SEQUENCE LISTING

A copy of the Sequence Listing is submitted with the specification electronically via EFS-Web as an ASCII formatted sequence listing with a file name of “074585-8001_SL_20210922-hxr_ST25”, a creation date of Sep. 22, 2021, and a size of about 442 Kb. The sequence listing contained this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to fusion proteins, pharmaceutical compositions thereof, and methods of using such to prevent and/or treat diseases.

BACKGROUND

Major biologically active fragment of Glucagon-Like Peptide-1 (GLP-1) is a 30 or 31 amino acid peptide fragment (amino acid 7-36 or 7-37 of GLP-1) deriving from the posttranslational processing of the proglucagon peptide. The initial GLP-1 product GLP-1 stimulates insulin synthesis and secretion and has been shown to prevent hyperglycemia in diabetics, especially type 2 diabetes. However, endogenous GLP-1 only has a half-life of approximately 2 minutes, which results in fasting plasma levels of GLP-1 of only 0-15 pmol/L.

Growth differentiation factor 15 (GDF15), also named as Macrophage inhibitory cytokine-1 or MIC-1, is a member of the transforming growth factor beta superfamily. The function of GDF15 is not fully understood. Existing studies show that GDF15 may have a role in regulating inflammatory pathways, participating in regulating apoptosis, cell repair and cell growth, which are biological processes observed in cardiovascular and neoplastic disorders. Other studies show that GDF15 possesses therapeutic utility in metabolic disorders such as obesity.

Metabolic disorders are commonly associated with insulin resistance, visceral adiposity, atherogenic dyslipidemia, etc., which pose major and escalating public health and clinical challenge worldwide. However, existing treatment for metabolic diseases faces problems such as short half-life and/or low efficacy.

Therefore, there is a need for an improved therapeutic solution for treating metabolic diseases.

SUMMARY OF THE INVENTION

Provided herein are fusion proteins and the conjugates thereof, and pharmaceutical compositions and methods of use for treating/preventing metabolic disorders.

In a first aspect, the present disclosure provides a fusion polypeptide comprising: a) a first polypeptide fragment comprising a Glucagon-like peptide-1 (GLP-1) receptor agonist; and b) a second polypeptide fragment comprising a GDNF receptor alpha like (GFRAL) receptor agonist; wherein the first polypeptide fragment and the second polypeptide fragment are linked to one another directly or via a linker.

In certain embodiments, the first polypeptide fragment comprises GLP-1 and/or the second polypeptide fragment comprises GDF15.

In certain embodiments, the GLP-1 comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 242 while retaining substantial biological activity of SEQ ID NO: 242, and/or the GDF15 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 248 while retaining substantial biological activity of SEQ ID NO: 248.

In certain embodiments, the GLP-1 comprises no more than 9, 8, 7, 6, 5, 4 substitutions relative to SEQ ID NO: 242 while retaining substantial biological activity of SEQ ID NO: 242, and/or the GDF15 comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 substitutions relative to SEQ ID NO: 248 while retaining substantial biological activity of SEQ ID NO: 248.

In certain embodiments, the GLP-1 comprises one or more mutations at a position selected from the group consisting of: A8, G22, K34, R36, and H7, or any combination thereof, relative to SEQ ID NO: 242.

In certain embodiments, the GLP-1 comprises or consists of one or more substitutions selected from the group consisting of: H7IA, H7IPA, A8G, A8Aib, K34R, G22E, and R36G, or any combination thereof.

In certain embodiments, the GDF15 comprises one or more mutations at a position selected from the group consisting of: A1, R2, N3, H6, P11, H18, T19, V20, R21, A30, M43, A47, R53, A54, M57, H66, R67, L68, A75, A81, P85, M86, Q90, T92, and L105, or any combination thereof.

In certain embodiments, the one or more mutations in GDF15 comprises:

1) mutation of N3 selected from the group consisting of: N3Q, N3S, N3E, N3A, N3T, N3P, N3G, N3V, N3H, N3Y and deletion of N3; and/or

2) substitution of M57 selected from the group consisting of: M57A, M57E and M57L; and/or

3) substitution of M86Lor M86A.

In certain embodiments, the one or more mutations in GDF15 are selected from the group consisting of: R2S, R2A, R2E, N3S, N3E, N3A, N3T, N3P, N3G, N3V, N3H, N3Y, N3Q, H6D, P11E, H18E, H18Q, T19S, V20L, R21E, A30E, M43L, M43E, A47E, R53E, A54E, M57A, M57E, M57L, H66E, R67E, L68E, A75E, A81E, P85E, M86F, M86A, M86L, Q90E, T92E, L105E, deletion of N3, and deletion of N-terminal 1-3 residues, or any combination thereof.

In certain embodiments, the fusion polypeptide comprises from N terminus to C terminus, the GLP-1, the linker and the GDF15.

