FGF21 compound / GLP-1R agonist combinations with optimized activity ratios

Abstract

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, with an optimized GLP-1R agonist / FGF21 compound activity ratio. The present 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, non-alcoholic steatohepatitis (NASH) and / or atherosclerosis.

Description

TECHNICAL FIELD

[0001] The present invention relates to combinations, pharmaceutical compositions and fusion molecules comprising a FGF21 (fibroblast growth factor 21) compound and a GLP-1R (glucagon-like peptide-1 receptor) agonist with an optimized GLP-1R agonist / FGF21 compound activity ratio. The present invention further 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, non-alcoholic steatohepatitis (NASH) and / or atherosclerosis. BACKGROUND

[0003] The administration of fibroblast growth factor 21 (FGF21) compounds, e.g. recombinantly produced FGF21 polypeptides, leads to a significant reduction of body weight, blood glucose and blood lipids and to an improvement of insulin sensitivity, as demonstrated, e.g., by Gaich et al. (2013) Cell Metab 18(3):333-340 and Dong et al. (2015) Br J Clin Pharmacol 80(5): 1051-1063. Glucagon-like peptide-1 receptor (GLP-1R) agonists are effective in lowering glucose and body weight in humans, as shown, e.g., by Astrup et al. (2012) Int J Obes (Lond) 36(6):843-854 and Nauck et al. (2013) Diabetes Obes Metab 15(3):204-212. The combination of the beneficial effects of FGF21 administration with the glucose-lowering effects of GLP-1 receptor agonists surprisingly results in a synergistic effect (see, e.g., WO 2011 / 089203 Al and WO 2014 / 037373 Al) that provides a more comprehensive treatment of diseases / conditions such as obesity, overweight, metabolic syndrome, diabetes, diabetic retinopathy, hyperglycemia, dyslipidemia, non-alcoholic steatohepatitis (NASH) and / or atherosclerosis. SUMMARY

[0005] The combination of FGF21 compounds and GLP-1R agonists, e.g. in the form of fusion proteins, can be used, e.g., for improving glycemic control in overweight to obese dyslipidemic patients with type 2 diabetes.

[0006] It is worth noting that FGF21 and GLP-1 (as a major GLP-1 R agonist) exert their pharmacological effects at different plasma concentrations. More particularly, FGF21 effects start to play at higher plasma levels compared to GLP-1 utility. Moreover, GLP-1 is known to have adverse effects at higher levels, such as nausea and vomiting. In summary, this means that when a combination of FGF21 compounds and GLP-1 R agonists is administered, e.g. in the form of a fusion protein, there is a risk of potential GLP-1 mediated adverse effects.

[0007] It is therefore an object of the present application to determine an optimized GLP-1 R agonist / FGF21 compound activity ratio in order to achieve beneficial effects while avoiding potential adverse effects, such as nausea and vomiting. It is a further object of the present application to provide respective combinations, pharmaceutical compositions and fusion molecules having an optimized GLP-1 R agonist / FGF21 compound activity ratio.

[0008] In one aspect, the present application relates to a combination comprising a FGF21 (fibroblast growth factor 21) compound and a GLP-1 R (glucagon-like peptide-1 receptor) agonist,

[0009] wherein the FGF21 compound has the same or essentially the same FGF21 activity as the FGF21 activity of native FGF21 and the FGF21 compound is a FGF21 variant comprising at least one mutation selected from the group consisting of:

[0010] - substitution of the amino acid residues at positions 98 to 101 of the N-terminus of native FGF21 from SEQ ID NO: 2 with the amino acid sequence EIRP (SEQ ID NO: 44);

[0011] - substitution of 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);

[0012] - substitution of 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 TGLEAN (SEQ ID NO: 46);

[0013] - substitution of the amino acid residue at position 170 of the N-terminus of native FGF21 from SEQ ID NO: 2 with the amino acid N;

[0014] - substitution of the amino acid residue at position 174 of the N-terminus of native FGF21 from SEQ ID NO: 2 with the amino acid N;

[0015] - substitution of the amino acid residue at position 180 from the N-terminus of native FGF21 of SEQ ID NO: 2 with the amino acid E and one or more mutations as defined above; and

[0016] - 1 to 10 amino acid residue mutations that reduce the immunogenicity of the FGF21 variant compared to native FGF21 of SEQ ID NO: 2, and

[0017] wherein the GLP-1 R agonistic activity of said GLP-1 R agonist is reduced 1 / 531 to 1 / 9 (or 1 / 531.0 to 1 / 9.449) compared to the GLP-1 R agonistic activity of native GLP-1 (7-36).

[0018] In one embodiment, the FGF21 activity refers to the activation of the FGF21 receptor. In one embodiment, the term refers to in vitro activity. In one embodiment, the activation of the FGF21 receptor is determined by measuring the autophosphorylation of the FGF21 receptor when contacted in vitro with the FGF21 compound. In one embodiment, the FGF21 activity is determined by using an In-Cell Western (ICW) assay, e.g. essentially as described in Example 3.

[0019] In one embodiment, the GLP-1 R agonistic activity refers to the activation of the GLP-1 receptor. In one embodiment, the term refers to in vitro agonistic activity. In one embodiment, the activation of the GLP-1 receptor is determined by assaying the cAMP response of cells stably expressing the GLP-1 receptor when contacted in vitro with the agonist. In one embodiment, the activation of the GLP-1 receptor is determined essentially as described in Example 4.

[0020] In one embodiment, the GLP-1 R agonist has a GLP-1 R agonistic activity that is reduced 1 / 482 to 1 / 9 (or 1 / 482.396 to 1 / 9.449) or 1 / 319 to 1 / 9 (or 1 / 319.311 to 1 / 9.449) or 1 / 121 to 1 / 9 (or 1 / 121.189 to 1 / 9.449) compared to the GLP-1 R agonistic activity of native GLP-1 (7-36).

[0021] In one embodiment, the GLP-1 R agonist has a GLP-1 R agonistic activity that is reduced 1 / 319 to 1 / 9 compared to the GLP-1 R agonistic activity of native GLP-1 (7-36).

[0022] In one embodiment, the GLP-1R agonist has a GLP-1R agonistic activity which is at most 1 / 9.4 or at most 1 / 9.45 or at most 1 / 9.5 of the GLP-1R agonistic activity of native GLP-1 (7-36).

[0023] In one embodiment, the GLP-1R agonist has a GLP-1R agonistic activity which is at most 1 / 10 of the GLP-1R agonistic activity of native GLP-1 (7-36).

[0024] In one embodiment, the GLP-1R agonist has a GLP-1R agonistic activity which is at least 1 / 482.4 or at least 1 / 482.35 of the GLP-1R agonistic activity of native GLP-1 (7-36).

[0025] In one embodiment, the GLP-1R agonist has a GLP-1R agonistic activity which is at least 1 / 482 of the GLP-1R agonistic activity of native GLP-1 (7-36).

[0026] In one embodiment, the GLP-1R agonist has a GLP-1R agonistic activity which is 1 / 482 to 1 / 10 of the GLP-1R agonistic activity of native GLP-1 (7-36).

[0027] In one embodiment, the GLP-1R agonist has a GLP-1R agonistic activity which is 1 / 319 to 1 / 10 of the GLP-1R agonistic activity of native GLP-1 (7-36).

[0028] In one embodiment, the GLP-1R agonist has a GLP-1R agonistic activity which is 1 / 100 to 1 / 90 of the GLP-1R agonistic activity of native GLP-1 (7-36).

[0029] In one embodiment, the GLP-1R agonist has a GLP-1R agonistic activity which is at most 1 / 18 (or at most 1 / 18.268) of the GLP-1R agonistic activity of native GLP-1 (7-36).

[0030] In one embodiment, the GLP-1R agonist has a GLP-1R agonistic activity which is reduced 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) compared to the GLP-1R agonistic activity of native GLP-1 (7-36).

[0031] In one embodiment, the GLP-1R agonist has a GLP-1R agonistic activity which is reduced 1 / 313 to 1 / 18 compared to the GLP-1R agonistic activity of native GLP-1 (7-36).

[0032] In one of the above embodiments, the GLP-1R agonist has a GLP-1R agonistic activity which is reduced at most 1 / 18.2 fold or at most 1 / 18.3 fold compared to the GLP-1R agonistic activity of native GLP-1 (7-36).

[0033] 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 FGF21.

[0034] In one embodiment, the FGF21 variant comprises or consists 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.

[0035] In one embodiment, the GLP-1R agonist comprises or consists of the amino acid sequence

[0036] H-G-E-G-T-F-T-S-D-X 10 -S-X 12 -Q-X 14 -X 15 -E-E-X 18 -V-X 20 -X 21 -F-I-E-W-L-X 27 -X 28 -X 29 -X 30 (SEQ ID NO: 37),

[0037] wherein

[0038] X 10 is L or K;

[0039] X 12 is K or I;

[0040] X 14 is L or M;

[0041] X 15 is E or D;

[0042] X 18 is A or R;

[0043] X 20 is R or Q;

[0044] X 21 is L or E;

[0045] X 27 is L, E, K or V;

[0046] X 28 is A, N or K;

[0047] X 29 is T or G;

[0048] X 30 is G or R;

[0049] wherein, optionally, the amino acid sequence comprises at its N-terminus at least one additional amino acid residue; and

[0050] wherein, optionally, the amino acid sequence comprises at its C-terminus a peptide extension consisting of up to 12, 11 or 10 amino acid residues.

[0051] In one embodiment, the GLP-1R agonist comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 9, 10, 12, 14, 15, 16, 17, 19 and 20.

[0052] In one embodiment, X 14 is L and X 28 is A.

[0053] In one embodiment, the GLP-1R agonist comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 9, 10, 12, 14, 16, 17, 19 and 20.

[0054] In another aspect, the present application relates to a pharmaceutical composition comprising an FGF21 (fibroblast growth factor 21) compound and a GLP-1R (glucagon-like peptide-1 receptor) agonist, and a pharmaceutically acceptable carrier and / or excipient,

[0055] wherein the FGF21 compound has the same or substantially the same FGF21 activity as the FGF21 activity of native FGF21, and the FGF21 compound is an FGF21 variant comprising at least one mutation selected from the group consisting of:

[0056] - substitution of the amino acid residues at positions 98 to 101 of the N-terminus of native FGF21 from SEQ ID NO: 2 with the amino acid sequence EIRP (SEQ ID NO: 44);

[0057] - substitution of 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);

[0058] - substitution of 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 TGLEAN (SEQ ID NO: 46);

[0059] - substitution of the amino acid residue at position 170 of the N-terminus of native FGF21 from SEQ ID NO: 2 with the amino acid N;

[0060] - substitution of the amino acid residue at position 174 of the N-terminus of native FGF21 from SEQ ID NO: 2 with the amino acid N;

[0061] - substitution of the amino acid residue at position 180 of the N-terminus of native FGF21 from SEQ ID NO: 2 with the amino acid E and one or more mutations as defined above; and

[0062] - 1 to 10 amino acid residue mutations that reduce the immunogenicity of the FGF21 variant compared to native FGF21 of SEQ ID NO: 2, and

[0063] wherein the GLP-1R agonist has GLP-1R agonistic activity that is reduced 1 / 531 to 1 / 9 (or 1 / 531.0 to 1 / 9.449) compared to the GLP-1R agonistic activity of native GLP-1 (7-36).

[0064] In yet another aspect, the present application relates to a fusion molecule comprising an FGF21 (fibroblast growth factor 21) compound and a GLP-1R (glucagon-like peptide-1 receptor) agonist,

[0065] wherein the FGF21 compound has the same or essentially the same FGF21 activity as the FGF21 activity of native FGF21 and the FGF21 compound is an FGF21 variant comprising at least one mutation selected from the group consisting of:

[0066] - substitution of the amino acid residues at positions 98 to 101 of the N-terminus of native FGF21 from SEQ ID NO: 2 with the amino acid sequence EIRP (SEQ ID NO: 44);

[0067] - substitution of 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);

[0068] - substitution of 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 TGLEAN (SEQ ID NO: 46);

[0069] - substitution of the amino acid residue at position 170 of the N-terminus of native FGF21 from SEQ ID NO: 2 with the amino acid N;

[0070] - substitution of the amino acid residue at position 174 of the N-terminus of native FGF21 from SEQ ID NO: 2 with the amino acid N;

[0071] - substitution of the amino acid residue at position 180 of the N-terminus of native FGF21 from SEQ ID NO: 2 with the amino acid E and one or more mutations as defined above; and

[0072] - 1 to 10 amino acid residue mutations that reduce the immunogenicity of the FGF21 variant compared to native FGF21 of SEQ ID NO: 2, and

[0073] wherein the GLP-1R agonist has GLP-1R agonistic activity that is reduced 1 / 531 to 1 / 9 (or 1 / 531.0 to 1 / 9.449) compared to the GLP-1R agonistic activity of native GLP-1 (7-36).

[0074] In one embodiment, the fusion molecule further comprises a hybrid Fc domain comprising a combination of partial Fc regions / domains of different immunoglobulins.

[0075] In one embodiment, the GLP-1R agonist and / or the FGF21 compound is as defined above.

[0076] In another aspect, the present application relates to a nucleic acid molecule encoding a fusion molecule as defined above.

[0077] In another aspect, the present application relates to a host cell comprising a nucleic acid molecule as defined above.

[0078] In another aspect, the present application 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.

[0079] In another aspect, the present application 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.

[0080] In another aspect, the present application 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, non-alcoholic steatohepatitis (NASH) and atherosclerosis.

[0081] In one embodiment, the disease or disorder is diabetes. In one embodiment, the diabetes is type 1 diabetes or type 2 diabetes.

[0082] In another aspect, the present application 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, non-alcoholic steatohepatitis (NASH) and atherosclerosis.

[0083] In one embodiment, the disease or disorder is diabetes. In one embodiment, the diabetes is type 1 diabetes or type 2 diabetes.

[0084] In another aspect, the present application relates to a method of treating a disease or disorder selected from the group consisting of obesity, overweight, metabolic syndrome, diabetes, diabetic retinopathy, hyperglycemia, dyslipidemia, non-alcoholic steatohepatitis (NASH) and atherosclerosis, 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.

