Liquid pharmaceutical compositions of polypeptide conjugates and methods of use thereof

Abstract

The present disclosure provides a polypeptide conjugate comprising a GLP-1 receptor agonist and a peptide linker, and a liquid pharmaceutical composition comprising the same. Methods of using such to treat disease are also provided.

Description

[Technical field]

[0001] The present disclosure relates generally to the field of therapeutic peptides, and more specifically to polypeptide conjugates, i.e., glucagon-like peptide 1 (GLP-1) compounds, pharmaceutical compositions thereof, and methods of using such, for preventing and / or treating metabolic disorders, such as diabetes. [Background technology]

[0002] Metabolic disorders or diseases are a group of pathologies characterized by the body's inability to properly convert food into energy and to utilize and / or store energy, the most prominent metabolic disorder being diabetes. Metabolic disorders are generally associated with insulin resistance, visceral adiposity, atherogenic dyslipidemia, etc., and pose a large and growing public health and clinical challenge worldwide.

[0003] Glucagon-like peptide-1 (GLP-1) is a proglucagon-derived peptide secreted from intestinal L-cells in response to nutrient intake.GLP-1 acts primarily as an incretin, an endocrine hormone, to globally regulate the concentration of glucagon, slow down gastric emptying, stimulate the biosynthesis of (pro-)insulin, increase sensitivity to insulin, stimulate the insulin-independent biosynthesis of glycogen, etc., thereby increasing the insulin response after oral ingestion of food.Since GLP-1 can rapidly lower glucose levels in both normal and diabetic subjects, there has been considerable interest in developing GLP-1-based pharmaceutical agents (i.e., hereafter referred to as GLP-1 compounds) to prevent and / or treat type 2 diabetes.

[0004] Currently, it is known that human GLP-1 in its native form (having 37 amino acid residues) is poorly active, and the two major naturally occurring and biologically active truncated versions of native GLP-1, which contain 30 and 31 amino acid peptide fragments derived from post-translational processing of the proglucagon peptide, namely GLP-1(7-36) or GLP-1(7-37), have extremely short in vivo half-lives, mainly due to N-terminal cleavage and inactivation by dipeptidyl peptidase DPP-IV. Although enormous efforts have been made to develop newer and better GLP-1 compounds with more favorable dosing regimens, existing GLP-1 compounds and their pharmaceutical compositions still fall far short of the ideal half-life, potency, and / or dosing frequency.

[0005] Thus, there is a need for improved designs for GLP-1 compounds and improved pharmaceutical compositions thereof. Summary of the Invention

[0006] Provided herein are pharmaceutical compositions and methods of use thereof for treating / preventing metabolic disorders.

[0007] In a first aspect, the disclosure provides a pharmaceutical composition comprising a polypeptide conjugate and a pharma- ceutically acceptable excipient.

[0008] The polypeptide conjugate essentially comprises a polypeptide portion and a conjugate portion. The polypeptide portion comprises a single bioactive peptide and a peptide linker. The bioactive peptide is attached to the N-terminus of the peptide linker and comprises a GLP-1 receptor agonist. The conjugate portion comprises a first clearance reducing moiety (CRM) conjugated to a first CRM residue in the peptide linker.

[0009] In some embodiments, the first CRM residue is at least 5 amino acid residues (excluding the CRM residue), e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 33, 35, 38, 40, 43, 45, 48, 50, 53, 55, 58, 60, 63, 65, 68, 70, 73, 75, or 78 amino acid residues away from the C-terminal amino acid residue of the biologically active peptide (e.g., a GLP-1 receptor agonist).

[0010] In some embodiments, the GLP-1 receptor agonist comprises GLP-1. As used herein, the term "GLP-1" refers to a molecule having the following characteristics: the molecule has substantially the biological activity of wild-type human GLP-1 (SEQ ID NO: 1), and the molecule comprises a polypeptide fragment having an amino acid sequence at least 50% identical to wild-type human GLP-1.

[0011] In some embodiments, the GLP-1 comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:1, while retaining substantial biological activity of SEQ ID NO:1.

[0012] In some embodiments, GLP-1 is 7 X 8 EGTFTSDVSSYLEX 22 X 23 AAX 26 X 27 FIX 30 WLVX 34 GX 36 G (SEQ ID NO:2), wherein X 7 is H, imidazole-4-acetate (IA), or imidazole propionic acid (IPA); X 8 is A, G, S, V, Aib, T, I, or L; X 22 is G or E;X 23 is Q, C, or K; X 26 is K, R, or C; X 27 is E, K, or C;X30 is A, C, or K; X 34 is R, K, or C; X 36 is R or G.

[0013] In some embodiments of GLP-1, X 7 is H;X 8 is G or Aib.

[0014] In some embodiments, the GLP-1 comprises or consists of one or more mutations at positions selected from the group consisting of A8, G22, K26, K34, and R36, or any combination thereof, compared to SEQ ID NO:1.

[0015] In some embodiments, the GLP-1 comprises or consists of one or more substitutions selected from the group consisting of A8Aib, G22E, K26R, K34R, and R36G, or any combination thereof.

[0016] In some embodiments, the GLP-1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:3-9.

[0017] In any of the embodiments of the polypeptide conjugates described above, the peptide linker may have a length of at least 10 amino acid residues, at least 12, 24, 32, 40, 48, 50, 60, 70, or 80 amino acid residues.

[0018] In some embodiments, the peptide linker consists of amino acid residues selected from the group consisting of G, Q, A, E, P, S, and T, except for the first CRM residue.

[0019] In some embodiments, the peptide linker comprises a first sequence and a second sequence. The first sequence comprises one or more repeats of the repeat sequence and is connected to the N-terminus of the second sequence. The first CRM residue is in the second sequence and is a lysine residue or a cysteine ​​residue.

[0020] In some embodiments, the repeat sequence in the first sequence of the peptide linker is selected from SEQ ID NO:10 (GQEPGAQP), SEQ ID NO:11 (GAQPGAQP), SEQ ID NO:12 (GQEP), SEQ ID NO:13 (GAQP), SEQ ID NO:14 (GAQPGQEPGAQP), SEQ ID NO:15 (GAQPGQEP), SEQ ID NO:16 (GEQP), SEQ ID NO:17 (GPQE), SEQ ID NO:18 (GPEQ), SEQ ID NO:19 (GSEP), SEQ ID NO:20 (GESP), SEQ ID NO:21 (GPSE), SEQ ID NO:22 (GPES), SEQ ID NO:23 (GQAP), SEQ ID NO:24 (GPAQ), SEQ ID NO:25 (GPQA), SEQ ID NO:26 (GPQA), SEQ ID NO:27 (GPQE), SEQ ID NO:28 (GPQE), SEQ ID NO:29 (GSEP), SEQ ID NO:30 (GESP), SEQ ID NO:31 (GPSE), SEQ ID NO:32 (GPES), SEQ ID NO:33 (GQAP), SEQ ID NO:34 (GPAQ), SEQ ID NO:35 (GPQA), SEQ ID NO:36 (GPQA), SEQ ID NO:37 (GPQE), SEQ ID NO:38 (GPQE), SEQ ID NO:39 (GPQE), SEQ ID NO:40 (GPQE), SEQ ID NO:41 (GPQE), SEQ ID NO:42 (GPQE), SEQ ID NO:43 (GPQE), SEQ ID NO:44 (GPQE), SEQ ID NO:45 (GPQE), SEQ ID NO:46 (GPQE), SEQ ID NO:47 (GPQE), SEQ ID NO:48 (GPQE), SEQ ID NO:49 (GPQE), SEQ ID NO:50 ( SEQ ID NO:26 (GSQP), SEQ ID NO:27 (GASP), SEQ ID NO:28 (GPAS), SEQ ID NO:29 (GPSA), SEQ ID NO:30 (GGGS), SEQ ID NO:31 (GSGS), SEQ ID NO:32 (GGGGS), SEQ ID NO:33 (GQEPGQAP), SEQ ID NO:34 (GQAPGQEP), SEQ ID NO:35 (SEPATSGSETPGTSESATPESGPGTSTEPSEG), SEQ ID NO:36 (SEPATS), SEQ ID NO:37 (GSETPG), SEQ ID NO:38 (TSESAT), SEQ ID NO:39 (PESGPG), SEQ ID NO:40 (TSTEPS), and GS.

[0021] In some embodiments, the repeat sequence in the first sequence of the peptide linker has the sequence of SEQ ID NO: 13 (GAQP), where optionally, the number of repeats of the repeat sequence is an integer between 1 and 30. In certain embodiments, the number of repeats of the repeat sequence is selected from the group consisting of 5, 7, 9, and 11.

[0022] In certain embodiments, the second sequence of the peptide linker has the sequence of SEQ ID NO: 41 (GQKP) or SEQ ID NO: 42 (GQCP).

[0023] In certain embodiments, the polypeptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-50 and 82-84.

[0024] In any of the embodiments of the polypeptide conjugates described above, the first CRM may comprise a plasma protein binding moiety, a polymer, an Fc, an HSA (albumin), an Xten sequence, or a PAS sequence.

[0025] In some embodiments, the first CRM comprises an albumin binding moiety.

[0026] In some embodiments, the albumin binding moiety comprises the structure *-ABCDE, where A, B, C, D, and E are interconnected via amide bonds and the * end of A is connected to a reactive group of a conjugable residue on the polypeptide complex.

[0027] where A is a bond,

[0028] [ka] wherein a, b, c, and d are independently integers from 0 to 4; 1 is hydrogen or -COOH.

[0029] where B is a bond,

[0030] [ka] wherein e is an integer from 1 to 4, and wherein the α position is linked to the α' position.

[0031] where C is a bond or

[0032] [ka] where R 2 Ha-CH 2 SO 3H or -COOH, f is an integer from 1 to 4, n is an integer from 1 to 25, and if B is not a bond, the β' position is linked to the β position, or if B is a bond, the β' position is linked to the α' position.

[0033] where D is a bond,

[0034] [ka] wherein g and h are independently 0 or 1; R 3 is H or -CH 2 COOH, where if B is not a bond and C is a bond, then the γ' position is linked to the β position; if C is not a bond, then the γ' position is linked to the γ position; and if B is a bond and C is a bond, then the γ' position is linked to the α' position.

[0035] where E is a group of formula:

[0036] [ka] is an acidic group having the formula In the formula, W is -(CR 4 R 5 ) l -, where R 4 and R 5 is independently selected from the group consisting of hydrogen, halogen, cyano, hydroxyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, hydroxyalkyl, amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy, and carboxamido; R 6 is hydroxyl or NR 7 R 8 wherein R 7 and R 8 are independently hydrogen, alkyl, hydroxyl, and

[0037] [ka] and l is an integer from 10 to 20. Here, when D is not a bond, the δ position is linked to the δ' position, when C is not a bond and D is a bond, the δ position is linked to the γ position, when B is not a bond, C is a bond, and D is a bond, the δ position is linked to the β position, and when A is not a bond and all of B, C, and D are bonds, the δ position is linked to the α' position.

[0038] In some embodiments of the first CRM, A is a bond or

[0039] [ka] In the formula, d is an integer from 1 to 4, and R 1 is hydrogen; B is a bond or

[0040] [ka] wherein e is an integer from 1 to 4, and wherein the α-position is linked to the α'-position;

[0041] [ka] where R 2 is -COOH, f is an integer from 1 to 4, n is an integer from 1 to 25, if B is not a bond, the β' position is linked to the β position, or if B is a bond, the β' position is linked to the α' position; D is a bond; with respect to E, R 4 and R 5 is hydrogen, and R 6 is hydroxyl, l is an integer from 10 to 20, and the δ position is linked to the γ position.

[0042] In some embodiments of the first CRM, d is 1, e is 2, f is 1, n is 1, and l is an integer from 14-20.

[0043] In some embodiments of the first CRM, 1 is 16.

[0044] In some embodiments of the polypeptide conjugate, the first CRM residue is a lysine residue, where: A is a bond and B is

[0045] [ka] It is.

[0046] In some embodiments, the first CRM comprises an A moiety (HOOC-(CH2)16-CO-gGlu-2XADO), the A moiety having the structure of the formula below:

[0047] [ka] has.

[0048] In some embodiments of the polypeptide conjugate, the first CRM residue is a cysteine ​​residue, where A is

[0049] [ka] and B is

[0050] [ka] It is.

[0051] In some embodiments, the first CRM comprises a B moiety (HOOC-(CH2)16-CO-gGlu-2XADO-EDA-CO-CH2), the B moiety having the structure of the formula below:

[0052] [ka] has.

[0053] In certain embodiments, the polypeptide conjugates provided herein are monoconjugated, having the CRM conjugated to a peptide linker rather than to the biologically active peptide (eg, GLP-1).

[0054] In some embodiments of a monoconjugated polypeptide conjugate, the first CRM residue is a lysine residue and the polypeptide conjugate includes only one lysine residue.

[0055] In some embodiments of a monoconjugated polypeptide conjugate, the first CRM residue is a cysteine ​​residue and the polypeptide conjugate includes only one cysteine ​​residue.

[0056] In some embodiments, the GLP-1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 5, 6, 8, and 9.

[0057] wherein, in some embodiments, the polypeptide portion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 52, 55, 56, 60-62, 66-71, and 75-80; and the first CRM residue is a lysine or cysteine ​​residue at a position selected from the group consisting of 60, 68, and 76.

[0058] The following list certain embodiments of the monoconjugated polypeptide conjugates disclosed herein:

[0059] the polypeptide portion comprises the amino acid sequence of SEQ ID NO:52 and is conjugated to a first CRM at 76K;

[0060] the polypeptide portion comprises the amino acid sequence of SEQ ID NO:55 and is conjugated to a first CRM at 68K;

[0061] the polypeptide portion comprises the amino acid sequence of SEQ ID NO:56 and is conjugated to a first CRM at 60K;

[0062] the polypeptide portion comprises the amino acid sequence of SEQ ID NO:60 and is conjugated to a first CRM at 76K;

[0063] the polypeptide portion comprises the amino acid sequence of SEQ ID NO:61 and is conjugated to a first CRM at 68K;

[0064] the polypeptide portion comprises the amino acid sequence of SEQ ID NO:62 and is conjugated to a first CRM at 60K;

[0065] the polypeptide portion comprises the amino acid sequence of SEQ ID NO:66 and is conjugated to a first CRM at 76C;

[0066] the polypeptide portion comprises the amino acid sequence of SEQ ID NO:67 and is conjugated to a first CRM at 68C;

[0067] the polypeptide portion comprises the amino acid sequence of SEQ ID NO:68 and is conjugated to the first CRM at 60C;

[0068] The polypeptide portion comprises the amino acid sequence of SEQ ID NO:69 and is conjugated to a first CRM at 76C;

[0069] the polypeptide portion comprises the amino acid sequence of SEQ ID NO:70 and is conjugated to a first CRM at 68C;

[0070] the polypeptide portion comprises the amino acid sequence of SEQ ID NO:71, and the first CRM is conjugated to the polypeptide portion at 60C;

[0071] The polypeptide portion comprises the amino acid sequence of SEQ ID NO: 75 and is conjugated to a first CRM at 76C;

[0072] the polypeptide portion comprises the amino acid sequence of SEQ ID NO:76 and is conjugated to a first CRM at 68C;

[0073] the polypeptide portion comprises the amino acid sequence of SEQ ID NO:77 and is conjugated to a first CRM at 60C;

[0074] The polypeptide portion comprises the amino acid sequence of SEQ ID NO: 78 and is conjugated to a first CRM at 76C;

[0075] the polypeptide portion comprises the amino acid sequence of SEQ ID NO:79 and is conjugated to a first CRM at 68C; or

[0076] The polypeptide portion comprises the amino acid sequence of SEQ ID NO:80 and is conjugated to the first CRM at 60C.

[0077] In certain embodiments of the polypeptide conjugate, the polypeptide portion comprises the amino acid sequence of SEQ ID NO:52 and the first CRM residue is 76K. More specifically, certain embodiments of the polypeptide conjugate have the structure shown below:

[0078] [ka] has.

[0079] In certain embodiments of the polypeptide conjugate, the polypeptide portion comprises the amino acid sequence of SEQ ID NO:60 and the first CRM residue is 76K. More specifically, certain embodiments of the polypeptide conjugate have the structure shown below:

[0080] [ka] has.

[0081] In some embodiments, the polypeptide conjugate is a dual-conjugated polypeptide conjugate, further comprising a second CRM conjugated to a second CRM residue.

[0082] In some embodiments of a dual-conjugated polypeptide conjugate, the first CRM residue and the second CRM residue are both lysine residues, and the polypeptide conjugate comprises only two lysine residues.

[0083] Wherein optionally the second CRM residue is in GLP-1 and is optionally selected from the group consisting of K23, K26, K27, K30, and K34.

[0084] In some embodiments, the second CRM residue is K26.

[0085] In some embodiments of a doubly conjugated polypeptide conjugate, the first CRM residue and the second CRM residue are both cysteine ​​residues, and the polypeptide conjugate comprises only two cysteine ​​residues.

[0086] Wherein optionally, the second CRM residue is in GLP-1 and is optionally selected from the group consisting of C23, C26, C27, C30, and C34.

[0087] In some embodiments, the second CRM residue is C26.

[0088] Here, the positions noted above (ie, 23, 26, 27, 30, and 34, as either K or C) are referred to relative to wild-type human GLP-1 (SEQ ID NO:1), beginning at the N-terminus thereof.

[0089] wherein, according to some embodiments, the second CRM is identical to the first CRM, hi some embodiments, the second CRM residues and the first CRM residues are both lysine residues or both cysteine ​​residues.

[0090] In some embodiments of the dual-conjugated polypeptide conjugate, the GLP-1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 6, and 7.

[0091] In some embodiments, the polypeptide portion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 51, 53, 54, 57-59, 63-65, and 72-74, 85, and 86; the first CRM residue is a lysine or cysteine ​​residue at a position selected from the group consisting of 68, 76, and 84; and the second CRM residue is a lysine or cysteine ​​residue at position 26.

[0092] The following list certain embodiments of the dual-conjugated polypeptide conjugates disclosed herein:

[0093] the polypeptide portion comprises the amino acid sequence of SEQ ID NO:51 and is conjugated to a first CRM and a second CRM at 26K and 76K, respectively;

[0094] the polypeptide portion comprises the amino acid sequence of SEQ ID NO:53 and is conjugated to a first CRM and a second CRM at 26K and 84K, respectively;

[0095] the polypeptide portion comprises the amino acid sequence of SEQ ID NO:54 and is conjugated to a first CRM and a second CRM at 26K and 68K, respectively;

[0096] the polypeptide portion comprises the amino acid sequence of SEQ ID NO:57 and is conjugated to a first CRM and a second CRM at 26K and 76K, respectively;

[0097] the polypeptide portion comprises the amino acid sequence of SEQ ID NO:58 and is conjugated to a first CRM and a second CRM at 26K and 84K, respectively;

[0098] the polypeptide portion comprises the amino acid sequence of SEQ ID NO:59 and is conjugated to a first CRM and a second CRM at 26K and 68K, respectively;

[0099] the polypeptide portion comprises the amino acid sequence of SEQ ID NO:85 and is conjugated to a first CRM and a second CRM at 26K and 96K, respectively;

[0100] the polypeptide portion comprises the amino acid sequence of SEQ ID NO:86 and is conjugated to a first CRM and a second CRM at 26K and 60K, respectively;

[0101] the polypeptide portion comprises the amino acid sequence of SEQ ID NO:63 and is conjugated to a first CRM and a second CRM at 26C and 84C, respectively;

[0102] the polypeptide portion comprises the amino acid sequence of SEQ ID NO:64, conjugated to a first CRM and a second CRM at 26C and 76C, respectively;

[0103] the polypeptide portion comprises the amino acid sequence of SEQ ID NO:65 and is conjugated to a first CRM and a second CRM at 26C and 68C, respectively;

[0104] the polypeptide portion comprises the amino acid sequence of SEQ ID NO:72, conjugated to a first CRM and a second CRM at 26C and 84C, respectively;

[0105] the polypeptide portion comprises the amino acid sequence of SEQ ID NO: 73 and is conjugated to a first CRM and a second CRM at 26C and 76C, respectively; or

[0106] The polypeptide portion comprises the amino acid sequence of SEQ ID NO: 74 and is conjugated to a first CRM and a second CRM at 26C and 68C, respectively.

