pro Leu Gly Val

1 5 10 15

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

20 25 30

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

35 40 45

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

50 55 60

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

65 70 75 80

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

85 90 95

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

100 105 110

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

115 120 125

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

130 135 140

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

145 150 155 160

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

165 170 175

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

180 185 190

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

195 200 205

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ala

210 215 220

Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg Asn Thr Gly Arg Gly Gly

225 230 235 240

Glu Glu Lys Lys Lys Glu Lys Glu Lys Glu Glu Gln Glu

245 250