Glp-1r agonist / fgf21 fusion proteins

Abstract

This invention relates to a fusion protein comprising a glucagon-like peptide-1 receptor 5 (GLP-1R) agonist peptide and a human fibroblast growth factor 21 (FGF21) variant. The invention further relates to the use of the fusion protein comprising the GLP-1R agonist peptide and the FGF21 variant as a pharmaceutical agent, particularly for treating subjects with obesity, overweight, metabolic syndrome, diabetes, diabetic retinopathy, hyperglycemia, dyslipidemia, nonalcoholic steatohepatitis (NASH), and / or atherosclerosis. The invention also relates to pharmaceutical compositions comprising a fusion protein containing the GLP-1R agonist peptide and the FGF21 variant.

Description

Technical Field

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

[0002] Using FGF21 and GLP-1R agonists as fusion proteins has drawbacks. The pharmacological effects of FGF21 have been observed at plasma levels higher than those of GLP-1 (the primary GLP-1R agonist), which exerts its pharmacological effects. Furthermore, at higher plasma levels, GLP-1 is known to have adverse effects, such as inducing nausea and vomiting. In summary, this indicates a risk of GLP-1-mediated adverse effects when the combination of FGF21 compounds and GLP-1R agonists is administered as a fusion protein. Therefore, there is a need for novel fusion proteins combining FGF21 and GLP-1R agonists, and their formulations. Summary of the Invention

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

[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 natural GLP-1 (7-36) comprising up to about 15 amino acid residues substituted in the amino acid sequence (SEQ ID NO:260) of natural GLP-1 (7-36).

[0006] In one embodiment, the human FGF21 functionally active variant 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, and contains

[0007] (i) Replace Q55C and P147C or replace Q55C and N149C, and

[0008] (ii) Substitution or deletion of G198 and / or P199

[0009] The amino acid residues are numbered according to SEQ ID NO:250.

[0010] In one embodiment, the GLP-1R agonist peptide and the human FGF21 functional active variant are linked via a linker molecule comprising a structure selected from L-Fc, Fc-L, L1-Fc-L2, and Fc, wherein L, L1, and L2 are independently selected from a single amino acid and peptide, and Fc is the Fc domain of an immunoglobulin or a variant thereof.

[0011] In one embodiment, the GLP-1R agonist peptide, which is part of the fusion protein, has a GLP-1R agonist activity that is reduced by about 9 to about 531 times compared to the GLP-1R agonist activity of natural GLP-1 (7-36).

[0012] In one embodiment, the GLP-1R agonist peptide, which is part of the fusion protein, has a GLP-1R agonist activity that is reduced by about 9 to about 482 times (or about 9.449 to about 482.396 times), or about 9 to about 319 times (or about 9.449 to about 319.311 times), or about 9 to about 121 times (or about 9.449 to about 121.189 times) compared to the GLP-1R agonist activity of native GLP-1 (7-36).

[0013] In one embodiment, the GLP-1R agonist peptide, which is part of the fusion protein, has a GLP-1R agonist activity that is reduced by about 9 to about 319 times compared to the GLP-1R agonist activity of natural GLP-1 (7-36).

[0014] In one embodiment, the GLP-1R agonist peptide, which is part of the fusion protein, has a GLP-1R agonist activity that is reduced by at least about 9.4 times, at least about 9.45 times, or at least about 9.5 times compared to the GLP-1R agonist activity of natural GLP-1 (7-36).

[0015] In one embodiment, the GLP-1R agonist peptide, which is part of the fusion protein, has at least about 10-fold reduced GLP-1R agonist activity compared to the GLP-1R agonist activity of natural GLP-1 (7-36).

[0016] In one embodiment, the GLP-1R agonist peptide, which is part of the fusion protein, has a GLP-1R agonist activity that is reduced by up to about 482.4 times or up to about 482.35 times compared to the GLP-1R agonist activity of natural GLP-1 (7-36).

[0017] In one embodiment, the GLP-1R agonist peptide, which is part of the fusion protein, has a GLP-1R agonist activity that is reduced by up to about 482 times compared to the GLP-1R agonist activity of native GLP-1 (7-36).

[0018] In one embodiment, the GLP-1R agonist peptide, which is part of the fusion protein, has a GLP-1R agonist activity that is reduced by about 10 to about 482 times compared to the GLP-1R agonist activity of natural GLP-1 (7-36).

[0019] In one embodiment, the GLP-1R agonist peptide, which is part of the fusion protein, has a GLP-1R agonist activity that is reduced by about 10 to about 319 times compared to the GLP-1R agonist activity of natural GLP-1 (7-36).

[0020] In one embodiment, the GLP-1R agonist peptide, which is part of the fusion protein, has a GLP-1R agonist activity that is reduced by about 90 to about 100 times compared to the GLP-1R agonist activity of natural GLP-1 (7-36).

[0021] In one embodiment, the GLP-1R agonist peptide, which is part of the fusion protein, has a GLP-1R agonist activity that is at least about 18-fold (or at least about 18.268-fold) lower than that of natural GLP-1 (7-36).

[0022] In one embodiment, the GLP-1R agonist peptide, which is part of the fusion protein, has a GLP-1R agonist activity that is reduced by about 18 to about 501 times (or about 18.268 to about 500.686 times), or about 18 to about 469 times (or about 18.268 to about 468.679 times), or about 18 to about 313 times (or about 18.268 to about 313.214 times), or about 18 to about 123 times (or about 18.268 to about 123.466 times) compared to the GLP-1R agonist activity of natural GLP-1 (7-36).

[0023] In one embodiment, the GLP-1R agonist peptide, which is part of the fusion protein, has a GLP-1R agonist activity that is reduced by about 18 to about 313 times compared to the GLP-1R agonist activity of natural GLP-1 (7-36).

[0024] In one of the above embodiments, the GLP-1R agonist peptide, which is part of the fusion protein, has a GLP-1R agonist activity that is reduced by at least about 18.2 times or at least about 18.3 times compared to the GLP-1R agonist activity of natural GLP-1 (7-36).

[0025] In one of the above embodiments, the GLP-1R agonist peptide, which is part of the fusion protein, has a GLP-1R agonist activity that is reduced by at least about 20 times, at least about 50 times, or at least about 100 times compared to the GLP-1R agonist activity of natural GLP-1 (7-36).

[0026] In one embodiment, the GLP-1R agonist peptide, as part of the fusion protein, exhibits GLP-1R agonist activity that is reduced by about 10-fold to about 500-fold compared to the GLP-1R agonist activity of native GLP-1 (7-36). In one embodiment, the GLP-1R agonist peptide, as part of the fusion protein, exhibits GLP-1R agonist activity that is reduced by about 15-fold to about 500-fold compared to the GLP-1R agonist activity of native GLP-1 (7-36). In one embodiment, the GLP-1R agonist peptide, as part of the fusion protein, exhibits GLP-1R agonist activity that is reduced by about 20-fold to about 500-fold compared to the GLP-1R agonist activity of native GLP-1 (7-36). In one embodiment, the GLP-1R agonist peptide, as part of the fusion protein, exhibits GLP-1R agonist activity that is reduced by about 50-fold to about 500-fold compared to the GLP-1R agonist activity of native GLP-1 (7-36). In one embodiment, the GLP-1R agonist peptide, as part of the fusion protein, exhibits GLP-1R agonist activity that is reduced by about 100-fold to about 500-fold compared to the GLP-1R agonist activity of native GLP-1 (7-36). In another embodiment, the GLP-1R agonist peptide, as part of the fusion protein, exhibits GLP-1R agonist activity that is reduced by about 100-fold to about 300-fold compared to the GLP-1R agonist activity of native GLP-1 (7-36).

[0027] In one embodiment, the fusion protein has GLP-1R agonist activity as defined above.

[0028] In one embodiment, the GLP-1R agonist peptide comprises or is composed of the following amino acid sequence:

[0029] 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 -X 22 -X 23-X 24 -X 25 -LX 27 -X 28 -X 29 -X 30 (SEQ ID NO:4077),

[0030] in

[0031] X1 is H, Y, or F.

[0032] X2 is G, S, T, or A.

[0033] X3 is either E or Q.

[0034] X 10 Is it K or L?

[0035] X 12 Is it K, I, or Q?

[0036] X 13 Is it Q or L?

[0037] X 14 Is it L, M, or C?

[0038] X 15 It is E, A, or D.

[0039] X 16 Is it E, K, or S?

[0040] X 17 Is it E, R, or Q?

[0041] X 18 Is it L, A, or R?

[0042] X 19 Is it V, A, or F?

[0043] X 20 It is R, H, Q, K, or I.

[0044] X 21 Is it L, E, H, or R?

[0045] X 22 Is it F or L?

[0046] X 23 Is it I, Y, or F?

[0047] X 24 Is it E, L, or Y?

[0048] X 25 Is it W or L?

[0049] X 27 Is it I, L, K, or E?

[0050] X 28 Is it A, K, N, or E?

[0051] X 29 It is G, T, K or V, and

[0052] X 30 It is either G or missing;

[0053] Optionally, the amino acid sequence further includes at least one additional amino acid residue at its N-terminus; and

[0054] Optionally, the amino acid sequence further includes a peptide extension consisting of up to about 12, about 11, or about 10 amino acid residues at its C-terminus.

[0055] 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.

[0056] In one embodiment, the peptide extension consists of an amino acid sequence selected from SEQ ID NO:4008 to 4063. In another embodiment, the peptide extension is a single amino acid residue, such as P.

[0057] In one embodiment, the GLP-1R agonist peptide comprises or consists of an amino acid sequence selected from or composed of said amino acid sequence.

[0058] In one embodiment, the GLP-1R agonist peptide comprises or is composed of the following amino acid sequence:

[0059] 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(SEQ ID NO:4078),

[0060] in

[0061] X1 is H, Y, or F.

[0062] X 10 Is it K or L?

[0063] X 12 Is it K, I, or Q?

[0064] X 13 Is it Q or L?

[0065] X 15 It is E, A, or D.

[0066] X 16 Is it E, K, or S?

[0067] X 17 Is it E, R, or Q?

[0068] X 18 Is it L, A, or R?

[0069] X 19 Is it V, A, or F?

[0070] X 20 It is R, H, Q, K, or I.

[0071] X 21 Is it L, E, H, or R?

[0072] X 23 Is it I, Y, or F?

[0073] X 27 Is it I, L, K, or E?

[0074] X 28 It is A, K, N or E, and

[0075] X 29 It is G, T, K, or V;

[0076] Optionally, the amino acid sequence further includes at least one additional amino acid residue at its N-terminus; and

[0077] Optionally, the amino acid sequence further includes a peptide extension consisting of up to about 12, about 11, or about 10 amino acid residues at its C-terminus.

[0078] 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.

[0079] In one embodiment, the peptide extension consists of an amino acid sequence selected from SEQ ID NO:4008 to 4063.

[0080] In one embodiment, the GLP-1R agonist peptide comprises or is composed of the following amino acid sequence:

[0081] HGEGTFTSDX 10 -SKQLEEEX 18 -VX 20 -LFIEWLKAX 29 -G(SEQID NO:4079),

[0082] in

[0083] X 10 Is it K or L?

[0084] X 18 Is it A or R?

[0085] X 20 It is R or Q, and

[0086] X 29 Is it G or T?

[0087] Optionally, the amino acid sequence further includes at least one additional amino acid residue at its N-terminus; and

[0088] Optionally, the amino acid sequence further includes a peptide extension consisting of up to 12, 11, or 10 amino acid residues at its C-terminus.

[0089] 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.

[0090] 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).

[0091] In one embodiment, the GLP-1R agonist peptide comprises or is composed of the amino acid sequence of SEQ ID NO:261 or 262.

[0092] In one embodiment, the GLP-1R agonist peptide does not contain or is not composed of any of the amino acid sequences of SEQ ID NO:4064 to 4076 and 553.

[0093] In one embodiment, the human FGF21 functionally active variant comprises a substitution or deletion selected from G198R, G198K, G198Y, and P199 deletion.

[0094] In one embodiment, the human FGF21 functional active variant comprises or consists of an amino acid sequence selected from or composed of said amino acid sequences, including SEQ ID NO: 253, 254, 255, and 256. In another embodiment, the human FGF21 functional active variant comprises or consists of an amino acid sequence, including or composed of said amino acid sequences, including SEQ ID NO: 253 or 254.

[0095] In one embodiment, the GLP-1R agonist peptide comprises or is composed of the amino acid sequence of SEQ ID NO:261, and the human FGF21 functional active variant comprises or is composed of the amino acid sequence of SEQ ID NO:253.

[0096] In one embodiment, the GLP-1R agonist peptide comprises or is composed of the amino acid sequence of SEQ ID NO:261, and the human FGF21 functional active variant comprises or is composed of the amino acid sequence of SEQ ID NO:254.

[0097] In one embodiment, the GLP-1R agonist peptide comprises or is composed of the amino acid sequence of SEQ ID NO:262, and the human FGF21 functional active variant comprises or is composed of the amino acid sequence of SEQ ID NO:253.

[0098] In one embodiment, the GLP-1R agonist peptide comprises or is composed of the amino acid sequence of SEQ ID NO:262, and the human FGF21 functional active variant comprises or is composed of the amino acid sequence of SEQ ID NO:254.

[0099] In one embodiment, the Fc domain of the immunoglobulin or a variant thereof comprises or is composed of an amino acid sequence selected from or consisting of said amino acid sequences. In one embodiment, the Fc domain of the immunoglobulin or a variant thereof comprises or is composed of the amino acid sequence of SEQ ID NO:257. In one embodiment, the Fc domain of the immunoglobulin or a variant thereof comprises or is composed of the amino acid sequence of SEQ ID NO:258. In one embodiment, the Fc domain of the immunoglobulin or a variant thereof comprises or is composed of the amino acid sequence of SEQ ID NO:259.

[0100] In one embodiment, the GLP-1R agonist peptide comprises or is composed of the amino acid sequence of SEQ ID NO:261, the human FGF21 functional active variant comprises or is composed of the amino acid sequence of SEQ ID NO:253, and the Fc domain of the immunoglobulin or a variant thereof comprises or is composed of the amino acid sequence of SEQ ID NO:257.

[0101] In one embodiment, the GLP-1R agonist peptide comprises or is composed of the amino acid sequence of SEQ ID NO:261, the human FGF21 functional active variant comprises or is composed of the amino acid sequence of SEQ ID NO:254, and the Fc domain of the immunoglobulin or a variant thereof comprises or is composed of the amino acid sequence of SEQ ID NO:257.

[0102] In one embodiment, the GLP-1R agonist peptide comprises or is composed of the amino acid sequence of SEQ ID NO:262, the human FGF21 functional active variant comprises or is composed of the amino acid sequence of SEQ ID NO:253, and the Fc domain of the immunoglobulin or a variant thereof comprises or is composed of the amino acid sequence of SEQ ID NO:257.

[0103] In one embodiment, the GLP-1R agonist peptide comprises or is composed of the amino acid sequence of SEQ ID NO:262, the human FGF21 functional active variant comprises or is composed of the amino acid sequence of SEQ ID NO:254, and the Fc domain of the immunoglobulin or a variant thereof comprises or is composed of the amino acid sequence of SEQ ID NO:257.