[0107] In certain embodiments, the polypeptide portion comprises the amino acid sequence of SEQ ID NO:57 and is conjugated to a first CRM and a second CRM at 26K and 76K, respectively. More specifically, certain embodiments of the polypeptide conjugate have the structure shown below:

[0108] [ka] has.

[0109] Now, to obtain a polypeptide conjugate in the pharmaceutical composition provided in the first aspect, a polynucleotide encoding the polypeptide part (or a fragment thereof) of the polypeptide conjugate can be designed and operably placed in a vector. The vector can be transferred to a host cell, such as a prokaryotic or eukaryotic cell, and the host cell can be cultured under conditions that allow expression of the polynucleotide, thereby obtaining the polypeptide part of the polypeptide conjugate. Optionally, the polypeptide part is now expressed as a soluble protein. After obtaining the polypeptide part, a CRM can be further conjugated to the polypeptide part, thereby obtaining a single-conjugated or double-conjugated polypeptide conjugate.

[0110] In another embodiment, the pharmaceutical compositions provided herein are liquid formulations.

[0111] In certain embodiments, the pharma- ceutically acceptable excipients include buffers and isotonicity agents.

[0112] 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.

[0113] In certain embodiments, the buffer is a phosphate buffer.

[0114] In certain embodiments, the phosphate buffer is present in the pharmaceutical composition at a concentration of 0.01 to 50 mM.

[0115] In certain embodiments, the phosphate buffer is present in the pharmaceutical composition at a concentration of 5-20 mM or 5-10 mM, optionally about 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, or 5 mM.

[0116] In certain embodiments, the phosphate buffer is selected from the group consisting of sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, or hydrates thereof.

[0117] In certain embodiments, the hydrate is a dodecahydrate or a dihydrate.

[0118] In certain embodiments, the phosphate buffer is disodium hydrogen phosphate dodecahydrate or disodium phosphate dehydrate.

[0119] In certain embodiments, disodium hydrogen phosphate dodecahydrate is present in the pharmaceutical composition at a concentration of about 0.1-15 mg / mL, 0.5-15 mg / mL, 1-15 mg / mL, 0.5-12 mg / mL, 0.5-10 mg / mL, 0.5-8 mg / mL, 0.5-7 mg / mL, or 0.5-5 mg / mL.

[0120] In certain embodiments, the disodium hydrogen phosphate dodecahydrate is present in the pharmaceutical composition at a concentration of about 2.87 mg / mL.

[0121] In certain embodiments, disodium phosphate dihydrate is present in the pharmaceutical composition at a concentration of about 0.1-15 mg / mL, 0.5-15 mg / mL, 1-15 mg / mL, 0.5-12 mg / mL, 0.5-10 mg / mL, 0.5-8 mg / mL, 0.5-7 mg / mL, or 0.5-5 mg / mL.

[0122] In one particular embodiment, disodium phosphate dihydrate is present at a concentration of about 1.42 mg / mL.

[0123] In certain embodiments, the buffer is a citrate buffer.

[0124] In certain embodiments, the citrate buffer is present at a concentration of about 1-50 mM.

[0125] In certain embodiments, the citrate buffer comprises a mixture of citric acid anhydrous and trisodium citrate (or a hydrate thereof, such as trisodium citrate dihydrate).

[0126] In certain embodiments, the citrate buffer comprises a mixture of 0.14 mg / mL anhydrous citric acid and 2.74 mg / mL trisodium citrate dihydrate.

[0127] In certain embodiments, the buffer is a histidine buffer.

[0128] In certain embodiments, the histidine buffer is present in the pharmaceutical composition at a concentration of about 1-70 mM, optionally about 5-50 mM, 5-20 mM, or 5-10 mM, or optionally about 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, or 5 mM.

[0129] In certain embodiments, the histidine buffer is present in the pharmaceutical composition at a concentration of about 0.5-10 mg / mL, 0.5-5 mg / mL, 1-5 mg / mL, or 1-3 mg / mL.

[0130] In certain embodiments, the histidine buffer is present in the pharmaceutical composition at a concentration of about 1.24 mg / mL.

[0131] In certain embodiments, the isotonicity agent is selected from the group consisting of sodium chloride, propylene glycol, sorbitol, sucrose, glycine, mannitol, lactose monohydrate, arginine, myo-inositol, and dimethylsulfone.

[0132] In certain embodiments, the isotonic agent is sodium chloride.

[0133] In certain embodiments, the sodium chloride is about 5-15 mg / mL.

[0134] In certain embodiments, the isotonicity agent is propylene glycol.

[0135] 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).

[0136] In certain embodiments, the isotonicity agent is mannitol.

[0137] In certain embodiments, mannitol is about 20 mg / mL to about 60 mg / mL (e.g., about 25 mg / mL to about 50 mg / mL, about 30 mg / mL to about 50 mg / mL, about 35 mg / mL to about 50 mg / mL).

[0138] In certain embodiments, the pharmaceutical excipient further comprises a preservative, a chelating agent, and / or a stabilizer.

[0139] In certain embodiments, the pharmaceutical composition has a pH of about 6.5 to about 8.3 (eg, about 6.5 to 7.4, or 7.4 to 8.3).

[0140] In certain embodiments, the pharmaceutical composition has a pH of about 7.4.

[0141] In certain embodiments, the polypeptide conjugate is a dual-conjugated polypeptide conjugate provided herein. In certain embodiments, the pharmaceutical composition has a polypeptide conjugate of about 1-80 mg / ml, 1-100 mg / ml, or 1-120 mg / ml, or optionally 5-40 mg / ml, 5-80 mg / ml, 5-100 mg / ml, or 5-120 mg / ml (e.g., about 5-90 mg / mL, about 5-70 mg / mL, about 5-60 mg / mL, about 5-50 mg / mL, about 5-30 mg / mL, about 5-20 mg / mL, about 5-10 mg / mL). In some embodiments of the pharmaceutical composition, the polypeptide portion of the polypeptide conjugate comprises the amino acid sequence of SEQ ID NO:57 and is conjugated to a first CRM and a second CRM at 26K and 76K, respectively.

[0142] In certain embodiments, the pharmaceutical composition comprises: (a) about 1-80 mg / mL, 1-100 mg / ml, or 1-120 mg / mL of a polypeptide conjugate, wherein the polypeptide portion of the polypeptide conjugate comprises the amino acid sequence of SEQ ID NO:57 and is conjugated to a first CRM and a second CRM at 26K and 76K, respectively; (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, propylene glycol, and mannitol; and (d) pH of about 6.5 to about 8.3 Includes.

[0143] In certain embodiments, the pharmaceutical composition comprises: (a) about 1-80 mg / mL, 1-100 mg / ml, or 1-120 mg / mL of a polypeptide conjugate, wherein the polypeptide portion of the polypeptide conjugate comprises the amino acid sequence of SEQ ID NO:57 and is conjugated to a first CRM and a second CRM at 26K and 76K, respectively; (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 5-15 mg / mL sodium chloride, 1-50 mg / mL propylene glycol, and 30-50 mg / mL mannitol; and (d) pH of about 6.5 to about 8.3 Includes.

[0144] In certain embodiments, the pharmaceutical composition comprises: (a) about 1-80 mg / mL, 1-100 mg / ml, or 1-120 mg / mL of a polypeptide conjugate, wherein the polypeptide portion of the polypeptide conjugate comprises the amino acid sequence of SEQ ID NO:57 and is conjugated to a first CRM and a second CRM at 26K and 76K, respectively; (b) about 0.5-5 mg / mL phosphate buffer, about 1-50 mM citrate buffer, or about 0.5-10 mg / mL histidine buffer; (c) an isotonicity agent selected from the group consisting of sodium chloride, propylene glycol, and mannitol; and (d) pH of about 6.5 to about 8.3 Includes.

[0145] In certain embodiments, the pharmaceutical composition comprises: (a) about 1-80 mg / mL, 1-100 mg / ml, or 1-120 mg / mL of a polypeptide conjugate, wherein the polypeptide portion of the polypeptide conjugate comprises the amino acid sequence of SEQ ID NO:57 and is conjugated to a first CRM and a second CRM at 26K and 76K, respectively; (b) about 0.5-5 mg / mL phosphate buffer, about 1-50 mM citrate buffer, or about 0.5-10 mg / mL histidine buffer; (c) an isotonicity agent selected from the group consisting of 5-15 mg / mL sodium chloride, 1-50 mg / mL propylene glycol, and 30-50 mg / mL mannitol; and (d) pH of about 6.5 to about 8.3 Includes.

[0146] In certain embodiments, the pharmaceutical composition comprises: (a) about 5-80 mg / mL, 5-100 mg / ml, or 5-120 mg / mL of a polypeptide conjugate, wherein the polypeptide portion of the polypeptide conjugate comprises the amino acid sequence of SEQ ID NO:57 and is conjugated to a first CRM and a second CRM at 26K and 76K, respectively; (b) about 0.5-5 mg / mL phosphate buffer, about 1-50 mM citrate buffer, or about 0.5-10 mg / mL histidine buffer; (c) an isotonicity agent selected from the group consisting of 5-15 mg / mL sodium chloride, 1-50 mg / mL propylene glycol, and 30-50 mg / mL mannitol; and (d) pH of about 6.5 to about 8.3 Includes.

[0147] In certain embodiments, the polypeptide conjugate is a monoconjugated polypeptide conjugate provided herein. In certain embodiments, the pharmaceutical composition has about 0.5-20 mg / ml, 0.5-50 mg / ml, or 0.5-80 mg / ml of the polypeptide conjugate. In certain embodiments of the pharmaceutical composition, the polypeptide portion of the polypeptide conjugate comprises the amino acid sequence of SEQ ID NO:60 and is conjugated to a first CRM at 76K. In certain embodiments of the pharmaceutical composition, the polypeptide portion of the polypeptide conjugate comprises the amino acid sequence of SEQ ID NO:52 and is conjugated to a first CRM at 76K.

[0148] In certain embodiments, the pharmaceutical composition comprises: (a) about 0.5-20 mg / mL, 0.5-50 mg / ml, or 0.5-80 mg / ml of a polypeptide conjugate, wherein the polypeptide portion of the polypeptide conjugate comprises the amino acid sequence of SEQ ID NO:60 or SEQ ID NO:52, and is conjugated to a first CRM at 76K; (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, propylene glycol, and mannitol; and (d) pH of about 6.5 to about 8.3 Includes.

[0149] In certain embodiments, the pharmaceutical composition comprises: (a) about 0.5-20 mg / mL, 0.5-50 mg / ml, or 0.5-80 mg / ml of a polypeptide conjugate, wherein the polypeptide portion of the polypeptide conjugate comprises the amino acid sequence of SEQ ID NO:60 or SEQ ID NO:52, and is conjugated to a first CRM at 76K; (b) about 0.5-5 mg / mL phosphate buffer, about 1-50 mM citrate buffer, or about 0.5-10 mg / mL histidine buffer; (c) an isotonicity agent selected from the group consisting of sodium chloride, propylene glycol, and mannitol; and (d) pH of about 6.5 to about 8.3 Includes.

[0150] In certain embodiments, the pharmaceutical composition comprises: (a) about 0.5-20 mg / mL, 0.5-50 mg / ml, or 0.5-80 mg / ml of a polypeptide conjugate, wherein the polypeptide portion of the polypeptide conjugate comprises the amino acid sequence of SEQ ID NO:60 or SEQ ID NO:52, and is conjugated to a first CRM at 76K; (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 5-15 mg / mL sodium chloride, 1-50 mg / mL propylene glycol, and 30-50 mg / mL mannitol; and (d) pH of about 6.5 to about 8.3 Includes.

[0151] In certain embodiments, the pharmaceutical composition comprises: (a) about 0.5-20 mg / mL, 0.5-50 mg / ml, or 0.5-80 mg / ml of a polypeptide conjugate, wherein the polypeptide portion of the polypeptide conjugate comprises the amino acid sequence of SEQ ID NO:60 or SEQ ID NO:52, and is conjugated to a first CRM at 76K; (b) about 0.5-5 mg / mL phosphate buffer, about 1-50 mM citrate buffer, or about 0.5-10 mg / mL histidine buffer; (c) an isotonicity agent selected from the group consisting of 5-15 mg / mL sodium chloride, 1-50 mg / mL propylene glycol, and 30-50 mg / mL mannitol; and (d) pH of about 6.5 to about 8.3 Includes.

[0152] In a second aspect, the 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 polypeptide conjugate of the first aspect.

[0153] In certain embodiments, the metabolic disorder is diabetes, obesity, overweight, nonalcoholic steatohepatitis (NASH), cardiovascular like dyslipidemia, atherosclerosis, alcoholic steatohepatitis (ASH), diabetic nephropathy, gestational diabetes, metabolic syndromes such as metabolic syndrome X, nonalcoholic fatty liver disease (NAFLD), end stage liver disease, hepatic steatosis (fatty liver), cirrhosis, or primary biliary cirrhosis (PBC), or Alzheimer's disease.

[0154] In certain embodiments, diabetes can be any form of diabetes, including, but not limited to, 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 levels of HbA1C.

[0155] In certain embodiments, the metabolic disorder is Alzheimer's disease. In certain embodiments, the present disclosure provides a method of preventing or treating Alzheimer's disease in a subject in need thereof, comprising administering a therapeutically effective amount of a polypeptide conjugate or pharmaceutical composition provided herein.

[0156] In certain embodiments, the present disclosure provides a method of preventing or treating Alzheimer's disease in a subject in need thereof comprising administering a pharmaceutical composition provided herein comprising a polypeptide conjugate provided herein, wherein the pharmaceutical composition is a liquid formulation.

[0157] In certain embodiments, the polypeptide conjugate comprises a polypeptide portion and a conjugate portion, the polypeptide portion comprising a single bioactive peptide and a peptide linker, and the conjugate portion comprising a first clearance reducing moiety (CRM) conjugated to a first CRM residue in the peptide linker. In certain embodiments, the polypeptide portion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 52, 55, 56, 60-62, 66-71, and 75-80; the first CRM residue is a lysine or cysteine ​​residue at position selected from the group consisting of 60, 68, and 76.

[0158] In some embodiments, the polypeptide conjugate is a dual-conjugated polypeptide conjugate and further comprises a second CRM conjugated to a second CRM residue. In some embodiments of dual-conjugated polypeptide conjugates, the first CRM residue and the second CRM residue are both lysine residues and the polypeptide conjugate comprises only two lysine residues. In some embodiments, the polypeptide portion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 51, 53, 54, 57-59, 63-65, and 72-74, 85, and 86; the first CRM residue is a lysine or cysteine ​​residue at a position selected from the group consisting of 68, 76, and 84; and the second CRM residue is a lysine or cysteine ​​residue at position 26. In some embodiments, the polypeptide portion comprises an amino acid sequence of SEQ ID NO: 57 and is conjugated to the first and second CRM at 26K and 76K, respectively.

[0159] In some embodiments, the CRM residue is a lysine residue and the CRM comprises an A moiety (HOOC-(CH2)16-CO-gGlu-2XADO), the A moiety having the structure of the formula below:

[0160] [ka] has.

[0161] In some embodiments, the CRM residue is a cysteine ​​residue and the CRM comprises a B moiety (HOOC-(CH2)16-CO-gGlu-2XADO-EDA-CO-CH2), which has the structure of the formula below:

[0162] [ka] has.

[0163] In a third aspect, the present disclosure provides a method of managing weight in a subject in need thereof comprising administering a therapeutically effective amount of a polypeptide conjugate provided herein, thereby managing the subject's weight.

[0164] In a fourth 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 polypeptide conjugate provided herein, thereby reducing the subject's food intake.

[0165] In a fifth aspect, the present disclosure provides a method of reducing weight in a subject in need thereof, comprising administering a therapeutically effective amount of a polypeptide conjugate provided herein, thereby reducing the subject's weight.

[0166] In any of the methods described above in the second to fifth aspects, the subject may be a human.

[0167] In certain embodiments, the subject has a fasting blood glucose level of 125 mg / dL or greater.

[0168] In certain embodiments, the subject has a body mass index (BMI) of at least 25 or greater.

[0169] In certain embodiments, the pharmaceutical compositions are administered in a dosing regimen no more frequent than twice a day, once a day, once every three days, or once a week, once every two weeks, once every three weeks, or once a month.

[0170] In certain embodiments, the pharmaceutical composition is administered twice a week, once a week, once every two weeks, once every three weeks, once a month, or once every two months.

[0171] In certain embodiments, the dosing regimen has a dosing interval ranging from about once every three days to about once per month, or from about once per week to about once per month.

[0172] In certain embodiments, the pharmaceutical composition is administered orally.

[0173] Throughout this disclosure, the articles "a," "an," and "the" are used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article. By way of example, "a fusion polypeptide" means one fusion polypeptide or more than one fusion polypeptide.

[0174] In 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 numerical value recited herein. Ranges provided herein are understood to include all values ​​within the range. For example, 1-10 is understood to include all 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.

[0175] As used herein, "about" or "approximately" is understood to include within 3 standard deviations of the mean or within the standard range of acceptance in a particular technical field. In certain embodiments, about is understood to vary by at most 0.5.

[0176] The articles "a" and "an" 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, "an element" means one type of element or more than one type of element.

[0177] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to." Similarly, "such as" is used herein to mean, and is used interchangeably with, the phrase "such as but not limited to."