[0085] In one embodiment, the disease or disorder is diabetes. In one embodiment, the diabetes is type 1 diabetes or type 2 diabetes. BRIEF DESCRIPTION OF DRAWINGS

[0086] Figure 1 is a plot showing EC50for adverse effects (Gastric emptying (GE) rate) and pharmacodynamics (i.e., HbAlc, triglycerides, fatty acids, non-HDL, fat mass) according to GLP-1 decay factor (12-month simulation):

[0087] • For GLP-1 decay factors greater than 9.449 (roundable to 9), the EC50for GLP-1 mediated gastrointestinal adverse effects (Gastric emptying; GE rate) is greater than the EC50for pharmacodynamic effects (i.e., HbAlc, fat mass, non-HDL, fatty acids, triglycerides).

[0088] • The maximum distance between the maximum of pharmacodynamics (HbAlc) normalized by the FGF21- (lipid) and GLP-1 mediated effects (HbAlc) and the adverse effect (GE rate) is 121.189; i.e., at 121.189 (roundable to 121), there is a maximum distance between the maximum of pharmacodynamic effects (HbAlc) and the adverse effect (GE rate) at the minimum distance between GLP-1 mediated effects (HbAlc) and FGF21 mediated average effects (i.e., fat mass, non-HDL, fatty acids, triglycerides) (see Figure 2 );

[0089] • The maximum distance between the maximum of pharmacodynamic effects (HbAlc) and the adverse effect (GE rate) is 319.311 (roundable to 319);

[0090] • The maximum distance between the average pharmacodynamic effects (i.e., HbAlc, fat mass, non-HDL, fatty acids, triglycerides) and the adverse effect (GE rate) is 482.396 (see Figure 2 ; roundable to 482);

[0091] • The maximum of gastric emptying rate is at 531.0;

[0092] (all: vertical lines).

[0093] Figure 2 is a plot showing EC50s for gastric emptying (GE) rate and mean pharmacodynamic effects (i.e., HbA1c, triglycerides, fatty acids, non-HDL, fat mass) according to GLP-1 decay factor (12-month simulation):

[0094] • The maximum distance between mean pharmacodynamics (i.e., HbA1c, fat mass, non-HDL, fatty acids, triglycerides) and adverse effect (GE rate) is 482.396 (right vertical line; can be rounded to 482);

[0095] • The maximum distance between maximum of pharmacodynamics (HbA1c) normalized by extending GLP-1 -mediated effect (HbA1c) and adverse effect (GE rate) is 121.189 (left vertical line; can be rounded to 121). The curve “(max-GE rate) / range” represents the ratio of the maximum distance between HbA1c and GE rate to the minimum distance between HbA1c and FGF21 -mediated mean effects (i.e., fat mass, non-HDL, fatty acids, triglycerides). At the minimum of the “(max-GE rate) / range” curve (i.e., at 121.189), there is a maximum distance between maximum of pharmacodynamic effect (HbA1c) and adverse effect (GE rate) at the minimum distance between GLP-1 -mediated effect (HbA1c) and FGF21 -mediated effects (i.e., fat mass, non-HDL, fatty acids, triglycerides).

[0096] Figure 3 is a plot showing EC50s for adverse effect (gastric emptying (GE) rate) and pharmacodynamics (HbA1c, triglycerides, fatty acids, non-HDL, fat mass) according to GLP-1 decay factor (3-month simulation):

[0097] • For GLP-1 decay factors greater than 18.268 (can be rounded to 18), the EC50 for GLP-1 -mediated adverse gastrointestinal effect (gastric emptying; GE rate) is greater than the EC50 for pharmacodynamic effects (i.e., HbA1c, fat mass, non-HDL, fatty acids, triglycerides);

[0098] • The maximum distance between the maximum of the pharmacodynamic (HbAlc) effect normalized by the FGF21 -(lipid) and GLP-1 mediated effect (HbAlc) and the adverse effect (GE rate) is 123.466; i.e., at 123.466 (roundable to 123), at the minimum distance between the GLP-1 mediated effect (HbAlc) and the FGF21 mediated average effect (i.e., fat mass, non-HDL, fatty acids, triglycerides), there is a maximum distance between the maximum of the pharmacodynamic effect (HbAlc) and the adverse effect (GE rate) (see Figure 4 );

[0099] • The maximum distance between the maximum of the pharmacodynamic (HbAlc) effect and the adverse effect (GE rate) is 313.214 (roundable to 313);

[0100] • The maximum distance between the average pharmacodynamic (i.e., HbAlc, fat mass, non-HDL, fatty acids, triglycerides) and the adverse effect (GE rate) is 468.679 (see Figure 4 ; roundable to 469);

[0101] • The maximum of the gastric emptying rate is at 500.686 (roundable to 501)

[0102] (all: vertical lines).

[0103] Figure 4 is a plot showing the EC50of the gastric emptying (GE) rate and the average pharmacodynamic effect (i.e., HbAlc, triglycerides, fatty acids, non-HDL, fat mass) according to the GLP-1 decay factor (3 month simulation):

[0104] • The maximum distance between the average pharmacodynamic (i.e., HbAlc, fat mass, non-HDL, fatty acids, triglycerides) and the adverse effect (GE rate) is 468.679 (right vertical line; roundable to 469);

[0105] • The maximum distance between the maximum of the pharmacodynamic (HbAlc) effect normalized by the extension of the FGF21 -(lipid) and GLP-1 mediated effect (HbAlc) and the adverse effect (GE rate) is 123.466 (left vertical line; rounded to 123). The curve "(max - GE rate) / range" represents the ratio of the maximum distance between HbAlc and GE rate over the minimum distance between HbAlc 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 (HbAlc) and the FGF21 mediated effect (i.e. fat mass, non-HDL, fatty acids, triglycerides), there is the maximum distance between the maximum of the pharmacodynamic effect (HbAlc) and the adverse effect (GE rate).

[0106] Figure 5 Dose response curves of (A) FGFR autophosphorylation or (B) ERK1 / 2-phosphorylation measured by In-Cell Western in CHO cells overexpressing human FGFRlc and β-klotho upon stimulation with mature human FGF21 (SEQ ID NO: 2). DETAILED DESCRIPTION

[0108] While the following describes the present application in detail, it is understood that the application is not limited to the particular methodology, protocols and reagents described herein as these can 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 application which is 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 to which this application belongs.

[0109] Hereinafter, certain elements of the present application will be described. These elements can be recited with specific embodiments, however it is understood that they can be combined in any manner and in any number to produce other embodiments. The differently described embodiments and preferred embodiments should not be viewed as limiting the application to only the explicitly described embodiments. The specification should be understood as supporting and encompassing embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, unless the context indicates otherwise, it is to be understood that the description of this application discloses any permutation and combination of all the elements described in this application.

[0110] The terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Ed., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0111] The practice of the present application will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques which are explained in the literature in the field (Sambrook, J. et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0112] 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 to the exclusion of any other member, integer or step or group of members, integers or steps to the subject on which they are used in some embodiments, such other member, integer or step or group of members, integers or steps can be excluded. The terms "a" and "an" and "the" and similar referents in the context of describing the application (especially in the context of the following claims) are to be construed to be inclusive rather than exclusive, unless otherwise indicated. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual 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 application and does not pose a limitation on the scope of the application otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.

[0113] In the body of this specification, several documents are cited. Each of the documents referred to herein, including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc., are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the application is not entitled to antedate the cited publications by virtue of prior application.

[0114] By using a systems pharmacology approach that integrates GLP-1 receptor signaling with key components of FGF21 production and action in the context of diabetes pathophysiology, the present inventors have successfully identified the optimal GLP-1R agonist / FGF21 compound activity ratio to achieve the beneficial effects of both active agents (e.g., in body weight, lipid, glycemic control) while avoiding potential adverse effects (e.g., nausea and emesis).

[0115] The term "combination" as used herein is intended to encompass the following ways: allowing the administration of an FGF21 compound and a GLP-1R agonist separately to a patient or in the form of a combination product in which the FGF21 compound and the GLP-1R agonist are present together (e.g., in one pharmaceutical composition) or in the form of a fusion molecule / protein comprising the FGF21 compound and the GLP-1R agonist. When administered separately, the administration can be simultaneous or in either order sequentially. The amounts of the FGF21 compound and the GLP-1R agonist and the relative timing of administration will be selected to achieve the desired combined therapeutic effect. Administration of the combination can be carried out concurrently in a single pharmaceutical composition including all active pharmaceutical ingredients; or in separate pharmaceutical compositions each including at least one active pharmaceutical ingredient. Alternatively, the combination can be administered separately in a sequential manner, where one therapeutic agent is administered first and the other therapeutic agent is administered afterwards, or vice versa. Such sequential administration can be in close temporal proximity or at longer time intervals. In one embodiment, the combination is provided in the form of a kit, e.g., a kit as defined herein.

[0116] The term "fibroblast growth factor 21" or "FGF21" as used herein refers to any FGF21 protein known in the art and specifically refers to human FGF21. In one embodiment, human FGF21 has the amino acid sequence of SEQ ID NO: 1.

[0117] The term "FGF21 compound" as used herein refers generally to a compound having FGF21 activity.

[0118] In one embodiment, the FGF21 compound is a peptide compound, i.e., a peptide or a protein.

[0119] The term "peptide" as used herein refers to a polymeric form of amino acids of any length, e.g., containing 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.

[0120] In one embodiment, the FGF21 compound is a natural FGF21 or an 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 natural FGF21.

[0121] The term "natural FGF21" as used herein refers to 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 FGF21"). The term "natural FGF21" as used herein also includes mature FGF21, i.e., naturally occurring FGF21 lacking the natural signal sequence (also referred to as signal peptide). In one embodiment, the natural FGF21 is mature human wild-type FGF21 lacking amino acids 1 to 28 (M1 to A28) of SEQ ID NO: 1 and is represented by SEQ ID NO: 2.

[0122] "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. In addition to manual methods, the optimal alignment of sequences for comparison can be produced, e.g., 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, 575 Science Drive, Madison, Wis.).

[0123] The FGF21 variant can be based on a deletion, addition and / or substitution of at least one amino acid residue in native FGF21 (e.g., SEQ ID NO: 1 or 2).

[0124] Such deletion, addition and / or substitution can contribute to an improved stability, e.g., proteolytic stability and / or thermal stability, 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.

[0125] 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, if their structure permits their D and L stereoisomers, to such stereoisomers. Amino acids are referred to herein by either their name, their commonly accepted three letter symbols, or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0126] The term "naturally occurring" when used in connection with an amino acid refers to the 20 common 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, pyrrolysine (PYL), and pyrroline-carboxylysine (PCL).

[0127] The term "non-natural amino acid" as used herein means an amino acid that is not encoded by the genetic code of any organism or is not found in any organism. It can be, for example, a purely synthetic compound. Examples of non-natural amino acids include, but are not limited to, hydroxyproline, gamma-carboxyglutamate, O-phosphoserine, azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminohexanoic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2,2'-diaminopimelic acid, t-butylglycine, 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-methylglycine, N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, iso-desmosine, allo-isoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthylalanine, norvaline, norleucine, ornithine, D-ornithine, D-arginine, p-aminophenylalanine, pentylglycine, pipecolic acid, and thioproline.

[0128] The term "amino acid analog" as used herein refers to a chemical compound having the same basic chemical structure as a naturally occurring amino acid. Amino acid analogs include natural and non-natural amino acids that are reversibly or irreversibly chemically blocked, or that have their C-terminal carboxyl group, their N-terminal amino group, and / or their side chain functional group chemically modified. Such analogs include, but are not limited to, methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine sulfoxide, S-(carboxymethyl)-cysteine sulfone, aspartic acid-(beta methyl ester), N-ethylglycine, alanine carboxamide, homoserine, norleucine, and methionine methylsulfonium.

[0129] The term "amino acid mimetic" as used herein refers to a chemical compound having a structure different from the general chemical structure of an amino acid, but that functions in a similar manner to a naturally occurring amino acid.

[0130] 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 a naturally occurring amino acid other than proline, a non-natural amino acid, an amino acid analog, and an amino acid mimetic. In one embodiment, the at least one additional amino acid is selected from the group consisting of G, A, N, and C. In a particular embodiment, the at least one additional amino acid is G.

[0131] Suitable FGF21 variants for use in the present application are described, for example, in 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.

[0132] In one embodiment, the FGF21 compound is an FGF21 variant comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 5 and 6.

[0133] In one embodiment, the FGF21 compound is an FGF21 variant comprising at least one mutation selected from the group consisting of:

[0134] - substitution of the amino acid residues at positions 98 to 101 of the N terminus of native FGF21 from SEQ ID NO: 2 with the amino acid sequence EIRP (SEQ ID NO: 44);

[0135] - substitution of 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);

[0136] - substitution of 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 TGLEAN (SEQ ID NO: 46);

[0137] - substitution of the amino acid residue at position 170 of the N terminus of native FGF21 from SEQ ID NO: 2 with the amino acid N;

[0138] - substitution of the amino acid residue at position 174 of the N terminus of native FGF21 from SEQ ID NO: 2 with the amino acid N;

[0139] - substitution of the amino acid residue at position 180 of the N terminus of native FGF21 from SEQ ID NO: 2 with the amino acid E and one or more mutations as defined above; and

[0140] - 1 to 10 amino acid residue mutations that reduce the immunogenicity of the FGF21 variant compared to native FGF21 of SEQ ID NO: 2.

[0141] The immunogenicity of a given FGF21 variant can be predicted by routine methods known in the art. For example, it can be predicted by using, for example, iTope TMand / or TCED TM Methods to screen proteins for potential immunogenicity. In addition, mutations to minimize immunogenicity can be designed by routine methods known in the art. For example, when immunogenicity is observed by performing EpiScreen TM analysis to assess potential immunogenicity, the amino acid sequences that induce immunogenicity can be identified by T cell epitope mapping, and immunogenicity-minimized mutants can be designed by computer prediction.

[0142] In one embodiment, the FGF21 compound is an 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.

[0143] The FGF21 compounds comprised in the combinations, pharmaceutical compositions, and fusion molecules of the present application 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 FGF21 activity of an FGF21 compound when not comprised in a fusion molecule as defined herein (not a component thereof) and / or when not further modified (see below).

[0144] The term "substantially the same" as used herein refers to FGF21 activity that is 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).