In certain embodiments, the linker comprises a polypeptide linker.

In certain embodiments, the polypeptide linker has a length of 4-172 amino acid residues (e.g. at least 4, 12, 24, 40, 50, 60, 70, 80, 90, 100, 110, or 120 amino acid resides) or more.

In certain embodiments, the polypeptide linker comprises at least one acidic amino acid residue.

In certain embodiments, the at least one acidic amino acid residue comprises Aspartic acid or glutamic acid.

In certain embodiments, the polypeptide linker comprises one or more repeats of a repeating sequence.

In certain embodiments, the repeating sequence consists of no more than 4, 5 or 6 types of amino acid residues selected from the group consisting of: G, Q, A, E, P, and S.

In certain embodiments, the repeating sequence comprises Q.

In certain embodiments, the repeating sequence consists of G, Q, A, E, and P.

In certain embodiments, the repeating sequence consists of a sequence selected from the group consisting of: SEQ ID NO: 213 (GQEPGAQP), SEQ ID NO: 214 (GAQPGAQP), SEQ ID NO: 215 (GQEP), SEQ ID NO: 216 (GAQP), SEQ ID NO: 217 (SEPATSGSETPGTSESATPESGPGTSTEPSEG), SEQ ID NO: 218 (GAQPGQEPGAQP), SEQ ID NO: 219 (GAQPGQEP), SEQ ID NO: 220 (GEQP), SEQ ID NO: 221 (GPQE), SEQ ID NO: 222 (GPEQ), SEQ ID NO: 223 (GSEP), SEQ ID NO: 224 (GESP), SEQ ID NO: 225 (GPSE), SEQ ID NO: 226 (GPES), SEQ ID NO: 227 (GQAP), SEQ ID NO: 228 (GPAQ), SEQ ID NO: 229 (GPQA), SEQ ID NO: 230 (GSQP), SEQ ID NO: 231 (GASP), SEQ ID NO: 232 (GPAS), SEQ ID NO: 233 (GPSA), SEQ ID NO: 234 (GGGS), SEQ ID NO: 235 (GSGS), SEQ ID NO: 236 (GGGGS), SEQ ID NO: 453 (GQEPGQAP), SEQ ID NO: 476: (GQAPGQEP) and GS.

In certain embodiments, the polypeptide linker comprises (Repeat1)r(Repeat2)s(Repeat3)x(Repeat4)y, wherein: Repeat1, Repeat2, Repeat3 and Repeat4 are linked via a peptide bond or via one or more amino acid residues, Repeat1, Repeat2, Repeat3 and Repeat4 independently, comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 213 (GQEPGAQP), SEQ ID NO: 214 (GAQPGAQP), SEQ ID NO: 215 (GQEP), SEQ ID NO: 216 (GAQP), SEQ ID NO: 217 (SEPATSGSETPGTSESATPESGPGTSTEPSEG), SEQ ID NO: 218 (GAQPGQEPGAQP), SEQ ID NO: 219 (GAQPGQEP), SEQ ID NO: 220 (GEQP), SEQ ID NO: 221 (GPQE), SEQ ID NO: 222 (GPEQ), SEQ ID NO: 223 (GSEP), SEQ ID NO: 224 (GESP), SEQ ID NO: 225 (GPSE), SEQ ID NO: 226 (GPES), SEQ ID NO: 227 (GQAP), SEQ ID NO: 228 (GPAQ), SEQ ID NO: 229 (GPQA), SEQ ID NO: 230 (GSQP), SEQ ID NO: 231 (GASP), SEQ ID NO: 232 (GPAS), SEQ ID NO: 233 (GPSA), SEQ ID NO: 234 (GGGS), SEQ ID NO: 235 (GSGS), SEQ ID NO: 236 (GGGGS), SEQ ID NO: 453 (GQEPGQAP), SEQ ID NO: 476: (GQAPGQEP) and GS, and r, s, x and y are independently an integer selected from 0 to 30, provided that r, s, x and y are not 0 at the same time.

In certain embodiments, x and y are 0, r, and s are independently an integer selected from 1 to 30, and Repeat1 and Repeat2 are a combination selected from the group consisting of:

Repeat1 comprises or consists of a sequence of SEQ ID NO: 213, Repeat2 comprises or consists of a sequence of SEQ ID NO: 216;

Repeat1 comprises or consists of a sequence of SEQ ID NO: 215, Repeat2 comprises or consists of a sequence of SEQ ID NO: 216;

Repeat1 comprises or consists of a sequence of SEQ ID NO: 216, Repeat2 comprises or consists of a sequence of SEQ ID NO: 215;

Repeat1 comprises or consists of a sequence of SEQ ID NO: 216, Repeat2 comprises or consists of a sequence of SEQ ID NO: 213;

Repeat1 comprises or consists of a sequence of SEQ ID NO: 218, Repeat2 comprises or consists of a sequence of SEQ ID NO: 216;

Repeat1 comprises or consists of a sequence of SEQ ID NO: 217, Repeat2 comprises or consists of a sequence of SEQ ID NO: 216;

Repeat1 comprises or consists of a sequence of SEQ ID NO: 216, Repeat2 comprises or consists of a sequence of SEQ ID NO: 453; and

Repeat1 comprises or consists of a sequence of SEQ ID NO: 217, Repeat2 comprises or consists of a sequence of SEQ ID NO: 215.