[0178] The term "or" is used inclusively herein to mean, and is used interchangeably with, the term "and / or," unless context clearly indicates otherwise. [Brief description of the drawings]

[0179] [Figure 1] Figures 1A-1B show the in vivo activity of test molecules in C57BL / 6 mice. To assess body weight, 10-week-old male C57BL / 6 mice were subcutaneously dosed once with test drug substance. Figure 1A shows the weight loss after treatment with molecule 012 or molecule 001 from day 1 to day 8. Figure 1B shows the weight loss after treatment with molecules 007, 008, 012, and 016 from day 1 to day 8. Data are presented as mean and standard error (SEM). Semaglutide was tested in parallel as a comparative control. [Figure 2 (1)] 2A-2E show the in vivo activity of molecules 001, 002, 012, and 019 in db / db mice. FIG. 2A shows fasting glucose after a single dose of molecule 001. FIG. 2B shows fasting glucose after a single dose of molecule 002. Data are shown as the mean and standard error of the mean (SEM). FIG. 2C shows non-fasting glucose after a single dose of molecule 012. FIG. 2D shows non-fasting glucose after a single dose of molecule 019, and FIG. 2E shows the area under the curve from 0 to 48 hours for non-fasting glucose after a single dose of molecule 019. To assess glucose, 10-week-old male db / db mice were subcutaneously administered the indicated GLP-1 polypeptide conjugate at the indicated dosage. Fasting glucose (Figures 2A and 2B) or non-fasting glucose (Figures 2C and 2D) were measured at various time points, and five animals were used for each group. Delta blood glucose is glucose subtracted from baseline levels. Data are presented as the mean and standard error of the mean (SEM). [Figure 2 (2)](Continued) Figures 2A-2E show the in vivo activity of molecules 001, 002, 012, and 019 in db / db mice. Figure 2A shows fasting glucose after a single dose of molecule 001. Figure 2B shows fasting glucose after a single dose of molecule 002. Data are shown as the mean and standard error of the mean (SEM). Figure 2C shows non-fasting glucose after a single dose of molecule 012. Figure 2D shows non-fasting glucose after a single dose of molecule 019, and Figure 2E shows the area under the curve from 0 to 48 hours for non-fasting glucose after a single dose of molecule 019. To assess glucose, 10-week-old male db / db mice were subcutaneously dosed with the indicated GLP-1 polypeptide conjugate at the doses shown. Fasting glucose (Figures 2A and 2B) or non-fasting glucose (Figures 2C and 2D) were measured at various time points, and five animals were used for each group. Delta blood glucose is glucose subtracted from baseline levels. Data are presented as the mean and standard error of the mean (SEM). [Diagram 3] Figures 3A-3B show in vivo efficacy in diet-induced obese (DIO) mice. To evaluate the effect of each GLP-1 polypeptide conjugate on body weight, food intake, and glucose levels, 22-week-old DIO mice (C57BL / 6 mice on a high-fat diet for 13 weeks) were subcutaneously administered various concentrations of the indicated GLP-1 polypeptide conjugate every other day for 25 days. Figure 3A shows the body weight change with dose titration of 10, 30, and 100 nmol / kg of molecule 012. Figure 3B shows the fasting glucose change upon treatment. Data are expressed as mean and standard error (SEM). [Figure 4(1)]Figures 4A-4E show in vivo efficacy in Alzheimer's disease (APP / PS1, APPswe / PSEN1dE9) mice. To evaluate the effect of each GLP-1 polypeptide conjugate on cognitive function (Morris water maze and Y-maze), brain Aβ plaque deposition, and hippocampal pyramidal neuron count, 6-month-old APP / PS1 male mice were administered various concentrations of the indicated GLP-1 polypeptide conjugate (molecule 012) subcutaneously for 10 weeks, once daily (QD) or every other day (Q2D). Figure 4A shows quadrant occupancy changes with 30 and 100 nmol / kg molecule 012, respectively. Figure 4B shows alternating percentage changes with 30 and 100 nmol / kg molecule 012, respectively. Figure 4C shows Aβ plaque deposition changes in the cerebral cortex with 30 and 100 nmol / kg molecule 012, respectively. Figure 4D shows the Aβ plaque deposition changes in the hippocampus with molecule 012 at 30 and 100 nmol / kg, respectively. Figure 4E shows the change in the number of neurons in the hippocampus with molecule 012 at 30 and 100 nmol / kg, respectively. Data are expressed as the mean and standard error of the mean (SEM). Vehicle-treated wild-type mice were used as controls. Vehicle-treated Alzheimer's disease (APP / PS1) mice were used as controls. Semaglutide-treated Alzheimer's disease (APP / PS1) mice were also used as controls. [Figure 4 (2)](Continued) Figures 4A-4E show in vivo efficacy in Alzheimer's disease (APP / PS1, APPswe / PSEN1dE9) mice. To evaluate the effect of each GLP-1 polypeptide conjugate on cognitive function (Morris water maze and Y-maze), brain Aβ plaque deposition, and hippocampal pyramidal neuron count, 6-month-old APP / PS1 male mice were administered various concentrations of the indicated GLP-1 polypeptide conjugate (molecule 012) subcutaneously for 10 weeks, once daily (QD) or every other day (Q2D). Figure 4A shows quadrant occupancy changes with 30 and 100 nmol / kg molecule 012, respectively. Figure 4B shows alternating percentage changes with 30 and 100 nmol / kg molecule 012, respectively. Figure 4C shows Aβ plaque deposition changes in the cerebral cortex with 30 and 100 nmol / kg molecule 012, respectively. Figure 4D shows the Aβ plaque deposition changes in the hippocampus with molecule 012 at 30 and 100 nmol / kg, respectively. Figure 4E shows the change in the number of neurons in the hippocampus with molecule 012 at 30 and 100 nmol / kg, respectively. Data are expressed as the mean and standard error of the mean (SEM). Vehicle-treated wild-type mice were used as controls. Vehicle-treated Alzheimer's disease (APP / PS1) mice were used as controls. Semaglutide-treated Alzheimer's disease (APP / PS1) mice were also used as controls. [Figure 5(1)] 5A-5E show all sequences disclosed in this disclosure (except SEQ ID NO:81). [Figure 5 (2)] (Continued) Figures 5A-5E show all sequences disclosed in this disclosure (except SEQ ID NO:81). [Figure 5 (3)] (Continued) Figures 5A-5E show all sequences disclosed in this disclosure (except SEQ ID NO:81). [Figure 5 (4)] (Continued) Figures 5A-5E show all sequences disclosed in this disclosure (except SEQ ID NO:81). [Figure 5 (5)](Continued) Figures 5A-5E show all sequences disclosed in this disclosure (except SEQ ID NO:81). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0180] The following description of the invention is intended to merely illustrate various embodiments of the invention. Therefore, the specific modifications discussed should not be interpreted as limitations on the scope of the invention. It is believed that various equivalents, variations, and modifications can be realized without departing from the scope of the invention, and it is understood that such equivalent embodiments should be included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0181] definition As used herein, the term "amino acid" refers to an amine (-NH 2 ) and carboxyl (-COOH) functional groups.

[0182] The term "naturally occurring" amino acid residue, as used herein, refers to an amino acid residue found in a natural protein or peptide, including all of its D and L stereoisomers, if their 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 The D stereoisomers include, but are not limited to, the twenty common amino acids, including D-amino acids, D-aspartic acid (D-aspartic acid or K), arginine (Arg or R), 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, and the like.

[0183] 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.

[0184] As used herein, "unnatural" 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 naturally occurring amino acids. Modified amino acids or mimetics can be created by adding chemical entities such as carbohydrate groups, phosphate groups, farnesyl groups, isofarmesyl groups, fatty acid groups, linkers for conjugation, functionalization, or other modifications. Unnatural 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), a-aminobutyric acid (Abu), tert-butylglycine (Tle), b-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-methyl-histidine, and the like. Examples of suitable carboxylic acids include, but are not limited to, 1-aminocyclopropyl-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.

[0185] Introduction of unnatural amino acids into a polypeptide can be accomplished 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 present 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.

[0186] "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) compared 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 to be identical residues. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be accomplished using publicly available tools such as, for example, BLASTN, BLASTp (available at the National Center for Biotechnology Information (NCBI) website, see also Altschul SF 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 may use the default parameters provided by the tool or may customize the parameters as needed for the alignment, for example by selecting an appropriate algorithm.

[0187] "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 (e.g., Met, Ala, Val, Leu, and Ile), between residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between residues with acidic side chains (e.g., Asp, Glu), between amino acids with basic side chains (e.g., His, Lys, and Arg), or between residues with aromatic side chains (e.g., Trp, Tyr, and Phe).As is known in the art, conservative substitution usually does not cause significant changes in protein conformation, and therefore can retain the biological activity of protein.

[0188] The term "functional form" as used herein refers to various forms of a parent molecule (such as variants, fragments, fusions, derivatives, and mimetics) that, despite having differences in amino acid sequence or chemical structure, still retain substantial biological activity of the parent molecule. As used herein, the phrase "retain substantial biological activity" means exhibiting at least a portion (e.g., at least no less than about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) 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.

[0189] The term "variant" as used herein refers to a polypeptide that has at least 70% sequence identity with a parent polypeptide. A variant may differ from a parent peptide by one or more amino acid residues. For example, a variant may have substitution, addition, deletion, insertion, or truncation of one or more amino acid residues of the parent polypeptide.

[0190] The term "fragment" as used herein refers to a subsequence of a parent polypeptide of any length. A fragment may still retain at least partial function of the parent polypeptide.

[0191] 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 can be, for example, alkylation, acylation, esterification, amidation, phosphorylation, glycosylation, labeling, methylation of one or more amino acids, or conjugation with one or more moieties.

[0192] The term "mimetic" as used herein refers to a molecular structure that serves as a substitute for an amino acid, peptide, polypeptide, or fusion polypeptide.For example, an amino acid mimic as 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 amides, methacryloyl or acryloyl derivatives of β-, γ-, δ-imino acids (such as piperidine-4-carboxylic acid), and the like.

[0193] 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 any combination thereof.

[0194] The term "vector" as used herein refers to a vehicle into which a polynucleotide encoding a protein can be operatively inserted to cause expression of the protein. A vector can be used to transform, transduce, or transfect a host cell to cause expression of the genetic element it carries in 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, herpes viruses (e.g., herpes simplex viruses), pox viruses, baculoviruses, papilloma viruses, and papova viruses (e.g., SV40). A vector 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 may contain origins of replication. Vectors may also contain materials that aid their entry into cells, including but not limited to viral particles, liposomes, or protein coatings. Vectors may be expression vectors or cloning vectors. The present disclosure provides vectors (e.g., expression vectors) that contain 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, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, p These 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, pEF-Bos, and the like.

[0195] As used herein, the phrase "host cell" refers to a cell into which an exogenous polynucleotide and / or vector has been introduced.

[0196] The term "pharmaceutical acceptable" indicates that the specified carrier, vehicle, diluent, excipient, and / or salt is generally chemically and / or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.

[0197] As used herein, the term "subject" or "individual" or "animal" or "patient" refers 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.

[0198] Polypeptide Conjugates In one embodiment, the present disclosure provides a polypeptide conjugate comprising a polypeptide portion and a conjugate portion. The polypeptide portion comprises a single bioactive peptide and a peptide linker. The bioactive peptide is attached to the N-terminus of the peptide linker and comprises a GLP-1 receptor agonist. The conjugate portion comprises a first clearance reducing moiety (CRM) conjugated to a first CRM residue in the peptide linker. Wherein the first CRM residue is at least 5 amino acid residues (excluding the CRM residue) away from the C-terminal amino acid residue of the bioactive peptide (e.g., the GLP-1 receptor agonist).

[0199] The terms "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 peptides or polypeptides provided herein can include naturally occurring or non-naturally occurring amino acid residues, or both. The polypeptides and peptides provided herein can include any suitable length of amino acid residues, for example, at least 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.

[0200] A polypeptide conjugate comprises a single, i.e., only one, bioactive peptide. "Single" in relation to bioactive peptide is intended to mean that a polypeptide conjugate does not contain two or more different bioactive peptides, each of which is attached to a peptide linker. However, a single bioactive peptide may comprise fragments or parts from different bioactive peptides fused together, for example, as a hybrid or chimera, as long as these parts are integrated as one peptide and are not each attached to a linker. However, when two different bioactive peptides are attached to the N-terminus and C-terminus of a linker, respectively, it is not a single bioactive peptide as used in the present disclosure.

[0201] The term "bioactive peptide" as used herein refers to a peptide that has a biological function or activity, such as a physiological function or a therapeutic function. In certain embodiments, a bioactive peptide has a therapeutic activity. A peptide that does not have a biological function when used alone is not a bioactive peptide. For example, a peptide linker, for example, when used alone, is not a bioactive peptide unless it has its own biological function or activity.

[0202] Bioactive peptides include glucagon-like peptide-1 (GLP-1) receptor agonists.

[0203] The term "glucagon-like peptide-1 (GLP-1) receptor" (also referred to as GLP1R) is a receptor protein found on beta cells in the pancreas and on neurons in the brain, which contains one extracellular domain and one transmembrane domain. The extracellular domain can bind to the C-terminal helix of GLP-1, and the transmembrane domain can bind to the N-terminal region of GLP-1. GLP-1 receptors are involved in the control of blood glucose levels by enhancing insulin secretion. When expressed in the brain, GLP-1 receptors can also be involved in the control of appetite.

[0204] As used herein, the term "glucagon-like peptide-1 (GLP-1) receptor agonist" or "GLP-1 receptor agonist" refers to a molecule capable of binding to and activating the GLP-1 receptor. A GLP-1 receptor agonist can elicit a GLP-1 receptor response that is the same as or a partial magnitude of the natural ligand.

[0205] The term "clearance modifying moiety" or "CRM" as used herein refers to a moiety that can modify one or more pharmacokinetic (PK) properties (e.g., increasing half-life in vivo). Examples of CRMs can include, but are not limited to, fatty acids, polyethylene glycol (PEG), glucuronic acid or other sugar-based linkers, polar, positively or negatively charged groups that can increase the rate of hydrolysis of the succinimidyl ring, reduce or minimize the rate of reverse Michael reaction, and thus reduce or minimize the rate of loss of drug, as well as the rate of loss of the linker group from bioactive peptides to other thiol-containing proteins and small molecules.

[0206] As used herein, a "CRM residue" refers to an amino acid residue that is conjugated to a CRM.

[0207] The term "conjugate" as used herein refers to a compound resulting from two or more molecules that are joined together to form one physical entity.For example, the conjugate of the present disclosure refers to a compound resulting from a polypeptide and one or more clearance modifying moieties that are joined together.The molecules may 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 may be attached together by covalent bonds.The bond may be permanent or reversible.In some embodiments, certain cleavable or non-cleavable linkages may be included.

[0208] Although conjugation of a CRM to a GLP-1 receptor agonist can extend the half-life of the GLP-1 receptor agonist, it can also adversely affect the biological activity of the GLP-1 receptor agonist, making it less active than its unconjugated counterpart. For example, the conjugated GLP-1 derivative semaglutide has a significantly extended half-life, but suffers from a 939-fold reduction in GLP-1 receptor binding in the presence of human serum albumin (I) compared to its unconjugated counterpart (J.Med.Chem.2015,58,7370-7380).

[0209] The inventors have unexpectedly found that certain CRM-conjugated polypeptides containing GLP-1 receptor agonists can have both extended half-life and retained biological activity when CRM is conjugated outside the GLP-1 receptor agonist on a peptide linker attached to its C-terminus.Interestingly, the inventors have found that the distance between the C-terminus of the GLP-1 receptor agonist and the conjugation site is particularly important.If CRM conjugation on the peptide linker is located close to the C-terminus of the GLP-1 receptor agonist, it is believed to significantly reduce biological activity, which can be even much lower than conjugation on the GLP-1 receptor agonist itself.However, if conjugation is located far enough away from the C-terminus of the GLP-1 receptor agonist, the CRM-conjugated GLP-1 receptor agonist can have both extended half-life and retained biological activity. Such unexpected effects are found with certain GLP-1 receptor agonists and certain CRMs.

[0210] Here, according to some embodiments of the polypeptide conjugate, the first CRM residue is at least 5 amino acid residues (excluding the CRM residue) away from the C-terminal amino acid residue of the biologically active peptide (e.g., a GLP-1 receptor agonist), such as 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, 33, 35, 38, 40, 43, 45, 48, 50, 53, 55, 58, 60, 63, 65, 68, 70, 73, 75, or 78 amino acid residues. In some embodiments of the polypeptide conjugate, the peptide linker may have a length of at least 10 amino acid residues, such as at least 12, 24, 32, 40, 48, 50, 60, 70, or 80 amino acid residues.

[0211] In certain embodiments, the first CRM residue is positioned within the peptide linker at a position proximal to the C-terminus of the peptide linker, which can be 20-50 amino acid residues away from the C-terminal amino acid residue of the biologically active peptide (e.g., a GLP-1 receptor agonist). Further details will be provided below.

[0212] According to some embodiments, the polypeptide conjugate is monoconjugated, i.e. the first CRM is the only conjugate, and the polypeptide conjugate is conjugated to the first CRM only at the first CRM residue (within the peptide linker).

[0213] According to some other embodiments, the polypeptide conjugate is dual conjugated, i.e., the polypeptide conjugate is conjugated to two conjugates: one such conjugate is a first CRM conjugated to the polypeptide conjugate at a first CRM residue (within the peptide linker), and the other such conjugate is a second CRM conjugated to the polypeptide conjugate at a second CRM residue located within the bioactive peptide (i.e., a GLP-1 receptor agonist such as GLP).

[0214] Further details regarding GLP-1 receptor agonists, CRMs and CRM residues, and polypeptide conjugates are provided below.

[0215] GLP-1 receptor agonists In certain embodiments, the GLP-1 receptor agonist comprises GLP-1. As used herein, the term "glucagon-like peptide-1" or "GLP-1" 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.

[0216] The term "native GLP-1 peptide" as used herein refers to native human glucagon-like peptide-1 (GLP-1(7-37)), the sequence of which is set forth in SEQ ID NO: 1. As used herein, when referring to specific amino acid residues in SEQ ID NO: 1 (i.e., GLP-1(7-37)), the numbering of GLP-1(1-37) is followed. In other words, SEQ ID NO: 1 corresponds to GLP-1(7-37), and thus the first residue in SEQ ID NO: 1, which is a histidine (H), is referred to as 7H, meaning that it corresponds to the seventh residue in GLP(1-37); the 31st residue in SEQ ID NO: 1, which is a glycine (G), is referred to as 37G, meaning that it corresponds to the 37th residue in GLP(1-37).

[0217] The functional form of the native GLP-1 peptide can activate the GLP-1 receptor at a level comparable to or at least 20% (or at least 30%, 40%, 50%, 60%, 70%, 80%, 90%) of that of the native GLP-1 peptide. Activation of the GLP-1 receptor typically initiates a signal transduction pathway that results in insulinotropic or other physiological effects, as known in the art. The functional form of the native GLP-1 peptide can contain one or more substitutions, additions, or deletions compared to SEQ ID NO: 1. Many functional forms of the native GLP-1 peptide are known in the art, including, but not limited to, liraglutide, semaglutide, dulaglutide, albiglutide, and those disclosed in WO2000055203A1, WO98 / 08871, WO2006 / 097537, the disclosures of which are incorporated herein in their entirety.

[0218] In certain embodiments, the GLP-1 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:1, while retaining substantial biological activity of SEQ ID NO:1.

[0219] In certain embodiments, the GLP-1 comprises at most 9, 8, 7, 6, 5, 4, 3, or 2 mutations (e.g., additions, deletions, substitutions) compared to SEQ ID NO: 1, while retaining substantial biological activity of SEQ ID NO: 1. In certain embodiments, the GLP-1 comprises at least 2, 3, 4, 5, 6, 7, 8, or 9 mutations (e.g., additions, deletions, substitutions) compared to SEQ ID NO: 1, while retaining substantial biological activity of SEQ ID NO: 1.

[0220] Those skilled in the art will understand that various amino acid substitutions, such as conservative amino acid substitutions, can be made in any of the sequences of the polypeptide fragments described herein without necessarily reducing their 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.