[0145] In one embodiment, the term "FGF21 activity" (or "FGF21 potency") as used herein refers to the activation of an FGF21 receptor (FGFR, e.g., FGFRlc). 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, the activation of an FGF21 receptor is determined by measuring the 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, the activity / potency is quantified by determining the EC50 value.

[0146] The term "In-Cell Western (ICW) assay" as used herein refers to an immuno cytochemical assay, more specifically a quantitative immunofluorescence assay, typically performed in microplates (e.g., in 96- or 384-well format). It 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): e9965). 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.

[0147] In one embodiment, an FGF21 compound having the same or essentially 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 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) can also be referred to herein as an FGF21 compound activating the FGF21 receptor with an EC50 of 2.40 to 7.20 nmol / L or 2.88 to 6.72 nmol / L or 3.12 to 6.48 nmol / L, respectively, in a pFGFR ICW assay, e.g., as essentially described in Example 3. In one embodiment, the EC50 value is given as EC50 ± SD. In one embodiment, the 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 a pFGFR ICW assay, e.g., as essentially described in Example 3. In one embodiment, the SD is 1.8 nmol / L.

[0148] According to the present application, the FGF21 compound can 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 the Fc region / domain of an immunoglobulin or a variant thereof, e.g., as further described below. In one embodiment, the FGF21 activity of the FGF21 compound referred to herein is the FGF21 activity of the FGF21 compound without said further modification, also referred to herein as "pure FGF21 compound".

[0149] The term "fused to" as used herein specifically refers to a genetic fusion, e.g. by recombinant DNA technology. The amino acid sequence of the (poly-)peptide half-life extension module can be introduced at any position within the variant amino acid sequence and can e.g. assume a loop shape within the structure of the encoded protein, or it can be an N-terminal or C-terminal fusion.

[0150] The term "conjugated to" as used herein specifically refers to a chemical and / or enzymatic conjugation leading to a stable covalent linkage between the (poly-)peptide and another molecule, e.g. the variant and the half-life extension module. Such conjugation can occur at the N- or C-terminus of the (poly-)peptide or at specific side chains, e.g. at lysine, cysteine, tyrosine or non-natural amino acid residues.

[0151] The term "GLP-1 R agonist" (short: "GLP-1 RA") as used herein generally refers to a compound that binds to and activates the GLP-1 receptor, such as GLP-1 (as the primary GLP-1 R agonist).

[0152] In one embodiment, the GLP-1 R agonist is a peptide compound, i.e. a peptide or a protein. In another embodiment, the GLP-1 R agonist is a small molecule, i.e. an organic compound with a molecular weight of less than 900 Da.

[0153] The GLP-1 R agonist comprised in the combinations, pharmaceutical compositions and fusion molecules of the present application exhibit a reduced GLP-1 R agonistic activity as compared to native GLP-1 (7-36) as defined herein. The value "x" in the expression "reduced x-fold" as used herein can refer to a "decay factor" or a "reduction factor". In one embodiment, the reduced GLP-1 R agonistic activity as compared to native GLP-1 (7-36) as defined herein is exhibited when the GLP-1 R agonist is a component of a fusion molecule as defined herein.

[0154] The term "native GLP-1 (7-36)" as used herein refers to a peptide having the amino acid sequence of SEQ ID NO: 7, which sequence optionally comprises an amide group at its C-terminus.

[0155] In one embodiment, the term "GLP-1 R agonistic activity" (or "GLP-1 R 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, the activation of the GLP-1 receptor is determined by measuring the cAMP response of 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 the human GLP-1 receptor. In one embodiment, the activation of the GLP-1 receptor is determined essentially as described in Example 4. In one embodiment, the activity / potency is quantified by determining the EC50 value.

[0156] In one embodiment, a GLP-1 R agonist having reduced GLP-1 R agonistic activity compared to the GLP-1 R agonistic activity of native GLP-1 (7-36) can be defined in terms of its EC50 value for GLP-1 receptor activation, e.g., as indicated in Table 4. For example, a GLP-1 R agonist having reduced GLP-1 R agonistic activity compared to the GLP-1 R agonistic activity of native GLP-1 (7-36) by a factor of 1 / 531 to 1 / 9 can also be referred to herein as a GLP-1 R agonist activating the GLP-1 receptor with an EC50 of 6.93 to 408.87 pmol / L, etc. In one embodiment, the EC50 value is determined as described above. In one embodiment, the EC50 value is given as EC50 ± SD. In one embodiment, the SD is the assay-dependent standard deviation.

[0157] Suitable GLP-1 R agonists having reduced GLP-1 R agonistic activity compared to the GLP-1 R agonistic activity of native GLP-1 (7-36) can be identified by the assays described herein for determining GLP-1 R agonistic activity, e.g., assays such as described in Example 4 or Xiao et al. (2001) Biochemistry. 40(9):2860-9 or Gault et al. (2013) J Biol Chem. 288(49):35581-91, e.g., analysis of GLP-1 R agonist-induced production of cytosolic cAMP, beta cell preservation (apoptosis), or glucose-stimulated insulin secretion (GSIS), etc. The above-mentioned suitable GLP-1 R agonists can be identified, e.g., by generating variants of known peptidic GLP-1 R agonists, such as native GLP-1 (7-36), e.g., by random or site-directed mutagenesis or chemical synthesis (see, e.g., Example 5), and subsequently determining their GLP-1 R agonistic activity using native GLP-1 (7-36) as a control as described herein. Alternatively, the above-mentioned suitable GLP-1 R agonists can be identified by screening small molecule libraries for GLP-1 R agonistic activity using native GLP-1 (7-36) as a control. These assays can all be performed in a high-throughput format.

[0158] A variant of a known peptidic GLP-1 R agonist, e.g., native GLP-1 (7-36), can be based on a deletion, addition and / or substitution of at least one amino acid residue in the amino acid sequence of the known peptidic GLP-1 R agonist or a deletion, addition and / or substitution of at least one amino acid residue to the amino acid sequence of the known peptidic GLP-1 R agonist.

[0159] 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.

[0160] In one embodiment, the GLP-1 R 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 amino acid residues in the native GLP-1 (7-36) sequence. In one embodiment, the substitutions are 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 legend of SEQ ID NOs: 8 to 20).

[0161] 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 a naturally occurring amino acid other than proline, a non-natural amino acid, an amino acid analogue, and an amino acid mimetic. 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, the at least one additional amino acid residue is (a single) G.

[0162] In some embodiments, the variant comprises a peptide extension at its C-terminus. The 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).

[0163] In one embodiment, the GLP-1 R agonist having reduced GLP-1 R agonistic activity compared to the GLP-1 R agonistic activity of native GLP-1 (7-36) as defined herein comprises or consists of the following amino acid sequence:

[0164] H-G-E-G-T-F-T-S-D-X 10 -S-X 12 -Q-X 14 -X 15 -E-E-X 18 -V-X 20 -X 21 -F-I-E-W-L-X 27 -X 28 -X 29 -X 30 (SEQ ID NO: 37)

[0165] wherein

[0166] X 10 is any amino acid, for example L or K;

[0167] X 12 is any amino acid, for example K or I;

[0168] X 14 is any amino acid, for example L or M;

[0169] X 15 is any amino acid, for example E or D;

[0170] X 18 is any amino acid, for example A or R;

[0171] X 20 is any amino acid, for example R or Q;

[0172] X 21 is any amino acid, for example L or E;

[0173] X 27 is any amino acid, for example L, E, K or V;

[0174] X 28 is any amino acid, for example A, N or K;

[0175] X 29 is any amino acid, for example T or G;

[0176] X 30 is any amino acid, for example G or R;

[0177] wherein, optionally, the amino acid sequence comprises at its N-terminus at least one additional amino acid residue; and

[0178] wherein, optionally, the amino acid sequence comprises at its C-terminus a peptide extension consisting of up to 12, 11 or 10 amino acid residues.

[0179] In one embodiment, X 27 is L, E or V, for example L. In one embodiment, X 28 is A or K, for example A.

[0180] 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, the at least one additional amino acid residue is (a single) G.

[0181] 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).

[0182] a modification as disclosed herein, such as the introduction of G or X 12 = I, leading to an appropriate reduction of GLP-1 R agonistic activity.

[0183] In one embodiment, the GLP-1 R agonist having reduced GLP-1 R agonistic activity compared to the GLP-1 R agonistic activity of native GLP-1 (7-36) as defined herein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 9, 10, 12, 14, 15, 16, 17, 19 and 20.

[0184] In one embodiment, X is L and X is A. 14 28 In one embodiment, X is L and X is A.

[0185] In one embodiment, the GLP-1 R agonist comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 9, 10, 12, 14, 16, 17, 19 and 20.

[0186] According to the present application, the GLP-1 R agonist can be further modified, e.g. as described above in connection with the FGF21 compound. For example, it can 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.

[0187] The pharmaceutical composition according to the present application 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 action of the active agents of the pharmaceutical composition.

[0188] The term "carrier" refers to an organic or inorganic component of natural or synthetic nature, wherein the active ingredient is combined to facilitate, enhance or achieve the application. According to the present application, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to a subject.

[0189] Carrier substances that can be used 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, naphthalenes and, in particular, biocompatible lactide polymers, lactide / glycolide copolymers or polyoxyethylene / polyoxypropylene copolymers.

[0190] The term "excipient" as used herein is intended to include all substances that can be present in a pharmaceutical composition and are not active ingredients, such as salts, binders (e.g. lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), fillers, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffer substances, flavorings or colorings.

[0191] ​Not all pharmaceutically acceptable salts are useful for the preparation of pharmaceutically acceptable salts and are included in the present application. Such pharmaceutically acceptable salts include, in a non-limiting manner, those 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 can also be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts, or calcium salts. Salts can be added to adjust ionic strength or tonicity.

[0192] Suitable preservatives for pharmaceutical compositions include antioxidants, citric acid, sodium citrate, benzalkonium chloride, chlorbutanol, cysteine, methionine, parabens, thiomersal, phenol, cresol, and mixtures thereof.

[0193] 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).

[0194] The pharmaceutical composition according to the present application is preferably sterile. The pharmaceutical composition can be provided in a unitary dosage form and can be prepared in a manner known per se. The pharmaceutical composition can be, for example, in the form of a solution or a suspension.

[0195] The pharmaceutical composition can also be formulated as a stable lyophilized product to be reconstituted with an appropriate diluent, optionally comprising one or more excipients as defined above.

[0196] The pharmaceutical composition according to the present application can further comprise at least one other active pharmaceutical ingredient.

[0197] 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 in the treatment or prevention of a disorder, e.g. a disease or a condition as defined herein. Exemplary pharmaceutically acceptable salts include (salts of) hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, maleic acid, malic acid, ascorbic acid, citric acid, tartaric acid, pamoic acid, lauric acid, stearic acid, palmitic acid, oleic acid, myristic acid, laurylsulfuric acid, naphthalenesulfonic acid, linoleic acid, linolenic acid, 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 and have the same meaning.

[0198] According to the present application, the active pharmaceutical ingredient is optionally selected from the group consisting of:

[0199] all drugs mentioned in the Rote Liste 2014, such as all antidiabetics mentioned in the Rote Liste 2014 Chapter 12, all weight loss drugs or appetite suppressants mentioned in the Rote Liste 2014 Chapter 06, all lipid-lowering drugs mentioned in the Rote Liste 2014 Chapter 58, all antihypertensive drugs mentioned in the Rote Liste 2014 Chapter 17, all kidney protecting drugs mentioned in the Rote Liste or all diuretics mentioned in the Rote Liste 2014 Chapter 36;

[0200] insulin and insulin derivatives, such as, for example: insulin glargine (e.g. Lantus® ), insulin glargine at a concentration higher than 100 U / mL, such as 270-330 U / mL insulin glargine or 300 U / mL insulin glargine (as disclosed in EP 2387989), insulin glulisine (e.g. Apidra® ), insulin detemir (e.g. Levemir® ), insulin lispro (e.g. Humalog® ), insulin degludec (e.g. Tresiba® Ideg Lira (NN9068)), insulin aspart and insulin aspart formulations (e.g. NovoLog® ), basal insulins and analogues (e.g. LY2605541, LY2963016, NN1436), pegylated insulin lispro (e.g. LY-275585), long-acting insulins (e.g. NN1436, Insumera (PE0139), AB-101, AB-102, Sensulin LLC), intermediate-acting insulins (e.g. Humulin® ), fast-acting and short-acting insulins (e.g. Novolin® PH20 insulin, NN1218, ), premixed insulins, NN1045, insulin plus PE-0139, ACP-002 hydrogel insulin and oral, inhalable, transdermal and buccal or sublingual insulins (e.g. Exubera® insulin, TPM-02 insulin, oral insulin, ORMD-0801, Oshadi oral insulin, NN1953, NN1954, NN1956, ). Those insulin derivatives which are bonded to albumin or another protein via a bifunctional linker are also suitable;

[0201] - glucagon-like peptide 1 (GLP-1), GLP-1 analogs and GLP-1 receptor agonists, such as: GLP-1 (7-37), GLP-1 (7-36) amide, lixisenatide (for example ), exenatide (for example Exendin-4, rExendin-4, Exenatide NexP), exenatide-LAR, liraglutide (for example ), semaglutide, taspoglutide, albiglutide, dulaglutide, Albugon, oxyntomodulin, geniproside, ACP-003, CJC-1131, CJC-1134-PC, GSK-2374697, PB-1023, TTP-054, ranexa- tide (HM-11260C), CM-3, GLP-1 Eligen, 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;

[0202] - dual GLP-1 / GIP agonists (for example RG-7697 (MAR-701), MAR-709, BHM081, BHM089, BHM098); dual GLP-1 / glucagon receptor agonists (for example BHM-034, OAP-189 (PF-05212389, TKS-1225), TT-401 / 402, ZP2929, LAPS-HMOXM25, MOD-6030);

[0203] - dual GLP-1 / gastrin agonists (for example ZP-3022);

[0204] - gastrointestinal peptides, such as peptide YY3-36 (PYY3-36) or analogs thereof and pancreatic polypeptide (PP) or analogs thereof;

[0205] - glucagon receptor agonists or antagonists, glucose-dependent insulinotropic polypeptide (GIP) receptor agonists or antagonists, ghrelin antagonists or reverse agonists, Xenopus laevis peptides and analogs thereof;

