Liquid pharmaceutical compositions of fusion polypeptides and methods of use thereof

Abstract

Fusion polypeptides are provided that include a nanobody and FGF21 linked by a polypeptide linker. Fusion polypeptides are also provided that include GLP-1, a nanobody, and FGF21, each linked by two polypeptide linkers. Fusion polypeptides are also provided that include a functional moiety conjugated to the FGF2. Liquid pharmaceutical compositions containing the same and methods for treating diseases using the same are also provided.

Description

[Technical Field]

[0001] The present disclosure relates generally to the field of therapeutic peptides, and more particularly to fusion polypeptides, pharmaceutical compositions thereof, and methods of using such to prevent and / or treat metabolic disorders such as diabetes. [Background technology]

[0002] Fibroblast growth factor 21 (FGF21) is a hormone synthesized in several metabolically active organs and regulates glucose and lipid homeostasis. The biology of FGF21 is inherently complex due to its diverse metabolic functions in multiple target organs. FGF21 has been reported to function in organs such as the liver, adipocytes, pancreas, hypothalamus, and muscle tissue (Fisher FM, Annu Rev Physiol, 2016, 78:223).

[0003] Glucagon-like peptide-1 (GLP-1) is a proglucagon-derived peptide secreted by intestinal L-cells in response to nutrient ingestion. GLP-1 primarily acts as an incretin, i.e., an endocrine hormone, generally enhancing the insulin response after oral food ingestion by regulating glucagon concentrations, delaying gastric emptying, stimulating (pro)insulin biosynthesis, improving insulin sensitivity, and stimulating insulin-independent glycogen biosynthesis. Because GLP-1 can rapidly lower glucose levels in both normal and diabetic subjects, there has been considerable interest in developing GLP-1-based pharmaceuticals (i.e., hereafter referred to as GLP-1 compounds) to prevent and / or treat type 2 diabetes. Currently, human GLP-1 in its native form (with 37 amino acid residues) is poorly active, and the two major naturally occurring biologically active truncated versions of native GLP-1 include 30- and 31-amino acid peptide fragments, namely GLP-1(7-36) or GLP-1(7-37), obtained by post-translational processing of the proglucagon peptide, which have extremely short half-lives in vivo due primarily to N-terminal cleavage and inactivation by the dipeptidyl peptidase DPP-IV.

[0004] Metabolic disorders or metabolic diseases are characterized by the body's inability to properly convert food into energy and to utilize and / or store energy, with diabetes being the most well-known metabolic disorder. Metabolic disorders are generally associated with insulin resistance, visceral adiposity, atherogenic dyslipidemia, etc., which pose a significant and growing public health and clinical challenge worldwide. Although significant efforts have been made to develop newer and better treatments with more favorable administration regimens, existing treatments for metabolic diseases face problems such as short half-lives and / or low efficacy.

[0005] Thus, there is a need for improved therapeutic solutions and pharmaceutical compositions thereof for treating metabolic diseases. Summary of the Invention

[0006] The present invention provides pharmaceutical compositions and methods of use thereof for treating / preventing metabolic disorders.

[0007] In a first aspect, the present disclosure provides a pharmaceutical composition comprising a polypeptide and a pharmaceutically acceptable excipient, wherein the polypeptide comprises a first fragment comprising a Nanobody domain capable of binding to serum albumin and a second fragment comprising a biologically active FGF21 domain, wherein the first fragment is connected to the N-terminus of the second fragment via a first linker.

[0008] As used herein, an FGF21 domain comprises an amino acid sequence that has at least 90% sequence identity with SEQ ID NO: 1 while substantially retaining its biological activity. Thus, an FGF21 domain may contain no more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residue mutations relative to SEQ ID NO: 1, so long as such mutation-containing FGF21 variants still retain the biological activity of FGF21. As used herein, amino acid residue mutations may be substitutions, insertions, or deletions.

[0009] In some embodiments of the polypeptide, the FGF21 domain may comprise one or more amino acid residue mutations, each at a position selected from positions 121, 168, 171, and 180 relative to SEQ ID NO: 1. Optionally, the one or more amino acid residue mutations in the FGF21 domain may comprise one, two, three, or four of the following three substitutions: N121Q, M168L, P171G, and A180E.

[0010] In some embodiments of the polypeptide, the FGF21 domain may include all three substitutions, N121Q, M168L, and A180E, and the amino acid sequence of the FGF21 domain is set forth in SEQ ID NO: 5. Other amino acid residues in the FGF21 domain may be additionally mutated in addition to the three mutations. In certain embodiments, the FGF21 domain further includes the substitution P171G. According to some embodiments of the polypeptide, the FGF21 domain includes the amino acid sequence of SEQ ID NO: 14.

[0011] In some embodiments of the polypeptide, the FGF21 domain further comprises a conjugable residue. As used herein, the conjugable residue may optionally be located at a position within the C-terminal fragment spanning positions 169 to 181 relative to SEQ ID NO: 1. According to certain embodiments, the conjugable residue is located at a position selected from the group consisting of 169, 170, 171, 172, 173, 174, 180, and 181 relative to SEQ ID NO: 1. According to some embodiments of the polypeptide, the FGF21 domain comprises the amino acid sequence of SEQ ID NOs: 2-5, 89-91, 14, and 102-105, except for the mutation to a conjugable residue at a position within the C-terminal fragment spanning positions 169 to 181 relative to SEQ ID NO: 1.

[0012] In some embodiments of the polypeptide, the FGF21 domain comprises the amino acid sequence of SEQ ID NOs: 6-13, 16-19, and 92.

[0013] In some embodiments, the polypeptide is conjugated to the functional moiety at a conjugable residue in the second fragment containing the FGF21 domain.

[0014] As used herein, the functional moiety conjugated to the FGF2 domain may optionally include a glycosyl moiety or a synthetic chemical moiety.

[0015] In some embodiments of the polypeptide, the functional moiety comprises a glycosyl moiety, and the conjugable residue can be an introduced residue that is glycosylatable, such as a threonine (T) or asparagine (N) residue. More specifically, the conjugable residue can optionally be an introduced T at position 172 or 173, or an introduced N residue at position 170 or 174, relative to SEQ ID NO: 1. According to some embodiments of the polypeptide, the FGF21 domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-19.

[0016] In some embodiments of the polypeptide, the functional moiety comprises a synthetic chemical moiety. Thus, the conjugable residue may optionally comprise an introduced cysteine ​​(C), and the synthetic chemical moiety may comprise the structure *-XYZ. Herein, X, Y, and Z are interconnected via a bond, and the * end of X is connected to a conjugable residue on the polypeptide. X is [ka] and Y may be [ka] and Z can be [ka] wherein the α position is linked to the α' position and the β position is linked to the β' position. As used herein, R1 can be hydrogen or -COOH, d can be 1, 2 or 3, a can be 1, 2 or 3, b can be 1, 2 or 3, c can be 1 or 2, d can be 1, 2 or 3, and e can be 1, 2 or 3.

[0017] In certain embodiments of the polypeptide, the synthetic chemical moiety has the following structure, also referred to herein as Ac-2XADO-EDA-CO—CH2-*: [ka] It has.

[0018] Optionally, the introduced cysteine ​​can be at a position selected from the group consisting of 169, 170, 171, 172, 173, 174, 180, and 181 relative to SEQ ID NO: 1. According to certain embodiments, the conjugable residue is at position 171 or 174 relative to SEQ ID NO: 1. According to some of these embodiments of the polypeptide, the FGF21 domain comprises the amino acid sequence of SEQ ID NOs: 6-13 and 92.

[0019] In some embodiments of the polypeptide, the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 8 with an introduced cysteine ​​at position 171, wherein the introduced cysteine ​​residue has the following structure: [ka] (also referred to herein as Ac-2XADO-EDA-CO—CH2*).

[0020] In some embodiments of the polypeptide, the FGF21 domain may comprise the amino acid sequence of SEQ ID NO: 92 with an introduced cysteine ​​at position 174, wherein the introduced cysteine ​​residue has the following structure: [ka] (also referred to herein as Ac-2XADO-EDA-CO—CH2*).

[0021] Certain embodiments of the polypeptide alternatively comprise: i) the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 6, with the introduced cysteine ​​at position 169; ii) the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 7, with the introduced cysteine ​​at position 170; iii) the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 8, with the introduced cysteine ​​at position 171; iv) the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 9, with the introduced cysteine ​​at position 172; v) the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 10, with the introduced cysteine ​​at position 173; vi) the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 11, with the introduced cysteine ​​at position 174; vii) the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 12, with the introduced cysteine ​​at position 180; or viii) the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 13, with the introduced cysteine ​​at position 181; ix) the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 92, with the introduced cysteine ​​at position 174; Optionally, the introduced cysteine ​​residue has the following structure: [ka] (also referred to herein as Ac-2XADO-EDA-CO—CH2*).

[0022] Additionally or alternatively, in some other embodiments of the polypeptide, the FGF21 domain further comprises a P171G substitution relative to SEQ ID NO: 1. In such embodiments, the FGF21 domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 92.

[0023] In any of the above polypeptide embodiments, the Nanobody domain may optionally comprise a VHH domain, and optionally the VHH domain may bind to human serum albumin (HSA).

[0024] Furthermore, optionally, the VHH domains may be humanized.

[0025] In some embodiments of the polypeptide, the VHH domain may comprise a complementarity determining region 1 (CDR1), a complementarity determining region 2 (CDR2), and a complementarity determining region 3 (CDR3). Herein, CDR1 may comprise the sequence of SEQ ID NO: 20 or a variant thereof having up to 3, 2, or 1 amino acid mutations, CDR2 may comprise the sequence of SEQ ID NO: 21 or a variant thereof having up to 3, 2, or 1 amino acid mutations, and / or CDR3 may comprise the sequence of SEQ ID NO: 22 or a variant thereof having up to 3, 2, or 1 amino acid mutations, wherein the VHH domain substantially retains binding specificity to serum albumin, optionally to human serum albumin.

[0026] In certain specific embodiments of the polypeptide, the VHH domain comprises a complementarity determining region 1 (CDR1) comprising the sequence of SEQ ID NO: 20, a CDR2 comprising the sequence of SEQ ID NO: 21, and a CDR3 comprising the sequence of SEQ ID NO: 22.

[0027] In certain specific embodiments of the polypeptide, the VHH domain comprises the amino acid sequence of SEQ ID NO: 23, or a variant thereof having at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 99%) identity to SEQ ID NO: 23, wherein the variant substantially retains binding specificity and / or affinity to serum albumin.

[0028] As used herein, optionally, a variant of SEQ ID NO:23 may have up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations relative to SEQ ID NO:23.

[0029] In any of the above embodiments, the Nanobody domain may optionally further comprise an N-terminal extension attached to the N-terminus of the VHH domain, wherein the N-terminal extension may optionally comprise an SG, AG, S or A amino acid residue.

[0030] In some specific embodiments of the polypeptide, the Nanobody domain comprises an amino acid sequence selected from SEQ ID NOs: 24-27.

[0031] In any of the above embodiments of the polypeptide, the first linker may have a length of at least four amino acid residues.

[0032] In some embodiments of the polypeptide, the first linker may not include any acidic amino acid residues, and more particularly, the first linker may not include any D or E residues.

[0033] Optionally, the first linker may comprise one or more units of a first repeat sequence, and according to some embodiments, the first repeat sequence may consist of no more than four or six types of amino acid residues selected from the group consisting of G, Q, A, P, T and S.

[0034] In some specific embodiments of the polypeptide, the first repeat sequence is G f S g (wherein each of f and g is independently an integer selected from 1 to 5), may comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NO:35 (GAQP), SEQ ID NO:36 (GQAP), SEQ ID NO:37 (GPAQ), SEQ ID NO:38 (GPQA), SEQ ID NO:39 (GSQP), SEQ ID NO:40 (GASP), SEQ ID NO:41 (GPAS), SEQ ID NO:42 (GPSA), SEQ ID NO:43 (GGGS), SEQ ID NO:44 (GSGS), SEQ ID NO:45 (GGGGS), SEQ ID NO:46 (GSAPGSPAGSPTGSAPGSPA), and SEQ ID NO:110 (GS).

[0035] In certain embodiments of the polypeptide, the first repeat sequence may have the amino acid sequence set forth in SEQ ID NO: 35 (GAQP), and the number of units may be an integer from 1 to 10.

[0036] In certain specific embodiments of the polypeptide, the first linker is selected from the group consisting of SEQ ID NO: 35 (GAQP), SEQ ID NO: 49 ((GAQP)2), SEQ ID NO: 50 ((GAQP)5), SEQ ID NO: 51 ((GAQP) 10), and SEQ ID NO: 48 (GGGGSGGGS).

[0037] In any of the above polypeptide embodiments, the polypeptide may further comprise a third fragment beyond the N-terminus of the first fragment, the third fragment comprising another functional domain, wherein the first fragment and the third fragment are connected via a second linker.

[0038] As used herein, another functional domain of the third fragment may optionally include a biologically active protein or a fragment thereof selected from the group consisting of glucagon-like peptide-1 (GLP-1), insulin, leptin, glucagon, gastrin, gastric inhibitory polypeptide (GIP), amylin, calcitonin, cholecystokinin, peptide YY, neuropeptide Y, bone morphogenetic protein-6 (BMP-6), bone morphogenetic protein-9 (BMP-9), oxyntomodulin, oxytocin, glucagon-like peptide-2 (GLP-2), irisin, fibronectin type III domain-containing protein 5 (FNDC5), apelin, adiponectin, Clq and tumor necrosis factor-related protein (CTRP family), resistin, visfatin, omentin, retinol-binding protein-4 (RBP-4), glicentin, angiopoietin, interleukin-22 (IL-22), exendin-4, and growth hormone.

[0039] In some embodiments of the polypeptide, another functional domain of the third fragment comprises a biologically active peptide of GLP-1 or a fragment thereof, and may comprise an amino acid sequence having at least 70% sequence identity to SEQ ID NO:28 while retaining substantial biological activity of SEQ ID NO:28.

[0040] As used herein, optionally, the alternative functional domain may comprise one or more mutations at positions 8, 22, 26, 34, and 36, or any combination thereof, relative to SEQ ID NO:28.

[0041] In some embodiments of the polypeptide, the one or more mutations in another functional domain of the third fragment of the polypeptide may include A8G, G22E, K26R, K34R, and R36G, or any combination thereof. According to some embodiments of the polypeptide, the one or more mutations in another functional domain of the third fragment of the polypeptide may include A8G, G22E, and R36G, or any combination thereof.

[0042] In certain specific embodiments of the polypeptide, another functional domain of the third fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NO:29 and SEQ ID NOs:31-34.

[0043] In any of the above embodiments, the polypeptide comprises a first fragment connected to the N-terminus of the first fragment via a second linker.

[0044] In certain embodiments, the second linker may have a length of at least 4 amino acid residues. In certain embodiments, the second linker may have a length of at least 8, 12, 16, or 20 amino acid residues.

[0045] As used herein, the second linker may comprise one or more units of a second repeat sequence, and the second repeat sequence may consist of 4 or 6 or less types of amino acid residues selected from the group consisting of G, Q, A, E, P, T and S.

[0046] In certain specific embodiments of the polypeptide, the second repeat sequence is G h S i(wherein each of h and i is independently an integer selected from 1 to 5), and may comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NO:35 (GAQP), SEQ ID NO:55 (GQEP), SEQ ID NO:56 (GEQP), SEQ ID NO:57 (GPQE), SEQ ID NO:58 (GPEQ), SEQ ID NO:59 (GSEP), SEQ ID NO:60 (GESP), SEQ ID NO:61 (GPSE), SEQ ID NO:62 (GPES), SEQ ID NO:36 (GQAP), SEQ ID NO:37 (GPAQ), SEQ ID NO:38 (GPQA), SEQ ID NO:39 (GSQP), SEQ ID NO:40 (GASP), SEQ ID NO:41 (GPAS), SEQ ID NO:42 (GPSA), SEQ ID NO:43 (GGGS), SEQ ID NO:44 (GSGS), SEQ ID NO:45 (GGGGS), SEQ ID NO:46 (GSAPGSPAGSPTGSAPGSPA), and SEQ ID NO:110 (GS).

[0047] In certain specific embodiments of the polypeptide, the second repeat sequence may have the amino acid sequence set forth in SEQ ID NO: 35 (GAQP), and the number of the one or more units may be an integer from 1 to 15.

[0048] In certain specific embodiments of the polypeptide, the second linker is selected from the group consisting of SEQ ID NO: 49 ((GAQP)2), SEQ ID NO: 50 ((GAQP)5), SEQ ID NO: 51 ((GAQP) 10 ), and SEQ ID NO: 52 ((GAQP) 14 ), and SEQ ID NO: 47 ((GGGGS)4).

[0049] In certain specific embodiments of a polypeptide consisting of a first and second fragment, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-68, 93, 99, 100, 101, and 107.

[0050] In certain specific embodiments of the polypeptide, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63, 68, and 100, and is conjugated to a synthetic chemical moiety provided herein at an introduced cysteine ​​residue at position 171 relative to SEQ ID NO: 1. In certain specific embodiments of the polypeptide, the polypeptide comprises the amino acid sequence of SEQ ID NO: 93, and is conjugated to a synthetic chemical moiety provided herein at an introduced cysteine ​​residue at position 174 relative to SEQ ID NO: 1. In these certain specific embodiments of the polypeptide, the synthetic chemical moiety has the following structure: [ka] (also referred to herein as Ac-2XADO-EDA-CO—CH2*).

[0051] In certain specific embodiments of the polypeptide, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 64-67, 93, and 107, and is not conjugated.

[0052] In certain specific embodiments of a polypeptide consisting of the first, second, and third fragments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 70, 74, 75, 79-83, 85, 94-98, and 108-109.

[0053] In certain specific embodiments of the polypeptide, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 74, 81-83, 85, and 96-97, and is conjugated to a synthetic chemical moiety provided herein at an introduced cysteine ​​residue at position 171 relative to SEQ ID NO: 1. In certain specific embodiments of the polypeptide, the polypeptide comprises the amino acid sequence of SEQ ID NO: 94, and is conjugated to a synthetic chemical moiety at an introduced cysteine ​​residue at position 174 relative to SEQ ID NO: 1. In certain of these specific embodiments of the polypeptide, the synthetic chemical moiety has the following structure: [ka] (also referred to herein as Ac-2XADO-EDA-CO—CH2*).

[0054] In certain specific embodiments of the polypeptide, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 70, 75, 79-80, 85, 94, 95, 98, and 108-109 and is not conjugated.

[0055] To obtain the polypeptide in the pharmaceutical composition provided herein in the first aspect, a polynucleotide encoding a polypeptide portion of the polypeptide (or a fragment thereof) may be designed, and the polynucleotide may be operably placed in a vector. The vector may be transferred into a host cell, such as a prokaryotic or eukaryotic cell, and the host cell may be cultured under conditions that allow expression of the polynucleotide, thereby obtaining the polypeptide.

[0056] Further provided herein is a process for producing the polypeptide described above, said process comprising: S100: Culturing the host cell described above under conditions that allow expression of the polynucleotide defined above, or a precursor thereof that further comprises a removable tag; and S200: Recovering and purifying the polypeptide or its precursor from the host cell.

[0057] In some embodiments of the process, the polypeptide is conjugated to a functional moiety at a conjugable residue in the second fragment, and the process, after step S200 of recovering and purifying the polypeptide from the host cell, comprises the steps of: S300: further comprising the step of conjugating a functional moiety to the polypeptide.

[0058] In some embodiments of the process, the host cell is E. coli, the vector comprises an E. coli compatible vector, and the polypeptide encoded by the polynucleotide in the vector is codon optimized for E. coli expression.

[0059] In some embodiments of the process, the step S200 of recovering and purifying the polypeptide from the host cell comprises the following substeps: S210: recovering the precursor of the polypeptide; S220: Refolding the precursor of the polypeptide; S230: treating the refolded precursor of the polypeptide to remove the tag, thereby obtaining the polypeptide; and S240: The method may include a step of purifying the polypeptide.

[0060] In some embodiments of the polypeptide production process, the process further comprises conjugating the purified polypeptide with a functional moiety. In certain embodiments, the functional moiety to be conjugated to the polypeptide is [ka] (Ac-2XADO-EDA-CO-CH2-*).

[0061] In another aspect, the pharmaceutical compositions provided herein are liquid formulations.

[0062] In certain embodiments, the pharmaceutically acceptable excipients include a buffering agent and an isotonicity agent.

[0063] In certain embodiments, the buffer is selected from the group consisting of a phosphate buffer, a citrate buffer, an acetate buffer, a histidine buffer, a glycine buffer, a carbonate buffer, a borate buffer, a glutamate buffer, a glycylglycine buffer, a lysine buffer, and an arginine buffer.

[0064] In certain embodiments, the pharmaceutical composition has a pH of about 6.0 to about 8.3 (e.g., about 6.5 to about 8.2, about 6.5 to about 8.0, about 6.5 to about 7.8, about 6.5 to about 7.4, about 6.5 to about 7.0, about 7.0 to about 7.8, about 7.0 to about 7.4, about 7.4 to about 8.2, about 7.4 to about 8.0, or about 7.4 to about 7.8).