[0221] Various substitutions have been introduced into native GLP-1 peptide and have been shown to retain or even improve its biological activity.In some embodiments of the polypeptide conjugates provided herein, GLP-1 comprises or consists of one or more mutations at positions selected from the group consisting of A8, G22, K26, K34, and R36, or any combination thereof, compared to SEQ ID NO:1.For example, substitutions at A8 are useful for preventing DPP4 enzymatic cleavage at this residue, substitutions at G22 are desirable for improving activity and solubility, and substitutions at R36 are useful for reducing immunogenicity.Examples of substitutions at these positions include, but are not limited to, A8G, A8Aib, A8T, G22E, K34R, R36G, and the substitutions described in U.S. Patent No. 8,273,854, which is incorporated herein in its entirety.

[0222] In some embodiments of the polypeptide conjugate, the GLP-1 comprises or consists of one or more substitutions selected from the group consisting of A8Aib, G22E, K26R, K34R, and R36G, or any combination thereof.

[0223] In some embodiments of the polypeptide conjugate, GLP-1 is a molecule comprising a polypeptide fragment that has substantially the biological activity of wild-type human GLP-1 (SEQ ID NO:1) and has an amino acid sequence with at least 50% (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) sequence identity to wild-type human GLP-1.

[0224] In some embodiments, GLP-1 comprises an amino acid sequence having at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, or 95%) sequence identity to SEQ ID NO:1, while retaining substantial biological activity of SEQ ID NO:1.

[0225] In some embodiments, GLP-1 is 7 X 8 EGTFTSDVSSYLEX 22 X 23 AAX 26 X 27 FIX 30 WLVX 34 GX 36 G (SEQ ID NO:2), wherein: X 7 is H, imidazole-4-acetate (IA), or imidazole propionic acid (IPA); X 8 is A, G, S, V, Aib, T, I, or L; X 22 is G or E;X 23 is Q, C, or K; X 26 is K, R, or C; X 27 is E, K, or C;X 30 is A, C, or K; X 34 is R, K, or C, and X 36 is R or G.

[0226] In some embodiments of GLP-1, X 7 is H and X 8 is G or Aib.

[0227] In certain embodiments, the GLP-1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 3-9 listed below (the genotype is indicated after the sequence identification number): SEQ ID NO:1 (WT GLP-1) SEQ ID NO: 3 (8Aib, 34R, 36G), SEQ ID NO: 4 (8Aib, 26C, 34R, 36G), SEQ ID NO:5 (8Aib, 26R, 34R, 36G), SEQ ID NO: 6 (8Aib, 22E, 34R, 36G), SEQ ID NO: 7 (8Aib, 22E, 26C, 34R, 36G), SEQ ID NO: 8 (8Aib, 22E, 26R, 34R, 36G), Sequence number 9 (8G, 22E, 26R, 34R, 36G).

[0228] In certain embodiments, the GLP-1 receptor agonist in the polypeptide conjugate comprises or is GLP-1 as provided herein. In some embodiments, the GLP-1 in the polypeptide conjugate provided herein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-9.

[0229] Peptide Linker In the polypeptide conjugates disclosed herein, the bioactive peptide, ie, a GLP-1 receptor agonist (eg, GLP-1), is attached to the N-terminus of a peptide linker.

[0230] wherein the GLP-1 receptor agonist can be optionally attached to a peptide linker via a direct bond (e.g., a covalent bond such as a peptide bond), and the peptide linker can be optionally composed of amino acid residues linked together by peptide bonds. Optionally, the peptide linker can further include one or more unnatural amino acids.

[0231] In some embodiments of the polypeptide conjugate, the peptide linker may have a length of at least 10 amino acid residues, such as at least 12, 24, 32, 40, 48, 50, 60, 70, or 80 amino acid residues.

[0232] Any suitable polypeptide linker may be used. For example, the polypeptide linker may 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), and lysine (K). In some embodiments, the polypeptide linker may be composed of a majority of amino acids that are sterically unhindered, such as glycine and alanine. In some embodiments, the linker is polyglycine, polyalanine, a combination of glycine and alanine (such as poly(Gly-Ala)), or a combination of glycine and serine (such as poly(Gly-Ser)).

[0233] In some embodiments, the peptide linker consists of amino acid residues selected from the group consisting of G, Q, A, E, P, S, and T, except for the first CRM residue.

[0234] In some embodiments, the peptide linker comprises a first sequence and a second sequence. The first sequence consists of one or more repeats of the repeat sequence and is connected to the N-terminus of the second sequence. The first CRM residue is in the second sequence and can optionally be a lysine or cysteine ​​residue.

[0235] In certain embodiments, the repeat sequence comprises or consists of a sequence selected from the group consisting of SEQ ID NOs: 10-40 and GS.

[0236] In some embodiments, the repeat sequence has the sequence of SEQ ID NO: 13 (GAQP).

[0237] Optionally, wherein the number of the one or more repeats of the repeat sequence is an integer from 1 to 30. In certain embodiments, the number of the one or more repeats of the repeat sequence is selected from the group consisting of 5, 7, 9, and 11.

[0238] In certain embodiments, the second sequence of the peptide linker has the sequence of SEQ ID NO: 41 (GQKP) or SEQ ID NO: 42 (GQCP).

[0239] In certain embodiments, the polypeptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 43-50 and 82-84.

[0240] It is noted that in addition to the above arrangement, where the peptide linker comprises a first sequence and a second sequence, and the first sequence is composed of one or more repeats of a repeat sequence, there can be other arrangements.In certain cases, the peptide linker can comprise or consist of more than one repeat sequence.For example, the polypeptide linker comprises or consists of two, three, or four different repeat sequences.In certain embodiments, the polypeptide linker comprises or consists of sequential or tandem repeats of different repeat sequences.

[0241] CRM residue Without wishing to be bound by any theory, it is believed that CRM (or fatty acid) conjugation to GLP-1 may reduce its activity in the presence of I. For example, a reduction in GLP-1 activity has been reported for semaglutide, which has a fatty acid conjugated to K26 of GLP-1 (8Aib, 36R) and showed a significant reduction in activity in the presence of I compared to its unconjugated counterpart. Since I is present in human blood and is unavoidable under physiological conditions, the reduction in GLP-1 receptor agonist activity in the presence of I is likely to impair the therapeutic activity of the protein conjugate. However, some of the polypeptide conjugates provided herein may retain most of their GLP-1 activity in the presence of I despite CRM conjugation. In such an embodiment, the polypeptide conjugates provided herein are monoconjugated and have a CRM conjugated to a peptide linker rather than to a biologically active peptide (e.g., GLP-1).

[0242] In some of these embodiments, such monoconjugated polypeptide conjugates provided herein have increased GLP-1 receptor agonist activity in the presence of human serum albumin (I) compared to a comparison polypeptide conjugate that does not have a peptide linker and has a CRM conjugated on GLP-1.In certain embodiments, the GLP-1 receptor agonist activity in the presence of I can be determined in an in vitro assay for GLP-1 receptor activation, either in a cell-free assay such as a cAMP assay or a cell-based assay such as a reporter cell assay, as known in the art.In certain embodiments, the comparison polypeptide conjugate is semaglutide.In certain embodiments, the monoconjugated polypeptide conjugates provided herein activate GLP-1 receptor in the presence of I with an EC50 of 50% or less (or 40%, 30%, 20%, 10%, 5%, or 3% or less) of that of semaglutide in the presence of human serum albumin (I), determined in the same or equivalent assay. In certain embodiments, the monoconjugated polypeptide conjugates provided herein activate the GLP-1 receptor in the absence of I with an EC50 comparable to (e.g., 20% to 300%) that of semaglutide in the absence of I, as determined in the same or equivalent assay. In certain embodiments, the amount of human serum albumin (I) allows for assessment of the effect of I binding on GLP-1 receptor activation. In certain embodiments, the in vitro assay for GLP-1 receptor activation is performed in the presence of at least 0.5%, 1%, 1.2%, 1.5%, 1.8%, or 2% I.

[0243] In certain embodiments, the monoconjugated polypeptide conjugates provided herein bind to I with a binding affinity (KD) comparable to that of semaglutide (e.g., about 70% to about 500%), as determined in the same or an equivalent assay.

[0244] In certain embodiments, the monoconjugated polypeptide conjugates provided herein have at least a comparable terminal half-life compared to a comparative polypeptide conjugate that does not have a peptide linker and has a CRM conjugated on GLP-1, such as semaglutide, determined under comparable in vivo investigation conditions and in the same type of experimental animal.Suitable animals for terminal half-life determination include, for example, mice, rats, minipigs, or monkeys.In certain embodiments, terminal half-life is determined in suitable animals after a single dose of intravenous, subcutaneous, or oral administration at a dose appropriate to provide therapeutic efficacy.

[0245] In certain embodiments, the polypeptide conjugate further comprises a second CRM conjugated to the second CRM residue.In other words, the present disclosure also provides a double-conjugated polypeptide conjugate.The inventors have unexpectedly found that the second CRM conjugation can further extend the half-life of the polypeptide conjugate.In certain embodiments, the double-conjugated polypeptide conjugate having both the first and second CRM conjugation can have an extended half-life that is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, or even longer than the corresponding polypeptide conjugate having only one CRM conjugation (e.g., semaglutide).

[0246] The second CRM residue can be in any suitable position. In certain embodiments, the second CRM residue is in the GLP-1 receptor agonist or in the peptide linker.

[0247] In certain embodiments, the second CRM residue is at K26 of GLP-1 peptide.It has been reported that conjugation with two fatty acid moieties on GLP-1 peptide can significantly reduce activity or even render the conjugated GLP-1 peptide ineffective.Therefore, it has been unexpectedly found by the present inventors that conjugation on both GLP-1 (e.g., K26) and on the peptide linker at a position sufficiently distant from the C-terminus of GLP-1 peptide can minimize the negative effect of fatty acid conjugation on the biological activity of GLP-1 polypeptide in the presence of I.

[0248] In some of these embodiments, such dual-conjugated polypeptide conjugates provided herein have comparable or acceptable GLP-1 receptor agonist activity in the presence of I compared to a comparison polypeptide conjugate having no peptide linker and a CRM conjugated onto GLP-1. In certain embodiments, a dual-conjugated polypeptide conjugate provided herein activates the GLP-1 receptor in the presence of I with an EC50 of 2000% or less (or 1500%, 1000%, 900%, 800%, 700%, 600%, or 500% or less) of that of a comparison polypeptide conjugate having no peptide linker and a CRM conjugated onto GLP-1, such as semaglutide, in the presence of I, as determined in the same or equivalent assay. In certain embodiments, the dual-conjugated polypeptide conjugates provided herein activate the GLP-1 receptor in the absence of I with an EC50 that is comparable to (e.g., 500%, 400%, 300%, or 200% or less than or even less than) that of a comparative polypeptide conjugate having no peptide linker and a CRM conjugated on GLP-1, such as semaglutide, as determined in the same or an equivalent assay. In certain embodiments, the dual-conjugated polypeptide conjugates provided herein activate the GLP-1 receptor with a first EC50 in the presence of I and with a second EC50 in the absence of I, the first EC50 being higher than the second EC50 but not greater than 1000-fold that of the second EC50, e.g., the first EC50 (in the presence of I) being not greater than 900-fold, 800-fold, 700-fold, 600-fold, or 500-fold that of the second EC50 (in the absence of I). In certain embodiments, I is present in an in vitro assay for GLP-1 receptor activation in an amount suitable to allow assessment of the effect of I binding on GLP-1 receptor activation.In certain embodiments, the in vitro assay for GLP-1 receptor activation is performed in the presence of a suitable amount of human serum albumin (I) that allows for the assessment of the effect of I binding on GLP-1 receptor activation. In certain embodiments, the in vitro assay for GLP-1 receptor activation is performed in the presence of at least 0.5%, 1%, 1.2%, 1.5%, 1.8%, or 2% I.

[0249] In certain embodiments, the dual-conjugated polypeptide conjugates provided herein bind to I with a higher binding affinity, as represented by a KD value that is significantly lower (e.g., 50%, 40%, 30%, or 20% or less) than that of a comparative polypeptide conjugate having no peptide linker and a CRM conjugated on GLP-1, such as semaglutide, determined in the same or an equivalent assay.

[0250] In certain embodiments, the dual-conjugated polypeptide conjugates provided herein have enhanced pharmacokinetic properties compared to a comparative polypeptide conjugate having no peptide linker and having a CRM conjugated onto a biologically active peptide (e.g., GLP-1), the pharmacokinetic properties being determined by measuring blood concentrations of the polypeptide conjugate following administration of a therapeutically effective dose to a subject.

[0251] In certain embodiments, the double-conjugated polypeptide conjugates provided herein have at least 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 180%, 200%, 300%, or 400% increased terminal half-life than that of a comparative polypeptide conjugate having no peptide linker and a CRM conjugated on GLP-1, such as semaglutide, determined under comparable in vivo investigation conditions and in the same type of experimental animal. Suitable animals for terminal half-life determination include, for example, mice, rats, minipigs, or monkeys. In certain embodiments, terminal half-life is determined in suitable animals after intravenous or subcutaneous administration of a single dose at a dose appropriate to provide therapeutic efficacy.

[0252] In certain embodiments, the doubly conjugated polypeptide conjugates provided herein have blood or plasma or serum concentrations that remain within the therapeutic window for the polypeptide conjugate for at least about 50% longer (60%, 70%, 80%, 90%, 100%, 120%, 150%, 180%, 200%, 300%, or 400% longer) than those of a comparative polypeptide conjugate that does not have a peptide linker and has a CRM conjugated on GLP-1, such as semaglutide, as determined under comparable in vivo study conditions and in the same type of experimental animals. The term "therapeutic window" as used herein refers to the range of concentration levels of the polypeptide conjugate in blood or plasma or serum that provides therapeutic benefit or efficacy for the disease condition being treated without unacceptable toxicity. The range of concentration levels can be from the minimum concentration that provides a therapeutic response to the maximum concentration that provides a therapeutic response without inducing unacceptable toxicity.

[0253] In certain embodiments, the double-conjugated polypeptide conjugates provided herein provide an extended duration of therapeutic efficacy compared to a comparative polypeptide conjugate having no peptide linker and a CRM conjugated on GLP-1, such as semaglutide, determined under comparable in vivo investigation conditions and in the same type of experimental animals.Such extended therapeutic efficacy can be characterized by the area under the curve (AUC) for a time-response curve, which can be plotted after a single dose or after repeated doses that are appropriate to provide the intended therapeutic effect in subjects with metabolic pathology (e.g., disease model animals).Extended therapeutic efficacy duration can also be characterized by the duration of therapeutic response.

[0254] In certain embodiments, the therapeutic response includes a reduction in body weight, a reduction in food intake, or a reduction in glucose levels (fasting glucose levels or non-fasting glucose levels). In certain embodiments, the dual-conjugated polypeptide conjugates provided herein have an increase in AUC for the time-response curve of at least 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 180%, 200% over that of a comparative polypeptide conjugate having no peptide linker and a CRM conjugated on GLP-1, such as semaglutide, determined under comparable in vivo study conditions and in the same type of experimental animal. In certain embodiments, the dual-conjugated polypeptide conjugates provided herein have an increase in the duration of therapeutic response of at least 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 180%, 200%, 300%, or 400% over that of a comparative polypeptide conjugate having no peptide linker and a CRM conjugated on GLP-1, such as semaglutide, determined under comparable in vivo study conditions and in the same type of experimental animal. In certain embodiments, the experimental animal for therapeutic response is a disease model animal, such as a db / db mouse or a diet-induced obese (DIO) animal.

[0255] CRM residue Within the scope of the disclosure provided herein, depending on the different CRMs used, different CRM residues may be used in the polypeptide conjugate, and one or two CRM residues may be present in the polypeptide conjugate that mediate conjugation of the CRM to the polypeptide conjugate.

[0256] In certain embodiments, the polypeptide conjugate may be mono-conjugated with a CRM.

[0257] In certain embodiments, the first CRM residue in the peptide linker is a lysine residue and the polypeptide conjugate comprises only one lysine residue. In such embodiments, the peptide linker comprises only one lysine residue and the GLP-1 receptor agonist comprises no lysine residues, and thus the naturally occurring residues found in the native GLP-1(1-37) sequence, i.e., K26 and K34, can be optionally substituted with non-lysine residues (e.g., R, Q, A, G, H, S, and T, etc.). In certain embodiments, the GLP-1 comprises substitutions of K26R and K34R.

[0258] It is noted that throughout this specification, when a CRM residue (e.g., K or C) is present in GLP-1, the position of the CRM residue is identified with reference to the amino acid sequence of GLP-1(1-37). For example, K26 indicates that the 26th position (corresponding to the 20th position in the amino acid sequence of GLP-1(7-37), i.e., SEQ ID NO:1) is K with reference to the amino acid sequence of GLP-1(1-37). Similarly, K34 indicates that the 34th position (corresponding to the 28th position in the amino acid sequence of GLP-1(7-37), i.e., SEQ ID NO:1) is K with reference to the amino acid sequence of GLP-1(1-37).

[0259] In certain embodiments, the first CRM residue in the peptide linker is a cysteine ​​residue, and the polypeptide conjugate contains only one cysteine ​​residue.In such embodiments, the peptide linker contains only one cysteine ​​residue, and the GLP-1 receptor agonist does not contain a cysteine ​​residue.In certain embodiments, the single bioactive peptide polypeptide in the polypeptide conjugate contains GLP-1.Natural GLP-1 does not contain any cysteine ​​residue, and therefore any GLP-1 derivative (including those provided herein) can be used as long as it does not contain a cysteine ​​residue.

[0260] In certain embodiments, the first CRM residue is a non-natural amino acid residue, and the polypeptide conjugate comprises only one non-natural amino acid residue as a CRM residue. Non-natural amino acids can contain a variety of functional or reactive groups that can provide additional functionality and / or reactivity. Particular non-natural amino acids that are useful for the purpose of conjugating moieties to the fusion polypeptides of the present disclosure include those with side chains that have an azide, an alkyne, an alkene, a cycloalkyne, or a halide.

[0261] In the monoconjugated polypeptide conjugate, when the first CRM residue in the peptide linker is a lysine residue, GLP-1 may comprise the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:8, where the peptide linker may optionally comprise an amino acid sequence selected from the group consisting of SEQ ID NO:43, SEQ ID NO:45, and SEQ ID NO:46. In certain embodiments, the polypeptide conjugate comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:52, 55, 56, and 60-62, as set forth below (the first CRM residue is listed after the sequence identification number): SEQ ID NO:52(76K), SEQ ID NO:55(68K), SEQ ID NO:56(60K), SEQ ID NO: 60 (76K), SEQ ID NO: 61 (68K), or Sequence number 62 (60K).

[0262] In a monoconjugated polypeptide conjugate, when the first CRM residue in the peptide linker is a cysteine ​​residue, GLP-1 may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 5, 6, and 9, where the peptide linker may optionally comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 47, SEQ ID NO: 49, and SEQ ID NO: 50. In certain embodiments, the polypeptide conjugate comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-71 and 75-80, as set forth below (the first CRM residue is listed after the sequence identification number): SEQ ID NO: 66(76C), SEQ ID NO: 67(68C), SEQ ID NO: 68(60C), SEQ ID NO: 69(76C), SEQ ID NO: 70(68C), SEQ ID NO:71(60C), SEQ ID NO: 75(76C), SEQ ID NO: 76(68C), SEQ ID NO: 77(60C), SEQ ID NO: 78(76C), SEQ ID NO: 79(68C), Sequence number 80 (60C).

[0263] In certain embodiments, the polypeptide conjugate further comprises a second CRM residue, i.e., the polypeptide conjugate is dual-conjugated, having two CRMs (i.e., a first CRM and a second CRM) conjugated at two CRM residues (i.e., a first CRM residue and a second CRM residue), respectively.

