GLP-1r agonist / FGF21 fusion proteins

Abstract

To provide fusion proteins with optimized GLP-1R agonist / FGF21 compound activity ratio.SOLUTION: The invention relates to a fusion protein comprising a glucagon-like peptide 1 receptor 5 (GLP-1R) agonistic peptide and a variant of human fibroblast growth factor 21 (FGF21). The invention further relates, in particular to the use of a fusion protein comprising a GLP-1R agonistic peptide and a variant of FGF21 as a medicament for treating obesity, overweight, metabolic syndrome, diabetes, diabetic retinopathy, hyper glycemia, dyslipidemia, non-alcoholic steatohepatitis (NASH) and / or atherosclerosis in a subject. The invention further relates to pharmaceutical compositions comprising a fusion protein comprising a GLP-1R agonistic peptide and a variant of FGF21.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to fusion proteins comprising a GLP-1R (glucagon-like peptide 1 receptor) agonist peptide and a variant of human fibroblast growth factor 21 (FGF21). The present invention further relates to the use of these fusion proteins as pharmaceuticals, particularly for the treatment of obesity, overweight, metabolic syndrome, diabetes, diabetic retinopathy, hyperglycemia, dyslipidemia, non-alcoholic steatohepatitis (NASH), and / or atherosclerosis. [Background technology]

[0002] The use of FGF21 and GLP-1R agonist as fusion protein has drawbacks.The pharmacological effect of FGF21 is observed at a higher plasma level than the plasma level at which GLP-1 (primary GLP-1R agonist) exerts its pharmacological effect.In addition, at higher plasma levels, GLP-1 is known to have adverse effects, for example, it induces nausea and vomiting.Together, this indicates the risk of GLP-1-mediated adverse effects when administering the combination of FGF21 compound and GLP-1R agonist in the form of fusion protein.Therefore, there is a need for a new fusion protein that combines FGF21 and GLP-1R agonist and its formulation. Summary of the Invention [Problem to be solved by the invention]

[0003] The object of the present invention is to provide a fusion protein with an optimized GLP-1R agonist / FGF21 compound activity ratio to achieve the beneficial effects of both active agents (e.g., in body weight, lipid, glycemic control, etc.) while avoiding potential adverse effects (e.g., nausea and vomiting, etc.). [Means for solving the problem]

[0004] In one aspect, the present invention relates to a fusion protein comprising a GLP-1R agonist peptide and a functionally active variant of human FGF21.

[0005] In one embodiment, the GLP-1R agonist peptide is a variant of native GLP-1(7-36) (SEQ ID NO: 260) that comprises substitutions of up to about 15 amino acid residues in the amino acid sequence of native GLP-1(7-36).

[0006] In one embodiment, the functionally active variant of human FGF21 comprises an amino acid sequence that is at least about 96% identical to the amino acid sequence of SEQ ID NO: 250 or SEQ ID NO: 251, (i) substitutions Q55C and P147C or substitutions Q55C and N149C, and (ii) containing a substitution or deletion of G198 and / or P199; Amino acid residue numbering is according to SEQ ID NO:250.

[0007] In one embodiment, the GLP-1R agonist peptide and a functionally active variant of human FGF21 are linked via a linker molecule comprising a structure selected from the group consisting of L-Fc, Fc-L, L1-Fc-L2, and Fc, where L, L1, and L2 are independently selected from the group consisting of single amino acids and peptides, and Fc is an Fc domain of an immunoglobulin or a variant thereof.

[0008] In one embodiment, the GLP-1R agonist peptide as part of the fusion protein has a GLP-1R agonist activity that is about 9 to about 53 times greater than that of native GLP-1(7-36). It has half the GLP-1R agonist activity.

[0009] In one embodiment, the GLP-1R agonist peptide as part of the fusion protein has GLP-1R agonist activity that is about 9 to about 482 fold (or about 9.449 to about 482.396 fold), or about 9 to about 319 fold (or about 9.449 to about 319.311 fold), or about 9 to about 121 fold (or about 9.449 to about 121.189 fold) less than the GLP-1R agonist activity of native GLP-1(7-36).

[0010] In one embodiment, the GLP-1R agonist peptide as part of a fusion protein has GLP-1R agonist activity that is about 9 to about 319 fold less than the GLP-1R agonist activity of native GLP-1(7-36).

[0011] In one embodiment, the GLP-1R agonist peptide as part of the fusion protein has GLP-1R agonist activity that is at least about 9.4-fold, or at least about 9.45-fold, or at least about 9.5-fold less than the GLP-1R agonist activity of native GLP-1(7-36).

[0012] In one embodiment, the GLP-1R agonist peptide as part of a fusion protein has GLP-1R agonist activity that is at least about 10-fold less than the GLP-1R agonist activity of native GLP-1(7-36).

[0013] In one embodiment, the GLP-1R agonist peptide as part of a fusion protein has GLP-1R agonist activity that is up to about 482.4-fold or up to about 482.35-fold less than the GLP-1R agonist activity of native GLP-1(7-36).

[0014] In one embodiment, the GLP-1R agonist peptide as part of a fusion protein has GLP-1R agonist activity that is up to about 482-fold less than the GLP-1R agonist activity of native GLP-1(7-36).

[0015] In one embodiment, the GLP-1R agonist peptide as part of a fusion protein has GLP-1R agonist activity that is about 10 to about 482 fold less than the GLP-1R agonist activity of native GLP-1(7-36).

[0016] In one embodiment, the GLP-1R agonist peptide as part of a fusion protein has GLP-1R agonist activity that is about 10 to about 319 fold less than the GLP-1R agonist activity of native GLP-1(7-36).

[0017] In one embodiment, the GLP-1R agonist peptide as part of a fusion protein has GLP-1R agonist activity that is about 90 to about 100 times less than the GLP-1R agonist activity of native GLP-1(7-36).

[0018] In one embodiment, the GLP-1R agonist peptide as part of the fusion protein has GLP-1R agonist activity that is at least about 18-fold (or at least about 18.268-fold) less than the GLP-1R agonist activity of native GLP-1(7-36).

[0019] In one embodiment, the GLP-1R agonist peptide as part of a fusion protein has about 18 to about 501 fold (or about 18,268 to about 500,686 fold) or about 18 to about 469 fold less GLP-1R agonist activity than native GLP-1(7-36). The GLP-1R agonist activity of the compound is about 1 / 2 (or about 18.268 to about 1468.679 fold) or about 18 to about 313 fold (or about 18.268 to about 1313.214 fold) or about 18 to about 123 fold (or about 18.268 to about 123.466 fold) that of the compound.

[0020] In one embodiment, the GLP-1R agonist peptide as part of a fusion protein has GLP-1R agonist activity that is about 18 to about 313 fold less than the GLP-1R agonist activity of native GLP-1(7-36).

[0021] In one of the above-described embodiments, the GLP-1R agonist peptide as part of the fusion protein has GLP-1R agonist activity that is at least about 18.2-fold or at least about 18.3-fold less than the GLP-1R agonist activity of native GLP-1(7-36).

[0022] In one of the above-described embodiments, the GLP-1R agonist peptide as part of the fusion protein has GLP-1R agonist activity that is at least about 20-fold less, or at least about 50-fold less, or at least about 100-fold less, than the GLP-1R agonist activity of native GLP-1(7-36).

[0023] In one embodiment, the GLP-1R agonist peptide as part of a fusion protein has GLP-1R agonist activity that is about 10- to about 500-fold less than the GLP-1R agonist activity of native GLP-1(7-36). In one embodiment, the GLP-1R agonist peptide as part of a fusion protein has GLP-1R agonist activity that is about 15- to about 500-fold less than the GLP-1R agonist activity of native GLP-1(7-36). In one embodiment, the GLP-1R agonist peptide as part of a fusion protein has GLP-1R agonist activity that is about 20- to about 500-fold less than the GLP-1R agonist activity of native GLP-1(7-36). In one embodiment, the GLP-1R agonist peptide as part of a fusion protein has GLP-1R agonist activity that is about 50- to about 500-fold less than the GLP-1R agonist activity of native GLP-1(7-36). In one embodiment, the GLP-1R agonist peptide as part of a fusion protein has GLP-1R agonist activity that is about 100- to about 500-fold less than the GLP-1R agonist activity of native GLP-1(7-36). In one embodiment, the GLP-1R agonist peptide as part of a fusion protein has GLP-1R agonist activity that is about 100- to about 300-fold less than the GLP-1R agonist activity of native GLP-1(7-36).

[0024] In one embodiment, the fusion protein has GLP-1R agonist activity as defined above.

[0025] In one embodiment, the GLP-1R agonist peptide has the amino acid sequence X1-X2-X3-GTFTSDX 10 -SX 12 -X 13 -X 14 -X 15 -X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -X22 -X 23 -X 24 -X 25 -LX 27 -X 28 -X 29 -X 30 (SEQ ID NO: 4077) comprising or consisting of During the ceremony X1 is H, Y, or F; X2 is G, S, T, or A; X3 is E or Q; X 10 is K or L, X 12 is K, I, or Q, X 13 is Q or L, X 14 is L, M, or C, X 15 is E, A, or D, X 16 is E, K, or S, X 17 is E, R, or Q, X 18 is L, A, or R, X 19 is V, A, or F, X 20 is R, H, Q, K, or I, X 21 is L, E, H, or R, X 22 is F or L, X 23 is I, Y, or F, X 24 is E, L, or Y, X 25 is W or L, X 27 is I, L, K, or E, X 28 is A, K, N, or E, X 29 is G, T, K, or V, X 30 is G or a deletion, Optionally, the amino acid sequence further comprises at least one additional amino acid residue at its N-terminus, Optionally, the amino acid sequence further comprises a peptide extension of up to about 12, about 11, or about 10 amino acid residues at its C-terminus.

[0026] In one embodiment, the at least one additional amino acid residue is G or A. In one embodiment, the at least one additional amino acid residue is a single amino acid residue. In one embodiment, the at least one amino acid residue is G.

[0027] In one embodiment, the peptide extension consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 4008 to 4063. In one embodiment, the peptide extension is a single amino acid residue, eg, P.

[0028] In one embodiment, the GLP-1R agonist peptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 261-565.

[0029] In one embodiment, the GLP-1R agonist peptide has the amino acid sequence X1-GEGTFTSDX 10 -SX 12 -X 13 -LX 15 -X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -FX 23 -EWLX 27 -X 28 -X 29 -G (sequence number 4078) comprising or consisting of During the ceremony X1 is H, Y, or F; X 10is K or L, X 12 is K, I, or Q, X 13 is Q or L, X 15 is E, A, or D, X 16 is E, K, or S, X 17 is E, R, or Q, X 18 is L, A, or R, X 19 is V, A, or F, X 20 is R, H, Q, K, or I, X 21 is L, E, H, or R, X 23 is I, Y, or F, X 27 is I, L, K, or E, X 28 is A, K, N, or E, X 29 is G, T, K, or V, Optionally, the amino acid sequence further comprises at least one additional amino acid residue at its N-terminus, Optionally, the amino acid sequence further comprises a peptide extension of up to about 12, about 11, or about 10 amino acid residues at its C-terminus.

[0030] In one embodiment, the at least one additional amino acid residue is G or A. In one embodiment, the at least one additional amino acid residue is a single amino acid residue. In one embodiment, the at least one amino acid residue is G.

[0031] In one embodiment, the peptide extension consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 4008-4063.

[0032] In one embodiment, the GLP-1R agonist peptide has the amino acid sequence HGEGTFTSDX 10 -SKQLEEEX 18 -VX 20 -LFIEWLKAX 29 -G (sequence number 4079), comprising or consisting of During the ceremony X 10 is K or L, X 18 is A or R, X 20 is R or Q, X 29 is G or T, Optionally, the amino acid sequence further comprises at least one additional amino acid residue at its N-terminus, Optionally, the amino acid sequence further comprises a peptide extension of up to 12, 11 or 10 amino acid residues at its C-terminus.

[0033] In one embodiment, the at least one additional amino acid residue is G or A. In one embodiment, the at least one additional amino acid residue is a single amino acid residue. In one embodiment, the at least one additional amino acid residue is G.

[0034] In one embodiment, the peptide extension is as defined above. In one embodiment, the peptide extension comprises or consists of the amino acid sequence of PSSGAPPPS (SEQ ID NO: 4047) or PKKIRYS (SEQ ID NO: 4040).

[0035] In one embodiment, the GLP-1R agonist peptide comprises or consists of the amino acid sequence of SEQ ID NO: 261 or 262.

[0036] In one embodiment, the GLP-1R agonist peptide does not comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 4064-4076 and 553.

[0037] In one embodiment, the functionally active mutant of human FGF21 comprises a substitution or deletion selected from the group consisting of G198R, G198K, G198Y, and a deletion of P199.

[0038] In one embodiment, the functionally active variant of human FGF21 is SEQ ID NO: 253, 2 In one embodiment, the functionally active variant of human FGF21 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 253, 254, 255, and 256. In one embodiment, the functionally active variant of human FGF21 comprises or consists of the amino acid sequence of SEQ ID NO: 253 or 254.

[0039] In one embodiment, the GLP-1R agonist peptide comprises or consists of the amino acid sequence of SEQ ID NO:261 and the functionally active variant of human FGF21 comprises or consists of the amino acid sequence of SEQ ID NO:253.

[0040] In one embodiment, the GLP-1R agonist peptide comprises or consists of the amino acid sequence of SEQ ID NO:261 and the functionally active variant of human FGF21 comprises or consists of the amino acid sequence of SEQ ID NO:254.

[0041] In one embodiment, the GLP-1R agonist peptide comprises or consists of the amino acid sequence of SEQ ID NO:262 and the functionally active variant of human FGF21 comprises or consists of the amino acid sequence of SEQ ID NO:253.

[0042] In one embodiment, the GLP-1R agonist peptide comprises or consists of the amino acid sequence of SEQ ID NO:262 and the functionally active variant of human FGF21 comprises or consists of the amino acid sequence of SEQ ID NO:254.

[0043] In one embodiment, the Fc domain of the immunoglobulin or variant thereof comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 257, 258, and 259. In one embodiment, the Fc domain of the immunoglobulin or variant thereof comprises or consists of the amino acid sequence of SEQ ID NO: 257. In one embodiment, the Fc domain of the immunoglobulin or variant thereof comprises or consists of the amino acid sequence of SEQ ID NO: 258. In one embodiment, the Fc domain of the immunoglobulin or variant thereof comprises or consists of the amino acid sequence of SEQ ID NO: 259.

[0044] In one embodiment, the GLP-1R agonist peptide comprises or consists of the amino acid sequence of SEQ ID NO: 261, the functionally active variant of human FGF21 comprises or consists of the amino acid sequence of SEQ ID NO: 253, and the Fc domain of an immunoglobulin or variant thereof comprises or consists of the amino acid sequence of SEQ ID NO: 257.

[0045] In one embodiment, the GLP-1R agonist peptide comprises or consists of the amino acid sequence of SEQ ID NO: 261, the functionally active variant of human FGF21 comprises or consists of the amino acid sequence of SEQ ID NO: 254, and the Fc domain of an immunoglobulin or variant thereof comprises or consists of the amino acid sequence of SEQ ID NO: 257.

[0046] In one embodiment, the GLP-1R agonist peptide comprises or consists of the amino acid sequence of SEQ ID NO: 262, the functionally active variant of human FGF21 comprises or consists of the amino acid sequence of SEQ ID NO: 253, and the Fc domain of an immunoglobulin or variant thereof comprises or consists of the amino acid sequence of SEQ ID NO: 257.

[0047] In one embodiment, the GLP-1R agonist peptide comprises or consists of the amino acid sequence of SEQ ID NO: 262, the functionally active variant of human FGF21 comprises or consists of the amino acid sequence of SEQ ID NO: 254, and the Fc domain of an immunoglobulin or variant thereof comprises or consists of the amino acid sequence of SEQ ID NO: 257.