[0104] In one embodiment, the GLP-1R agonist peptide comprises or is composed of the amino acid sequence of SEQ ID NO:261, the human FGF21 functional active variant comprises or is composed of the amino acid sequence of SEQ ID NO:253, and the Fc domain of the immunoglobulin or a variant thereof comprises or is composed of the amino acid sequence of SEQ ID NO:258.

[0105] In one embodiment, the GLP-1R agonist peptide comprises or is composed of the amino acid sequence of SEQ ID NO:261, the human FGF21 functional active variant comprises or is composed of the amino acid sequence of SEQ ID NO:254, and the Fc domain of the immunoglobulin or a variant thereof comprises or is composed of the amino acid sequence of SEQ ID NO:258.

[0106] In one embodiment, the GLP-1R agonist peptide comprises or is composed of the amino acid sequence of SEQ ID NO:262, the human FGF21 functional active variant comprises or is composed of the amino acid sequence of SEQ ID NO:253, and the Fc domain of the immunoglobulin or a variant thereof comprises or is composed of the amino acid sequence of SEQ ID NO:258.

[0107] In one embodiment, the GLP-1R agonist peptide comprises or is composed of the amino acid sequence of SEQ ID NO:262, the human FGF21 functional active variant comprises or is composed of the amino acid sequence of SEQ ID NO:254, and the Fc domain of the immunoglobulin or a variant thereof comprises or is composed of the amino acid sequence of SEQ ID NO:258.

[0108] In one embodiment, the GLP-1R agonist peptide comprises or is composed of the amino acid sequence of SEQ ID NO:261, the human FGF21 functional active variant comprises or is composed of the amino acid sequence of SEQ ID NO:253, and the Fc domain of the immunoglobulin or a variant thereof comprises or is composed of the amino acid sequence of SEQ ID NO:259.

[0109] In one embodiment, the GLP-1R agonist peptide comprises or is composed of the amino acid sequence of SEQ ID NO:261, the human FGF21 functional active variant comprises or is composed of the amino acid sequence of SEQ ID NO:254, and the Fc domain of the immunoglobulin or a variant thereof comprises or is composed of the amino acid sequence of SEQ ID NO:259.

[0110] In one embodiment, the GLP-1R agonist peptide comprises or is composed of the amino acid sequence of SEQ ID NO:262, the human FGF21 functional active variant comprises or is composed of the amino acid sequence of SEQ ID NO:253, and the Fc domain of the immunoglobulin or a variant thereof comprises or is composed of the amino acid sequence of SEQ ID NO:259.

[0111] In one embodiment, the GLP-1R agonist peptide comprises or is composed of the amino acid sequence of SEQ ID NO:262, the human FGF21 functional active variant comprises or is composed of the amino acid sequence of SEQ ID NO:254, and the Fc domain of the immunoglobulin or a variant thereof comprises or is composed of the amino acid sequence of SEQ ID NO:259.

[0112] In another aspect, the present invention relates to 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).

[0113] The GLP-1R agonist peptide comprises or is composed of an amino acid sequence selected from or consisting of the amino acid sequences SEQ ID NO:261 to 565;

[0114] The human FGF21 functionally active variant comprises or consists of an amino acid sequence selected from or composed of said amino acid sequences, SEQ ID NO: 253, 254, 255, and 256; and

[0115] The GLP-1R agonist peptide and the human FGF21 functional active variant are linked via a linker molecule comprising a structure selected from L-Fc, Fc-L, L1-Fc-L2, and Fc, wherein L, L1, and L2 are independently selected from a single amino acid and a peptide, and Fc is the Fc domain of an immunoglobulin or a variant thereof, wherein the Fc domain or a variant thereof comprises or is composed of an amino acid sequence selected from or consisting of the amino acid sequences of SEQ ID NO: 257, 258, and 259.

[0116] In yet another aspect, the present invention relates to a fusion protein or a functionally active variant thereof, said fusion protein comprising or consisting of an amino acid sequence selected from SEQ ID NO:1-8, 16-31, 33-229 and 566-4007, said functionally active variant comprising or consisting of an amino acid sequence that is at least about 96% identical to or consists of an amino acid sequence selected from SEQ ID NO:1-8, 16-31, 33-229 and 566-4007.

[0117] In another aspect, the present invention relates to a fusion protein or a functionally active variant thereof, said fusion protein comprising, or consisting of, an amino acid sequence selected from or composed of said amino acid sequence, SEQ ID NO: 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, said functionally active variant comprising, and constitutes an amino acid sequence selected from SEQ ID NO: 1-8, 18-31, 39, 40, 42-72, 74, 76, 78-84, 88-90, 92-97, 100-102, 105-109, 112, 116, 217 and 219-229, said amino acid sequence, said functionally active variant comprising, and constitutes an amino acid sequence selected from SEQ ID NO: 1-8, 18-31, 39, 40, 42-72, 74, 76, 78-84, 88-90, 92-97, 100-102, 105-109, 112, 116, 217 The amino acid sequences of 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 are at least about 96% identical or consist of said amino acid sequences.

[0118] In another aspect, the present invention relates to a fusion protein or a functionally active variant thereof, the fusion protein comprising or consisting of an amino acid sequence selected from SEQ ID NO:2, 7 and 8, and the functionally active variant thereof comprising or consisting of an amino acid sequence that is at least about 96% identical to or consists of an amino acid sequence selected from SEQ ID NO:2, 7 and 8.

[0119] In one embodiment, a fusion protein (or a functionally active variant thereof) as defined above activates human GLP-1R at an EC50 concentration 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 substantially as described in Example 4.

[0120] In one embodiment, the fusion protein (or its functionally active variant) as defined above induces (i) autophosphorylation of human FGF receptor 1c (FGFR1c) 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 (e.g., EC50 of about 10 nmol / L to about 50 nmol / L, or about 15 nmol / L to about 50 nmol / L). / L, or about 15 nmol / L to about 45 nmol / L); and / or (ii) 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 (e.g., 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, autophosphorylation of human FGFR1c and / or phosphorylation of MAPK ERK1 / 2 are determined, for example, substantially as described in Example 3, by using In-Cell Western blotting (ICW).

[0121] In one embodiment, the melting temperature and / or aggregation temperature of the fusion protein (or its functionally active variant) as defined above is 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 substantially as described in Example 5.

[0122] In one embodiment, the fusion protein (or its functionally active variant) as defined above has a terminal plasma half-life of at least about 15 hours or at least about 20 hours in non-human primates. In one embodiment, the fusion protein (or its functionally active variant) as 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 mice. In one embodiment, the terminal half-life is determined after a single subcutaneous administration of about 0.3 mg / kg of the fusion protein solution to a non-human primate (e.g., a cynomolgus monkey) or to a mouse (e.g., a C57Bl / 6 mouse). In one embodiment, the terminal half-life is determined by substantially the method described in Example 6.

[0123] In another aspect, the present invention relates to a nucleic acid molecule that encodes a fusion protein as defined above.

[0124] In another aspect, the present invention relates to a host cell containing nucleic acid molecules as defined above.

[0125] In another aspect, the present invention relates to a method for producing a fusion protein as defined above, the method comprising culturing a host cell as defined above and isolating the fusion protein.

[0126] 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.

[0127] 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.

[0128] In another aspect, the present invention relates to fusion proteins as defined above, nucleic acid molecules as defined above, host cells as defined above, or pharmaceutical compositions as defined above, which are used as pharmaceutical agents.

[0129] In another aspect, the present invention relates to fusion proteins as defined above, nucleic acid molecules as defined above, host cells as defined above, or pharmaceutical compositions as defined above, for use in treating diseases or disorders selected from obesity, overweight, metabolic syndrome, diabetes, diabetic retinopathy, hyperglycemia, dyslipidemia, NASH, and atherosclerosis.

[0130] In one embodiment, the disease or disorder is diabetes. In one embodiment, the diabetes is type 1 diabetes or type 2 diabetes.

[0131] In another aspect, the present invention relates to the use of fusion proteins as defined above, nucleic acid molecules as defined above, host cells as defined above, or pharmaceutical compositions as defined above in the manufacture of an agent for treating diseases or disorders selected from: obesity, overweight, metabolic syndrome, diabetes, diabetic retinopathy, hyperglycemia, dyslipidemia, NASH, and atherosclerosis.

[0132] In one embodiment, the disease or disorder is diabetes. In one embodiment, the diabetes is type 1 diabetes or type 2 diabetes.

[0133] In another aspect, the present invention relates to a method for treating a disease or disorder selected from obesity, overweight, metabolic syndrome, diabetes, diabetic retinopathy, hyperglycemia, dyslipidemia, NASH, and atherosclerosis, the method comprising administering to a subject in need 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.

[0134] In one embodiment, the disease or disorder is diabetes. In one embodiment, the diabetes is type 1 diabetes or type 2 diabetes.

[0135] In another aspect, the present invention relates to fusion proteins as defined above, nucleic acid molecules as defined above, host cells as defined above, or pharmaceutical compositions as defined above for improving glycemic control in overweight to obese patients with type 2 diabetes and dyslipidemia. Attached Figure Description

[0136] Figure 1 This is a graph showing the EC50 of adverse effects (gastric emptying (GE) rate) and pharmacodynamics (i.e., HbA1c, triglycerides, fatty acids, non-HDL, fat mass) based on the GLP-1 decay factor (12-month simulation):

[0137] • For GLP-1 attenuation factors greater than 9.449 (which can be rounded to 9), the EC50 of GLP-1-mediated adverse gastrointestinal effects (gastric emptying; GE rate) is greater than the EC50 of pharmacodynamic effects (i.e., HbA1c, fat mass, non-HDL, fatty acids, triglycerides).

[0138] The maximum distance between the maximum pharmacodynamic (HbA1c) normalized by extending the effects of FGF21-mediated (lipids) and GLP-1-mediated (HbA1c) and the adverse effect (GE rate) is 121.189; that is, at 121.189 (which can be rounded to 121), at the minimum distance between the GLP-1-mediated effect (HbA1c) and the average effect mediated by FGF21 (i.e., fat mass, non-HDL, fatty acids, triglycerides), there exists the maximum distance between the maximum pharmacodynamic effect (HbA1c) and the adverse effect (GE rate) (see [reference]). Figure 2 );

[0139] The maximum distance between the maximum value of pharmacodynamic (HbA1c) and the adverse effect (GE rate) is 319.311 (which can be rounded to 319);

[0140] 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 [reference]). Figure 2 (It can be rounded to 482);

[0141] The maximum gastric emptying rate was 531.0.

[0142] (All: Vertical lines).

[0143] Figure 2 This is a graph showing the EC50 of gastric emptying (GE) rate and mean pharmacodynamic effects (i.e., HbA1c, triglycerides, fatty acids, non-HDL, and fat mass) based on the GLP-1 decay factor (12-month simulation):

[0144] • The maximum distance between mean pharmacodynamics (i.e., HbA1c, fat mass, non-HDL, fatty acids, triglycerides) and adverse effects (GE rate) is 482.396 (right vertical line; can be rounded to 482);

[0145] The maximum distance between the maximum value of the pharmacodynamic (HbA1c) normalized by extending the effects of FGF21-mediated (lipids) and GLP-1-mediated (HbA1c) and the adverse effect (GE rate) is 121.189 (left vertical line; can be rounded to 121). The curve “(maximum value - GE rate) / range” represents the ratio of the maximum distance between HbA1c and the GE rate to the minimum distance between HbA1c and the average effect mediated by FGF21 (i.e., fat mass, non-HDL, fatty acids, triglycerides). At the minimum value of the “(maximum value - GE rate) / range” curve (i.e., at 121.189), at the minimum distance between the GLP-1-mediated effect (HbA1c) and the FGF21-mediated effect (i.e., fat mass, non-HDL, fatty acids, triglycerides), there exists the maximum distance between the maximum value of the pharmacodynamic effect (HbA1c) and the adverse effect (GE rate).

[0146] Figure 3 This is a graph showing the EC50 of adverse effects (gastric emptying (GE) rate) and pharmacodynamics (HbA1c, triglycerides, fatty acids, non-HDL, fat mass) based on the GLP-1 decay factor (3-month simulation):

[0147] • For GLP-1 attenuation factors greater than 18.268 (which can be rounded to 18), the EC50 of GLP-1-mediated adverse gastrointestinal effects (gastric emptying; GE rate) is greater than the EC50 of pharmacodynamic effects (i.e., HbA1c, fat mass, non-HDL, fatty acids, triglycerides).

[0148] The maximum distance between the maximum pharmacodynamic (HbA1c) and the adverse effect (GE rate) normalized by extending the effects of FGF21-mediated (lipids) and GLP-1-mediated (HbA1c) is 123.466; that is, at 123.466 (which can be rounded to 123), at the minimum distance between the GLP-1-mediated effect (HbA1c) and the average effect (i.e., fat mass, non-HDL, fatty acids, triglycerides), there exists the maximum distance between the maximum pharmacodynamic effect (HbA1c) and the adverse effect (GE rate) (see [reference]). Figure 4 );

[0149] The maximum distance between the maximum value of pharmacodynamics (HbA1c) and the adverse effects (GE rate) is 313.214 (which can be rounded to 313);

[0150] 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 [reference]). Figure 4 (It can be rounded to 469);

[0151] The maximum gastric emptying rate is 500.686 (which can be rounded to 501).

[0152] (All: Vertical lines).

[0153] Figure 4 This is a graph showing the EC50 of gastric emptying (GE) rate and mean pharmacodynamic effects (i.e., HbA1c, triglycerides, fatty acids, non-HDL, and fat mass) based on the GLP-1 decay factor (3-month simulation):

[0154] • The maximum distance between mean pharmacodynamics (i.e., HbA1c, fat mass, non-HDL, fatty acids, triglycerides) and adverse effects (GE rate) is 468.679 (right vertical line; can be rounded to 469);

[0155] The maximum distance between the maximum value of the pharmacodynamic (HbA1c) normalized by extending the effects of FGF21-mediated (lipids) and GLP-1-mediated (HbA1c) and the adverse effect (GE rate) is 123.466 (left vertical line; can be rounded to 123). The curve “(maximum value - GE rate) / range” represents the ratio of the maximum distance between HbA1c and the GE rate to the minimum distance between HbA1c and the average effect mediated by FGF21 (i.e., fat mass, non-HDL, fatty acids, triglycerides). At the minimum value of the “(maximum value - GE rate) / range” curve (i.e., at 123.466), at the minimum distance between the GLP-1-mediated effect (HbA1c) and the FGF21-mediated effect (i.e., fat mass, non-HDL, fatty acids, triglycerides), there exists the maximum distance between the maximum value of the pharmacodynamic effect (HbA1c) and the adverse effect (GE rate).

[0156] Figure 5 (A and B) are graphs showing the results of in vitro cell Western spectroscopy (ICW) assays of human FGF21 receptor efficacy in CHO cells. pFGFR is depicted in (A) and pERK is depicted in (B).

[0157] Figure 6 (A through D) are figures showing the results of in vitro cell assays of the efficacy of different GLP-1R agonists on the human glucagon-like peptide-1 (GLP-1) receptor in HEK-293 cells. SEQ ID NO:2 is depicted in (A), SEQ ID NO:7 in (B), SEQ ID NO:8 in (C), and SEQ ID NO:2, 7, and 8 in (D).