[0206] - dipeptidyl peptidase-IV (DPP-4) inhibitors, such as: alogliptin (for example ), linagliptin (e.g. Trajenta® ), saxagliptin (e.g. Ondexila® Komboglyze ), sitagliptin (e.g. Januvia® Janumet ), anagliptin, trelagliptin (e.g. Tanzeum® ), teneligliptin, vildagliptin (e.g. Galvus® ), gemigliptin, omarigliptin, evogliptin, dutogliptin, DA-1229, MK-3102, KM-223, KRP-104, PBL-1427, pioigliptin hydrochloride and Ari-2243;

[0207] - sodium-dependent glucose transporter 2 (SGLT-2) inhibitors, such as: canagliflozin, dapagliflozin, remogliflozin, ipragliflozin, sergliflozin, empagliflozin, ipi'agliptin, tologliflozin, lusogliflozin, eptagliflozin, EGT-0001442, LIK-066, SBM-TFC-039 and KGA-3235 (DSP-3235);

[0208] - dual SGLT-2 and SGLT-1 inhibitors (e.g. LX-4211, LIK066);

[0209] - SGLT-1 inhibitors (e.g. LX-2761, KGA-3235) or SGLT-1 inhibitors in combination with anti-obesity drugs, such as inhibitors of the ileal bile acid transporter (IBAT) (e.g. GSK-1614235 + GSK-2330672);

[0210] - biguanides (e.g. metformin, buformin, phenformin);

[0211] - thiazolidinediones (e.g. pioglitazone, rosiglitazone), glitzone analogues (e.g. lobeglitazone);

[0212] - peroxisome proliferator-activated receptor (PPAR-) (alpha, gamma or alpha / gamma) agonists or modulators (e.g. saroglitazar (e.g. ), GFT-505) or PPAR gamma partial agonists (e.g. Int-131);

[0213] - sulfonylureas (e.g. tolbutamide, glibenclamide, glimepiride, gliclazide) and meglitinides (e.g. nateglinide, repaglinide, mitiglinide);

[0214] - alpha-glucosidase inhibitors (e.g. acarbose, miglitol, voglibose);

[0215] - amylin and amylin analogs (e.g. pramlintide, ) ;

[0216] - 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-Chem1) ;

[0217] - GPR40 agonists (e.g. TUG-424, P-1736, P-11187, JTT-851, GW9508, CNX-011-67, AM-1638, AM-5262) ;

[0218] - GPR120 agonists and GPR142 agonists ;

[0219] - systemic or low-absorption TGR5 (GPBAR1 = G-protein coupled bile acid receptor 1) agonists (e.g. INT-777, XL-475, SB756050) ;

[0220] - diabetes immunotherapeutics, e.g. oral C-C chemokine receptor type 2 (CCR-2) antagonists (e.g. CCX-140, JNJ-41443532), interleukin 1 beta (IL-1 beta) antagonists (e.g. AC-201) or oral monoclonal antibodies (MoA) (e.g. Acipimox, VVP808, PAZ-320, P-1736, PF-05175157, PF-04937319) ;

[0221] - anti-inflammatory agents for the treatment of metabolic syndrome and diabetes, e.g. nuclear factor kappa B inhibitors (e.g. ) ;

[0222] - adenosine monophosphate-activated protein kinase (AMPK) stimulators, e.g. I meglimin (PXL-008), Debio-0930 (MT-63-78), R-118;

[0223] - 11-beta-hydroxysteroid dehydrogenase 1 (11-beta-HSD-1) inhibitors (e.g. LY2523199, BMS770767, RG-4929, BMS816336, AZD-8329, HSD-016, BI-135585) ;

[0224] Activators of glucosamine kinase (e.g., PF-04991532, TTP-399 (GK1-399), GKM-001 (ADV-1002401), ARRY-403 (AMG-151), TAK-329, TMG-123, ZYGK1);

[0225] Inhibitors of diacylglycerol O-acyltransferase (DGAT) (e.g., pradigastat (LCQ-908)), protein tyrosine phosphatase 1 inhibitors (e.g., trodusquemine), glucose-6-phosphatase inhibitors, fructose-1,6-bisphosphatase inhibitors, glycogen phosphorylase inhibitors, phosphoenolpyruvate carboxyl kinase inhibitors, glycogen synthase kinase inhibitors, and pyruvate dehydrogenase kinase inhibitors.

[0226] - Regulators of glucose transporter-4 and somatostatin receptor 3 agonists (e.g., MK-4256);

[0227] One or more lipid-lowering agents are also suitable as combination partners, such as: 3-hydroxy-3-methylglutaryl-CoA-reductase (HMG-CoA-reductase) inhibitors, such as svastatin (e.g.) ), atorvastatin (e.g.) ), rosuvastatin (e.g.) ), pravastatin (e.g.) ), fluvastatin (e.g.) ), pitavastatin (e.g.) lovastatin (e.g.) ), mevastatin (e.g.) ), livastatin, silivastatin fibrates (such as bezafibrate) Delaying agents), cyclopropionic acid (e.g.) ), fenofibrate (e.g.) ), Gefitinib (e.g.) (e.g., etoposide, difibrate, clonicotinamide, crifibrate), clobemide, niacin and its derivatives (e.g., nicotinic acid, including sustained-release formulations of nicotinic acid), niacin receptor 1 agonists (e.g., GSK-256073), PPAR-δ agonists, acetyl-CoA-acetyltransferase (ACAT) inhibitors (e.g., avamidib), cholesterol absorption inhibitors (e.g., ezetimibe, ...) S-556971), bile acid conjugates (e.g., cholestyramine, cholesvelam), ileal bile acid transporter (IBAT) inhibitors (e.g., GSK-2330672, LUM-002), microsomal triglyceride transfer protein (MTP) inhibitors (e.g., lometabine (AEGR-733), SLx-4090, glantapa), proprotein convertase subtilisin / kexin Modulators of PCSK9 type 9 (e.g., alizumab (REGN727 / SAR236553), AMG-145, LGT-209, PF-04950615, MPSK3169A, LY3015014, ALD-306, ALN-PCS, BMS-962476, SPC5001, ISIS-394814, 1B20, LGT-210, 1D05, BMS-PCSK9Rx-2, SX-PCK9, RG7652), and LDL receptor upregulators (e.g., liver-selective thyroid hormone receptor β agonists (e.g., illotirol (KB-2115), MB07811, subitilol (QRX)). -431), VIA-3196, ZYT1), and HDL-raising compounds, such as: cholesterol ester transfer protein (CETP) inhibitors (e.g., acetrapip (MK0859), dacetrapip, estrapip, 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., dareradil, ... Varenafil (Varenafil, Relapaldine), ApoA-1 enhancers (e.g., RVX-208, CER-001, MDCO-216, CSL-112), cholesterol synthesis inhibitors (e.g., ETC-1002), lipid metabolism regulators (e.g., BMS-823778, TAP-301, DRL-21994, DRL-21995), and ω-3 fatty acids and their derivatives (e.g., ethyl eicosapentaenoate (AMR101)). AKR-063, NKPL-66, PRC-4016, CAT-2003);

[0228] - Bromocriptine (e.g.) ), phentermine and phentermine formulations or combinations (e.g., Adipex-P, phentermine, ... ), benzyphenamine (e.g.) ), amphetamine (e.g.) ), benzoyltracin (e.g.) ), bupropion and combinations (e.g. Wellbutrin ), sibutramine (e.g. ), topiramate (e.g. ), zonisamide (e.g. ), tesofensine, opioid antagonists (such as naltrexone (e.g. Naltrexone + bupropion)), 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. velneperit, S-234462), NPY4 agonists (e.g. PP-1420), beta-3-adrenergic receptor agonists, leptin or leptin mimetics, serotonin 2c (5HT2c) receptor agonists (e.g. lorcaserin, ), pramlintide / metreleptin, lipase inhibitors (such as orlistat (e.g. ), sibutramine (e.g. )), 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 (such as fluoxetine (e.g. ), duloxetine (e.g. )), dual or triple monoamine uptake inhibitors (dopamine, norepinephrine and serotonin reuptake) (such as sertraline (e.g. ), tesofensine), methionine aminopeptidase 2 (MetAP2) inhibitors (e.g. beloranib) and antisense oligonucleotides directed against fibroblast growth factor receptor 4 (FGFR4) (e.g. ISIS-FGFR4Rx) or somatostatin-targeting peptides-1 (e.g. );

[0229] - nitric oxide donors, AT1 antagonists or angiotensin II (AT2) receptor antagonists (such as telmisartan (e.g. ), candesartan (e.g. ), valsartan (e.g. ), losartan (e.g. ), eprosartan (e.g. ), irbesartan (e.g. ), olmesartan (e.g. ), tasosartan, azilsartan (e.g. )) dual angiotensin receptor blockers (dual ARBs), angiotensin converting enzyme (ACE) inhibitors, ACE-2 activators, renin inhibitors, prorenin 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 mineralocorticoid / CCB, centrally acting antihypertensives, neutral endopeptidase inhibitors, aminopeptidase-A inhibitors, vasopeptidase inhibitors, dual vasopeptidase inhibitors (e.g., renin-angiotensin-aldosterone system (RAAS) inhibitors or renin-angiotensin-aldosterone system (RAAS) inhibitors), dual AT1 / ETA antagonists), advanced glycation end product (AGE) breakers, recombinant renin, blood pressure vaccines (e.g., anti-RAAS (renin-angiotensin-aldosterone system) vaccines, AT1-vaccines or AT2-vaccines), drugs based on hypertension pharmacogenomics (e.g., modulators of genetic polymorphisms with anti-hypertensive response, platelet aggregation inhibitors and others), or combinations thereof.

[0230] The term "fusion molecule" generally refers to a molecule that results from the joining (particularly covalent joining) of two or more different molecules (e.g., proteins and / or peptides), resulting in a single molecule with 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 technology) or by chemical and / or enzymatic conjugation. Two or more different molecules can also be joined by a suitable linker molecule, e.g., a peptide linker or a non-peptide polymer such as polyethylene glycol (PEG).

[0231] In general, 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 acids are selected from glycine and serine, i.e. the peptide linker consists only of 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 can further comprise one or more specific protease cleavage sites.

[0232] In one embodiment, the fusion molecule is a fusion protein. In general, the fusion protein of the present application comprises a GLP-1 R agonist, a linker molecule, and a FGF21 compound. In one embodiment, the fusion protein further comprises an Fc region / domain of an immunoglobulin (e.g. IgGl, IgG2, IgG3, IgG4, or IgD) or a variant thereof. In one embodiment, the variant of the 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 mutations are selected from the group consisting of amino acid substitutions and deletions, e.g. N- or C-terminal deletions. In one embodiment, the variant of the Fc region / domain of IgG4 (also referred to as "IgG4 Fc variant") comprises or consists of the amino acid sequence of SEQ ID NO: 21.

[0233] In one embodiment, the fusion protein further comprises an Fc region / domain of an immunoglobulin (e.g. IgGl, IgG2, IgG3, IgG4, or IgD) or a variant thereof. In one embodiment, the variant of the 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 mutations are selected from the group consisting of amino acid substitutions and deletions, e.g. N- or C-terminal deletions. In one embodiment, the variant of the Fc region / domain of IgG4 (also referred to as "IgG4 Fc variant") comprises or consists of the amino acid sequence of SEQ ID NO: 21.

[0234] In one embodiment, the variant of the Fc region / domain is a hybrid Fc region / domain. Such hybrid Fc regions / domains are described, e.g., in WO 2016 / 114633 Al, WO 2017 / 074117 Al, WO 2017 / 074123 Al, and WO 2018 / 088838 Al, 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., IgGl, IgG2, IgG3, IgG4, or IgD). In one embodiment, the hybrid Fc region / domain comprises partial Fc regions / domains of IgG4 and IgD (also referred to as "IgG4 / IgD hybrid Fc region / domain"), preferably human IgG4 and IgD. In one embodiment, the hybrid Fc region / domain comprises the partial hinge sequence and CH2 of an IgD Fc region / domain, and the 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 NOs: 65, 66, 67, 68, 69, and 70.

[0235] In one embodiment, the FGF21 compound and the GLP-1 R agonist are linked via an Fc region / domain of an immunoglobulin or a variant thereof. In one embodiment, the FGF21 compound and the GLP-1 R agonist are linked via a linker molecule comprising a structure selected from the group consisting of L - Fc, Fc - L, L1 - Fc - L2, and Fc, wherein L, L1, and L2 are peptide linkers (L1 and L2 are the same or different) as defined herein, and Fc is an Fc region / domain of an immunoglobulin or a variant thereof.

[0236] In one embodiment, the fusion protein comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 26, 28, 30, 31, 32, 33, 35, and 36.

[0237] Further features of the fusion proteins according to the application are described, e.g., in WO 2014 / 037373 Al and WO 2017 / 093465 Al, which are incorporated herein by reference.

[0238] According to the application, a "nucleic acid molecule" is preferably a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). According to the application, the nucleic acid molecule can be in the form of a single-stranded or double-stranded linear or covalently closed molecule to form a circle.

[0239] The term "DNA" refers to a molecule comprising deoxyribonucleotide residues and preferably consisting entirely or essentially of deoxyribonucleotide residues. A "deoxyribonucleotide" refers to a nucleotide lacking a hydroxyl group at the 2'-position of the beta-D-ribofuranosyl group. The term "DNA" includes isolated DNA, such as partially or completely purified DNA, substantially pure DNA, synthetic DNA, and recombinantly produced DNA, and includes modified DNA that differs from naturally occurring DNA by addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations can include the addition of non-nucleotide material, such as to one or both ends of a DNA or to an internal position, e.g., at one or more nucleotides of the DNA. Nucleotides in a DNA molecule can also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides. These altered DNAs can be referred to as analogs or analogs of naturally occurring DNA. The term "naturally occurring," when used in connection with nucleotides, refers to the bases adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U).

[0240] The term "RNA" refers to a molecule comprising ribonucleotide residues and preferably consisting entirely or essentially of ribonucleotide residues. A "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2'-position of the beta-D-ribofuranosyl group. The term "RNA" includes isolated RNA, such as partially or completely purified RNA, substantially pure RNA, synthetic RNA, and recombinantly produced RNA, and includes modified RNA that differs from naturally occurring RNA by addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations can include the addition of non-nucleotide material, such as to one or both ends of a RNA or to an internal position, e.g., at one or more nucleotides of the RNA. Nucleotides in a RNA molecule can also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally occurring RNA. According to the present application, "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 stability (e.g., half-life) of the RNA. Such modifications are known to those of skill in the art and include, e.g., a 5'-cap or 5' cap analog.