[0048] In one embodiment, the GLP-1R agonist peptide comprises or consists of the amino acid sequence of SEQ ID NO: 261, and the functionally active variant of human FGF21 comprises or consists of the amino acid sequence of SEQ ID NO: 253, and the Fc domain of the immunoglobulin or variant thereof comprises or consists of the amino acid sequence of SEQ ID NO:258.

[0049] In one embodiment, the GLP-1R agonist peptide comprises or consists of the amino acid sequence of SEQ ID NO: 261, the functionally active variant of human FGF21 comprises or consists of the amino acid sequence of SEQ ID NO: 254, and the Fc domain of an immunoglobulin or variant thereof comprises or consists of the amino acid sequence of SEQ ID NO: 258.

[0050] In one embodiment, the GLP-1R agonist peptide comprises or consists of the amino acid sequence of SEQ ID NO: 262, the functionally active variant of human FGF21 comprises or consists of the amino acid sequence of SEQ ID NO: 253, and the Fc domain of an immunoglobulin or variant thereof comprises or consists of the amino acid sequence of SEQ ID NO: 258.

[0051] In one embodiment, the GLP-1R agonist peptide comprises or consists of the amino acid sequence of SEQ ID NO: 262, the functionally active variant of human FGF21 comprises or consists of the amino acid sequence of SEQ ID NO: 254, and the Fc domain of an immunoglobulin or variant thereof comprises or consists of the amino acid sequence of SEQ ID NO: 258.

[0052] In one embodiment, the GLP-1R agonist peptide comprises or consists of the amino acid sequence of SEQ ID NO: 261, the functionally active variant of human FGF21 comprises or consists of the amino acid sequence of SEQ ID NO: 253, and the Fc domain of an immunoglobulin or variant thereof comprises or consists of the amino acid sequence of SEQ ID NO: 259.

[0053] In one embodiment, the GLP-1R agonist peptide comprises or consists of the amino acid sequence of SEQ ID NO: 261, the functionally active variant of human FGF21 comprises or consists of the amino acid sequence of SEQ ID NO: 254, and the Fc domain of an immunoglobulin or variant thereof comprises or consists of the amino acid sequence of SEQ ID NO: 259.

[0054] In one embodiment, the GLP-1R agonist peptide comprises or consists of the amino acid sequence of SEQ ID NO: 262, the functionally active variant of human FGF21 comprises or consists of the amino acid sequence of SEQ ID NO: 253, and the Fc domain of an immunoglobulin or variant thereof comprises or consists of the amino acid sequence of SEQ ID NO: 259.

[0055] In one embodiment, the GLP-1R agonist peptide comprises or consists of the amino acid sequence of SEQ ID NO: 262, the functionally active variant of human FGF21 comprises or consists of the amino acid sequence of SEQ ID NO: 254, and the Fc domain of an immunoglobulin or variant thereof comprises or consists of the amino acid sequence of SEQ ID NO: 259.

[0056] In another aspect, the present invention provides a fusion protein comprising a GLP-1R (Glucagon-like peptide 1 receptor) agonist peptide and a functionally active variant of human FGF21 (Fibroblast Growth Factor 21), The GLP-1R agonist peptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 261 to 565; A functionally active variant of human FGF21 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 253, 254, 255, and 256; The GLP-1R agonist peptide and the functionally active variant of human FGF21 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 independently selected from the group consisting of single amino acids and peptides, and Fc is an immunoglobulin or immunoglobulin comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 257, 258, and 259. or a variant thereof, Concerning fusion proteins.

[0057] In yet another aspect, the present invention relates to a fusion protein comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8, 16-31, 33-229, and 566-4007, or a functionally active variant thereof comprising or consisting of an amino acid sequence that is at least about 96% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8, 16-31, 33-229, and 566-4007.

[0058] In another aspect, the present invention relates to a fusion protein comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8, 18-31, 39, 40, 42-72, 74, 76, 78-84, 88-90, 92-97, 100-102, 105-109, 112, 113, 115, 116, 118, 120-124, 126-130, 132-136, 139, 142-148, 150-153, 155-158, 161-172, 174-177, 180-188, 190, 192-209, 211, 212, 216, 217, and 219-229. and functionally active variants thereof, comprising or consisting of an amino acid sequence that is at least about 96% identical to an amino acid sequence selected from the group consisting of: 8, 18-31, 39, 40, 42-72, 74, 76, 78-84, 88-90, 92-97, 100-102, 105-109, 112, 113, 115, 116, 118, 120-124, 126-130, 132-136, 139, 142-148, 150-153, 155-158, 161-172, 174-177, 180-188, 190, 192-209, 211, 212, 216, 217, and 219-229.

[0059] In another aspect, the present invention relates to a fusion protein comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 7, and 8, or a functionally active variant thereof comprising or consisting of an amino acid sequence that is at least about 96% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 7, and 8.

[0060] In one embodiment, the fusion protein (or functionally active variant thereof) as defined above activates human GLP-1R with an EC50 of about 15 pmol / L to about 400 pmol / L, or about 20 pmol / L to about 400 pmol / L, or about 50 pmol / L to about 400 pmol / L, or about 100 pmol / L to about 400 pmol / L, as determined, for example, by measuring the cAMP response of cells stably expressing human GLP-1R. In one embodiment, activation of human GLP-1R is determined essentially as described in Example 4.

[0061] In one embodiment, the fusion protein (or functionally active variant thereof) as defined above (i) exhibits an EC50 of about 250 nmol / L or less, or about 200 nmol / L or less, or about 150 nmol / L or less, or about 100 nmol / L or less, or about 75 nmol / L or less, or about 50 nmol / L or less (e.g., an EC50 of about 10 nmol / L to about 50 nmol / L, or about 15 nmol / L to about 50 nmol / L, or about 15 nmol / L to about 45 nmol / L) in humans. and / or (ii) induces the phosphorylation of mitogen-activated protein kinase (MAPK) ERK1 / 2 at an EC50 of about 100 nmol / L or less, or about 75 nmol / L or less, or about 50 nmol / L or less, or about 25 nmol / L or less, or about 20 nmol / L or less, or about 15 nmol / L or less (e.g., at an EC50 of about 2.5 nmol / L to about 15 nmol / L or about 4 nmol / L to about 12 nmol / L). In one embodiment, the autophosphorylation of human FGFR1c and / or the phosphorylation of MAPK ERK1 / 2 is induced by, for example, , determined by using an In-Cell Western (ICW) essentially as described in Example 3.

[0062] In one embodiment, the fusion protein (or functionally active variant thereof) defined above has a melting temperature and / or aggregation temperature of at least about 45° C., or at least about 50° C., or at least about 55° C., or at least about 60° C. In one embodiment, the melting temperature and / or aggregation temperature is determined essentially as described in Example 5.

[0063] In one embodiment, the fusion protein (or functionally active variant thereof) defined above has a terminal plasma half-life of at least about 15 hours or at least about 20 hours in a non-human primate. In one embodiment, the fusion protein (or functionally active variant thereof) defined above has a terminal plasma half-life of at least about 8 hours, at least about 10 hours, or at least about 12 hours in a mouse. In one embodiment, the terminal half-life is determined after a single subcutaneous administration of about 0.3 mg / kg of the fusion protein in solution to a non-human primate, such as a cynomolgus monkey, or a mouse, such as a C57BI / 6 mouse. In one embodiment, the terminal half-life is determined essentially by the method described in Example 6.

[0064] In another aspect, the present invention relates to a nucleic acid molecule encoding a fusion protein as defined above.

[0065] In another aspect, the present invention relates to a host cell containing a nucleic acid molecule as defined above.

[0066] In another aspect, the present invention relates to a method for producing a fusion protein as defined above, comprising culturing a host cell as defined above and isolating the fusion protein.

[0067] In another aspect, the present invention relates to a pharmaceutical composition comprising a fusion protein as defined above, a nucleic acid molecule as defined above, or a host cell as defined above.

[0068] In another aspect, the present invention relates to a kit comprising a fusion protein as defined above, a nucleic acid molecule as defined above, a host cell as defined above, or a pharmaceutical composition as defined above.

[0069] In another aspect, the present invention relates to a fusion protein as defined above, a nucleic acid molecule as defined above, a host cell as defined above, or a pharmaceutical composition as defined above, for use as a medicament.

[0070] In another aspect, the present invention relates to a fusion protein as defined above, a nucleic acid molecule as defined above, a host cell as defined above, or a pharmaceutical composition 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, NASH, and atherosclerosis.

[0071] In one embodiment, the disease or disorder is diabetes. In one embodiment, the diabetes is type 1 diabetes or type 2 diabetes.

[0072] In another aspect, the present invention relates to a fusion protein as defined above, a nucleic acid molecule as defined above, a host cell as defined above, or a pharmaceutical composition as defined above in the manufacture of a medicament for the treatment of a disease or disorder selected from the group consisting of obesity, overweight, metabolic syndrome, diabetes, diabetic retinopathy, hyperglycemia, dyslipidemia, NASH, and atherosclerosis.

[0073] In one embodiment, the disease or disorder is diabetes. In one embodiment, the diabetes is type 1 diabetes or type 2 diabetes.

[0074] In another aspect, the present invention relates to a method for treating a disease or disorder selected from the group consisting of obesity, overweight, metabolic syndrome, diabetes, diabetic retinopathy, hyperglycemia, dyslipidemia, NASH, and atherosclerosis, comprising administering to a subject in need thereof a fusion protein as defined above, a nucleic acid molecule as defined above, a host cell as defined above, or a pharmaceutical composition as defined above.

[0075] In one embodiment, the disease or disorder is diabetes. In one embodiment, the diabetes is type 1 diabetes or type 2 diabetes.

[0076] In another aspect, the present invention relates to a fusion protein as defined above, a nucleic acid molecule as defined above, a host cell as defined above, or a pharmaceutical composition as defined above for improving glycemic control in overweight, obese, dyslipidemic patients with type 2 diabetes. [Brief explanation of the drawings]

[0077] [Figure 1]Figure 1 shows the EC50s of adverse effects (gastric emptying (GE) rate) and pharmacodynamics (i.e., HbA1c, triglycerides, fatty acids, non-HDL, fat mass) depending on the GLP-1 decay rate (12-month simulation): · For GLP-1 decay rates greater than 9.449 (which can be rounded to 9), the EC50 of GLP-1-mediated gastrointestinal adverse effects (gastric emptying; GE rate) was greater than the EC50 of pharmacodynamic effects (i.e., HbA1c, fat mass, non-HDL, fatty acids, triglycerides); · The maximum distance between the maximum pharmacodynamic (HbA1c) and adverse effects (GE rate) normalized by the evolution of FGF21- (lipids) and GLP-1-mediated effects (HbA1c) was 121.189, i.e., at 121.189 (which can be rounded to 121), there is a maximum distance between the maximum pharmacodynamic (HbA1c) and adverse effects (GE rate) at the minimum distance between the GLP-1-mediated effects (HbA1c) and the mean FGF21-mediated effects (i.e., fat mass, non-HDL, fatty acids, triglycerides) (see Figure 2); The maximum distance between the maximum pharmacodynamic (HbA1c) and adverse effects (GE rate) was 319.311 (which can be rounded to 319); The maximum distance between mean pharmacodynamics (i.e., HbA1c, fat mass, non-HDL, fatty acids, triglycerides) and adverse effects (GE rate) was 482.396 (see Figure 2; can be rounded to 482); the maximum gastric emptying rate was 531.0; (all: vertical lines). [Figure 2]Graphs showing the EC50 of gastric emptying (GE) rate and mean pharmacodynamic effects (i.e., HbA1c, triglycerides, fatty acids, non-HDL, fat mass) depending on the GLP-1 decay rate (12-month simulation): The maximum distance between the mean pharmacodynamics (i.e., HbA1c, fat mass, non-HDL, fatty acids, triglycerides) and the adverse effect (GE rate) was 482.396 (right vertical line; can be rounded to 482). The maximum distance between the maximum pharmacodynamics (HbA1c) normalized by the evolution of FGF21- (lipids) and GLP-1-mediated effects (HbA1c) and the adverse effect (GE rate) was 121.189 (left vertical line; can be rounded to 121). The curve "(Max GE Rate) / Range" represents the ratio between the maximum distance between HbA1c and GE rate and the minimum distance between HbA1c and the mean FGF21-mediated 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), at the minimum distance between the GLP-1-mediated effects (HbA1c) and the FGF21-mediated effects (i.e., fat mass, non-HDL, fatty acids, triglycerides), there is the maximum distance between the maximum pharmacodynamic effect (HbA1c) and the adverse effect (GE rate). [Figure 3]Graph showing the EC50s of adverse effects (gastric emptying (GE) rate) and pharmacodynamics (HbA1c, triglycerides, fatty acids, non-HDL, fat mass) depending on the GLP-1 decay rate (3-month simulation): · For GLP-1 decay rates greater than 18.268 (which can be rounded to 18), the EC50 of GLP-1-mediated gastrointestinal adverse effects (gastric emptying; GE rate) was greater than the EC50 of pharmacodynamic effects (i.e., HbA1c, fat mass, non-HDL, fatty acids, triglycerides); · The maximum distance between the maximum pharmacodynamic (HbA1c) and adverse effect (GE rate) normalized by the evolution of FGF21-(lipids) and GLP-1-mediated effects (HbA1c) was 123.466, i.e., at 123.466 (which can be rounded to 123), there is a maximum distance between the maximum pharmacodynamic (HbA1c) and adverse effect (GE rate) at the minimum distance between the GLP-1-mediated effect (HbA1c) and the mean FGF21-mediated effects (i.e., fat mass, non-HDL, fatty acids, triglycerides) (see Figure 4); The maximum distance between the maximum pharmacodynamic (HbA1c) and adverse effect (GE rate) was 313.214 (which can be rounded to 313); The maximum distance between mean pharmacodynamics (i.e., HbA1c, fat mass, non-HDL, fatty acids, triglycerides) and adverse effects (GE rate) was 468.679 (see Figure 4; can be rounded to 469); the maximum gastric emptying rate was 500.686 (can be rounded to 501); (all: vertical lines). [Figure 4]Graphs showing the EC50 of gastric emptying (GE) rate and mean pharmacodynamic effects (i.e., HbA1c, triglycerides, fatty acids, non-HDL, fat mass) depending on the GLP-1 decay rate (3-month simulation): · The maximum distance between the mean pharmacodynamics (i.e., HbA1c, fat mass, non-HDL, fatty acids, triglycerides) and the adverse effect (GE rate) was 468.679 (right vertical line; can be rounded to 469); · The maximum distance between the maximum pharmacodynamics (HbA1c) normalized by the evolution of FGF21- (lipids) and GLP-1-mediated effects (HbA1c) and the adverse effect (GE rate) was 123.466 (left vertical line; can be rounded to 123). The curve "(Max GE Rate) / Range" represents the ratio between the maximum distance between HbA1c and GE rate and the minimum distance between HbA1c and the mean FGF21-mediated effects (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 effects (HbA1c) and the FGF21-mediated effects (i.e., fat mass, non-HDL, fatty acids, triglycerides), there is the maximum distance between the maximum pharmacodynamic effect (HbA1c) and the adverse effect (GE rate). [Figure 5] (A and B) Graphs showing the results of an in vitro cell assay (In-Cell Western, ICW) for the efficacy of human FGF21 receptor in CHO cells. pFGFR is shown in (A) and pERK is shown in (B). [Figure 6] (A-D) Graphs showing the results of an in vitro cell assay for the efficacy of different GLP-1R agonists at the human glucagon-like peptide 1 (GLP-1) receptor in HEK-293 cells. SEQ ID NO: 2 is shown in (A), SEQ ID NO: 7 is shown in (B), SEQ ID NO: 8 is shown in (C), and SEQ ID NOs: 2, 7, and 8 are shown in (D). [Figure 7-1](A-F) Graphs showing plasma concentrations of GLP-1R agonist / FGF21 Fc fusion protein after a single subcutaneous administration of a 0.3 mg / kg solution to female C57B1 / 6 mice or male cynomolgus monkeys using three different bioanalytical methods: (A) shows SEQ ID NO: 2 in mice, (B) shows SEQ ID NO: 2 in monkeys, (C) shows SEQ ID NO: 7 in mice, (D) shows SEQ ID NO: 7 in monkeys, (E) shows SEQ ID NO: 8 in mice, and (F) shows SEQ ID NO: 8 in monkeys. [Figure 7-2] Continued from Figure 7-1. [Figure 8] 1 is a graph showing plasma concentrations of GLP-1R agonist / FGF21 Fc fusion protein, and G-FGF21 (SEQ ID NO: 252) following a single subcutaneous administration of a 0.3 mg / kg solution to female C57B1 / 6 mice using a bioanalytical method for quantification of the intact full-length fusion protein. [Figure 9] 1 is a graph showing the evolution of body weight in female diet-induced obese (DIO) mice dosed with GLP-1RA / FGF21 Fc fusion protein and control once a week for 28 days. [Figure 10] 1 is a graph showing the evolution of cumulative food intake in female DIO mice dosed with GLP-1RA / FGF21 Fc fusion protein and control once a week for 28 days. [Figure 11] (A and B) Graphs showing 24-hour blood glucose profiles of db / db mice after the first treatment with GLP-1RA / FGF21 Fc fusion protein and control on day 1 (A) or after the fourth treatment starting on day 22 (B). Data are mean ± standard error of the mean, n=8 per group. [Figure 12] 1 is a graph showing plasma HbA1c content in female db / db mice dosed with GLP-1RA / FGF21 Fc fusion protein and control once weekly for 36 days. [Figure 13-1](A-B) Graphs showing the evolution of liver weight and lipid content in DIO NASH mice after 8 weeks of weekly dosing with GLP-1RA / FGF21 Fc fusion protein and control. (A) Liver weight and lipid levels, (B) liver cholesterol and liver triglyceride levels. [Figure 13-2] Continuation of Figure 13-1. [Figure 14] Graphs showing the development of fibrosis and the evolution of non-alcoholic fatty liver disease (NAFLD) activity scores in DIO NASH mice after 8 weeks of weekly dosing with GLP-1RA / FGF21 Fc fusion protein and control. [Figure 15] 1 shows graphs depicting the number of animals with high, the same, or low fibrosis and NAFLD activity scores in DIO NASH mice after 8 weeks of weekly dosing with GLP-1RA / FGF21 Fc fusion protein and control. DETAILED DESCRIPTION OF THE INVENTION