[0158] Figure 7(A through F) are graphs showing the plasma concentrations of the GLP-1R agonist / FGF21 Fc fusion protein obtained using three different bioanalytical methods after a single subcutaneous administration of 0.3 mg / kg solution to female C57Bl / 6 mice or male cynomolgus monkeys. (A) depicts SEQ ID NO:2 in mice, (B) depicts SEQ ID NO:2 in monkeys, (C) depicts SEQ ID NO:7 in mice, (D) depicts SEQ ID NO:7 in monkeys, (E) depicts SEQ ID NO:8 in mice, and (F) depicts SEQ ID NO:8 in monkeys.

[0159] Figure 8 This is a graph showing the plasma concentrations of GLP-1R agonist / FGF21 Fc fusion protein and G-FGF21 (SEQ ID NO:252) obtained using a bioanalytical method for quantifying the intact full-length fusion protein after a single subcutaneous administration of 0.3 mg / kg solution to female C57Bl / 6 mice.

[0160] Figure 9 This is a graph showing the weight development of female diet-induced obese (DIO) mice that were administered GLP-1RA / FGF21 Fc fusion protein once a week and control mice that underwent a 28-day diet.

[0161] Figure 10 This is a graph showing the development of cumulative food intake in female DIO mice that were administered GLP-1RA / FGF21 Fc fusion protein once a week for 28 days, compared to a control group.

[0162] Figure 11 (A and B) are graphs showing the 24-hour blood glucose profiles of db / db mice after the first treatment with GLP-1RA / FGF21 Fc fusion protein on day 1 and the control (A), or after the fourth treatment on day 22 (B). Data are mean ± SEM; n = 8 mice / group.

[0163] Figure 12 This is a graph showing the plasma HbA1c levels in female db / db mice that were administered GLP-1RA / FGF21 Fc fusion protein once a week and as a control for 36 days.

[0164] Figure 13 (A and B) are graphs showing the development of liver weight and lipid content in DIO NASH mice after weekly administration of GLP-1RA / FGF21 Fc fusion protein and control for 8 weeks. (A) depicts liver weight and lipid levels, and (B) depicts liver cholesterol and liver triglyceride levels.

[0165] Figure 14A graph depicts the development of fibrosis and non-alcoholic fatty liver disease (NAFLD) activity scores in DIO NASH mice after 8 weeks of weekly administration of GLP-1RA / FGF21 Fc fusion protein and control.

[0166] Figure 15 A graph depicts the number of DIO NASH mice with higher, same, or lower fibrosis and NAFLD activity scores after 8 weeks of weekly administration of the GLP-1RA / FGF21 Fc fusion protein and control. Detailed Implementation

[0167] Although the invention is described in detail below, it should be understood that the invention is not limited to the specific methods, schemes, and reagents described herein, as these methods, schemes, and reagents can 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 invention, which will be defined only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0168] Certain elements of the invention will be described below. These elements can be listed in specific embodiments; however, it should be understood that they can be combined in any manner and in any number to produce other embodiments. The differentiated descriptions of embodiments and exemplary embodiments should not be construed as limiting the invention to only the explicitly described embodiments. This specification should be understood to support and cover embodiments combining the explicitly described embodiments with any number of disclosed and / or exemplary elements. Furthermore, unless the context otherwise indicates, any permutation and combination of the elements described herein should be considered.

[0169] The terminology used in this article is as follows: “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, H.G. Leuenberger, B. Nagel, and H. As defined in Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0170] Unless otherwise indicated, the practice of this invention will be carried out using conventional methods of chemistry, biochemistry, cell biology, immunology and recombinant DNA technology, as explained in the literature in the art (Sambrook, J. et al. (2001) Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0171] Throughout this specification and the following claims, unless the context otherwise requires, the word "comprise" and variations such as "comprises" and "comprising" should be understood to imply inclusion of the stated member, integer, or step, or a group of members, integers, or steps, but not to exclude any other member, integer, or step, or a group of members, integers, or steps. However, in some embodiments, such other members, integers, or steps, or groups of members, integers, or steps may be excluded, i.e., the subject matter is to include the stated member, integer, or step, or a group of members, integers, or steps. Unless otherwise indicated herein or clearly contradicted by the context, the terms "a" and "an," as well as "the" and similar references used in the context of describing the invention (especially in the context of the claims), should be interpreted to cover both the singular and plural. The range of values ​​enumerated herein is intended only as a shorthand for individually referring to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into this specification as if it were individually enumerated herein. Unless otherwise indicated herein or otherwise clearly contradicted by the context, all methods described herein may be performed in any suitable order. The use of any and all examples or exemplary language (e.g., “such as”) provided herein is intended only to better illustrate the invention and does not impose any limitation on the scope of the originally claimed invention. No language in this specification should be construed as indicating that any unclaimed element is necessary for the practice of the invention.

[0172] Numerous references are cited throughout this specification. Every reference cited herein, whether above or below (including all patents, patent applications, scientific publications, manufacturer's instructions, guidelines, etc.), is incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to any prior disclosure.

[0173] The term "fusion protein" generally refers to a protein produced by linking (particularly covalently linking) two or more distinct proteins (e.g., proteins and / or peptides) to create a single molecule having functional properties derived from each of the original proteins. Typically, the fusion proteins of this invention exhibit GLP-1R agonist activity and FGF21 activity. Fusion proteins can be produced by genetic fusion (e.g., via recombinant DNA technology) or by chemical and / or enzymatic conjugation. In the fusion protein according to the invention, the components of the fusion protein can be arranged in sequence (from N-terminus to C-terminus) ABC or CBA, where A is a GLP-1R agonist peptide, B is a linker molecule, and C is a human FGF21 functional variant.

[0174] As used herein, the term “GLP-1R agonist peptide” refers to a peptide that binds to and activates the GLP-1 receptor, such as GLP-1 (as the primary GLP-1R agonist). GLP-1R agonist peptide may also be referred to simply as “GLP-1R agonist” in this document.

[0175] The term "peptide" generally refers to a polymeric form of amino acids of any length, comprising, for example, about two or more, or about three or more, or about four or more, or about six or more, or about eight or more, or about nine or more, or about ten or more, or about thirteen or more, or about sixteen or more, or about 21 or more amino acids covalently linked by peptide bonds. A peptide can, for example, consist of up to 100 amino acids. The term "polypeptide" refers to a macropeptide. 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.

[0176] In one embodiment, the GLP-1R agonist peptide is a variant of natural GLP-1 (7-36). As used herein, the term "natural GLP-1 (7-36)" refers to a peptide having the amino acid sequence of SEQ ID NO:260, which optionally includes an amide group at its C-terminus.

[0177] Typically, variants of natural GLP-1(7-36) can be based on the deletion, addition, and / or substitution of at least one amino acid residue in the amino acid sequence of natural GLP-1(7-36).

[0178] 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 (SEQ ID NO:260) of natural GLP-1 (7-36).

[0179] As used herein, the term “amino acid” or “amino acid residue” refers to naturally occurring amino acids, non-natural amino acids that function in a manner similar to that of naturally occurring amino acids, amino acid analogs and amino acid mimics, in their D and / or L stereoisomer forms, if their structure allows for such stereoisomer forms. Amino acids are referred to herein by their names, their three-letter symbols known in the art, or the single-letter symbols recommended by the IUPAC-IUB Committee on Biochemical Nomenclature.

[0180] When used in conjunction with amino acids, the term "naturally occurring" refers to the 20 common 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 selenocysteine, pyrrolidone (PYL), and pyrrolidone-carboxylysine (PCL).

[0181] As used herein, the term "non-natural amino acid" is intended to refer to an amino acid that is not naturally encoded or found in the genetic code of any organism. It can be, for example, a purely synthetic compound. Examples of non-natural amino acids include, but are not limited to, hydroxyproline, γ-carboxyglutamic acid, O-phosphoserine, azacyclobutanecarboxylic acid, 2-aminohexanoic acid, 3-aminohexanoic acid, β-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminohexanoic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, tert-butylglycine, 2,4-diaminoisobutyric acid, desmodium, 2,2'-diaminopimelic acid, and 2,3-diaminopropionic acid. N-Ethylglycine, N-Methylglycine, N-Ethylasparagine, Hoproline, Hydroxylysine, Allohydroxylysine, 3-Hydroxyproline, 4-Hydroxyproline, Isodesin, Alloisoleucine, N-Methylalanine, N-Methylglycine, N-Methylisoleucine, N-Methylpentylglycine, N-Methylvaline, Naphthylalanine, Ornithine, Ornithine, D-Ornithine, D-Arginine, p-Aminophenylalanine, Pentylglycine, Piperidinic acid, and Thioproline.

[0182] As used herein, the term "amino acid analog" refers to a compound having the same basic chemical structure as naturally occurring amino acids. Amino acid analogs include both natural and non-natural amino acids that have been 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-(β-methyl ester), N-ethylglycine, alanine formamide, homoserine, ortholeucine, and methionine methylsulfonium.

[0183] As used herein, the term "amino acid analogue" refers to a chemical compound that has a structure different from the general chemical structure of amino acids but functions in a manner similar to that of naturally occurring amino acids.

[0184] In some embodiments, the variant includes 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 other than proline; non-natural amino acids; amino acid analogs; and amino acid mimics. In one embodiment, the at least one additional amino acid residue is selected from 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.

[0185] In some embodiments, the variant includes a peptide extension at its C-terminus. The peptide extension may consist, for example, of 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 SEQ ID NO: 4008 to 4063. In one embodiment, the peptide extension is a single amino acid residue, such as P.

[0186] In one embodiment, the GLP-1R agonist peptide component of the fusion protein of the present invention exhibits reduced GLP-1R agonist activity compared to the GLP-1R agonist activity of native GLP-1 (7-36) as defined herein. The expression "GLP-1R agonist peptide as part of a fusion protein" means that the reduced GLP-1R agonist peptide exhibits this activity when it is a component of the fusion protein and not necessarily in its isolated form (i.e., when it is not a component of the fusion protein).

[0187] In one embodiment, as used herein, the term "GLP-1R agonistic activity" (or "GLP-1R agonistic potency") refers to the activation of the GLP-1 receptor. In one embodiment, the term refers to in vitro agonistic activity / potency. In another embodiment, the term refers to in vivo agonistic activity / potency. In one embodiment, the activation of the GLP-1 receptor is determined by measuring the cAMP response of cells stably expressing the GLP-1 receptor upon in vitro contact with an agonist. In one embodiment, the cells are derived from the HEK-293 cell line. In one embodiment, the GLP-1 receptor is the human GLP-1 receptor. In one embodiment, the activation of the GLP-1 receptor is determined substantially as described in Example 4. In one embodiment, the activity / potency is quantified by determining an EC50 value.

[0188] As used herein, the term "fibroblast growth factor 21" or "FGF21" refers to any FGF21 protein known in the art, and particularly 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 to 28 (M1 to 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.

[0189] In one embodiment, the human FGF21 functional active variant comprises an amino acid sequence that is at least about 96%, 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, and contains

[0190] (i) Replace Q55C and P147C or replace Q55C and N149C, and

[0191] (ii) Substitution or deletion of G198 and / or P199

[0192] The amino acid residues are numbered according to SEQ ID NO:250.

[0193] 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.

[0194] The “sequence identity” between two amino acid sequences indicates the percentage of identical amino acids between the sequences. Besides manual comparison, the best alignment of sequences for comparison can be performed using the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482; the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443; the similarity search method of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444; or using computer programs that employ these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Avenue, Madison, Wisconsin).

[0195] When used in conjunction with FGF21 (e.g., human FGF21), the term “functionally active variant” refers to a protein that has FGF21 activity.

[0196] In one embodiment, as used herein, the term "FGF21 activity" (or "FGF21 potency") refers to the activation of the FGF21 receptor (FGFR, e.g., FGFR1c). In one embodiment, the FGF21 receptor is the 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, the activation of the FGF21 receptor is determined by measuring the autophosphorylation of the FGF21 receptor and / or the phosphorylation of MAPK ERK1 / 2 upon in vitro contact with an FGF21 compound. In one embodiment, the autophosphorylation of human FGFR1c and / or the phosphorylation of MAPK ERK1 / 2 are determined, for example, by using in-Cell Western blotting (ICW), substantially as described in Example 3. In one embodiment, activity and / or potency are quantified by determining an EC50 value.

[0197] As used herein, the term “In-Cell Western (ICW) assay” refers to an immunocytochemical assay typically performed in microplates (e.g., in 96-well or 384-well formats), more specifically a quantitative immunofluorescence assay. It combines the specificity of Western blotting with the reproducibility and throughput of ELISA (see, for example, Aguilar HN et al. (2010) PLoS ONE 5(4):e9965). Suitable ICW assay systems are commercially available (e.g., from LI-COR Biosciences, USA). In one embodiment, anti-pFGFR and / or anti-pERK are used in the ICW assay.

[0198] In one embodiment, a functionally active variant of human FGF21 exhibits the same or substantially the same FGF21 activity as wild-type human FGF21 (e.g., SEQ ID NO: 250 or 251 or 252), wherein the FGF21 activity refers to the FGF21 activity of a separate functionally active variant of human FGF21 (i.e., when it is not included in the fusion protein of the present invention or modified in any other way).

[0199] When used in conjunction with FGF21 (e.g., human FGF21), the term "substantially identical" means that the FGF21 activity is in the range of 50% to 150%, 60% to 140%, or 65% to 135% of the FGF21 activity of FGF21 (e.g., wild-type human FGF21 (e.g., SEQ ID NO: 250 or 251 or 252)).

[0200] Optionally, the functionally active variant of human FGF21 further comprises substitutions for G141S and / or P174L, which are naturally occurring mutations in human FGF21, wherein the amino acid residues are numbered according to SEQ ID NO:250. G141S in SEQ ID NO:250 corresponds to G113S in SEQ ID NO:251; P174L in SEQ ID NO:250 corresponds to P146L in SEQ ID NO:251.

[0201] Other suitable FGF21 variants for use in this invention are described, for example, in PCT / EP2016 / 079551, which is incorporated herein by reference.

[0202] In one embodiment, the GLP-1R agonist peptide and the human FGF21 functional active variant are linked via a linker molecule comprising a structure selected from L-Fc, Fc-L, L1-Fc-L2, and Fc, wherein L, L1, and L2 are independently selected from a single amino acid and peptide, and Fc is the Fc domain of an immunoglobulin or a variant thereof.

[0203] In one embodiment, the Fc domain (also referred to as the Fc region) is the Fc domain of immunoglobulin IgG1 or IgG4. In one embodiment, the variant of the Fc domain contains up to about 6, about 5, or about 4 mutations compared to the wild-type sequence of the Fc domain. In one embodiment, the mutations are selected from amino acid substitutions, amino acid additions, and amino acid deletions, such as N-terminal or C-terminal deletions. In one embodiment, the Fc domain or its variants may have greater than about 50%, about 60%, about 70%, about 80%, about 90%, about 93%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the wild-type sequence of the IgG1 Fc region (e.g., the human IgG1 Fc region), or may have about 100% sequence identity with it. In one embodiment, the Fc domain or a variant thereof may have greater than about 50%, about 60%, about 70%, about 80%, about 90%, about 93%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the wild-type sequence of the IgG4 Fc region (e.g., the human IgG4 Fc region), or may have about 100% sequence identity with it. In one embodiment, the Fc domain of the immunoglobulin or a variant thereof comprises or consists of an amino acid sequence selected from or composed of said amino acid sequences of SEQ ID NO: 257, 258, and 259.