In certain embodiments, r, x and y are 0, s is an integer selected from 1 to 30, and Repeat1 comprises or consists of a sequence of SEQ ID NO: 213.

In certain embodiments, y is 0, r, s and x are independently an integer selected from 1 to 30, and Repeat1, Repeat2 and Repeat3 are a combination selected from the group consisting of:

Repeat1 comprises or consists of a sequence of SEQ ID NO: 216, Repeat2 comprises or consists of a sequence of SEQ ID NO: 218; and Repeat3 comprises or consists of a sequence of SEQ ID NO: 216;

Repeat1 comprises or consists of a sequence of SEQ ID NO: 216, Repeat2 comprises or consists of a sequence of SEQ ID NO: 215; and Repeat3 comprises or consists of a sequence of SEQ ID NO: 216;

Repeat1 comprises or consists of a sequence of SEQ ID NO: 217, Repeat2 comprises or consists of a sequence of SEQ ID NO: 215; Repeat3 comprises or consists of a sequence of SEQ ID NO: 216;

Repeat1 comprises or consists of a sequence of SEQ ID NO: 217, Repeat2 comprises or consists of a sequence of SEQ ID NO: 216; Repeat3 comprises or consists of a sequence of SEQ ID NO: 215;

Repeat1 comprises or consists of a sequence of SEQ ID NO: 213, Repeat2 comprises or consists of a sequence of SEQ ID NO: 227; Repeat3 comprises or consists of a sequence of SEQ ID NO: 216,

Repeat1 comprises or consists of a sequence of SEQ ID NO: 216, Repeat2 comprises or consists of a sequence of SEQ ID NO: 453; Repeat3 comprises or consists of a sequence of SEQ ID NO: 213,

Repeat1 comprises or consists of a sequence of SEQ ID NO: 216, Repeat2 comprises or consists of a sequence of SEQ ID NO: 213; Repeat3 comprises or consists of a sequence of SEQ ID NO: 216, and

Repeat1 comprises or consists of a sequence of SEQ ID NO: 216, Repeat2 comprises or consists of a sequence of SEQ ID NO: 213; Repeat3 comprises or consists of a sequence of SEQ ID NO: 453.

In certain embodiments, r, s, x and y are independently an integer selected from 1 to 30, and Repeat1, Repeat2, Repeat 3 and Repeat 4 are a combination selected from the group consisting of:

Repeat1 comprises or consists of a sequence of SEQ ID NO: 216, Repeat2 comprises or consists of a sequence of SEQ ID NO: 219; Repeat3 comprises or consists of a sequence of SEQ ID NO: 215, and Repeat4 comprises or consists of a sequence of SEQ ID NO: 216;

Repeat1 comprises or consists of a sequence of SEQ ID NO: 213, Repeat2 comprises or consists of a sequence of SEQ ID NO: 453; Repeat3 comprises or consists of a sequence of SEQ ID NO: 213, and Repeat4 comprises or consists of a sequence of SEQ ID NO: 216,

Repeat1 comprises or consists of a sequence of SEQ ID NO: 216, Repeat2 comprises or consists of a sequence of SEQ ID NO: 453; Repeat3 comprises or consists of a sequence of SEQ ID NO: 213, and Repeat4 comprises or consists of a sequence of SEQ ID NO: 216,

Repeat1 comprises or consists of a sequence of SEQ ID NO: 216, Repeat2 comprises or consists of a sequence of SEQ ID NO: 227; Repeat3 comprises or consists of a sequence of SEQ ID NO: 216, and Repeat4 comprises or consists of a sequence of SEQ ID NO: 213, and
Repeat1 comprises or consists of a sequence of SEQ ID NO: 216, Repeat2 comprises or consists of a sequence of SEQ ID NO: 213; Repeat3 comprises or consists of a sequence of SEQ ID NO: 476, and Repeat4 comprises or consists of a sequence of SEQ ID NO: 453.

In certain embodiments, the polypeptide linker comprises an amino acid sequence selected from the group consisting of: a) SEQ ID NOs: 1-59, 61-88, 90-197, 199-212, and 454-471, or a variant thereof; b) SEQ ID NOs: 1-59, 61-88, 90-93, 454-459, and 463-471, or a variant thereof; c) SEQ ID NOs: 94-156, or a variant thereof; d) SEQ ID NOs: 210-212, and 460-462, or a variant thereof, or e) SEQ ID NOs: 157-197, and 199-209, or a variant thereof.