[0065] In certain embodiments, the buffer is a phosphate buffer. Optionally, the pharmaceutical composition has a pH of about 6.5 to about 8.0, about 6.5 to about 7.4, about 7.0 to about 7.8, about 7.0 to about 7.4, or about 7.4 to about 7.8.

[0066] In certain embodiments, the phosphate buffer is present in the pharmaceutical composition at a concentration of 0.01 to 50 mM.

[0067] In certain embodiments, the phosphate buffer is present in the pharmaceutical composition at a concentration of 5-20 mM, optionally at a concentration of about 8 mM.

[0068] In certain embodiments, the phosphate buffer is selected from the group consisting of sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, or hydrates thereof.

[0069] In certain embodiments, the hydrate is a dodecahydrate or a dihydrate.

[0070] In certain embodiments, the phosphate buffer is disodium hydrogen phosphate dodecahydrate or disodium phosphate dihydrate.

[0071] In certain embodiments, disodium hydrogen phosphate dodecahydrate is present at a concentration of about 0.01 to 17 mg / mL (e.g., about 0.01 to 15 mg / mL, about 0.05 to 12 mg / mL, about 0.05 to 10 mg / mL, about 0.1 to 10 mg / mL, about 1 to 5 mg / mL, or about 1 to 3 mg / mL).

[0072] In certain embodiments, disodium hydrogen phosphate dodecahydrate is present in the pharmaceutical composition at a concentration of about 2.87 mg / mL.

[0073] In certain embodiments, disodium hydrogen phosphate dihydrate is present at a concentration of about 0.01 to 8 mg / mL (e.g., about 0.01 to 5 mg / mL, about 0.05 to 4 mg / mL, about 0.05 to 3 mg / mL, about 0.1 to 3 mg / mL, or about 1 to 3 mg / mL).

[0074] In certain embodiments, disodium hydrogen phosphate dihydrate is present at a concentration of about 1.42 mg / mL.

[0075] In certain embodiments, the buffer is a citrate buffer.

[0076] In certain embodiments, the citrate buffer is present at a concentration of about 0.05 to 20 mg / mL, or optionally, 0.05 to 10 mg / mL.

[0077] In certain embodiments, the citrate buffer comprises a mixture of anhydrous citric acid and trisodium citrate (or a hydrate thereof, e.g., trisodium citrate dihydrate). Optionally, the pharmaceutical composition has a pH of about 6.0 to about 6.8, or optionally, about 6.5.

[0078] In certain embodiments, the anhydrous citric acid / trisodium citrate dihydrate is present at a concentration of about 0.05-10 mg / mL. In certain embodiments, the citrate buffer comprises a mixture of about 0.14 mg / mL anhydrous citric acid and about 2.74 mg / mL trisodium citrate dihydrate.

[0079] In certain embodiments, the buffering agent is a histidine buffering agent. Optionally, the pharmaceutical composition has a pH of about 6.0 to about 6.8, or optionally, about 6.5.

[0080] In certain embodiments, the histidine buffer is present at a concentration of about 0.5-10 mg / mL (eg, about 1-5 mg / mL, or about 1-3 mg / mL).

[0081] In certain embodiments, the histidine buffer is present at a concentration of about 1.55 mg / mL.

[0082] In certain embodiments, the isotonicity agent is selected from the group consisting of sodium chloride, glycerol, sorbitol, sucrose, propylene glycol, mannitol, glycine, lactose monohydrate, arginine, myo-inositol, and dimethyl sulfone. In certain embodiments, the isotonicity agent is not sodium chloride.

[0083] In certain embodiments, the isotonicity agent is glycerol.

[0084] In certain embodiments, the glycerol is about 5-30 mg / mL.

[0085] In certain embodiments, the glycerol is about 20 mg / mL.

[0086] In certain embodiments, the isotonic agent is propylene glycol.

[0087] In certain embodiments, the propylene glycol is about 1 mg / mL to about 50 mg / mL (eg, about 5 mg / mL to about 25 mg / mL, about 8 mg / mL to about 16 mg / mL).

[0088] In certain embodiments, the propylene glycol is about 14 mg / mL.

[0089] In certain embodiments, the isotonic agent is sodium chloride.

[0090] In certain embodiments, the sodium chloride is about 5-15 mg / mL.

[0091] In certain embodiments, the sodium chloride is about 8.25 mg / mL.

[0092] In certain embodiments, the isotonic agent is mannitol.

[0093] In certain embodiments, mannitol is about 20 mg / mL to about 100 mg / mL (eg, about 25 mg / mL to about 70 mg / mL, about 30 mg / mL to about 60 mg / mL, about 35 mg / mL to about 55 mg / mL).

[0094] In certain embodiments, the mannitol is about 45 mg / mL.

[0095] In certain embodiments, the isotonicity agent is sorbitol.

[0096] In certain embodiments, the sorbitol is about 20 mg / mL to about 100 mg / mL (eg, about 40 mg / mL to about 50 mg / mL).

[0097] In certain embodiments, the isotonic agent is sucrose.

[0098] In certain embodiments, the sucrose is about 5 mg / mL to about 150 mg / mL (eg, about 45 mg / mL to about 100 mg / mL).

[0099] In certain embodiments, the pharmaceutically acceptable excipient further comprises a non-ionic surfactant.

[0100] In certain embodiments, the non-ionic surfactant is polysorbate 80.

[0101] In certain embodiments, the polysorbate 80 is about 0.05 mg / mL to about 5 mg / mL (eg, about 0.1 mg / mL to about 0.5 mg / mL).

[0102] In certain embodiments, the pharmaceutical excipient further comprises a preservative, a chelating agent, and / or a stabilizer.

[0103] In certain embodiments, the pharmaceutical composition has about 1 to 100 mg / mL (e.g., about 1 to 90 mg / mL, about 1 to 80 mg / mL, about 1 to 70 mg / mL, about 1 to 60 mg / mL, about 1 to 50 mg / mL, about 1 to 40 mg / mL, about 1 to 30 mg / mL, about 1 to 20 mg / mL, about 1 to 10 mg / mL) of a polypeptide provided herein. In certain embodiments of the pharmaceutical composition, the polypeptide comprises the amino acid sequence of SEQ ID NO: 85 and is conjugated to a synthetic chemical moiety provided herein at an introduced cysteine ​​residue at position 171 relative to SEQ ID NO: 1. In these particular specific embodiments of the polypeptide, the synthetic chemical moiety has the following structure: [ka] (also referred to herein as Ac-2XADO-EDA-CO-CH2*). In certain embodiments of the pharmaceutical composition, the polypeptide comprises the amino acid sequence of SEQ ID NO: 96 and is conjugated to a synthetic chemical moiety at an introduced cysteine ​​residue located at position 171 relative to SEQ ID NO: 1. In certain of these specific embodiments of the polypeptide, the synthetic chemical moiety has the following structure: [ka] (also referred to herein as Ac-2XADO-EDA-CO—CH2*).

[0104] In some embodiments, the present disclosure provides: (a) a polypeptide provided herein (optionally, the polypeptide comprises the amino acid sequence of SEQ ID NO: 85 or SEQ ID NO: 96); (b) a buffer selected from the group consisting of a phosphate buffer, a citrate buffer, an acetate buffer, a histidine buffer, a glycine buffer, a carbonate buffer, a borate buffer, a glutamate buffer, a glycylglycine buffer, a lysine buffer, and an arginine buffer; (c) an isotonicity agent selected from the group consisting of sodium chloride, glycerol, sorbitol, sucrose, propylene glycol, mannitol, glycine, lactose monohydrate, arginine, myo-inositol, and dimethyl sulfone; and (d) Provide a pharmaceutical composition having a pH of about 6.5 to about 8.2.

[0105] In certain embodiments, the pharmaceutical composition comprises: (a) about 1 to 100 mg / mL of a polypeptide provided herein (optionally, the polypeptide comprises the amino acid sequence of SEQ ID NO: 85 or SEQ ID NO: 96); (b) a buffer selected from the group consisting of a phosphate buffer, a citrate buffer, and a histidine buffer; (c) an isotonicity agent selected from the group consisting of sodium chloride, glycerol, sorbitol, sucrose, propylene glycol, and mannitol; and (d) The pH is about 6.5 to about 8.2.

[0106] In certain embodiments, the pharmaceutical composition comprises: (a) about 1 to 100 mg / mL of a polypeptide provided herein (optionally, the polypeptide comprises the amino acid sequence of SEQ ID NO: 85 or SEQ ID NO: 96); (b) about 0.1 to 10 mg / mL of a phosphate buffer, about 0.05 to 10 mg / mL of a citrate buffer, or about 0.5 to 10 mg / mL of a histidine buffer; (c) an isotonicity agent selected from the group consisting of sodium chloride, glycerol, sorbitol, sucrose, propylene glycol, and mannitol; and (d) The pH is about 6.5 to about 8.2.

[0107] In certain embodiments, the pharmaceutical composition comprises: (a) about 1 to 100 mg / mL of a polypeptide provided herein (optionally, the polypeptide comprises the amino acid sequence of SEQ ID NO: 85 or SEQ ID NO: 96); (b) a buffer selected from the group consisting of a phosphate buffer, a citrate buffer, and a histidine buffer; (c) an isotonic agent selected from the group consisting of 5 to 15 mg / mL sodium chloride, 5 to 30 mg / mL glycerol, 20 to 100 mg / mL sorbitol, 5 to 150 mg / mL sucrose, 1 to 50 mg / mL propylene glycol, and 20 to 100 mg / mL mannitol; and (d) The pH is about 6.5 to about 8.2.

[0108] In certain embodiments, the pharmaceutical composition comprises: (a) about 1 to 100 mg / mL of a polypeptide provided herein (optionally, the polypeptide comprises the amino acid sequence of SEQ ID NO: 85 or SEQ ID NO: 96); (b) about 0.1 to 10 mg / mL of a phosphate buffer, about 0.05 to 10 mg / mL of a citrate buffer, or about 0.5 to 10 mg / mL of a histidine buffer; (c) an isotonic agent selected from the group consisting of 5 to 15 mg / mL sodium chloride, 5 to 30 mg / mL glycerol, 20 to 100 mg / mL sorbitol, 5 to 150 mg / mL sucrose, 1 to 50 mg / mL propylene glycol, and 20 to 100 mg / mL mannitol; and (d) The pH is about 6.5 to about 8.2.

[0109] In certain embodiments, the pharmaceutical composition comprises: (a) about 1 to 100 mg / mL of a polypeptide provided herein (optionally, the polypeptide comprises the amino acid sequence of SEQ ID NO: 85 or SEQ ID NO: 96); (b) about 1.55 mg / mL histidine buffer; and (c) an isotonicity agent comprising about 45 mg / mL mannitol; and (d) The pH is about 6.5.

[0110] In certain embodiments, the pharmaceutical composition further comprises a non-ionic surfactant having a concentration of polysorbate 80 of about 0.1 to 0.5 mg / mL.

[0111] In a second 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 a pharmaceutical composition provided herein.

[0112] In certain embodiments, the metabolic disorder is diabetes, obesity, non-alcoholic steatohepatitis (NASH), cardiovascular-like dyslipidemia, arteriosclerosis, alcoholic steatohepatitis (ASH), diabetic nephropathy, gestational diabetes, metabolic syndromes such as metabolic syndrome X, non-alcoholic fatty liver disease (NAFLD), end-stage liver disease, hepatic steatosis (fatty liver), cirrhosis, primary biliary cirrhosis (PBC), or severe hypertriglyceridemia (SHTG).

[0113] In certain embodiments, diabetes can be any form of diabetes, including, without limitation, hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin dependent diabetes, MODY (maturity-onset diabetes of the young), gestational diabetes, and elevated HbA1C levels.

[0114] In a third aspect, the present disclosure provides a method for managing weight in a subject in need thereof, comprising managing the subject's weight by administering a therapeutically effective amount of a pharmaceutical composition provided herein.

[0115] In a fourth aspect, the present disclosure provides a method of reducing food intake in a subject in need thereof, comprising reducing the food intake of the subject by administering a therapeutically effective amount of a pharmaceutical composition provided herein.

[0116] In a fifth aspect, the present disclosure provides a method of reducing weight in a subject in need thereof, comprising reducing the subject's weight by administering a therapeutically effective amount of a pharmaceutical composition provided herein.

[0117] In any of the methods described above in the second to fifth aspects, the subject may be a human.

[0118] In certain embodiments, the subject has a fasting blood glucose level of 125 mg / dL or greater.

[0119] In certain embodiments, the subject has a body mass index (BMI) of at least 25 or higher.

[0120] In certain embodiments, the pharmaceutical composition is administered in a dosing regimen that is no more frequent than once daily, once every three days, once a week, or once every two weeks.

[0121] In certain embodiments, the pharmaceutical composition is administered twice weekly, once weekly, or once every two weeks.

[0122] In certain embodiments, the dosing regimen has a dosing interval of about once every three days to about once every two weeks.

[0123] In certain embodiments, the pharmaceutical composition is administered by parenteral administration.

[0124] In certain embodiments, the pharmaceutical composition is administered subcutaneously, intravenously, or intramuscularly.

[0125] Throughout this 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, "polypeptide" means one polypeptide or more than one polypeptide.

[0126] At all occurrences in this application where there is a series of recited numerical values, it should be understood that any of the recited numerical values ​​may be the upper or lower limit of the numerical range. It should be further understood that the present invention encompasses all such numerical ranges, i.e., ranges having a combination of upper and lower numerical limits, and the numerical values ​​for each of the upper and lower limits may be any of the numerical values ​​recited herein. Ranges provided herein are understood to include all values ​​within the range. For example, 1 to 10 is understood to include all values ​​of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, as well as decimal values, where appropriate. Similarly, ranges delimited by "at least" are understood to include the lower value provided and all higher numbers.

[0127] As used herein, "about" is understood to include within 3 standard deviations of the mean or within the normal range of acceptance in a particular technical field. In certain embodiments, about is understood to mean a variation of no more than 0.5.

[0128] Terms such as "comprise," "comprising," "contain," "containing," or the like, unless otherwise noted, should be construed as open-ended terms (i.e., meaning "including, but not limited to").

[0129] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to." Similarly, the term "such as" is used herein to mean, and is used interchangeably with, the phrase "such as but not limited to."

[0130] The term "or" is used inclusively herein to mean, and is used interchangeably with, the term "and / or," unless the context clearly indicates otherwise. [Brief explanation of the drawings]

[0131] [Figure 1A] 1A-1B show polypeptides according to two different embodiments of the present disclosure. [Figure 1B] (the above) [Figure 2A] 2A-2B show the in vitro FGF21 activity of molecules MLC#9, MLC#10, MLC#14, control#6, and the reference compound YH-dual. [Figure 2B] (the above) [Figure 3A] 3A and 3B show the efficacy of molecular control #2, control #6, MLC#9, and MLC#10 in reducing body weight (FIG. 3A) and suppressing food intake (FIG. 3B) in DIO rats. [Figure 3B] (the above) [Figure 4A] Figures 4A-4L show the efficacy of molecules MLC#9, MLC#14, MLC#16, and MLC#17, and reference compounds semaglutide, YH-dual, and tirzepatide in reducing body weight (Figure 4A), glucose control (Figure 4B), plasma triglyceride (Figure 4C), LDL-C (Figure 4D), total cholesterol (Figure 4E), and ALT (Figure 4F) concentrations, as well as reducing fat weight (Figure 4G) and liver weight (Figure 4H), along with improving liver TG (Figure 4I), liver TC (Figure 4J), and insulin sensitivity (Figure 4K), and increasing adiponectin levels (Figure 4L) in DIO mice. [Figure 4B] (the above) [Figure 4C] (the above) [Figure 4D] (the above) [Figure 4E] (the above) [Figure 4F] (the above) [Figure 4G] (the above) [Figure 4H] (the above) [Figure 4I] (the above) [Figure 4J] (the above) [Figure 4K] (the above) [Figure 4L] (the above) [Figure 5A] Figures 5A-5M show the efficacy of various dosages of molecules MLC#14 and MLC#16 compared to reference compounds semaglutide, YH-dual, and tirzepatide in reducing body weight (Figure 5A), glucose control (Figure 5B), plasma triglyceride (Figure 5C), LDL-C (Figure 5D), total cholesterol (Figure 5E), ALT (Figure 5F), and AST (Figure 5G) concentrations, as well as reducing fat weight (Figure 5H) and liver weight (Figure 5I), along with improving liver TG (Figure 5J), liver TC (Figure 5K), and insulin sensitivity (Figure 5L), and increasing adiponectin levels (Figure 5M) in DIO mice. [Figure 5B] (the above) [Figure 5C] (the above) [Figure 5D] (the above) [Figure 5E] (the above) [Figure 5F] (the above) [Figure 5G] (the above) [Figure 5H] (the above) [Figure 5I] (the above) [Figure 5J] (the above) [Figure 5K] (the above) [Figure 5L] (the above) [Figure 5M] (the above) [Figure 6A] Figures 6A-6H show the efficacy of various dosages of molecules MLC#16, MLC#19, MLC#6, and MLC#23 compared to the reference compound semaglutide in reducing serum triglyceride (Figure 6C), LDL-C (Figure 6D), and total cholesterol (Figure 6E) concentrations in plasma, as well as fat weight (Figure 6F) and liver weight (Figure 6G), along with improving liver TG (Figure 6H). [Figure 6B] (the above) [Figure 6C] (the above) [Figure 6D] (the above) [Figure 6E] (the above) [Figure 6F] (the above) [Figure 6G] (the above) [Figure 6H] (the above) [Figure 7-1] FIG. 7 shows all the sequences disclosed in this disclosure. [Figure 7-2] (the above) [Figure 7-3] (the above) [Figure 7-4] (the above) [Figure 7-5] (the above) [Figure 7-6] (the above) [Figure 7-7] (the above) [Figure 7-8] (the above) [Figure 7-9] (the above) [Figure 7-10] (the above) [Figure 7-11] (the above) [Figure 7-12] (the above) [Figure 7-13] (the above) [Figure 7-14] (the above) [Figure 7-15] (the above) [Figure 7-16] (the above) [Figure 7-17] (the above) [Figure 7-18] (the above) DETAILED DESCRIPTION OF THE INVENTION

[0132] The following description of the present invention is intended to merely illustrate various embodiments of the present invention. As such, the specific modifications discussed should not be construed as limitations on the scope of the present invention. It will be apparent to those skilled in the art that various equivalents, variations, and modifications can be made without departing from the scope of the present invention, and it is understood that such equivalent embodiments are to be included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0133] definition The terms "protein," "peptide," and "polypeptide" are used interchangeably herein and refer to a polymer of amino acid residues linked by covalent bonds, such as peptide bonds. The proteins or polypeptides provided herein may contain naturally occurring or non-naturally occurring amino acid residues, or both. The polypeptides, peptides, and proteins provided herein may be of any suitable length, for example, at least 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more amino acid residues in length.

[0134] As used herein, the term "amino acid" refers to an organic compound containing an amine (-NH2) and a carboxyl (-COOH) functional group, as well as a side chain characteristic of each amino acid.

[0135] As used herein, the term "naturally occurring" amino acid residue refers to an amino acid residue found in a natural protein or peptide, including all of its D and L stereoisomers, if its structure allows for such stereoisomeric forms. Examples of naturally occurring amino acid residues include glycine (Gly or G), alanine (Ala or A), valine (Val or V), leucine (Leu or L), isoleucine (Ile or I), serine (Ser or S), cysteine ​​(Cys or C), threonine (Thr or T), methionine (Met or M), proline (Pro or P), phenylalanine (Phe or F), tyrosine (Tyr or Y), tryptophan (Trp or W), histidine (His or H), lysine (L), and threonine (Thr or T). D-stereoisomers include, but are not limited to, the 20 standard amino acids, including D-aspartic acid (Asp or D), glutamic acid (Glu or E), asparagine (Asn or N), and glutamine (Gln or Q), as well as naturally occurring analogs thereof, such as canavanine, pyrrolysine (PYL), selenocysteine, pyrroline-carboxy-lysine (PCL), sarcosine, beta-alanine, phosphoserine, gamma-carboxyglutamic acid, and ornithine. Examples of naturally occurring amino acid residues in their D-stereoisomers include, for example, D-aspartic acid, D-serine, D-cysteine, D-alanine, D-glutamic acid, etc.

[0136] An "amino acid analog" is a compound that has the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but will retain the same basic chemical structure as a naturally occurring amino acid.