[0204] 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. Typically, peptide linkers are designed to provide flexibility and protease resistance. In one embodiment, the peptide linker is a glycine-serine-rich linker, wherein, for example, 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 consists only 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 or composed of said amino acid sequences. 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.

[0205] When used in conjunction with the fusion proteins of the present invention, the term "functionally active variant" refers to a fusion protein having GLP-1R agonist activity and FGF21 activity within the ranges defined herein.

[0206] In one embodiment, the functionally active variant comprises or consists of an amino acid sequence that is at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to or composed of the amino acid sequence of the fusion protein from which it is derived.

[0207] In one embodiment, the deviation of the amino acid sequence from the amino acid sequence of the fusion protein from which the functionally active variant is derived is based solely on mutations (e.g., substitutions, deletions, and / or additions of one or more amino acids) occurring in regions of the fusion protein that are not involved in its GLP-1R agonist activity and / or FGF21 activity. In one embodiment, the mutation occurs only outside the amino acid sequence of one or more amino acid sequences of the GLP-1R agonist peptide and / or the human FGF21 functionally active variant contained in the fusion protein. In one embodiment, the deviation of the amino acid sequence of the functionally active variant from the amino acid sequence of the fusion protein from which it is derived is based solely on conserved amino acid substitutions.

[0208] Conservative amino acid substitution involves replacing an amino acid with another amino acid from the same amino acid family (i.e., an amino acid that is related to it on its side chain, for example, in terms of charge and / or size). Naturally occurring amino acids are generally classified 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.

[0209] According to the present invention, a "nucleic acid molecule" is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). According to the present invention, the nucleic acid molecule can be in molecular form, and said molecule can be single-stranded or double-stranded. The nucleic acid molecule according to the present invention can be linear or covalently closed to form a loop.

[0210] The term "DNA" refers to a molecule that contains deoxyribonucleotide residues and is optionally composed entirely or substantially of deoxyribonucleotide residues. A "deoxyribonucleotide" is a nucleotide lacking a hydroxyl group at the 2' position of the β-D-furanose group. The term "DNA" includes isolated DNA, such as partially or completely purified DNA, substantially pure DNA, synthetic DNA, and recombinant DNA. The term "DNA" also includes modified DNA, which differs from naturally occurring DNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations can include the addition of non-nucleotide material, such as adding to one or more ends of the DNA or adding it internally, for example, at one or more nucleotides in 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.

[0211] The term "RNA" refers to a molecule that contains ribonucleotide residues and is optionally composed entirely or substantially of ribonucleotide residues. "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-furanose ribonucleotide. The term "RNA" includes isolated RNA, such as partially or completely purified RNA, substantially pure RNA, synthetic RNA, and recombinant RNA. The term "RNA" also includes modified RNA, which differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may include the addition of non-nucleotide material, such as adding to one or more ends of the RNA or adding it internally, for example, at one or more nucleotides of the RNA. Nucleotides in the RNA molecule may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. Modified RNA molecules may be referred to as analogs or analogs of naturally occurring RNA. According to the invention, "RNA" refers to single-stranded RNA or double-stranded RNA. In one embodiment, the RNA is mRNA, such as in vitro transcribed RNA (IVT RNA) or synthetic RNA. RNA may also be modified, for example, by having one or more modifications that increase the stability of the RNA (e.g., half-life). Such modifications are known to those skilled in the art and include, for example, 5'-caps or 5'-cap analogues.

[0212] When used in conjunction with nucleotides, the term “naturally occurring” refers to the bases adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U).

[0213] The nucleic acid molecules according to the invention can be contained in / encapsulated in a vector. As used herein, the term "vector" includes all vectors known to those skilled in the art, including plasmid vectors, granular vectors, phage vectors (e.g., λ phage vectors), viral vectors (e.g., adenovirus or baculovirus vectors), or artificial chromosome vectors (e.g., bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC)). The vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors and typically contain the desired coding sequence and appropriate DNA sequences required to express the operatively 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 typically used to engineer and amplify a desired DNA fragment and may lack the functional sequences required to express the desired DNA fragment.

[0214] Alternatively, the nucleic acid molecules according to the invention can be integrated into the genome (e.g., the genome of a host cell). The means and methods for integrating specific nucleic acid molecules into the genome are well known to those skilled in the art.

[0215] In some exemplary embodiments, the terms "cell" or "host cell" refer to an intact cell, i.e., a cell with an intact membrane that has not yet released its normal intracellular components (such as enzymes, organelles, or genetic material). In some exemplary embodiments, an intact cell is a living cell, i.e., a living cell capable of performing its normal metabolic functions. In some exemplary embodiments, a cell or host cell is any cell that can be transfected or transformed with exogenous nucleic acids. In some exemplary embodiments, when transfected or transformed with exogenous nucleic acids and transferred to a recipient, the cell can express the nucleic acid in the recipient's body.

[0216] 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 strains of: Gram-negative bacteria such as *Escherichia coli*, *Proteus*, and *Pseudomonas*; and Gram-positive bacteria such as *Bacillus*, *Streptomyces*, *Staphylococcus*, and *Lactococcus*. Suitable fungal cells include, but are not limited to, cells from species of the genera *Trichoderma*, *Neurospora*, and *Aspergillus*. Suitable yeast cells include cells from species of the following genera: *Saccharomyces* (e.g., *Saccharomyces cerevisiae*), *Schizosaccharomyces* (e.g., *Schizosaccharomyces pombe*), *Pichia* (e.g., *Pichia pastoris* and *Pichia amethanolica*), and *Hansenula*. Suitable mammalian cells include, but are not limited to, CHO cells, BHK cells, HeLa cells, COS cells, HEK-293 cells, etc. In one embodiment, HEK-293 cells are used. However, amphibian cells, insect cells, plant cells, and any other cells used in the art for expressing heterologous proteins may also be used. In some exemplary embodiments, adoptive transfer is performed using mammalian cells (e.g., cells from humans, mice, hamsters, pigs, goats, or primates). Cells can originate 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) and other cell types.

[0217] As used herein, an "antigen-presenting cell" is a cell that displays an antigen on its surface against the background of the major histocompatibility complex. T cells can recognize this complex using their T cell receptors (TCRs). The cell or host cell can be isolated or part of a tissue or organism (particularly a "non-human object").

[0218] As used herein, the term “non-human object” is intended to include non-human primates or other animals, such as mammals, such as cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, and rodents (e.g., mice, rats, guinea pigs, or hamsters).

[0219] The pharmaceutical compositions according to the present invention comprise one or more carriers and / or excipients, all of which are pharmaceutically acceptable. As used herein, the term "pharmaceutically acceptable" means that the material is non-toxic and, in some exemplary embodiments, does not interact with the active agent of the pharmaceutical composition.

[0220] As used herein, the term "carrier" refers to an organic or inorganic component of natural or synthetic nature, wherein the active components are combined to facilitate, enhance, or achieve application. According to the invention, the term "carrier" also includes one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to a subject.

[0221] Suitable carrier materials for parenteral administration include, but are not limited to, sterile water, Ringer's solution, lactated Ringer's solution, physiological saline, antibacterial saline (e.g., saline containing 0.9% benzyl alcohol), phosphate-buffered saline (PBS), Hankley's solution, polyalkylene glycols, hydrogenated naphthalene, and particularly biocompatible lactide polymers, lactide / glycolic acid copolymers, or polyoxyethylene / polyoxypropylene copolymers.

[0222] As used herein, the term "excipient" is intended to include all substances that may be present in a pharmaceutical composition and are not active ingredients, such as salts, binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), fillers, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, flavoring agents, or coloring agents.

[0223] Not all pharmaceutically acceptable salts can be used to prepare pharmaceutically acceptable salts, and are included in this invention. Such pharmaceutically acceptable salts include, in a non-limiting manner, those prepared from acids such as 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 or alkaline earth metal salts, such as sodium, potassium, or calcium salts. Salts can be added to adjust the ionic strength or tonicity of the pharmaceutical composition.

[0224] Suitable preservatives for use in pharmaceutical compositions include, but are not limited to, antioxidants, citric acid, sodium citrate, benzalkonium chloride, chlorobutanol, cysteine, methionine, p-hydroxybenzoate, thimerosal, phenol, cresol, and mixtures thereof.

[0225] Suitable buffering substances for use in pharmaceutical compositions include, but are not limited to, acetates, citrates, borates, phosphates, and tris(hydroxymethyl)aminomethane (Tris, THAM, tromethamine).

[0226] In some exemplary embodiments, the pharmaceutical composition according to the invention is sterile. The pharmaceutical composition may be provided in a consistent dosage form and may be prepared in a manner known to those skilled in the art. The pharmaceutical composition may, for example, be in the form of a solution or suspension.

[0227] The pharmaceutical composition may also be formulated as a stable lyophilized product reconstituted with an appropriate diluent, which optionally contains one or more excipients as defined above.

[0228] The pharmaceutical composition according to the invention may further comprise at least one other active pharmaceutical ingredient.

[0229] As used herein, the term “active pharmaceutical ingredient” (API) includes any pharmaceutically active chemical or biological compound and any pharmaceutically acceptable salt thereof and any mixture thereof, which provides some pharmacological effect and is intended to treat or prevent a condition, such as a disease or disorder as defined herein.

[0230] Exemplary pharmaceutically acceptable salts include, but are not limited to, salts prepared from one or more of the following acids: hydrochloric acid (e.g., chloride), sulfuric acid (e.g., sulfate), nitric acid (e.g., nitrate), phosphoric acid (e.g., phosphate), hydrobromic acid (e.g., hydrobromide), maleic acid (e.g., maleate), malic acid (e.g., malate), ascorbic acid, citric acid (e.g., citrate), tartaric acid (e.g., tartrate), bis(hydroxynaphthyl)ic acid (e.g., papoate or embonate), lauric acid (e.g., laurate), stearic acid (e.g., stearate), palmitic acid (e.g., palmitate), oleic acid, myristic acid (e.g., myristate), lauryl acid, naphthalenesulfonic acid, linolenic acid (e.g., linoleate), etc.

[0231] As used in this article, the terms “active pharmaceutical ingredient,” “active agent,” “active component,” “active substance,” “therapeutic active compound,” and “drug” are intended to be synonymous, that is, to have the same meaning.

[0232] According to the present invention, the active pharmaceutical ingredient is optionally selected from:

[0233] - All medications mentioned in Rote Liste 2014, such as all antidiabetic drugs mentioned in Chapter 12 of Rote Liste 2014, all weight loss drugs or appetite suppressants mentioned in Chapter 6 of Rote Liste 2014, all lipid-lowering drugs mentioned in Chapter 58 of Rote Liste 2014, all antihypertensive drugs mentioned in Chapter 17 of Rote Liste 2014, all kidney-protecting drugs mentioned in Rote Liste, or all diuretics mentioned in Chapter 36 of Rote Liste 2014;

[0234] - Insulin and insulin derivatives, such as: glargine insulin (e.g., ), insulin glargine at concentrations higher than 100 U / mL (e.g., insulin glargine at 270-330 U / mL or insulin glargine at 300 U / mL (as disclosed in EP 2387989)), insulin glutares (e.g., ), detemir insulin (e.g., ), lispro insulin (e.g., ), degludec insulin (e.g., IdegLira (NN9068)), insulin aspart and insulin aspart formulations (e.g., ), basal insulin and analogues (e.g., LY2605541, LY2963016, NN1436), pegylated lispro insulin (e.g., LY-275585), long-acting insulin (e.g., NN1436, Insumera (PE0139), AB-101, AB-102, Sensulin LLC), intermediate-acting insulin (e.g., N、 N), rapid-acting and short-acting insulin (e.g., N), R, R, PH20 insulin, NN1218, ), premixed insulin, NN1045, Insulin Plus PE-0139, ACP-002 hydrogel insulin, and oral, inhalable, transdermal, and buccal or sublingual insulins (e.g., tregopil insulin, TPM-02 insulin, Oral insulin, ORMD-0801, Oshadi oral insulin, NN1953, NN1954, NN1956, Insulin derivatives that bind to albumin or another protein via bifunctional linkers are also suitable;

[0235] - Glucagon-like peptide-1 (GLP-1), GLP-1 analogs and GLP-1 receptor antagonists, such as: GLP-1(7-37), GLP-1(7-36) amide, lixilate (e.g., ), exenatides (e.g., kinin-4, rExendin-4, Exenatide NexP), exenatide-LAR, liraglutide (e.g., Semaglutide, taspoglutide, abiglutide, dulaglutide, albugon, gastrin, genipin, ACP-003, CJC-1131, CJC-1134-PC, GSK-2374697, PB-1023, TTP-054, langlenatide (HM-11260C), CM-3, GLP-1 Eligen, AB-201, ORM D-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 analogues;

[0236] - 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-05212389, TKS-1225), TT-401 / 402, ZP2929, LAPS-HMOXM25, MOD-6030);

[0237] - Dual GLP-1 / gastrin agonists (e.g., ZP-3022);

[0238] - Gastrointestinal peptides, such as peptide YY 3-36 (PYY3-36) or analogues thereof and pancreatic polypeptide (PP) or analogues thereof;

[0239] - Glucagon receptor agonists or antagonists, glucose-dependent insulinotropic peptide (GIP) receptor agonists or antagonists, orexin antagonists or inverse agonists, xenin and its analogues.

[0240] - Dipeptidyl peptidase-IV (DPP-4) inhibitors, such as alogliptin (e.g., ), linagliptin (e.g., ), saxagliptin (e.g., Komboglyze ), Sigliptin (e.g., Janumet ), alagliptin, ticagliptin (e.g., ), tregliptin, vildagliptin (e.g., ), Giglitazone, Ogliptin, Evogliptin, Dutogliptin, DA-1229, MK-3102, KM-223, KRP-104, PBL-1427, Pinoxacin hydrochloride, and Ari-2243;

[0241] - Sodium-dependent glucose transporter 2 (SGLT-2) inhibitors, such as: canagliflozin, dapagliflozin, remogliflozin, remogliflozin etopolate, serpagliflozin, empagliflozin, ioggliflozin, tolagliflozin, ruggliflozin, eoggliflozin, EGT-0001442, LIK-066, SBM-TFC-039 and KGA-3235 (DSP-3235);

[0242] - Dual inhibitors of SGLT-2 and SGLT-1 (e.g., LX-4211, LIK066).