In certain embodiments, the fusion polypeptide comprises up to two conjugatable residue.

In certain embodiments, the fusion polypeptide comprises a first conjugatable residue which is present in one of the GLP-1, the GDF15, and the polypeptide linker.

In certain embodiments, the fusion polypeptide comprises a second conjugatable residue which is present in one of the GLP-1, the GDF15, and the polypeptide linker.

In certain embodiments, the fusion polypeptide comprises a first conjugatable residue and a second conjugatable residue, wherein:1) both conjugatable residues are present in the linker,2) both conjugatable residues are present in the GLP-1,3) both conjugatable residues are present in the GDF15,4) the first conjugatable residue is present in the GLP-1 and the second conjugatable residue is present in the linker,5) the first conjugatable residue is present in the GDF15 and the second conjugatable residue is present in the linker, or6) the first conjugatable residue is present in the GLP-1 and the second conjugatable residue is present in the GDF15.

In certain embodiments, the GLP-1 comprises up to two conjugatable residues or up to one conjugatable residue.

In certain embodiments, the conjugatable residue(s) in the GLP-1 comprises or consists of lysine.

In certain embodiments, the conjugatable residue(s) in the GLP-1 comprises or consists of K26 and/or K34.

In certain embodiments, the GLP-1 further comprises substitution of K26 and/or K34 to a non-conjugatable residue.

In certain embodiments, the GLP-1 comprises substitution of K26 and/or K34 to a residue selected from the group consisting of Arginine (R), Glutamine (Q), Alanine (A), Glycine (G), Histidine (H), Serine (S), and Threonine (T).

In certain embodiments, the substitution of K26 is selected from K26R and K26Q, and/or the substitution at K34 is selected from K34R and K34Q.

In certain embodiments, the conjugatable residue(s) in the GLP-1 comprises an introduced residue, optionally by substitution of a non-conjugatable residue to a conjugatable residue, or by insertion of a conjugatable residue.

In certain embodiments, the conjugatable residue(s) in the GLP-1 comprises or consists of lysine and is introduced by a substitution selected from the group consisting of: E27K, and R36K relative to SEQ ID NO: 242.

In certain embodiments, the conjugatable residue(s) in the GLP-1 comprises or consists of cysteine or a non-natural amino acid.

In certain embodiments, the conjugatable residue(s) in the GLP-1 comprises or consists of cysteine and is introduced by a substitution at a position selected from the group consisting of: K26C, K34C, E27C and R36C, relative to SEQ ID NO: 242.

In certain embodiments, the GLP-1 comprises no conjugatable residue.

In certain embodiments, the GLP-1 comprises an amino acid sequence of X7X8EGTFTSDVSSYLEX22QAAX26X27FIAWLVX34GX36G (SEQ ID NO: 325), wherein: the X7is H, imidazole-4-acetate (IA), imidazolepropionic acid (IPA); the X8is A, G, S, V, Aib, T, I, or L; the X22is G, or E; the X26is K, R, or C; the X27is E, K, or C; the X34is R, K, or C, and the X36is K, R, G, or C.

In certain embodiments, the X7is H, X8is G, the X22is E; the X26is K, R or C; the X27is E; the X34is K or R, and the X36is K, or G.

In certain embodiments, the GLP-1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 336, SEQ ID NO: 448 and SEQ ID NO: 478.

In certain embodiments, the GDF15 comprises no conjugatable residue.

In certain embodiments, the conjugatable residue comprises or consists of lysine.

In certain embodiments, the GDF15 comprises or consists substitutions of K91 and K107, relative to SEQ ID NO: 248.

In certain embodiments, the GDF15 comprises substitutions at K91, and K107, to a non-conjugatable residue optionally selected from the group consisting of Arginine (R), Glutamine (Q), Alanine (A), Glycine (G), Histidine (H), Serine (S), and Threonine (T).

In certain embodiments, the GDF15 further comprises substitutions at K62 and/or K69.

In certain embodiments, the GDF15 comprises K62 and/or K69.

In certain embodiments, the GDF15 comprises up to two or up to one conjugatable residue.

In certain embodiments, the conjugatable residue in GDF15 comprises K91, K107, or an introduced conjugatable residue selected from the group consisting of lysine, cysteine and non-natural amino acid. In certain embodiments, the GDF15 comprises an amino acid sequence of SEQ ID NO: 291 or SEQ ID NO: 358.

In certain embodiments, the fusion polypeptide comprises or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 349 and 350.