[0137] As used herein, a "non-naturally occurring" amino acid residue refers to any amino acid residue not found in nature, including, but not limited to, modified amino acid residues and / or amino acid mimetics that are not one of the known naturally occurring amino acids but function in a manner similar to a naturally occurring amino acid. Modified amino acids or mimetics can be created by adding chemical entities such as carbohydrate groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers, etc. for conjugation, functionalization, or other modification. Non-naturally occurring amino acids can also refer to amino acids produced by chemical synthesis. Exemplary unnatural amino acids are 2-aminoisobutyric acid (Aib), imidazole-4-acetate (IA), imidazolepropionic acid (IPA), α-aminobutyric acid (Abu), tert-butylglycine (Tle), β-alanine, 3-aminomethylbenzoic acid, anthranilic acid, des-amino-histidine (abbreviated as DesaminoHis, alternatively named imidazopropionic acid, abbreviated as lmpr), beta analogs of amino acids such as β-alanine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, Nα-acetyl-histidine, α-fluoro-methylhistidine, α-amino- ... Examples of suitable amino acids include, but are not limited to, 1-amino-histidine, α-methyl-histidine, α,α-dimethyl-glutamic acid, m-CF3-phenylalanine, α,β-diaminopropionic acid (abbreviated as Dap), 3-pyridylalanine, 2-pyridylalanine or 4-pyridylalanine, (1-aminocyclopropyl)carboxylic acid, (1-aminocyclobutyl)carboxylic acid, (1-aminocyclopentyl)carboxylic acid, (1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl)carboxylic acid, and (1-aminocyclooctyl)carboxylic acid.

[0138] Introduction of unnatural amino acids into polypeptides can be achieved by techniques described in Wang et al., Science 292:498-500, 2001; Deiters et al., J Am Chem Soc 125:11782-11783, 2003; Wang and Schultz, Science 301:964-967, 2003; Zhang et al., Science 303:371-373, 2004, or U.S. Patent No. 7,083,970. Briefly, some of these expression systems involve site-directed mutagenesis to introduce stop codons, such as amber (UAG), ochre (UAA), and opal (UGA) codons, into an open reading frame encoding a fusion polypeptide of the disclosure. Other codons, such as four-base codons (e.g., AGGA, AGGU, CGGU, CGCU, CGAU, CCCU, CUCU, CUAU, and GGGU), five-base codons, six-base codons, etc., can also be introduced into expression systems for unnatural amino acids. Such expression vectors are then introduced into hosts that can utilize tRNAs specific for the introduced stop or other codon and carrying the unnatural amino acid of choice. As another example, unnatural amino acids can be chemically synthesized and inserted or attached to a polypeptide by a chemical reaction, such as acylation.

[0139] The terms "fusion" or "fused," when used in reference to amino acid sequences (e.g., peptides, polypeptides, or proteins), refer to the combination of two or more amino acid sequences into a single amino acid sequence that does not occur in nature, for example, by chemical linkage or recombinant means. A fused amino acid sequence can be produced by genetic recombination of two encoding polynucleotide sequences and can be expressed by introducing a construct containing the recombinant polynucleotide into a host cell.

[0140] "Percent (%) sequence identity" is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum number of identical amino acids (or nucleic acids). In other words, the percent (%) sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of identical amino acid residues (or bases) relative to the reference sequence to which it is compared by the total number of amino acid residues (or bases) in the candidate sequence or in the reference sequence, whichever is shorter. Conservative substitutions of amino acid residues are not considered identical residues. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be achieved using publicly available tools such as, for example, BLASTN, BLASTp (available at the National Center for Biotechnology Information (NCBI) website; see also Altschul S F et al, J. Mol. Biol., 215:403-410 (1990); Stephen F. et al, Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available at the European Bioinformatics Institute website; see also Higgins D Get al, Methods in Enzymology, 266:383-402 (1996); Larkin MA et al, Bioinformatics (Oxford, England), 23(21):2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. One skilled in the art can use the default parameters provided by the tool or can customize the parameters as needed for the alignment, for example by selecting an appropriate algorithm.

[0141] " Conservative substitution " in relation to amino acid sequence refers to replacing an amino acid residue with a different amino acid residue that has a side chain with similar physicochemical properties.For example, conservative substitution can be made between amino acid residues with hydrophobic side chains (for example, Met, Ala, Val, Leu and Ile), between residues with neutral hydrophilic side chains (for example, Cys, Ser, Thr, Asn and Gln), between residues with acidic side chains (for example, Asp, Glu), between amino acids with basic side chains (for example, His, Lys and Arg), or between residues with aromatic side chains (for example, Trp, Tyr and Phe).As is known in the art, conservative substitution usually does not cause significant changes in protein conformation, and therefore can maintain the biological activity of protein.

[0142] As used herein, the term "functional form" refers to various forms of a parent molecule (such as variants, fragments, fusions, derivatives, and mimetics) that, despite differences in amino acid sequence or chemical structure, still retain substantial biological activity of the parent molecule. As used herein, the phrase "retaining substantial biological activity" means exhibiting at least some (e.g., about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more) or all of the biological activity of the parent molecule. Functional forms of a parent polypeptide can include both naturally occurring variant forms and non-naturally occurring forms, such as those obtained by recombinant methods or chemical synthesis. Functional forms can contain non-natural amino acid residues.

[0143] As used herein, the term "variant" refers to a polypeptide that has at least 70% sequence identity with a parent polypeptide and retains at least partial function of the parent polypeptide. A variant may differ from the parent polypeptide in one or more amino acid residues. For example, a variant may have one or more amino acid residues substituted, added, deleted, inserted, or truncated in the parent polypeptide.

[0144] As used herein, the term "fragment" refers to a subsequence of a parent polypeptide of any length. A fragment may still retain at least partial function of the parent polypeptide.

[0145] The term "derivative" as used herein refers to a chemically modified polypeptide or fusion polypeptide in which one or more clearly defined number of substituents are covalently attached to one or more specific amino acid residues of the polypeptide or fusion polypeptide. Exemplary chemical modifications may be, for example, alkylation, acylation, esterification, amidation, phosphorylation, glycosylation, labeling, methylation, or conjugation with one or more moieties of one or more amino acids.

[0146] The term "mimetic" as used herein refers to a molecular structure that serves as a substitute for amino acid, peptide, polypeptide, or fusion polypeptide.For example, the amino acid mimic used herein may or may not be an amino acid, but may be a synthetic structure (known or unknown) that retains the functional properties of the parent amino acid, while the structure of the amino acid mimic is different from the structure of the parent amino acid.Examples include amide, methacryloyl or acryloyl derivatives of β-, γ-, δ-imino acid (such as piperidine-4-carboxylic acid), etc.

[0147] As used herein, "treating" or "treatment" of a condition includes preventing or alleviating the condition, slowing the onset or progression of the condition, reducing the risk of developing the condition, preventing or delaying the onset of symptoms associated with the condition, reducing or terminating symptoms associated with the condition, causing complete or partial regression of the condition, curing the condition, or some combination thereof.

[0148] As used herein, the term "vector" refers to a vehicle into which a polynucleotide encoding a protein can be operably inserted to result in the expression of that protein. A vector can be used to transform, transduce, or transfect a host cell to result in the expression of the genetic elements it carries within the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. Categories of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Vectors can contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, vectors can contain origins of replication.Vectors can also contain materials that aid their entry into cells, including but not limited to viral particles, liposomes, or protein coatings.Vector can be an expression vector or a cloning vector.The present disclosure provides a vector (e.g., expression vector) that contains the nucleic acid sequence provided herein that encodes a fusion polypeptide, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker.Examples of vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papova viruses (e.g., SV40), lambda phage, and M13 phage, and the plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, p Examples of such vectors include, but are not limited to, EGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, and pEF-Bos.

[0149] As used herein, the phrase "host cell" refers to a cell into which an exogenous polynucleotide and / or vector has been introduced.

[0150] The term "pharmaceutically acceptable" indicates that the specified carrier, vehicle, diluent, excipient, and / or salt is generally chemically and / or physically compatible with the other ingredients that make up the formulation, and physiologically compatible with the recipient thereof.

[0151] As used herein, the terms "subject" or "individual" or "animal" or "patient" refer to a human or non-human animal, including a mammal or primate, in need of diagnosis, prognosis, amelioration, prevention, and / or treatment of a disease or disorder. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, bears, etc.

[0152] FGF21 domain The polypeptides disclosed herein comprise an FGF21 domain. For example, the first and second embodiments of the polypeptides disclosed herein and shown in Figures 1A and 1B each comprise an FGF21 domain.

[0153] 1A and 1B show two main embodiments of the polypeptides provided in the present disclosure. As shown in FIG. 1A, the first embodiment of polypeptide 001A comprises a first fragment 100 and a second fragment 200 connected in an N-terminal to C-terminal direction via a first linker 10. The first fragment 100 substantially comprises a Nanobody domain capable of binding to serum albumin, such as human serum albumin (HSA). The second fragment 200 comprises a biologically active FGF21 domain substantially comprising a functional form of FGF21, and according to certain specific embodiments of the present disclosure, comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1 while substantially retaining its biological activity.

[0154] Optionally, the polypeptide may further comprise an additional functional domain in addition to the Nanobody domain in the first fragment 100 and the FGF21 domain in the second fragment 200. As shown in FIG. 1B, in addition to the first fragment 100 and second fragment 200 in the first embodiment shown in FIG. 1A, this second embodiment of polypeptide 001B further comprises a third fragment 300 through the N-terminus of first fragment 100. The third fragment 300 comprises an additional functional domain that may exert biological activity additive or synergistic with the FGF21 domain in the second fragment 200. The first fragment 100 and the third fragment 300 are connected via a second linker 20. According to some embodiments of the present disclosure, the additional functional domain in the third fragment 300 is a biologically active glucagon-like peptide-1 (GLP-1) domain that substantially comprises a functional form of GLP-1. According to some embodiments, it comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 28 while substantially retaining its biological activity.

[0155] As used herein, the term "FGF21" refers to and is an abbreviation for "fibroblast growth factor 21" and is intended to broadly encompass native human FGF21 as well as all functional forms thereof, including functional variants, fragments, fusions, derivatives, or mimetics. Native human FGF21 consists of 209 amino acid residues, with amino acid residues 1-28 being a signal polypeptide and amino acid residues 29-209 being a mature polypeptide of 181 residues (accession number Q9NSA1 in the Uniprot database). The phrase "biologically active FGF21 domain" provided in this disclosure refers to a functional form of FGF21, which may be the mature polypeptide or a functional variant, fragment, fusion, derivative, or mimetic thereof. The mature polypeptide of human FGF21 is included herein as SEQ ID NO: 1. As used herein, the numbering of residues in FGF21 refers to the sequence of SEQ ID NO: 1, beginning with His at position 1 and Ser at position 181.

[0156] The functional form of the mature FGF21 polypeptide can activate the FGF21 receptor at a level equivalent to or at about 20% or more (or 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) that of the mature polypeptide of native human FGF21. Activation of the FGF21 receptor can lead to biological activities such as the ability to activate glucose uptake in adipocytes, the ability to lower blood glucose and triglyceride levels, or the ability to lower body weight (Tezze C et al., Front Physiol. 2019, 10:419). Many functional forms of the mature polypeptide of FGF21 are known, including, but not limited to, WO2019043457A2, WO2018088838A1, WO2018039081A1, WO2017220706A1, WO2017180988A2, WO2017116207A1, WO2017093465, WO2017059371A1, WO2016102562A1, WO2016065326A, WO20131731A, the disclosures of which are incorporated herein by their entireties. 58A1, WO2013052311A1, WO2013033452A2, WO2012066075A1, WO2012059873A2, WO2012010553A1, WO2011140086A2, WO2010084169A2, WO2010065439A1, WO2008121563A2, WO2006028595A2, WO2006028714A1, WO2005113606A2, WO2016102562A are known in the art.

[0157] In certain embodiments, the FGF21 domains provided herein comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1 while retaining substantial biological activity thereof.

[0158] In certain embodiments, the FGF21 domain comprises no more than 12, 11, 10, 9, 8, 7 amino acid mutations (e.g., substitutions, insertions, or deletions) relative to SEQ ID NO: 1, while retaining substantial biological activity of SEQ ID NO: 1. In certain embodiments, the FGF21 domain comprises the amino acid sequence of SEQ ID NOs: 2-14, 16-19, 89-92, and 102-105.

[0159] In certain embodiments, FGF21 comprises one or more mutations. In certain embodiments, the one or more mutations comprise conservative substitutions. In certain embodiments, the one or more mutations are at positions selected from the group consisting of 121, 168, 171, and 180 relative to SEQ ID NO: 1.

[0160] In certain embodiments, one or more mutations in the FGF21 domain are selected from N121Q, M168L, P171G, and A180E, or any combination thereof.For example, the FGF21 domain in the polypeptide disclosed herein can optionally contain one of four mutations, two of four mutations, three of four mutations, or four of mutations.

[0161] In certain embodiments, the FGF21 domain is 1) N121Q, 2) M168L, 3) A180E, 4) N121Q and M168L, 5) N121Q and A180E, 6) M168L and A180E, 7) N121Q, P171G and A180E, 8) N121Q, M168L, and P171G, 9) M168L, P171G and A180E, and 10) Contains a combination of mutations relative to SEQ ID NO: 1 selected from the group consisting of N121Q, M168L, P171G and A180E.

[0162] In certain embodiments, the FGF21 domain may comprise the amino acid sequence set forth in SEQ ID NO:2 (N121Q), SEQ ID NO:3 (M168L), SEQ ID NO:4 (A180E), SEQ ID NO:5 (N121Q, M168L, and A180E), SEQ ID NO:89 (N121Q and M168L), SEQ ID NO:90 (N121Q and A180E), SEQ ID NO:91 (M168L and A180E), or SEQ ID NO:14 (N121Q, M168L, P171G, and A180E).

[0163] In certain embodiments, the FGF21 domain in the polypeptide further comprises a conjugable residue or at most one conjugable residue to which the functional moiety is conjugated.

[0164] As used herein, the term "conjugable residue" refers to an amino acid residue at a specific position in a polypeptide that not only has a functional group that can be chemically or enzymatically conjugated to a chemical moiety, but also makes its position in the polypeptide more susceptible to such conjugation in a conjugation reaction for that specific functional group.As used herein, "conjugation" refers to the reaction of joining two molecules together to form a physical entity.For example, a covalent bond that connects two molecules can be formed in conjugation.The conjugable residue can be a natural amino acid residue, a non-natural amino acid residue, a modified amino acid residue, or an amino acid mimic.Examples of conjugable residues include, but are not limited to, lysine, cysteine, or a non-natural amino acid residue.

[0165] Those skilled in the art will understand that whether a residue is a conjugable residue will depend on a given conjugation reaction and / or the functional group to be conjugated. For example, if the functional group to be conjugated is a thiol group and / or the conjugation reaction is specific or selective for thiol groups, then lysine (i.e., without a functional thiol group in its side chain) would not be considered a conjugable residue regardless of its position, whereas an unpaired cysteine ​​residue that does not form a disulfide bond (whether intrachain or interchain) may be a conjugable residue.

[0166] In certain embodiments, the polypeptide comprises a conjugable residue for a thiol group-specific or selective conjugation reaction (e.g., a maleimide reaction, or a reaction for forming a disulfide bond). When referring to an amino acid residue, the term "conjugable" is intended to mean that the residue at a particular position in the polypeptide is sufficiently available for conjugation. For example, a cysteine ​​residue as part of a disulfide bridge is not a conjugable residue in the present disclosure.

[0167] In certain embodiments, the conjugable residue is a cysteine ​​residue, preferably a free cysteine ​​residue that is not part of a disulfide bridge.

[0168] In certain embodiments, the conjugable residues CR in the FGF21 domain of the polypeptides provided herein may be located at a position within the C-terminal fragment of the FGF21 domain, for example, spanning positions 169 to 181 relative to SEQ ID NO: 1. Further optionally, the conjugable residue may be located at a position selected from the group consisting of positions 169, 170, 171, 172, 173, 174, 180, and 181 relative to SEQ ID NO: 1. Without wishing to be bound by any theory, the present inventors have found that conjugation at the C-terminal fragment of the FGF21 domain reduces C-terminal degradation of the FGF21 domain.

[0169] In certain embodiments, the FGF21 domain in the polypeptide has a combination of mutations: 1) N121Q and M168L, 2) N121Q, M168L and P171G, 3) N121Q, M168L and A180E, or 4) N121Q, M168L, P171G and A180E, and further comprises a conjugable residue (e.g., a cysteine ​​residue) at position 169, 170, 171, 172, 173, 174, or 180, 181 relative to SEQ ID NO: 1.

[0170] In certain embodiments, the FGF21 domain in the polypeptide has a combination of N121Q and M168L mutations, or a combination of N121Q, M168L and P171G mutations, and further comprises a conjugable residue (e.g., a cysteine ​​residue) at position 180 relative to SEQ ID NO:1.

[0171] In certain embodiments, the FGF21 domain in the polypeptide has a combination of mutations N121Q, P171G and M168L, or a combination of mutations N121Q, M168L, P171G and A180E, and further comprises an introduced cysteine ​​residue (e.g., by substitution) at the following conjugable residues in relation to SEQ ID NO:1: 169, 170, 172, 173 or 174.

[0172] In certain embodiments, the FGF21 domain in the polypeptide has an amino acid sequence that includes an introduced cysteine ​​residue at position 169 (e.g., SEQ ID NO: 6), 170 (e.g., SEQ ID NO: 7), 171 (e.g., SEQ ID NO: 8), 172 (e.g., SEQ ID NO: 9), 173 (e.g., SEQ ID NO: 10), 174 (e.g., SEQ ID NO: 11 or SEQ ID NO: 92), 180 (e.g., SEQ ID NO: 12), or 181 (e.g., SEQ ID NO: 13). In certain embodiments, the FGF21 comprises the amino acid sequences of SEQ ID NOs: 2-5, 89-91, 14, and 102-105, except for one or more amino acid residue mutations at positions selected from 121, 168, 171, and 180, respectively, relative to SEQ ID NO: 1.

[0173] In certain embodiments, the FGF21 domain in the polypeptide has an introduced G at position 171 (e.g., SEQ ID NO: 14). Without wishing to be bound by any theory, the present inventors have found that the introduction of 171G in the FGF21 domain may also be useful in reducing C-terminal degradation of the FGF21 domain.

[0174] In certain embodiments, the FGF21 domain in the polypeptide has a combination of mutations N121Q, P171G and M168L, or a combination of mutations N121Q, M168L, P171G and A180E, and further comprises a conjugable residue at the positions listed below relative to SEQ ID NO:1: an introduced T at positions 172, 173, 174, where the positions are relative to SEQ ID NO:1, or an introduced N at positions 170 or 174, where the positions are relative to SEQ ID NO:1.

[0175] In certain embodiments, the FGF21 domain in the polypeptide has an introduced T at position 172 (e.g., SEQ ID NO: 16) or 173 (e.g., SEQ ID NO: 17), or an introduced N at position 170 (e.g., SEQ ID NO: 18) or 174 (e.g., SEQ ID NO: 19), relative to SEQ ID NO: 1. In certain embodiments, the FGF21 domain in the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-19.

[0176] Functional moieties on the FGF21 domain 1A and 1B, either or both of polypeptides 001A and 001B may be optionally conjugated to a functional moiety 800 at conjugable residues CR (indicated by "*" in both figures) in second fragment 200 (i.e., the FGF21 domain), thereby forming a polypeptide conjugate. In certain other embodiments, the polypeptide is not conjugated to a functional moiety, for example, if the polypeptide has an introduced G at position 171.

[0177] The term "conjugate" as used herein refers to a compound resulting from two or more molecules joined together to form one physical entity.For example, the polypeptide of the present disclosure can form a polypeptide conjugate, which is essentially a compound resulting from the polypeptide and functional moiety joined together, according to certain embodiments.The molecules (e.g., functional moiety and polypeptide) can be attached together by covalent bonds, non-covalent bonds, linkers, chemical modifications, or protein fusion, or by any means known to those skilled in the art.Preferably, the molecules can be attached together by covalent bonds.Attachment can be permanent or reversible.In some embodiments, certain cleavable or non-cleavable linkers can be included.

[0178] As used herein, the term "functional moiety" refers to a moiety that can functionally alter biological, pharmacokinetic (PK), or pharmacodynamic (PD) properties (e.g., enhance biological activity, increase in vitro stability, increase in vivo half-life, or enhance binding to a target receptor). The functional moiety conjugated to the FGF2 domain can optionally include a glycosyl moiety or a synthetic chemical moiety.

[0179] Optionally, the functional moiety 800 may comprise a synthetic chemical moiety, and the conjugable residue CR may be an introduced residue that can be conjugated with the synthetic chemical moiety.

[0180] According to some embodiments, the conjugable residue CR may be an introduced cysteine ​​residue at any of the above-mentioned positions 169, 170, 171, 172, 173, 174, 180 and 181 relative to SEQ ID NO: 1. As used herein, the synthetic chemistry moiety may optionally comprise the structure *-XYZ, where X, Y and Z are interconnected via a bond, and the * end of X is connected to a conjugable residue on the polypeptide. As used herein, X is [ka] and Y may be [ka] and Z may be [ka] wherein the α position is linked to the α' position and the β position is linked to the β' position. As used herein, R1 can be hydrogen or -COOH, d can be 1, 2, or 3, a can be 1, 2, or 3, b can be 1, 2, or 3, c can be 1 or 2, d can be 1, 2, or 3, and e can be 1, 2, or 3.