[0241] A nucleic acid molecule according to the application can be contained in / comprised in a vector. The term "vector" as used herein encompasses all vectors known to the skilled person, including plasmid vectors, cosmid vectors, phage vectors (such as lambda phage), viral vectors (such as adenoviral or baculoviral vectors) or artificial chromosome vectors (such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC) or P1 artificial chromosomes (PAC)). The vectors include expression as well as cloning vectors. Expression vectors comprise plasmids as well as viral vectors and typically contain the desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g. bacteria, yeast, plants, insects or mammals) or in an in vitro expression system. Cloning vectors are typically used for engineering and amplification of a certain desired DNA fragment and can lack functional sequences required for expression of the desired DNA fragment.

[0242] Alternatively, a nucleic acid molecule according to the application can be integrated into a genome (e.g. of a host cell). Means and methods for integrating a particular nucleic acid molecule into a genome are known to the person skilled in the art.

[0243] The term "cell" or "host cell" preferably relates to an intact cell, i.e. a cell having an intact membrane and which has not released its normal intracellular components, such as enzymes, organelles, or genetic material. The intact cell is preferably a viable cell, i.e. a living cell which is able to carry out its normal metabolic functions. Preferably, according to the present application, the term relates to any cell which 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, Proteus, and Pseudomonas, as well as Gram-positive bacterial strains, such as strains of Bacillus, Streptomyces, Staphylococcus, and Lactococcus. Suitable fungal cells include cells from species of Trichoderma, Neurospora, and Aspergillus. Suitable yeast cells include cells from species of Saccharomyces (e.g. Saccharomyces cerevisiae), Schizosaccharomyces (e.g. Schizosaccharomyces pombe), Pichia (e.g. Pichia pastoris and Pichia methanolica), and 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 used in the art for expression of heterologous proteins can also be used. Mammalian cells are particularly preferred for adoptive transfer, such as cells from humans, mice, hamsters, pigs, goats, and primates. The cells can be derived from a number 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 which display antigens in the context of major histocompatibility complexes on their surface. T cells can recognize this complex using their T cell receptors (TCRs).A "cell" or "host cell" can be an isolated cell or part of a tissue or organism, in particular a "non-human organism".

[0244] 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.

[0245] The term "kit of parts" (short: 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 can be filled with one or more of the above-mentioned agents (reagents). Other containers can be included in the kit, which contain, for example, diluents, buffers and other reagents. The data carrier can be a non-electronic data carrier, for example a graphic data carrier, such as a leaflet, a 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 can allow access to a database, for example an internet database, a centralized database or a distributed database. The data carrier can contain instructions for using the agents of the application, such as combinations, pharmaceutical compositions and fusion molecules as described herein and related agents, such as nucleic acid molecules and host cells.

[0246] The agents and compositions described herein can be administered by any conventional route, for example, orally, pulmonarily, by inhalation or parenterally, including by injection or infusion. In one embodiment, parenteral administration is used, for example, intravenously, intra-arterially, subcutaneously, intradermally or intramuscularly. The agents and compositions described herein can also be administered by sustained release administration.

[0247] Pharmaceutical compositions suitable for parenteral administration contain or consist of a sterile aqueous or non-aqueous preparation of the active compound, which is preferably isotonic with the blood of the recipient. Examples of suitable 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 non-volatile oils can be used as solutions or suspensions in the medium.

[0248] The agents and compositions described herein are generally administered in a therapeutically effective amount. A "therapeutically effective amount" means an amount which alone, or together with further doses, achieves the desired therapeutic response or desired therapeutic effect, preferably without causing unacceptable side effects. In the context of treating a particular disease or particular condition, the desired response preferably relates to inhibition of the disease progression. This includes slowing down the disease progression, and in particular interrupting or reversing the disease progression. The desired response in the treatment of a disease or condition can also be a delay in the onset of the disease or the condition, or prevention of the onset of the disease or the condition. The effective amount of an agent or composition described herein will depend on the condition to be treated, the disease severity, the individual parameters of the subject, including age, physiological condition, size, and weight, the duration of treatment, the types of concomitant treatment, if any, the specific route of administration, and similar factors. Accordingly, the dosage of an agent described herein can depend on a plurality of such parameters. In case the initial dosage elicits an insufficient response in the subject, higher dosages (or effectively higher dosages achieved by different, more local routes of administration) can be used.

[0249] According to the present application, the term "disease or disorder" refers to any pathological or unhealthy state, in particular obesity, overweight, metabolic syndrome, diabetes, diabetic retinopathy, hyperglycemia, dyslipidemia, non-alcoholic steatohepatitis (NASH) and / or atherosclerosis.

[0250] The term "obesity" refers to a medical condition in which an excess amount of body fat has accumulated to the extent that it can have a negative impact on health. For human (adult) subjects, obesity can be defined as a body mass index (BMI) greater than or equal to 30 kg / m 2 (BMI > 30 kg / m 2 ).

[0251] The term "overweight" refers to a medical condition in which the amount of body fat is higher than what is optimally healthy. For human (adult) subjects, obesity can be defined as a body mass index (BMI) greater than or equal to 25 kg / m 2 (e.g., 25 kg / m 2 < 30 kg / m 2 ).

[0252] BMI is a simple index of weight-for-height that is commonly used to classify overweight and obesity in adults. It is defined as a person's weight in kilograms divided by the square of his height in meters (kg / m 2 ).

[0253] "Metabolic syndrome" can be defined as the clustering of at least three of the following medical conditions: abdominal (central type) obesity (e.g., defined as waist circumference > 94 cm for European men, and > 80 cm for European women, with ethnically specific values for other populations), elevated blood pressure (e.g., 130 / 85 mm Hg or higher), elevated fasting plasma glucose (e.g., at least 100 mg / dL), high serum triglycerides (e.g., at least 150 mg / dL), and low high-density lipoprotein (HDL) levels (e.g., less than 40 mg / dL for men, and less than 50 mg / dL for women).

[0254] "Diabetes mellitus" (also simply "diabetes") refers to a group of metabolic diseases characterized by high blood sugar levels resulting from defects in insulin production, insulin action, or both. In one embodiment, the diabetes is selected from the group consisting of type 1 diabetes, type 2 diabetes, gestational diabetes, Latent Autoimmune Diabetes in Adults (LADA), Maturity Onset Diabetes of the Young (MODY), and other types of diabetes due to specific genetic conditions, drugs, malnutrition, infections, and other diseases.

[0255] The current WHO diagnostic criteria for diabetes are as follows: fasting plasma glucose > 7.0 mmol / l (126 mg / dL) or 2-h plasma glucose > 11.1 mmol / l (200 mg / dL).

[0256] "Type 1 diabetes" (also known as "insulin-dependent diabetes mellitus" (IDDM) or "juvenile diabetes") is a condition characterized by high blood sugar levels resulting from a complete lack of insulin. This occurs when the body's immune system attacks the beta cells in the pancreas that produce insulin and destroys them. The pancreas then produces little or no insulin. Pancreas removal or disease can also cause loss of insulin-producing beta cells. Type 1 diabetes accounts for 5-10% of diabetes cases.

[0257] "Type 2 diabetes" (also known as "non-insulin-dependent diabetes mellitus" (NIDDM) or "maturity onset diabetes") is a condition characterized by too much glucose in the blood, despite the availability of insulin, because of insufficient glucose clearance (insulin action). Type 2 diabetes can account for about 90% to 95% of all diagnosed diabetes cases.

[0258] "Gestational diabetes" is a condition in which a woman who has not been previously diagnosed with diabetes exhibits high blood sugar levels during pregnancy, particularly during the last three months of pregnancy. Gestational diabetes affects 3-10% of pregnancies, depending on the population studied.

[0259] "Latent autoimmune diabetes in adults (LADA)" (also known as "slow- onset type 1 diabetes") is a form of type 1 diabetes that occurs in adults, usually with a slower onset process.

[0260] "Adult-onset diabetes mellitus of the young (MODY)" refers to an inherited form of diabetes caused by a mutation in a dominant autosomal gene that disrupts insulin production.

[0261] "Diabetic retinopathy" is an eye disease induced by metabolic disorder occurring in a diabetic patient and results in progressive loss of vision.

[0262] The term "hyperglycemia" refers to an excess of sugar (glucose) in the blood.

[0263] The term "dyslipidemia" refers to a disorder of lipoprotein metabolism, including overproduction ("hyperlipidemia") or deficiency ("hypolipidemia") of lipoproteins. Dyslipidemia can manifest as elevated concentrations of total cholesterol, low-density lipoprotein (LDL) cholesterol, and / or triglycerides in the blood, and / or decreased concentrations of high-density lipoprotein (HDL) cholesterol.

[0264] Nonalcoholic steatohepatitis (NASH) is a liver disease characterized by accumulation of fat (lipid droplets) and degradation and inflammation of liver cells. Once it occurs, the disease is accompanied by a high risk of cirrhosis, a condition that changes liver function and can progress to liver insufficiency. Thereafter, NASH usually progresses to liver cancer.

[0265] "Atherosclerosis" is a vascular disease characterized by irregular distribution of lipid deposits called plaques in the intima of large and medium-sized arteries, which can cause stenosis of the arterial lumen and progress to fibrosis and calcification. Lesions are usually local and progress slowly and indirectly. Sometimes plaque rupture occurs, causing obstruction of blood flow, thus causing death of tissues distal to the obstruction. Limited blood flow explains most of the clinical manifestations, which vary with the distribution and severity of the obstruction.

[0266] The term "drug" as used herein refers to a substance / composition used in therapy, i.e. for the treatment of a disease or disorder.

[0267] "Treatment" means administering a compound or composition or a combination of compounds or compositions to the subject to prevent or eliminate the disease or disorder; to arrest or slow the progression of the disease or disorder in the subject; to inhibit or slow the progression of a new disease or disorder in the subject; to reduce the frequency or severity of symptoms and / or relapses in a subject who currently has or who previously has had the disease or disorder; and / or to prolong (i.e., increase) the life of the subject.

[0268] In particular, the term "treating / treatment of a disease or disorder" includes curing the disease or disorder or its symptoms, shortening its duration, ameliorating, preventing, slowing or inhibiting the progression or worsening of the disease or disorder or its symptoms, or preventing or delaying the onset of the disease or disorder or its symptoms.

[0269] According to the present application, the term "subject" means a subject for treatment, in particular a diseased subject (also referred to as "patient"), including a human, a non-human primate or other animal, in particular a mammal, such as a bovine, equine, porcine, ovine, caprine, canine, feline, lagomorph, or rodent, e.g. a mouse, rat, guinea pig, and hamster. In one embodiment, the subject / patient is a human.

[0270] The present application will now be further described with reference to the following examples, which are intended to illustrate, rather than limit, the scope of the present application. Example

[0271] Example 1 : Determination of optimal GLP-1 RA / FGF21 activity ratio by systems pharmacology modeling

[0272] The optimal GLP-1 RA / FGF21 potency ratio was identified using improved mechanistic understanding of the pharmacological effects of GLP-1 RA / FGF21 fusion proteins in humans. A mechanistic systems pharmacology 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):216-220; Deacon et al. (2011) Rev Diabet Stud 8(3):293-306; Kim et al. (2008) Pharmacol Rev 60(4):470-512; Kharitonenkov et al. (2014) Mol Metab 3(3):221-229).

[0273] The model represents relevant pathways of 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 the therapeutic response to simulated drug treatments (e.g., GLP-1 RA / FGF21 fusion proteins, liraglutide, FGF21 analog LY2405319). See Kharitonenkov et al. (2013) PLoS ONE 8(3):e58575 for LY2405319.

[0274] The model covers key aspects of glucose homeostasis controlled by the hormones insulin, glucagon, and incretins (GLP-1, GIP). The main model endpoint with respect to glycemic control is HbAlc. HbAlc is a clinical endpoint commonly used to estimate the average blood glucose concentration over the previous months. HbAlc is estimated in the model using a linear relationship between average blood glucose and HbAlc as reported in Nathan et al. (2008) Diabetes Care 31(8): 1473-1478.

[0275] The model incorporates triglyceride and fatty acid metabolism at a level suitable for manipulating basal lipid metabolism, including a representation of cholesterol. HDL and non-HDL (i.e., LDL plus VLDL cholesterol) are circulating lipoproteins. The representation of lipid metabolism allows for simulation of the effects of FGF21 compounds on lipids and interaction with statins. FGF21 compounds have a significant effect on lipid concentrations (Gaich et al. (2013) Cell Metab 18(3):333-340; Fisher et al. (2011) Endocrinology 152(8):2996-3004).

[0276] Weight loss or gain in the model is measured as a change in body fat mass. There is a direct relationship between fat mass and body weight (Broyles et al. (2011) Br J Nutr 105(8): 1272-1276). Food intake is based on basal and resting metabolic rates (Amirkalali et al. (2008) Indian J Med Sci 62(7):283-290). Body fat mass remains constant when energy expenditure equals caloric intake. The formulation of Gobel et al. (2014) Obesity (Silver Spring) 22(10):2105-2108 is used in the model to implement the therapeutic effect on food intake.

[0277] Food is considered as carbohydrates (glucose equivalents), fats (fatty acid equivalents), and proteins (amino acid equivalents). All nutrients enter the stomach, pass through a delay junction, and then pass through a three-compartment gastrointestinal tract. The gastrointestinal tract design is based on work by Bastianelli et al. (1996) J Anim Sci 74(8): 1873-1887; Worthington (1997) Med Inform (Lond) 22(1):35-45) and food digestion and absorption.