[0264] In certain embodiments, just like the first CRM residue, the second CRM residue is within the peptide linker. In such embodiments, the second CRM residue can be at any suitable distance from the first CRM residue, so long as both CRM residues can be properly conjugated.

[0265] In certain embodiments, the second CRM residue is in a GLP-1 receptor agonist, such as GLP-1. In certain embodiments, the second CRM residue is a naturally occurring residue found in a native GLP-1 sequence, or an introduced residue, for example, by substitution of a naturally occurring residue or by insertion of a new residue.

[0266] In certain embodiments, the first and second CRM residues are both lysine residues, and the polypeptide conjugate comprises only two lysine residues. In certain embodiments, the second lysine residue is in GLP-1 and is selected from the group consisting of K23, K26, K27, K30, and K34. In certain embodiments, the second CRM residue is a naturally occurring residue found in the native GLP-1 sequence, such as K26 or K34. In certain embodiments, the second CRM residue is an introduced residue. The CRM residue can be introduced into the GLP-1 sequence at any suitable position, for example, by substitution, so long as such substitution does not substantially reduce the GLP-1R agonist activity of GLP-1. The second CRM residue can be introduced, for example, by substitution of Q23K, E27K, or A30K. In certain embodiments, all lysine residues other than the CRM residues in GLP-1 are replaced with non-lysine residues, so that the polypeptide conjugate does not comprise additional lysine residues except the CRM residues. For example, if K26 is the second CRM residue, then K34 is substituted with a non-lysine residue, or vice versa. The non-lysine residue may be selected by one of skill in the art, and examples include arginine I, glutamine (Q), alanine (A), glycine (G), histidine (H), serine (S), or threonine (T).

[0267] In a dual-conjugated polypeptide conjugate, where both the first and second CRM residues are lysine residues, GLP-1 may comprise the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:6, where the peptide linker may optionally comprise an amino acid sequence selected from the group consisting of SEQ ID NOs:43-45 or 83. In certain embodiments, the polypeptide conjugate comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:51, 53, 54, 57-59, 85, and 86, as set forth below (where the first and second CRM residues are listed after the sequence identification number): SEQ ID NO:51 (26K, 76K), SEQ ID NO:53 (26K, 84K), SEQ ID NO:54 (26K, 68K), SEQ ID NO:57 (26K, 76K), SEQ ID NO:58 (26K, 84K), SEQ ID NO:59 (26K, 68K), SEQ ID NO:85 (26K, 96K), or Sequence number 86 (26K, 60K).

[0268] In certain embodiments, the first and second CRM residues are both cysteine ​​residues, and the polypeptide conjugate contains only two cysteine ​​residues. In certain embodiments, the second cysteine ​​residue is in GLP-1. In certain embodiments, the second cysteine ​​residue is in the peptide linker.

[0269] In certain embodiments, the first and second CRM residues are both unnatural amino acid residues, and the polypeptide conjugate comprises only two unnatural amino acid residues as CRM residues.

[0270] In certain embodiments, a cysteine ​​residue or non-natural amino acid residue in GLP-1 is introduced by substitution at a position selected from the group consisting of Q23, K26, E27, A30, and K34 relative to SEQ ID NO: 1. In certain embodiments, a cysteine ​​residue or NNAA in GLP-1 is introduced by substitution at position K26 or E27 relative to SEQ ID NO: 1. In certain embodiments, the second CRM residue in GLP-1 is a cysteine ​​and is introduced by substitution at a position selected from the group consisting of Q23C, K26C, E27C, A30C, and K34C.

[0271] In a dual-conjugated polypeptide conjugate, where both the first and second CRM residues are cysteine ​​residues, GLP-1 may comprise the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 7, where the peptide linker may optionally comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-49. In certain embodiments, the polypeptide conjugate comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 63-65 and 72-74, as set forth below (where the first and second CRM residues are listed after the sequence identification number): SEQ ID NO: 63 (26C, 84C), SEQ ID NO: 64 (26C, 76C), SEQ ID NO: 65 (26C, 68C), SEQ ID NO: 72 (26C, 84C), SEQ ID NO: 73 (26C, 76C), Sequence number 74 (26C, 68C).

[0272] CRM In certain embodiments, each of the first CRM and / or the second CRM (or the CRMs collectively) comprises a plasma protein binding moiety, a polymer, Fc, human serum albumin (HSA) and functional fragments thereof, an Xten sequence, or a PAS sequence. In certain embodiments, the Xten sequence is an extended recombinant polypeptide sequence having an amino acid sequence as described in WO2007103515, WO2009023270, WO2010091122, WO2011123813, WO2013130683, WO2017146979, WO2011084808, WO2013040093, WO2013122617, WO2014011819, WO2013184216, WO2014164568, WO2015023891, WO2016077505, and WO2017040344, the disclosures of which are incorporated by reference in their entireties. In certain embodiments, the term "PAS," which may also be used interchangeably with the term "APS," refers to an amino acid repeat consisting of Ala, Ser, and Pro residues, as described in US8563521B2, the disclosure of which is incorporated by reference in its entirety.

[0273] In certain embodiments, the CRM comprises an albumin binding moiety. The term "albumin binding moiety" refers to any functional moiety that can bind, preferably non-covalently, with sufficient specificity to albumin (e.g., human serum albumin) or any functional fragment thereof. The albumin binding moiety attached to the therapeutic fusion polypeptide, polypeptide, or polypeptide complex typically has an affinity for human serum albumin of less than 10 μM, preferably less than 1 pM. The albumin binding moiety may include, but is not limited to, an albumin binding domain, an albumin binding sequence derived from a synthetic peptide, and an albumin binding chemical moiety. For example, the albumin binding moiety is selected from an albumin binding domain derived from streptococcal protein G, an albumin binding domain derived from Peptostreptococcus magnus protein PAB, an albumin binding peptide having the core sequence DICLPRWGCLW (SEQ ID NO: 81). Some small peptides that are albumin binding moieties are described in J. Biol Chem. 277, 38 (2002) 35035-35043. In another example, the albumin binding moiety is selected from linear and branched lipohophillic moieties containing 4-40 carbon atoms, compounds having a cyclopentanophenanthrene skeleton, etc. For example, the albumin binding moiety can be of the formula CH 3 (CH 2 ) v CO-NHCH(COOH)(CH 2 ) 2 CO— group, in which v is an integer of 10 to 24.

[0274] In certain embodiments, the albumin binding moiety comprises the structure *-ABCDE, where A, B, C, D, and E are interconnected via amide bonds and the * end of A is connected to a reactive group of a conjugable residue on the polypeptide complex, where: A is a bond,

[0275] [ka] wherein a, b, c, and d are independently integers from 0 to 4; 1 is hydrogen or -COOH; B is a bond,

[0276] [ka] wherein e is an integer from 1 to 4, and wherein the α position is linked to the α' position; C is a bond or

[0277] [ka] where R 2 Ha-CH 2 SO 3 H or -COOH, f is an integer from 1 to 4, n is an integer from 1 to 25, and if B is not a bond, then the β' position is linked to the β position, or if B is a bond, then the β' position is linked to the α' position; D is a bond,

[0278] [ka] wherein g and h are independently 0 or 1; R 3 is H or -CH 2 COOH, when B is not a bond and C is a bond, then the γ' position is linked to the β position; when C is not a bond, the γ' position is linked to the γ position; when B is a bond and C is a bond, the γ' position is linked to the α' position; E is a group of the formula:

[0279] [ka] is an acidic group having the formula In the formula, W is -(CR 4 R 5 ) l -, where R4 and R 5 is independently selected from the group consisting of hydrogen, halogen, cyano, hydroxyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, hydroxyalkyl, amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy, and carboxamido; R 6 is hydroxyl or NR 7 R 8 wherein R 7 and R 8 are independently hydrogen, alkyl, hydroxyl, and

[0280] [ka] wherein l is an integer from 10 to 20; when D is not a bond, the δ position is linked to the δ' position; when C is not a bond and D is a bond, the δ position is linked to the γ position; when B is not a bond, C is a bond and D is a bond, then the δ position is linked to the β position; When A is not a bond and B, C, and D are all bonds, then the δ position is linked to the α' position.

[0281] In certain embodiments, A is a bond or

[0282] [ka] In the formula, d is an integer from 1 to 4, and R 1 is hydrogen; B is a bond or

[0283] [ka] wherein e is an integer from 1 to 4, and wherein the α position is linked to the α' position; C is

[0284] [ka] where R 2 is -COOH, f is an integer from 1 to 4, and n is an integer from 1 to 25, and if B is not a bond, then the β' position is linked to the β position, or if B is a bond, then the β' position is linked to the α' position; D is a bond; Regarding E, R 4 and R 5 Each of R is hydrogen. 6 is hydroxyl, l is an integer from 10 to 20, and the δ position is linked to the γ position.

[0285] Further optionally, d is 1, e is 2, f is 1, n is 1, and l is an integer from 14 to 20.

[0286] In certain embodiments, the CRM is conjugated to a lysine residue, optionally within the peptide linker or within GLP-1, where A can be a bond and B can be

[0287] [ka] It could be.

[0288] In such embodiments, the CRM has the formula:

[0289] [ka] (also referred to as -HOOC-(CH2)16-CO-gGlu-2XADO, where 2XADO refers to two consecutive ADO moieties and ADO is an abbreviation for 8-amino-3,6-dioxaoctanoic acid).

[0290] In certain embodiments, the CRM is conjugated to a cysteine ​​residue, optionally within the peptide linker or within GLP-1, where A is

[0291] [ka] and B is

[0292] [ka] where the α position is linked to the α' position.

[0293] In such embodiments, the CRM has the formula:

[0294] [ka] (also referred to as HOOC-(CH2)16-CO-gGlu-2XADO-EDA-CO-CH2 or HOOC-(CH2)20-CO-gGlu-2XADO-EDA-CO-CH2).

[0295] Further, according to some embodiments, the CRM has the structure of the following formula (also referred to as HOOC-(CH2)16-CO-gGlu-2XADO-EDA-CO-CH2):

[0296] [ka] Includes.

[0297] Polypeptide Conjugates In certain embodiments, the polypeptide conjugates provided herein comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 51-80 and 85-88.

[0298] In certain embodiments, the polypeptide conjugates provided herein are monoconjugated, i.e., a single CRM is conjugated at a single CRM residue in the polypeptide portion of the polypeptide conjugate, wherein GLP-1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 5, 6, 8, and 9, wherein the polypeptide portion of the polypeptide conjugate may comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 52, 55, 56, 60-62, 66-71, and 75-80, the CRM residue may be a lysine or cysteine ​​residue in the peptide linker of the polypeptide portion at a position selected from the group consisting of 60, 68, and 76, and the CRM may comprise the structure of the A portion (HOOC-(CH2)16-CO-gGlu-2XADO) for lysine conjugation, or the B portion (HOOC-(CH2)16-CO-gGlu-2XADO-EDA-CO-CH2) for cysteine ​​conjugation.

[0299] In the monoconjugated polypeptide conjugates where the CRM residue is a lysine residue, various embodiments of the polypeptide conjugates are listed below, each indicated by the amino acid sequence relative to the polypeptide moiety and the position relative to the CRM residue: SEQ ID NO:52 (76K); SEQ ID NO:55 (68K); SEQ ID NO:56 (60K); SEQ ID NO:60 (76K); SEQ ID NO:61 (68K); and SEQ ID NO:62 (60K). In certain embodiments, the polypeptide conjugate comprises a polypeptide moiety comprising or consisting of the amino acid sequence of SEQ ID NO:52, and the first CRM residue is 76K. In certain embodiments, the polypeptide conjugate comprises a polypeptide moiety comprising or consisting of the amino acid sequence of SEQ ID NO:60, and the first CRM residue is 76K. Each polypeptide conjugate has an A moiety (HOOC-(CH2)16-CO-gGlu-2XADO) as the CRM.

[0300] In monoconjugated polypeptide conjugates where the CRM residue is a cysteine ​​residue, various embodiments of the polypeptide conjugates are listed below, each indicated by the amino acid sequence relative to the polypeptide moiety and the position relative to the CRM residue: SEQ ID NO:66 (76C); SEQ ID NO:67 (68C); SEQ ID NO:68 (60C); SEQ ID NO:69 (76C); SEQ ID NO:70 (68C); SEQ ID NO:71 (60C); SEQ ID NO:75 (76C); SEQ ID NO:76 (68C); SEQ ID NO:77 (60C); SEQ ID NO:78 (76C); SEQ ID NO:79 (68C); and SEQ ID NO:80 (60C). In certain embodiments, the polypeptide conjugate comprises a polypeptide moiety comprising or consisting of the amino acid sequence of SEQ ID NO:57, conjugated to a first CRM and a second CRM at 26K and 76K, respectively. Each polypeptide conjugate has a B moiety (HOOC-(CH2)16-CO-gGlu-2XADO-EDA-CO-CH2) as the CRM.

[0301] In certain embodiments, the polypeptide conjugates provided herein are dual-conjugated, i.e., two CRMs are conjugated to two CRM residues of the polypeptide moiety of the polypeptide conjugate, one to GLP-1 and the other to a peptide linker, respectively, where GLP-1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 6, and 7.

[0302] wherein the polypeptide portion of the polypeptide conjugate may comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 51, 53, 54, 57-59 and 63-65, 72-74, 85, and 86. The two CRM residues may be a lysine or cysteine ​​residue, one at position 26 and the other at a position selected from the group consisting of 68, 76, and 84. The two CRMs are identical and comprise the structure of the A portion (HOOC-(CH2)16-CO-gGlu-2XADO) for lysine conjugation or the B portion (HOOC-(CH2)16-CO-gGlu-2XADO-EDA-CO-CH2) for cysteine ​​conjugation.

[0303] In the double-conjugated polypeptide conjugates where the two CRM residues are both lysine residues, various embodiments of the polypeptide conjugates are listed below, each indicated by the amino acid sequence for the polypeptide moiety and the position for the two CRM residues: SEQ ID NO:51 (26K, 76K); SEQ ID NO:53 (26K, 84K); SEQ ID NO:54 (26K, 68K); SEQ ID NO:57 (26K, 76K); SEQ ID NO:58 (26K, 84K); SEQ ID NO:59 (26K, 68K); SEQ ID NO:85 (26K, 96K); and SEQ ID NO:86 (26K, 60K). Each polypeptide conjugate has an A moiety (HOOC-(CH2)16-CO-gGlu-2XADO) as the two CRMs.

[0304] In the double-conjugated polypeptide conjugates where the two CRM residues are both cysteine ​​residues, various embodiments of the polypeptide conjugates are listed below, each indicated by the amino acid sequence for the polypeptide moiety and the position for the two CRM residues: SEQ ID NO:63 (26C, 84C); SEQ ID NO:64 (26C, 76C); SEQ ID NO:65 (26C, 68C); SEQ ID NO:72 (26C, 84C); SEQ ID NO:73 (26C, 76C); and SEQ ID NO:74 (26C, 68C). Each polypeptide conjugate has the B moiety (HOOC-(CH2)16-CO-gGlu-2XADO-EDA-CO-CH2) as the two CRMs.

[0305] In certain embodiments, the CRM is conjugated to a non-natural amino acid residue in the fusion polypeptide. In certain embodiments, the CRM is conjugated to a non-natural amino acid residue in the polypeptide.

[0306] Table 1 below shows detailed information about certain exemplary polypeptide conjugates, including the sequence numbers of the polypeptide moiety, GLP-1, and peptide linker, as well as CRM residues. The mutations in GLP-1, and the repeat sequence and number of repeats in the peptide linker sequence are also shown.

[0307] [Table 1-1] [Table 1-2] [Table 1-3]

[0308] In certain embodiments, the polypeptide conjugate comprises the structures shown below (including mono-conjugated molecules 002 and 019, and doubly conjugated molecule 012), in which the amino acid residues are represented as single-letter abbreviations in circles. Molecule 002:

[0309] [ka] Or molecule 019:

[0310] [ka] , or molecule 012:

[0311] [ka]

[0312] Method of preparation The present disclosure provides isolated nucleic acids or polynucleotides that encode the polypeptide portion (or a fragment thereof) of the polypeptide conjugates provided herein.

[0313] The term "nucleic acid" or "polynucleotide" as used herein refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof, 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 a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular polynucleotide sequence also 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)).

[0314] The nucleic acid or polynucleotide encoding the polypeptide (or its fragment) provided herein can be constructed using recombinant techniques.For this purpose, the DNA encoding the GLP-1 receptor agonist (such as GLP-1) and the DNA encoding the peptide linker can be obtained and operably linked to allow transcription and expression in host cells to produce a fusion polypeptide.If necessary, the polynucleotide sequence encoding one or more linkers can also be operably linked to allow expression of the desired product.

[0315] 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 or polypeptide complex is feasible and under proper control.

[0316] The coding polynucleotide sequence can be inserted into a vector for further cloning (amplification of 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.

[0317] Vectors and host cells In another aspect, the disclosure provides a vector comprising a polynucleotide provided herein.

[0318] 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 or eukaryotic cell.

[0319] The host cell suitable for cloning or expressing the DNA in the vector herein is mainly prokaryote.Prokaryote suitable for this purpose includes 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, host cell is eukaryote, such as yeast and mammalian cell (e.g., immortalized mammalian cell).

[0320] The vector comprising the polynucleotide sequence provided herein can be introduced into host cells using any suitable method known to those skilled in the art, such as transformation, transfection or transduction.In one example, the polynucleotide sequence encoding GLP-1 polypeptide can be subcloned into an expression vector, which is expressed as inclusion bodies in host cells.The vector can be a viral vector, and any suitable viral vector can be used in this capacity.

[0321] In another aspect, the present disclosure provides a host cell comprising the vector provided herein.The host cell is a prokaryotic or eukaryotic cell.The host cell transformed with the expression or cloning vector described above can be cultured in conventional nutrient medium, which is modified as necessary to induce promoter, select transformant, or amplify cloning vector.

[0322] In another aspect, the disclosure provides a method of producing a polypeptide conjugate provided herein, comprising culturing a host cell provided herein under conditions allowing expression of a polynucleotide provided herein to obtain a polypeptide portion of the polypeptide conjugate.

[0323] For the production of the polypeptide portion provided herein, the host cell transformed with the expression vector 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 necessary 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 GENTAMYCIN™ drug), 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 one 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 one of skill in the art.

[0324] In one aspect, the disclosure provides a method of expressing a polypeptide portion of a polypeptide conjugate provided herein, comprising culturing a host cell provided herein under conditions in which the polypeptide portion is expressed. In certain embodiments, the polypeptide portion is expressed as a soluble polypeptide.

[0325] When using recombinant techniques, the polypeptides provided 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.In brief, cell paste is melted 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 expression systems is generally first concentrated using a commercially available protein concentration filter, for example, 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.

[0326] In certain embodiments, the method further comprises isolating the polypeptide.

[0327] The polypeptides provided 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.

[0328] 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.

[0329] In certain embodiments, the method further comprises conjugating the CRM to the polypeptide. The polypeptide can be conjugated, for example, at a lysine residue, a cysteine ​​residue, or an unnatural amino acid by a suitable conjugation reaction.