[0181] According to some embodiments, the synthetic chemical moiety can be Ac-2XADO-EDA-CO—CH2*, where the * terminus is [ka] is connected to a conjugable cysteine ​​residue on a polypeptide having the structure:

[0182] In each of the above embodiments of FGF21 domains containing an introduced cysteine ​​residue at their respective positions as indicated above, the sequences of which are set forth in SEQ ID NOs: 6-13 and 92, a synthetic chemical moiety such as Ac-2XADO-EDA-CO-CH2-* may be conjugated at the introduced cysteine ​​residue (e.g., by substitution), thereby obtaining a polypeptide conjugate bearing the synthetic chemical moiety.

[0183] In a polypeptide bearing a conjugated functional moiety, the functional moiety 800 may optionally comprise a glycosyl moiety, and accordingly the conjugable residue CR may be an introduced residue that is glycosylatable (i.e., capable of being glycosylated), which may be an introduced T at position 172 or 173, or an introduced N residue at position 170 or 174, relative to SEQ ID NO: 1.

[0184] In each of the above embodiments of the FGF21 domain, whose sequences are set forth in SEQ ID NOs: 16-19, containing an introduced T or N at their respective positions as indicated above, a glycosyl moiety can be conjugated at the introduced cysteine ​​residue, thereby obtaining a polypeptide conjugate bearing the glycosyl moiety.

[0185] In certain embodiments, the FGF21 domain comprises an introduced G at position 171 relative to SEQ ID NO: 1 and is not conjugated to a functional moiety. In certain embodiments, such an FGF21 domain may comprise the amino acid sequence of SEQ ID NO: 14.

[0186] Nanobody Domain Each of the first and second embodiments of the polypeptides disclosed herein and shown in Figures 1A and 1B comprises a Nanobody domain.

[0187] As used herein, the term "nanobody" is considered interchangeable with "single-domain antibody" and refers to an antibody fragment containing a single variable domain of a heavy chain or a single variable domain of a light chain. Nanobodies contain three complementarity-determining regions (CDRs). In certain embodiments, nanobodies can bind to a specific antigen (e.g., serum albumin).

[0188] The term "serum albumin" as used herein refers to albumin (a type of globular protein) found in vertebrate blood. Serum albumin is produced by the liver, exists dissolved in plasma, and is the most abundant blood protein in mammals. Serum albumin typically has a half-life of around 3 weeks, and is primarily regulated by fetal Fc receptor (FcRn). FcRn binds serum albumin with high affinity, diverting it from the lysosomal pathway and returning it to the extracellular compartment, thereby protecting it from intracellular degradation. In some embodiments, the serum albumin is selected from human serum albumin (HSA), cynomolgus monkey serum albumin, and mouse serum albumin. In some embodiments, the serum albumin provided herein is HSA.

[0189] As used herein, the term "antibody" includes any immunoglobulin that binds to a specific antigen. Conventional antibodies (e.g., antibodies of human or mouse origin) contain two heavy (H) chains and two light (L) chains. Heavy chains are classified as α, δ, ε, γ, and μ, with each heavy chain consisting of a variable domain (VH domain) and a first, second, third, and optionally a fourth constant region (CH1, CH2, CH3, and CH4, respectively). Light chains are classified as λ or κ, with each light chain consisting of a variable domain (VL domain) and a constant domain. Antibodies have a "Y" shape, with the stem of the Y consisting of the second and third constant domains of two heavy chains linked together via disulfide bonds. Each arm of the Y contains the variable domain and first constant domain of a single heavy chain linked to the variable and constant domains of a single light chain. The variable domains of the light and heavy chains are involved in antigen binding. The variable domains in both chains generally contain three hypervariable regions called complementarity determining regions (CDRs, ie CDR1, CDR2 and CDR3 in the light or heavy chain).CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the Kabat, IMGT, Chothia, or Al-Lazikani conventions (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927(1997); Chothia, C. et al., J. Mol. Biol. Dec 5, 186(3):651-63(1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196, 901(1987); Chothia, C. et al., Nature. Dec 21-28, 342(6252):877-83(1989); Kabat EA et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991); Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27:55-77 (2003); Marie-Paule Lefranc et al., Immunome Research, 1(3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (second edition), chapter 26, 481-514, (2015)). The three CDRs are interposed between adjacent stretches known as framework regions (FRs, i.e., FR1, FR2, FR3, and FR4 of the light or heavy chain), which are more highly conserved than the CDRs and form a scaffold supporting the highly variable loops. The constant domains of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Conventional antibodies are assigned to classes based on the amino acid sequence of the constant domain of their heavy chains.

[0190] The single variable domain may be derived from the variable domain of a camelid antibody (VHH domain) or the variable domain of a cartilaginous antibody (VNAR domain). Both camelid and cartilaginous antibodies naturally lack light chains and consist of a pair of heavy chains. Alternatively, the single variable domain may be derived from the variable domain of a conventional antibody (e.g., human or mouse-derived) heavy chain (VH domain) or the variable domain of a common antibody light chain (VL domain). Single domain antibodies are contemplated to be fairly small in size, e.g., having a molecular weight of 25 kD or less, 20 kD or less, 15 kD or less, or 10 kD or less.

[0191] It should be noted that the term "nanobody" or "single-domain antibody" is used herein in its broadest sense and is not limited to a specific biological source or to a specific method of preparation. For example, single-domain antibodies can be obtained, for example, by (1) isolating the VHH or VNAR domain of a naturally occurring heavy chain antibody, (2) by expressing a nucleotide sequence encoding a naturally occurring VHH or VNAR domain, (3) by "humanizing" a naturally occurring VHH or VNAR domain (described below) or by expressing a nucleic acid encoding such a humanized VHH or VNAR domain, (4) by "camelizing" a naturally occurring VH domain from any animal species, particularly a mammalian species, such as from humans, or by expressing a nucleic acid encoding such a camelized VH domain, 5) using synthetic or semi-synthetic techniques to prepare proteins, polypeptides, or other amino acid sequences, and / or (6) any combination of the foregoing. Suitable methods and techniques for carrying out the foregoing will be apparent to those skilled in the art.

[0192] In some embodiments, the single domain antibodies described herein comprise VHH domains derived from antibodies made in Camelidae species, such as camel, dromedary, alpaca, and guanaco. Single domain antibodies comprising VHH domains are highly soluble and stable to heat, pH, proteases, and other denaturing agents or conditions.

[0193] In some embodiments, the first polypeptide fragment comprises one or more single domain antibodies capable of specifically binding to serum albumin.

[0194] The terms "binding specificity," "specific binding," or "specifically binds" in relation to the interaction of a binding molecule, such as an antibody, and its binding partner, such as an antigen, mean that the interaction depends on the presence of a specific structure, such as an antigenic determinant or epitope, on the binding partner. In other words, an antibody preferentially binds to or recognizes its binding partner even when the binding partner is present in a mixture of other molecules or organisms. An antibody or fragment thereof that immunospecifically binds to an antigen may be cross-reactive with related antigens bearing the same epitope. "Binding specificity" is generally measured against nonspecific background binding. Typically, an antibody is considered specific if it binds to a target antigen at least 10 times more than background binding.

[0195] The serum-binding single domain antibodies envisaged herein can bind to or associate with serum albumin such that the binding of serum albumin to FcRn is not (significantly) reduced or inhibited (i.e., compared to the binding of said albumin molecule to FcRn when the single domain antibody is not bound to it). In this embodiment of the present invention, "not significantly reduced or inhibited" means that the binding affinity of serum albumin to FcRn (measured using a suitable assay such as SPR) is not reduced by more than 50%, preferably not reduced by more than 30%, even more preferably not reduced by more than 10%, for example not reduced by more than 5%, or essentially not reduced at all. In this embodiment, "not significantly reduced or inhibited" can also mean that the half-life of the serum albumin molecule is not significantly reduced (e.g., not reduced by more than 50%, preferably not reduced by more than 30%, even more preferably not reduced by more than 10%, for example not reduced by more than 5%, or essentially not reduced at all, as measured using a suitable technique known per se). In some embodiments, single domain antibodies may bind to amino acid residues on serum albumin that are not involved in the binding of serum albumin to FcRn.

[0196] In some embodiments, the single domain antibody described herein binds to serum albumin selected from HSA, cynomolgus monkey serum albumin, and mouse serum albumin.In some embodiments, the binding affinity to mouse serum albumin is approximately weaker than that to human or cynomolgus monkey serum albumin.In some embodiments, the single domain antibody specifically binds to HSA.

[0197] In some embodiments, the single domain antibodies described herein bind to serum albumin with sufficient binding affinity. As used herein, the term "affinity" refers to the strength of non-covalent interactions between an immunoglobulin molecule (i.e., an antibody) or fragment thereof and an antigen. Affinity can be expressed numerically using a "Kd" value. Generally, a lower Kd value corresponds to stronger binding. Kd can be determined by any conventional method known in the art, including, but not limited to, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), microscale thermophoresis, HPLC-MS, and flow cytometry (such as FACS). In some embodiments, the antibodies disclosed herein bind to serum albumin with sufficient binding affinity to a specific antigen. d Value ≦ 10 -6 M (e.g., ≦5×10 -7 M, ≤ 2 × 10 -7 M, ≤10 -7 M, ≤ 5 × 10 -8 M, ≤ 2 × 10 -8 M, ≤10 -8 M, ≤ 5 × 10 -9 M, ≤ 4 × 10 -9 M, ≤ 3 × 10 -9 M, ≤ 2 × 10 -9 M, or ≤ 10 -9 M).

[0198] In certain embodiments, the single domain antibodies provided herein comprise 10 -5 M~1×10 -12 Under M, 10 -7 M~1×10 -12 Less than M or 10 -8 M~1×10 -12 In some embodiments, the Kd is less than 1×10 -7 M or less (e.g., 5 × 10 -7 M or less, 2×10 -7 M or less, 10 -7 M or less, 5×10 -8 M or less, 2×10 -8 M or less, 10-8 M or less, 5×10 -9 M or less, 4×10 -9 M or less, 3×10 -9 M or less, 2×10 -9 M or less, or 10 -9 M or less).

[0199] In some embodiments, the single domain antibodies provided herein are humanized antibodies. As used herein, the term "humanized" means that the single domain antibody comprises CDRs derived from a non-human animal and FR regions derived from a human. Humanized antibody polypeptides are desirable for their reduced immunogenicity in humans. Humanized antibody polypeptides are chimeric in their variable regions because non-human CDR sequences are grafted onto human or substantially human FR sequences. Humanization of antibody polypeptides can be essentially performed by substituting non-human (e.g., camelid) CDR genes for the corresponding human CDR genes in human immunoglobulin genes (see, e.g., Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536).

[0200] Various single domain antibodies capable of specifically binding to HSA with high affinity are known in the art, such as fully human domain antibodies isolated using phage display, VHH antibodies developed from the camelidae family, and VNAR antibodies developed from cartilaginous fish, see Zorzi, A et al, Med Chem Commun, 2019, 10, 1068.Exemplary HSA-binding single domain antibodies are described in US8188223B2, US9067991B2, US9321832B2, PCT applications WO2008028977A2, WO2008043822A2, WO2020099871A1, G. Winter, et al., Annu. Rev. Immunol., 1994, 12, 433-455., L. J. Holt, et al., Protein Eng., Des. Sel., 2008, 21(5), 283-288, A. Walker, et al., Protein Eng., Des. Sel., 2010, 23(4), 271-278, L. J. Goodall, et al., PLoS One,2015,10(9),e0137065, RLO'Connor-Semmes,et al.,Clin.Pharmacol.Ther.,2014,96(6),704-712,C.Read,et al.,Basic Clin.Pharmacol.Toxicol.,2019,1-8,R.Adams,et al. al.,mAbs,2016,8(7),1336-1346, E.Dave,et al.,mAbs,2016,8(7),1319-1335, S.Steeland,et al.,Drug Discovery Today,2016,21(7),1076-1113, K.Coppieters,et al.,Arthritis Rheum.,2006,54(6),1856-1866, M. Van Roy,,et al.,Arthritis Res. Ther., 2015, 17, 135; C. McMahon, et al., Nat. Struct. Mol. Biol., 2018, 25(3), 289-296; M.R. Muller, et al., mAbs, 2012, 4(6), 673-685, all of which are contemplated within the scope of the present disclosure and incorporated by reference.

[0201] In some embodiments, the single domain antibody comprises a VHH domain. In some embodiments, the VHH domain is humanized.

[0202] In some embodiments, the VHH domain comprises complementarity determining region 1 (CDR1), CDR2 and CDR3, wherein CDR1 comprises the sequence of SEQ ID NO: 20 (SFGMS) or a variant thereof having up to 3, 2 or 1 amino acid mutations, CDR2 comprises the sequence of SEQ ID NO: 21 (SISGSGSDTLYADSVKG) or a variant thereof having up to 3, 2 or 1 amino acid mutations, and / or CDR3 comprises the sequence of SEQ ID NO: 22 (GGSLSR) or a variant thereof having up to 3, 2 or 1 amino acid mutations, and wherein the VHH domain retains binding specificity to serum albumin, optionally to human serum albumin.

[0203] In some embodiments, the VHH domain comprises a complementarity determining region 1 (CDR1) comprising the sequence of SEQ ID NO:20, a CDR2 comprising the sequence of SEQ ID NO:21, and a CDR3 comprising the sequence of SEQ ID NO:22.

[0204] In some embodiments, the VHH domain comprises a complementarity determining region 1 (CDR1) consisting of the sequence of SEQ ID NO:20, a CDR2 consisting of the sequence of SEQ ID NO:21, and a CDR3 consisting of the sequence of SEQ ID NO:22.

[0205] In some embodiments, the VHH domain comprises the sequence of SEQ ID NO: 23, or a variant thereof having at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 99%) identity to SEQ ID NO: 23, wherein said variant retains binding specificity and / or affinity for serum albumin.

[0206] In some embodiments, a variant of SEQ ID NO:23 has up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations relative to SEQ ID NO:23.

[0207] In certain embodiments of the polypeptide depicted in Figure 1A, the serum albumin binding Nanobody domain further comprises an N-terminal extension attached to the VHH domain. In certain embodiments, the N-terminal extension comprises amino acid residues SG, AG, S, or A, and thus may comprise an amino acid sequence selected from SEQ ID NOs: 24-27.

[0208] In certain embodiments, the N-terminal extension comprises a tag, optionally a cleavable tag. Without wishing to be bound by any theory, it is believed that certain N-terminal extensions may be useful for expression and post-translational processing.

[0209] In certain embodiments, the serum albumin binding Nanobody domain does not comprise an N-terminal extension attached to the VHH domain, for example, the VHH domain may be attached to a cleavable tag that is no longer present in the final product after cleavage.

[0210] In some embodiments, the first fragment may comprise one or more serum albumin binding Nanobody domains.

[0211] GLP-1 domain A second embodiment of the polypeptide disclosed herein and shown in FIG. 1B further comprises a third fragment 300 that comprises, in addition to the above-described FGF21 domain in the second fragment 200 and the serum albumin binding Nanobody domain in the first fragment 100, an additional functional domain.

[0212] According to some embodiments, the additional functional domain comprises a biologically active GLP-1 domain.

[0213] The term "glucagon-like peptide-1" or "GLP-1" as used herein is intended to broadly encompass the native GLP-1 peptide and all its functional forms, such as its functional variants, fragments, fusions, derivatives, and mimetics.

[0214] As used herein, the term "native GLP-1 peptide" refers to native human glucagon-like peptide-1 (GLP-1(7-37)), the sequence of which is set forth in SEQ ID NO: 28. The numbering of residues in GLP-1 begins with the H residue at position 7 and ends with the G residue at position 37, with reference to the sequence of SEQ ID NO: 28.

[0215] A functional form of a native GLP-1 peptide may activate the GLP-1 receptor at a level equivalent to or about 20% or more (or 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) of that of the native GLP-1 peptide. Activation of the GLP-1 receptor typically initiates a signal transduction pathway that leads to insulinotropic activity or other physiological effects, as known in the art. Many functional forms of GLP-1 peptides are known in the art, including, but not limited to, liraglutide, semaglutide, dulaglutide, albiglutide, and those disclosed in WO2000055203A1, WO98 / 08871, WO2006 / 097537, WO2007139589A1, WO1998019698A1, WO2001098331A2, WO2003040309A2, WO2005000892A2, WO2015000942A1, WO2016083499A1, the disclosures of which are incorporated herein in their entireties.

[0216] In certain embodiments, the GLP-1 domain provided herein comprises an amino acid sequence having at least 70% (e.g., at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) sequence identity to SEQ ID NO:28 while substantially retaining the biological activity of SEQ ID NO:28.

[0217] In certain embodiments, the GLP-1 domain comprises no more than 9, 8, 7, 6, 5, 4, 3, or 2 substitutions relative to SEQ ID NO: 28, while retaining substantial biological activity of SEQ ID NO: 28. In certain embodiments, the GLP-1 domain comprises at least 2, 3, 4, 5, 6, 7, 8, or 9 substitutions relative to SEQ ID NO: 28, while retaining substantial biological activity of SEQ ID NO: 28.

[0218] In certain embodiments, the GLP-1 domain further comprises one or more mutations. Examples of amino acid substitutions include substituting an L-amino acid for its corresponding D-amino acid, substituting cysteine ​​for homocysteine ​​or other unnatural amino acids with thiol-containing side chains, substituting lysine for homolysine, diaminobutyric acid, diaminopropionic acid, ornithine, or other unnatural amino acids with amino-containing side chains, or substituting alanine for norvaline or the like.

[0219] Various substitutions have been introduced into native GLP-1 peptides, and have been shown to be able to maintain or even improve its biological activity.In certain embodiments, GLP-1 comprises one or more mutations at positions selected from the group consisting of A8, G22, K34, R36 and H7, and / or any combination thereof, relative to SEQ ID NO: 28.For example, substitution at A8 is useful for preventing DPP4 enzymatic cleavage at this residue, substitution at G22 is desirable for improving activity and solubility, and substitution at R36 is likely to be useful for reducing immunogenicity.In certain embodiments, GLP-1 comprises one or more mutations at positions selected from the group consisting of A8, G22, K26, K34 and R36, and / or any combination thereof, relative to SEQ ID NO: 28. Examples of substitutions at these positions include, but are not limited to, A8G, A8S, A8V, A8Aib, A8T, A8I, A8L, G22E, K26R, K34R, R36G, or any combination thereof, as well as the substitutions described in U.S. Patent No. 8,273,854, which is incorporated herein by its entirety. In certain embodiments, the one or more additional substitutions comprise conservative substitutions.

[0220] In certain embodiments, the GLP-1 domain comprises an A8 substitution selected from the group consisting of A8G, A8S, A8V, A8Aib, A8T, and A8L. In certain embodiments, the GLP-1 domain comprises a G22E substitution. In certain embodiments, the GLP-1 domain comprises an R36G n substitution. In certain embodiments, the GLP-1 domain comprises a K26 substitution that is K26R. In certain embodiments, the GLP-1 domain comprises a K34 substitution that is K34R.

[0221] In certain embodiments, GLP-1 comprises or consists of one or more substitutions selected from the group consisting of A8G, K26R, K34R, G22E, and R36G. In certain embodiments, GLP-1 comprises or consists of one or more substitutions selected from the group consisting of A8G, G22E, and R36G.

[0222] In the present disclosure, at least the following embodiments of the biologically active GLP-1 domain in the third fragment 300 in FIG. 1B are provided: (1) SEQ ID NO: 28, which represents the wild-type functional form of GLP-1; (2) SEQ ID NO: 29, which represents a three-substituted functional form of GLP-1, containing substitutions A8G, G22E, and R36G; (3) SEQ ID NO: 31, which represents a 4-substituted functional form of GLP-1, containing substitutions A8G, G22E, K34R, and R36G; (4) SEQ ID NO: 32, which represents a 5-substituted functional form of GLP-1, containing substitutions A8G, G22E, K26R, K34R, and R36G; (4) SEQ ID NO: 33, which represents a 5-substituted functional form of GLP-1, containing the substitutions A8G, G22E, K26R, K34R, and R36K; and (5) SEQ ID NO: 34, representing a four-substituted functional form of GLP-1, containing substitutions A8G, G22E, K26R, and R36G.

[0223] In addition to GLP-1, additional functional domains of the third fragment 300 shown in FIG. 1B include insulin, C-peptide, leptin, glucagon, gastrin, gastric inhibitory polypeptide (GIP), amylin, calcitonin, cholecystokinin, peptide YY, neuropeptide Y, bone morphogenetic protein-6 (BMP-6), bone morphogenetic protein-9 (BMP-9), oxyntomodulin, oxytocin, glucagon-like peptide-2 (GLP-2), irisin, fibronectin, and erythrocyte serine monophosphate (CREB). It should be noted that the phospholipids may alternatively include biologically active forms (i.e., functional forms) of phospholipid type III domain-containing protein 5 (FNDC5), apelin, adiponectin, Clq and tumor necrosis factor-related proteins (CTRP family), resistin, visfatin, omentin, retinol-binding protein-4 (RBP-4), glicentin, angiopoietin, interleukin-22 (IL-22), exendin-4, or growth hormone.