[0078] The present invention will be described in detail below, but it should be understood that the present invention is not limited to the specific methods, protocols, and reagents described herein, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0079] The following describes certain elements of the present invention. While these elements are listed according to specific embodiments, it should be understood that they can be combined in any manner and in any number to create additional embodiments. The variously described examples and exemplary embodiments should not be construed as limiting the invention to only the specifically described embodiments. The description should also be understood to support and encompass embodiments that combine the specifically described embodiment with any number of disclosed and / or exemplary elements. Furthermore, all sequences and combinations of all described elements in this application should be construed as disclosed by the description of this application, unless otherwise indicated by context.

[0080] The terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", edited by H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0081] The practice of the present invention will employ, unless otherwise indicated, conventional methods in chemistry, biochemistry, cell physiology, immunology, and recombinant DNA technology as described in the art (Sambrook, J. et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0082] Throughout the following specification and claims, unless the context otherwise requires, the word "comprise" and variations thereof, such as "comprises" and "comprising," imply the inclusion of not only the stated member, integer, or step, but also any other member, integer, or step, or group of members, integers, or steps; however, it should also be understood that in some embodiments, such other member, integer, or step, or group of members, integers, or steps, may be excluded, i.e., the subject matter consists of the inclusion of the stated member, integer, or step, or group of members, integers, or steps. The terms "a," "an," and "the" and similar referents used in the context of describing the invention (particularly in the context of the claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values ​​herein merely serves as a shorthand method of individually referring to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or 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 illustrate the invention and does not impose limitations on the scope of the invention unless otherwise asserted. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0083] Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instruction manuals, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the disclosure is not entitled to antedate such disclosure by virtue of prior invention.

[0084] The term "fusion protein" generally refers to a protein created by linking, particularly covalently bonding, two or more different proteins (e.g., proteins and / or peptides) resulting in a single molecule possessing functional properties derived from each of the original proteins. Generally, the fusion proteins of the present invention exhibit GLP-1R agonist activity and FGF21 activity. Fusion proteins can be created by genetic fusion (e.g., by recombinant DNA technology) or by chemical and / or enzymatic conjugation. In the fusion proteins according to the present invention, the components of the fusion protein can be arranged (from N-terminus to C-terminus) in the order ABC or CBA, where A is a GLP-1R agonist peptide, B is a linker molecule, and C is a functionally active variant of human FGF21.

[0085] The term "GLP-1R agonist peptide" as used herein refers to a peptide that binds to and activates a GLP-1 receptor, e.g., GLP-1 (as a primary GLP-1R agonist). A GLP-1R agonist peptide may also be simply referred to herein as a "GLP-1R agonist."

[0086] The term "peptide" generally refers to a polymeric form of amino acids of any length, including, for example, about 2 or more, or about 3 or more, or about 4 or more, or about 6 or more, or about 8 or more, or about 9 or more, or about 10 or more, or about 13 or more, or about 16 or more, or about 21 or more amino acids covalently linked by peptide bonds. Peptides consist, for example, of up to about 100 amino acids. The term "polypeptide" refers to large peptides. In one embodiment, the term "polypeptide" refers to a peptide having more than about 100 amino acid residues. The terms "polypeptide" and "protein" are used interchangeably herein.

[0087] In one embodiment, the GLP-1R agonist peptide is a variant of native GLP-1(7-36). The term "native GLP-1(7-36)" as used herein refers to a peptide having the amino acid sequence of SEQ ID NO: 260, optionally including an amide group at its C-terminus.

[0088] In general, variants of native GLP-1(7-36) may be based on the deletion, addition and / or substitution of at least one amino acid residue in / to the amino acid sequence of native GLP-1(7-36).

[0089] In one embodiment, the variant comprises substitutions of up to about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, or about 5 amino acid residues in the amino acid sequence of native GLP-1(7-36) (SEQ ID NO: 260).

[0090] The term "amino acid" or "amino acid residue," as used herein, refers to naturally occurring amino acids, unnatural amino acids, amino acid analogs, and amino acid mimetics that function in a manner similar to the naturally occurring amino acids, in their D and / or L stereoisomers, if their structure allows for such stereoisomeric forms. Amino acids are referred to herein by either their names or their three-letter symbols known in the art, or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0091] When used in reference to amino acids, the term "naturally occurring" refers to any of the 20 conventional amino acids (i.e., alanine (Ala or A), cysteine ​​(Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y)), as well as selenium. The term refers to lysine, pyrrolysine (PYL), and pyrroline-carboxylysine (PCL).

[0092] The term "unnatural amino acid" as used herein refers to an amino acid that is not naturally encoded or found in the genetic code of any organism.It may be, for example, a purely synthetic compound.Examples of unnatural 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-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, tert-butylglycine, 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, and 2,3-diaminopropionic acid. , N-ethylglycine, N-methylglycine, N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline, norleucine, ornithine, D-ornithine, D-arginine, p-aminophenylalanine, pentylglycine, pipecolic acid, and thioproline.

[0093] The term "amino acid analog" as used herein refers to a compound that has the same basic chemical structure as a naturally occurring amino acid.Amino acid analogs include natural and unnatural amino acids that are reversibly or irreversibly chemically blocked or chemically modified, for example, at one or any combination of their C-terminal carboxyl group, their N-terminal amino group, and / or their side chain functional groups.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.

[0094] The term "amino acid mimetic," as used herein, refers to a chemical compound that has a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0095] 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 a single amino acid residue. In one embodiment, the at least one additional amino acid residue is selected from naturally occurring amino acids, non-natural amino acids, amino acid analogs, and amino acid mimetics excluding proline. In one embodiment, the at least one additional amino acid residue is selected from the group consisting of G, A, N, and C. In one embodiment, the at least one additional amino acid residue is G or A. In one embodiment, the at least one additional amino acid residue is G.

[0096] In some embodiments, the variant comprises a peptide extension at its C-terminus. The peptide extension can consist of, for example, up to about 12, about 11, about 10, or about 9 amino acid residues (e.g., about 7, about 8, or about 9 amino acid residues). In one embodiment, the peptide extension consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 4008-4063. In one embodiment, the peptide extension is a single amino acid residue, e.g., P.

[0097] In one embodiment, the GLP-1R agonist peptide component of the fusion protein of the invention exhibits reduced GLP-1R agonist activity compared to that of native GLP-1(7-36) as defined herein. The phrase "GLP-1R agonist peptide as part of a fusion protein" means that the reduced GLP-1R agonist activity is exhibited when the GLP-1R agonist peptide is a component of a fusion protein, and not necessarily in its isolated form (i.e., when not a component of a fusion protein).

[0098] In one embodiment, the term "GLP-1R agonist activity" (or "GLP-1R agonist efficacy"), as used herein, refers to activation of the GLP-1 receptor. In one embodiment, the term refers to in vitro agonist activity / efficacy. In another embodiment, the term refers to in vivo agonist activity / efficacy. In one embodiment, the activity 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 derived from the HEK-293 cell line. In one embodiment, the GLP-1 receptor is a human GLP-1 receptor. In one embodiment, the activity of the GLP-1 receptor is determined essentially as described in Example 4. In one embodiment, the activity / efficacy is quantified by determining the EC50 value.

[0099] The term "fibroblast growth factor 21" or "FGF21," as used herein, refers to any FGF21 protein known in the art, and in particular to human FGF21. In one embodiment, human FGF21 has the amino acid sequence of SEQ ID NO: 250 (full-length human wild-type FGF21). Mature human wild-type FGF21, i.e., human wild-type FGF21 lacking amino acids 1-28 (M1-A28) of SEQ ID NO: 250 (i.e., the signal sequence / peptide), is represented by SEQ ID NO: 251. Mature human wild-type FGF21 with an additional N-terminal Gly is represented by SEQ ID NO: 252 and is referred to herein as G-FGF21.

[0100] In one embodiment, the functionally active variant of human FGF21 comprises an amino acid sequence that is at least about 96%, or at least about 97%, or at least about 98% identical to the amino acid sequence of SEQ ID NO: 250 or SEQ ID NO: 251, (i) substitutions Q55C and P147C or substitutions Q55C and N149C, and (ii) containing a substitution or deletion of G198 and / or P199; Amino acid residue numbering is according to SEQ ID NO:250.

[0101] Q55C in SEQ ID NO: 250 corresponds to Q27C in SEQ ID NO: 251, P147C in SEQ ID NO: 250 corresponds to P119C in SEQ ID NO: 251, N149C in SEQ ID NO: 250 corresponds to N121C in SEQ ID NO: 251, G198 in SEQ ID NO: 250 corresponds to G170 in SEQ ID NO: 251, and P199 in SEQ ID NO: 250 corresponds to P171 in SEQ ID NO: 251.

[0102] "Sequence identity" between two amino acid sequences indicates the percentage of identical amino acids between the sequences. Optimal alignment of sequences for comparison can be performed manually or 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 (Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Wisconsin, USA). GAP, BESTFIT, FASTA, BLAST in Madison, Wis. P, BLAST N, and TFASTA).

[0103] When used in reference to FGF21 (eg, human FGF21), the term "functionally active variant" refers to a protein that has FGF21 activity.

[0104] In one embodiment, the term "FGF21 activity" (or "FGF21 potency"), as used herein, refers to the activity of an FGF21 receptor (FGFR, e.g., FGFR1c). In one embodiment, the FGF21 receptor is a human FGF21 receptor. In one embodiment, the term refers to in vitro activity / potency. In another embodiment, the term refers to in vivo activity / potency. In one embodiment, activation of the FGF21 receptor is determined by measuring FGF21 receptor autophosphorylation and / or phosphorylation of MAPK ERK1 / 2 upon contact with an FGF21 compound in vitro. In one embodiment, autophosphorylation of human FGFR1c and / or phosphorylation of MAPK ERK1 / 2 are determined, for example, by using an In-Cell Western (ICW), essentially as described in Example 3. In one embodiment, activity and / or potency are quantified by determining an EC50 value.

[0105] The term "In-Cell Western (ICW) assay" as used herein refers to immunocytochemical assay, more specifically, quantitative immunofluorescence assay, which is usually carried out in microplate (for example, 96-well format or 384-well format).It combines the specificity of Western blot with the reproducibility and throughput of ELISA (for example, see Aguilar HN et al. (2010) PLoS ONE 5(4):e9965).Suitable ICW assay systems are commercially available (for example, LI-COR Biosciences, USA).In one embodiment, anti-pFGFR and / or anti-pERK are used in ICW assay.

[0106] In one embodiment, the functionally active variant of human FGF21 exhibits the same or substantially the same FGF21 activity as that of wild-type human FGF21 (e.g., SEQ ID NO: 250 or 251 or 252), and the FGF21 activity refers to the FGF21 activity of an isolated functionally active variant of human FGF21, i.e., when it is not contained in a fusion protein of the present invention or modified by any other means.

[0107] When used in reference to FGF21 (e.g., human FGF21), the term "substantially the same" refers to FGF21 activity that is within 50-150%, or 60-140%, or 65-135% of the FGF21 activity of FGF21 (e.g., wild-type human FGF21 (e.g., SEQ ID NO: 250 or 251 or 252)).

[0108] Optionally, the functionally active variant of human FGF21 further comprises substitutions G141S and / or P174L, which are naturally occurring mutations in human FGF21, where the numbering of amino acid residues is according to SEQ ID NO: 250. G141S in SEQ ID NO: 250 corresponds to G113S in SEQ ID NO: 251, and P174L in SEQ ID NO: 250 corresponds to P146L in SEQ ID NO: 251.

[0109] Further suitable FGF21 variants for use in the present invention are described, for example, in PCT / EP2016 / 079551, which is incorporated herein by reference.

[0110] In one embodiment, the functional relationship between a GLP-1R agonist peptide and human FGF21 is The variants active against an immunoglobulin are linked via a linker molecule comprising a structure selected from the group consisting of L-Fc, Fc-L, L1-Fc-L2, and Fc, where L, L1, and L2 are independently selected from the group consisting of single amino acids and peptides, and Fc is the Fc domain of an immunoglobulin or variant thereof.

[0111] In one embodiment, the Fc domain (also referred to as Fc region) is an Fc domain of immunoglobulin IgG1 or IgG4. In one embodiment, the variant of the Fc domain contains up to about six, about five, or about four mutations compared to the wild-type sequence of the Fc domain. In one embodiment, the mutations are selected from the group consisting of amino acid substitutions, amino acid additions, and amino acid deletions, e.g., N- or C-terminal deletions. In one embodiment, the Fc domain or variant thereof may have greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 93%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, or greater than about 99% sequence identity, or about 100% sequence identity, with the wild-type sequence of an IgG1 Fc region, e.g., a human IgG1 Fc region. In one embodiment, the Fc domain or variant thereof may have greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 93%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, or greater than about 99% sequence identity, or may have about 100% sequence identity, to the wild-type sequence of an IgG4 Fc region, e.g., a human IgG4 Fc region. In one embodiment, the Fc domain of the immunoglobulin or variant thereof comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 257, 258, and 259.

[0112] In one embodiment, the peptide in the linker molecule (also referred to herein as a "peptide linker") has a length of about 2 to about 100 amino acid residues, or about 2 to about 90 amino acid residues, or about 2 to about 80 amino acid residues, or about 2 to about 70 amino acid residues, or about 2 to about 60 amino acid residues, or about 2 to about 50 amino acid residues, or about 2 to about 40 amino acid residues, or about 2 to about 30 amino acid residues, or about 2 to about 25 amino acid residues, or about 2 to about 20 amino acid residues. In one embodiment, the peptide linker contains at least about 5 amino acid residues. Generally, peptide linkers are designed to provide flexibility and protease resistance. In one embodiment, the peptide linker is a glycine-serine-rich linker, e.g., at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 85% of the amino acids are glycine or serine residues, respectively. In another embodiment, the amino acids are selected from glycine and serine, i.e., the peptide linker is composed exclusively of glycine and serine (referred to as a glycine-serine linker). In one embodiment, the peptide linker further comprises an alanine residue at its C-terminus. The peptide linker may further comprise one or more specific protease cleavage sites. In one embodiment, the peptide linker comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 231-245. L1 and L2 may be the same or different. In one embodiment, L1 and L2 are different. In one embodiment, L1 comprises or consists of the amino acid sequence of SEQ ID NO: 232, and L2 comprises or consists of the amino acid sequence of SEQ ID NO: 231, or vice versa.