[0243] - SGLT-1 inhibitors (e.g., LX-2761, KGA-3235) or combinations of SGLT-1 inhibitors with anti-obesity drugs, such as ileal bile acid transport (IBAT) inhibitors (e.g., GSK-1614235+GSK-2330672);

[0244] - Biguanides (e.g., metformin, buformin, phenformin);

[0245] -Thiazolidinediones (e.g., pioglitazone, rosiglitazone), glitazone analogs (e.g., lobeglitazone);

[0246] - Peroxisome proliferator-activated receptor (PPAR-) (α, γ, or α / γ) agonists or modulators (e.g., saroglitazar) (GFT-505) or PPARγ partial agonists (e.g., Int-131);

[0247] - Sulfonylureas (e.g., tolbutamide, glibenclamide, glimepiride, ... Glipizide and chloroanisole (e.g., nateglinide, repaglinide, miglitol);

[0248] -α-glucosidase inhibitors (e.g., acarbose, miglitol, voglibose);

[0249] - Amylin and amylin analogues (e.g., pramlintide, ... );

[0250] -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);

[0251] -GPR40 agonists (e.g., TUG-424, P-1736, P-11187, JTT-851, GW9508, CNX-011-67, AM-1638, AM-5262);

[0252] -GPR120 agonist and GPR142 agonist;

[0253] - Systemic or poorly absorbable TGR5 (GPBAR1 = G protein-coupled bile acid receptor 1) agonists (e.g., INT-777, XL-475, SB756050);

[0254] -Immunotherapy agents for diabetes, such as: oral type 2 CC chemokine receptor (CCR-2) antagonists (e.g., CCX-140, JNJ-41443532), interleukin-1β (IL-1β) antagonists (e.g., AC-201) or oral monoclonal antibodies (MoA) (e.g., methazozamide, VVP808, PAZ-320, P-1736, PF-05175157, PF-04937319);

[0255] - Anti-inflammatory drugs used to treat metabolic syndrome and diabetes, such as nuclear factor κB inhibitors (e.g., );

[0256] -Adenosine monophosphate activated protein kinase (AMPK) stimulators, such as: Imeglimin (PXL-008), Debio-0930 (MT-63-78), R-118;

[0257] -11-β-hydroxysteroid dehydrogenase 1 (11-β-HSD-1) inhibitors (e.g., LY2523199, BMS770767, RG-4929, BMS816336, AZD-8329, HSD-016, BI-135585);

[0258] - Glucokinase activators (e.g., PF-04991532, TTP-399 (GK1-399), GKM-001 (ADV-1002401), ARRY-403 (AMG-151), TAK-329, TMG-123, ZYGK1);

[0259] α-Diacylglycerol O-acyltransferase (DGAT) inhibitors (e.g., pradigastat (LCQ-908)), protein tyrosine phosphatase 1 inhibitors (e.g., trodusquemine)), glucose-6-phosphatase inhibitors, fructose-1,6-bisphosphatase inhibitors, glycogen phosphorylase inhibitors, phosphoenolpyruvate carboxykinase inhibitors, glycogen synthase kinase inhibitors, and pyruvate dehydrogenase kinase inhibitors;

[0260] - Glucose transporter-4 regulators, somatostatin receptor 3 agonists (e.g., MK-4256);

[0261] One or more lipid-lowering drugs are also suitable as combination partners, such as: 3-hydroxy-3-methylglutaryl-CoA-reductase (HMG-CoA-reductase) inhibitors such as simvastatin (e.g., ), atorvastatin (e.g., ), rosuvastatin (e.g., ), pravastatin (e.g., ), fluvastatin (e.g., ), pitavastatin (e.g., lovastatin (e.g.) ), mevastatin (e.g., ), rivastatin, silivastatin Fibrates such as benzalofop-p-ethyl (e.g., Delaying agents), cyclopropofol (e.g., ), fenofibrate (e.g., ), gemfibrozil (e.g., ), etoposide, difibrate, clonicafibrate, crifibrate, clobemide, niacin and its derivatives (e.g., niacin, including sustained-release formulations of niacin), niacin receptor 1 agonists (e.g., GSK-256073), PPAR-δ agonists, acetyl-CoA-acetyltransferase (ACAT) inhibitors (e.g., avamidib), cholesterol absorption inhibitors (e.g., ezetimibe, ... S-556971), bile acid conjugates (e.g., cholestyramine, cholesvelam), ileal bile acid transporter (IBAT) inhibitors (e.g., GSK-2330672, LUM-002), microsomal triglyceride transporter (MTP) inhibitors (e.g., lometabine (AEGR-733), SLx-4090, granotapide), proprotein convertase subtilisin / kexin Type 9 (PCSK9) modulators (e.g., alikumab (REGN727 / SAR236553), AMG-145, LGT-209, PF-04950615, MPSK3169A, LY3015014, ALD-306, ALN-PCS, BMS-962476, SPC5001, ISIS-394814, 1B20, LGT-210, 1D05, BMS-PCSK9Rx-2, SX-PCK9, RG7652), LDL receptor upregulators such as hepatic selective thyroid hormone receptor beta agonists (e.g., illotirone (KB-2115), MB07811, sobutirone (QRX-431), V... IA-3196, ZYT1), and HDL-elevating compounds such as: cholesterol ester transporter (CETP) inhibitors (e.g., acetrapib (MK0859), dacetrapib, 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) modulators, lipid metabolism modulators (e.g., BMS-823778, TAP-301, DRL-21994, DRL-21995), and phospholipase A2 (PLA2) inhibitors (e.g., darapladib). Varespladib, relapadib, 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 ω-3 fatty acids and their derivatives (e.g., ethyl eicosapentaenoate (AMR101)). AKR-063, NKPL-66, PRC-4016, CAT-2003);

[0262] - Bromocriptine (e.g., ), phentermine and phentermine preparations or combinations (e.g., Adipex-P, phentermine (lonamin), ), benzylphenamine (e.g., ), amfepramone (e.g., ), benzotriazine (for example, ), bupropion and combinations (e.g., Wellbutrin ), Sibutramine (for example, ), topiramate (e.g., ), zonisamide (e.g., ), tersofencin, opioid antagonists (such as naltrexone (e.g., Naltrexone + bupropion), cannabinoid receptor 1 (CB1) antagonists (e.g., TM-38837), melanin condensing 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., velifibrate, S-234462), NPY4 agonists (e.g., PP-1420), β-3-adrenergic receptor agonists, leptin or leptin mimics, serotonin 2c (5HT2c) receptor agonists (e.g., lorcaserin, ... ), pramlintide / metriptine, lipase inhibitors (such as neolisistat (e.g., Orlistat (e.g.) Angiogenesis inhibitors (e.g., ALS-L1023), β-histidine and histamine H3 antagonists (e.g., HPP-404), AgRP (spiced mouse-associated protein) inhibitors (e.g., TTP-435), and serotonin reuptake inhibitors (such as fluoxetine (e.g., ), duloxetine (e.g., ), dual or triple monoamine reuptake inhibitors (dopamine, norepinephrine, and serotonin reuptake) (such as sertraline (e.g., ), tersofensin, methionine aminopeptidase 2 (MetAP2) inhibitors (e.g., beloranib), and antisense oligonucleotides targeting fibroblast growth factor receptor 4 (FGFR4) (e.g., ISIS-FGFR4Rx) or antiproliferative protein targeting peptide-1 (e.g., tersofensin), ... );

[0263] - Nitric oxide donors, AT1 antagonists, or angiotensin II (AT2) receptor antagonists (such as telmisartan, e.g., ), candesartan (e.g., ), valsartan (e.g., Co- ), losartan (e.g., ), eprosartan (e.g., ), Irbesartan (e.g., ), Olmesartan (e.g., ), tasosartan, azisartan (e.g., Dual angiotensin receptor blockers (dual ARBs), angiotensin-converting enzyme (ACE) inhibitors, ACE-2 activators, renin inhibitors, prorenin inhibitors, endothelin-converting enzyme (ECE) inhibitors, endothelin receptor (ET1 / ETA) blockers, endothelin antagonists, diuretics, aldosterone antagonists, aldosterone synthase inhibitors, alpha-blockers, alpha-2 adrenergic receptor antagonists, beta-blockers, mixed alpha / beta-blockers, calcium channel blockers, calcium channel blockers (CCBs), nasal formulations of the calcium channel blocker diltiazem (e.g., CP-404), dual mineralocorticoids / CCBs, central effects Antihypertensive drugs, neutral endopeptidase inhibitors, aminopeptidase-A inhibitors, angiopeptide inhibitors, dual angiopeptide inhibitors (such as enkephalin-ACE inhibitors or enkephalin-ECE inhibitors, dual AT receptor-enkephalin inhibitors, dual AT1 / ETA antagonists), advanced glycation end products (AGE) degraders, recombinant renal enzymes, blood pressure vaccines (such as anti-RAAS (renin-angiotensin-aldosterone-system) vaccines, AT1 vaccines or AT2 vaccines), drugs based on the genomics of hypertension pharmacopoeia (such as modulators of genetic polymorphisms with antihypertensive responses, platelet aggregation inhibitors, and others), or combinations thereof are suitable.

[0264] As used herein, the term "kit of parts" (abbreviated as "kit") refers to an article of manufacture comprising one or more containers and optionally a data carrier. The one or more containers may contain one or more of the agents described above, such as fusion proteins, pharmaceutical compositions, and related agents, such as nucleic acid molecules and host cells. The kit may contain additional containers containing, for example, diluents, buffers, and other reagents. The data carrier may be a non-electronic data carrier, such as a graphic data carrier, such as a leaflet, information sheet, barcode, or access code; or an electronic data carrier, such as an optical disc (CD), digital multifunction optical disc (DVD), microchip, or another semiconductor-based electronic data carrier. The access code may allow access to a database, such as an internet database, a centralized database, or a decentralized database. The data carrier may contain instructions for using the agents of the present invention, such as fusion proteins, pharmaceutical compositions, and related agents, such as nucleic acid molecules and host cells, as described herein.

[0265] The pharmaceutical agents and compositions described herein can be administered via any conventional route, such as oral, pulmonary, inhalation, or parenteral administration, including by injection or infusion. In one embodiment, parenteral administration is used, such as intravenous, intra-arterial, subcutaneous, intradermal, or intramuscular administration. The pharmaceutical agents and compositions described herein can also be administered via sustained-release administration.

[0266] Pharmaceutical compositions suitable for parenteral administration typically comprise sterile aqueous or non-aqueous formulations of the active compound, optionally isotonic with the recipient's blood. Examples of compatible carriers / solvents / diluents are sterile water, Ringer's solution, lactated Ringer's solution, physiological saline, antibacterial saline (e.g., saline containing 0.9% benzyl alcohol), phosphate-buffered saline (PBS), and Hank's solution. Additionally, sterile non-volatile oils can often be used as solution or suspension media.

[0267] The pharmaceuticals and compositions described herein are typically administered in therapeutically effective amounts. A “therapeutically effective amount” is an amount, alone or in combination with other doses, that achieves the desired therapeutic response or effect, optionally without causing unacceptable side effects. In the treatment of a specific disease or condition, the desired response is related to inhibiting the course of the disease. This includes slowing the progression of the disease, and particularly interrupting or reversing its progression. A desired response in the treatment of a disease or condition may also be the delay of the onset of said disease or condition or the prevention of its onset. The effective amount of the pharmaceuticals or compositions described herein will depend on the condition being treated, the severity of the disease, the individual parameters of the subject (including age, physiological condition, body size, and weight), the duration of treatment, the type of concomitant therapy (if present), the specific route of administration, and similar factors. Therefore, the dosage of the pharmaceuticals described herein may depend on several such parameters. In cases where the initial dose results in an insufficient response in the subject, a higher dose (or a higher effective dose achieved through a different, more localized route of administration) may be used.

[0268] According to the present invention, the term "disease or disorder" refers to any pathological or unhealthy condition, particularly obesity, overweight, metabolic syndrome, diabetes, diabetic retinopathy, hyperglycemia, dyslipidemia, nonalcoholic steatohepatitis (NASH), and / or atherosclerosis.

[0269] The term "obesity" refers to a medical condition characterized by the accumulation of excessive body fat to a degree that may negatively impact health. For human (adult) subjects, obesity can be defined as a body mass index (BMI) greater than or equal to 30 kg / m². 2 (BMI≥30kg / m 2 ).

[0270] The term "overweight" refers to a medical condition where the amount of body fat exceeds the optimal level of health. For human (adult) subjects, obesity can be defined as a body mass index (BMI) greater than or equal to 25 kg / m². 2 (For example, 25kg / m 2 <BMI<30kg / m 2 ).

[0271] BMI is a simple index of weight to height, commonly used to classify overweight and obesity in adults. It is defined as an individual's weight (in kilograms) divided by the square of their height (in meters) (kg / m²). 2 ).

[0272] Metabolic syndrome can be defined as a cluster of at least three of the following medical conditions: abdominal (central) obesity (e.g., defined as waist circumference ≥94 cm for European men and ≥80 cm for European women, with ethnic-specific values ​​for other ethnic groups), elevated blood pressure (e.g., 130 / 85 mmHg or higher), elevated fasting plasma glucose (e.g., at least 100 mg / dL), high serum triglycerides (e.g., at least 150 mg / dL), and low high-density lipoprotein (HDL) levels (e.g., less than 40 mg / dL for men and less than 50 mg / dL for women).

[0273] "Diabetes mellitus" (also simply "diabetes") refers to a group of metabolic diseases characterized by high blood sugar levels due to defects in insulin production, insulin action, or both. In one implementation, diabetes is selected from type 1 diabetes, type 2 diabetes, gestational diabetes, late-onset autoimmune diabetes in adults (LADA), adult-onset diabetes mellitus (MODY) with juvenile onset, and other types of diabetes caused by specific genetic conditions, medications, malnutrition, infections, and other diseases.

[0274] 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).

[0275] Type 1 diabetes (also known as insulin-dependent diabetes mellitus (IDDM) or juvenile diabetes) is a condition characterized by high blood sugar levels caused by a complete lack of insulin. This occurs when the body's immune system attacks and destroys the beta cells in the pancreas that produce insulin. The pancreas then produces very little or no insulin. Pancreatic removal or disease can also lead to the loss of insulin-producing beta cells. Type 1 diabetes accounts for between 5% and 10% of all diabetes cases.

[0276] Type 2 diabetes (also known as non-insulin-dependent diabetes mellitus (NIDDM) or adult-onset diabetes) is a condition characterized by the production of too much glucose despite the availability of insulin, resulting in persistently high circulating glucose levels due to insufficient glucose clearance (the action of insulin). Type 2 diabetes may account for approximately 90% to 95% of all diagnosed diabetes cases.

[0277] Gestational diabetes mellitus (GDM) is a condition in which women who were not previously diagnosed with diabetes exhibit high blood sugar levels during pregnancy, particularly in the later stages. GDM affects 3%–10% of pregnancies, depending on the population studied.

[0278] Late-onset autoimmune diabetes in adults (LADA) (also known as slow-onset type 1 diabetes) is a form of type 1 diabetes that occurs in adults and typically has a slower onset process.

[0279] "Adult-onset diabetes mellitus (MODY)" refers to a form of inherited diabetes caused by mutations in autosomal dominant genes that disrupt insulin production.

[0280] As used in this article, "diabetic retinopathy" is an eye disease induced by metabolic disorders in diabetic patients, which leads to a progressive decline in vision.

[0281] The term "hyperglycemia" refers to an excess of sugar (glucose) in the blood.