In certain embodiments, the GDF15 comprises or consists of combination of substitutions selected from the group consisting of: 1) N3Q, M57L, K69R, K107R, and K91R; 2) N3Q, K69R, K107R, and K91R; 3) K69R, K107R, and K91R; 4) N3Q, M57L, K107R, and K91R; 5) N3Q, K107R, and K91R; 6) K107R, and K91R; 7) K62R, K69R, K107R, and K91R, N3Q, and M57L; 8) K62R, K69R, K107R, K91R, and N3Q; 9) K62R, K69R, K107R, and K91R, and 10) N3Q, M57L, K69R, and K91R, 11) del 1-3 and M57L, 12) N3Q, M57L, M86L, K69R, K107R, K91R, 13) N3Q and M57L, 14) M57L, and 15) M57L, K69R, K107R, K91R.

In certain embodiments, the GDF15 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NOs: 290-297, SEQ ID NO: 358, SEQ ID NO: 271, and SEQ ID NO: 451, SEQ ID NO: 479 and SEQ ID NO: 480.

In certain embodiments, the polypeptide linker comprises a first conjugatable residue, and optionally, a second conjugatable residue.

In certain embodiments, the first conjugatable residue and/or the second conjugatable residue in the polypeptide linker is/are at least 2 amino acid residue away from the most N-terminal residue of the GDF15.

In certain embodiments, the first conjugatable residue and/or the second conjugatable residue is/are at least 2, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61. 62, 63, 64, 65, 66, 67, 68, 69. 70, 71, 72, 73, 74, 75, or 76 amino acid residue away (inclusive of the conjugatable residue) from the most N-terminal residue of the GDF15, and optionally the first conjugatable residue and/or the second conjugatable residue is/are in the polypeptide linker.

In certain embodiments, the polypeptide linker comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-59, 61-88, 90-93, 454-459, and 463-471, except for one or two substitution to the conjugatable residue at a position ranging from the 1stto the 78th, 1stto the 72nd, 1stto the 60th, 1stto the 50th, 1stto the 40th, 1stto the 38th, 1stto the 32nd, 1stto the 24th, 1stto the 16th, 4thto the 72nd, 4thto the 60th, 4thto the 50th, 4thto the 40th, 4thto the 38th, 4thto the 32nd, 4thto the 24th, 4thto the 16th, 16thto the 72nd, 16thto the 60th, 16thto the 50th, 16thto the 40th, 16thto the 38th, 16thto the 32nd, 16thto the 24th, 24thto the 72nd, 24thto the 60th, 24thto the 50th, 24thto the 40th, 24thto the 38th, 24thto the 32nd, the 5thto the 73rd, the 17thto the 73rd, the 25thto the 73rd, the 33rdto the 73rd, the 39thto the 73rd, the 41stto the 73rd, the 51stto the 73rd, the 5thto the 51st, the 17thto the 51st, the 25thto the 51st, the 33rdto the 51st, the 39thto the 51st, the 41stto the 51st, the 5thto the 41st, the 17thto the 41st, the 25thto the 41st, the 33rdto the 41st, the 39thto the 41st, the 5thto the 39th, the 17thto the 39th, the 25thto the 39th, the 33rdto the 39th, the 5thto the 33rd, the 17thto the 33rd, the 25thto the 33rd, the 5thto the 25th, the 17thto the 25th, or the 5thto the 17thresidue in the amino acid sequence.

In certain embodiments, the polypeptide linker comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 94-156, except for one or two substitution to the conjugatable residue at a position ranging from the 1stto the 58th, 1stto the 52nd, 1stto the 45th, 1stto the 40th, 1stto the 30th, 1stto the 25th, or 1stto the 20thresidue in the amino acid sequence.

In certain embodiments, the polypeptide linker comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 157-197 and 199-209, except for one or two substitutions to the conjugatable residue at a position ranging from the 1stto the 38th, 1stto the 32nd, 1stto the 30th, 1stto the 25th, or 1stto the 20thresidue in the amino acid sequence.

In certain embodiments, the polypeptide linker comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 210-212, 460-462, and 458-459, except for one or two substitution to the conjugatable residue at a position at least 2, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28, 29, or 30 amino acid residues away from the most N-terminal residue of the GDF15.

In certain embodiments, the conjugatable residue in the polypeptide linker comprises lysine, cysteine, or a non-natural amino acid.

In certain embodiments, the polypeptide linker comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 298-324, 60, 89, 198, 332, 333, 334, 342-346, 409-447, 475 and 483, or a variant thereof in which the lysine residue in the polypeptide linker is substituted with a cysteine residue or a non-natural amino acid, or in which the cysteine residue in the polypeptide linker is substituted with a lysine residue or a non-natural amino acid.

In certain embodiments, the conjugatable residue in the polypeptide linker comprises lysine, and the polypeptide linker comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 298-324, 60, 89, 198, 332, 333, 334, 342-346, 409-410, 414-417, and 426-429.