[0113] The term "functionally active variant" when used in connection with the fusion proteins of the present invention refers to a fusion protein that has GLP-1R agonist activity and FGF21 activity within the ranges defined herein.

[0114] In one embodiment, a functionally active variant is an amino acid sequence of the fusion protein from which it is derived. The amino acid sequence may comprise or consist of an amino acid sequence that is at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identical to the amino acid sequence of the target polypeptide.

[0115] In one embodiment, deviations in the amino acid sequence of a functionally active variant from the amino acid sequence of the fusion protein from which it is derived are based exclusively on mutations (e.g., substitutions, deletions, and / or additions of one or more amino acids) that occur in regions of the fusion protein that are not involved in its GLP-1R agonist activity and / or FGF21 activity. In one embodiment, mutations occur exclusively outside the amino acid sequence of the functionally active variant of the GLP-1R agonist peptide and / or human FGF21 contained in the fusion protein. In one embodiment, deviations in the amino acid sequence of a functionally active variant from the amino acid sequence of the fusion protein from which it is derived are based exclusively on conservative amino acid substitutions.

[0116] Conservative amino acid substitutions involve replacing one amino acid with another from the same amino acid family, i.e., amino acids related in their side chains (e.g., in terms of charge and / or size). Naturally occurring amino acids are generally divided into four families: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids.

[0117] A "nucleic acid molecule" according to the present invention is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). A nucleic acid molecule according to the present invention may be in the form of a single-stranded or double-stranded molecule. A nucleic acid molecule according to the present invention may be linear or may be covalently closed to form a circle.

[0118] The term "DNA" refers to a molecule containing, or in some cases consisting entirely or substantially of, deoxyribonucleotide residues. "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, e.g., partially or completely purified DNA, essentially pure DNA, synthetic DNA, and DNA produced by genetic recombination. The term "DNA" also includes modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications can include the addition of non-nucleotide material, such as to one or more nucleotides at the end or within the DNA. Nucleotides in a DNA molecule can also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides. Modified DNA molecules can be referred to as analogs or analogs of naturally occurring DNA.

[0119] The term "RNA" refers to a molecule comprising, and in some cases entirely or substantially composed of, ribonucleotide residues. "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a beta-D-ribofuranosyl group. The term "RNA" includes isolated RNA, e.g., partially or completely purified RNA, essentially pure RNA, synthetic RNA, and RNA produced by genetic recombination. The term "RNA" also includes modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications can include the addition of non-nucleotide material, such as to the end or within the RNA, e.g., to one or more nucleotides of the RNA. Nucleotides in an RNA molecule can be non-standard nucleotides, e.g., non-naturally occurring nucleotides or chemically synthesized nucleotides. The modified RNA molecule may also contain nucleotides, nucleotides, or deoxynucleotides. The modified RNA molecule may be referred to as an analog or an analog of naturally occurring RNA. According to the present invention, "RNA" refers to single-stranded RNA or double-stranded RNA. In one embodiment, the RNA is mRNA, for example, in vitro transcribed RNA (IVT RNA), or synthetic RNA. The RNA can be modified, for example, by one or more modifications that increase the stability (e.g., half-life) of the RNA. Such modifications are known to those skilled in the art and include, for example, a 5'-cap or a 5'-cap analog.

[0120] The term "naturally occurring" when used in conjunction with nucleotides refers to the bases adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U).

[0121] The nucleic acid molecule according to the present invention can be contained in a vector. As used herein, the term "vector" includes all vectors known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors (e.g., lambda phage vectors), viral vectors (e.g., adenovirus or baculovirus vectors), or artificial chromosome vectors (e.g., bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or P1 artificial chromosomes (PACs)). These vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors and generally contain a desired coding sequence and appropriate DNA sequences required for expression of the operably linked coding sequence in a specific host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are generally used to design and amplify a specific desired DNA fragment and may lack functional sequences required for expression of the desired DNA fragment.

[0122] Alternatively, the nucleic acid molecule according to the invention may be integrated into a genome, for example the genome of a host cell. Means and methods for integrating particular nucleic acid molecules into a genome are well known to those skilled in the art.

[0123] In certain exemplary embodiments, the term "cell" or "host cell" refers to an intact cell, i.e., a cell with an intact membrane that has not released its normal intracellular components, such as enzymes, organelles, or genetic material. In certain exemplary embodiments, an intact cell is a viable cell, i.e., a living cell that is capable of carrying out its normal metabolic functions. In certain exemplary embodiments, a cell or host cell is any cell that can be transfected or transformed with an exogenous nucleic acid. In certain exemplary embodiments, a cell that has been transfected or transduced with an exogenous nucleic acid and transferred to a recipient is capable of expressing the nucleic acid in the recipient.

[0124] The term "cell" includes prokaryotic cells, such as bacterial cells, and eukaryotic cells, such as yeast cells, fungal cells, or mammalian cells. Suitable bacterial cells include, but are not limited to, cells from gram-negative bacterial strains, such as Escherichia coli, Proteus, and Pseudomonas, and gram-positive bacterial strains, such as Bacillus, Streptomyces, Staphylococcus, and Lactococcus. Suitable fungal cells include, but are not limited to, cells from species of Trichoderma, Neurospora, and Aspergillus. Yeast cells include cells derived from Saccharomyces species (e.g., Saccharomyces cerevisiae), Schizosaccharomyces species (e.g., Schizosaccharomyces pombe), Pichia species (e.g., Pichia pastoris and Pichia methanolica), and Hansenula species. Suitable mammalian cells include, but are not limited to, CHO cells, BHK cells, HeLa cells, COS cells, HEK-293 cells, and the like. In one embodiment, HEK-293 cells are used. However, amphibian cells, insect cells, plant cells, and any other cells used in the art for the expression of heterologous proteins can also be used. In certain exemplary embodiments, mammalian cells (e.g., cells derived from a human, mouse, hamster, pig, goat, or primate) are used for adoptive transfer. These cells can be derived from many tissue types and include primary cells and cell lines, such as cells of the immune system (e.g., antigen-presenting cells, such as dendritic cells and T cells, stem cells, such as hematopoietic stem cells and mesenchymal stem cells), as well as other cell types.

[0125] Antigen-presenting cells, as used herein, are cells that present 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" may be isolated or may be part of a tissue or organism, particularly a "non-human body."

[0126] The term "non-human organism," as used herein, is meant to include non-human primates or other animals, e.g., mammals, such as cows, horses, pigs, sheep, goats, dogs, cats, rabbits, and rodents (e.g., mice, rats, guinea pigs, or hamsters).

[0127] Pharmaceutical compositions according to the present invention include one or more carriers and / or excipients, all of which are pharmaceutically acceptable. The term "pharmaceutically acceptable," as used herein, in certain exemplary embodiments, refers to a non-toxic material that does not interact with the action of the active agent of the pharmaceutical composition.

[0128] The term "carrier" as used herein refers to a natural or synthetic organic or inorganic component that is combined with an active ingredient to facilitate, enhance, or enable application. According to the present invention, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents, or encapsulating substances that are suitable for administration to a subject.

[0129] Suitable carrier materials for parenteral administration include, but are not limited to, sterile water, Ringer's solution, lactated Ringer's solution, normal saline, bacteriostatic saline (e.g., saline containing 0.9% benzyl alcohol), phosphate-buffered saline (PBS), Hank's solution, polyalkylene glycols, hydrogenated naphthalenes, and biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxy-propylene copolymers, among others.

[0130] The term "excipient," as used herein, is intended to include all substances that are not active ingredients but may be present in a pharmaceutical composition, such as, for example, salts, binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), fillers, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffer substances, flavorings, or coloring agents, etc.

[0131] Pharmaceutically unacceptable salts may be used to prepare pharmaceutically acceptable salts and are included in the present invention. Pharmaceutically acceptable salts of this type include, but are not limited to, 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, etc. 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 the ionic strength or tonicity of the pharmaceutical composition.

[0132] Suitable preservatives for use in the pharmaceutical compositions include, but are not limited to, antioxidants, citric acid, sodium citrate, benzalkonium chloride, chlorobutanol, cysteine, methionine, parabens, thimerosal, phenol, cresol, and mixtures thereof.

[0133] Suitable buffering substances for use in pharmaceutical compositions include, but are not limited to, acetic acid in a salt, citric acid in a salt, boric acid in a salt, phosphoric acid in a salt, and tris(hydroxymethyl)aminomethane (Tris, THAM, trometamol).

[0134] In certain exemplary embodiments, the pharmaceutical composition according to the present invention is sterile. The pharmaceutical composition can be provided in a uniform dosage form and can be manufactured in a manner known to those skilled in the art. The pharmaceutical composition can be, for example, in the form of a solution or suspension.

[0135] The pharmaceutical compositions may also be formulated as stable lyophilized products to be reconstituted with a suitable diluent, optionally containing one or more excipients as defined above.

[0136] The pharmaceutical composition according to the present invention may further comprise at least one other active pharmaceutical ingredient.

[0137] The term "active pharmaceutical ingredient" (API), as used herein, includes any pharmaceutically active chemical or biological compound, as well as any pharmaceutically acceptable salts thereof, and any mixtures thereof, that provides some pharmacological effect and is used, for example, to treat or prevent a condition, such as a disease or disorder, as defined herein.

[0138] Exemplary pharmaceutically acceptable salts include, but are not limited to, salts made from one or more of the following acids: hydrochloric acid (e.g., chloride salts), sulfuric acid (e.g., sulfate salts), nitric acid (e.g., nitrate salts), phosphoric acid (e.g., phosphate salts), hydrobromic acid (e.g., hydrobromide salts), maleic acid (e.g., maleate salts), malic acid (e.g., malate salts), ascorbic acid, citric acid (e.g., citrate salts), tartaric acid (e.g., tartrate salts), pamoic acid (e.g., pamoate salts), lauric acid (e.g., laurate salts), stearic acid (e.g., stearate salts), palmitic acid (e.g., palmitate salts), oleic acid, myristic acid (e.g., myristate salts), lauric acid, naphthalenesulfonic acid, linolenic acid (e.g., linolenic acid), and the like.

[0139] As used herein, the terms "active pharmaceutical ingredient," "active agent," "active ingredient," "active substance," "therapeutically active compound," and "drug" are intended to be synonymous, i.e., have the same meaning.