[0278] Nutrients, hormones, drugs, and disease conditions can cause a delay in gastric emptying. In healthy conditions, the rate of gastric emptying depends 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): 769-772; Fouillet et al. (2009) Am J Physiol Regul Integr Comp Physiol 297(6): R1691-1705). Individuals with diabetes mellitus often have a delayed glucose absorption, as seen by oral glucose tolerance tests or meal tests (Bharucha et al. (2009) Clin Endocrinol (Oxf) 70(3): 415-420; Chang et al. (2012) Diabetes Care 35(12): 2594-2596). This delay is attributed to a slowing of gastric emptying. The delay between the stomach and the small intestine was increased in the model to account for the delayed gastric emptying in diabetic subjects. Drugs and hormones (GLP-1) can affect the vagal tone of the stomach, which reduces mechanical mixing and / or peristalsis, and this also slows gastric emptying (Jelsing et al. (2012) Diabetes Obes Metab 14(6): 531-538; Little et al. (2006) J Clin Endocrinol Metab 91(5): 1916-1923; Nauck et al. (2011) Diabetes 60(5): 1561-1565; van Can et al. (2013) Int J Obes (Lond) 38(6): 784-93).

[0279] One objective of this study was to prevent GLP-1 related adverse effects, i.e. nausea and vomiting (Lean et al. (2014) Int J Obes (Lond) 38(5): 689-697). Gastric emptying measurements provide an estimate of adverse events like nausea and vomiting, which are associated with low gastric emptying rates. Therefore, a marker of gastric adverse events in the model is the sum of gastric emptying rates.

[0280] Different virtual patients representing healthy and different disease stages of type 2 diabetes patients were implemented in the model platform. Moreover, the virtual patients cover different degrees of obesity and dyslipidemia. The virtual patients represent the variability in disease severity and pathophysiology as well as the variability in phenotypes observed in the clinic.

[0281] Several therapies are implemented in the model, i.e., GLP-1 RA / FGF21 fusion protein, liraglutide, FGF21 analog LY2405319, metformin, atorvastatin, sitagliptin, human insulin. These therapies can be turned on or off in the simulation. The virtual patient is assumed to have a metformin and atorvastatin background when GLP-1 RA / FGF21 fusion protein is administered.

[0282] A virtual GLP-1 RA / FGF21 fusion protein is implemented in the model. The fusion protein contains both FGF21 and GLP-1 agonist activity, and it 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 liraglutide (Geiser et al. (2016) Clin Pharmacokinet 55(5):625-34).

[0283] The model was validated by comparison with multiple datasets. The simulation results were consistent in nature with relevant data and knowledge, e.g., Hellerstein et al. (1997) J Clin Invest 100(5): 1305-1319; Muscelli et al. (2008) Diabetes 57(5): 1340-1348. The model matched relevant quantitative test data, e.g., Aschner et al. (2006) Diabetes Care 29(12): 2632-2637; Dalla Man, Caumo et al. (2005) Am J Physiol Endocrinol Metab 289(5): E909-914; Dalla Man et al. (2005) Diabetes 54(11): 3265-3273; Fiallo-Scharer (2005) J Clin Endocrinol Metab 90(6): 3387-3391; Hahn et al. (2011) Theor 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): 876-886 and J Clin Endocrinol Metab 91(11): 4612-4619; 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): 415-419; Sikaris (2009) J Diabetes Sci Technol 3(3): 429-438; Vicini and Cobelli (2001) Am J Physiol Endocrinol Metab 280(1): E179-186; Vollmer et al. (2008) Diabetes 57(3): 678-687.

[0284] Existing therapies were implemented in the model for direct comparison, including FGF21 analogs and GLP-1 receptor agonists. The effects of FGF21 analogs were validated with clinical data, e.g., Gaich et al. 2013. The GLP-1 receptor agonist liraglutide is a direct competitor for the target, and its implementation was compared to a variety of clinical data, e.g., Jacobsen et al. (2009) Br J Clin Pharmacol 68(6):898-905; Elbrond et al. (2002) Diabetes Care 25(8): 1398-1404; Chang et al. (2003) Diabetes 52(7): 1786-1791; Kolterman et al. (2003) J Clin Endocrinol Metab 88(7):3082-3089; Degn et al. (2004) Diabetes 53(5): 1187-1194; Kolterman et al. (2005) Am J Health Syst Pharm 62(2): 173-181; Vilsboll et al. (2008) Diabet Med 25(2): 152-156; Buse et al. (2009) Lancet 374(9683):39-47; Jelsing 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 Pract 65(4):397-407; Nauck et al. (2013) Diabetes Obes Metab 15(3): 204-212; Flint et al. (2011) Adv Ther 28(3):213-226; Kapitza et al. (2011) Adv Ther 28(8):650-660; Astrup et al. (2012) Int J Obes (Lond) 36(6):843-854.

[0285] The model platform allows to simulate the beneficial and adverse effects of virtual GLP-1 RA / FGF21 fusion proteins at varying activity ratios. The effective FGF21 -mediated EC50 value is set to a constant value derived from Gaich et al. (2013) Cell Metab 18(3):333-340. The effective GLP-1 -mediated EC50 value is reduced by a factor of 2 to 600 increments of 1 relative to endogenous GLP-1 (Table 1).

[0286] Table 1: GLP-1 R agonist / FGF21 fusion protein pharmacodynamics (EC50 values).

[0287]

[0288] * relative to endogenous GLP-1

[0289] ** The FGF21 EC50 value is set according to Gaich et al. (2013) Cell Metab 18(3):333-340 assuming a half-maximal effect.

[0290] For each virtual fusion protein, exposure-response relationships are simulated for the relevant pharmacodynamic endpoints (i.e. HbA1 c, triglycerides, fatty acids, non-HDL cholesterol and fat mass). Gastric emptying rate is used as a marker for GLP-1 -mediated adverse events. A 52-week treatment of a virtual patient with general obesity, dyslipidemia type 2 diabetes with a GLP-1 RA / FGF21 fusion protein is simulated for a wide dose range. After 52 weeks of treatment, steady state is expected to be reached for all relevant pharmacodynamic endpoints. For each endpoint, the half-maximal effective concentration (EC50 value) is determined from the exposure-response curve. Especially for the main GLP-1 -mediated endpoints HbA1 c and gastric emptying rate, the EC50 value varies with the activity ratio. Figure 1 EC50 values are plotted as a function of the GLP-1 attenuation factor. An increasing GLP-1 attenuation factor indicates a decreasing GLP-1 R agonistic activity.

[0291] This procedure allows to identify relevant activity ratios for which the adverse effects act at higher plasma levels compared to the pharmacodynamic effects. For GLP-1 attenuation factors greater than 9, the EC50 for GLP-1 -mediated gastrointestinal adverse effects is greater than the EC50 for the pharmacodynamic effects. Thus, the gastric adverse effects act at higher plasma levels than the pharmacodynamic effects. It is possible to find a dose that provides all desired pharmacodynamic effects while avoiding GLP-1 -mediated gastrointestinal adverse effects. Therefore, activity ratios below 1 : 10 are not relevant.

[0292] The maximum EC50 value for gastric emptying rate is reached at a decay factor of 531. The maximum distance between adverse effects and mean pharmacodynamic effects is reached at a decay factor of 482 Figure 2 ). Therefore, activity ratios beyond 1 :482 are not relevant. The maximum distance between the maximum of pharmacodynamics (HbAlc) and adverse effects is 319. The maximum distance between the maximum of pharmacodynamics (HbAlc) normalized by the GLP-1 mediated effect (HbAlc) and adverse effects is 121 by extending FGF21-(lipid).

[0293] GLP-1 RA / FGF21 fusion proteins with potency ratios of 1 :10 to 1 :482 are predicted to be most beneficial in improving lipid profile, body weight and glucose metabolism and, based on the gastric emptying response, are likely not to cause severe adverse events. Lower potency ratios can not be good candidates based on their predicted strong inhibition of gastric emptying and the likelihood of adverse events. Higher potency ratios can not be potent enough and therefore not competitive.

[0294] Furthermore, a 12-week treatment of a virtual patient with general obesity, dyslipidemia type 2 diabetes with a GLP-1 RA / FGF21 fusion protein is simulated over a wide dose range, because the primary GLP-1 mediated parameter HbAlc reaches clinical steady state after 12 weeks of treatment.

[0295] Figure 3 The EC50 values depending on the GLP-1 decay factor of the 12-week simulation are depicted. For GLP-1 decay factors larger than 18, the EC50 of the GLP-1 mediated adverse gastrointestinal effects is larger than the EC50 of the pharmacodynamic effects. The maximum EC50 value for gastric emptying rate is reached at a decay factor of 501. The maximum distance between adverse effects and mean pharmacodynamic effects is reached at a decay factor of 469 Figure 4 ). The maximum distance between the maximum of pharmacodynamics (HbAlc) and adverse effects is 313. The maximum distance between the maximum of pharmacodynamics (HbAlc) normalized by the GLP-1 mediated effect (HbAlc) and adverse effects is 123 by extending FGF21-(lipid).

[0296] The system pharmacology approach is used to investigate the efficacy and potential of GLP-1 RA / FGF21 fusion proteins with different activity ratios on adverse events. Fusion proteins with a presumably calculated ideal potency ratio are identified, which are predicted to be beneficial in improving lipid profile, body weight and glycemic control, while, based on the gastric emptying response, are likely not to cause severe adverse GLP-1 RA related effects. Therefore, compounds with the selected model informed potency ratio are predicted to provide a good efficacy to risk profile.

[0297] Example 2: Expression of GLP1RA-FGF21 fusion proteins in HEK293 cells

[0298] The FGF21 protein of SEQ ID NO: 2 was directly fused 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 was inserted, the GLP1RA was fused N-terminally to the linker sequence and the FGF21 was fused C-terminally to the linker sequence. The DNA sequence of the GLP1RA-FGF21 fusion protein was fused N-terminally to an IL2 signal sequence, followed by a histidine-rich sequence (His-tag) and a Tev cleavage site. The GLP1RA-FGF21 fusion proteins were produced by transient transfection of HEK293 cells. The signal sequence was 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 could be cleaved by addition of Tev protease. For construct screening purposes, the His-tag was cleaved by direct addition of Tev protease to the incubation medium used for GLP1RA activity assay. The incubation time before starting the assay was 10-60 minutes to ensure complete cleavage of the His-tag. Constructs with GLP1RA activity in the desired range were produced on a larger scale. The GLP1RA-Fc-FGF21 fusion proteins were 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 His-tag cleavage, the cleavage reaction solution was passed a second time over the IMAC column (cOmplete His-tag Purification Resin (Roche)) to collect the flow-through fraction (without his-tag). 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°C until further use. TM His-tag Purification Resin (Roche)) to collect the flow-through fraction (without his-tag). 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°C until further use.

[0299] Example 3: In vitro cell assay for human FGF21 receptor efficacy in CHO cells (intracellular Western)

[0300] The in vitro cellular efficacy of mature human FGF21 (SEQ ID NO:2) or FGF21 variants was measured using an in-cell Western blotting (ICW) assay, which is specific and highly sensitive. The ICW assay is an immunocytochemical assay, typically performed in microplate form. FGF21 receptor autophosphorylation was measured using CHOFlp-ln cells (Invitrogen, Darmstadt, Germany) stably expressing human FGFR1c (=FGF receptor 1c isotype) and human β-Klotho (KLB) cells (Aguilar HN et al. (2010) PLoS ONE 5(4):e9965). To determine the receptor autophosphorylation level or downstream activation of MAP kinase ERK1 / 2, 2 x 10⁻⁶ cells were used. 4 Cells were seeded per well into 96-well plates and allowed to grow for 48 hours. Cells were starved of serum for 3–4 hours with Ham's F-12 nutrient mix (Gibco, Darmstadt, Germany) containing GlutaMAX. Cells were then treated with increasing concentrations of mature human FGF21 (SEQ ID NO: 2) at 37°C for 5 minutes. After incubation, the medium was discarded, and cells were fixed in 3.7% freshly prepared paraformaldehyde for 20 minutes. Cells were infiltrated with 0.1% Triton-X-100 in PBS for 20 minutes. Blocking was performed at room temperature for 2 hours with Odyssey blocking buffer (LICOR, Bad Homburg, Germany). Primary antibodies (anti-pFGFR Tyr653 / 654 (New England Biolabs, Frankfurt, Germany) or anti-pERK phosphate p44 / 42 MAP kinase Thr202 / Tyr204 (cell signaling)) were added and incubated overnight at 4°C. After incubation with the primary antibody, cells were washed with PBS containing 0.1% Tween 20. Secondary antibody (mouse 800CW antibody, LICOR, Bad Homburg, Germany) was added and incubated at room temperature for 1 hour. Subsequently, cells were washed again with PBS containing 0.1% Tween 20, and infrared dye signals were quantified using an Odyssey imager (LICOR, Bad Homburg, Germany). Results were normalized by quantifying DNA using TO-PRO3 dye (Invitrogen, Karlsruhe, Germany). Data were obtained in arbitrary units (AU), and EC50 values ​​were obtained from the dose-response curve and summarized in Table 2. Figure 5 Results of ICW from CHO cells overexpressing human FGFR1c plus KLB are shown.

[0301] Table 2: EC50 values of mature human FGF21 (SEQ ID NO: 2) measured by ICW pFGFR or ICW pERK in CHO cells overexpressing human FGFRlc and KLB.

[0302]

[0303] Example 4: In vitro cell assay for human GLP-1 receptor efficacy

[0304] Agonistic effects of compounds on the human glucagon-like peptide-1 (GLP-1) receptor were determined by functional assay measuring the cAMP response in a HEK-293 cell line stably expressing the human GLP-1 receptor.

[0305] The cAMP content of the cells was determined using a kit from Cisbio Corp. (cat. no. 62AM4PEC) based on HTRF (homogeneous time-resolved fluorescence). For preparation, cells were distributed into T175 culture flasks and grown overnight in culture medium (DMEM / 10% FBS) to near confluency. The culture medium was then removed and the cells were washed with PBS lacking calcium and magnesium, followed by protease treatment with Accutase (Sigma-Aldrich cat. no. A6964). The detached cells were washed and resuspended in assay buffer (lx HBSS; 20 mM HEPES, 0.1% BSA, 2 mM IBMX) and the cell density was determined. They were then diluted to 4x10 5 cells / mL and 25 μL aliquots were distributed into the wells of a 96-well plate. For measurement, 25 μL of test compound in assay buffer were added to the wells, followed by incubation for 30 min at room temperature. After addition of the HTRF reagents diluted in lysis buffer (kit component), the plate was incubated for 1 h before the fluorescence ratio at 665 / 620 nm was measured. The in vitro potency of agonists was quantified by determining the concentration (EC 50 ) causing 50% activation of the maximal response. The results are summarized in Table 3.