[0224] First and / or second linkers Each of the first and second embodiments of the polypeptide disclosed herein and shown in Figures 1A and 1B utilizes a first linker 10 to connect a first fragment 100 (comprising a Nanobody domain capable of binding serum albumin) and a second fragment 200 (comprising a biologically active FGF21 domain), and the second embodiment of the polypeptide shown in Figure 1B utilizes a second linker 20 to connect the first fragment 100 and a third fragment 300 (comprising a biologically active additional functional domain, e.g., a GLP-1 domain, that has an additive or synergistic effect on the FGF21 domain).

[0225] The term "linker" as used herein, such as first linker 10 and / or second linker 20, generally refers to a "polypeptide linker," which can be any suitable polypeptide that can bind two entities, thereby forming one molecule, or that is in sufficient proximity to maintain the association of the two entities, but without substantial interference with the biological activity of each of the two entities.

[0226] Linkers may be incorporated into the resulting linked molecule or structure. Herein, a first linker 10 operably separates the Nanobody domain 100 and the FGF21 domain 200 without substantial interference with the biological activity of each of the two functional domains, and a second linker 20 operably separates the GLP-1 domain and the Nanobody domain 100 without substantial interference with the biological activity of each of the two functional domains. The linker may be composed of amino acid residues linked together by peptide bonds, but may optionally further comprise one or more unnatural amino acids.

[0227] Generally, each of the first linker 10 and the second linker 20 has a length of at least four amino acid residues. Thus, in certain embodiments, each linker has a length of at least 4, 8, 10, 20, 24, 28, 30, 40, 48, 50, 60, 70, 80, 90, 100, 110, 120, or more amino acid residues. Without wishing to be bound by any theory, it is believed that the appropriate length of the linker can further improve the biological activity, stability, or pharmacokinetic parameters of each of the two functional domains so linked by it in the overall polypeptide molecule.

[0228] Any suitable polypeptide can be used as a linker. For example, a polypeptide linker can comprise or consist of amino acid residues selected from the amino acids glycine (G), serine (S), alanine (A), methionine (M), asparagine (N), glutamine (Q), cysteine ​​(C), proline (P), glutamic acid (E), threonine (T), and lysine (K). In some embodiments, a polypeptide linker can be composed of a majority of sterically unhindered amino acids, such as glycine and alanine. In some embodiments, the linker is polyglycine, polyalanine, a combination of glycine and alanine (e.g., poly(Gly-Ala)), or a combination of glycine and serine (e.g., poly(Gly-Ser)).

[0229] In certain embodiments, each of the first linker and the second linker comprises or consists of one or more repeats of the repeat sequence. In certain embodiments, the polypeptide linker comprises or consists of 1, 2, 3, 4, 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, or 30 repeats of the repeat sequence, or any number within the range defined by any two of the numbers listed above.

[0230] Based on the experimental data presented in the Examples below, the following configurations are provided herein, which are nevertheless optional and non-limiting in scope.

[0231] Regarding the first linker, it may not contain an acidic amino acid residue (e.g., D or E) according to some embodiments. The first linker may optionally contain one or more units of a first repeat sequence, and the first repeat sequence may consist of 4 or 6 or less types of amino acid residues, which may be selected from the group consisting of G, Q, A, P, T and S. According to some embodiments of the polypeptide, the first repeat sequence may consist of G, Q, A, P, T and S. f S g(each of f and g is independently an integer selected from 1 to 5), SEQ ID NO:35 (GAQP), SEQ ID NO:36 (GQAP), SEQ ID NO:37 (GPAQ), SEQ ID NO:38 (GPQA), SEQ ID NO:39 (GSQP), SEQ ID NO:40 (GASP), SEQ ID NO:41 (GPAS), SEQ ID NO:42 (GPSA), SEQ ID NO:43 (GGGS), SEQ ID NO:44 (GSGS), SEQ ID NO:45 (GGGGS), SEQ ID NO:46 (GSAPGSPAGSPTGSAPGSPA), and SEQ ID NO:110 (GS). In certain embodiments of the polypeptide, the first repeat sequence has the amino acid sequence set forth in SEQ ID NO:35 (GAQP), and the number of the one or more units is an integer between 1 and 10. In certain embodiments, the first linker comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:35 (GAQP), SEQ ID NO:49 ((GAQP)2), SEQ ID NO:50 ((GAQP)5), SEQ ID NO:51 ((GAQP) 10 ), and SEQ ID NO: 48 (GGGGSGGGS).

[0232] With respect to the second linker, it may similarly comprise one or more units of a second repeat sequence, and the second repeat sequence may consist of no more than four or six types of amino acid residues selected from the group consisting of G, Q, A, E, P, T and S. In certain embodiments of the polypeptide, the second repeat sequence may consist of G h S i(each of h and i is independently an integer selected from 1 to 5), comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:35 (GAQP), SEQ ID NO:55 (GQEP), SEQ ID NO:56 (GEQP), SEQ ID NO:57 (GPQE), SEQ ID NO:58 (GPEQ), SEQ ID NO:59 (GSEP), SEQ ID NO:60 (GESP), SEQ ID NO:61 (GPSE), SEQ ID NO:62 (GPES), SEQ ID NO:36 (GQAP), SEQ ID NO:37 (GPAQ), SEQ ID NO:38 (GPQA), SEQ ID NO:39 (GSQP), SEQ ID NO:40 (GASP), SEQ ID NO:41 (GPAS), SEQ ID NO:42 (GPSA), SEQ ID NO:43 (GGGS), SEQ ID NO:44 (GSGS), SEQ ID NO:45 (GGGGS), SEQ ID NO:46 (GSAPGSPAGSPTGSAPGSPA), and SEQ ID NO:110 (GS). In certain embodiments, the second repeat sequence has the amino acid sequence set forth in SEQ ID NO: 35 (GAQP), and the number of one or more units is an integer between 1 and 15, optionally 1, 2, 5, 10, or 14. In certain embodiments, the second linker has the amino acid sequence set forth in SEQ ID NO: 49 ((GAQP)2), SEQ ID NO: 50 ((GAQP)5), SEQ ID NO: 51 ((GAQP) 10 ), and SEQ ID NO: 52 ((GAQP) 14 ), and SEQ ID NO: 47 ((GGGGS)4).

[0233] In certain embodiments, each of the first linker and the second linker can comprise or consist of more than one repeat sequence.For example, one such linker can comprise or consist of two, three, or four different repeat sequences.In certain embodiments, one such linker can comprise or consist of consecutive or tandem repeats of different repeat sequences.The number of repeats of each repeat sequence can be independently selected from the group consisting of 1, 2, 3, 4, 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, or more.

[0234] Other notes With respect to each of the FGF21 domains, Nanobody domains, GLP-1 domains, and first / second linkers described above, the following should be noted: Those skilled in the art will understand that various amino acid substitutions, e.g., conservative amino acid substitutions, can be made in the sequence of any of the polypeptide fragments described herein without necessarily reducing its activity. Examples of amino acid substitutions include substituting an L-amino acid for its corresponding D-amino acid, substituting cysteine ​​for homocysteine ​​or other non-natural amino acids with thiol-containing side chains, substituting lysine for homolysine, diaminobutyric acid, diaminopropionic acid, ornithine, or other non-natural amino acids with amino-containing side chains, or substituting alanine for norvaline or the like.

[0235] The following full length sequences are provided for certain embodiments of the first embodiment (shown in FIG. 1A) and the second embodiment (shown in FIG. 1B).

[0236] Certain embodiments of the polypeptide are substantially fusion polypeptides between a serum albumin-binding Nanobody domain and an FGF21 domain, from N- to C-terminal, and may have an amino acid sequence selected from SEQ ID NOs: 63-68, 93, 99-101, and 107. In some of these embodiments, the polypeptide is conjugated to a synthetic chemical moiety provided herein (see, for example, Figure 1A). The synthetic chemical moiety may be, for example, Ac-2XADO-EDA-CO-CH2-*, which is conjugated to an introduced cysteine ​​residue at position 171 or 174 relative to SEQ ID NO: 1 for a polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 63, 68, 93, and 100. In certain alternative embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 64-67, 69, 99, and 108 and is not conjugated.

[0237] Table 1A below shows the SEQ ID NOs for exemplary fusion polypeptide sequences, as well as the SEQ ID NOs for the Nanobody, FGF21, and first and second polypeptide linkers contained in the fusion polypeptide.

[0238] [Table 1A]

[0239] Certain embodiments of the polypeptide are substantially fusion polypeptides between, from N- to C-terminal, a GLP-1 domain, a serum albumin-binding Nanobody domain, and an FGF21 domain, which may have an amino acid sequence selected from SEQ ID NOs: 70, 74, 75, 79-83, 85, 94-98, and 108-109. In some of these embodiments, the polypeptide is conjugated to a synthetic chemical moiety provided herein (see, e.g., Figure IB). The synthetic chemical moiety may be, for example, Ac-2XADO-EDA-CO-CH2-*, which is conjugated to an introduced cysteine ​​residue at position 171 or 174 relative to SEQ ID NO: 1 for an amino acid sequence selected from the group consisting of SEQ ID NOs: 74, 81-83, 85, 94, 96, and 97. In certain alternative embodiments, the polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 70, 75, 79, 80, 95, 98, and 108-109 and is not conjugated.

[0240] Table 1B below shows the SEQ ID NOs for exemplary fusion polypeptide sequences, as well as the SEQ ID NOs for the GLP-1 domain, first polypeptide linker, Nanobody, FGF21 domain, and first and second polypeptide linkers contained in the fusion polypeptide. Mutations in FGF21 and GLP-1, as well as multiple repeats in the repeat sequence and peptide linker sequence, are also shown.

[0241] [Table 1B-1] [Table 1B-2]

[0242] Polynucleotides encoding polypeptides In yet another aspect, the present disclosure further provides methods for preparing the polypeptides (or optionally, polypeptide conjugates) described above. To this end, the following are provided in the present disclosure:

[0243] First, the disclosure provides isolated nucleic acids or polynucleotides that encode the fusion polypeptides described herein.

[0244] As used herein, the term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and its polymers, either in single-stranded or double-stranded form.Unless specifically limited, the term encompasses polynucleotides that contain known analogs of natural nucleotides, which have similar binding properties as reference nucleic acids and are metabolized in the same manner as naturally occurring nucleotides.Unless otherwise indicated, a specific polynucleotide sequence implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0245] The nucleic acid or polynucleotide encoding the fusion polypeptide described herein can be constructed using recombinant technology.For this purpose, the DNA encoding serum albumin binding Nanobody, the DNA encoding FGF21 domain, and optionally the DNA encoding GLP-1 domain can be obtained, and can be operably linked to allow transcription and expression in host cell, so as to produce polypeptide.The polynucleotide sequence encoding polypeptide linker can also be operably linked to allow expression of desired product.

[0246] The coding polynucleotide sequence may further be operably linked to one or more regulatory sequences, optionally in an expression vector, so that expression or production of the fusion polypeptide is feasible and under proper control.

[0247] The coding polynucleotide sequence can be inserted into a vector for further cloning (amplification of the DNA) or for expression using recombinant techniques known in the art. Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., prokaryotic promoters such as T7, T7lac, Sp6, araBAD, trp, lac, tac, pLm, A3, lac, lpp, npr, pac, syn, trc, and T3, or eukaryotic promoters such as SV40, CMV, and EF-1α), and a transcription termination sequence.

[0248] Vectors and host cells Second and third, the present disclosure further provides vectors comprising the polynucleotides provided above, and host cells comprising the vectors described herein.

[0249] As used herein, the term "vector" refers to a vehicle into which a polynucleotide encoding a protein can be operably inserted to result in the expression of that protein. A vector can be used to transform, transduce, or transfect a host cell to result in the expression of the genetic elements it carries within the host cell. Non-limiting examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. Categories of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Vectors can contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication. The vector may also contain materials that support its entry into cells, including but not limited to viral particles, liposomes, or protein coatings. The vector may be an expression vector or a cloning vector. The present disclosure provides a vector (e.g., an expression vector) that contains the nucleic acid sequence provided herein that encodes a fusion polypeptide, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker.Examples of vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papova viruses (e.g., SV40), lambda phage, and M13 phage, and the plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, p Examples of such vectors include, but are not limited to, EGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, and pEF-Bos.

[0250] As used herein, the phrase "host cell" refers to a cell into which an exogenous polynucleotide and / or vector has been introduced.

[0251] The vector comprising the polynucleotide sequence provided herein can be introduced into host cells for cloning or gene expression.As used herein, the term "host cell" refers to the cell into which exogenous polynucleotide and / or vector is introduced.In other embodiments, the vector is extrachromosomal.If desired, the host cell can be isolated.In certain embodiments, the host cell is a prokaryotic cell, and in some other embodiments, the host cell is a eukaryotic cell.

[0252] Suitable host cells for cloning or expressing the DNA in the vectors herein are primarily prokaryotes. Suitable prokaryotes for this purpose include eubacteria, such as gram-negative or gram-positive organisms, for example, Escherichia, Enterobacteriaceae, such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescans and Shigella, and Bacilli, such as B. subtilis and B. licheniformis, Pseudomonas, such as P. aeruginosa, and Streptomyces. In some embodiments, the host cell is a eukaryote, such as yeast and mammalian cells (e.g., immortalized mammalian cells).

[0253] The vector comprising the polynucleotide sequence provided herein can be introduced into host cells by any suitable method known to those skilled in the art, such as transformation, transfection or transduction.In one example, the polynucleotide sequence encoding fusion polypeptide can be subcloned into an expression vector, which is expressed as inclusion body in host cells.The vector can be a viral vector, and any suitable viral vector can be used in this capacity.

[0254] As used herein, host cells can be prokaryotic or eukaryotic cells. Host cells transformed with the above-described expression or cloning vectors can be cultured in conventional nutrient media, modified as necessary to induce promoters, select transformants, or amplify cloning vectors.

[0255] In another aspect, the present disclosure provides a method of producing a fusion polypeptide described herein, comprising culturing a host cell provided herein under conditions that allow expression of the fusion polypeptide described herein.

[0256] To produce the fusion polypeptides described herein, host cells transformed with expression vectors can be cultured in a variety of media. Commercially available bacterial growth media such as Terrific Broth, LB Broth, LB Agar, M9 Minimal Medium, MagiaMedia Medium, and ImMedia Medium (ThermoFisher) are suitable for culturing bacterial host cells. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium (DMEM) (Sigma) are suitable for culturing eukaryotic host cells. Any of these media may be supplemented, as needed, with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug GENTAMYCIN™), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those of skill in the art. Culture conditions, such as temperature, pH, etc., will be those previously used with the host cell selected for expression and will be apparent to those of skill in the art.

[0257] Methods for Producing Polypeptides In one aspect, the present disclosure provides a method of expressing a fusion polypeptide described herein, comprising culturing a host cell provided herein under conditions in which the fusion polypeptide described herein is expressed.

[0258] In certain embodiments, the fusion polypeptide is expressed as inclusion bodies. In certain embodiments, the method further comprises renaturing the fusion polypeptide from the inclusion bodies.

[0259] When using recombinant techniques, the fusion polypeptides described herein can be produced intracellularly in the periplasmic space, or directly secreted into the medium.If the product is produced intracellularly, as a first step, particulate debris, whether host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration.Carter et al., Bio / Technology 10:163-167 (1992) describes a procedure for isolating proteins secreted into the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes.Cell debris can be removed by centrifugation.If the product is secreted into the medium, the supernatant from such an expression system is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0260] In certain embodiments, the method further comprises isolating the fusion polypeptide.

[0261] The fusion polypeptides described herein prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography.

[0262] Other techniques for protein purification, such as fractionation on ion exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium phosphate precipitation, can also be used depending on the protein of interest to be recovered.

[0263] Also provided in the present disclosure is a method or process for producing the above-described polypeptide, which essentially comprises the following two steps: S100: Culturing the host cell described above under conditions that allow expression of the polynucleotide defined above, or a precursor thereof that further comprises a removable tag; and S200: Recovering and purifying the polypeptide or its precursor from the host cells.

[0264] In certain embodiments, the polypeptide is expressed as inclusion bodies. In certain embodiments, the method further comprises renaturing the polypeptide from the inclusion bodies to allow it to refold. Furthermore, according to some embodiments, the host cell is E. coli, the vector comprises an E. coli-compatible vector, and the polypeptide encoded by the polynucleotide in the vector is codon-optimized for E. coli expression.

[0265] When using recombinant techniques, the polypeptides described herein can be produced intracellularly in the periplasmic space, or can be directly secreted into the medium.If the product is produced intracellularly, as a first step, particulate debris, whether host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration.

[0266] According to some embodiments, the step S200 of recovering and purifying the polypeptide from the host cell may include the following substeps: S210: recovering the precursor of the polypeptide; S220: Refolding the precursor of the polypeptide; S230: treating the refolded precursor of the polypeptide to remove the tag, thereby obtaining the polypeptide; and S240: Purifying the polypeptide.

[0267] In certain embodiments, the host cells are lysed and the polypeptide or precursor of the polypeptide is obtained from the insoluble fraction containing the polypeptide or precursor of the polypeptide.

[0268] According to some embodiments of the polypeptide production process, the process further comprises conjugating the purified polypeptide with a functional moiety. In certain embodiments, the functional moiety to be conjugated to the polypeptide is: [ka] (Ac-2XADO-EDA-CO-CH2-*).

[0269] Pharmaceutical Composition In another aspect, the present disclosure also provides pharmaceutical compositions comprising the polypeptides provided herein. In certain embodiments, the pharmaceutical composition is a liquid composition for parenteral administration.

[0270] As used herein, the polypeptide essentially functions as the active ingredient (API) in the pharmaceutical composition. In addition to the polypeptide, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0271] The term "pharmaceutically acceptable" indicates that the specified excipient salt is generally chemically and / or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.

[0272] "Pharmaceutically acceptable excipient" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is biologically active and acceptable to a subject and non-toxic.

[0273] Examples of pharmaceutically acceptable excipients for liquid formulations may include pharmaceutically acceptable liquids, gels, solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, sequestrants or chelating agents, diluents, adjuvants, excipients, or other non-toxic auxiliary substances, or other components known in the art, or various combinations thereof.

[0274] Suitable pharmaceutically acceptable excipients for liquid formulations include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection; non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents in bacteriostatic or fungistatic concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; The carrier may include suspending and dispersing agents such as carboxymethylcellulose, hydroxypropylmethylcellulose, or polyvinylpyrrolidone; emulsifiers such as polysorbate 80 (TWEEN-80); and sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents utilized as carriers, including phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride, may be added to pharmaceutical compositions in multidose containers. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable nontoxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.

[0275] In embodiments, pharmaceutical compositions are formulated into injectable compositions.Injectable pharmaceutical compositions can be prepared in any conventional form, such as liquid solution, suspension, emulsion, or suitable solid form for making liquid solution, suspension or emulsion.Injectable preparations can include sterile and / or non-pyrogenic solution ready for injection, sterile dry soluble product, such as lyophilized powder, ready to be combined with solvent immediately before use, including hypodermic tablets, sterile suspension ready for injection, sterile dry insoluble product ready to be combined with vehicle immediately before use, and sterile and / or non-pyrogenic emulsion.Solution can be either aqueous or non-aqueous.

[0276] In certain embodiments, unit-dose parenteral preparations are packaged in an ampoule, vial, or syringe with a needle. All preparations for parenteral administration must be sterile and non-pyrogenic, as known and practiced in the art.

[0277] In certain embodiments, sterile, lyophilized powders are prepared by dissolving a polypeptide disclosed herein in a suitable solvent. The solvent may contain excipients that improve the stability or other pharmacological components of the powder or a reconstituted solution prepared from the powder. Excipients that can be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may contain a buffer, such as citrate, sodium or potassium phosphate, or other such buffers known to those of skill in the art, in one embodiment at approximately neutral pH. Subsequent sterile filtration of the solution, followed by lyophilization under standard conditions known to those of skill in the art, provides the desired formulation. In one embodiment, the resulting solution will be apportioned into vials for lyophilization. Each vial may contain a single or multiple doses of a polypeptide (e.g., a conjugated polypeptide) or composition thereof provided herein. Overfilling the vial by a small amount (e.g., about 10%) beyond that needed for a single dose or series of doses is permissible to facilitate accurate sample withdrawal and accurate dosing. The lyophilized powder may be stored under appropriate conditions, such as at about 4°C to room temperature.

[0278] Reconstitution of the lyophilized powder with water for injection provides a formulation for use in parenteral administration. In one embodiment, sterile and / or non-pyrogenic water or other liquid suitable carrier is added to the lyophilized powder for reconstitution. The exact amount depends on the selected therapy being administered and can be empirically determined.

[0279] Administration of the pharmaceutical compositions described herein can be via any route known to be effective by a physician of ordinary skill. One example is peripheral parenteral administration by a sterile syringe or some other mechanical device, such as an infusion pump. In certain embodiments, the peripheral parenteral route is an intravenous, intramuscular, or subcutaneous route of administration.