[0140] In accordance with the present invention, the active pharmaceutical ingredient is optionally selected from: - All drugs mentioned in the Rote Liste 2014, e.g. All antidiabetic drugs mentioned in Rote Liste 2014, chapter 12, all weight loss or appetite suppressants mentioned in Rote Liste 2014, chapter 06, all lipid-lowering drugs mentioned in Rote Liste 2014, chapter 58, Rote Liste 2014 chapter 17, all antihypertensives mentioned in the Rote Liste, all nephroprotectives mentioned in the Rote Liste, or all diuretics mentioned in Rote Liste 2014 chapter 36; - insulin and insulin derivatives, for example: insulin glargine (e.g. Lantus®), insulin glargine concentrated to more than 100 U / mL, for example 270-330 U / mL insulin glargine or 300 U / mL insulin glargine (disclosed in EP 2387989), insulin glulisine (e.g. Apidra®), insulin detemir (e.g. Levemir®), insulin lispro (e.g. Humalog®, Liprolo®), g®), insulin degludec (e.g., DegludecPlus®, IdegLira (NN9068)), insulin aspart and aspart formulations (e.g., NovoLog®), basal insulins and analogs (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® N, Novolin® N), rapid- and short-acting insulins (e.g., Humulin® R, Novolin® R, Linjeta® (VIAject®), PH20 insulin, NN1218, HinsBet®), premixed insulin, SuliXen®, NN1045, insulin + Symlin®, PE-0139, ACP-002 insulin Hydrogel insulins, and oral, inhalable, transdermal, and buccal or sublingual insulins (e.g., Exubera®, Nasulin®, Afrezza®, insulin tregopil, TPM-02 insulin, Capsulin®, Oral-lyn®, Cobalamin®, oral insulin, ORMD-0801, Oshadi oral insulin, NN1953, NN1954, NN1956, VIAtab®). Derivatives of these insulins linked to albumin or other proteins by bifunctional linkers are also suitable; - glucagon-like peptide 1 (GLP-1), GLP-1 analogues and GLP-1 receptor agonists, such as: GLP-1(7-37), GLP-1(7-36)amide, lixisenatide (e.g., Lyxumia®), exenatide (e.g., exendin-4, rexendin-4, Byetta®, Bydureon®, exenatide NexP), exenatide-LAR, liraglutide (e.g., Victoza®), semaglutide, taspoglutide, albiglutide, dulaglutide, albumon, oxyntomodulin, geniproside, ACP-003, CJC-1131, CJC-1 134-PC, GSK-2374697, PB-1023, TTP-054, langrenatide (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; - Dual GLP-1 / GIP agonists (e.g., RG-7697 (MAR-701), MAR-709, BHM081, BHM089, BHM098); dual GLP-1 / glucagon receptor agonists (e.g., BHM-034, OAP-189 (PF-05212) 389, TKS-1225), TT-401 / 402, ZP2929, LAPS-HMOXM25, MOD-6030); - Dual GLP-1 / gastrin agonists (e.g., ZP-3022); - gastrointestinal peptides, such as peptide YY3-36 (PYY3-36) or an analog thereof and pancreatic polypeptide (PP) or an analog thereof; - glucagon receptor agonists or antagonists, glucose-dependent insulinotropic polypeptide (GIP) receptor agonists or antagonists, ghrelin antagonists or inverse agonists, xenin and its analogs; - dipeptidyl peptidase-IV (DPP-4) inhibitors, for example: alogliptin (e.g. Nesina®, Kazano®), linagliptin (e.g. Ondero®, Trajenta®, Tradjenta®, Trayenta®), saxagliptin (e.g. Onglyza®, Komboglyze XR®), sitagliptin (e.g. Januvia®, Xelevia®, Tesavel®, Janumet®, Velmetia®, Juvisync®, Janumet®), XR®), anagliptin, teneligliptin (e.g., Tenelia®), trelagliptin, vildagliptin (e.g., Galvus®, Galvumet®), gemigliptin, omarigliptin, evogliptin, dutogliptin, DA-1229, MK-3102, KM-223, KRP-104, PBL-1427, pinoxacin hydrochloride, and Ari-2243; - sodium-dependent glucose transporter 2 (SGLT-2) inhibitors, such as: canagliflozin, dapagliflozin, remogliflozin, remogliflozin etabonate, sergliflozin, empagliflozin, ipragliflozin, tofogliflozin, luseogliflozin, ertugliflozin, EGT-0001442, LIK-066, SBM-TFC-039, and KGA-3235 (DSP-3235); - Dual inhibitors of SGLT-2 and SGLT-1 (e.g., LX-4211, LIK066). - anti-obesity drugs, for example SGLT-1 inhibitors (e.g. LX-2761, KGA-3235) or SGLT-1 inhibitors in combination with ileal bile acid transporter (IBAT) inhibitors (e.g. GSK-1614235+GSK-2330672); - Biguanides (e.g. metformin, buformin, phenformin); - thiazolidinediones (e.g., pioglitazone, rosiglitazone), glitazone analogues (e.g., lobeglitazone); - peroxisome proliferator-activated receptor (PPAR-) (alpha, gamma, or alpha / gamma) agonists or modulators (e.g., saroglitazar (e.g., Lipaglyn®), GFT-505), or PPAR gamma partial agonists (e.g., Int-131); sulfonylureas (e.g., tolbutamide, glibenclamide, glimepiride, Amaryl®, glipizide) and meglitinides (e.g., nateglinide, repaglinide, mitiglinide); - alpha-glucosidase inhibitors (e.g., acarbose, miglitol, voglibose); - amylin and amylin analogues (e.g., pramlintide, Symlin®); - 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); - GPR40 agonists (e.g., TUG-424, P-1736, P-11187, JTT-851, GW9508, CNX-011-67, AM-1638, AM-52 62); - GPR120 agonists and GPR142 agonists; - systemic or poorly absorbed TGR5 (GPBAR1 = G protein-coupled bile acid receptor 1) agonists (e.g., INT-777, XL-475, SB756050); - diabetes immunotherapy, for example: oral CC chemokine receptor type 2 (CCR-2) antagonists (e.g., CCX-140, JNJ-41443532), interleukin-1 beta (IL-1β) antagonists (e.g., AC-201), or oral monoclonal antibodies (MoA) (e.g., methazolamide, VVP808, PAZ-320, P-1736, PF-05175157, PF-04937319); - anti-inflammatory agents for the treatment of metabolic syndrome and diabetes, such as: nuclear factor kappa B inhibitors (e.g. Triolex®); - adenosine monophosphate-activated protein kinase (AMPK) stimulators, such as: Imeglimin (PXL-008), Debio-0930 (MT-63-78), R-118; - inhibitors of 11-beta-hydroxysteroid dehydrogenase 1 (11-beta-HSD-1) (e.g., LY2523199, BMS770767, RG-4929, BMS816336, AZD-8329, HSD-016, BI-135585); - glucokinase activators (e.g., PF-04991532, TTP-399 (GK1-399), GKM-001 (ADV-1002401), ARRY-403 (AMG-151), TAK-329, TMG-123, ZYGK1); - inhibitors of diacylglycerol O-acyltransferase (DGAT) (e.g., pradigastat (LCQ-908)), inhibitors of protein tyrosine phosphatase 1 (e.g., trodusquemine), inhibitors of glucose-6-phosphatase, inhibitors of fructose-1,6-bisphosphatase, inhibitors of glycogen phosphorylase, inhibitors of phosphoenolpyruvate carboxykinase, inhibitors of glycogen synthase kinase, inhibitors of pyruvate dehydrogenase kinase; - Glucose transporter-4 modulators, somatostatin receptor 3 agonists (e.g., MK-4256); - one or more lipid-lowering agents are also suitable as combination partners, for example: 3-hydroxy-3-methylglutaryl-coenzyme-A-reductase (HMG-CoA-reductase) inhibitors, for example simvastatin (e.g. Zocor®, Inegy®, Simcor®), atorvastatin (e.g. Sortis®, Caduet®), rosuvastatin (e.g. Crestor®), pravastatin (e.g., Lipostat®, Selipran®), fluvastatin (e.g., Lescol®), pitavastatin (e.g., Livazo®, Livalo®), lovastatin (e.g., Mevacor®, Advicor®), mevastatin (e.g., Compactin®), rivastatin, cerivastatin (Lipobay®), fibrates, e.g., bezafibrate (e.g., Cedur®, Retard), ciprofibrate (e.g., Hyperlipen®), fenofibrate (e.g., Antara®, Lipofen®, Lipanthyl®), gemfibrozil (e.g., Lopid®, Gevilon®), etofibrate, simfibrate, lonifibrate, clinofibrate, clofibrate, nicotinic acid and its derivatives (e.g., Nia, niacin, such as sustained-release formulations of niacin), nicotinic acid receptor 1 agonists (e.g., GSK-256073), PPAR-delta agonists, acetyl-CoA-acetyltransferase (ACAT) inhibitors (e.g., avasimibe), cholesterol absorption inhibitors (e.g., ezetimibe, Ezetrol®, Zetia®, Liptruzet®, Vytorin®, S-556971), bile acid binders (e.g., cholestyramine, resevelam), ileal bile acid transporter (IBAT) inhibitors (e.g., GSK-2330672, LUM-002), microsomal triglyceride transfer protein (MTP) inhibitors (e.g., lomitapide (AEGR-733), SLx-4090, granotapide), proprotein convertase subtilisin / kexin type 9 (PCSK9) modulators (e.g., alirocumab (REGN727 / SAR236553), AMG-145, LGT-209, PF-04950615, MPSK3169) A, LY3015014, ALD-306, ALN-PCS, BMS-962476, SPC5001, ISIS-394814, 1B20, LGT-210, 1D05, BMS-PCSK9Rx-2, SX-PCK9, RG7652), LDL receptor upregulators, e.g., liver-selective thyroid hormone receptor beta agonists (e.g., eprotirom (KB-2115), MB07811, sobetirom (QRX-431), VIA-3196, ZYT1), HDL-raising compounds compounds), such as: cholesteryl ester transfer protein (CETP) inhibitors (e.g., anacetrapib (MK0859), dalcetrapib, evacetrapib, 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) regulators, lipid metabolism modulators (e.g., BMS-823778, TAP-301, DRL-21994, DRL-21995), phospholipase A2 (PLA2) inhibitors (e.g., dalcetrapib, evacetrapib, JTT-302, DRL-17822, TA-8995, R-1658, LY-2484595, DS-1442), lapladib, Tyrisa®, varespladib, rilapladib), ApoA-I enhancers (e.g., RVX-208, CER-001, MDCO-216, CSL-112), cholesterol synthesis inhibitors (e.g., ETC-1002), lipid metabolism modulators (e.g., BMS-823778, TAP-301, DRL-21994, DRL-21995), and omega-3 fatty acids and their derivatives (e.g., ethyl icosapentate (AMR101), Epanova®, AKR-063, NKPL-66, PRC-4016, CAT-2003); Bromocriptine (e.g., Cyclotet®, Parlodel®), phentermine and phentermine formulations or combinations (e.g., Adipex-P, Ionamin, Qsymia®), benzphetamine (e.g., Didrex®), diethylpropion (e.g., Tenuate®), phendimetrazine (e.g., Adipost®, Bontril®), bupropion and combinations (e.g., Zyban®, Wellbutrin®), XL®, Contrave®, Empatic®), sibutramine (e.g., Reductil®, Meridia®), topiramate (e.g., Topamax®), zonisamide (e.g., Zonegran®), tesofensine, opioid antagonists such as naltrexone (e.g., Naltrexin®, naltrexone plus 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., Verne®, Perito, S-234462), NPY4 agonists (e.g., PP-1420), beta-3-adrenergic receptor agonists, leptin or leptin mimetics, agonists of the 5-hydroxytryptamine 2c (5HT2c) receptor (e.g., lorcaserin, Belviq®), pramlintide / metreleptin, lipase inhibitors, e.g., cetilistat (e.g., Cametor®), orlistat Stats (e.g., Xenical®, Calobalin®), angiogenesis inhibitors (e.g., ALS-L1023), beta-histidine and histamine H3 antagonists (e.g., HPP-404), AgRP (agouti-related protein) inhibitors (e.g., TTP-435), serotonin reuptake inhibitors, e.g., fluoxetine (e.g., Fluctine®), duloxetine anti-inflammatory drugs (e.g., Cymbalta®), dual or triple monoamine uptake inhibitors (dopamine, norepinephrine, and serotonin reuptake inhibitors), such as sertraline (e.g., Zoloft®), tesofensine, methionine aminopeptidase-2 (MetAP2) inhibitors (e.g., beloranib), and antisense oligonucleotides against the production of fibroblast growth factor receptor 4 (FGFR4) (e.g., ISIS-FGFR4Rx) or prohibitin targeting peptide-1 (e.g., Adipotide®); nitrate oxide donors, AT1 antagonists or angiotensin II (AT2) receptor antagonists, such as telmisartan (e.g., Kinzal®, Micardis®), candesartan (e.g., Atacand®, Blopress®), valsartan (e.g., Diovan®, Co-Diovan®), losartan (e.g., Cosaar®), eprosartan (e.g., Teveten®), irbesartan (e.g., For example, Aprovel®, CoAprovel®), olmesartan (e.g., Votum®, Olmetec®), tasosartan, azilsartan (e.g., Edarbi®), 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, aldo steroid antagonists, aldosterone synthase inhibitors, alpha-blockers, alpha-2 adrenoceptor antagonists, beta-blockers, mixed alpha / beta-blockers, calcium antagonists, calcium channel blockers (CCBs), intranasal calcium channel blocker diltiazem (e.g., CP-404), dual mineralocorticoids / CCBs, centrally acting antihypertensives, inhibitors of neutral endopeptidase, aminopeptidase A inhibitors, vasopeptide inhibitors, dual vasopeptide inhibitors, e.g., neprilysin Suitable drugs include ACE inhibitors or neprilysin-ECE inhibitors, dual-acting AT1 receptor-neprilysin inhibitors, dual AT1 / ETA antagonists, advanced glycation end products (AGE) degraders, recombinant renalase, blood pressure vaccines, e.g., anti-RAAS (renin-angiotensin-aldosterone-system) vaccines, AT1- or AT2-vaccines, hypertension pharmacogenomics-based drugs, e.g., modulators of genetic polymorphisms with antihypertensive responses, thrombocyte aggregation inhibitors, and others or combinations thereof.

[0141] As used herein, the term "kit of parts" (briefly: kit) refers to a product comprising one or more containers and, optionally, a data medium. The one or more containers can be filled with one or more of the agents of the present invention, e.g., fusion proteins, pharmaceutical compositions, and related agents, e.g., nucleic acid molecules and host cells. The kit may also include additional containers containing, for example, diluents, buffers, and additional reagents. The data medium may be a non-electronic data medium, e.g., a graphic data medium, e.g., an information leaflet, information sheet, bar code, or access code, or an electronic data medium, e.g., a compact disc (CD), digital versatile disc (DVD), microchip, or another semiconductor-based electronic data medium. The access code allows access to a database, e.g., an internet database, a centralized database, a distributed database, etc. The data medium may also include instructions for use of the agents of the present invention, e.g., fusion proteins, pharmaceutical compositions, and related agents, such as the nucleic acid molecules and host cells described herein.

[0142] The agents and compositions described herein can be administered by any conventional route, for example, orally, pulmonary, inhalation, or parenterally by injection or infusion. In one embodiment, parenteral administration is used intravenously, intraarterially, subcutaneously, intradermally, or intramuscularly. The agents and compositions described herein can also be administered by sustained release administration. It can also be administered by

[0143] Pharmaceutical compositions suitable for parenteral administration usually comprise sterile aqueous or non-aqueous preparations of active compounds, which are optionally isotonic with the recipient's blood. Examples of compatible carriers / solvents / diluents include sterile water, Ringer's solution, lactated Ringer's solution, physiological saline, bacteriostatic saline (e.g., physiological saline containing 0.9% benzyl alcohol), phosphate-buffered saline (PBS), and Hank's solution. In addition, sterile non-volatile oils can usually be used as a solution or suspension medium.

[0144] The agents and compositions described herein are usually administered in a therapeutically effective amount. "Therapeutically effective amount" refers to an amount that achieves a desired therapeutic response or a desired therapeutic effect, either alone or together with further doses, possibly without causing unacceptable side effects. In the case of the treatment of a specific disease or a specific condition, the desired response relates to the inhibition of the course of the disease. This includes slowing down the progression of the disease, and particularly halting or reversing the progression of the disease. The desired response in the treatment of a disease or condition may be the delay or prevention of the onset of the disease or condition. The effective amount of the agents and compositions described herein will depend on the condition being treated, the severity of the disease, individual parameters of the subject, such as age, physiological condition, size, and weight, the duration of treatment, the type of concomitant therapy (if any), the specific route of administration, and similar factors. Therefore, the dose of the agents described herein to be administered depends on various such parameters. If the response in the subject is not sufficient with the initial dose, a higher dose (or a higher dose effectively achieved by another, more localized route of administration) can be used.

[0145] According to the present invention, the term "disease or disorder" refers to any pathological or unhealthy condition, in particular obesity, overweight, metabolic syndrome, diabetes, diabetic retinopathy, hyperglycemia, dyslipidemia, non-alcoholic steatohepatitis (NASH), and / or atherosclerosis.

[0146] The term "obesity" refers to a medical condition in which excess body fat has accumulated to an extent that can have negative effects on health. For human (adult) subjects, obesity is defined as a weight gain greater than 30 kg / m 2 Body mass index (BMI) or higher (BMI ≥ 30 kg / m 2 ) can be defined as

[0147] The term "overweight" refers to a medical condition in which the amount of body fat exceeds that which is optimally healthy. For human (adult) subjects, obesity is defined as an average body weight of 25 kg / m2 Body mass index (BMI) of 25 kg / m or more (e.g., 25 kg / m 2 BMI < 30 kg / m 2 ) can be defined as

[0148] BMI is a simple measure of weight-to-height ratio commonly used to classify overweight and obesity in adults. It is defined as a person's weight in kilograms divided by the square of their height in meters (kg / m 2 ).

[0149] "Metabolic syndrome" can be defined as a clustering of at least three of the following medical conditions: abdominal (central) obesity (e.g., waist circumference ≥ 94 cm for Caucasian men and ≥ 80 cm for Caucasian women, with other groups defined as having ethnic-specific values), high blood pressure (e.g., ≥ 130 / 85 mmHg), high fasting plasma glucose (e.g., at least 100 mg / dL), high serum triglycerides (at least 150 mg / dL), and low high-density lipoprotein (HDL) levels (e.g., < 40 mg / dL for men and < 50 mg / dL for women).

[0150] "Diabetes mellitus" (also simply "diabetes") refers to a group of metabolic diseases characterized by high levels of glucose in the blood 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, slow-onset autoimmune diabetes in adults (LADA), maturity-onset diabetes of the young (MODY), and other types of diabetes resulting from certain genetic conditions, drugs, malnutrition, infections, and other illnesses.

[0151] The current WHO diagnostic criteria for diabetes are as follows: fasting plasma glucose ≥ 7.0 mmol / l (126 mg / dL) or 2-hour plasma glucose ≥ 11.1 mmol / l (200 mg / dL).

[0152] "Type 1 diabetes" (also known as "insulin-dependent diabetes mellitus (IDDM)" or "juvenile diabetes") is a condition characterized by high blood glucose levels caused by a complete lack of insulin. It occurs when the body's immune system attacks and destroys insulin-producing beta cells in the pancreas. The pancreas produces little or no insulin. Pancreas removal or pancreatic disease can also result in a deficiency of insulin-producing beta cells. Type 1 diabetes accounts for between 5% and 10% of diabetes cases.

[0153] "Type 2 diabetes" (also known as "non-insulin-dependent diabetes mellitus (NIDDM)" or "adult-onset diabetes") is a condition characterized by excessively high circulating glucose levels as a result of excessive glucose production independent of insulin availability and inadequate glucose clearance (insulin action). Type 2 diabetes accounts for approximately 90 to 95% of all diagnosed cases of diabetes.

[0154] "Gestational diabetes" is a condition in which women without previously diagnosed diabetes have high blood glucose levels during pregnancy, particularly in the third trimester. Gestational diabetes affects 3-10% of pregnancies, depending on the population studied.

[0155] "Slow-onset autoimmune diabetes in adults (LADA)" (also called "late-onset type 1 diabetes") is a form of type 1 diabetes that occurs in adults and often has a slower onset course.

[0156] "Maturity-onset diabetes of the young (MODY)" refers to a genetic form of diabetes caused by mutations in autosomal dominant genes that interfere with insulin production.

[0157] "Diabetic retinopathy" is an eye disease caused by metabolic derangements that occurs in diabetic patients and leads to progressive loss of vision.

[0158] The term "hyperglycemia" refers to excess sugar (glucose) in the blood.

[0159] The term "dyslipidemia" refers to a disorder of lipoprotein metabolism, including lipoprotein overproduction (hyperlipidemia) or deficiency (hypolipidemia). Dyslipidemia is manifested by elevated blood total cholesterol, low-density lipoprotein (LDL) cholesterol and / or triglyceride levels, and / or decreased high-density lipoprotein (HDL) cholesterol levels.