[0306] Table 3: EC50 values of GLP-1 receptor agonists (SEQ ID NO: 7 and 24-36) measured by detection of the cAMP response in a HEK-293 cell line stably expressing the human GLP-1 receptor. The corresponding ratio of GLP-1 R agonistic activity to native GLP-1 (7-36) is also shown (native GLP-1 (7-36) / GLP-1 R agonist). Ratio X means that the GLP-1 R agonistic activity is reduced to 1 / X compared to the GLP-1 R agonistic activity of native GLP-1 (7-36).

[0307]

[0308] Table 4: Selected ratios of GLP-1 R agonistic activity (native GLP-1 (7-36) / GLP-1 R agonist) and corresponding calculated EC50 values (based on results obtained above). Ratio X means that the GLP-1 R agonistic activity is reduced to 1 / X compared to the GLP-1 R agonistic activity of native GLP-1 (7-36).

[0309]

[0310] Example 5: Synthesis of the peptide compounds

[0311] The fusion proteins were produced by recombinant methods (see Example 2), whereas the isolated peptide GLP-1 R agonists were chemically synthesized.

[0312] More specifically, the peptides were synthesized by the following artificial synthesis procedure:

[0313] A 0.3 g oven-dried Rink Amide MBHA resin (0.66 mmol / g) was placed in a polyethylene vessel equipped with a polypropylene filter. The resin was swelled in DCM (15 ml) for 1 h and in DMF (15 ml) for 1 h. The Fmoc group on the resin was deprotected by treating it twice (5 min and 15 min) with a 20% (v / v) piperidine / DMF solution. The resin was washed with DMF / DCM / DMF (6:6:6 each). Removal of the Fmoc from the solid support was confirmed using the Kaiser test (quantitative method). The C-terminal Fmoc-amino acid (5 eq excess corresponding to the resin loading) in dry DMF was added to the deprotected resin and the coupling of the next Fmoc-amino acid was initiated with 5 eq excess of DIC and HOBT in DMF. The concentration of each reactant in the reaction mixture was about 0.4 M. The mixture was rotated on a rotator for 2 h at room temperature. The resin was filtered and washed with DMF / DCM / DMF (6:6:6 each). The Kaiser test on a small aliquot of the peptide resin was negative (no color on the resin) after the coupling was complete. After the attachment of the first amino acid, the unreacted amino groups in the resin, if present, were capped / ended using acetic anhydride / pyridine / DCM (1 :8:8) for 20 min to avoid any deletions of the sequence. After capping / ending, the resin was washed with DCM / DMF / DCM / DMF (6 / 6 / 6 / 6 each). The Fmoc group on the C-terminal amino acid of the peptide-based resin attachment was deprotected by treating it twice (5 min and 15 min) with a 20% (v / v) piperidine / DMF solution. The resin was washed with DMF / DCM / DMF (6:6:6 each). The Kaiser test on a small aliquot of the peptide resin was positive after the Fmoc deprotection was complete.

[0314] The remaining amino acids in the target sequence were sequentially coupled on Rink amide MBHA resin using the Fmoc AA / DIC / HOBt method using a 5 equivalent excess over resin loading in DMF. The concentration of each reactant in the reaction mixture was about 0.4 M. The mixture was rotated on a rotator for 2 hours at room temperature. The resin was filtered and washed with DMF / DCM / DMF (6:6:6 each). After each coupling step and Fmoc deprotection step, a Kaiser test was performed to confirm completion of the reaction.

[0315] After completion of the linear sequence, the epsilon amino group of lysine used as branching point or modification point was deprotected by using 2.5% hydrazine hydrate in DMF for 15 minutes x 2 and washed with DMF / DCM / DMF (6:6:6 each). The gamma carboxylic end of glutamic acid was attached to the epsilon amino group of Lys using Fmoc-Glu(OH)-OtBu and DIC / HOBt method (5 equivalent excess over resin loading) in DMF. The mixture was rotated on a rotator for 2 hours at room temperature. The resin was filtered and washed with DMF / DCM / DMF (6 x 30 ml each). The Fmoc group on the glutamic acid was deprotected by treating it with 20% (v / v) piperidine / DMF solution twice for 5 minutes and 15 minutes (25 ml each). The resin was washed with DMF / DCM / DMF (6:6:6 each). After completion of the Fmoc deprotection, a Kaiser test on a small aliquot of the peptide resin was positive.

[0316] If the side chain branching also contains another gamma-glutamic acid, a second Fmoc-Glu(OH)-OtBu was attached to the free amino group of the gamma-glutamic acid using the DIC / HOBt method (5 equivalent excess over resin loading) in DMF. The mixture was rotated on a rotator for 2 hours at room temperature. The resin was filtered and washed with DMF / DCM / DMF (6 x 30 ml each). The Fmoc group on the gamma-glutamic acid was deprotected by treating it with 20% (v / v) piperidine / DMF solution twice for 5 minutes and 15 minutes (25 ml each). The resin was washed with DMF / DCM / DMF (6:6:6 each). After completion of the Fmoc deprotection, a Kaiser test on a small aliquot of the peptide resin was positive.

[0317] Final cleavage of the peptide from the resin:

[0318] Peptide-resins synthesized by manual synthesis were washed with DCM (6 x 10 ml), MeOH (6 x 10 ml) and ether (6 x 10 ml) and dried in a vacuum desiccator overnight. Cleavage of the peptide from the solid support was achieved by treating the peptide-resin with a reagent cocktail (80% TFA / 5% benzyl mercaptide / 5% phenol / 2.5% EDT / 2.5% DMS / 5% DCM) at room temperature for 3 hours. The cleavage mixture was collected by filtration and the resin was washed with TFA (2 ml) and DCM (2 x 5 ml). The excess TFA and DCM were concentrated to a small volume under nitrogen and a small amount of DCM (5-10 ml) 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 allowed to cool to 0°C 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.