[0282] The term "dyslipidemia" refers to a disorder of lipoprotein metabolism, including excessive production of lipoproteins ("hyperlipidemia") or deficiency of lipoproteins ("hypolipidemia"). Dyslipidemia can manifest as elevated levels of total cholesterol, low-density lipoprotein (LDL) cholesterol, and / or triglycerides in the blood, and / or decreased levels of high-density lipoprotein (HDL) cholesterol.

[0283] Nonalcoholic steatohepatitis (NASH) is a liver disease characterized by the accumulation of fat (lipid droplets), along with inflammation and hepatocyte degradation. Once it occurs, the disease is associated with a high risk of cirrhosis, a condition characterized by altered liver function that can progress to liver failure. Subsequently, NASH often progresses to liver cancer.

[0284] Atherosclerosis is a vascular disease characterized by the irregular distribution of 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. Lesions are usually localized and progress slowly and intermittently. Sometimes plaques rupture, leading to blood flow obstruction and causing tissue death away from the site of the obstruction. Blood flow restriction explains most clinical presentations, which vary depending on the distribution and severity of the obstruction.

[0285] As used herein, the term "medicine" refers to a substance / composition used in therapy (i.e., for the treatment of a disease or disorder).

[0286] "Treatment" means administering a compound or composition or a combination of compounds or compositions to a subject in order to: prevent, improve or eliminate a disease or disorder in the subject; stop or slow a disease or disorder in the subject; inhibit or slow the development of a new disease or disorder in the subject; reduce the frequency or severity of symptoms and / or relapses in a subject who currently has or has previously had a disease or disorder; and / or prolong (i.e., increase) the lifespan of the subject.

[0287] Specifically, the phrases “treat a disease or disorder” and “treatment of a disease or disorder” include curing a disease or disorder or its symptoms, shortening the duration of a disease or disorder or its symptoms, improving, preventing, slowing or inhibiting the progression or worsening of a disease or disorder or its symptoms, or preventing or delaying the onset of a disease or disorder or its symptoms.

[0288] According to the present invention, the term "subject" refers to a subject used for treatment, particularly a sick subject (also referred to as a "patient"), including but not limited to humans, non-human primates or other animals such as cattle, horses, pigs, sheep, goats, dogs, cats, rabbits or rodents (e.g., mice, rats, guinea pigs or hamsters). In one embodiment, the subject or patient is a human.

[0289] The invention will now be further described with reference to the following embodiments, which are intended to illustrate rather than limit the scope of the invention.

[0290] Example

[0291] Example 1: Determining the optimal GLP-1RA / FGF21 activity ratio through systems pharmacology modeling

[0292] The optimal GLP-1RA / FGF21 potency ratio was identified using improved mechanistic understanding of the pharmacological effects of the GLP-1RA / FGF21 fusion protein in humans. A mechanistic systems pharmacology model was developed to describe the effects of GLP-1 and FGF21 on glucose, lipid, and energy metabolism in humans (Cuevas-Ramos et al. (2009) Curr Diabetes Rev 5(4):216-220; Deacon et al. (2011) Rev Diabet Stud 8(3):293-306; Kim et al. (2008) Pharmacol Rev60(4):470-512; Kharitonenkov et al. (2014) Mol Metab 3(3):221-229).

[0293] The model represents the relevant pathways of GLP-1 and FGF21 effects. Glucose 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 captured to assess the therapeutic response to simulated drug treatments (e.g., GLP-1RA / FGF21 fusion protein, liraglutide, FGF21 analog LY2405319). For LY2405319, see Kharitonenkov et al. (2013) PLoSONE 8(3):e58575.

[0294] The model covers key aspects of glucose homeostasis controlled by the hormones insulin, glucagon, and certain intestinal hypoglycemic agents (e.g., GLP-1, GIP). The primary model endpoint for glycemic control is HbA1c, a common clinical endpoint used to estimate mean plasma glucose concentration over previous months. HbA1c is estimated within 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.

[0295] The model incorporates triglyceride and fatty acid metabolism at levels suitable for operating basal lipid metabolism (including a representative of cholesterol). HDL and non-HDL (i.e., LDL plus VLDL cholesterol) are circulating lipoproteins. The representative of lipid metabolism allows for the simulation of the effects of FGF21 compounds on lipids and their interactions with statins. FGF21 compounds have a significant effect on lipid concentrations (Gaich et al. (2013) Cell Metab 18(3):333-340; Fisher et al. (2011) Endocrinology 152(8):2996-3004).

[0296] Weight loss or gain in the model was measured as a change in body fat mass. A direct relationship exists between body fat mass and weight (Broyles et al. (2011) Br J Nutr 105(8):1272-1276). Food intake was based on basal and resting metabolic rates (Amirkalali et al. (2008) Indian J Med Sci 62(7):283-290). Body fat mass remained constant when energy expenditure equaled caloric intake. The therapeutic effects of food intake were assessed in the model using a formulation from Gobel et al. (2014) (Obesity (Silver Spring) 22(10):2105-2108).

[0297] Food is considered as carbohydrates (glucose equivalents), fats (fatty acid equivalents), and proteins (amino acid equivalents). All nutrients enter the stomach, pass through the delayed colon, and then enter the three-chambered gastrointestinal tract. The gastrointestinal tract design is based on the work of Bastianelli et al. (1996) (J Anim Sci 74(8):1873-1887) and Worthington (1997) (Med Inform (Lond) 22(1):35-45) on food digestion and absorption.

[0298] Nutrients, hormones, medications, and disease conditions can cause delayed gastric emptying. In healthy individuals, the rate of gastric emptying depends on the amount of food consumed, 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 typically have delayed glucose absorption, as observed by oral glucose tolerance tests or dietary trials (Bharucha et al. (2009) Clin Endocrinol (Oxf) 70(3):415-420; Chang et al. (2012) Diabetes Care 35(12):2594-2596). This delay is attributed to slowed gastric emptying. In the model of this example, a delay in the journey between the stomach and small intestine is added to account for the delayed gastric emptying in diabetic subjects. Drugs and hormones (e.g., GLP-1) may affect gastric vagal tone, which reduces mechanical mixing and / or peristalsis and also slows gastric emptying (Jelsing et al. (2012) Diabetes Obes Metab 14(6):531-538; Little et al. (2006) J Clin Endocrinol Metab 91(5):1916-1923; Nauck et al. (2011) Diabetes 60(5):1561-1565; vanCan et al. (2013) Int J Obes (Lond) 38(6):784-93).

[0299] One objective of the fusion protein described in this article is 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 provide estimates of adverse events such as nausea and vomiting, which are associated with low gastric emptying rates. Therefore, the gastric adverse events in the model are characterized by the sum of gastric emptying rates.

[0300] The model platform implemented different virtual patients representing healthy individuals and those at different stages of type 2 diabetes. Furthermore, the virtual patients covered varying degrees of obesity and dyslipidemia. The virtual patients represented clinically observed disease severity, pathophysiological variability, and phenotypic variability.

[0301] Several therapies were administered in the model: GLP-1RA / FGF21 fusion protein, liraglutide, the FGF21 analog LY2405319, metformin, atorvastatin, sitagliptin, and human insulin. These therapies could be turned on or off in the simulation. The virtual patient was assumed to have a background of metformin and atorvastatin administration when receiving the GLP-1RA / FGF21 fusion protein.

[0302] In the model described in this embodiment, a virtual GLP-1RA / FGF21 fusion protein is implemented. The fusion protein contains both FGF21 and GLP-1 agonist activities and has the same effects as both FGF21 and GLP-1 receptor agonists. It is assumed that the pharmacokinetic profile of the virtual fusion protein is similar to that of duraglutide (Geiser et al. (2016) Clin Pharmacokinet 55(5):625-34).

[0303] The model was validated by comparison with multiple datasets. The simulation results were qualitatively consistent with relevant data and knowledge, such as Hellerstein et al. (1997) J Clin Invest 100(5):1305-1319; Muscelli et al. (2008) Diabetes 57(5):1340-1348. The model is matched with relevant quantitative test data, such as Aschner et al. (2006) Diabetes Care 29(12):2632-2637; Dalla Man, Caumo et al. (2005) Am J Physiol Endocrinol Metab 289(5):E909-914; Dalla Man et al. (2005) Diabetes 54(11):3265-3273; Fiallo-Scharer (2005) J Clin Endocrinol Metab 90(6):3387-3391; Hahn et al. (2011) Theor Biol Med Model 8:12; Herman et al. (2005) Clin Pharmacol Ther 78(6):675-688; Herman et al. (2006) J Clin Pharmacol 46(8):876-886 and J Clin Endocrinol Metab 91(11):4612-4619; Hojlund et al. (2001) Am J Physiol Endocrinol Metab 280(1):E50-58; Monauni et al. (2000) Diabetes 49(6):926-935; Nauck et al. (2009) Diabetes Care 32(1):84-90; Nauck et al. (1993) J Clin Invest 91(1):301-307; Nauck et al. (2004) Regul Pept 122(3):209-217; Tzamaloukas et al. (1989) West J Med 150(4):415-419; Sikaris (2009) J Diabetes Sci Technol 3(3):429-438; Vicini and Cobelli (2001) Am J Physiol Endocrinol Metab 280(1):E179-186; Vollmer et al. (2008) Diabetes 57(3):678-687.

[0304] Existing therapies, including FGF21 analogs and GLP-1 receptor agonists, were implemented in the model for direct comparison. The effects of the FGF21 analogs were validated with clinical data, such as those from Gaich et al. (2013) CellMetab 18(3):333-340.The GLP-1 receptor agonist liraglutide is a direct competitor to the target, and its implementation has been compared with various clinical data, such as those described in the following literature: 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) Diabetes Med 25(2):152-156; Buse et al. (2009) Lancet 374(9683):39-47; Jelsing et al. (2012) Diabetes Obes Metab 14(6):531-538; Hermansen et al. (2013) Diabetes Obes Metab 15(11):1040-1048; Suzuki et al. (2013) Intern Med 52(10):1029-1034; van Can et al. (2013) Int J Obes (Lond) 38(6):784-93); Zinman et al. (2009) Diabetes Care 32(7):1224-1230; Russell-Jones et al. (2009) Diabetologia 52(10):2046-2055; Pratley et al. (2011) Int J Clin Pract 65(4):397-407; Nauck et al. (2013) Diabetes Obes Metab 15(3):204-212; Flint et al. (2011) Adv Ther 28(3):213-226; Kapitza et al. (2011) Adv Ther 28(8):650-660; and Asstrup et al. (2012) Int J Obes (Lond) 36(6):843-854.

[0305] The model platform allows for the simulation of the beneficial and adverse effects of the virtual GLP-1RA / FGF21 fusion protein with varying activity ratios. Effective FGF21-mediated EC50 values ​​were set constant relative to those derived from Gaich et al. (2013) Cell Metab 18(3):333-340. Effective GLP-1-mediated EC50 values ​​decreased by 2 to 600-fold relative to endogenous GLP-1, in increments of 1 (Table 1).

[0306] Table 1: Pharmacodynamics of GLP-1R agonist / FGF21 fusion protein (EC50 values).

[0307]

[0308] * Compared to endogenous GLP-1

[0309] ** The FGF21 EC50 values ​​were set according to Gaich et al. (2013) Cell Metab 18(3):333-340, assuming a half-maximal effect. For each virtual fusion protein, the exposure-response relationship was simulated for relevant pharmacodynamic endpoints, namely 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 simulation of treatment with the GLP-1RA / FGF21 fusion protein in a typical obese patient with dyslipidemia and type 2 diabetes was conducted over a wide dose range. Steady-state was expected for all relevant pharmacodynamic endpoints after 52 weeks of treatment. For each endpoint, the half-maximal effective concentration (EC50 value) was determined based on the exposure-response curve. In particular, for the major GLP-1-mediated endpoints HbA1c and gastric emptying rate, the EC50 value varied with the activity ratio. Figure 1 EC50 values ​​dependent on the GLP-1 attenuation factor were plotted. An increased GLP-1 attenuation factor indicates decreased GLP-1R agonist activity.

[0310] This procedure allows for the identification of relevant activity ratios, for which adverse effects are observed at higher plasma levels compared to the plasma levels mediating the pharmacodynamic effect. For GLP-1 attenuation factors greater than 9, the EC50 of GLP-1-mediated gastrointestinal adverse effects is greater than the EC50 of the pharmacodynamic effect. Therefore, gastrointestinal adverse effects occur at plasma levels higher than those required to achieve the pharmacodynamic effect. Doses that provide all desired pharmacodynamic effects while avoiding GLP-1-mediated gastrointestinal adverse effects can be described.

[0311] The maximum EC50 value for gastric emptying rate was reached at an attenuation factor of 531. The maximum distance between adverse effects and the mean pharmacodynamic effect was reached at an attenuation factor of 482. Figure 2Therefore, activity ratios exceeding 1:482 are irrelevant. The maximum distance between the pharmacodynamic (HbA1c) maximum and adverse effects is 319. The maximum distance between the pharmacodynamic (HbA1c) maximum and adverse effects normalized by extending FGF21-mediated effects (lipids) and GLP-1-mediated effects (HbA1c) is 121.

[0312] GLP-1RA / FGF21 fusion proteins with predicted power ratios between 1:10 and 1:482 are most beneficial in improving lipid distribution, body weight, and glucose metabolism, and are unlikely to cause serious adverse events based on gastric emptying response. Lower power ratios may not be good candidates based on the predicted strong inhibition of gastric emptying and the likelihood of adverse events. Higher power ratios are considered insufficiently effective and therefore not competitive.

[0313] This study simulated a 12-week treatment regimen of a wide-dose-range dapoxetine with type 2 diabetes mellitus and dyslipidemia using the GLP-1RA / FGF21 fusion protein, as the primary GLP-1-mediated parameter, HbA1c level, reached clinical steady state after 12 weeks of treatment with a known GLP-1 receptor agonist and an FGF21 agent.

[0314] Figure 3 The EC50 values ​​obtained relative to the GLP-1 decay factor over a 12-week simulation period were plotted. For GLP-1 decay factors greater than 18, the EC50 for GLP-1-mediated gastrointestinal adverse effects was greater than the EC50 for the pharmacodynamic effects. The maximum EC50 value for gastric emptying rate was reached at a decay factor of 501. The maximum distance between adverse effects and the mean pharmacodynamic effect was reached at a decay factor of 469. Figure 4 The maximum distance between the pharmacodynamic (HbA1c) value and the adverse effect is 313. The maximum distance between the pharmacodynamic (HbA1c) value and the adverse effect is 123 after normalization by extending the FGF21-mediated effect (lipid) and GLP-1-mediated effect (HbA1c).

[0315] The efficacy and potential for adverse events of GLP-1RA / FGF21 fusion proteins with different activity ratios were investigated using a described systems pharmacology approach. Fusion proteins with a theoretically calculated ideal potency ratio were identified, predicting their potential benefits in improving lipid distribution, body weight, and glycemic control, while, based on gastric emptying response, they were unlikely to cause serious adverse GLP-1RA-related effects. Compounds with selected model-informed potency ratios were predicted to provide a favorable efficacy-risk profile.

[0316] Example 2: Expression of homodimeric GLP-1RA / FGF21 fusion protein in HEK-293, CHO and E. coli cells and chemical synthesis of isolated GLP-1R agonist peptide.