[0160] Nonalcoholic steatohepatitis (NASH) is a liver disease characterized by the accumulation of fat (lipid droplets) accompanied by inflammation and degeneration of liver cells. Once affected, the disease NASH is associated with a high risk of cirrhosis, a condition that alters liver function and can progress to liver failure. NASH then often progresses to liver cancer.

[0161] "Atherosclerosis" is a vascular disease characterized by irregularly distributed lipid deposits, called plaques, in the intima of large and medium-sized arteries, which can cause narrowing of the arterial lumen and progress to fibrosis and calcification. The lesions are usually localized and progress slowly and intermittently. Occasionally, plaque rupture occurs, causing impaired blood flow and consequent tissue death distal to the obstruction. Blood flow restriction is the primary cause of most clinical symptoms, which vary depending on the distribution and severity of the obstruction.

[0162] The term "medicament" as used herein refers to a substance / composition used in therapy, i.e., in the treatment of diseases and disorders.

[0163] "Treatment" means administering a compound or composition or combination of compounds or compositions to a subject to prevent, ameliorate, or eliminate a disease or disorder in the subject; to halt or slow the development of a disease or disorder in the subject; to inhibit or slow the development of new diseases or disorders in the subject; to reduce the frequency or severity and / or recurrence of symptoms in a subject with a current or previous disease or disorder; and / or to prolong, i.e., increase, the longevity of the subject.

[0164] In particular, the phrases "treating a disease or disorder" and "treatment of a disease or disorder" include curing, shortening the duration, amelioration, prevention, slowing or inhibiting the progression or deterioration, or preventing or delaying the onset of a disease or disorder or symptoms thereof.

[0165] The term "subject," as used herein, refers to a subject for treatment, particularly an affected subject (also referred to as a "patient"), including, but not limited to, a human, a non-human primate, or other animal, such as a cow, horse, pig, sheep, goat, dog, cat, rabbit, or rodent (e.g., a mouse, rat, guinea pig, or hamster). In one embodiment, the subject or patient is a human.

[0166] The present invention will now be further described by reference to the following examples which are illustrative but not intended to limit the scope of the invention. [Example]

[0167] Determining the optimal GLP-1RA / FGF21 activity ratio through systems pharmacology modeling Improved mechanistic insight into the pharmacological effects of GLP-1RA / FGF21 fusion proteins in humans was used to identify optimal GLP-1RA / FGF21 potency ratios. A mechanistic systems pharmacology model was developed to explain 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).

[0168] This model represented the relevant pathways for the effects of GLP-1 and FGF21. Glycemic control (i.e., HbA1c, fasting plasma glucose, postprandial blood glucose), lipid parameters (i.e., plasma triglycerides, fatty acids, cholesterol), and energy balance (i.e., body weight, food intake, energy expenditure) were acquired and simulated drug treatments (e.g., GLP-1RA / FGF21 fusion protein, liraglutide, FGF21 analog L) were administered. We evaluated the therapeutic response to LY2405319 (Kharitonenkov et al. (2013) PLoS ONE 8(3):e58575).

[0169] This model encompassed important aspects of glucose homeostasis, which are controlled by the hormones insulin, glucagon, and certain incretins (e.g., GLP-1, GIP). The primary model endpoint for glycemic control was HbA1c, a common clinical endpoint used to estimate mean plasma glucose concentrations over the next several months. HbA1c was estimated in the model using the linear correlation between mean plasma glucose and HbA1c, as reported by Nathan et al. (2008) Diabetes Care 31(8):1473-1478.

[0170] This model incorporated triglyceride and fatty acid metabolism at a level appropriate for handling basic lipid metabolism, including cholesterol presentation. HDL and non-HDL, i.e., LDL + VLDL cholesterol, are circulating lipoproteins. Presentation of lipid metabolism allowed us to simulate the effects of FGF21 compounds on lipids and their interactions with statins. FGF21 compounds had significant effects on lipid concentrations (Gaich et al. (2013) Cell Metab 18(3):333-340; Fisher et al. (2011) Endocrinology 152(8):2996-3004).

[0171] Weight loss or gain in this model was measured as the change in body fat mass. There was a direct relationship between fat mass and body weight (Broyles et al. (2011) Br J Nutr 105(8):1272-1276). Food intake was based on basal and resting metabolic rate (Amirkalali et al. (2008) Indian J Med Sci 62(7):283-290). When energy expenditure was equal to calorie intake, body fat mass remained constant. The effect of treatment on food intake was implemented in the model using the equation from Gobel et al. (2014) (Obesity (Silver Spring) 22(10):2105-2108).

[0172] Food was considered to be carbohydrates (glucose equivalents), fats (fatty acid equivalents), and proteins (amino acid equivalents). All nutrients entered the stomach, passed through the delayed node, and then entered the three-compartment digestive tract. The design of the digestive tract was based on work done by Bastianelli et al. (1996) J Anim Sci 74(8):1873-1887; Worthington (1997) Med Inform (Lond) 22(1):35-45) on food digestion and absorption.

[0173] Nutritional, hormonal, drug and disease states can cause delayed gastric emptying. Under healthy conditions, the gastric emptying rate is a function of the size of the meal, 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 often have delayed glucose absorption observed in 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 due to slowed gastric emptying. A delay in transit between the stomach and small intestine was added to this example model to explain the delayed gastric emptying in diabetic subjects. Drugs and hormones (e.g., GLP-1) can affect gastric vagal tone, which reduces mechanical mixing and / or peristalsis and also delays gastric emptying (Jelsing et al. (2012) Diabetes Obes Metab 14(6):531-538; Little et al. (2006) J Clin Endocrinol Meta b91(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).

[0174] One purpose of the fusion proteins described herein was to prevent or reduce GLP-1-related adverse effects, namely nausea and vomiting (Lean et al. (2014) Int J Obes (Lond) 38(5):689-697). Gastric emptying measurements provided estimates of adverse events such as nausea and vomiting, which correlate with low gastric emptying rates. Therefore, the marker for gastric adverse events in the model was the sum of gastric emptying rates.

[0175] Different virtual patients representing healthy individuals and type 2 diabetes patients at different stages of the disease were implemented in the model platform. Furthermore, the virtual patients encompassed different degrees of obesity and dyslipidemia. The virtual patients represented the variability of disease severity as well as the pathophysiological and phenotypic variability observed in the clinic.

[0176] Several therapeutic agents were implemented in the model: GLP-1RA / FGF21 fusion protein, liraglutide, the FGF21 analog LY2405319, metformin, atorvastatin, sitagliptin, and human insulin. These agents could be switched on and off in the simulation. Virtual patients were assumed to be on a background of metformin and atorvastatin when administered the GLP-1RA / FGF21 fusion protein.

[0177] A hypothetical GLP-1RA / FGF21 fusion protein was tested in the model described in this example. The fusion protein contains both FGF21 agonist activity and GLP-1 agonist activity, and has the same effect as both an FGF21 receptor agonist and a GLP-1 receptor agonist. The pharmacokinetic properties of the hypothetical fusion protein were assumed to be similar to those of dulaglutide (Geiser et al. (2016) Clin Pharmacokinet 55(5):625-34).

[0178] The model was validated by comparison with many data sets, and the simulation results were quantitatively consistent 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 was based on relevant quantitative study 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):41 5-419;Sikaris(2009)J Diabetes Sci Technol3(3):429-438;Vicini and Cobelli(2001)Am J Physiol Endocrinol Metab 280(1):E179-186; adapted from Vollmer et al. (2008) Diabetes 57(3):678-687.

[0179] Existing therapeutic agents, including FGF21 analogs and GLP-1 receptor agonists, were tested in the model for direct comparison. The effects of FGF21 analogs were validated with clinical data, e.g., Gaich et al. (2013) Cell Metab 18(3):333-340.The GLP-1 receptor agonist riraglutide is a direct competitor for the target and its performance is supported by various 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 Med25(2):152-156; Buse et al. (2009) Lancet374(9683):39-47; Jelsing et al. (2012) Diabetes Obes Metab14(6):531-538; Hermansen et al. (2013) Diabetes Obes Metab15(11):1040-1048; Suzuki et al. (2013) Intern Med52(10):1029-1034; van Can et al. (2013) Int J Obes(Lond)38(6):784-93; Zinman et al. (2009) Diabetes Care32(7):1224-1230; Russell-Jones et al. (2009) Diabetologia52(10):2046-2055; Pratley et al. (2011) Int J Comparisons were made with data from 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; and Astrup et al. (2012) Int J Obes(Lond) 36(6):843-854.

[0180] The model platform allowed for simulation of beneficial and adverse effects of a hypothetical GLP-1RA / FGF21 fusion protein by varying the activity ratio. The effective FGF21-mediated EC50 values ​​were set to a constant relative to those derived from Gaich et al. (2013) Cell Metab 18(3):333-340. The effective GLP-1-mediated EC50 values ​​decreased by a factor of 2-600 with an increase of 1 relative to endogenous GLP-1 (Table 1).

[0181] [Table 1]

[0182] For each hypothetical fusion protein, exposure-response relationships were simulated for relevant pharmacodynamic endpoints: HbA1c, triglycerides, fatty acids, non-HDL cholesterol, and fat mass. Gastric emptying rate was used as a marker for GLP-1-mediated adverse events. A 52-week treatment with GLP-1RA / FGF21 fusion protein was simulated over a wide dose range in a hypothetical patient with average obesity, dyslipidemic type 2 diabetes. After 52 weeks of treatment, all relevant pharmacodynamic endpoints were expected to reach steady state. For each endpoint, the half-maximal effective concentration (EC50 value) was determined from the exposure-response curve. EC50 values ​​varied with activity ratio, particularly for HbA1c and gastric emptying rate, which are primarily GLP-1-mediated endpoints. Figure 1 shows the EC50 values, which depend on the GLP-1 decay rate. An increase in the GLP-1 decay rate indicated decreased GLP-1R agonist activity.

[0183] This procedure allowed for the identification of relevant activity ratios where adverse effects were observed at higher plasma levels relative to those that mediated the pharmacodynamic effects. 9 For higher GLP-1 decay rates, the EC50 for GLP-1-mediated gastrointestinal adverse effects was higher than the EC50 for the pharmacodynamic effects. Thus, gastric adverse effects occurred at plasma levels higher than those required to achieve the pharmacodynamic effects. It was possible to identify doses that provided all the desired pharmacodynamic effects while avoiding GLP-1-mediated gastrointestinal adverse effects.

[0184] The maximum EC50 value for gastric emptying rate was reached at a decay rate of 531. The maximum distance between adverse effects and the mean pharmacodynamic effect was reached at a decay rate of 482 (Figure 2). Therefore, activity ratios of 1:482 or greater were not relevant. The maximum distance between the maximum pharmacodynamic (HbA1c) and adverse effects was 319. The maximum distance between the maximum pharmacodynamic (HbA1c) and adverse effects normalized by the evolution of FGF21-(lipid) and GLP-1-mediated effects (HbA1c) was 121.

[0185] GLP-1RA / FGF21 fusion proteins with potency ratios between 1:10 and 1:482 were predicted to be most beneficial in improving lipid profiles, body weight, and glucose metabolism, and were unlikely to produce significant adverse events based on gastric emptying responses. Lower potency ratios were unlikely to be good candidates based on their predicted strong inhibition of gastric emptying and potential for adverse events. Higher potency ratios were unlikely to be sufficiently effective and therefore unlikely to be competitive.

[0186] A 12-week treatment of an average obese, dyslipidemic, type 2 diabetic hypothetical patient with GLP-1RA / FGF21 fusion protein was not simulated over a wide dose range, primarily because the GLP-1-mediated parameter HbA1c level clinically reaches a steady state after 12 weeks of treatment with GLP-1 receptor agonists and FGF21 agents known in the art.

[0187] Figure 3 shows the EC50 values ​​obtained for GLP-1 decay over the 12-week simulation period. For GLP-1 decay rates higher than 18, the EC50 for GLP-1-mediated gastrointestinal adverse effects was higher than the EC50 for pharmacodynamic effects. The maximum EC50 value for gastric emptying rate was reached at a decay rate of 501. The maximum distance between the adverse effect and the mean pharmacodynamic effect was reached at a decay rate of 469 (Figure 4). The maximum distance between the maximum pharmacodynamic (HbA1c) and adverse effect was 313. The maximum distance between the maximum pharmacodynamic (HbA1c) and adverse effect normalized by the evolution of FGF21-(lipid) and GLP-1-mediated effects (HbA1c) was 123.

[0188] The efficacy and potential for adverse events for GLP-1RA / FGF21 fusion proteins with different activity ratios were investigated using a described systems pharmacology approach. Fusion proteins with a presumably calculated ideal efficacy ratio were identified and predicted to be beneficial for improving lipid profiles, body weight, and glycemic control while avoiding significant adverse GLP-1RA-related effects based on gastric emptying responses. Compounds with efficacy ratios informed by the selected model were predicted to provide favorable efficacy versus risk profiles. [Example]

[0189] Expression of homodimeric GLP-1RA / FGF21 fusion proteins in HEK-293, CHO, and E. coli cells and chemical synthesis of isolated GLP-1R agonist peptides GLP-1RA / FGF21 Fc fusion protein was produced by transient transfection in HEK-293 or CHO cells. The DNA sequence of the fusion protein was fused to the IL2 signal sequence (SEQ ID NO: 246) at the N-terminus, followed by a histidine-rich sequence (His tag) and a TEV protease cleavage site (SEQ ID NO: 247 or 248). The signal sequence was required for secretion of the desired protein into the culture medium. The protein was purified from the culture supernatant using immobilized metal ion affinity chromatography (IMAC) (cOmplete His-Tag Purification Column™, Roche). After elution from the IMAC column, the N-terminal His tag was optionally cleaved by adding TEV protease. After His tag cleavage, the cleavage reaction solution was passed twice over an IMAC column (cOmplete His-Tag Purification Column™, Roche), and the flow-through fraction (without the His tag) was collected. The protein was further purified using Protein A affinity chromatography (rProtein A Sepharose, GE Healthcare) and a gel filtration column with phosphate-buffered saline (PBS, Gibco) as the running buffer. Fractions containing the desired protein were collected, pooled, concentrated, and stored at -80°C until further use.

[0190] The FGF21 protein of SEQ ID NO: 252 (mature human wild-type FGF21 with an N-terminal Gly addition, herein referred to as G-FGF21) was expressed in E. coli. The DNA sequence of the FGF21 protein was fused to a histidine-rich sequence (His tag) and a TEV or SUMO protease cleavage site (SEQ ID NO: 248 or 249) at the N-terminus. The desired protein was purified using immobilized metal ion affinity chromatography (IMAC) (HisTrap HP, GE Healthcare), followed by cleavage of the N-terminal His tag by adding TEV or SUMO protease. After cleavage of the s-tag, the cleavage reaction solution was purified using an ion exchange column (Source 15, GE Healthcare) followed by a gel filtration column (Superdex 75, GE Healthcare) using phosphate-buffered saline (PBS, Gibco) as the running buffer. Fractions containing the desired protein were collected, pooled, concentrated, and stored at -80°C until further use.

[0191] In an alternative approach, the fusion protein was produced by expression in E. coli inclusion bodies, followed by a refolding step in which the inclusion bodies were unfolded in a Tris-buffered guanidium chloride solution and refolded by dilution into a buffer without chaotropic salts to obtain the folded fusion protein. The fusion protein was purified using protein A affinity chromatography (MabSelect SuRe, GE Healthcare), followed by cleavage of the N-terminal presequence by addition of TEV protease. The cleavage reaction solution was then transferred to an anion exchange column (POROS 50 The purified fractions were collected and pooled using a PBS (Gibco). The final buffer conditions and protein concentration were established by an ultrafiltration / diafiltration step using PBS (Gibco). The sample was stored at -80°C until further use.

[0192] Fusion proteins were produced by recombinant methods (see above), while isolated peptide GLP-1R agonists were chemically synthesized.