[0330] For example, a polypeptide having one or more CRM residues, such as lysine, may be reacted with an amino-reactive agent. In certain embodiments, the CRM is conjugated to the lysine residue via an acyl group in an acylation reaction. Exemplary methods of acylation reactions are described, for example, in WO2009083549 and WO2010029159, the contents of which are incorporated herein in their entirety. The CRM to be conjugated in an acylation reaction may contain, among others, a carboxylic acid group, an α,ω-fatty diacid residue, an activated ester, or an activated N-hydroxyimide ester. Examples of activated esters include O-succinimide reagents, such as N-hydroxysuccinimidyl (NHS) or sulfo-NHS ester, and imidoester compounds, such as Traut's reagent, which may react with the ε-amino group of a conjugable lysine residue to form an amide or amidine bond. Additional examples of suitable amino-reactive agents include O-acylisourea, N-hydroxytriazole esters, anhydrides, phenyl active esters, P-hydroxamic acid active esters, acylimidazoles, acylbenzotriazoles, acyl azides, acid hylides, phosphonium salts, aminium / uronium salts.

[0331] As another example, a polypeptide having one or more CRM residues, such as cysteine, can be linked to a thiol-reactive agent. In certain embodiments, the CRM is conjugated to the cysteine ​​residue in an alkylation reaction. In certain embodiments, the CRM is conjugated to the conjugable cysteine ​​residue via a maleimide or iodoacetamide to form a carbon-sulfur bond. In certain embodiments, the CRM is conjugated to the conjugable cysteine ​​residue via a disulfide to form a disulfide bond. Additional examples of suitable thiol-reactive groups include dienylsulfones, α-haloacyls, or other thiol-reactive conjugation partners. For details, see Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London; Means (1990) Bioconjugate Chem. 1:2; Hermanson, G. in Bioconjugate Techniques (1996) Academic Press, San Diego, pp. 40-55, 643-671.

[0332] For example, a polypeptide having one or more CRM residues, such as a non-natural amino acid residue (NNAA), can be conjugated to a CRM, thereby forming a stable linkage between the NNAA and the CRM of the fusion polypeptide. For example, an NNAA containing a keto group or an aldehyde or β-diketo moiety can react with a hydrazide- or O-alkylhydroxylamine-, hydroxylamine-containing agent to form a hydrazone or O-alkylated oxime linkage. As another example, an NNAA containing an azide group can react with an alkyne derivative to form a stable triazole linker by copper(I)-catalyzed [3+2] cycloaddition (or vice versa). As another example, an NNAA containing an azide group can be ligated with a suitable water-soluble phosphine-containing agent to form an amide linkage by Staudinger ligation. Additionally, a thioester moiety in an NNAA can react with an amine-containing agent to form an amide linkage. The fusion polypeptides provided herein incorporating NNAA can be conjugated to agents via cycloaddition reactions, such as (4+2) cycloaddition between dienes and dienophiles (Diels-Alder reaction), (3+2) cycloaddition via 1,3-dipolar Huisgen cycloaddition, and (3+2) cycloaddition via nitrone-olefin cycloaddition. Cycloaddition methods suitable for antibody conjugation are described, for example, in WO05003294, US20120004183, WO06009901, WO07130453, and U.S. Patent No. 6,737,236.

[0333] As another example, a polypeptide can be conjugated to biotin, which is then indirectly conjugated to a CRM that is conjugated to avidin. As yet another example, a fusion polypeptide or polypeptide complex can be linked to a coupling agent, which further links to a CRM. Examples of coupling agents include bifunctional moieties such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suherate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and his-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agents include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et al., Biochem. J. 173:723-737 (1978)) and N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP), which provide a disulfide linkage.

[0334] Additional methods for conjugation of CRMs to polypeptides can be found, for example, in U.S. Patent No. 5,208,020; U.S. Patent No. 6,4411,163; WO2005037992; WO2005081711; and WO2006 / 034488, which are incorporated herein by reference in their entireties. Specific examples of methods for preparing the conjugates of the present disclosure are also included in the experimental section of this disclosure.

[0335] Pharmaceutical Compositions In another aspect, the present disclosure also provides a pharmaceutical composition comprising a polypeptide conjugate provided herein. In certain embodiments, the pharmaceutical composition is a liquid composition for parenteral administration.

[0336] Here, the polypeptide conjugate essentially serves as the active pharmaceutical ingredient (API) in the pharmaceutical composition. In addition to the polypeptide conjugate, the pharmaceutical composition further comprises one or more pharma- ceutically acceptable excipients.

[0337] The term "pharmaceutical acceptable" indicates that a specified excipient is generally chemically and / or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.

[0338] "Pharmaceutically acceptable excipient" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is bioactive and acceptable to a subject and non-toxic.

[0339] Examples of pharma- ceutically acceptable excipients for liquid formulations may include pharma- ceutically acceptable liquids, gels, solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, sequestering or chelating agents, diluents, adjuvants, excipients, or other non-toxic auxiliary substances, or other components known in the art, or various combinations thereof.

[0340] Suitable pharma- ceutically acceptable carriers 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; sodium carboxymethylcellulose (sodium The carrier may include suspending and dispersing agents such as carboxymethylcelluose, hydroxypropylmethylcellulose, or polyvinylpyrrolidone, emulsifying agents such as polysorbate 80 (TWEEN-80), 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 may be added to the pharmaceutical compositions in multi-dose containers, including phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. 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.

[0341] In an embodiment, the pharmaceutical composition is formulated into an injectable composition.The injectable pharmaceutical composition can be prepared in any conventional form, such as liquid solution, suspension, emulsion, or solid form suitable for making liquid solution, suspension, or emulsion.The preparation for injection can include sterile and / or non-pyrogenic solution ready for injection, sterile dry soluble product such as lyophilized powder ready for mixing with solvent immediately before use, including hypodermic tablets, sterile suspension ready for injection, sterile dry insoluble product ready for mixing with vehicle immediately before use, and sterile and / or non-pyrogenic emulsion.The solution can be either aqueous or non-aqueous.

[0342] 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 nonpyrogenic, as known and practiced in the art.

[0343] In certain embodiments, a sterile lyophilized powder is prepared by dissolving the polypeptide conjugate disclosed herein in a suitable solvent. The solvent may contain excipients that improve the stability or other pharmacological components of the powder or the reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbital, 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 the fusion polypeptide, polypeptide complex, or conjugate, or compositions thereof, provided herein. Overfilling the vial with a small amount (e.g., about 10%) beyond that needed for a dose or series of doses is permissible to facilitate accurate sample draws and accurate dosing. The lyophilized powder may be stored under appropriate conditions, such as at about 4° C. to room temperature.

[0344] Reconstitution of the lyophilized powder with water for injection provides a formulation for use in parenteral administration.In one embodiment, for reconstitution, sterile and / or nonpyrogenic water or other liquid suitable carrier is added to the lyophilized powder.The exact amount depends on the selected therapy being given and can be empirically determined.

[0345] 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, subcutaneous, or intraperitoneal route of administration.

[0346] In certain embodiments, the polypeptide conjugates 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.

[0347] 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).

[0348] 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 can 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.

[0349] In certain embodiments, a pharmaceutical composition provided herein comprises a polypeptide conjugate and a pharma- ceutically acceptable excipient.

[0350] In certain embodiments, the pharma- ceutically acceptable excipients include buffers and isotonicity agents.

[0351] 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.

[0352] In certain embodiments, the buffer is a phosphate buffer.

[0353] In certain embodiments, the phosphate buffer is present in the pharmaceutical composition at a concentration of 0.01-50 mM. For example, the phosphate buffer is present in the pharmaceutical composition at a concentration of 0.01-45 mM, 0.01-40 mM, 0.01-35 mM, 0.01-30 mM, 0.01-25 mM, 0.01-20 mM, 0.01-15 mM, 0.1-30 mM, 1-25 mM, 1-20 mM, 1-15 mM, 5-30 mM, 5-25 mM, 5-20 mM, or 5-15 mM.

[0354] In certain embodiments, the phosphate buffer is present in the pharmaceutical composition at a concentration of 5-20 mM or 5-10 mM, optionally about 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, or 5 mM.

[0355] In certain embodiments, the phosphate buffer is selected from the group consisting of sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, or hydrates thereof.

[0356] In certain embodiments, the hydrate is a dodecahydrate or a dihydrate.

[0357] In certain embodiments, the phosphate buffer is disodium hydrogen phosphate dodecahydrate or disodium phosphate dihydrate.

[0358] In certain embodiments, disodium hydrogen phosphate dodecahydrate is present in the pharmaceutical composition at a concentration of about 0.1-15 mg / mL, 0.5-15 mg / mL, 1-15 mg / mL, 0.5-12 mg / mL, 0.5-10 mg / mL, 0.5-8 mg / mL, 0.5-7 mg / mL, or 0.5-5 mg / mL. In certain embodiments, disodium hydrogen phosphate dodecahydrate is present in the pharmaceutical composition at a concentration of about 2.87 mg / mL.

[0359] In certain embodiments, disodium phosphate dihydrate is present in the pharmaceutical composition at a concentration of about 0.1-15 mg / mL, 0.5-15 mg / mL, 1-15 mg / mL, 0.5-12 mg / mL, 0.5-10 mg / mL, 0.5-8 mg / mL, 0.5-7 mg / mL, or 0.5-5 mg / mL. In certain embodiments, disodium phosphate dihydrate is present at a concentration of about 1.42 mg / mL.

[0360] In certain embodiments, the buffer is a citrate buffer.

[0361] In certain embodiments, the citrate buffer is present at a concentration of about 1-50 mM.

[0362] In certain embodiments, the citrate buffer comprises a mixture of anhydrous citric acid and trisodium citrate (or a hydrate thereof, such as trisodium citrate dihydrate).

[0363] In certain embodiments, the citrate buffer comprises a mixture of 0.14 mg / mL anhydrous citric acid and 2.74 mg / mL trisodium citrate dihydrate.

[0364] In certain embodiments, the buffer is a histidine buffer.

[0365] In certain embodiments, the histidine buffer is present in the pharmaceutical composition at a concentration of about 1-70 mM, optionally about 5-50 mM, 5-20 mM, or 5-10 mM, or optionally about 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, or 5 mM.

[0366] In certain embodiments, the histidine buffer is present in the pharmaceutical composition at a concentration of about 0.5-10 mg / mL, 0.5-5 mg / mL, 1-5 mg / mL, or 1-3 mg / mL.

[0367] In certain embodiments, the histidine buffer is present in the pharmaceutical composition at a concentration of about 1.24 mg / mL.

[0368] In certain embodiments, the isotonicity agent is selected from the group consisting of sodium chloride, propylene glycol, sorbitol, sucrose, glycine, mannitol, lactose monohydrate, arginine, myo-inositol, and dimethylsulfone.

[0369] In certain embodiments, the isotonic agent is sodium chloride.

[0370] In certain embodiments, the sodium chloride is about 1-20 mg / mL, 1-18 mg / mL, 1-16 mg / mL, 1-15 mg / mL, 2-20 mg / mL, 3-20 mg / mL, 4-20 mg / mL, or 5-20 mg / mL. In certain embodiments, the sodium chloride is about 5-15 mg / mL.

[0371] In certain embodiments, the isotonicity agent is propylene glycol.

[0372] In certain embodiments, 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).

[0373] In certain embodiments, the isotonicity agent is mannitol.

[0374] In certain embodiments, mannitol is about 20 mg / mL to about 60 mg / mL (e.g., about 25 mg / mL to about 50 mg / mL, about 30 mg / mL to about 50 mg / mL, about 35 mg / mL to about 50 mg / mL).

[0375] In certain embodiments, the pharmaceutical excipient further comprises a preservative, a chelating agent, and / or a stabilizer.

[0376] In certain embodiments, the pharmaceutical composition has a pH of about 6.5 to about 8.3 (eg, about 6.5 to 7.4, or 7.4 to 8.3).

[0377] In certain embodiments, the pharmaceutical composition has a pH of about 7.4.

[0378] In certain embodiments, the polypeptide conjugate is dual-conjugated. In certain embodiments of the pharmaceutical composition, the polypeptide conjugate is dual-conjugated, and the conjugate portion of the polypeptide conjugate further comprises a second CRM conjugated to a second CRM residue in the polypeptide portion. Optionally, the second CRM residue is a lysine residue, and further optionally, each of the first CRM and the second CRM comprises an A moiety (HOOC-(CH2)16-CO-gGlu-2XADO), the A moiety having the structure of the formula below:

[0379] [ka] has.

[0380] In some embodiments of the pharmaceutical composition, the polypeptide portion of the polypeptide conjugate comprises the amino acid sequence of SEQ ID NO:57 and is conjugated to a first CRM and a second CRM at 26K and 76K, respectively. In certain embodiments, the pharmaceutical composition has about 1-80 mg / ml, 1-100 mg / ml, or 1-120 mg / ml, or optionally 5-40 mg / ml, 5-80 mg / ml, 5-100 mg / ml, or 5-120 mg / ml (e.g., about 5-90 mg / mL, about 5-70 mg / mL, about 5-60 mg / mL, about 5-50 mg / mL, about 5-30 mg / mL, about 5-20 mg / mL, about 5-10 mg / mL).

[0381] In certain embodiments, the pharmaceutical composition comprises: (a) about 1-80 mg / mL, 1-100 mg / ml, or 1-120 mg / mL of a polypeptide conjugate, wherein the polypeptide portion of the polypeptide conjugate comprises the amino acid sequence of SEQ ID NO:57 and is conjugated to a first CRM and a second CRM at 26K and 76K, respectively; (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, propylene glycol, and mannitol; and (d) pH of about 6.5 to about 8.3 Includes.

[0382] In certain embodiments, the pharmaceutical composition comprises: (a) about 1-80 mg / mL, 1-100 mg / ml, or 1-120 mg / mL of a polypeptide conjugate, wherein the polypeptide portion of the polypeptide conjugate comprises the amino acid sequence of SEQ ID NO:57 and is conjugated to a first CRM and a second CRM at 26K and 76K, respectively; (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 5-15 mg / mL sodium chloride, 1-50 mg / mL propylene glycol, and 30-50 mg / mL mannitol; and (d) pH of about 6.5 to about 8.3 Includes.

[0383] In certain embodiments, the pharmaceutical composition comprises: (a) about 1-80 mg / mL, 1-100 mg / ml, or 1-120 mg / mL of a polypeptide conjugate, wherein the polypeptide portion of the polypeptide conjugate comprises the amino acid sequence of SEQ ID NO:57 and is conjugated to a first CRM and a second CRM at 26K and 76K, respectively; (b) about 0.5-5 mg / mL phosphate buffer, about 1-50 mM citrate buffer, or about 0.5-10 mg / mL histidine buffer; (c) an isotonicity agent selected from the group consisting of sodium chloride, propylene glycol, and mannitol; and (d) pH of about 6.5 to about 8.3 Includes.

[0384] In certain embodiments, the pharmaceutical composition comprises: (a) about 1-80 mg / mL, 1-100 mg / ml, or 1-120 mg / mL of a polypeptide conjugate, wherein the polypeptide portion of the polypeptide conjugate comprises the amino acid sequence of SEQ ID NO:57 and is conjugated to a first CRM and a second CRM at 26K and 76K, respectively; (b) about 0.5-5 mg / mL phosphate buffer, about 1-50 mM citrate buffer, or about 0.5-10 mg / mL histidine buffer; (c) an isotonicity agent selected from the group consisting of 5-15 mg / mL sodium chloride, 1-50 mg / mL propylene glycol, and 30-50 mg / mL mannitol; and (d) pH of about 6.5 to about 8.3 Includes.

[0385] In certain embodiments, the pharmaceutical composition comprises: (a) about 5-80 mg / mL, 5-100 mg / ml, or 5-120 mg / mL of a polypeptide conjugate, wherein the polypeptide portion of the polypeptide conjugate comprises the amino acid sequence of SEQ ID NO:57 and is conjugated to a first CRM and a second CRM at 26K and 76K, respectively; (b) about 0.5-5 mg / mL phosphate buffer, about 1-50 mM citrate buffer, or about 0.5-10 mg / mL histidine buffer; (c) an isotonicity agent selected from the group consisting of 5-15 mg / mL sodium chloride, 1-50 mg / mL propylene glycol, and 30-50 mg / mL mannitol; and (d) pH of about 6.5 to about 8.3 Includes.

[0386] In certain embodiments, the Polypeptide Conjugate is monoconjugated and the first CRM is the only CRM in the conjugate portion of the Polypeptide Conjugate. Optionally, the first CRM residue is a lysine residue, and further optionally, the first CRM comprises an A moiety (HOOC-(CH2)16-CO-gGlu-2XADO), the A moiety having the structure of the formula below:

[0387] [ka] has.

[0388] In certain embodiments of the pharmaceutical composition, the polypeptide portion of the polypeptide conjugate comprises the amino acid sequence of SEQ ID NO: 60 and is conjugated to a first CRM at 76 K. In certain embodiments of the pharmaceutical composition, the polypeptide portion of the polypeptide conjugate comprises the amino acid sequence of SEQ ID NO: 52 and is conjugated to a first CRM at 76 K. In certain embodiments, the pharmaceutical composition has about 0.5-20 mg / ml, 0.5-50 mg / ml, or 0.5-80 mg / ml of the polypeptide conjugate.

[0389] In certain embodiments, the pharmaceutical composition comprises: (a) about 0.5-20 mg / mL, 0.5-50 mg / ml, or 0.5-80 mg / ml of a polypeptide conjugate, wherein the polypeptide portion of the polypeptide conjugate comprises the amino acid sequence of SEQ ID NO:60 or SEQ ID NO:52, and is conjugated to a first CRM at 76K; (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, propylene glycol, and mannitol; and (d) pH of about 6.5 to about 8.3 Includes.

[0390] In certain embodiments, the pharmaceutical composition comprises: (a) about 0.5-20 mg / mL, 0.5-50 mg / ml, or 0.5-80 mg / ml of a polypeptide conjugate, wherein the polypeptide portion of the polypeptide conjugate comprises the amino acid sequence of SEQ ID NO:60 or SEQ ID NO:52, and is conjugated to a first CRM at 76K; (b) about 0.5-5 mg / mL phosphate buffer, about 1-50 mM citrate buffer, or about 0.5-10 mg / mL histidine buffer; (c) an isotonicity agent selected from the group consisting of sodium chloride, propylene glycol, and mannitol; and (d) pH of about 6.5 to about 8.3 Includes.

[0391] In certain embodiments, the pharmaceutical composition comprises: (a) about 0.5-20 mg / mL, 0.5-50 mg / ml, or 0.5-80 mg / ml of a polypeptide conjugate, wherein the polypeptide portion of the polypeptide conjugate comprises the amino acid sequence of SEQ ID NO:60 or SEQ ID NO:52, and is conjugated to a first CRM at 76K; (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 5-15 mg / mL sodium chloride, 1-50 mg / mL propylene glycol, and 30-50 mg / mL mannitol; and (d) pH of about 6.5 to about 8.3 Includes.

[0392] In certain embodiments, the pharmaceutical composition comprises: (a) about 0.5-20 mg / mL, 0.5-50 mg / ml, or 0.5-80 mg / ml of a polypeptide conjugate, wherein the polypeptide portion of the polypeptide conjugate comprises the amino acid sequence of SEQ ID NO:60 or SEQ ID NO:52, and is conjugated to a first CRM at 76K; (b) about 0.5-5 mg / mL phosphate buffer, about 1-50 mM citrate buffer, or about 0.5-10 mg / mL histidine buffer; (c) an isotonicity agent selected from the group consisting of 5-15 mg / mL sodium chloride, 1-50 mg / mL propylene glycol, and 30-50 mg / mL mannitol; and (d) pH of about 6.5 to about 8.3 Includes.