In certain embodiments, the GDF15 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 246, SEQ ID NOs: 290-293, 295-297, SEQ ID NO: 358, SEQ ID NO: 451, SEQ ID NO: 479 and SEQ ID NO: 480, and/or the GLP-1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 448 and SEQ ID NO: 478.

In certain embodiments, the fusion polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 252, 254-260, 262-265, 273-289, 327-330, 337-341, 354, 356-357, 359-361, 363-365, 370-373, and 385-386, 390-391.

In certain embodiments, the conjugatable residue in the polypeptide linker comprises cysteine, and the polypeptide linker comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NOs: 411-413, 418-425, 430-447 475, and 483.

In certain embodiments, the GDF15 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 294, and SEQ ID NO: 271, and/or the GLP-1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 336, SEQ ID NO: 448 and SEQ ID NO: 478.

In certain embodiments, the fusion polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 367-369, 374-381, 392-400, 402-408, 472-473, 477, 481 and 482.

In certain embodiments, the polypeptide linker comprises no conjugatable residue.

In certain embodiments, the polypeptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 33, 51, 58, 64, 18, 91, 157, 158, 159, 116, 210, 211 and 461.

In certain embodiments, fusion polypeptide comprises an amino acid sequence comprising or consisting of: SEQ ID NOs: 249, 266-270, 347, 383 and 387.

In certain embodiments, the fusion polypeptide comprises up to one conjugatable residue for acylation, and comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 249, 251-260, 262-265, 273-289, 326, 329-331, 337-341, 349, 350, 354-357, 359-365, 370-373, 385-386, and 390-391.

In certain embodiments, the fusion polypeptide comprises up to one conjugatable residue for alkylation, and comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 366-369, 374-381, 392-408, 472-473, 477, 481 and 482.

In certain embodiments, the fusion polypeptide comprises up to two conjugatable residues, and comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 327-328, 352-353, and 361.

In certain embodiments, the GLP-1 comprises an amino acid sequence of SEQ ID NO: 243 or SEQ ID NO: 336, the GDF15 comprises an amino acid sequence of SEQ ID NO: 247, and a polypeptide linker.

In certain embodiments, the fusion polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 249, 266, 267, 268, 269, 270, 347, 251, 260, 263-265, 273, 277, 329-330, 359, 369, 374, 376-381, and 392-408.

In a second aspect, the present disclosure provides a polypeptide complex comprising a dimer of the fusion polypeptide of the first aspect of the present disclosure.

In certain embodiments, the polypeptide complex is a homo-dimer.

In certain embodiments, the dimer is associated with a disulfide bond.

In certain embodiments, the disulfide bond is formed between the second polypeptide fragments comprising the GDF15.

In a third aspect, the present disclosure provides a polynucleotide encoding the fusion polypeptide of the first aspect of the present disclosure or the polypeptide complex of the second aspect of the present disclosure.

In a fourth aspect, the present disclosure provides a vector comprising the polynucleotide of the third aspect of the present disclosure.

In a fifth aspect, the present disclosure provides a host cell comprising the vector of the fourth aspect of the present disclosure.

In certain embodiments, the host cell is a prokaryotic cell or eukaryotic cell.

In a sixth aspect, the present disclosure provides a method of producing the polypeptide complex of the second aspect of the present disclosure, comprising culturing the host cell of the fifth aspect of the present disclosure under a condition that allows expression of the polynucleotide of the third aspect of the present disclosure.

In certain embodiments, the host cell is prokaryotic cell or a eukaryotic cell.

In certain embodiments, the polypeptide complex is expressed as inclusion bodies.

In certain embodiments, the method further comprises renaturing the polypeptide complex from the inclusion bodies.

In a seventh aspect, the present disclosure provides a conjugate comprising the fusion polypeptide of the first aspect and at least one clearance-reducing moiety (CRM), wherein the at least one CRM is conjugated to at least one conjugatable residue on the fusion polypeptide.

In certain embodiments, the fusion polypeptides comprises two or no more than two conjugatable residues, each of which is conjugated to a CRM.

In an eighth aspect, the present disclosure provides a conjugate comprising the polypeptide complex of the second aspect and at least two clearance-reducing moieties (CRMs), wherein the at least two CRM are conjugated respectively to one of the conjugatable residues on the polypeptide complex.

In certain embodiments, the polypeptide complex comprises four and no more than four conjugatable residues, each of which is conjugated to a CRM.

In certain embodiments, the CRM comprises a plasma protein-binding moiety, a polymer, Fc, HSA (albumin), Xten sequence, or PAS sequence.

In certain embodiments, the CRM comprises an albumin-binding moiety.