[0193] More specifically, the peptide was synthesized using the following manual synthesis procedure: 0.3 g of dried Rink amide MBHA resin (0.66 mmol / g) was placed in a polyethylene container equipped with a polypropylene filter. The resin was swollen in DCM (15 ml) for 1 hour and DMF (15 ml) for 1 hour. The Fmoc group on the resin was deprotected by treating it twice with a 20% (v / v) piperidine / DMF solution for 5 and 15 minutes. The resin was washed with DMF / DCM / DMF (6:6:6 times each). The Kaiser test (quantitative method) was used to determine the Fmoc removal from the solid support. A C-terminal Fmoc amino acid (5 equivalents 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 equivalents excess of DIC and HOBT in DMF. The concentration of each reactant in the reaction mixture was approximately 0.4 M. The mixture was rotated on a rotor at room temperature for 2 hours. The resin was filtered and washed with DMF / DCM / DMF (6:6:6 times each). A Kaiser test performed on an aliquot of the peptide resin upon completion of coupling was negative (the resin was colorless). After attachment of the first amino acid, any unreacted amino groups in the resin, if any, were capped using acetic anhydride / pyridine / DCM (1:8:8) for 20 minutes to avoid any loss of sequence. After capping, the resin was washed with DCM / DMF / DCM / DMF (6 / 6 / 6 / 6 hours each). The Fmoc group of the C-terminal amino acid-attached peptidyl resin was deprotected by treatment with 20% (v / v) piperidine / DMF solution twice for 5 and 15 minutes. The resin was washed with DMF / DCM / DMF (6:6:6 times each). A Kaiser test performed on an aliquot of the peptide resin upon completion of Fmoc deprotection was positive.

[0194] The remaining amino acids in the target sequence on the Rink amide MBHA resin were sequentially coupled using the Fmoc AA / DIC / HOBt method, using a 5-equivalent excess corresponding to the resin loading in DMF. The concentration of each reactant in the reaction mixture was approximately 0.4 M. The mixture was rotated on a rotor at room temperature for 2 hours. The resin was filtered and washed with DMF / DCM / DMF (6:6:6 times each). After each coupling step and Fmoc deprotection step, a Kaiser test was performed to confirm the completeness of the reaction.

[0195] After the linear sequence is completed, the ε-amino group of lysine, which is used as a branching or modification point, is Deprotection was performed using 2.5% hydrazine hydrate in DMF twice for 15 min and washed with DMF / DCM / DMF (6:6:6 times each). The γ-carboxyl terminus of glutamic acid was attached to the ε-amino group of Lys using Fmoc-Glu(OH)-OtBu by the DIC / HOBt method in DMF (using a 5 equivalent excess relative to the resin load). The mixture was rotated on a rotor for 2 h at room temperature. The resin was filtered and washed with DMF / DCM / DMF (6 × 30 mL each). The Fmoc group of glutamic acid was deprotected by treating with 20% (v / v) piperidine / DMF solution twice for 5 and 15 min (25 mL each). The resin was washed with DMF / DCM / DMF (6:6:6 times each). Upon completion of Fmoc deprotection, a Kaiser test of an aliquot of the peptide resin was positive.

[0196] If the side-chain branch also contained one additional γ-glutamic acid, a second Fmoc-Glu(OH)-OtBu was used to attach to the free amino group of the γ-glutamic acid using the DIC / HOBt method in DMF (5 equivalents excess relative to resin loading). The mixture was rotated on a rotor at room temperature for 2 hours. The resin was filtered and washed with DMF / DCM / DMF (6 × 30 mL each). The Fmoc group of the γ-glutamic acid was deprotected by treating twice with 20% (v / v) piperidine / DMF solution for 5 and 15 minutes (25 mL). The resin was washed with DMF / DCM / DMF (6:6:6 times each). Upon completion of Fmoc deprotection, a Kaiser test on an aliquot of the peptide resin was positive.

[0197] Final cleavage of the peptide from the resin: Peptidyl resin prepared by manual synthesis was washed with DCM (6 × 10 mL), MeOH (6 × 10 mL), and ether (6 × 10 mL) and dried overnight in a vacuum desiccator. Cleavage of the peptide from the solid support was achieved by treating the peptide-resin with a reagent cocktail (80% TFA / 5% thioanisole / 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 × 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. This process was repeated 3–4 times to remove most of the volatile impurities. The residue was cooled to 0 °C, and anhydrous ether was added to precipitate the peptide. The precipitated peptide was centrifuged, the supernatant ether removed, fresh ether 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. [Example]

[0198] In vitro cell assay (In-Cell Western) for the availability of human FGF21 receptor in CHO cells The cellular in vitro efficacy of G-FGF21 (SEQ ID NO: 252) and the fusion protein of the present invention was measured using a specific and highly sensitive In-Cell Western (ICW) assay. ICW assays are immunocytochemical assays that are usually performed using a microplate format. For the FGF21 receptor autophosphorylation ICW assay (Aguilar et al. (2010) PLoS ONE 5(4): e9965), CHO Flp-In cells (Invitrogen, Darmstadt, Germany) stably expressing human FGFR1c together with human beta-Klotho (KLB) were used. To determine the receptor autophosphorylation level or downstream activation of MAP kinase ERK1 / 2, 2 × 10 4Cells / well were seeded into 96-well plates and grown for 48 hours. Cells were serum-starved for 3-4 hours with serum-free medium (Ham's F-12 Nutrient Mix with GlutaMAX, Gibco, Darmstadt, Germany). Cells were then treated with increasing concentrations of either G-FGF21 (SEQ ID NO: 252) or the indicated fusion proteins for 5 minutes at 37°C. After incubation, the medium was discarded and cells were fixed in 3.7% freshly prepared paraformaldehyde for 20 minutes. Cells were permeabilized with 0.1% Triton-X-100 in PBS for 10 min. Blocking was performed with Odyssey blocking buffer (LICOR, Bad Homburg, Germany) at room temperature for 2 h. Primary antibodies, anti-pFGFR Tyr653 / 654 (New England Biolabs, Frankfurt, Germany) or anti-pERK phospho-p44 / 42 MAP kinase Thr202 / Tyr204 (Cell Signaling), were added and incubated overnight at 4°C. After primary antibody incubation, cells were washed with PBS + 0.1% Tween 20. Cells were then incubated with secondary anti-mouse 800CW antibody (LICOR, Bad Homburg, Germany) at room temperature for 1 h. Subsequently, cells were washed again with PBS + 0.1% Tween 20. Infrared dye signals were quantified using an Odyssey image display device (LICOR, Bad Homburg, Germany). Results were normalized by DNA quantification with TO-PRO3 dye (Invitrogen, Karlsruhe, Germany). Data were obtained as arbitrary units (AU), and EC50 values ​​were obtained from dose-response curves (summarized in Tables 2 and 3). Figure 5 shows the results of ICW with CHO cells overexpressing human FGFR1c+KLB.

[0199] [Table 2]

[0200] [Table 3] [Example]

[0201] In vitro cellular assay for human glucagon-like peptide 1 (GLP-1) receptor availability The agonism of the compounds for the human glucagon-like peptide 1 (GLP-1) receptor was determined by a functional assay measuring the cAMP response in a HEK-293 cell line stably expressing the human GLP-1 receptor.

[0202] Recombinant HEK-293 cells were grown to near confluence in medium (DMEM containing 10% FBS) in a T175 culture flask at 37°C, and 1–5 × 10 cells were cultured in a 2 mL vial. 7 The cells were collected in cell culture medium containing 10% DMSO at a concentration of 1.8 mL / mL. Each vial contained 1.8 mL of cell suspension. The vials were slowly frozen to -80°C in an isopropanol chamber and then transferred to liquid nitrogen for long-term storage.

[0203] Before their use, frozen cells were quickly thawed at 37°C, washed with 20 mL of cell buffer (1x HBSS, 20 mM HEPES, 0.1% BSA) and centrifuged at 900 rpm for 5 min. Cells were resuspended in assay buffer (cell buffer + 2 mM IBMX) and diluted to 1x10 6 The cell density was adjusted to 100 cells / mL. For the measurement, 5 μL of the cell suspension (final 5 × 10 3 Cells / well) and 5 μL of test compound were added to wells of a 384-well plate, followed by incubation at room temperature for 30 minutes. Human GLP-1(7-36) amide (SEQ ID NO: 260) obtained from Bachem (Bubendorf, Switzerland, H-6795) was obtained as a control. Cellular cAMP content was measured using Homogenous Time Based on High Temperature Resolved Fluorescence (HTRF), Cisbio The in vitro efficacy of agonists was determined using a kit from Biochem., Inc. (Cat. No. 62AM4PEC). After adding HTRF reagent diluted in lysis buffer (a component of the kit), the plates were incubated for 1 hour, followed by measurement of the fluorescence ratio at 665 / 620 nm. Force is calculated as 50% of the maximum response (EC 50 ) was quantified by determining the concentration that caused

[0204] The results are summarized in Table 4 and the dose-response curves are shown in FIG.

[0205] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Example]

[0206] Conformational and thermal stability analysis of GLP-1RA / FGF21 Fc fusion protein Conformational stability and aggregation tendency were analyzed using UNit (Unchained Labs, Kali The fluorescence intensity of a GLP-1RA / FGF21 Fc fusion protein was simultaneously determined using the UNit (University of California, USA). UNit combines analysis of the intrinsic fluorescence of proteins to detect protein unfolding with static light scattering (SLS) measurements to investigate aggregation behavior.

[0207] Data were acquired for fusion proteins formulated at a concentration of 5 mg / mL in pH 7.4 phosphate buffer. A volume of 9 μL of each sample was loaded into a UNit capillary holder and analyzed in triplicate in UNit. The temperature was increased from 20 to 95°C at a constant linear rate of 0.3°C / min. BaryCentric Mean (BCM), representing the intrinsic fluorescence and SLS signal detected with a 266 nm laser, was plotted against the applied temperature to obtain the melting temperature (Tm) and aggregation onset temperature (Tagg). Data were analyzed using UNit Analysis software v. 2.1 and are summarized in Table 5.

[0208] Furthermore, for some proteins, a thermal shift assay was applied to analyze the thermal stability, mimicking the differential scanning calorimetry (DSF or ThermoFluor™) assay (Ahmad S. et al. (2012) Protein Science 21: 433-446; Pantoliano et al. (2001) J. Biomol. Screen 6: 429-440; Niesen et al. (2007) Nat. Protoc. 2: 2212-21). This assay is based on the observation that hydrophobic fluorescent dyes, such as Sypro™ Orange (Life Technologies, Cat. No. S6651), increase their fluorescence when bound to hydrophobic patches of proteins. Such hydrophobic patches become exposed in proteins when the proteins are unfolded by heating, and the increase in fluorescence can therefore be used as a measure of the degree of unfolding and, therefore, the thermal stability of the protein.

[0209] Proteins were tested by mixing a solution of each protein in PBS (Gibco) with a 160x solution of Sypro™ Orange (diluted in water from a 5000x DMSO stock provided by the supplier). The sample volume was adjusted to 20 μL with PBS. Typical conditions included 0.8 mg / mL protein and 8x Sypro™ Orange in the final mixture, although protein concentrations could vary from 0.4 mg / mL to 1.2 mg / mL. Samples were dispensed into a 96-well PCR plate (BioRad Semi-Skirt 96, white) and briefly centrifuged to remove air bubbles. The plate was inserted into a BioRad iQ5 real-time PCR instrument and subjected to a thermal gradient from 10 to 90°C at a ramp rate of 1°C / min. Filters with wavelengths of 485 nm and 575 nm were selected for excitation and fluorescence quantification. BioRad iQ5 Standard Edition software (v. 2.0.148.60623) was used for data processing. In the curve of fluorescence intensity versus temperature, the inflection point was selected as a measure of the melting temperature (Tm).

[0210] [Table 5] [Example]

[0211] Pharmacokinetics in mice and non-human primates Plasma concentrations and pharmacokinetic parameters of the GLP-1RA / FGF21 Fc fusion protein were determined using three different methods after a single subcutaneous administration of a 0.3 mg / kg solution to female C57Bl / 6 mice or male cynomolgus monkeys. Blood samples were collected between 30 minutes and 168 hours after dosing.

[0212] a.) Bioanalytical screening method for quantification of the intact FGF21 portion of GLP-1RA / FGF21 Fc fusion proteins Plasma samples were analyzed for the intact FGF21 portion of the fusion protein using an ELISA kit (F1231-K01, Eagle Biosciences, USA). The assay utilized a two-site sandwich technique using two selected antibodies that bound to different epitopes on human intact FGF21. One antibody specifically bound to the N-terminal amino acids (amino acids 29-35) of human FGF21, and the other antibody specifically bound to the C-terminus (amino acids 203-209) of human FGF21. Assay standards, controls, and unknown samples were added directly to wells of a microplate coated with anti-human FGF21 (amino acids 29-35)-specific antibody. At the same time, horseradish peroxidase-conjugated anti-human FGF21 (amino acids 203-209)-specific antibody was added to each well. After the first incubation period, the antibody on the wall of the microtiter well captured human FGF21 in the sample, and unbound proteins from each microtiter well were washed away. A "sandwich" of "anti-FGF21 antibody-human intact FGF21-HRP conjugated tracer antibody" was formed. Unbound tracer antibody was removed in a subsequent washing step. To detect the immune complexes, the wells were then incubated with a substrate solution for a fixed reaction time, followed by measurement using a spectrophotometric microplate reader. The enzymatic activity of the immune complexes bound to human intact FGF21 on the wall of the microtiter well was directly proportional to the amount of intact FGF21 in the sample.

[0213] b.) Bioanalytical screening methods for quantification of intact, full-length fusion proteins The concentration of full-length GLP-1RA / FGF21 Fc fusion protein in plasma was determined using an ELISA method. The N-terminus of the fusion protein was captured by a mouse monoclonal anti-GLP1 antibody (Mesoscale Discovery, MSD). The plate was blocked with 1 μL of Blocker A (MSD) for 1 hour at room temperature (RT) with gentle shaking. After washing three times with 300 μL of wash buffer, 50 μL of diluted plasma samples (standards and PK study samples) were added to each well, and the plate was incubated for 1 hour at room temperature with gentle shaking. After washing three times with 300 μL of wash buffer, 50 μL of primer detection antibody (C-terminal rabbit anti-FGF21 antibody, Pineda Antikorper-Service, Berlin, Germany) was added to each well, and the plate was incubated for 1 hour at room temperature. After washing three times with 300 μL of wash buffer, 25 μL of goat anti-rabbit antibody (Sulfo-tagged, MSD) diluted in PBS-Tween 0.05% (PBS-T) was added to each well, and the plate was incubated for 1 hour at room temperature. After washing three times with 300 μL of PBS-T, 150 μL of read buffer was added to the wells.

[0214] c.) Bioanalytical screening method for quantification of the intact GLP-1 moiety of GLP-1 FGF21 Fc fusion proteins. Plasma samples were analyzed for the intact GLP-1 portion of the fusion protein using a GLP-1 ELISA method. ELISA plates were coated with a mouse monoclonal anti-GLP-1 antibody (Mesoscale Discovery, MSD). After blocking with 150 μL of Blocker A (MSD) for 1 hour at room temperature (RT) with gentle shaking and washing three times with 300 μL of PBS-T, 50 μL of diluted plasma samples (standards and PK study samples) were added to each well, and the plate was incubated for 1 hour at room temperature with gentle shaking. After washing three times with 300 μL of PBS-T, 25 μL of goat anti-human IgG (Sulfo-tagged, MSD) diluted (1 / 3,333) in PBS-T was added to each well, and the plate was incubated for 1 hour at room temperature. After washing three times with 300 μL of PBS-T, 150 μL of read buffer was added to the wells.

[0215] Pharmacokinetic parameters were calculated using the program WinNonlin 6.4 using a non-compartmental model and linear trapezoidal interpolation calculations. The results are shown in Figures 7 and 8 and Table 6. The results show that the novel GLP-1RA / FGF21 Fc fusion protein maintained plasma levels in the ng / mL range with a half-life of up to 20-40 hours.