[0393] Treatment Method 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 polypeptide conjugate or pharmaceutical composition provided herein.

[0394] Also provided are methods of treatment that include administering to a subject in need thereof a therapeutically effective amount of a polypeptide conjugate provided herein, thereby treating or preventing the condition or disorder. In certain embodiments, the subject has been identified as having a disorder or condition that may be responsive to a polypeptide conjugate provided herein.

[0395] In certain embodiments, the metabolic disorder is diabetes, obesity, overweight, 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, or primary biliary cirrhosis (PBC), or Alzheimer's disease.

[0396] In certain embodiments, the diabetic condition 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 levels of HbA1C.

[0397] In certain embodiments, the diabetic condition includes diabetic complications such as vascular disease.

[0398] In certain embodiments, the present disclosure provides a method for preventing or treating Alzheimer's disease in a subject in need thereof, comprising administering a therapeutically effective amount of a polypeptide conjugate or pharmaceutical composition provided herein. The term "Alzheimer's disease" as used herein is intended to encompass all forms and stages of Alzheimer's disease. Alzheimer's disease is a progressive degenerative brain disorder that gradually impairs memory, cognitive function, and behavior. There are five stages of Alzheimer's disease, including preclinical Alzheimer's disease, mild cognitive impairment caused by Alzheimer's disease, mild dementia caused by Alzheimer's disease, moderate dementia linked to Alzheimer's disease, and severe dementia associated with Alzheimer's disease. The term "dementia" is used to refer to a collection of symptoms that severely affect intellectual and social abilities and ultimately interfere with daily functioning. There are two main forms of Alzheimer's disease: familial Alzheimer's disease and sporadic or late-onset Alzheimer's disease. Familial Alzheimer's disease is typically caused by inherited genetic mutations, including mutations in one of three genes: APP, PSEN1, or PSEN2. This form of the disease is a rare and devastating illness, and onset may occur in middle age. The second and by far most common form of the disease is sporadic or late-onset Alzheimer's disease, which is the most common form and occurs without any known genetic cause. In some embodiments, the subject with or suffering from Alzheimer's disease may have symptoms selected from the group of cognitive defects, including memory impairment, language impairment, and visuospatial ability; functional impairment that may extend to occupational and social problems (e.g., activities of daily living) as the severity of the disease progresses; and behavioral symptoms may also appear, including depression, anxiety, aggression, and psychosis.

[0399] In certain embodiments, the subject has preclinical Alzheimer's disease, mild cognitive impairment caused by Alzheimer's disease, mild dementia caused by Alzheimer's disease, moderate dementia linked to Alzheimer's disease, or severe dementia associated with Alzheimer's disease.

[0400] In certain embodiments, the subject has familial Alzheimer's disease or sporadic Alzheimer's disease.

[0401] It has been reported that conjugated GLP-1 agonists, such as semaglutide, may be useful for treating Alzheimer's disease, see, for example, published U.S. patent application US2022 / 0280612A1, the entirety of which is incorporated herein. The GLP-1 agonist semaglutide has been reported to reduce measures of neuroinflammation, which may affect cognition and function, in animal studies related to Alzheimer's disease. Furthermore, real-world evidence suggests a possible link between reduced risk of dementia and treatment with GLP-1 agonists. The rate of dementia development was significantly lower in subjects treated with GLP-1 agonists.

[0402] This disclosure shows that the polypeptide conjugate provided herein and the pharmaceutical composition provided herein are effective for treating Alzheimer's disease.In certain embodiments, the polypeptide conjugate provided herein and the pharmaceutical composition provided herein are effective for improving cognitive function such as memory and attention, and / or delaying the onset or progression of Alzheimer's disease.In certain embodiments, the polypeptide conjugate provided herein is more effective than semaglutide in treating Alzheimer's disease.

[0403] In another aspect, the present disclosure provides a method of managing weight in a subject in need thereof, comprising administering a therapeutically effective amount of a polypeptide conjugate provided herein.

[0404] 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 polypeptide conjugate provided herein.

[0405] In another aspect, the disclosure provides a method of reducing weight in a subject in need thereof, comprising administering a therapeutically effective amount of a polypeptide conjugate provided herein.

[0406] In certain embodiments, the conditions or metabolic disorders that can be treated or improved using the polypeptide conjugates provided herein include conditions in which a human subject has a fasting blood glucose level of 125 mg / dL or more, for example, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, or more than 200 mg / dL. The blood glucose level can be determined in a fed or fasted state, or randomly. The metabolic conditions or disorders can also include conditions in which the 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.

[0407] In certain embodiments, conditions or metabolic disorders that may be treated or ameliorated using the polypeptide conjugates provided herein include conditions in which a human subject has a body mass index (BMI) of at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, 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 ranging from 25-30, 26-30, 27-30, 28-30, 25-29, or 25-28.

[0408] The therapeutically effective amount of the polypeptide conjugates 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 potential for cross-reactions, allergies, sensitivities, and adverse side effects, as well as the route of administration and the extent of disease progression.Dosage amounts 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 may be the amount of fusion polypeptides, polypeptide complexes, and conjugates provided herein that elicits the biological or pharmaceutical response in a tissue system, animal, or human that is sought by a researcher, physician, or other clinician, including alleviating or ameliorating the symptoms of the disease or disorder being treated, i.e., the amount that supports an observable level of one or more desired biological or pharmaceutical responses, such as lowering blood glucose, insulin, triglyceride, or cholesterol levels; lowering body weight; or improving glucose tolerance, energy expenditure, or insulin sensitivity.

[0409] In certain embodiments, the polypeptide conjugates 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 certain of these embodiments, the polypeptide conjugates provided herein are administered at a dosage of about 50 mg / kg or less, and in certain 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 dosage administered may vary over the course of treatment. For example, in certain embodiments, the dosage administered initially may be higher than the dosage administered subsequently. In certain embodiments, the dosage administered may vary over the course of treatment depending on the subject's response.

[0410] In certain embodiments, the polypeptide conjugate provided herein can be administered to a subject (e.g., a human) with a dosing regimen that is less frequent than once a day, once every 3 days, once a week, once every 2 weeks, once every 3 weeks, or once a month.In certain embodiments, the polypeptide conjugate provided herein can be administered to a subject (e.g., a human) with a dosing interval of twice a week, once a week, once every 2 weeks, once every 3 weeks, once a month, or once every 2 months.The therapeutic efficacy with low dosing frequency has the potential to improve patient compliance and long-term treatment success.Semaglutide, which is currently available treatment, is administered once a week.

[0411] Without wishing to be bound by any theory, certain polypeptide conjugates provided herein have a significantly extended half-life and are suitable for less frequent dosing than semaglutide, for example less frequent dosing than once a week (e.g., once every 8 days, once every 9 days, once every 10 days, once every 11 days, once every 12 days, once every 13 days, once every 14 days, once every 18 days, once every 3 weeks, once every 24 days, once every 4 weeks, or once every month) for treating metabolic conditions.In certain embodiments, the dosing regimen is a continuous dosing regimen selected from twice weekly dosing, once weekly dosing, once every 2 weeks dosing, once every 3 weeks dosing, once monthly dosing, or once every 2 months dosing. In certain embodiments, the dosing regimen has a dosing interval ranging from about once every three days to about once per month, or from about once per week to about once per month.

[0412] 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.

[0413] The pharmaceutical compositions provided herein may be administered orally.

[0414] The polypeptide conjugates may be administered alone or in combination with one or more additional therapeutic procedures or agents.

[0415] In certain embodiments, when used to treat metabolic disease, the polypeptide conjugates 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 administration simultaneously as part of the same pharmaceutical composition, simultaneously as separate compositions, or 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 the phrase is used herein, even if the composition and the second agent are administered via different routes. When possible, the additional therapeutic agent administered in combination with the fusion polypeptide, polypeptide complex, or conjugate provided herein is administered according to the schedule listed in the product information sheet of the additional therapeutic agent, or according to the Physicians' Desk Reference (Physicians' Desk Reference, 70th Ed (2016)) or protocols well known in the art. A non-limiting list of examples of compounds that may be administered in combination with a pharmaceutical composition comprising a polypeptide conjugate provided herein includes rosiglitizone, pioglitazone, repaglinide, nateglitinide, metformin, exenatide, sitagliptin, pramlintide, glipizide, glimepriride, acarbose, and miglitol.

[0416] kit Also provided is a kit for carrying out the disclosed method.Such kit can include the nucleic acid encoding the polypeptide conjugate provided herein, the vector and cell containing such nucleic acid, and pharmaceutical composition such as those described herein, including pharmaceutical composition containing such nucleic acid-containing compound, which can be provided in a sterile container.Optionally, instructions on how to use the pharmaceutical composition provided in the treatment of metabolic disorder can also be included or made available to patients or medical providers.

[0417] In one embodiment, the kit comprises: (a) a pharmaceutical composition comprising a therapeutically effective amount of the polypeptide conjugate or variant form thereof provided herein; and (b) one or more containers for the pharmaceutical composition. Such kit may also comprise instructions for its use; 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 comprises instructions for administering to a patient 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 syndrome such as metabolic syndrome X, non-alcoholic fatty liver disease (NAFLD), end-stage liver disease, hepatic steatosis (fatty liver), liver cirrhosis, or primary biliary cirrhosis (PBC), or Alzheimer's disease.

[0418] 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 container of the kit or a component thereof (e.g., associated with the 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., CD-ROM, diskette, or the like. In yet other embodiments, the actual instructions are not present in the kit, but a means is provided for obtaining the instructions from a remote source, such as through 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 the instructions can be downloaded. Often, it will be desirable for some or all of the components of the kit to be packaged in suitable packaging that maintains sterility. The components of the kit may be packaged in a kit storage element that creates a single, easily handled unit, and 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 components of the kit. EXAMPLES

[0419] [Example 1] Recombinant expression and purification of GLP-1 protein The GLP-1 fusion polypeptides listed in Table 1 (i.e., SEQ ID NOs: 51-80, 85-88) were produced from a bacterial E. coli expression system using the BL21(DE3) derivative strain. DNA encoding the tagged GLP-1 fusion polypeptides was codon-optimized for E. coli expression, de novo synthesized, and subcloned into a PET derivative expression vector (Novagen). Amino acid substitutions were accomplished by modification of the corresponding genetic code. Overexpression of the tagged GLP-1 fusion polypeptides was induced with 0.5 mM isopropyl bd-thiogalactoside (IPTG) when the cell density reached an OD600 of 2.0 in Terrific Broth (TB) medium. After protein induction at 37°C for 20-22 hours, the cells were harvested. The cells were harvested and lysed in 20 mM Tris pH 8.0, 0.15 M NaCl buffer by cell grinding (900 bar, 2 times). The soluble fraction containing the tagged GLP-1 fusion polypeptide was collected by centrifugation (8,000×g, 30 min). After removing the tag with protease, the GLP-1 fusion polypeptide was purified by reversed-phase chromatography. Samples at each step were characterized by LC / MS to confirm the correct molecular weight.

[0420] [Example 2] Incorporation of non-proteinogenic amino acids in recombinant proteins The N-terminal His-Aib-Glu-Gly tetrapeptide or His-Aib dipeptide was dissolved in an organic solvent and added to a solution of the GLP-1 fusion polypeptide in the organic solvent. The reaction solution was stirred at room temperature for 3 hours. Then piperidine was added to the reaction solution to remove the Fmoc protecting group.

[0421] [Example 3] Preparation of GLP-1 Polypeptide Conjugates To a solution of GLP-1 fusion polypeptide obtained in Example 2 in NaOH, CRM reagent (e.g., HOOC-(CH2)16-CO-gGlu-2XADO, HOOC-(CH2)16-CO-gGlu-2XADO-EDA-CO-CH2, etc.) in organic solvent was added dropwise. The reaction was stirred at room temperature for 1 hour. The product was then applied to reverse phase chromatography. This provided the GLP-1 polypeptide conjugates listed in Table 1 shown above.

[0422] The GLP-1 polypeptide conjugates 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.

[0423] [Example 4a] In vitro activity Methods: In vitro GLP-1 activity 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 GLP-1 polypeptide conjugates were measured at 1 nM or 100 nM as the top concentration in the absence or presence of 1% HSA using 3-fold serial dilutions. After treating the cells with the molecules for 4 hours, luciferase activity was measured by Steadylite plus kit (Perkin Elmer, 6066751).

[0424] The activity of each GLP-1 polypeptide conjugate was expressed by an EC50 derived from non-linear regression analysis.

[0425] Conclusion: Almost all molecules (i.e., GLP-1 polypeptide conjugates) show comparable or even better efficacy than semaglutide in assays without HSA supplementation. However, different molecules show different degrees of reduction in GLP-1 activity (i.e., EC 50The data in Tables 2a and 2b suggested that the linker length, fatty acid position, and number of conjugated fatty acid moieties may be related to GLP-1 activity. As shown in Table 2, molecules (whether mono- or double-acylated) with short linker lengths (i.e., 12 amino acid residues) had significantly reduced GLP-1 activity compared to those molecules with longer linker lengths. As the distance between the C-terminal residue of GLP-1 and the CRM residues (i.e., lysine in the linker) increased, the GLP-1 activity of the molecules in the presence of HSA appeared to steadily improve.

[0426] As shown in Table 2a, the monoacylated molecules 002, 010 and 011 showed much higher activity than semaglutide in the presence of 1% HSA. As for the dual acylated molecules 012, 016, 004, 006, 007, 005, 001 and 061, all showed relatively lower GLP-1 activity in the presence of 1% HSA than semaglutide, but still within the acceptable range.

[0427] [Table 2a]

[0428] Example 4b In vitro activity Methods: In vitro GLP-1 activity was measured using a CHO 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 GLP-1 polypeptide conjugates were measured at 30 nM or 600 nM as the top concentration in the absence or presence of 1% HSA using 3-fold serial dilutions. After treating the cells with the molecules for 5 hours, luciferase activity was measured by Bright-Glo Luciferase Assay System (Promega, E2620). The activity of each GLP-1 polypeptide conjugate was expressed by EC50 derived from nonlinear regression analysis.

[0429] Conclusions: As shown in Table 2b, monoacylated molecules 002 (1006) and 019 (1023) showed much higher activity than semaglutide in the presence of 1% HSA.

[0430] [Table 2b]

[0431] [Example 5] In vivo activity in C57 lean mice Methods: Ten-week-old male C57BL / 6 mice were injected subcutaneously with protein once on day 1. Body weight was measured daily and five animals were used for each treatment group. Body weight was monitored for each individual animal. % BW loss = 100 * (BW on day n - BW on day 1) / (BW on day 1). Data are presented as mean and standard error of the mean (SEM). Area under the curve for % body weight loss from day 0 to day 8 (AUC ΔBW 0-8d) was calculated.

[0432] Conclusion: Molecules 001 and 012 showed dose-dependent efficacy on body weight control (Figure 1A and Table 3). As shown in Figure 1D and Table 3, molecules 007 and 008 had lower efficacy than molecules 012 and 016, which is consistent with the in vitro activity data. Molecules 001, 016, and 012 with dual fatty acids exhibited more sustainable efficacy than semaglutide, suggesting that molecules 001, 016, and 012 may have a longer half-life (Figures 1A and 1B).

[0433] [Table 3]

[0434] [Example 6a] In vivo activity in db / db mice Methods: 10-week-old male db / db mice were given a single subcutaneous injection of GLP-1 polypeptide conjugate. Fasting glucose was measured at various time points, and three animals were used for each group. Delta blood glucose is glucose subtracted from baseline levels. Data are presented as mean and standard error (SEM).

[0435] Conclusion: Both molecules 001 and 002 showed dose-dependent efficacy on glucose control (Figures 2A and 2B). Molecule 001 with dual fatty acids exhibited more sustainable efficacy than semaglutide, suggesting that molecule 001 may have a longer half-life.

[0436] Methods: Ten-week-old male db / db mice were given a single subcutaneous injection of GLP-1 polypeptide conjugate. Nonfasting glucose was measured at various time points, and five animals were used for each group. Delta blood glucose is glucose subtracted from baseline levels. Data are presented as mean and standard error of mean (SEM). Area under the curve for delta blood glucose from 0 to 192 h (AUC ΔBG 0-192h) was calculated.

[0437] Conclusion: Molecule 012 showed dose-dependent efficacy on glucose control (Figure 2C, Table 4). Molecule 012 with dual fatty acids exhibited more sustainable efficacy than semaglutide.

[0438] [Table 4]

[0439] [Example 6b] In vivo activity in db / db mice Methods: Ten-week-old male db / db mice were given a single subcutaneous injection of molecule 019. Random blood glucose was measured at various time points, with five animals used in each group. Delta blood glucose is glucose subtracted from baseline levels. Data are presented as mean and standard error of the mean (SEM).

[0440] Conclusions: Molecule 019 demonstrated dose-dependent efficacy on glucose control (Figures 2D and 2E) and significantly greater efficacy than semaglutide.

[0441] [Example 7] Pharmacokinetic measurements Methods: Male C57BL / 6 mice aged 6-8 weeks were administered a single subcutaneous dose of 30 nmol / kg protein (n=3 / group). Plasma samples were collected pre-dose (-5 min), 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, and 192 h after injection. Concentrations of GLP-1 polypeptide conjugates in plasma were measured by ELISA assay. Pharmacokinetic parameters were calculated by WinNonlin based on graphs showing plasma concentration vs. time for each GLP-1 polypeptide conjugate after subcutaneous injection.

[0442] Conclusions: Molecules 001 and 012 showed a longer half-life in mice than semaglutide and molecule 002 (Table 5), consistent with their in vivo efficacy.

[0443] [Table 5]

[0444] Methods: Male SD rats aged 6-8 weeks were administered a single subcutaneous dose of 15 nmol / kg GLP-1 polypeptide conjugate (n=3 / group) and a single intravenous dose of 15 nmol / kg GLP-1 polypeptide conjugate (n=3 / group), respectively. Plasma samples were collected pre-dose (-5 min), 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 24 h, 32 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, 192 h, 216 h, and 240 h after subcutaneous administration. Plasma samples were collected before dosing (-5 min), 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 24 h, 32 h, 48 h, 72 h, 96 h, and 120 h after intravenous administration. The concentrations of the polypeptide conjugates in plasma were measured by LC-MS / MS method. Pharmacokinetic parameters were calculated by WinNonlin based on the graphs showing the plasma concentration vs. time of each polypeptide conjugate after subcutaneous injection.

[0445] Conclusion: As shown in Table 6, molecules 001 and 012 exhibit a longer half-life than semaglutide, which has a T of 12 hours in rats. 1 / 2 (sc administration, reported in nonclinical review by the FDA).

[0446] [Table 6]

[0447] Methods: 10-month-old male Bama minipigs were administered a single subcutaneous dose of 5 nmol / kg GLP-1 polypeptide conjugate (n=2 / group) and a single intravenous dose of 5 nmol / kg protein (n=2 / group), respectively. Plasma samples for the molecule 012 and semaglutide groups were collected pre-dose (-5 min), 0.5 h, 1 h, 3 h, 8 h, 24 h, 48 h, 72 h, 96 h, 168 h, 336 h, 504 h, and 672 h after subcutaneous administration. Plasma samples for the molecule 012 and semaglutide groups were collected pre-dose (-5 min), 0.083 h, 0.5 h, 1 h, 3 h, 8 h, 24 h, 48 h, 72 h, 96 h, 168 h, 336 h, and 504 h after intravenous administration. For the semaglutide group, the plasma concentration of GLP-1 polypeptide conjugate was measured by ELISA assay. For the molecule 012 group, the plasma protein concentration was measured by LC-MS / MS method. Based on the graph showing the plasma concentration of each GLP-1 polypeptide conjugate versus time after administration, the pharmacokinetic parameters were calculated by WinNonlin.