In certain embodiments, the albumin-binding moiety comprises a structure of: *-A-B-C-D-E, wherein A, B, C, D and E are interconnected via amide bonds, and the * end of A is connected to a reactive group of the conjugatable residue on the polypeptide complex, and wherein:

A is selected from a bond,


a, b, c and d are independently an integer from 0 to 4, R1is hydrogen or —COOH;
B is selected from a bond,


e is an integer from 1 to 4, wherein position α is linked to the position α′,
C is a bond or


R2is —CH2SO3H or —COOH, f is an integer from 1 to 4, n is an integer from 1 to 25, wherein when B is not bond, then position β′ is linked to position β, or when B is bond, then position β′ is linked to position α′;
D is selected from a bond,


g and h are independently 0 or 1, and R3is H or —CH2COOH, wherein:when B is not a bond and C is a bond, then position γ′ is linked to position β;when C is not a bond, then position γ′ is linked to position γ; andwhen B is a bond and C is a bond, then position γ′ is linked to position α′; E is an acidic group having a formula:


wherein W represents —(CR4R5)l—,
R4and R5are independently selected from the group consisting of hydrogen, halogen, cyano, hydroxyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, hydroxyalkyl, amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy, and carboxamide,
R6is selected from hydroxyl or NR7R8;
R7and R8are independently selected from the group consisting of hydrogen, alkyl, hydroxyl, and


and l is an integer from 10 to 20, and wherein:when D is not a bond, then position δ is linked to position δ′,when C is not a bond and D is a bond, then position δ is linked to position γ,when B is not a bond, C is a bond and D is a bond, then position δ is linked to position β,when A is not a bond, and all of B, C, and D are bond, then position δ is linked to position α′.

In certain embodiments, wherein the CRM is conjugated to a lysine residue, optionally the lysine residue is in the polypeptide linker or in the GLP-1 or in the GDF15.

In certain embodiments, A is a bond.

In certain embodiments, A is a bond, and B is a bond or


wherein e is 1, 2 or 3.
In certain embodiments, A is a bond, B is


and C is


wherein e is 1, 2 or 3, f is 1, 2, or 3, and n is 1 or 2, wherein position β′ is linked to position β. In certain embodiments, D is a bond, and E is an acidic group having a formula:


wherein position δ is linked to position γ. In certain embodiments, R2is —COOH, and R6is hydroxyl. In certain embodiments, W represents —(CR4R5)l—, R4and R5are independently hydrogen, l is an integer from 10 to 20.

In certain embodiments, A is a bond, B is a bond, and C is a bond.

In certain embodiments, A is a bond, B is a bond, and C is


wherein f is 1, 2, or 3, and n is 1 or 2, and the position β′ is linked to position β.

In certain embodiments, A is


wherein a is 1, 2 or 3, b is 1, 2 or 3, and c is 1 or 2.

In certain embodiments, A is


and B is


wherein a is 1, 2 or 3, b is 1, 2 or 3, c is 1 or 2, and e is 1, 2 or 3, wherein position α is linked to the position α′.

In certain embodiments, A is


B is


wherein position α is linked to the position α′, and C is


wherein a is 1, 2 or 3, b is 1, 2 or 3, c is 1 or 2, e is 1, 2 or 3, f is 1, 2, or 3, and n is 1 or 2, wherein position β′ is linked to position β.

In certain embodiments, A is


B is


wherein position α is linked to the position α′, and C is bond, wherein a is 1, 2 or 3, b is 1, 2 or 3, c is 1 or 2, and e is 1, 2 or 3.

In certain embodiments, A is


and B is a bond, wherein a is 1, 2 or 3, b is 1, 2 or 3, and c is 1 or 2.

In certain embodiments, A is


B is a bond, and C is


wherein a is 1, 2 or 3, b is 1, 2 or 3, c is 1 or 2, f is 1, 2, or 3, and n is 1 or 2, wherein position β′ is linked to position α′.

In certain embodiments, A is


B is a bond, and C is a bond, wherein a is 1, 2 or 3, b is 1, 2 or 3, and c is 1 or 2.

In certain embodiments, D is a bond.

In certain embodiments, A is


B is


wherein position α is linked to the position α′, C is


wherein position β′ is linked to position β, and D is a bond, wherein a is 1, 2 or 3, b is 1, 2 or 3, c is 1 or 2, e is 1, 2 or 3, f is 1, 2, or 3, and n is 1 or 2.

In certain embodiments, D is


wherein g is 0 or 1, and h is 0 or 1.

In certain embodiments, A is


B is a bond or


wherein when B is not a bond, position α is linked to the position α′, C is a bond, and D is


wherein a is 1, 2 or 3, b is 1, 2 or 3, c is 1 or 2, e is 1, 2 or 3, g is 0 or 1, and h is 0 or 1, wherein when B is not a bond, then position γ′ is linked to position β, or when B is a bond, then position γ′ is linked to position α′.