[0317] GLP-1RA / FGF21 Fc fusion protein was generated by transient transfection into HEK-293 or CHO cells. The DNA sequence of the fusion protein was fused at the N-terminus to an IL2 signal sequence (SEQ ID NO: 246), followed by a histidine-rich sequence (His tag) and a TEV protease cleavage site (SEQ ID NO: 247 or 248). The desired protein was secreted into the culture medium containing the required signal sequence. Immobilized metal ion affinity chromatography (IMAC) was used. TM The protein was purified from the culture supernatant by 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 solution was passed through the IMAC column a second time. TM Roche collected (His-tagged) fractions. Proteins were further purified using protein A affinity chromatography (rProtein A Sepharose, GE Healthcare) and gel filtration columns with phosphate-buffered saline (PBS, Gibco) as the run buffer. Fractions containing the desired proteins were collected, combined, concentrated, and stored at -80°C until further use.

[0318] The FGF21 protein of SEQ ID NO:252 (mature human wild-type FGF21 with an additional N-terminal Gly; referred to herein as G-FGF21) was expressed in *E. coli*. The DNA sequence of the FGF21 protein was fused at the N-terminus to a histidine-rich sequence (His tag) and a TEV or SUMO protease cleavage site (SEQ ID NO:248 or 249). 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 His tag cleavage, the lysis reaction solution was purified using an ion exchange column (Source 15, GE Healthcare) followed by a gel filtration column (Superdex 75, GE Healthcare) with phosphate-buffered saline (PBS, Gibco) as the run buffer. Fractions containing the desired protein were collected, combined, concentrated, and stored at -80°C until further use.

[0319] In an alternative method, a fusion protein is generated by expression in *E. coli* inclusion bodies followed by a refolding step, in which the folded fusion protein is obtained by unfolding the inclusion bodies in a Tris-buffered guanidine hydrochloride solution and diluting the refolded protein in a buffer without dissociation salts. The fusion protein is purified using protein A affinity chromatography (MabSelectSuRe, GE Healthcare), followed by cleavage of the N-terminal pre-sequence with the addition of a TEV protease. The cleavage reaction solution is purified using an anion exchange column (POROS 50HQ, ThermoFisher). Fractions containing the desired protein are collected and combined. Final buffer conditions and protein concentrations are established using an ultrafiltration / percolation step with PBS (Gibco). Samples are stored at -80°C until further use.

[0320] Fusion proteins are produced through recombinant methods (see above), but chemically synthesized GLP-1R agonists are isolated.

[0321] More specifically, peptides are synthesized using the following artificial synthesis procedure:

[0322] 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 one hour and in DMF (15 mL) for one hour. The Fmoc groups on the resin were deprotected by treating it twice with a 20% (v / v) piperidine / DMF solution (5 min and 15 min). The resin was washed with DMF / DCM / DMF (6:6:6 times each). The removal of Fmoc from the solid support was confirmed using the Kaiser assay (quantitative method). C-terminal Fmoc-amino acids (5 equivalents excess, corresponding to the resin loading) in anhydrous DMF were added to the deprotected resin, and the coupling of the next Fmoc-amino acid was initiated with 5 equivalents excess 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 vortex mixer at room temperature for 2 hours. The resin was filtered and washed with DMF / DCM / DMF (6:6:6 times each). A Kaiser test on aliquots of the peptide resin after coupling was negative (no color on the resin). After the first amino acid attachment, unreacted amino groups (if present) in the resin were capped for 20 minutes with acetic anhydride / pyridine / DCM (1:8:8) to avoid any sequence deletion. After capping, the resin was washed with DCM / DMF / DCM / DMF (6 / 6 / 6 / 6 times each). The Fmoc groups on the C-terminal amino acid-attached peptide resin were deprotected by treating it twice with a 20% (v / v) piperidine / DMF solution (5 minutes and 15 minutes). The resin was washed with DMF / DCM / DMF (6:6:6 times each). A Kaiser test on aliquots of the peptide resin after Fmoc deprotection was positive.

[0323] Using the Fmoc AA / DIC / HOBt method, the remaining amino acids in the target sequence on Rink amide MBHA resin were sequentially coupled using a 5-equivalent excess in DMF (corresponding to resin loading). The concentration of each reactant in the reaction mixture was approximately 0.4 M. The mixture was rotated on a vortex mixer at room temperature for 2 hours. The resin was filtered and washed with DMF / DCM / DMF (6:6:6 times each). A Kaiser test was performed after each coupling step and the Fmoc deprotection step to confirm the completion of the reaction.

[0324] After linear sequencing, the ε-amino group of lysine was used as a branching or modification point and deprotected twice by using 2.5% hydrazine hydrate in DMF, 15 min each time, followed by washing with DMF / DCM / DMF (6:6:6 times each). The γ-carboxyl terminus of glutamic acid was attached to the ε-amino group of Lysine using Fmoc-Glu(OH)-OtBu in DMF with the DIC / HOBt method (5 equivalences excess relative to resin loading). The mixture was rotated on a vortex mixer at room temperature for 2 h. The resin was filtered and washed with DMF / DCM / DMF (6 x 30 mL each). The Fmoc group on glutamic acid was deprotected by treating it twice with 20% (v / v) piperidine / DMF solution (5 min and 15 min, 25 mL each). The resin was washed with DMF / DCM / DMF (6:6:6 times each). Kaiser tests on aliquots of the peptide resin after Fmoc deprotection were positive.

[0325] If the side chain branch also contains another γ-glutamic acid, a second Fmoc-Glu(OH)-OtBu is attached to the free amino group of the γ-glutamic acid using the DIC / HOBt method in DMF (with 5 equivalences relative to the resin loading). The mixture is rotated on a vortex mixer at room temperature for 2 hours. The resin is filtered and washed with DMF / DCM / DMF (6 x 30 mL each). The Fmoc group on the γ-glutamic acid is deprotected by treating it twice with a 20% (v / v) piperidine / DMF solution (5 min and 15 min, 25 mL each). The resin is washed with DMF / DCM / DMF (6:6:6 each). A Kaiser test on an aliquot of the peptide resin after Fmoc deprotection is positive.

[0326] Final cleavage of peptides from resin:

[0327] The synthesized peptide-based resin was washed with DCM (6 x 10 mL), MeOH (6 x 10 mL), and ether (6 x 10 mL) and dried overnight in a vacuum desiccator. The peptides were lysed from the solid support by treating the peptide-resin at room temperature for 3 hours with a reagent mixture (80% TFA / 5% anisole / 5% phenol / 2.5% EDT / 2.5% DMS / 5% DCM). The lysate was collected by filtration and the resin was washed with TFA (2 mL) and DCM (2 x 5 mL). 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 peptides. The precipitated peptides were centrifuged, and the ether in the supernatant was removed. Fresh ether was added to the peptides, and the mixture was centrifuged again. The crude sample was purified by preparative HPLC and lyophilized. The peptide identity was confirmed by LCMS.

[0328] Example 3: In vitro cell assay of human FGF21 receptor efficacy in CHO cells (In-Cell Western blotting) The in vitro cell efficacy of the G-FGF21 (SEQ ID NO:252) or fusion protein of the present invention was measured using a specific and highly sensitive In-Cell Western blotting (ICW) assay. The ICW assay is an immunocytochemical assay that is typically performed in microplate form. The FGF21 receptor autophosphorylation ICW assay was performed using CHO Flp-In cells (Invitrogen, Darmstadt, Germany) stably expressing human FGFR1c and human β-Klotho (KLB). (Aguilar et al. (2010) PLoS ONE 5(4):e9965). To determine the level of receptor autophosphorylation or downstream activation of MAP kinase ERK1 / 2, 2 × 10⁻⁶ cells were used. 4Cells were seeded per well into 96-well plates and allowed to grow for 48 hours. Cells were starved for 3–4 hours with serum-free medium (Ham's F-12 nutrient mix containing GlutaMAX, Gibco, Darmstadt, Germany). Cells were then treated with increasing concentrations of G-FGF21 (SEQ ID NO: 252) or the indicated fusion protein at 37°C for 5 minutes. After incubation, the medium was discarded, and cells were fixed in 3.7% freshly prepared paraformaldehyde for 20 minutes. Cells were permeabilized for 20 minutes with 0.1% Triton-X-100 in PBS. Blocking was performed for 2 hours at room temperature with Odyssey blocking buffer (LICOR, Bad Homburg, Germany). As primary antibodies, anti-pFGFR Tyr653 / 654 (New England Biolabs, Frankfurt, Germany) or anti-pERK phosphate-p44 / 42 MAP kinase Thr202 / Tyr204 (Cell Signaling) was added, and incubation was performed overnight at 4°C. After incubation with the primary antibody, cells were washed with PBS containing 0.1% Tween 20. Cells were then incubated with anti-mouse 800CW secondary antibody (LICOR, Bad Homburg, Germany) at room temperature for 1 hour. Subsequently, cells were washed again with PBS containing 0.1% Tween 20. Infrared dye signals were quantified using an Odyssey imager (LICOR, Bad Homburg, Germany). Results were normalized by quantifying DNA using TO-PRO3 dye (Invitrogen, Karlsruhe, Germany). Data were obtained in arbitrary units (AU), and EC50 values ​​were obtained from dose-response curves (summarized in Tables 2 and 3). Figure 5 The results of ICW performed on CHO cells overexpressing human FGFR1c plus KLB are shown.

[0329] Table 2: EC50 values ​​of G-FGF21 (SEQ ID NO:252) and GLP-1RA / FGF21 Fc fusion proteins measured by ICW pFGFR in CHO cells.

[0330]

[0331]

[0332] Table 3: EC50 values ​​of G-FGF21 (SEQ ID NO:252) and GLP-1RA / FGF21 Fc fusion proteins measured by ICW pERK in CHO cells.

[0333]

[0334] Example 4: In vitro cell assay of efficacy against human glucagon-like peptide-1 (GLP-1) receptor

[0335] The agonistic effect of the compound on the human glucagon-like peptide-1 (GLP-1) receptor was determined by functional assays of cAMP response in the HEK-293 cell line stably expressing the human GLP-1 receptor.

[0336] Recombinant HEK-293 cells were grown to near confluence in DMEM medium (containing 10% FBS) in T175 culture flasks at 37°C, and at a growth rate of 1–5 × 10⁻⁶ cells / year. 7 Cells / mL concentrations were collected in 2 mL vials of cell culture medium containing 10% DMSO. Each vial contained 1.8 mL of cell suspension. The vials were slowly frozen to -80°C in the isopropanol chamber and then transferred to liquid nitrogen for long-term storage.

[0337] Before use, rapidly thaw frozen cells at 37°C, wash with 20 mL of cell buffer (1×HBSS; 20 mM HEPES, 0.1% BSA), and centrifuge at 900 rpm for 5 minutes. Resuspend the cells in assay buffer (cell buffer plus 2 mM IBMX) and adjust to 1×10⁻⁶. 6 Cell density of cells / mL. For measurement, 5 μL of cell suspension (finally 5 × 10⁶ cells / mL) was prepared. 3 5 μL of the test compound (1 cell / well) was added to each well of a 384-well plate, followed by incubation at room temperature for 30 min. Human GLP-1(7-36)amide (SEQ ID NO:260) from Bachem (Bubbendorf, Switzerland, H-6795) was used as a control. Cellular cAMP levels were determined using a kit from Cisbio Corp. (catalog number 62AM4PEC) based on homogeneous time-resolved fluorescence (HTRF). After adding the HTRF reagent (kit component) diluted in rehydration buffer, the plate was incubated for 1 h, followed by measurement of the fluorescence ratio at 665 / 620 nm. The in vitro potency of the agonist was quantified by determining the concentration that elicited the 50% activation that caused the maximum response (EC50).

[0338] The results are summarized in Table 4, and the dose-response curves are shown in Table 4. Figure 6 middle.

[0339] Table 4: EC50 values ​​of human GLP-1(7-36) (SEQ ID NO:260), GLP-1RA / FGF21 Fc fusion protein and several single GLP-1R agonist peptides as measured by HTRF cAMP in HEK-293 cells.

[0340]

[0341]

[0342]

[0343]

[0344]

[0345] nd: Undetermined

[0346] Example 5: Analysis of the conformation and thermal stability of the GLP-1RA / FGF21 Fc fusion protein

[0347] The conformational stability and aggregation tendency of the GLP-1RA / FGF21 Fc fusion protein were simultaneously determined using UNit (Unchained Labs, California, USA). UNit combines the analysis of the intrinsic fluorescence of the unfolded protein with static light scattering (SLS) measurements to study aggregation behavior.

[0348] Data were obtained for a fusion protein prepared at a concentration of 5 mg / mL in phosphate buffer at pH 7.4. Each 9 μL sample was loaded into a UNit capillary fixator and analyzed in triplicate on UNit. The temperature was increased from 20 °C to 95 °C at a constant linear rate of 0.3 °C / min. The barycentric mean (BCM) of intrinsic fluorescence and SLS signal detected by a 266 nm laser was plotted against the applied temperature to obtain the melting temperature (Tm) and aggregation initiation temperature (Tagg). Data were analyzed using UNit analysis software v.2.1 and are summarized in Table 5.

[0349] In addition, for some proteins, differential scanning fluorometry (DSF or ThermoFluor) is used. TM Thermal stability was analyzed using thermal displacement measurements in simulations (Ahmad S. et al. (2012) Protein Science 21:433-446; Pantolian et al. (2001) J. Biomol. Screen 6:429-440; Niesen et al. (2007) Nat. Protoc. 2:2212-21). This measurement was based on the observation that hydrophobic fluorescent dyes (such as Sypro) TMOrange (LifeTechnologies, catalog number S6651) increases their fluorescence when they bind to hydrophobic patches on proteins. These hydrophobic patches are exposed to the protein when it unfolds upon heating, so the increase in fluorescence can be used as a measure of the degree of unfolding, and thus as a measure of the protein's thermal stability.

[0350] By mixing each protein's solution in PBS (Gibco) with 160x Sypro TM To test for proteins, mix an Orange solution (diluted in water with 5000X DMSO stock solution as provided by the supplier). Adjust the sample volume to 20 μL with PBS. Typical conditions include 0.8 mg / mL protein and 8x Sypro in the final mixture. TM Orange, but protein concentrations can vary between 0.4 mg / mL and 1.2 mg / mL. Samples were dispensed into 96-well PCR plates (BioRad Semi-Skirt96 white) and briefly centrifuged to remove air bubbles. The plates were then inserted into a BioRad iQ5 real-time PCR instrument and subjected to a thermal gradient from 10°C to 90°C at a rate of 1°C / min. Filters at wavelengths of 485 nm and 575 nm were selected for excitation and quantification of fluorescence. Data processing was performed using BioRad iQ5 Standard Edition software (v.2.0.148.60623). The inflection point in the fluorescence intensity versus temperature curve was selected as a measure of the melting temperature (Tm).

[0351] Table 5: Demothering and aggregation temperatures of G-FGF21 (SEQ ID NO:252) and the selected GLP-1RA / FGF21 Fc fusion protein.