[0448] Conclusions: Molecule 012 showed a longer half-life than semaglutide in minipigs (Table 7).

[0449] [Table 7]

[0450] [Example 8a] Efficacy studies in disease models Selected molecules are assessed in disease animal models (such as db / db mice) to determine body weight, food intake, glucose potency, and dose response in chronic studies. Several biomarkers are also measured, including fasting insulin, plasma triglycerides, cholesterol, liver triglycerides, and inflammatory biomarkers (ALT, AST, and CRP).

[0451] method: 22-week-old DIO male C57BL / 6 mice (approximately 50 g) were injected subcutaneously every other day (Q2D) with the indicated GLP-1 polypeptide conjugate (i.e., molecule 012) 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 each individual animal, while food intake for each group animal 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); cumulative food intake loss is calculated by -100*(cumulative food intake-cumulative food intake of vehicle) / cumulative food intake of vehicle.

[0452] Conclusion: In the DIO study, molecule 012 demonstrated dose-dependent efficacy on weight loss, food intake reduction, and blood glucose control, as shown in Figures 3A, 3B, and Table 8.

[0453] [Table 8]

[0454] [Example 8b] Efficacy studies in disease models Selected molecules will be assessed in Alzheimer's disease animal models (APP / PS1, APPswe / PSEN1dE9 mice) to determine cognitive function (Morris water maze and Y-maze), brain Aβ plaque deposition, and hippocampal pyramidal neuron numbers with respect to dose response in the study.

[0455] Morris water maze and Y-maze were used to assess the cognitive function of animals. The effect of molecules on beta-amyloid plaque deposition in the cerebral cortex and hippocampus, the brain regions affected in Alzheimer's disease, was also measured. In addition, the number of pyramidal neurons in the hippocampus was assessed as a measure of the potential neuroprotective effect of the drug. These methods allowed a comprehensive evaluation of the efficacy of molecules in alleviating the pathological changes associated with Alzheimer's disease in a preclinical setting.

[0456] Methods: Six-month-old APP / PS1 male mice were injected subcutaneously for 10 weeks with the indicated GLP-1 polypeptide conjugates (molecule 012 at 20 mg / mL, 2.87 mg / mL disodium hydrogen phosphate dodecahydrate, and 8.25 mg / ml sodium chloride, pH 7.4) once daily (QD) or every other day (Q2D). 15 animals were used for each treatment group. Body weight and food intake were monitored weekly. Cognitive function behavioral experiments, including Morris water maze and Y-maze, were performed after dosing. Brain tissues were harvested for immunohistochemical staining to evaluate Aβ content in the cerebral cortex and hippocampus. The number of hippocampal pyramidal neurons was measured by HE staining. Data are presented as mean and standard error (SEM). Statistical analysis was performed by one-way ANOVA. Semaglutide was used as a positive control.

[0457] Conclusion: In the APP / PS1 study, molecule 012 demonstrated dose-dependent efficacy in improving cognitive function, reducing brain Aβ plaque deposition, and protecting hippocampal pyramidal neurons, as shown in Figures 4A, 4B, 4C, 4D, and 4E. Molecule 012 was found to be effective in increasing quadrant occupancy, which refers to the percentage of time spent in the target quadrant during the navigation task, and in increasing alternation percentage, which refers to the frequency of switching back and forth between different locations during the maze task. Additionally, molecule 012 was observed to reduce Aβ plaque deposition in both the cerebral cortex and hippocampus regions of the brain that are typically affected in Alzheimer's disease. The number of neurons in the hippocampus, an area crucial for memory, was also improved by molecule 012. These findings suggest that molecule 012 has a positive impact on cognitive function and reduces pathological changes associated with Alzheimer's disease. Molecule 012 showed significantly greater efficacy than semaglutide in treating Alzheimer's disease in animal studies.

[0458] [Example 8c] Efficacy studies in disease models Selected molecules are assessed in the Alzheimer's disease animal model for sporadic Alzheimer's dementia, which is the senescence-accelerated mouse prone 8 (SAMP8) model.SAMP8 mice exhibit age-related cognitive decline and neuropathological changes similar to those observed in human Alzheimer's disease, including beta-amyloid plaque deposition and neuroinflammation.They also exhibit behavioral abnormalities and reduced synaptic plasticity.

[0459] Seven-month-old SAMP8 mice were injected subcutaneously every other day (Q2D) with the indicated GLP-1 polypeptide conjugates (molecule 012) at 30 nmol / kg, 100 nmol / kg, and 300 nmol / kg, respectively, for 16 weeks. Fifteen animals were used for each treatment group. Body weight and food intake were monitored weekly. Cognitive function behavioral experiments, including Morris water maze and Y-maze, were performed after dosing. Brain tissues were harvested for immunohistochemical staining to evaluate Aβ content in the cerebral cortex and hippocampus. The number of hippocampal pyramidal neurons was measured by HE staining. Data are presented as mean and standard error (SEM). Statistical analysis was performed by one-way ANOVA. Semaglutide and donepezil were used as positive controls.

[0460] Molecule 012 shows dose-dependent efficacy in improving cognitive function, reducing brain Aβ plaque deposition, and protecting hippocampal pyramidal neurons in SAMP8 mice. Molecule 012 also shows significantly better efficacy than the positive control in treating Alzheimer's disease in SAMP8 mice.

[0461] [Example 9a] PK study in non-human primates The pharmacokinetics of selected molecules are assessed in monkeys. Both subcutaneous and intravenous injections are performed.

[0462] Methods: Male cynomolgus monkeys aged 3-5 years were administered a single subcutaneous dose of 5 nmol / kg GLP-1 polypeptide conjugate (n=2 / group). Plasma samples for the semaglutide group were collected pre-dose (-5 min), 2, 4, 6, 8, 24, 48, 72, 96, 120, 144, 216, 288, 360, 432, and 504 hours after injection. Plasma samples for the molecule 012 group were collected pre-dose (-5 min), 0.5, 1, 2, 4, 6, 8, 24, 48, 96, 120, 144, 168, 192, 216, 288, 432, and 504 hours after injection. For the semaglutide group, the plasma protein concentration was measured by ELISA assay. For the molecule 012 group, the plasma GLP-1 polypeptide conjugate concentration was measured by LC-MS / MS method. Pharmacokinetic parameters were calculated by WinNonlin based on the graph showing the plasma concentration of each protein versus time after subcutaneous injection.

[0463] Conclusions: Molecule 012 showed a longer half-life in monkeys than semaglutide and molecule 002 (Table 9a).

[0464] [Table 9a]

[0465] [Example 9b] PK study in non-human primates Methods: Male cynomolgus monkeys aged 3-5 years were administered a single intravenous dose of 4 nmol / kg and 5 nmol / kg for semaglutide and molecule 019, respectively (n=2 / group). Plasma samples for the semaglutide group were collected pre-dose (-5 min), 0.083 h, 1 h, 2 h, 4 h, 8 h, 24 h, 36 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h after injection. Plasma samples for the molecule 012 group were collected pre-dose (-5 min), 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h after injection. The protein concentrations in plasma were measured by LC-MS / MS. Pharmacokinetic parameters were calculated by WinNonlin based on the graphs showing the plasma concentration of each protein versus time after intravenous administration.

[0466] Conclusions: Molecule 019 exhibits a similar half-life to semaglutide in monkeys (Table 9b), which potentially supports once-weekly dosing in humans. Furthermore, given the potent GLP-1 activity of molecule 019 (10-fold higher than semaglutide), molecule 019 is expected to be a promising candidate not only to provide better efficacy than semaglutide, but also to support the desired once-weekly dosing frequency (or potentially even less frequently).

[0467] [Table 9b]

[0468] [Example 10] Immunogenicity assessment Selected GLP-1 polypeptide conjugates are also assessed for immunogenicity by in silico (iTope and TCED methods) and ex vivo (EpiScreen) methods.

[0469] [Example 11] Human serum albumin binding Methods: Binding of molecules to serum albumin was characterized by surface plasmon resonance on a Biacore 8K instrument. Serum albumin from different species was covalently coupled to the CM5 sensor chip surface until 4000RU was reached. The chip was blocked with 1M ethanolamine for 420 seconds at a flow rate of 10 μL / min. 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 the bound molecules for 30 seconds.

[0470] Conclusion: Molecules 004, 001, 006 and 012 show higher binding affinity for human serum albumin than semaglutide, molecule 019 and molecule 002 (see Table 10), which is consistent with the PK data.

[0471] [Table 10]

[0472] [Example 12] Preparation of Liquid Compositions Unless otherwise specified, molecule 012 was prepared by diluting it into a formulation buffer consisting of a buffering agent (e.g., disodium hydrogen phosphate dodecahydrate or disodium phosphate dihydrate were used for pH 7.4-8.3, and histidine or anhydrous citric acid / trisodium citrate dihydrate were used for pH 6.5) and an isotonicity agent (sodium chloride, propylene glycol, or mannitol). 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.

[0473] Compositions containing molecule 012 were tested in this experiment. As shown in Table 11, the test compositions had propylene glycol (14 mg / mL), sodium chloride (8.25 mg / mL), or mannitol (46.4 mg / mL) as isotonicity agents combined with various pH and protein concentrations. For composition numbers 1-6, 8-11, 13-16, and 18-23, 2.87 mg / mL of disodium hydrogen phosphate dodecahydrate was used as a pH buffer. For composition numbers 7, 12, and 17, 0.14 mg / mL of anhydrous citric acid and 2.74 mg / mL of trisodium citrate dihydrate were used as pH buffers. For composition number 24, 1.24 mg / mL of histidine was used as a pH buffer.

[0474] [Table 11]

[0475] [Example 13] Stability testing of molecule 012 composition Twenty-four compositions listed in Example 12 were prepared and stored at 25° C. for 6 months. Impurities, high molecular weight products (HMWPs), and relative biological activity were investigated using RP-UPLC, SEC-UPLC, and hGLP1-R expressing cell assays at 0, 2, and 6 months. RP-UPLC was performed using a Kinetex C18 column (Phenomenex) and UPLC H-class (Waters). SEC-UPLC was performed using a TSKgel UP-SW2000 (Tosoh) and UPLC H-class (Waters). Relative biological activity was determined using human GLP-1 receptor expressing CHO cells by detecting cAMP activity and calculated using the formula (100*(EC50 標準 / EC50 サンプル))%. The results for composition numbers 1-8, 11, and 21-23 are shown in Tables 12 and 13, and the results for composition numbers 9, 10, 12-20, and 24 are not shown. All compositions were tested, and the results show that they are stable over the test period. As shown in Tables 12 and 13, all compositions were stable at 25° C. for at least 2 or 6 months.

[0476] [Table 12]

[0477] [Table 13]

Claims

1. A liquid pharmaceutical composition comprising a polypeptide conjugate and a pharmaceutically acceptable excipient, A polypeptide conjugate comprises a polypeptide portion and a conjugate portion. The polypeptide portion comprises a single bioactive peptide and a peptide linker, the bioactive peptide being attached to the N-terminus of the peptide linker and containing GLP-1; The conjugate portion includes a first CRM conjugated to a first clearance reduction (CRM) residue in the peptide linker, and a second CRM conjugated to a second CRM residue in the polypeptide portion. Both the first and second CRM residues are lysine residues, and the polypeptide conjugate contains only two lysine residues. The polypeptide portion contains the amino acid sequence of SEQ ID NO: 51 and is conjugated with a first CRM and a second CRM at 26K and 76K, respectively; The polypeptide portion contains the amino acid sequence of SEQ ID NO: 53 and is conjugated with a first CRM and a second CRM at 26K and 84K, respectively; The polypeptide portion contains the amino acid sequence of SEQ ID NO: 54 and is conjugated with a first CRM and a second CRM at 26K and 68K, respectively; The polypeptide portion contains the amino acid sequence of SEQ ID NO: 57 and is conjugated with a first CRM and a second CRM at 26K and 76K, respectively; The polypeptide portion contains the amino acid sequence of SEQ ID NO: 58 and is conjugated with a first CRM and a second CRM at 26K and 84K, respectively; The polypeptide portion contains the amino acid sequence of SEQ ID NO: 59 and is conjugated with a first CRM and a second CRM at 26K and 68K, respectively; The polypeptide portion comprises the amino acid sequence of SEQ ID NO: 85 and is conjugated with a first CRM and a second CRM at 26K and 96K, respectively; or The polypeptide portion contains the amino acid sequence of SEQ ID NO: 86 and is conjugated with a first CRM and a second CRM at 26K and 60K, respectively; Both the first and second CRMs have the structure shown in the following formula: 【Chemistry 1】 Having, Liquid pharmaceutical composition.

2. Polypeptide conjugates have the structure shown below: 【Chemistry 2】 A pharmaceutical composition according to claim 1, having the following characteristics.

3. Pharmaceutically acceptable excipients include buffers and isotonic agents. Optionally, the buffering agent may be selected from the group consisting of phosphate buffering agents, citrate buffering agents, acetate buffering agents, histidine buffering agents, glycine buffering agents, carbonate buffering agents, borate buffering agents, glutamate buffering agents, glycylglycine buffering agents, lysine buffering agents, and arginine buffering agents. Optionally, the isotonic agent is selected from the group consisting of sodium chloride, propylene glycol, sorbitol, sucrose, glycine, mannitol, lactose monohydrate, arginine, myo-inositol, and dimethyl sulfone. The pharmaceutical composition according to claim 1.

4. The buffer is a phosphate buffer, and optionally, the phosphate buffer is present in the pharmaceutical composition at a concentration of 0.01 to 50 mM, optionally 5 to 20 mM or 5 to 10 mM, and optionally 8 mM. The pharmaceutical composition according to claim 3.

5. The pharmaceutical composition according to claim 4, wherein the phosphate buffer is disodium hydrogen phosphate dodecahydrate, and the disodium hydrogen phosphate dodecahydrate is present in the pharmaceutical composition at a concentration of about 0.01 to 15 mg / mL (for example, about 0.5 to 15 mg / mL, about 0.5 to 12 mg / mL, about 0.5 to 10 mg / mL, about 0.5 to 5 mg / mL, or about 2.87 mg / mL).

6. The pharmaceutical composition according to claim 4, wherein the phosphate buffer is disodium phosphate dihydrate, and the disodium phosphate dihydrate is present at a concentration of about 0.1 to 15 mg / mL (for example, about 0.5 to 15 mg / mL, about 0.5 to 12 mg / mL, about 0.5 to 10 mg / mL, about 0.5 to 5 mg / mL, or about 1.42 mg / mL).

7. The pharmaceutical composition according to claim 3, wherein the buffer is a citrate buffer, and optionally the citrate buffer is present in a concentration of about 1 to 50 mM.

8. The buffer is a histidine buffer. Optionally, the histidine buffer may be present at concentrations of 1–70 mM, 5–50 mM, 5–20 mM, 5–10 mM, or 8 mM. Optionally, a histidine buffer may be present at a concentration of approximately 0.5–10 mg / mL (e.g., approximately 0.5–5 mg / mL, approximately 1–5 mg / mL, approximately 1–3 mg / mL, or approximately 1.24 mg / mL). The pharmaceutical composition according to claim 3.

9. The isotonic agent is sodium chloride, and the concentration is optional, ranging from approximately 5 to 15 mg / mL. Optionally, the isotonic agent is propylene glycol, and optionally, the concentration of propylene glycol is approximately 1 mg / mL to approximately 50 mg / mL (for example, approximately 5 mg / mL to approximately 25 mg / mL, approximately 8 mg / mL to approximately 16 mg / mL). The pharmaceutical composition according to claim 3.

10. The pharmaceutical composition according to claim 3, wherein the isotonic agent is mannitol, and optionally the mannitol concentration is about 30 mg / mL to about 50 mg / mL.

11. The pharmaceutical composition according to claim 3, wherein the pharmaceutical excipients further comprise a preservative, a chelating agent, and / or a stabilizer.

12. The pharmaceutical composition according to claim 3, having a pH of approximately 6.5 to approximately 8.3 (for example, approximately 6.5 to 7.4, or 7.4 to 8.3), and optionally having a pH of approximately 7.

4.

13. The pharmaceutical composition according to claim 3, having a polypeptide conjugate of approximately 1 to 80 mg / ml, 1 to 100 mg / ml, or 1 to 120 mg / ml, or optionally 5 to 40 mg / ml, 5 to 80 mg / ml, 5 to 100 mg / ml, or 5 to 120 mg / ml (for example, having a polypeptide conjugate of approximately 5 to 90 mg / mL, approximately 5 to 70 mg / mL, approximately 5 to 60 mg / mL, approximately 5 to 50 mg / mL, approximately 5 to 30 mg / mL, approximately 5 to 20 mg / mL, or approximately 5 to 10 mg / mL).

14. Polypeptide conjugate; A buffer selected from the group consisting of phosphate buffers, citrate buffers, acetate buffers, histidine buffers, glycine buffers, carbonate buffers, borate buffers, glutamate buffers, glycylglycine buffers, lysine buffers, and arginine buffers; An isotonic 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 pH of approximately 6.5 to 8.3 The pharmaceutical composition according to claim 3, comprising:

15. Polypeptide conjugates with a concentration of approximately 5-80 mg / mL, 5-100 mg / mL, or 5-120 mg / mL, wherein the polypeptide portion of the polypeptide conjugate contains the amino acid sequence of SEQ ID NO: 57 and is conjugated with a first CRM and a second CRM at 26K and 76K, respectively; Approximately 0.5–5 mg / mL of phosphate buffer, approximately 1–50 mM of citrate buffer, or approximately 0.5–10 mg / mL of histidine buffer; An isotonic agent selected from the group consisting of 5-15 mg / mL sodium chloride, 1-50 mg / mL propylene glycol, and 30-50 mg / mL mannitol; and pH of approximately 6.5 to 8.3 The pharmaceutical composition according to claim 3, comprising:

16. A pharmaceutical composition according to any one of claims 1 to 15, for use in a method of preventing or treating a metabolic disorder in a subject requiring the same, wherein the method comprises administering the pharmaceutical composition.

17. Metabolic disorders include diabetes, obesity, overweight, non-alcoholic steatohepatitis (NASH), cardiovascular 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. Diabetes mellitus includes 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 (young-onset adult-onset diabetes), gestational diabetes, and elevated HbA1c levels. The pharmaceutical composition according to claim 16.

18. A pharmaceutical composition according to any one of claims 1 to 15, for use in a method of managing weight, reducing food intake, or reducing weight in a subject requiring such management, wherein the method comprises administering the pharmaceutical composition.

19. The pharmaceutical composition according to any one of claims 16 to 18, wherein the pharmaceutical composition is administered in a dosage regimen less frequently than once a day, once every three days, or once a week, once every two weeks, once every three weeks, or once a month.

20. The pharmaceutical composition according to any one of claims 16 to 18, wherein the pharmaceutical composition is administered by parenteral administration, and optionally, the pharmaceutical composition is administered subcutaneously, intravenously, or intramuscularly.

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