[0280] In certain embodiments, the polypeptides described herein are formulated into a solid formulation, such as lyophilized or spray-dried, which is then reconstituted in an appropriate diluent solution prior to administration.

[0281] Standard pharmaceutical formulation techniques may be employed, such as those described in Remington: The Science and Practice of Pharmacy (DB Troy, Editor, 21st Edition, Lippincott, Williams & Wilkins, 2006).

[0282] Liquid formulations In another aspect, the pharmaceutical composition provided herein is a liquid formulation.In certain embodiments, the liquid formulation is an aqueous solution.The aqueous solution may contain, for example, at least 70% w / w, at least 75% w / w, at least 80%, at least 85% w / w, at least 90% w / w, or at least 95% w / w water.

[0283] In certain embodiments, pharmaceutical compositions provided herein comprise a polypeptide provided herein and a pharmaceutically acceptable excipient.

[0284] In certain embodiments, the pharmaceutically acceptable excipients include a buffering agent and an isotonicity agent.

[0285] In certain embodiments, the buffer is selected from the group consisting of a phosphate buffer, a citrate buffer, an acetate buffer, a histidine buffer, a glycine buffer, a carbonate buffer, a borate buffer, a glutamate buffer, a glycylglycine buffer, a lysine buffer, and an arginine buffer.

[0286] In certain embodiments, the pharmaceutical composition has a pH of about 6.0 to about 8.3 (e.g., about 6.5 to about 8.2, about 6.5 to about 8.0, about 6.5 to about 7.8, about 6.5 to about 7.4, about 6.5 to about 7.0, about 7.0 to about 7.8, about 7.0 to about 7.4, about 7.4 to about 8.2, about 7.4 to about 8.0, or about 7.4 to about 7.8).

[0287] In certain embodiments, the buffer is a phosphate buffer. Optionally, the pharmaceutical composition has a pH of about 6.5 to about 8.0, about 6.5 to about 7.4, about 7.0 to about 7.8, about 7.0 to about 7.4, or about 7.4 to about 7.8.

[0288] In certain embodiments, the phosphate buffer is present in the pharmaceutical composition at a concentration of 0.01 to 50 mM.

[0289] In certain embodiments, the phosphate buffer is present in the pharmaceutical composition at a concentration of 5-20 mM, optionally about 8 mM.

[0290] In certain embodiments, the phosphate buffer is selected from the group consisting of sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, or hydrates thereof.

[0291] In certain embodiments, the hydrate is a dodecahydrate or a dihydrate.

[0292] In certain embodiments, the phosphate buffer is disodium hydrogen phosphate dodecahydrate or disodium phosphate dihydrate.

[0293] In certain embodiments, disodium hydrogen phosphate dodecahydrate is present at a concentration of about 0.01 to 17 mg / mL (e.g., about 0.01 to 15 mg / mL, about 0.05 to 12 mg / mL, about 0.05 to 10 mg / mL, about 0.1 to 10 mg / mL, about 1 to 5 mg / mL, or about 1 to 3 mg / mL).

[0294] In certain embodiments, disodium hydrogen phosphate dodecahydrate is present in the pharmaceutical composition at a concentration of about 2.87 mg / mL.

[0295] In certain embodiments, disodium hydrogen phosphate dihydrate is present at a concentration of about 0.01 to 8 mg / mL (e.g., about 0.01 to 5 mg / mL, about 0.05 to 4 mg / mL, about 0.05 to 3 mg / mL, about 0.1 to 3 mg / mL, or about 1 to 3 mg / mL).

[0296] In certain embodiments, disodium hydrogen phosphate dihydrate is present at a concentration of about 1.42 mg / mL.

[0297] In certain embodiments, the buffer is a citrate buffer.

[0298] In certain embodiments, the citrate buffer is present at a concentration of about 0.05 to 20 mg / mL, or optionally, 0.05 to 10 mg / mL.

[0299] In certain embodiments, the citrate buffer comprises a mixture of anhydrous citric acid and trisodium citrate (or a hydrate thereof, e.g., trisodium citrate dihydrate). Optionally, the pharmaceutical composition has a pH of about 6.0 to about 6.5, or optionally, about 6.5.

[0300] In certain embodiments, the anhydrous citric acid / trisodium citrate dihydrate is present at a concentration of about 0.05-10 mg / mL. In certain embodiments, the citrate buffer comprises a mixture of about 0.14 mg / mL anhydrous citric acid and about 2.74 mg / mL trisodium citrate dihydrate.

[0301] In certain embodiments, the buffering agent is a histidine buffering agent. Optionally, the pharmaceutical composition has a pH of about 6.0 to about 6.8, or optionally, about 6.5.

[0302] In certain embodiments, the histidine buffer is present at a concentration of about 0.5-10 mg / mL (eg, about 1-5 mg / mL, or about 1-3 mg / mL).

[0303] In certain embodiments, the histidine buffer is present at a concentration of about 1.55 mg / mL.

[0304] In certain embodiments, the isotonicity agent is selected from the group consisting of sodium chloride, glycerol, sorbitol, sucrose, propylene glycol, mannitol, glycine, lactose monohydrate, arginine, myo-inositol, and dimethyl sulfone. In certain embodiments, the isotonicity agent is not sodium chloride.

[0305] In certain embodiments, the isotonicity agent is glycerol.

[0306] In certain embodiments, the glycerol is about 5-30 mg / mL.

[0307] In certain embodiments, the glycerol is about 20 mg / mL.

[0308] In certain embodiments, the isotonic agent is propylene glycol.

[0309] In certain embodiments, the propylene glycol is about 1 mg / mL to about 50 mg / mL (eg, about 5 mg / mL to about 25 mg / mL, about 8 mg / mL to about 16 mg / mL).

[0310] In certain embodiments, the propylene glycol is about 14 mg / mL.

[0311] In certain embodiments, the isotonic agent is sodium chloride.

[0312] In certain embodiments, the sodium chloride is about 5-15 mg / mL.

[0313] In certain embodiments, the sodium chloride is about 8.25 mg / mL.

[0314] In certain embodiments, the isotonic agent is mannitol.

[0315] In certain embodiments, mannitol is about 20 mg / mL to about 100 mg / mL (eg, about 25 mg / mL to about 70 mg / mL, about 30 mg / mL to about 60 mg / mL, about 35 mg / mL to about 55 mg / mL).

[0316] In certain embodiments, the mannitol is about 45 mg / mL.

[0317] In certain embodiments, the isotonicity agent is sorbitol.

[0318] In certain embodiments, the sorbitol is about 20 mg / mL to about 100 mg / mL (eg, about 40 mg / mL to about 50 mg / mL).

[0319] In certain embodiments, the isotonic agent is sucrose.

[0320] In certain embodiments, the sucrose is about 5 mg / mL to about 150 mg / mL (eg, about 45 mg / mL to about 100 mg / mL).

[0321] In certain embodiments, the pharmaceutically acceptable excipient further comprises a non-ionic surfactant.

[0322] In certain embodiments, the non-ionic surfactant is polysorbate 80.

[0323] In certain embodiments, the polysorbate 80 is about 0.05 mg / mL to about 5 mg / mL (eg, about 0.1 mg / mL to about 0.5 mg / mL).

[0324] In certain embodiments, the pharmaceutical excipient further comprises a preservative, a chelating agent, and / or a stabilizer.

[0325] In certain embodiments, the pharmaceutical composition has about 1 to 100 mg / mL (e.g., about 1 to 90 mg / mL, about 1 to 80 mg / mL, about 1 to 70 mg / mL, about 1 to 60 mg / mL, about 1 to 50 mg / mL, about 1 to 40 mg / mL, about 1 to 30 mg / mL, about 1 to 20 mg / mL, about 1 to 10 mg / mL) of a polypeptide provided herein. In certain embodiments of the pharmaceutical composition, the polypeptide comprises the amino acid sequence of SEQ ID NO: 85 and is conjugated to a synthetic chemical moiety provided herein at an introduced cysteine ​​residue at position 171 relative to SEQ ID NO: 1. In these particular specific embodiments of the polypeptide, the synthetic chemical moiety has the following structure: [ka] (also referred to herein as Ac-2XADO-EDA-CO-CH2*). In certain embodiments of the pharmaceutical composition, the polypeptide comprises the amino acid sequence of SEQ ID NO: 96 and is conjugated to a synthetic chemical moiety at an introduced cysteine ​​residue located at position 171 relative to SEQ ID NO: 1. In certain of these specific embodiments of the polypeptide, the synthetic chemical moiety has the following structure: [ka] (also referred to herein as Ac-2XADO-EDA-CO—CH2*).

[0326] In some embodiments, the present disclosure provides: (b) a polypeptide provided herein; and (b) a buffer selected from the group consisting of a phosphate buffer, a citrate buffer, an acetate buffer, a histidine buffer, a glycine buffer, a carbonate buffer, a borate buffer, a glutamate buffer, a glycylglycine buffer, a lysine buffer, and an arginine buffer; (c) an isotonicity agent selected from the group consisting of sodium chloride, glycerol, sorbitol, sucrose, propylene glycol, mannitol, glycine, lactose monohydrate, arginine, myo-inositol, and dimethyl sulfone; and (d) Provide a pharmaceutical composition having a pH of about 6.5 to about 8.2.

[0327] In certain embodiments, the pharmaceutical composition comprises: (b) about 1 to 100 mg / mL of a polypeptide provided herein; (b) a buffer selected from the group consisting of a phosphate buffer, a citrate buffer, and a histidine buffer; (c) an isotonicity agent selected from the group consisting of sodium chloride, glycerol, sorbitol, sucrose, propylene glycol, and mannitol; and (d) The pH is about 6.5 to about 8.2.

[0328] In certain embodiments, the pharmaceutical composition comprises: (a) about 1 to 100 mg / mL of a polypeptide provided herein; (b) about 0.1 to 10 mg / mL of a phosphate buffer, about 0.05 to 10 mg / mL of a citrate buffer, or about 0.5 to 10 mg / mL of a histidine buffer; (c) an isotonicity agent selected from the group consisting of sodium chloride, glycerol, sorbitol, sucrose, propylene glycol, and mannitol; and (d) The pH is about 6.5 to about 8.2.

[0329] In certain embodiments, the pharmaceutical composition comprises: (a) about 1 to 100 mg / mL of a polypeptide provided herein; (b) a buffer selected from the group consisting of a phosphate buffer, a citrate buffer, and a histidine buffer; (c) an isotonic agent selected from the group consisting of 5 to 15 mg / mL sodium chloride, 5 to 30 mg / mL glycerol, 20 to 100 mg / mL sorbitol, 5 to 150 mg / mL sucrose, 1 to 50 mg / mL propylene glycol, and 20 to 100 mg / mL mannitol; and (d) The pH is about 6.5 to about 8.2.

[0330] In certain embodiments, the pharmaceutical composition comprises: (a) about 1 to 100 mg / mL of a polypeptide provided herein; (b) about 0.1 to 10 mg / mL of a phosphate buffer, about 0.05 to 10 mg / mL of a citrate buffer, or about 0.5 to 10 mg / mL of a histidine buffer; (c) an isotonic agent selected from the group consisting of 5 to 15 mg / mL sodium chloride, 5 to 30 mg / mL glycerol, 20 to 100 mg / mL sorbitol, 5 to 150 mg / mL sucrose, 1 to 50 mg / mL propylene glycol, and 20 to 100 mg / mL mannitol; and (d) The pH is about 6.5 to about 8.2.

[0331] In certain embodiments, the pharmaceutical composition comprises: (a) about 1 to 100 mg / mL of a polypeptide provided herein; (b) about 1.55 mg / mL histidine buffer; and (c) an isotonicity agent that is about 45 mg / mL mannitol; and (d) The pH is about 6.5.

[0332] In certain embodiments, the pharmaceutical composition further comprises a non-ionic surfactant having a concentration of polysorbate 80 of about 0.1 to 0.5 mg / mL.

[0333] Treatment methods In another 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 a pharmaceutical composition provided herein.

[0334] Also provided are therapeutic methods comprising administering a therapeutically effective amount of the pharmaceutical compositions provided herein to a subject in need thereof to treat or prevent a condition or disorder. In certain embodiments, the subject has been identified as having a disorder or condition that may respond to the pharmaceutical compositions provided herein.

[0335] In certain embodiments, the metabolic disorder is diabetes, obesity, non-alcoholic steatohepatitis (NASH), cardiovascular-like dyslipidemia, atherosclerosis, alcoholic steatohepatitis (ASH), diabetic nephropathy, gestational diabetes, metabolic syndromes such as metabolic syndrome X, non-alcoholic fatty liver disease (NAFLD), end-stage liver disease, hepatic steatosis (fatty liver), cirrhosis, primary biliary cirrhosis (PBC), or severe hypertriglyceridemia (SHTG).

[0336] In certain embodiments, the condition diabetes includes all forms of diabetes, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin dependent diabetes, MODY (maturity-onset diabetes of the young), gestational diabetes, and / or elevated HbA1C levels.

[0337] In another aspect, the present disclosure provides a method for managing weight in a subject in need thereof, comprising administering a therapeutically effective amount of a pharmaceutical composition provided herein.

[0338] In another aspect, the present disclosure provides a method of reducing food intake in a subject in need thereof, comprising administering a therapeutically effective amount of a pharmaceutical composition provided herein.

[0339] In another aspect, the present disclosure provides a method of reducing weight in a subject in need thereof, comprising administering a therapeutically effective amount of a pharmaceutical composition provided herein.

[0340] In certain embodiments, conditions or metabolic disorders that can be treated or improved using the pharmaceutical compositions provided herein include conditions in which a human subject has a fasting blood glucose level of 125 mg / dL or higher, for example, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, or 200 mg / dL or higher. Blood glucose levels can be determined in a fed or fasted state, or randomly. Metabolic conditions or disorders can also include conditions in which a subject is at increased risk of developing a metabolic condition. For human subjects, such conditions include a fasting blood glucose level of 100 mg / dL.

[0341] In certain embodiments, conditions or metabolic disorders that can be treated or ameliorated using the pharmaceutical compositions provided herein include conditions in which a human subject has a body mass index (BMI) of at least or greater than 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40. In certain embodiments, a human subject has a BMI of 25-30, 26-30, 27-30, 28-30, 25-29, or 25-28.

[0342] The therapeutically effective amount of the pharmaceutical compositions provided herein will depend on various factors known in the art, such as the subject's weight, age, medical history, current medications, health status, and the potential for cross-reactivity, allergies, sensitivities, and adverse side effects, as well as the route of administration and the extent of disease progression. Dosages may be proportionally reduced or increased by a skilled artisan (e.g., a physician or veterinarian) as indicated by these and other circumstances or requirements. A therapeutically effective amount can be the amount of the fusion polypeptides and pharmaceutical compositions herein that elicits the biological or pharmaceutical response in a tissue system, animal, or human being sought by a researcher, physician, or other clinician, including alleviation or amelioration of the symptoms of the disease or disorder being treated, i.e., an amount that supports an observable level of one or more desired biological or pharmaceutical responses, such as reduced blood glucose, insulin, triglyceride, or cholesterol levels, reduced body weight, or improved glucose tolerance, energy expenditure, or insulin sensitivity.

[0343] In certain embodiments, a polypeptide or pharmaceutical composition provided herein may be administered at a therapeutically effective dosage of about 0.01 mg / kg to about 100 mg / kg (e.g., about 0.01 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, or about 100 mg / kg). In some of these embodiments, the polypeptide or pharmaceutical composition provided herein is administered at a dosage of about 50 mg / kg or less, and in some of these embodiments, the dosage is 10 mg / kg or less, 5 mg / kg or less, 1 mg / kg or less, 0.5 mg / kg or less, or 0.1 mg / kg or less. In certain embodiments, the administered dosage may vary over the course of treatment. For example, in certain embodiments, the initial administered dosage may be higher than subsequent administered dosages. In certain embodiments, the administered dosage may vary over the course of treatment depending on the subject's response.

[0344] In certain embodiments, provided herein is a polypeptide can be administered to a subject (for example, a human) with a dosing regimen of once a day, once every 3 days, once a week, or once every 2 weeks or less.In certain embodiments, provided herein is a polypeptide can be administered to a subject (for example, a human) with a dosing interval of twice a week, once a week, or once every 2 weeks.The therapeutic efficacy at low dosing frequency has the potential to improve patient compliance and long-term treatment success rate.Currently available treatment, semaglutide, is administered once a week.

[0345] Without being bound by any theory, it is believed that certain polypeptides provided herein have significantly extended half-life, and are more suitable than semaglutide for less frequent administration in the treatment of metabolic conditions, for example, less frequent administration than once a week (for example, once every 8 days, once every 9 days, once every 10 days, once every 11 days, once every 12 days, once every 13 days or once every 14 days).In certain embodiments, the administration regimen is a continuous administration regimen selected from twice a week administration, once a week administration or once every 2 weeks administration.In certain embodiments, the administration regimen has an administration interval of about once every 3 days to about once every 2 weeks.

[0346] Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single dose can be administered, or several divided doses can be administered over time.

[0347] A polypeptide or pharmaceutical composition provided herein can be administered by any route known in the art, such as, for example, parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.

[0348] In certain embodiments, a polypeptide or pharmaceutical composition provided herein can be administered to a subject by parenteral administration.

[0349] The polypeptides can be administered alone or in combination with one or more additional therapeutic procedures or agents.

[0350] In certain embodiments, when used to treat metabolic disease, the polypeptides provided herein can be administered in combination with any other therapeutic agent for use in the treatment of metabolic disease or any related medical disorder.As used herein, "administered in combination" includes simultaneous administration as part of the same pharmaceutical composition, simultaneous administration as separate compositions, or administration at different times as separate compositions.A composition that is administered before or after another agent is considered to be administered "in combination" with that agent, as this phrase is used herein, even if the composition and the second agent are administered via different routes.If possible, the additional therapeutic agent administered in combination with the polypeptides provided herein is administered according to the schedule listed on the product information sheet of the additional therapeutic agent, or according to the Physicians' Desk Reference (Physicians' Desk Reference, 70th Edition (2016)) or a protocol well known in the art.

[0351] kit Also provided is a kit for carrying out the method for administering the pharmaceutical composition described above.Such a kit can include the pharmaceutical composition described herein, which can be provided in a sterile container.Optionally, instructions on how to use the provided pharmaceutical composition in the treatment of metabolic disorders can also be included, or can be made available to patients or healthcare providers.

[0352] Such a kit may include (a) a pharmaceutical composition comprising a therapeutically effective amount of the polypeptide provided herein, and (b) one or more containers for the pharmaceutical composition. Such a kit may also include instructions for use, and the instructions may be tailored to the exact metabolic disorder being treated. The instructions may describe the use and properties of the materials provided in the kit. In certain embodiments, the kit includes instructions for patients to administer to treat metabolic disorders such as elevated glucose levels, elevated insulin levels, diabetes, obesity, non-alcoholic steatohepatitis (NASH), cardiovascular-like dyslipidemia, atherosclerosis, alcoholic steatohepatitis (ASH), diabetic nephropathy, metabolic syndromes such as metabolic syndrome X, non-alcoholic fatty liver disease (NAFLD), end-stage liver disease, hepatic steatosis (fatty liver), cirrhosis, primary biliary cirrhosis (PBC) or severe hypertriglyceridemia (SHTG).

[0353] The instructions may be printed on a substrate such as paper or plastic and may be present in the kit as a package insert, on a label on the kit's container or a component thereof (e.g., associated with packaging), or the like. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer-readable storage medium, e.g., a CD-ROM, diskette, etc. In still other embodiments, the actual instructions are not present in the kit, but means are provided for obtaining the instructions from a remote source, such as via the Internet. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which they can be downloaded. Often, it will be desirable for some or all components of the kit to be packaged in appropriate packaging that maintains sterility. The components of the kit may be packaged in a kit storage element that creates a single, easily handled unit; the kit storage element, e.g., a box or similar structure, may or may not be a hermetically sealed container, e.g., to further maintain sterility for some or all of the kit's components. [Example]

[0354] Example 1: Recombinant expression of Nanobody-FGF21 proteins and GLP-1-Nanobody-FGF21 The GLP-1-Nanobody-FGF21 proteins or Nanobody-FGF21 fusion proteins listed in Tables 1A, 1B, and 1C were produced in a bacterial E. coli expression system using the BL21(DE3) derivative strain. DNA encoding the GLP-1-Nanobody-FGF21 fusion precursor or Nanobody-FGF21 fusion protein was codon-optimized for E. coli expression, de novo synthesized, and subcloned into a PET derivative expression vector (Novagen). Amino acid substitutions were performed by modifying the corresponding genetic code. When the cell density reached an OD600 of 2.0 in Terrific Broth (TB) medium, overexpression of the GLP-1-Nanobody-FGF21 fusion precursor or Nanobody-FGF21 fusion protein was induced with 0.5 mM isopropyl bd-thiogalactoside (IPTG). After 20–22 h of protein induction at 37°C, cells were harvested.