[0319] Table 5: Sequence Listing

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330] Sequence Listing <110> Sanofi <120> FGF21 compound / GLP-1R agonist combinations with optimized activity ratio <130> 589-264 PCT2 <150> EP 18 305 784.3 <151> June 21, 2018 <160> 70 <170> PatentIn version 3.5 <210> 1 <211> 209 <212> PRT <213> 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 Gin 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 Gin Pro Pro Asp Val Gly Ser Ser Asp 180 185 190 Pro Leu Ser Met Val Gly Pro Ser Gin 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 Gin Phe Gly Gly Gin Val 1 5 10 15 Arg Gin Arg Tyr Leu Tyr Thr Asp Asp Ala Gin Gin Thr Glu Ala His 20 25 30 Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gin Ser 35 40 45 Pro Glu Ser Leu Leu Gin Leu Lys Ala Leu Lys Pro Gly Val Ile Gin 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 lie Pro Asp Ser Ser Pro Leu Leu Gin Phe Gly Gly Gin Val 1 5 10 15 Arg Gin Arg Tyr Leu Tyr Thr Asp Asp Ala Gin Gin Thr Glu Cys His 20 25 30 Leu Glu lie Arg Glu Asp Gly Thr Val Gly Cys Ala Ala Asp Gin Ser 35 40 45 Pro Glu Ser Leu Leu Gin Leu Lys Ala Leu Lys Pro Gly Val lie Gin 50 55 60 lie Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gin 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 Gin 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 lie Leu Ala Pro Gin Pro Pro Asp Val 145 150 155 160 Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gin 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 Gin Phe Gly Gly Gin Val 1 5 10 15 Arg Gin Arg Tyr Leu Tyr Thr Asp Asp Ala Cys Gin Thr Glu Ala His 20 25 30 Leu Gin Ile Arg Gin Asp Gin Thr Val Gly Gly Ala Ala Asp Gin Ser 35 40 45 Pro Gin Ser Leu Leu Gin Leu Lys Ala Leu Lys Pro Gly Val Ile Gin 50 55 60 Ile Leu Gly Val Lys Thr Ser Gin Phe Leu Cys Gin Arg Pro Asp Gly 65 70 75 80 Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Gin Ala Cys Ser Phe Arg 85 90 95 Glu Leu Leu Leu Gin Asp Gin Tyr Gin Val Tyr Gin 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 lie Pro Asp Ser Ser Pro Leu Leu Gin Phe Gly Gly Gin Val 1 5 10 15 Arg Gin Arg Tyr Leu Tyr Thr Asp Asp Ala Cys Gin Thr Glu Ala His 20 25 30 Leu Glu lie Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gin Ser 35 40 45 Pro Gin Ser Leu Leu Gin Leu Lys Ala Leu Lys Pro Gly Val lie Gin 50 55 60 lie Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gin 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 Gin 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 lie Leu Ala Pro Gin Pro Pro Asp Val 145 150 155 160 Gly Ser Ser Asp Pro Leu Ser Met Val Gly Ser Gin Gly Arg Ser Pro 165 170 175 Ser Tyr Ala Ser 180 <210> 7 <211> 30 <212> PRT <213> 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 Gin 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 lie Gin Leu Glu 1 5 10 15 Glu Glu Ala Val Arg Leu Phe lie 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 Gin Leu Glu Glu 1 5 10 15 Glu Ala Val Gin Leu Phe lie Glu Trp Leu Leu Ala Thr Gly Pro Ser 35T,R36G,insPSSGEPPPES20 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 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> 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 Gly 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 Gin Glu Glu Met Thr Lys Asn Gin 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 Gin 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 Gin Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gin 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 linker (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 linker (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]_IgG4 Fc_variant[7GS] FGF21 (His29-Ser209)_A59C, G71C <400> 24 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin 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 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 Gin 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]_IgG4 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 Gin Phe Asn Ser Thr Tyr Arg Val Val Ser 130 135 140 Val Leu Thr Val Leu His Gin 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 Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro 180 185 190 Pro Ser Gin Glu Glu Met Thr Lys Asn Gin 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 Gin 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 Gin Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 260 265 270 His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly 275 280 285 Gly Ser Gly Ser Ala His Pro lie Pro Asp Ser Ser Pro Leu Leu Gin 290 295 300 Phe Gly Gly Gin Val Arg Gin Arg Tyr Leu Tyr Thr Asp Asp Ala Cys 305 310 315 320 Gln Thr Glu Ala His Leu Glu lie Arg Glu Asp Gly Thr Val Gly Gly 325 330 335 Ala Ala Asp Gin Ser Pro Glu Ser Leu Leu Gin Leu Lys Ala Leu Lys 340 345 350 Pro Gly Val lie Gin lie 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 lie 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]_IgG4 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]_IgG4 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 Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu 180 185 190 Pro Pro Ser Gin Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys 195 200 205 Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala Val Glu Trp Glu Ser 210 215 220 Asn Gly Gin 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 Gin Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 260 265 270 Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Leu Gly Gly 275 280 285 Gly Gly Ser Gly Ser Ala His Pro He Pro Asp Ser Ser Pro Leu Leu 290 295 300 Gln Phe Gly Gly Gin Val Arg Gin Arg Tyr Leu Tyr Thr Asp Asp Ala 305 310 315 320 Cys Gin Thr Glu Ala His Leu Glu He Arg Glu Asp Gly Thr Val Gly 325 330 335 Gly Ala Ala Asp Gin Ser Pro Glu Ser Leu Leu Gin Leu Lys Ala Leu 340 345 350 Lys Pro Gly Val He Gin He Leu Gly Val Lys Thr Ser Arg Phe Leu 355 360 365 Cys Gin 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 Gin 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 He Leu Ala 435 440 445 Pro Gin Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met Val Gly 450 455 460 Ser Gin 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]_Ig4G 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 Gin Phe Asn Ser Thr Tyr Arg Val Val 130 135 140 Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr 145 150 155 160 Lys Gin Gin Pro Arg Glu Glu Gin Phe Asn Ser Thr Tyr Arg Val Val 165 170 175 Ile Ser Lys Ala Lys Gin Gin Pro Arg Glu Glu Gin Phe Asn Ser Thr 180 185 190 Pro Pro Ser Gin Glu Glu Met Thr Lys Asn Gin 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 Gin 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 Gin Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 260 265 270 Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Leu Gly Gly 275 280 285 Gly Gly Ser Gly Ser Ala His Pro lie Pro Asp Ser Ser Pro Leu Leu 290 295 300 Gln Phe Gly Gly Gin Val Arg Gin Arg Tyr Leu Tyr Thr Asp Asp Ala 305 310 315 320 Cys Gin Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro 325 330 335 Gly Ala Ala Asp Gin Ser Pro Glu Ser Leu Leu Gin Leu Lys Ala Leu 340 345 350 Lys Pro Gly Val lie Gin lie Leu Gly Val Lys Thr Ser Arg Phe Leu 355 360 365 Cys Gin 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 Gin 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 lie Leu Ala 435 440 445 Pro Gin Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Met Val Gly 450 455 460 Ser Gin 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]_IgG4 Fc_variant [7GS]_FGF21(His29-Ser209) Q55C, N149C, G198Y <400> 30 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Leu Glu Glu 1 5 10 15 Glu Ala Val Gin Leu Phe lie 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 Gin 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 Gin Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 260 265 270 Leu His Asn His Tyr Thr Gin 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 Gin Val Arg Gin Arg Tyr Leu Tyr Thr Asp Asp Ala 305 310 315 320 Cys Gin Thr Glu Ala His Leu Glu He Arg Glu Asp Gly Thr Val Gly 325 330 335 Gly Ala Ala Asp Gin Ser Pro Glu Ser Leu Leu Gin Leu Lys Ala Leu 340 345 350 Lys Pro Gly Val He Gin He Leu Gly Val Lys Thr Ser Arg Phe Leu 355 360 365 Cys Gin 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 Ser 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 Gin Val Tyr Thr Leu Pro Pro Ser Gin 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 Gin 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 Gin Glu Gly Asn Val Phe Ser Cys 245 250 255 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin 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 Gin Phe Gly Gly Gin Val Arg Gin Arg Tyr 290 295 300 Leu Tyr Thr Asp Asp Ala Cys Gin Thr Glu Ala His Leu Glu Ile Arg 305 310 315 320 Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gin Ser Pro Glu Ser Leu 325 330 335 Leu Gin Leu Lys Ala Leu Lys Pro Gly Val He Gin He Leu Gly Val 340 345 350 Lys Thr Ser Arg Phe Leu Cys Gin 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 Gin 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 He Leu Ala Pro Gin Pro Pro Asp Val Gly Ser Ser Asp 435 440 445 Pro Leu Ser Met Val Tyr Pro Ser Gin 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 Gin Leu Glu Glu 1 5 10 15 Glu Ala Val Gin Leu Phe lie 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 lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 85 90 95 Val Ser Gin Glu Asp Pro Glu Val Gin Phe Asn Trp Tyr Val Asp Gly 100 105 110 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Phe Asn 115 120 125 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin 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 Gin Val Tyr Thr Leu Pro Pro Ser Gin 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 Gin 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 Gin Glu Gly Asn Val Phe Ser Cys 245 250 255 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin 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 Gin Phe Gly Gly Gin Val Arg Gin Arg Tyr 290 295 300 Leu Tyr Thr Asp Asp Ala Cys Gin Thr Glu Ala His Leu Glu Ile Arg 305 310 315 320 Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gin Ser Pro Glu Ser Leu 325 330 335 Leu Gin Leu Lys Ala Leu Lys Pro Gly Val Ile Gin Ile Leu Gly Val 340 345 350 Lys Thr Ser Arg Phe Leu Cys Gin 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 Gin 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 Gin Pro Pro Asp Val Gly Ser Ser Asp 435 440 445 Pro Leu Ser Met Val Tyr Pro Ser Gin 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 Gin Leu Glu Glu 1 5 10 15 Glu Ala Val Gin Leu Phe lie 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 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 Gin Ile Leu Gly Val Lys Thr Ser Arg Phe Leu 355 360 365 Cys Gin Arg Pro Asp Gly Ala Leu Tyr Gin 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 Gin Ser Glu Ala His Gin Leu Pro Leu His Leu Pro Gin 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 Gin Leu Pro Pro Ala Pro Pro Gin Pro Pro Gin Ile Leu Gin 435 440 445 Pro Gin Pro Pro Asp Val Gin Ser Ser Asp Pro Leu Ser Met Val Gin 450 455 460 Ser Gin Gin 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]_IgG4 Fc_variant [7GS]_FGF21 (His29-Ser209) Q55C, N149C, delP199 <400> 35 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gin Leu Glu Glu 1 5 10 15 Glu Ala Val Gin Leu Phe lie 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 lie Ser Arg Thr 85 90 95 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gin Glu Asp Pro Glu 100 105 110 Val Gin Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 115 120 125 Thr Lys Pro Arg Glu Glu Gin Phe Asn Ser Thr Tyr Arg Val Val Ser 130 135 140 Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys 145 150 155 160 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser lie Glu Lys Thr lie 165 170 175 Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro 180 185 190 Pro Ser Gin Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu 195 200 205 Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn 210 215 220 Gly Gin 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 Gin Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 260 265 270 His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly 275 280 285 Gly Ser Gly Ser Ala His Pro lie Pro Asp Ser Ser Pro Leu Leu Gin 290 295 300 Phe Gly Gly Gin Val Arg Gin Arg Tyr Leu Tyr Thr Asp Asp Ala Cys 305 310 315 320 Gln Thr Glu Ala His Leu Glu lie Arg Glu Asp Gly Thr Val Gly Gly 325 330 335 Ala Ala Asp Gin Ser Pro Glu Ser Leu Leu Gin Leu Lys Ala Leu Lys 340 345 350 Pro Gly Val lie Gin lie 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 lie 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-1 RA, generic sequence <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 variant <400> 47 His Pro lie Pro Asp Ser Ser Pro Leu Leu Gin Phe Gly Gly Gin Val 1 5 10 15 Arg Gin Arg Tyr Leu Tyr Thr Asp Asp Ala Gin Gin Thr Gin Ala His 20 25 30 Leu Gin lie Arg Gin Asp Gly Thr Val Gly Gly Ala Ala Asp Gin Ser 35 40 45 Pro Gin Ser Leu Leu Gin Leu Lys Ala Leu Lys Pro Gly Val lie Gin 50 55 60 lie Leu Gly Val Lys Thr Ser Gin Phe Leu Gin Gin Arg Pro Asp Gly 65 70 75 80 Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Gin Ala Cys Ser Phe Gin 85 90 95 Glu Gin lie Arg Pro Asp Gly Tyr Gin Val Tyr Gin Ser Gin Ala His 100 105 110 Gly Leu Pro Leu His Leu Pro Gly Gin Lys Ser Pro Gin Arg Asp Pro 115 120 125 Ala Pro Gin Gly Pro Ala Gin Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140 Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gin Pro Pro Asp Val 145 150 155 160 Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gin Gly Arg Ser 165 170 175 Pro Ser Tyr Ala Ser 180 <210> 48 <211> 182 <212> PRT <213> Artificial Sequence <220> <223> FGF21 variant <400> 48 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gin Phe Gly Gly Gin Val 1 5 10 15 Arg Gin Arg Tyr Leu Tyr Thr Asp Asp Ala Gin Gin Thr Gin Ala His 20 25 30 Leu Gin Ile Arg Gin Asp Gly Thr Val Gly Gly Ala Ala Asp Gin Ser 35 40 45 Pro Gin Ser Leu Leu Gin Leu Lys Ala Leu Lys Pro Gly Val Ile Gin 50 55 60 Ile Leu Gly Val Lys Thr Ser Gin Phe Leu Gin Gin Arg Pro Gin Gly 65 70 75 80 Ala Leu Tyr Gin Ser Leu His Phe Asp Pro Gin Ala Cys Ser Phe Gin 85 90 95 Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gin 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 Gin 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 variant <400> 49 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gin Phe Gly Gly Gin Val 1 5 10 15 Arg Gin Arg Tyr Leu Tyr Thr Asp Asp Ala Gin Gin Thr Glu Ala His 20 25 30 Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gin Ser 35 40 45 Pro Glu Ser Leu Leu Gin Leu Lys Ala Leu Lys Pro Gly Val He Gin 50 55 60 Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gin 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 Gin 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 He Leu Ala Pro Gin 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 variant <400> 50 His Pro lie Pro Asp Ser Ser Pro Leu Leu Gin Phe Gly Gly Gin Val 1 5 10 15 Arg Gin Arg Tyr Leu Tyr Thr Asp Asp Ala Gin Gin Thr Glu Ala His 20 25 30 Leu Glu lie Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gin Ser 35 40 45 Pro Glu Ser Leu Leu Gin Leu Lys Ala Leu Lys Pro Gly Val lie Gin 50 55 60 lie Leu Gly Val Lys Thr Ser Arg Phe Leu Cy s Gin 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 Gin 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 lie Leu Ala Pro Gin Pro Pro Asp Val 145 150 155 160 Gly Ser Ser Asp Pro Leu Ser Met Val Asn Pro Ser Gin Gly Arg Ser 165 170 175 Pro Ser Tyr Ala Ser 180 <210> 51 <211> 181 <212> PRT <213> Artificial Sequence <220> <223> FGF21 variant <400> 51 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gin Phe Gly Gly Gin Val 1 5 10 15 Arg Gin Arg Tyr Leu Tyr Thr Asp Asp Ala Gin Gin Thr Gin Ala His 20 25 30 Leu Gin Ile Arg Gin Asp Gly Thr Val Gin Gin Ala Ala Asp Gin Ser 35 40 45 Pro Gin Ser Leu Leu Gin Leu Lys Ala Leu Lys Pro Gly Val Ile Gin 50 55 60 Ile Leu Gin Val Lys Thr Ser Gin Phe Leu Gin Gin Arg Pro Gin Gly 65 70 75 80 Ala Leu Tyr Gin Ser Leu His Phe Asp Pro Gin Ala Cys Ser Phe Gin 85 90 95 Glu Leu Leu Leu Gin Asp Gly Tyr Gin Val Tyr Gin Gin Gin Gin Gin 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 variant <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 Gin 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 Gin 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 Gin 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 variant <400> 53 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gin Phe Gly Gly Gin Val 1 5 10 15 Arg Gin Arg Tyr Leu Tyr Thr Asp Asp Ala Gin Gin Thr Glu Ala His 20 25 30 Leu Glu lie Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gin Ser 35 40 45 Pro Glu Ser Leu Leu Gin Leu Lys Ala Leu Lys Pro Gly Val lie Gin 50 55 60 lie Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gin 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 lie Arg Pro Asp Gly Tyr Asn Val Tyr Gin 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 lie Leu Ala Pro Gin 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 variant <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 variant <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 variant <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 sequences <220> <223> FGF21 variants <400> 57 His Pro lie Pro Asp Ser Ser Pro Leu Leu Gin Phe Gly Gly Gin Val 1 5 10 15 Arg Gin Arg Tyr Leu Tyr Thr Asp Asp Ala Gin Gin Thr Glu Ala His 20 25 30 Leu Glu lie Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gin Ser 35 40 45 Pro Glu Ser Leu Leu Gin Leu Lys Ala Leu Lys Pro Gly Val lie Gin 50 55 60 Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Tyr Gin 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 Gin 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 Gin 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 variant <400> 58 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gin Phe Gly Gly Gin Val 1 5 10 15 Arg Gin Arg Tyr Leu Tyr Thr Asp Asp Ala Gin Gin Thr Glu Ala His 20 25 30 Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gin Ser 35 40 45 Pro Glu Ser Leu Leu Gin Leu Lys Ala Leu Lys Pro Gly Val Ile Gin 50 55 60 Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gin 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 Gin 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 Gin 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 variant <400> 59 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gin Phe Gly Gly Gin Val 1 5 10 15 Arg Gin Arg Tyr Leu Tyr Thr Asp Asp Ala Gin Gin Thr Glu Ala His 20 25 30 Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gin Ser 35 40 45 Pro Glu Ser Leu Leu Gin Leu Lys Ala Leu Lys Pro Gly Val He Gin 50 55 60 Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gin 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 Gin 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 He Leu Ala Pro Gin Pro Pro Asp Val 145 150 155 160 Gly Ser Ser Asp Pro Leu Ser Met Val Asn Pro Ser Gin Gly Arg Ser 165 170 175 Pro Ser Tyr Glu Ser 180 <210> 60 <211> 181 <212> PRT <213> Artificial Sequence <220> <223> FGF21 variant <400> 60 His Pro lie Pro Asp Ser Ser Pro Leu Leu Gin Phe Gly Gly Gin Val 1 5 10 15 Arg Gin Arg Tyr Leu Tyr Thr Asp Asp Ala Gin Gin Thr Glu Ala His 20 25 30 Leu Glu lie Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gin Ser 35 40 45 Pro Glu Ser Leu Leu Gin Leu Lys Ala Leu Lys Pro Gly Val lie Gin 50 55 60 lie Leu Gly Val Lys Thr Ser Arg Phe Leu Tyr Gin 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 Gin 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 lie Leu Ala Pro Gin Pro Pro Asp Val 145 150 155 160 Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gin Asn Arg Ser 165 170 175 Pro Ser Tyr Glu Ser 180 <210> 61 <211> 182 <212> PRT <213> Artificial Sequence <220> <223> FGF21 variant <400> 61 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gin Phe Gly Gly Gin Val 1 5 10 15 Arg Gin Arg Tyr Leu Tyr Thr Asp Asp Ala Gin Gin Thr Glu Ala His 20 25 30 Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gin Ser 35 40 45 Pro Glu Ser Leu Leu Gin Leu Lys Ala Leu Lys Pro Gly Val Ile Gin 50 55 60 Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Tyr Gin 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 Gin 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 variant <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 Gin 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 Gin 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 Gin 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 variant <400> 63 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gin Phe Gly Gly Gin Val 1 5 10 15 Arg Gin Arg Tyr Leu Tyr Thr Asp Asp Ala Gin Gin Thr Glu Ala His 20 25 30 Leu Glu lie Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gin Ser 35 40 45 Pro Glu Ser Leu Leu Gin Leu Lys Ala Leu Lys Pro Gly Val lie Gin 50 55 60 lie Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gin 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 lie Arg Pro Asp Gly Tyr Asn Val Tyr Gin 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 lie Leu Ala Pro Gin Pro Pro Asp Val 145 150 155 160 Gly Ser Ser Asp Pro Leu Ser Met Val Asn Pro Ser Gin Gly Arg Ser 165 170 175 Pro Ser Tyr Glu Ser 180 <210> 64 <211> 181 <212> PRT <213> Artificial Sequence <220> <223> FGF21 variant <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 variant <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 Gin 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 Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro 115 120 125 Pro Ser Gin Glu Glu Met Thr Lys Asn Gin 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 Gin 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 Gin Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 195 200 205 His Asn His Tyr Thr Gin 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 variant <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 Gin Glu Asp Pro Glu Val Gin 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 Gin Val Tyr Thr Leu Pro Pro Ser Gin Glu Glu Met 130 135 140 Thr Lys Asn Gin 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 Gin 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 Gin Glu Gly Asn Val 195 200 205 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin 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 variant <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 Gin Phe Asn Ser Thr Tyr Arg Val Val Ser 100 105 110 Val Leu Thr Val Leu His Gin 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 Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro 145 150 155 160 Pro Ser Gin Glu Glu Met Thr Lys Asn Gin 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 Gin 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 Gin Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 225 230 235 240 His Asn His Tyr Thr Gin 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 variant <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 variant <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

Claims

1. A fusion molecule comprising an FGF21 (fibroblast growth factor 21) compound and a GLP-1R (glucagon-like peptide-1 receptor) agonist, wherein the amino acid sequence of the fusion molecule comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 25, 26, 28, 30, 31, 32, 33, 35, and 36.

2. A pharmaceutical composition comprising the fusion molecule of claim 1 and a pharmaceutically acceptable carrier and / or excipient.

3. A nucleic acid molecule encoding the fusion molecule of claim 1.

4. A host cell containing the nucleic acid molecule of claim 3.

5. A kit comprising the fusion molecule of claim 1, the pharmaceutical composition of claim 2, the nucleic acid molecule of claim 3, or the host cell of claim 4.

6. Use of the fusion molecule of claim 1, the pharmaceutical composition of claim 2, the nucleic acid molecule of claim 3, or the host cell of claim 4 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, hyperglycemia, dyslipidemia, nonalcoholic steatohepatitis (NASH), and atherosclerosis.

7. The use of claim 6, wherein the diabetes is type 1 diabetes or type 2 diabetes.

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