[0216] [Table 6] [Example]

[0217] In vivo efficacy in mouse models a.) Multiple-dose, diet-induced obese (DIO) mice Female C57BL / 6N Charles River mice were group-housed in a specific pathogen-free barrier facility with a 12-hour light / dark cycle and free access to a standard diet or a high-fat diet (ssniff-regulated fat diet E15797). After 20 weeks of pre-feeding on the high-fat diet, mice were stratified into treatment groups (n = 8) according to body weight, ensuring that each group had a similar mean body weight. An age-matched group with free access to a standard diet (ssniff R / MH, V1534-0) was included as a standard control group. A dulaglutide-treated group was also included for comparison. Prior to the start of treatment, mice were subcutaneously (sc) injected with vehicle solution, weighed, and allowed to acclimate to the procedure for 3 days.

[0218] 1) Acute effects on blood glucose in female DIO mice after a meal: The first blood sample was collected immediately before the first administration (subcutaneous) of vehicle (phosphate buffer solution) or GLP-1RA / FGF21 Fc fusion protein (dissolved in phosphate buffer), respectively. The administration volume was 5 or 10 mL / kg, depending on the concentration of the stock solution. Animals had free access to water and their corresponding diet throughout the experiment. Blood glucose levels were measured at t = 0 h, t = 1 h, t = 2 h, t = 3 h, t = 4 h, t = 6 h, and t = 24 h (method: Accu-Check blood glucose meter). Blood samples were collected by tail incision without anesthesia.

[0219] 2) Chronic effects on body weight in female DIO mice: Mice were treated with either vehicle or test compound once a week for 4 weeks, on the morning of day 8, at the beginning of the light cycle. Body weight and food intake were recorded daily. Total fat mass was measured by nuclear magnetic resonance (NMR) 2 days before the start of treatment and on day 26.

[0220] The effects of the fusion proteins on body weight and food intake are shown in Figures 9 and 10, respectively. Animals treated with the fusion proteins of SEQ ID NO:8 or SEQ ID NO:7 cumulatively ate more food than animals treated with vehicle or dulaglutide by the end of the study. They had significantly greater weight loss than vehicle- or dulaglutide-treated animals, clearly demonstrating the balance between the GLP-1 receptor activity and FGF21 mimetic activity of both molecules SEQ ID NO:7 and SEQ ID NO:8, since suppression of food intake was not required to achieve their effect on weight loss.

[0221] b.) Blood glucose lowering effect of multiple subcutaneous administrations in postprandial female diabetic db / db mice Animals, study design (pre-dose phase, dosing phase), pharmacological intervention Healthy female lean (BKS.Cg-(lean) / OlaHsd or BKS.Cg-Dock7(m)+ / + Lepr(db)J) and diabetes-prone obese db / db (BKS.Cg-+Leprdb / +Leprdb / OlaHsd or BKS.CG-m+ / + Lepr(db) / J) mice were ordered from Envigo RMS Inc. or Charles River Laboratories. All animals were group-housed in shoebox cages with wood chip bedding and allowed to acclimate for approximately 2–3 weeks before the dosing phase.

[0222] Mice were housed under ecological conditions including a 12-hour light / dark cycle (light phase 4:00 AM to 4:00 PM), a room temperature of 20-26°C, and a relative humidity of 30-70%. All animals had free access to Greenfield tap water and Purina Fomulab Diet 5008. At the start of the study, mice were approximately 10-12 weeks old.

[0223] Pre-medication phase (15 days) Blood was collected by tail clip on day 9 for HbA1c and blood glucose measurements. Blood glucose concentrations were measured using an AlphaTRAK wide-range blood glucose meter (Code 29 strips). Blood glucose meter readings were taken before any other vital activity and in duplicate. If values ​​differed by more than 20 mg / dL (calculated blood glucose meter value), a third value was recorded. Obesity measurements were collected on days 9 and 15. HbA1c and obesity values ​​were used for block randomization. On day 15, animals were assigned to treatment groups (n = 8 animals / group) and new cages and cagemates (n = 4 animals / cage) according to the results of block randomization. The low-fat group was included in the study as an age-matched healthy reference group.

[0224] Medication Formulations and Administration On days 1, 8, 15, 22, and 27 of the dosing phase, animals were treated with either vehicle (sterile PBS), dulaglutide, SEQ ID NO: 8, or SEQ ID NO: 7 via subcutaneous injection in a volume of 5 ml / kg. Dosing was completed between 10:00 AM and 12:00 PM and adjusted to each animal's most recent obesity reading. Trulicity (dulaglutide pen) injection solutions were prepared by adding sterile PBS to the stock solution or pen formulation to achieve the appropriate concentration.

[0225] Medication phase (36 days) 1) Blood glucose concentrations in animals after breakfast: Animals had unlimited access to water and food throughout the experiment. Blood glucose was measured between 10:00 AM and 12:00 PM on days 1, 2, 8, 9, 15, 16, 22, 23, 27, and 28, and 24 hours after dosing on days 2, 9, 16, 23, and 28, prior to any other vital activity. Additionally, on days 1 and 22, blood was collected 1, 2, 3, 4, 6, and 24 hours after dosing (Figure 11). Approximately 5 μL of blood was collected via tail clip, and blood glucose measurements were performed using an AlphaTRAK wide-range blood glucose monitor (Code 29 strips). Duplicate measurements were performed using the RIMS (R = 0.01) and RIMS (R = 0.01). If values ​​differed by more than 20 mg / dL (calculated glucose value), a third value was recorded. Area under the curve (AUC) was calculated by the trapezoid method for each individual and time point indicated.

[0226] 2) HbA1c analysis: Blood was collected via tail clip on day 9 of the pre-dosing phase and day 36 of the dosing phase. 5 μL of blood was collected into an additive-free microcapillary tube and immediately placed into a centrifuge tube containing hemolyzed blood. The tube was vigorously shaken to mix the hemolyzed blood with the blood and placed on a rocker to ensure complete mixing of the blood and reagents. Plasma HbA1c levels at the start and end of the study are shown in Figure 12.

[0227] Statistical Analysis: Data are presented as mean ± standard error of the mean. For statistical analysis, one-way analysis of variance (ANOVA) and multiple comparisons (Dunnett's test) were performed to compare the group of diabetic, obese db / db vehicle mice (n = 8) with the group of diabetic, obese db / db test article-treated mice (n = 8). Differences in the means of the two groups were considered statistically significant if they exceeded 0.05. Data from the non-diabetic low-fat vehicle group are shown in Figures 11 and 12 and serve as the reference data set for the non-obese, non-diabetic condition.

[0228] In animals treated with the fusion protein of SEQ ID NO:8 or SEQ ID NO:7, the lowering effect on blood glucose levels was significantly greater than that in vehicle- or dulaglutide-treated animals ( FIG. 11 ). The highest dose of fusion protein of SEQ ID NO:8 even resulted in a reduction in blood glucose levels to normal, non-diabetic animal levels across nearly the entire 24-hour blood glucose profile measured on day 22 of treatment. Furthermore, animals treated with the fusion protein of SEQ ID NO:8 or SEQ ID NO:7 showed a more pronounced suppression of HbA1c increase by the end of the study than animals treated with vehicle or dulaglutide, as shown in FIG.

[0229] c.) DIO-NASH mouse model Animals and experimental set-up All animal experiments conformed to internationally accepted principles for the care and use of laboratory animals.

[0230] Five-week-old male C57Bl / 6J mice were obtained from JanVier (JanVier labs, France) and housed in groups of five animals per cage under a 12 / 12-h dark / light cycle. Room temperature was controlled at 22°C ± 1°C with 50% ± 10% humidity. Animals were fed a high-fat diet (40%, of which 18% was trans fat), 40% carbohydrate (20% fructose), and 2% cholesterol (D09100301, Research Diet, USA) previously described as the AMLN diet (Clapper et al. (2013) Am J Physiol Gastrointest Liver Physiol 305:G483-G495) or regular rodent chow (Altromin 1324, Brogaarden, Denmark) and tap water ad libitum (low-fat diet, n = 10–12 animals). After 26 weeks, liver biopsies were performed for histological assessment of fibrosis and steatosis staging in individuals at baseline.

[0231] Mice were pretreated with enrofloxacin (Bayer, Germany) (5 mg / mL / 1 mL / kg) one day before biopsy. Before biopsy, mice were anesthetized with isoflurane (2%–3%) in 100% oxygen. A small abdominal incision was made in the midline to expose the left lateral lobe of the liver. Cone-shaped pieces of liver tissue (50–100 mg) were excised from the distal part of the lobe and fixed in 4% paraformaldehyde for histology. Previously described by Clapper et al. A modified version of the previously described biopsy procedure was performed using bipolar electrocoagulation of the cut surface of the liver using an ERBE VIO 100C electrosurgical instrument (ERBE, USA). The liver was returned to the abdominal cavity, the abdominal wall was sutured, and the skin was stapled. Carprofen (Pfizer, USA) (5 mg / mL - 0.01 mL / 10 g) and enrofloxacin (5 mg / mL - 1 mL / kg) were administered intraperitoneally at the time of surgery and on postoperative days 1 and 2, respectively, to relieve postoperative pain and control infection. After the biopsy procedure, animals were housed singly and allowed to recover on an AMLN diet for 3 weeks. Stratification and randomization into study groups of 10–12 animals were based on individual disease staging assessed by baseline liver biopsy.

[0232] The animals were then treated with 50 mg / kg of GLP-1RA / FGF21 Fc fusion protein, 0.6 mg / kg of dulaglutide, or vehicle (PBS) by subcutaneous injection once a week for an additional 8 weeks on either the AMLN or normal diet. Subsequently, the animals were euthanized, liver weights were determined, and liver tissue was collected for histological and biochemical analysis (see Figure 13).

[0233] Histological evaluation and digital image analysis Baseline liver biopsies and end-stage samples were taken from the left lateral lobe (approximately 100 mg) and fixed overnight in 4% paraformaldehyde. Liver tissues were embedded in paraffin and sectioned (3 μm thick). To evaluate liver morphology and fibrosis, sections were stained with hematoxylin and eosin and Sirius Red, respectively, and then analyzed using Visiomorph software (Visiopharm, Denmark). Histological evaluation and scoring were performed by a pathologist blinded to the study. NAFLD activity score (NAS) (fatty liver, inflammation, balloon degeneration) and fibrosis stage were performed using the clinical criteria outlined by Kleiner et al. (2005) Hepatology 41: 1313-1321. The data are presented in two different formats in Figure 14 and Figure 15.

[0234] The fusion protein of SEQ ID NO: 8 clearly demonstrated effects on liver weight, liver total lipid content, liver cholesterol and triglyceride content, and NAFLD activity score that were superior to those of GLP-1 agonism alone, as exemplified by the effects of dulaglutide.

Claims

1. A fusion protein comprising a GLP-1R (Glucagon-like peptide 1 receptor) agonist peptide and a functionally active variant of human FGF21 (Fibroblast Growth Factor 21), the GLP-1R agonist peptide is a variant of native GLP-1(7-36) (SEQ ID NO: 260) comprising substitutions of up to about 15 amino acid residues in the amino acid sequence of native GLP-1(7-36); The functionally active variant of human FGF21 comprises an amino acid sequence that is at least about 96% identical to the amino acid sequence of SEQ ID NO: 250 or SEQ ID NO: 251; (i) substitutions Q55C and P147C or substitutions Q55C and N149C, and (ii) comprising a substitution or deletion of G198 and / or P199; Amino acid residue numbering is according to SEQ ID NO: 250 The GLP-1R agonist peptide and the functionally active variant of human FGF21 include L-Fc, Fc-L, L １ -Fc-L ２ and Fc, wherein L, L １ , and L ２ are independently selected from the group consisting of a single amino acid and a peptide, and Fc is an Fc domain of an immunoglobulin or a variant thereof.

2. The fusion protein of claim 1, which has a GLP-1R agonist activity that is about 9 to about 531 times lower than the GLP-1R agonist activity of native GLP-1(7-36).

3. The fusion protein of claim 1 or 2, wherein the GLP-1R agonist peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 261-565.

4. The GLP-1R agonist peptide has the amino acid sequence H-G-E-G-T-F-T-S-D-X １０ -S-K-Q-L-EEE-X １８ -V-X ２０ -L-F-I-E-W-L-K-A-X ２９ -G (SEQ ID NO: 4079) wherein X １０ is K or L, X １８ is A or R, X ２０ is R or Q, X ２９ is G or T, Optionally, the amino acid sequence further comprises at least one additional amino acid residue at its N-terminus; 4. The fusion protein of claim 1, wherein the amino acid sequence optionally further comprises a peptide extension of up to about 12, about 11, or about 10 amino acid residues at its C-terminus.

5. The fusion protein of any one of claims 1 to 4, wherein the GLP-1R agonist peptide comprises the amino acid sequence of SEQ ID NO: 261 or 262.

6. The fusion protein according to any one of claims 1 to 5, wherein the functionally active mutant of human FGF21 comprises a substitution or deletion selected from the group consisting of G198R, G198K, G198Y, and deletion of P199.

7. The fusion protein of any one of claims 1 to 6, wherein the functionally active mutant of human FGF21 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 253, 254, 255, and 256.

8. The fusion protein of any one of claims 1 to 7, wherein the Fc domain of the immunoglobulin or variant thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 257, 258, and 259.

9. 100-102, 105-109, 112, 113, 115, 116, 118, 120-124, 126-130, 132-136, 139, 142-148, 150-153, 155-158, 161-172, 174-177, 180-188, 190, 192-209, 211, 212, 216, 217, and 219-229, or a fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8, 18-31, 39, 40, 42-72, 74, 76, 78-84, 88-90, 92-97, 100-102, 105-109, 112, 113, 115, 116, 118, 120-124, 126-130, 132-136, 139, 142-148, 150-153, 155-158, 161-172, 174-177, 180-188, 190, 192-209, 211, 212, 216, 217, and 219-229. 31, 39, 40, 42-72, 74, 76, 78-84, 88-90, 92-97, 100-102, 105-109, 112, 113, 115, 116, 118, 120-124, 126-130, 132-136, 139, 142-148, 150-153, 155-158, 161-172, 174-177, 180-188, 190, 192-209, 211, 212, 216, 217, and 219-229, or a functionally active variant thereof.

10. A fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 7, and 8, or a functionally active variant thereof comprising an amino acid sequence that is at least about 96% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 7, and 8.

11. 11. The fusion protein of any one of claims 1 to 10, wherein the fusion protein is capable of activating human GLP-1R with an EC50 of about 15 pmol / L to about 400 pmol / L, or about 20 pmol / L to about 400 pmol / L, or about 50 pmol / L to about 400 pmol / L, or about 100 pmol / L to about 400 pmol / L, as determined by measuring the cAMP response of cells stably expressing human GLP-1R.

12. 12. The fusion protein of any one of claims 1 to 11, wherein the fusion protein (i) is capable of inducing autophosphorylation of human FGF receptor lc (FGFRlc) with an EC50 of about 250 nmol / L or less, or about 200 nmol / L or less, or about 150 nmol / L or less, or about 100 nmol / L or less, or about 75 nmol / L or less, or about 50 nmol / L or less; and / or (ii) is capable of inducing phosphorylation of mitogen-activated protein kinase (MAPK) ERK1 / 2 with an EC50 of about 100 nmol / L or less, or about 75 nmol / L or less, or about 50 nmol / L or less, or about 25 nmol / L or less, or about 20 nmol / L or less, or about 15 nmol / L or less, or about 10 nmol / L or less.

13. A nucleic acid molecule encoding the fusion protein of any one of claims 1 to 12.

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

15. A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 12, the nucleic acid molecule of claim 13, or the host cell of claim 14.

16. A kit comprising the fusion protein of any one of claims 1 to 12, the nucleic acid molecule of claim 13, the host cell of claim 14, or the pharmaceutical composition of claim 15.

17. A fusion protein according to any one of claims 1 to 12 for use as a medicament. A nucleic acid molecule according to claim 13, a host cell according to claim 14, or a pharmaceutical composition according to claim 15.

18. 16. The fusion protein of any one of claims 1 to 12, the nucleic acid molecule of claim 13, the host cell of claim 14, or the pharmaceutical composition of claim 15, 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.

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

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