[0355] [Table 1C]

[0356] Example 2: Purification of Nanobody-FGF21 protein The cells were harvested and lysed in 20 mM Tris pH 8.0, 0.15 M NaCl buffer using a cell disrupter (900 bar, 2 times). The insoluble fraction containing the nanobody-FGF21 fusion protein was collected by centrifugation (8,000 × g, 30 min). After refolding, the fusion protein was purified by anion exchange chromatography. Samples at each step were characterized by LC / MS to confirm the correct molecular weight.

[0357] Example 3: Purification of GLP-1-Nanobody-FGF21 Cells were harvested and lysed in 20 mM Tris pH 8.0, 0.15 M NaCl buffer using a cell disrupter (900 bar, 2 passes). The insoluble fraction containing the GLP-1-Nanobody-FGF21 fusion precursor was collected by centrifugation (8,000 × g, 30 min). After refolding, the fusion protein precursor was captured by anion exchange chromatography. After tag removal with protease, the protein was purified by hydrophobic interaction chromatography. Samples at each step were characterized by LC / MS to confirm the correct molecular weight.

[0358] Example 4 Preparation of Nanobody-FGF21 and GLP-1-Nanobody-FGF21 fusion protein conjugates To a solution of Nanobody-FGF21 or GLP-1-Nanobody-FGF21 fusion protein in Tris buffer, Ac-2XADO-EDA-CO-CH2-Br in organic solvent was added dropwise. The reaction was stirred at room temperature for 1 hour. The product was then subjected to anion exchange chromatography. This provided the compounds listed in Tables 1A, 1B, and 1C shown above.

[0359] The conjugated fusion proteins were detected and characterized by LC-MS methods using a Waters BioAccord LC-MS system or by UPLC using a Waters Acquity UPLC system, using conditions optimized for the different conjugates according to the supplier's manual.

[0360] [Example 5] In vitro activity Methods: In vitro GLP-1 activity of the fusion proteins was measured using a BHK cell line overexpressing the human GLP-1 receptor and a CRE luciferase reporter in the presence or absence of 1% human serum albumin (HSA). Test fusion proteins were measured at a top concentration of 100 nM in the presence of 1% HSA using a 3-fold serial dilution. After treating the cells with the molecules for 4 hours, luciferase activity was measured using a Steadylite plus kit (Perkin Elmer, 6066751).

[0361] The activity of each fusion protein was expressed as an EC50 derived from nonlinear regression analysis.

[0362] The in vitro FGF21 activity of the fusion protein was assessed using a HEK293 cell line overexpressing human beta-Klotho. The test fusion protein conjugate was measured at a top concentration of 400 nM using a 4-fold serial dilution in the presence of 1% HSA. After treating the cells with the fusion protein conjugate for 12 minutes, p-ERK levels were measured using a p-ERK kit (Cisbio, 64ERKPEH).

[0363] The activity of each fusion protein was calculated using the EC 50 It is expressed by

[0364] Conclusion: As shown in Tables 2 and 3, all fusion proteins exhibited efficacy comparable to that of native FGF21. However, the fusion molecules (MLC#9, MLC#10, MLC#12, MLC#13, MLC#14, MLC#15, MLC#16, MLC#17, MLC#19, and MLC#21) exhibited different GLP-1 activity. Molecules MLC#14, MLC#16, MLC#17, MLC#19, and MLC#21 exhibited significantly higher GLP-1 activity than MLC#9, MLC#12, MLC#13, and MLC#15. Compared with the other fusion molecules tested, MLC#15 has a relatively shorter second linker and lower GLP-1 activity. Taken together, this may indicate that fusion molecules with longer second linkers may have higher GLP-1 activity. As shown in Figures 2A and 2B, the fusion molecules (MLC#9, MLC#10, control#6, and MLC#14) exhibited significantly higher FGF21 potency than the YH-dual molecule (i.e., the molecule disclosed as SEQ ID NO: 66 in WO2017 / 074123).

[0365] [Table 2]

[0366] [Table 3]

[0367] [Example 6] Efficacy study in disease models Selected molecules will be assessed in disease animal models (such as db / db mice and diet-induced obese (DIO) mice) to determine body weight, food intake, and glucose efficacy in relation to dose response in chronic studies. Several biomarkers will also be measured, including plasma insulin, plasma triglycerides, plasma cholesterol, plasma LDL-c, plasma adiponectin, liver triglycerides, liver cholesterol, and liver function indices (ALT, AST).

[0368] A) Food intake and weight loss Methods: 22-week-old DIO male C57BL / 6 mice (approximately 50 g) were injected subcutaneously every other day (Q2D) with the indicated polypeptide conjugates (i.e., molecule #9 and molecule #10) for 25 days. Food intake and body weight were measured twice a week, and fasting blood glucose was measured once a week. Five animals were used for each treatment group. Body weight and fasting blood glucose were monitored for individual animals, but food intake for each group of animals was measured together. Days 1 and 25 represent the first and last days of molecule dosing. Data are presented as mean and standard error (SEM) or pooled values. Statistical analysis was performed by one-way ANOVA. Body weight loss on day 25 is calculated by -1*(% BW loss-% BW loss of vehicle group) and cumulative food intake reduction is calculated by -100*(cumulative food intake-cumulative food intake of vehicle) / cumulative food intake of vehicle.

[0369] Conclusion: In the DIO study, molecular control #2, control #6, MLC #9, and MLC #10 have significant effects on weight loss and food intake suppression, as shown in Figures 3A, 3B, and Table 4.

[0370] [Table 4]

[0371] B) Metabolic parameters in DIO animal models Methods: Sixteen-week-old DIO male C57BL / 6 mice (35-40 g) were injected subcutaneously once daily (QD) with the indicated GLP-1 / FGF21 conjugates (i.e., MLC#9, MLC#14, MLC#16, and MLC#17) for 21 days. Food intake and body weight were measured daily, and non-fasting blood glucose was measured every three days. Five animals were used for each treatment group. Body weight and blood glucose were monitored for each individual animal, but food intake for each group of animals was measured together. Days 1 and 21 represent the first and last days of treatment, respectively. Terminal blood was collected, and EDTA-K3 plasma was prepared and frozen at -80°C for biomarker measurements (LDL-C, TC, TG, ALT, insulin, and adiponectin). Liver and adipose tissue were also collected, frozen in liquid nitrogen, and stored at -80°C. Data are shown as mean and standard error (SEM) or pooled values. Statistical analysis was performed by one-way ANOVA. Body weight loss on day 21 is calculated by -1*(BW loss%-BW loss% of vehicle group), and cumulative food intake reduction is calculated by -100*(cumulative food intake-cumulative food intake of vehicle) / cumulative food intake of vehicle.

[0372] Conclusion: In a DIO mouse study, the molecules MLC#9, MLC#14, MLC#16, and MLC#17 demonstrated significantly better efficacy in reducing body weight compared to semaglutide, tirzepatide, and YH-dual (SEQ ID NO: 66 in WO2017 / 074123), as shown in Figure 4A. MLC#9, MLC#14, MLC#16, and MLC#17 induced approximately 30-35% weight loss in the study. In contrast, semaglutide, tirzepatide, and YH-dual induced approximately 20-25% weight loss.

[0373] In Figure 4B, the MLC#9, MLC#14, MLC#16, and MLC#17 groups demonstrated better glucose control than semaglutide, tirzepatide, and YH-dual. MLC#9, MLC#14, MLC#16, and MLC#17 reduced non-fasting glucose levels to below 7 mmol / L. In contrast, semaglutide reduced non-fasting glucose levels to approximately 7.5–10 mmol / L.

[0374] MLC#9, MLC#14, MLC#16, and MLC#17 induced improvements in hepatic TG (Figure 4I), hepatic TC (Figure 4J), and insulin sensitivity (Figure 4K), as well as decreases in plasma triglyceride (Figure 4C), LDL-C (Figure 4D), total cholesterol (Figure 4E), and ALT (Figure 4F) concentrations, as well as decreases in fat mass (Figure 4G) and liver weight (Figure 4H).

[0375] In vivo, FGF21 can induce the secretion of adiponectin, which is reported to be an insulin sensitizer. The fusion molecules MLC#9, MLC#14, MLC#16, and MLC#17 showed better effects on increasing adiponectin levels than YH-dual, which showed better FGF21 activity (Figure 4L).

[0376] C) Metabolic parameters in the ob / ob animal model at different dosages Methods: Ten-week-old ob / ob male mice (42-55 g) were injected subcutaneously once daily (QD) with the indicated GLP-1 / FGF21 conjugates (i.e., MLC#14 and MLC#16) for 14 days. Food intake and body weight were measured daily. Non-fasting blood glucose was measured every 3 days. Plasma triglyceride (TG) levels were measured weekly. Five or six animals were used in each treatment group. Body weight and blood glucose were monitored individually, but food intake for each group was measured together. Days 1 and 14 were the first and last days of molecule dosing. On day 14 after treatment, mice were sacrificed by cardiac puncture under anesthesia. Terminal blood was collected and frozen at -80°C for biomarker measurements (LDL-C, TC, TG, ALT / AST, insulin, and adiponectin). Liver and adipose tissue were also collected, frozen in liquid nitrogen, and stored at -80°C. Data are shown as mean and standard error (SEM) or pooled values. Statistical analysis was performed by one-way ANOVA. Body weight loss on day 14 was calculated by -1*(BW loss%-BW loss% of vehicle group), and cumulative food intake loss was calculated by -100*(cumulative food intake-cumulative food intake of vehicle) / cumulative food intake of vehicle.

[0377] Conclusion: In ob / ob mice, the fusion molecules MLC#14 and MLC#16 demonstrated dose-dependent efficacy in weight loss (FIG. 5A), glucose reduction, and biomarker changes, as shown in Figures 5A-5M. In Figure 5A, the molecules MLC#14 and MLC#16 demonstrated significantly better efficacy in weight loss than the same doses of semaglutide, tirzepatide, and YH-dual (SEQ ID NO: 66 in WO2017 / 074123). In Figure 5B, the MLC#14 and MLC#16 groups achieved similar effects on glucose control as the same doses of semaglutide. As shown in Figure 5C, MLC#14 and MLC#16 induced better reductions in plasma triglycerides than the same doses of semaglutide, tirzepatide, and YH-dual. MLC#14 and MLC#16 improved liver TG (Figure 5J), liver TC (Figure 5K), and insulin sensitivity (Figure 5L), as well as reduced plasma LDL-C (Figure 5D), total cholesterol (Figure 5E), ALT (Figure 5F), and AST (Figure 5G) levels, as well as reduced fat mass (Figure 5H) and liver weight (Figure 5I). The fusion molecules MLC#14 and MLC#16 showed significantly better effects on increasing adiponectin levels than the same dosage of YH-dual, which showed better FGF21 activity (Figure 5M).

[0378] D) Metabolic parameters in DIO animal models Methods: Sixteen-week-old DIO male C57BL / 6 mice (35-50 g) were injected subcutaneously once daily (QD) with the indicated GLP-1 / FGF21 conjugates and FGF21 conjugates (i.e., MLC#16, MLC#19, MLC#6, and MLC#23) for 22 days. Food intake and body weight were measured every three days, and fasting blood glucose was measured weekly. Five animals were used for each treatment group. Body weight and blood glucose were monitored for individual animals, but food intake for each group of animals was measured together. Days 1 and 22 represent the first and last days of treatment, respectively. Terminal blood was collected, and EDTA-K3 plasma was prepared and frozen at -80°C for biomarker measurements (LDL-C, TC, TG). Liver and adipose tissue were also collected, frozen in liquid nitrogen, and stored at -80°C. Data are presented as mean and standard error (SEM) or pooled values. Statistical analysis was performed by one-way ANOVA.

[0379] Conclusion: In a DIO mouse study, GLP-1-nanobody-FGF21 fusion protein conjugates (MLC#16 and MLC#19) and nanobody-FGF21 fusion conjugates (MLC#6 and MLC#23) demonstrated favorable efficacy for weight loss, as shown in Figure 6A. In Figure 6B, the MLC#16, MLC#19, MLC#6, and MLC#23 groups demonstrated better glucose control than semaglutide, and induced reductions in serum triglyceride (Figure 6C), LDL-C (Figure 6D), and total cholesterol (Figure 6E) concentrations in plasma, as well as reductions in fat weight (Figure 6F) and liver weight (Figure 6G), along with improved liver TG (Figure 6H).

[0380] [Example 7] Pharmacokinetic study in rats Methods: Male SD rats aged 6–8 weeks were administered a single subcutaneous dose of 15 nmol / kg of protein MLC#9 (conjugated) or MLC#10 (unconjugated) (n=3 / group). Plasma samples were collected pre-dose (-5 min) and 0.5, 1, 2, 4, 6, 8, 12, 24, 32, 48, 72, and 96 h after subcutaneous administration. Plasma concentrations of the polypeptide conjugates were measured by ELISA. Pharmacokinetic parameters were calculated using WinNonlin based on the plasma concentration versus time graphs of each polypeptide conjugate after subcutaneous injection.

[0381] Conclusion: MLC#9 has a T half-life of 14.3 hours in rats. 1 / 2 It has a half-life T of 9.5 hours 1 / 2 It shows a longer half-life than MLC#10, which has

[0382] [Example 8] PK study in minipigs The pharmacokinetics of selected molecules will be assessed in minipigs. Both subcutaneous and intravenous injections will be performed.

[0383] [Example 9] PK study in non-human primates The pharmacokinetics of selected molecules will be assessed in monkeys. Both subcutaneous and intravenous injections will be performed.

[0384] Methods: Male cynomolgus monkeys aged 4-5 years were administered a single subcutaneous dose of 5 mg / kg (123 nmol / kg) MLC#16 (n=2 / group). Plasma samples were collected pre-dose (-5 min) and at 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, 72, 96, 120, 144, and 168 hours after subcutaneous administration. MLC#16 in plasma was measured by LC-MS / MS. Pharmacokinetic parameters were calculated using WinNonlin based on the graph showing the plasma concentration of MLC#16 versus time after subcutaneous injection.

[0385] Conclusion: MLC#16 exhibited a half-life of 54.3 hours in monkeys, which potentially supports a once-weekly dosing frequency in humans (Table 5).

[0386] [Table 5]

[0387] Example 10: Immunogenicity assessment Selected GLP-1 polypeptide conjugates will also be assessed for immunogenicity by in silico (iTope and TCED methods) and ex vivo (EpiScreen) methods.

[0388] Example 11: Stability assessment To test the stability, various GLP-1 polypeptide conjugates were formulated in buffers with different compositions (pH 6, 7, 7.4, and 8.0) and stored at different temperatures (e.g., 4°C and 25°C) for 2 to 4 weeks. %HMWP and %LMW were analyzed by size-exclusion chromatography (SEC)-HPLC. Concentration and modification were analyzed by reverse-phase (RP)-UPLC and LC / MS.

[0389] [Example 12] Human serum albumin binding Methods: Binding of molecules to serum albumin was characterized by surface plasmon resonance on a Biacore 8K instrument. Human serum albumin was covalently coupled to the surface of a CM5 sensor chip until 4000 RU was reached. The chip was blocked with 1 M ethanolamine at a flow rate of 10 μL / min for 420 seconds. Each molecule sample was diluted and injected at a flow rate of 30 μL / min to allow binding to the albumin bound to the chip for 120 seconds and dissociation for 300 seconds. Binding buffer without molecules was pumped over the chip at a flow rate of 20 seconds to allow spontaneous dissociation of bound molecules for 30 seconds.

[0390] Conclusion: All fusion molecules (MLC#9, MLC#10, Control#4, Control#5, Control#6, MLC#12, MLC#16, and MLC#19) show similar binding affinity to human serum albumin (see Table 6).

[0391] [Table 6]

[0392] [Example 13] FAP enzymatic cleavage on fusion protein Methods: Fibroblast activation protein (FAP) is a serine protease. This enzyme has been reported to regulate the degradation of FGF21. To test the C-terminal degradation of fusion proteins by FAP enzyme, fusion proteins (MLC#23, MLC#25) and FAP enzyme were incubated at a ratio of 200:1 at 37°C for 20 hours. LC-MS was performed to analyze the percentage degradation of the fusion proteins.

[0393] Conclusion: The unconjugated fusion proteins (MLC#25, MLC#18, and MLC#21) showed 50.6–65.2% C-terminal degradation upon incubation with FAP enzyme for 20 hours at 37° C. However, the fusion protein conjugates (MLC#23, MLC#16, and MLC#19) and the fusion protein with the 171G substitution (MLC#10) showed resistance to FAP enzyme.

[0394] [Table 7]

[0395] [Example 14] Preparation of liquid composition Unless otherwise specified, MLC#16 or MLC#19 was prepared by diluting MLC#16 or MLC#19 into a formulation buffer consisting of a buffering agent (e.g., disodium hydrogen phosphate dodecahydrate or disodium hydrogen phosphate dihydrate for pH 7.4-8.2, and histidine or anhydrous citric acid / trisodium citrate dihydrate for pH 6.5) and an isotonicity agent (e.g., sodium chloride, glycerol, sorbitol, sucrose, propylene glycol, or mannitol). Nonionic surfactants (e.g., polysorbate 80) were added to some buffers. The pH was adjusted to the relevant value with sodium hydroxide and / or hydrochloric acid. The composition was filtered through a 0.22 μm sterile filter.

[0396] Compositions containing MLC#16 or MLC#19 were tested in this experiment. The compositions tested contained glycerol (20 mg / ml), propylene glycol (14 mg / ml), sodium chloride (8.25 mg / ml), mannitol (40-50 mg / ml), sorbitol (40-50 mg / ml), or sucrose (45-100 mg / ml) as an isotonicity agent and had various pH values ​​as shown in Table 8. For compositions 1-7, 20-25, and 36-48, 2.87 mg / ml of disodium hydrogen phosphate dodecahydrate was used as a pH buffer. For compositions 8-17 and 26-35, 1.55 mg / ml of histidine was used as a pH buffer. For compositions 18 and 19, 0.14 mg / ml of anhydrous citric acid and 2.74 mg / ml of trisodium citrate dihydrate were used as pH buffers. For compositions Nos. 12, 19, 30 and 45, an additional 0.20 mg / ml of polysorbate 80 was added.

[0397] [Table 8-1] [Table 8-2]

[0398] Example 15: First round of stability testing of the composition Forty-eight compositions listed in Example 14 were prepared and stored at 25°C for up to 6 months. Impurities and high molecular weight products were analyzed by LC-MS and SEC-HPLC at 0, 3, and 6 months. LC-MS was performed using an Acquity BEH C4 column (Waters) and a UPLC I-class (Waters). SEC-HPLC was performed using an Xbridge BEH 200A SEC 3.5 μm 7.8 × 150 mm (Waters) and an Arc-HPLC (Waters). Some compositions (i.e., numbers 1–5, 7, 18–19, 21, 24–30, 37, 40–41, 43–45, and 47–48) are listed in Table 9. All compositions in Table 9 were tested, and the results showed that they were stable during the test period, except for compositions 25 and 48. Compositions with a pH above 8.0 (i.e., Nos. 7, 24, 25, and 48) had relatively higher HMWP and total impurities than compositions with a pH between 6.5 and 8.0, indicating that pH between 6.5 and 8.0 is more beneficial for molecular stability. Compositions containing sodium chloride as an isotonicity agent had relatively higher HMWP and total impurities than compositions containing other isotonicity agents (e.g., Nos. 24 vs. 25), indicating that isotonicity agents such as glycerol, propylene glycol, mannitol, sorbitol, and sucrose are more beneficial for molecular stability.

[0399] [Table 9]

Claims

1. A liquid pharmaceutical composition comprising a polypeptide and a pharmaceutically acceptable excipient, The polypeptide comprises a first fragment comprising a Nanobody domain capable of binding to serum albumin and a second fragment comprising a biologically active FGF21 domain, wherein the first fragment is connected to the N-terminus of the second fragment via a first linker.

2. the FGF21 domain comprises one or more amino acid residue mutations, each at a position selected from the group consisting of positions 121, 168, 171, and 180 relative to SEQ ID NO: 1; Optionally, 2. The pharmaceutical composition of claim 1, wherein the one or more amino acid residue mutations in the FGF21 domain are selected from the group consisting of N121Q, M168L, P171G and A180E, or any combination thereof.

3. The pharmaceutical composition of claim 1 or 2, wherein the FGF21 domain further comprises a conjugable residue.

4. wherein the conjugable residue is at a position within the C-terminal fragment spanning positions 169 to 181 relative to SEQ ID NO: 1, and optionally 4. The pharmaceutical composition of claim 3, wherein the conjugable residue is at a position selected from the group consisting of positions 169, 170, 171, 172, 173, 174, 180 and 181 relative to SEQ ID NO:

1.

5. The pharmaceutical composition of claim 3 or 4, wherein the FGF21 domain comprises the amino acid sequence of SEQ ID NOs: 6-13, 16-19, and 92.

6. 6. The pharmaceutical composition of claim 3, wherein the polypeptide is conjugated to a functional moiety at the conjugable residue in the second fragment.

7. The pharmaceutical composition of claim 6 , wherein the functional moiety comprises a glycosyl moiety or a synthetic chemical moiety.