In certain embodiments, D is



In certain embodiments, A is


B is


wherein position α is linked to the position α′, C is a bond or


wherein when C is not a bond, then position β′ is linked to position β; and D is


wherein a is 1, 2 or 3, b is 1, 2 or 3, c is 1 or 2, e is 1, 2 or 3, f is 1, 2, or 3, and n is 1 or 2, wherein when C is not a bond, then position γ′ is linked to position γ, or when C is a bond, then position γ′ is linked to position β.

In certain embodiments, the CRM comprises the structure of below formula:



In certain embodiments, the CRM is conjugated to a cysteine residue, optionally the cysteine residue is in the polypeptide linker or in the GLP-1 or in the GDF15.

In certain embodiments, A is


and B is


wherein position α is linked to the position α′, wherein a is 1, 2 or 3, b is 1, 2 or 3, c is 1 or 2, d is 1, 2, or 3, e is 1, 2 or 3, R1is hydrogen or —COOH.

In certain embodiments, A is


B is


wherein position α is linked to the position α′, and C is


wherein β′ is linked to position β, wherein a is 1, 2 or 3, b is 1, 2 or 3, c is 1 or 2, d is 1, 2, or 3, f is 1, 2, or 3, and n is 1 or 2. In certain embodiments, D is a bond, and E is an acidic group having a formula:


wherein position δ is linked to position γ. In certain embodiments, R2is —COOH, and R6is hydroxyl. In certain embodiments, W represents —(CR4R5)l—, R4and R5are independently hydrogen, l is an integer from 10 to 20.

In certain embodiments, A is


B is


wherein position α is linked to the position α′, C is


wherein position β′ is linked to position β, and D is a bond, wherein a is 1, 2 or 3, b is 1, 2 or 3, c is 1 or 2, d is 1, 2, or 3, f is 1, 2, or 3, and n is 1 or 2.

In certain embodiments, A is


and B is


wherein d is 1, 2, or 3, and e is 1, 2 or 3, wherein position α is linked to the position α′.

In certain embodiments, A is


B is


wherein position α is linked to the position α′, and C is a bond, wherein d is 1, 2, or 3, and e is 1, 2 or 3.

In certain embodiments, A is


B is


wherein position α is linked to the position α′, C is a bond, and D is


wherein position γ′ is linked to position β, wherein d is 1, 2, or 3, e is 1, 2 or 3, g is 0 or 1, and h is 0 or 1.

In certain embodiments, the CRM comprises the structure of below formula:



In a ninth aspect, the present disclosure provides a pharmaceutical composition comprising the fusion polypeptide according to the first aspect, the polypeptide complex according to the second aspect, or the conjugate according to the seventh or eighth aspect, and a pharmaceutically acceptable salt.

In a tenth aspect, the present disclosure provides a method of producing the conjugate of the seventh or eighth aspect, comprising conjugating a clearance-reducing moiety to the fusion polypeptide of the first aspect or the polypeptide complex of the second aspect.

In a eleventh aspect, the present disclosure provides a method of preventing or treating a metabolic disorder in a subject in need thereof, comprising administering a therapeutically effective amount of the fusion polypeptide of the first aspect, the polypeptide complex of the second aspect, or the conjugate of the seventh or eighth aspect.

In certain embodiments, the metabolic disorder is diabetes, obesity, non-alcoholic steatohepatitis (NASH), cardiovascular disorders like dyslipidemia, atherosclerosis, alcoholic steatohepatitis (ASH), diabeticnephropathy, gestational diabetes, metabolic syndrome such as metabolic syndrome X, nonalcoholic fatty liver disease (NAFLD), end-stage liver disease, hepatic steatosis (fatty liver), liver cirrhosis, or primary biliary cirrhosis (PBC).

Throughout the present disclosure, the articles “a,” “an,” and “the” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “a fusion polypeptide” means one fusion polypeptide or more than one fusion polypeptides.

In all occurrences in this application where there are a series of recited numerical values, it is to be understood that any of the recited numerical values may be the upper limit or lower limit of a numerical range. It is to be further understood that the invention encompasses all such numerical ranges, i.e., a range having a combination of an upper numerical limit and a lower numerical limit, wherein the numerical value for each of the upper limit and the lower limit can be any numerical value recited herein. Ranges provided herein are understood to include all values within the range. For example, 1-10 is understood to include all of the values 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and fractional values as appropriate. Similarly, ranges delimited by “at least” are understood to include the lower value provided and all higher numbers.

As used herein, “about” is understood to include within three standard deviations of the mean or within standard ranges of tolerance in the specific art. In certain embodiments, about is understood a variation of no more than 0.5.

The articles “a” and “an” are used herein to refer to one or more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”. Similarly, “such as” is used herein to mean, and is used interchangeably, with the phrase “such as but not limited to”.

The term “or” is used inclusively herein to mean, and is used interchangeably with, the term “and/or,” unless context clearly indicates otherwise.