[0352]

[0353]

[0354] * Data generated via DSF; nd: undetermined

[0355] Example 6: Pharmacokinetics in mice and non-human primates

[0356] Plasma concentrations and pharmacokinetic parameters of the GLP-1RA / FGF21 Fc fusion protein were determined using three different methods following a single subcutaneous administration of 0.3 mg / kg solution to female C57Bl / 6 mice or male cynomolgus monkeys. Blood samples were obtained at time points from 30 minutes to 168 hours post-administration.

[0357] a.) Bioanalytical screening methods for quantifying the intact FGF21 moiety of the GLP-1RA / FGF21 Fc fusion protein

[0358] The intact FGF21 moiety of the fusion protein in plasma samples was analyzed using an ELISA kit (F1231-K01, Eagle Biosciences, USA). The assay utilized a two-site sandwich technique employing two selected antibodies that bind to different epitopes of the intact human FGF21. One antibody specifically binds to the N-terminal amino acids (aa 29-35) of human FGF21, and the other antibody specifically binds to the C-terminus (aa 203-209) of human FGF21. Assay standards, controls, and unknown samples were added directly to the wells of a microplate coated with an anti-human FGF21 (aa 29-35) specific antibody. Simultaneously, a horseradish peroxidase-conjugated anti-human FGF21 (aa 203-209) specific antibody was added to each well. After the first incubation period, the antibody on the walls of the microtiter wells captured human FGF21 in the samples, and unbound protein in each microtiter well was washed away. A sandwich of "anti-FGF21 antibody - human intact FGF21 - HRP conjugated tracer antibody" is formed. Unbound tracer antibody is removed in a subsequent washing step. To detect the immune complex, the wells are then incubated with the substrate solution in a timed reaction, followed by measurement using a spectrophotometer. The enzyme activity of the immune complex of human intact FGF21 bound to the walls of the microtiter wells is proportional to the amount of intact FGF21 in the sample.

[0359] b.) Bioanalytical screening methods for quantifying intact, full-length fusion proteins

[0360] The concentration of the 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 mouse monoclonal anti-GLP1 antibody (Mesoscale Discovery, MSD). After blocking the plate with 150 μL Blocker A (MSD) for 1 hour at room temperature (RT) with gentle shaking and washing three times with 300 μL wash buffer, 50 μL of diluted plasma sample (standard and PK study sample) was added to each well, and the plate was incubated at room temperature with gentle shaking for 1 hour. After washing three times with 300 μL wash buffer, 50 μL of primer detection antibody (C-terminal rabbit anti-FGF21 antibody, Pineda) was added. Add 25 μL of goat anti-rabbit antibody (Sulfo-tagged, MSD) diluted in 0.05% PBS-Tween (PBS-T) to each well and incubate the plate at room temperature for 1 hour. After washing three times with 300 μL of wash buffer, add 150 μL of read buffer to each well.

[0361] c.) Bioanalytical screening methods for quantifying the intact GLP-1 moiety of the GLP-1FGF21 Fc fusion protein

[0362] The complete GLP-1 moiety of the fusion protein in plasma samples was analyzed using a GLP-1 ELISA method. ELISA plates were coated with mouse monoclonal anti-GLP-1 antibody (Mesoscale Discovery, MSD). After blocking with 150 μL Blocker A (MSD) for 1 hour at room temperature (RT) with gentle shaking and washing three times with 300 μL PBS-T, 50 μL of diluted plasma sample (standards and PK study samples) was added to each well, and the plate was incubated at room temperature with gentle shaking for 1 hour. After washing three times with 300 μL 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 at room temperature for 1 hour. After washing three times with 300 μL PBS-T, 150 μL of read buffer was added to each well.

[0363] Pharmacokinetic parameters were calculated using a non-compartmental model and linear trapezoidal interpolation in WinNonlin 6.4. The results are presented in... Figure 7 and Figure 8 And as shown in Table 6. The results showed that the novel GLP-1RA / FGF21 Fc fusion protein maintained its plasma level within the ng / mL range, with a half-life of 20-40 hours.

[0364] Table 6: Terminal half-life of the selected GLP-1RA / FGF21 Fc fusion protein and G-FGF21 (SEQ ID NO:252) after subcutaneous injection of 0.3 mg / kg in mice and non-human primates.

[0365]

[0366]

[0367] nd: Undetermined

[0368] Example 7: In vivo efficacy in a rodent model

[0369] a.) Diet-induced obesity (DIO) mice

[0370] Female C57Bl / 6N Charles River mice were housed in specific pathogen-free barrier facilities under a 12-hour light / dark cycle, with free access to water and either a standard or high-fat diet (ssniff-regulated fat diet E15797). After 20 weeks of pre-feeding on the high-fat diet, mice were stratified by body weight into treatment groups (n=8) to ensure similar average body weights in each group. An age-matched group with free access to a standard diet (ssniff R / MH, V1534-0) served as a standard control group. A duraglutide treatment group was also included as a comparison group. Mice were subcutaneously injected with the carrier solution and weighed for 3 days prior to the start of treatment to acclimatize them to the procedure.

[0371] 1) Acute effects on blood glucose in fed female DIO mice: Initial blood samples were collected immediately before the first administration of the mediator (phosphate-buffered saline) or the first administration of the GLP-1RA / FGF21 Fc fusion protein (dissolved in phosphate-buffered saline). The administration volume was 5 or 10 mL / kg, depending on the concentration of the stock solution. Animals had access to water and their appropriate diet during the experiment. Blood glucose levels were measured at t=0, t=1, t=2, t=3, t=4, t=6, and t=24 hours (method: Accu-Check blood glucose meter). Blood samples were taken by tail slit without anesthesia.

[0372] 2) Chronic effects on body weight in female DIO mice: Mice were treated weekly with the medium or test compound every 8 days at the start of the light phase for 4 weeks. Body weight and food intake were recorded daily. Total fat mass was measured by nuclear magnetic resonance (NMR) two days before the start of treatment and on day 26.

[0373] The effects of fusion proteins on body weight and food intake are shown in the figures below. Figure 9 and Figure 10 Although by the end of the study, animals treated with the fusion proteins of SEQ ID NO:8 or SEQ ID NO:7 accumulated more food consumption than animals treated with the mediator or duraglutide, they also lost significantly more body weight than animals treated with the mediator or duraglutide. This clearly demonstrates the balance of GLP-1 receptor activity of the two molecules, SEQ ID NO:7 and SEQ ID NO:8, relative to the activity of the FGF21 mimic, as their effect on weight loss does not require inhibition of food intake.

[0374] b.) Hypoglycemic effect of multiple subcutaneous doses in female diabetic db / db mice

[0375] Animals, study design (pre-drug administration phase, dosing phase), pharmacological intervention

[0376] Female, healthy, lean (BKS.Cg-(lean) / OlaHsd or BKS.Cg-Dock7(m)+ / +Lepr(db)J) mice and diabetic, 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 housed in groups in shoebox cages with wood shavings bedding and acclimatized for approximately 2 to 3 weeks prior to the administration phase.

[0377] Mice were housed in animal husbandry conditions including a 12-hour light / dark cycle (light phase from 4:00 AM to 4:00 PM), room temperature between 20°C and 26°C, and relative humidity between 30% and 70%. All animals had free access to Greenfield municipal water and Purina Fomulab 5008 diet. At the start of the study, the mice were approximately 10–12 weeks old.

[0378] Pre-drug administration phase (15 days)

[0379] Blood was collected via tail clip on day 9 for HbA1c and blood glucose measurements. Blood glucose concentrations were measured using an extended-range AlphaTRAK blood glucose meter (code 29 test strips). Blood glucose measurements were taken before any other vital activities and were performed in duplicate. A third value was recorded if the values ​​differed by more than 20 mg / dL (calculated blood glucose meter value). Body weight measurements were collected on days 9 and 15. Block randomization was performed using HbA1c and body weight values. Based on the block randomization results, animals were assigned to the treatment group (n = 8 animals / group) and to new and paired cages (n = 4 animals / cage) on day 15. The lean group was included in the study as an age-matched healthy reference group.

[0380] Dosage preparation and administration

[0381] Animals were treated once per subcutaneous injection on days 1, 8, 15, 22, and 27 of the dosing phase, using a medium (sterile PBS), duraglutide, SEQ ID NO:8, or SEQ ID NO:7 at a dose of 5 ml / kg. Dosing was performed between 10:00 AM and 12:00 PM, adjusted to the most recent recorded weight for each individual. To achieve the appropriate concentration, an injectable solution containing Trulicity (duraglutide Pen) was prepared by adding sterile PBS to a stock solution or Pen formulation.

[0382] Dosing phase (36 days)

[0383] 1) Blood glucose concentrations in animals fed in the morning: Animals had unrestricted access to water and feed during the experiment. Blood glucose was measured on days 1, 2, 8, 9, 15, 16, 22, 23, 27, and 28 between 10:00 AM and 12:00 PM before any other vital activities, and on days 2, 9, 16, 23, and 28 24 hours after administration. Additionally, blood samples were collected on days 1 and 22 at 1, 2, 3, 4, 6, and 24 hours after administration. Figure 11 Approximately 5 μL of blood was collected via the tail clip and blood glucose was measured in duplicate using an extended-range AlphaTRAK blood glucose meter (code 29 test strips). If the values ​​differed by more than 20 mg / dL (the calculated blood glucose value), a third value was recorded. The area under the curve (AUC) for each individual and time period was calculated using the trapezoidal method.

[0384] 2) HbA1c analysis: Blood was collected via tail clip on day 9 of the pre-dose phase and day 36 of the dosing phase. Blood was collected into 5 μL additive-free microcapillaries and immediately placed in centrifuge tubes containing the hemolysate. The tubes were vigorously shaken to mix the hemolysate and blood, and placed on a shaker to ensure complete mixing of blood and reagents. Plasma HbA1c levels at the start of the study and at the end of the study are shown below. Figure 12 middle.

[0385] Statistical analysis: Data are plotted as mean ± SEM. For statistical analysis, one-way ANOVA and multiple comparisons (Dunnet's method) were performed to compare diabetic obese db / db mediator mice (n=8) with diabetic obese db / db experimental mouse mice (n=8). A difference greater than 0.05 between the means of the two groups was considered statistically significant. Data from the non-diabetic, lean mediator group are plotted as... Figure 11 and Figure 12 It is used as a reference dataset for non-obese, non-diabetic states.

[0386] In animals treated with the fusion protein of SEQ ID NO:8 or SEQ ID NO:7, the effect on reducing blood glucose levels was significantly greater than that in animals treated with the mediator or duraglutide. Figure 11 The highest dose of the fusion protein in SEQ ID NO:8 even resulted in a reduction in blood glucose levels to those of normal non-diabetic animals throughout almost the entire 24-hour blood glucose profile measured on day 22 of treatment. Furthermore, as... Figure 12 As shown, by the end of the study, animals treated with the fusion protein of SEQ ID NO:8 or SEQ ID NO:7 showed a more significant inhibition of HbA1c increase compared to animals treated with the mediator or duraglutide.

[0387] c.) DIO-NASH mouse model

[0388] Animals and experimental apparatus

[0389] All animal experiments were conducted in accordance with internationally recognized principles of laboratory animal care and use.

[0390] Five-week-old male C57Bl / 6J mice were obtained from JanVier (JanVier labs, France), and five animals per cage were housed in each group under a 12 / 12-hour dark-light cycle. The room temperature was maintained at 22°C ± 1°C, and the humidity at 50% ± 10%. Animals were given an optional diet high in fat (40%, of which 18% was trans fat), 40% carbohydrates (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 a conventional rodent diet (Altromin 1324, Brogaarden, Denmark) and tap water (for lean chow diets, n = 10-12). At 26 weeks, liver biopsy was performed to histologically assess individual fibrosis and steatosis staging at baseline.

[0391] Mice were pretreated with enrofloxacin (Bayer, Germany) (5 mg / mL / 1 mL / kg) the day before the biopsy. Mice were anesthetized with isoflurane (2%–3%) in 100% oxygen prior to the biopsy. A small abdominal incision was made along the midline, exposing the left lateral lobe of the liver. A conical wedge of liver tissue (50–100 mg) was excised from the distal portion of the lobe and fixed in 4% paraformaldehyde for histological purposes. The biopsy procedure, previously described by Clapper et al., was improved using bipolar electrocoagulation on the liver cutting surface with the aid of an ERBE VIO 100C electrosurgical unit (ERBE, USA). The liver was returned to the abdominal cavity, the abdominal wall was sutured, and the skin was stapled. Carbofen (Pfizer, USA) (5 mg / mL–0.01 mL / 10 g) and enrofloxacin (5 mg / mL–1 mL / kg) were administered intraperitoneally during the operation and on the first and second postoperative days to control postoperative pain relief and infection, respectively. Following the biopsy procedure, the animals were kept in isolation and fed an AMLN diet for three weeks to allow for recovery. Stratified and randomized groups of 10–12 animals were based on individual disease staging as assessed by baseline liver biopsy.

[0392] Animals were then treated weekly with subcutaneous injections of 50 mg / kg GLP-1RA / FGF21 Fc fusion protein, 0.6 mg / kg duraglutide, or a carrier (PBS) for an additional 8 weeks, fed an AMLN diet or a regular food diet. Subsequently, the animals were euthanized, liver weight was determined, and visceral liver tissue was collected for histological and biochemical analysis (see [link to relevant documentation]). Figure 13 ).

[0393] Histological evaluation and digital image analysis

[0394] Baseline liver biopsies and terminal samples (approximately 100 mg) were collected from the left lateral lobe and fixed overnight in 4% paraformaldehyde. Liver tissue was paraffin-embedded and sectioned (3 μm thick). To assess liver morphology and fibrosis, sections were stained with hematoxylin and eosin, and Sirius red, respectively, and subsequently analyzed using Visiomorph software (Visiopharm, Denmark). Histological evaluation and scoring were performed by pathologists unaware of the study. NAFLD activity scoring (NAS) (steatodegenesis, inflammation, ballooning degeneration) and fibrosis staging were performed using the clinical criteria outlined in Kleiner et al. (2005) Hepatology 41:1313-1321. Data are presented in two distinct formations. Figure 14 and Figure 15 middle.

[0395] The fusion protein of SEQ ID NO:8 clearly showed effects on liver weight, total liver lipid content, liver cholesterol and triglyceride content, and NAFLD activity score, which were superior to those effects of GLP-1 agonists alone, as exemplified by the effect of duraglutide.

Claims

1. A fusion protein, wherein the amino acid sequence of the fusion protein is selected from SEQ ID NO: 2, 7 and 8.

2. A nucleic acid molecule encoding the fusion protein according to claim 1.

3. A host cell comprising the nucleic acid molecule according to claim 2.

4. A pharmaceutical composition comprising the fusion protein according to claim 1 or the nucleic acid molecule according to claim 2.

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

6. Use of the fusion protein of claim 1, the nucleic acid molecule of claim 2, the host cell of claim 3, or the pharmaceutical composition of claim 4 in the preparation of a medicament for treating a disease or disorder selected from obesity, type 2 diabetes, dyslipidemia, and non-alcoholic steatohepatitis (NASH).

7. The use according to claim 6, wherein the disease or disorder is overweight.

8. The use according to claim 6, wherein the disease or disorder is hyperglycemia.

Images