8. 8. The pharmaceutical composition of claim 6, wherein the functional moiety comprises a glycosyl moiety and the conjugable residue is an introduced residue that is glycosylatable.

9. 9. The pharmaceutical composition of claim 8, wherein the conjugable residue comprises an introduced T at position 172 or 173, or an introduced N residue at position 170 or 174 relative to SEQ ID NO:

1.

10. The pharmaceutical composition of claim 6 , wherein the functional moiety comprises a synthetic chemical moiety.

11. The pharmaceutical composition of claim 10, wherein the conjugable residue comprises an introduced cysteine ​​residue.

12. the synthetic chemistry moiety comprises the structure *-X-Y-Z, where X, Y, and Z are interconnected via a bond and the * end of X is connected to the conjugable residue on the polypeptide, wherein: X is 【Chemistry 1】 and Y may be 【Chemistry 2】 and Z may be 【Transformation 3】 It can be, wherein the α position is linked to the α' position, the β position is linked to the β' position, R1 is hydrogen or -COOH, d is 1, 2, or 3, a is 1, 2, or 3, b is 1, 2, or 3, c is 1 or 2, d is 1, 2, or 3, and e is 1, 2, or 3.

13. The synthetic chemistry moiety has the following structure: 【Chemistry 4】 13. The pharmaceutical composition of claim 12, having the formula:

14. 14. The pharmaceutical composition of any one of claims 11 to 13, wherein the introduced cysteine ​​is at a position selected from the group consisting of 169, 170, 171, 172, 173, 174, 180 and 181 relative to SEQ ID NO:

1.

15. The introduced cysteine ​​is at position 171, and the synthetic chemistry moiety has the following structure: 【Transformation 5】 15. The pharmaceutical composition of claim 14, having the formula:

16. 16. The pharmaceutical composition of claim 15, wherein the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 8, and optionally the introduced cysteine ​​is at position 171.

17. i) the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 6, and the introduced cysteine ​​is at position 169; ii) the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 7, and the introduced cysteine ​​is at position 170; iii) the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 9, and the introduced cysteine ​​is at position 172; iv) the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 10, and the introduced cysteine ​​is at position 173; v) the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 11, and the introduced cysteine ​​is at position 174; vi) the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 12, and the introduced cysteine ​​is at position 180; vii) the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 13 and the introduced cysteine ​​is at position 181; or viii) The pharmaceutical composition of claim 14, wherein the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 92 and the introduced cysteine ​​is at position 174.

18. 15. The pharmaceutical composition of claim 1, wherein the FGF21 domain further comprises the substitution P171G relative to SEQ ID NO:

1.

19. 10. The pharmaceutical composition of any preceding claim, wherein the Nanobody domain comprises a VHH domain.

20. 20. The pharmaceutical composition of claim 19, wherein the VHH domain is humanized.

21. the VHH domain comprises a complementarity determining region 1 (CDR1), a complementarity determining region 2 (CDR2), and a complementarity determining region 3 (CDR3); the CDR1 comprises the sequence of SEQ ID NO: 20, or a variant thereof having up to 3, 2, or 1 amino acid mutations; said CDR2 comprises the sequence of SEQ ID NO: 21 or a variant thereof having up to 3, 2 or 1 amino acid mutations; and / or the CDR3 comprises the sequence of SEQ ID NO: 22 or a variant thereof having up to 3, 2, or 1 amino acid mutations; 21. The pharmaceutical composition of claim 19 or 20, wherein the VHH domain substantially retains binding specificity to serum albumin, optionally to human serum albumin.

22. The pharmaceutical composition of claim 21, wherein the VHH domain comprises a complementarity determining region 1 (CDR1) comprising the sequence of SEQ ID NO: 20, a CDR2 comprising the sequence of SEQ ID NO: 21, and a CDR3 comprising the sequence of SEQ ID NO:

22.

23. 23. The pharmaceutical composition of any one of claims 19 to 22, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO: 23, or a variant thereof having at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 99%) identity to SEQ ID NO: 23, wherein the variant substantially retains binding specificity and / or affinity to serum albumin.

24. 24. The pharmaceutical composition of claim 23, wherein the variant of SEQ ID NO: 23 has up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations relative to SEQ ID NO:

23.

25. 25. The pharmaceutical composition of any one of claims 19 to 24, wherein the Nanobody domain further comprises an N-terminal extension attached to the N-terminus of the VHH domain.

26. 26. The pharmaceutical composition of claim 25, wherein the N-terminal extension comprises an SG, AG, S, or A amino acid residue.

27. 27. The pharmaceutical composition of claim 26, wherein the Nanobody domain comprises an amino acid sequence selected from SEQ ID NOs: 24-27.

28. 10. The pharmaceutical composition of claim 1, wherein the first linker has a length of at least four amino acid residues.

29. 29. The pharmaceutical composition of claim 28, wherein the first linker does not contain any acidic amino acid residues.

30. 29. The pharmaceutical composition of claim 28, wherein the first linker does not contain any D or E residues.

31. 29. The pharmaceutical composition of claim 28, wherein the first linker comprises one or more units of a first repeat sequence.

32. 32. The pharmaceutical composition of claim 31, wherein the first repeat sequence consists of no more than 4 or 6 types of amino acid residues selected from the group consisting of G, Q, A, P, T and S.

33. The first repeat sequence is G ｆ S ｇ , SEQ ID NO:35 (GAQP), SEQ ID NO:36 (GQAP), SEQ ID NO:37 (GPAQ), SEQ ID NO:38 (GPQA), SEQ ID NO:39 (GSQP), SEQ ID NO:40 (GASP), SEQ ID NO:41 (GPAS), SEQ ID NO:42 (GPSA), SEQ ID NO:43 (GGGS), SEQ ID NO:44 (GSGS), SEQ ID NO:45 (GGGGGS), SEQ ID NO:46 (GSAPGSPAGSPTGSAPGSPA), and SEQ ID NO:110 (GS), wherein each of f and g is independently an integer selected from 1 to 5.

34. 34. The pharmaceutical composition of claim 33, wherein the first repeat sequence has the amino acid sequence set forth in SEQ ID NO: 35 (GAQP), and the number of the one or more units is an integer from 1 to 10.

35. The first linker is selected from the group consisting of SEQ ID NO: 35 (GAQP), SEQ ID NO: 49 (GAQP) ２ ), SEQ ID NO: 50 ((GAQP) ５ ), SEQ ID NO: 51 ((GAQP) １０ 35. The pharmaceutical composition of claim 34, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 48 (GGGGSGGGGS).

36. 1. A pharmaceutical composition further comprising a third fragment beyond the N-terminus of said first fragment, said third fragment comprising another functional domain, 10. The pharmaceutical composition of claim 1, wherein the first fragment and the third fragment are connected via a second linker.

37. 37. The pharmaceutical composition of claim 36, wherein the additional functional domain of the third fragment comprises a biologically active peptide of GLP-1 or a fragment thereof, and the additional functional domain comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO:28 while retaining substantial biological activity thereof.

38. 38. The pharmaceutical composition of claim 37, wherein the additional functional domain comprises one or more mutations at positions 8, 22, 26, 34, and 36, or any combination thereof, relative to SEQ ID NO:

28.

39. 39. The pharmaceutical composition of claim 38, wherein the one or more mutations comprise A8G, G22E, K26R, K34R, R36G, or any combination thereof.

40. 40. The pharmaceutical composition of claim 39, wherein the additional functional domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 29 and SEQ ID NOs: 31-34.

41. 41. The polypeptide of any one of claims 36 to 40, wherein the second linker has a length of at least 8 amino acid residues (e.g., at least 12, 16, or 20 amino acid residues).

42. 42. The polypeptide of claim 41, wherein the second linker comprises one or more units of a second repeat sequence.

43. 43. The polypeptide of claim 42, wherein the second repeat sequence consists of no more than four or six types of amino acid residues selected from the group consisting of G, Q, A, E, P, T and S.

44. The second repeat sequence is G ｈ S ｉ , SEQ ID NO:35 (GAQP), SEQ ID NO:55 (GQEP), SEQ ID NO:56 (GEQP), SEQ ID NO:57 (GPQE), SEQ ID NO:58 (GPEQ), SEQ ID NO:59 (GSEP), SEQ ID NO:60 (GESP), SEQ ID NO:61 (GPSE), SEQ ID NO:62 (GPES), SEQ ID NO:36 (GQAP), SEQ ID NO:37 (GPAQ), SEQ ID NO:38 (GPQA), SEQ ID NO:39 (GSQP), SEQ ID NO:40 (GASP), SEQ ID NO:41 (GPAS), SEQ ID NO:42 (GPSA), SEQ ID NO:43 (GGGS), SEQ ID NO:44 (GSGS), SEQ ID NO:45 (GGGGS), SEQ ID NO:46 (GSAPGSPAGSPTGSAPGSPA), and SEQ ID NO:110 (GS), wherein each of h and i is independently an integer selected from 1 to 5.

45. 45. The polypeptide of claim 44, wherein the second repeat sequence has the amino acid sequence set forth in SEQ ID NO: 35 (GAQP), and the number of the one or more units is an integer from 1 to 15.

46. The second linker is selected from the group consisting of SEQ ID NO: 49 ((GAQP) ２ ), SEQ ID NO: 50 ((GAQP) ５ ), SEQ ID NO: 51 ((GAQP) １０ ), and SEQ ID NO: 52 ((GAQP) １４ ), and SEQ ID NO: 47 ((GGGGS) ４ 42. The polypeptide of claim 41, comprising an amino acid sequence selected from the group consisting of:

47. 10. The polypeptide of any preceding claim, comprising an amino acid sequence selected from SEQ ID NOs: 63-68, 93, 99, 100, 101 and 107.

48. 48. The polypeptide of any one of claims 36 to 47, comprising an amino acid sequence selected from SEQ ID NOs: 70, 74, 75, 79-83, 85, 94-98, and 108-109.

49. 49. The pharmaceutical composition of any one of claims 1 to 48, wherein the pharmaceutically acceptable excipients comprise a buffering agent and an isotonicity agent.

50. 50. The pharmaceutical composition of claim 49, wherein the buffer is selected from the group consisting of a phosphate buffer, a citrate buffer, an acetate buffer, a histidine buffer, a glycine buffer, a carbonate buffer, a borate buffer, a glutamate buffer, a glycylglycine buffer, a lysine buffer, and an arginine buffer.

51. 51. The pharmaceutical composition of any one of claims 49-50, having a pH of about 6.0 to about 8.0 (e.g., about 6.5 to about 7.8, about 6.5 to about 7.4, about 6.5 to about 7.0, about 7.0 to about 7.8, about 7.0 to about 7.4, about 7.4 to about 8.2, about 7.4 to about 8.0, or about 7.4 to about 7.8).

52. 52. The pharmaceutical composition of claim 50 or 51, wherein the buffering agent is a phosphate buffer, and optionally the pharmaceutical composition has a pH of about 6.5 to about 8.0, or optionally about 6.5 to about 7.4, about 7.0 to about 7.8, about 7.0 to about 7.4, or about 7.4 to about 7.

8.

53. 53. The pharmaceutical composition of claim 52, wherein the phosphate buffer is present in the pharmaceutical composition at a concentration of 0.01 to 50 mM, optionally 5 to 20 mM.

54. 54. The pharmaceutical composition of any one of claims 52 to 53, wherein the phosphate buffer is selected from the group consisting of sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, or hydrates thereof.

55. 55. The pharmaceutical composition of claim 54, wherein the phosphate buffer is disodium hydrogen phosphate dodecahydrate or disodium phosphate dihydrate.

56. 56. The pharmaceutical composition of claim 55, wherein the disodium hydrogen phosphate dodecahydrate is present at a concentration of about 0.01 to 17 mg / mL (e.g., about 0.01 to 15 mg / mL, about 0.05 to 12 mg / mL, about 0.05 to 10 mg / mL, about 0.1 to 10 mg / mL, about 1 to 5 mg / mL, about 1 to 3 mg / mL, or about 2.87 mg / mL).

57. 56. The pharmaceutical composition of claim 55, wherein the disodium hydrogen phosphate dihydrate is present at a concentration of about 0.01 to 8 mg / mL (e.g., about 0.01 to 5 mg / mL, about 0.05 to 4 mg / mL, about 0.05 to 3 mg / mL, about 0.1 to 3 mg / mL, about 1 to 3 mg / mL, or about 1.42 mg / mL).

58. 52. The pharmaceutical composition of claim 50 or 51, wherein the buffer is a citrate buffer.

59. 59. The pharmaceutical composition of claim 58, wherein the citrate buffer is present at a concentration of about 0.05 to 20 mg / mL.

60. 60. The pharmaceutical composition of any one of claims 58-59, wherein the citrate buffer is anhydrous citric acid / trisodium citrate dihydrate, and optionally the pharmaceutical composition has a pH of about 6.0 to 6.8, or optionally about 6.

5.

61. 61. The pharmaceutical composition of claim 60, wherein the anhydrous citric acid / trisodium citrate dihydrate is present at a concentration of about 0.05 to 10 mg / mL.

62. 52. The pharmaceutical composition of claim 50 or 51, wherein the buffer is a histidine buffer, and optionally the pharmaceutical composition has a pH of about 6.0 to 6.8, or optionally about 6.

5.

63. 63. The pharmaceutical composition of claim 62, wherein the histidine buffering agent is present at a concentration of about 0.5-10 mg / mL (e.g., about 1-5 mg / mL, about 1-3 mg / ml, about 1.55 mg / ml).

64. 64. The pharmaceutical composition of any one of claims 49 to 63, wherein the isotonicity agent is selected from the group consisting of sodium chloride, glycerol, sorbitol, sucrose, propylene glycol, mannitol, glycine, lactose monohydrate, arginine, myo-inositol, and dimethyl sulfone.

65. 65. The pharmaceutical composition of claim 64, wherein the isotonicity agent is glycerol, and optionally the glycerol is at about 5-30 mg / mL.

66. 65. The pharmaceutical composition of claim 64, wherein the isotonicity agent is propylene glycol, and optionally the propylene glycol is from about 1 mg / mL to about 50 mg / mL (e.g., from about 5 mg / mL to about 25 mg / mL, from about 8 mg / mL to about 16 mg / mL).

67. 65. The pharmaceutical composition of claim 64, wherein the isotonicity agent is mannitol, and optionally the mannitol is at about 20 mg / mL to about 100 mg / mL (e.g., about 25 mg / mL to about 70 mg / mL, about 30 mg / mL to about 60 mg / mL, about 35 mg / mL to about 55 mg / mL, about 45 mg / mL).

68. 65. The pharmaceutical composition of claim 64, wherein the isotonicity agent is sorbitol, and optionally the sorbitol is at about 20 mg / mL to about 100 mg / mL (e.g., about 40 mg / mL to about 50 mg / mL).

69. 65. The pharmaceutical composition of claim 64, wherein the isotonicity agent is sucrose, and optionally the sucrose is from about 5 mg / mL to about 150 mg / mL (e.g., from about 45 mg / mL to about 100 mg / mL).

70. 70. The pharmaceutical composition of any one of claims 49 to 69, wherein the pharmaceutically acceptable excipient further comprises a non-ionic surfactant.

71. 71. The pharmaceutical composition of claim 70, wherein the non-ionic surfactant is polysorbate 80, and optionally the polysorbate 80 is from about 0.05 mg / mL to about 5 mg / mL (e.g., from about 0.1 mg / mL to about 0.5 mg / mL).

72. 72. The pharmaceutical composition of any one of claims 49 to 71, wherein the pharmaceutical excipient further comprises a preservative, a chelating agent, and / or a stabilizer.

73. 73. The pharmaceutical composition of claims 1-72, wherein the pharmaceutical composition has about 1-100 mg / mL (e.g., about 1-90 mg / mL, about 1-80 mg / mL, about 1-70 mg / mL, about 1-60 mg / mL, about 1-50 mg / mL, about 1-40 mg / mL, about 1-30 mg / mL, about 1-20 mg / mL, about 1-15 mg / mL, about 1-10 mg / mL) of the polypeptide.

74. the polypeptide (optionally, the polypeptide comprises the amino acid sequence of SEQ ID NO:85 or SEQ ID NO:96); a buffer selected from the group consisting of phosphate buffer, citrate buffer, acetate buffer, histidine buffer, glycine buffer, carbonate buffer, borate buffer, glutamate buffer, glycylglycine buffer, lysine buffer, and arginine buffer; an isotonicity agent selected from the group consisting of glycerol, sorbitol, sucrose, propylene glycol, mannitol, glycine, lactose monohydrate, arginine, myo-inositol, and dimethyl sulfone; 74. The pharmaceutical composition of claims 1 to 73, wherein the pH is from about 6.5 to about 8.

0.

75. about 1-100 mg / mL of the polypeptide, optionally wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:85 or SEQ ID NO:96; a buffer selected from the group consisting of phosphate buffers, citrate buffers, and histidine buffers; an isotonicity agent selected from the group consisting of glycerol, sorbitol, sucrose, propylene glycol, and mannitol; 74. The pharmaceutical composition of claims 1 to 73, wherein the pH is from about 6.5 to about 8.

0.

76. about 1-100 mg / mL of the polypeptide, optionally wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:85 or SEQ ID NO:96; about 0.1-10 mg / mL phosphate buffer, about 0.05-10 mg / mL citrate buffer, or about 0.5-10 mg / mL histidine buffer; an isotonicity agent selected from the group consisting of glycerol, sorbitol, sucrose, propylene glycol, and mannitol; 74. The pharmaceutical composition of claims 1 to 73, wherein the pH is from about 6.5 to about 8.

0.

77. about 1-100 mg / mL of the polypeptide, optionally wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:85 or SEQ ID NO:96; a buffer selected from the group consisting of phosphate buffers, citrate buffers, and histidine buffers; an isotonicity agent selected from the group consisting of 5-30 mg / mL glycerol, 20-100 mg / mL sorbitol, 5-150 mg / mL sucrose, 1-50 mg / mL propylene glycol, and 20-100 mg / mL mannitol; 74. The pharmaceutical composition of claims 1 to 73, wherein the pH is from about 6.5 to about 8.

0.

78. about 1-100 mg / mL of the polypeptide, optionally wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:85 or SEQ ID NO:96; about 0.1-10 mg / mL phosphate buffer, about 0.05-10 mg / mL citrate buffer, or about 0.5-10 mg / mL histidine buffer; an isotonicity agent selected from the group consisting of 5-30 mg / mL glycerol, 20-100 mg / mL sorbitol, 5-150 mg / mL sucrose, 1-50 mg / mL propylene glycol, and 20-100 mg / mL mannitol; 74. The pharmaceutical composition of claims 1 to 73, wherein the pH is from about 6.5 to about 8.

0.

79. 79. The pharmaceutical composition of any one of claims 74 to 78, further comprising a non-ionic surfactant at about 0.1 to 0.5 mg / mL of Polysorbate 80.

80. 80. A method for preventing or treating a metabolic disorder in a subject in need thereof, comprising administering the pharmaceutical composition of any one of claims 1 to 79.

81. 81. The method of claim 80, wherein the metabolic disorder is diabetes, obesity, overweight, non-alcoholic steatohepatitis (NASH), cardiovascular-like dyslipidemia, atherosclerosis, alcoholic steatohepatitis (ASH), diabetic nephropathy, gestational diabetes, metabolic syndrome such as metabolic syndrome X, non-alcoholic fatty liver disease (NAFLD), end-stage liver disease, hepatic steatosis (fatty liver), cirrhosis, primary biliary cirrhosis (PBC), or Alzheimer's disease.

82. 82. The method of claim 81, wherein the diabetes comprises one or more conditions selected from the group consisting of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin dependent diabetes, MODY (maturity-onset diabetes of the young), gestational diabetes, and elevated HbA1C levels.

83. 80. A method of managing weight in a subject in need thereof comprising administering the pharmaceutical composition of any one of claims 1 to 79.

84. 80. A method of reducing food intake in a subject in need thereof comprising administering the pharmaceutical composition of any one of claims 1 to 79.

85. 80. A method of reducing weight in a subject in need thereof comprising administering the pharmaceutical composition of any one of claims 1 to 79.

86. 86. The method of any one of claims 80 to 85, wherein the subject is a human.

87. 87. The method of claim 86, wherein the subject has a fasting blood glucose level of 125 mg / dL or greater.

88. 88. The method of claim 86 or 87, wherein the subject has a body mass index (BMI) of at least 25 or higher.

89. 89. The method of any one of claims 80-88, wherein the pharmaceutical composition is administered in a dosing regimen that is no more frequent than once daily, once every three days, once weekly, or once every two weeks.

90. 89. The method of any one of claims 80-88, wherein the pharmaceutical composition is administered twice weekly, once weekly, or once every two weeks.

91. 89. The method of any one of claims 80 to 88, wherein the dosing regimen has a dosing interval of about once every three days to about once every two weeks.

92. 89. The method of any one of claims 80 to 88, wherein the pharmaceutical composition is administered by parenteral administration.

93. 89. The method of any one of claims 80 to 88, wherein the pharmaceutical composition is administered subcutaneously, intravenously, or intramuscularly.

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