Polypeptide conjugate and method of use

Abstract

Polypeptide conjugates comprising a GLP-1 receptor agonist and a peptide linker, and pharmaceutical compositions comprising the same, are provided. Methods of using such to treat disease are also provided.

Description

Technical Field

[0001] The present invention relates to polypeptide conjugates, pharmaceutical compositions thereof, and methods of using the same for preventing and / or treating diseases.

Background Art

[0002] The main biologically active fragment of glucagon-like peptide-1 (GLP-1) is a 30 or 31 amino acid peptide fragment (amino acids 7-36 or 7-37 of GLP-1) generated by post-translational processing of proglucagon peptide. The initial GLP-1 product GLP-1 has been shown to stimulate insulin synthesis and secretion, and prevent hyperglycemia in diabetes, particularly type 2 diabetes. However, endogenous GLP-1 only has a half-life of approximately two minutes, which results in fasting plasma levels of GLP-1 of only 0-15 pmol / L.

[0003] Metabolic disorders are generally associated with insulin resistance, visceral adiposis, atherosclerotic dyslipidemia, etc., which pose a large and growing public health and clinical challenge worldwide. However, existing treatments for metabolic diseases face problems such as short half-life and / or low efficacy.

[0004] Furthermore, existing GLP-1 compounds are mainly administered by injection, and when the dosing frequency is too frequent, patients may have fear of the injection itself.

[0005] Therefore, there is a need for improved therapeutic solutions for treating metabolic diseases and also for reducing dosing frequency.

Summary of Invention

[0006] Provided herein are polypeptide conjugates for treating / preventing metabolic disorders, pharmaceutical compositions comprising the same, and methods of use.

[0007] In a first embodiment, the disclosure provides a polypeptide conjugate comprising a single bioactive peptide attached to the N-terminus of a peptide linker, and a first CRM conjugated to a first clearance reduction (CRM) residue in the peptide linker, wherein the bioactive peptide comprises a GLP-1 receptor agonist, and the first CRM residue is located at least 5 amino acid residues (excluding the CRM residue) away from the C-terminal amino acid residue of the GLP-1 receptor agonist.

[0008] In some embodiments, the first CRM residue is located at least 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 35, 38, 40, 45, 46, 50, 55, 58, 60, 65, 70, 75, and 78 amino acid residues (excluding the CRM residue) away from the C-terminal amino acid of the bioactive peptide.

[0009] In certain embodiments, the GLP-1 receptor agonist in the polypeptide conjugate has a length of at most 70, 60, or 50 amino acid residues. In certain embodiments, the GLP-1 receptor agonist in the polypeptide conjugate has a length of at most 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, or 30 amino acid residues.

[0010] In some embodiments, the GLP-1 receptor agonist contains GLP-1.

[0011] In some embodiments, the bioactive peptide does not have a conjugated CRM.

[0012] In certain embodiments, the polypeptide conjugates provided herein are monoconjugated and have a CRM conjugated to a peptide linker rather than to a bioactive peptide (e.g., GLP-1). 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 (HSA) compared to comparative polypeptide conjugates that do not have a peptide linker and have a CRM conjugated on GLP-1. In certain embodiments, the monoconjugated polypeptide conjugates provided herein activate the GLP-1 receptor in the presence of HSA 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 HSA, as 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 HSA, with an EC50 comparable to that of semaglutide in the absence of HSA (e.g., about 20% to about 300%), as determined in the same or equivalent assay.

[0013] In a particular embodiment, HSA is present in an appropriate amount in an in vitro assay for GLP-1 receptor activation, enabling assessment of the effect of HSA binding on GLP-1 receptor activation.

[0014] In certain embodiments, the monoconjugated polypeptide conjugates provided herein bind to HSA with binding affinity (KD) comparable to that of semaglutide (e.g., about 70% to about 500%), as determined in the same or equivalent assay.

[0015] In certain embodiments, the monoconjugated polypeptide conjugates provided herein have at least equivalent terminal half-life to comparative polypeptide conjugates having a CRM conjugated on GLP-1 and lacking a peptide linker, such as semaglutide, as determined under equivalent in vivo conditions and in the same type of experimental animals.

[0016] In some embodiments, the polypeptide conjugate is a double-conjugated polypeptide conjugate, further comprising a second CRM conjugated to a second CRM residue.

[0017] In some embodiments, the second CRM residue is located within the bioactive peptide or within the peptide linker. In a particular embodiment, the second CRM residue is located at K26 of the GLP-1 peptide.

[0018] In some of these embodiments, the double-conjugated polypeptide conjugates provided herein have equivalent or acceptable GLP-1 receptor agonist activity in the presence of HSA compared to comparative polypeptide conjugates that lack a peptide linker and have a CRM conjugated on GLP-1. In certain embodiments, the double-conjugated polypeptide conjugates provided herein activate the GLP-1 receptor in the presence of HSA with an EC50 of 2000% or less (or 1500%, 1000%, 900%, 800%, 700%, 600%, or 500% or less) of comparative polypeptide conjugates that lack a peptide linker and have a CRM conjugated on GLP-1, such as semaglutide, as determined in the same or equivalent assay. In certain embodiments, the double-conjugated polypeptide conjugates provided herein activate the GLP-1 receptor in the absence of HSA at an EC50 comparable to that of a comparative polypeptide conjugate having a CRM without a peptide linker, such as semaglutide, and conjugated on GLP-1, as determined in the presence of HSA (e.g., 500%, 400%, 300%, or 200%, or even less) in the presence of HSA. In certain embodiments, the double-conjugated polypeptide conjugate provided herein activates the GLP-1 receptor at a first EC50 in the presence of HSA and at a second EC50 in the absence of HSA, where the first EC50 is higher than the second EC50 but no more than 1000 times that of the second EC50, for example, the first EC50 (in the presence of HSA) is 900 times, 800 times, 700 times, 600 times, or 500 times less than the second EC50 (in the absence of HSA). In certain embodiments, HSA is present in an in vitro assay for GLP-1 receptor activation in an appropriate amount that allows for assessment of the effect of HSA binding on GLP-1 receptor activation.

[0019] In certain embodiments, the double-conjugated polypeptide conjugates provided herein bind to HSA with a higher binding affinity, represented by a significantly lower KD value (e.g., 50%, 40%, 30%, or 20% or less) than comparative polypeptide conjugates having a CRM without a peptide linker and conjugated on GLP-1, such as semaglutide, as determined in the same or equivalent assay.

[0020] In certain embodiments, the double-conjugated polypeptide conjugates provided herein have at least a 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 180%, 200%, 300%, and 400% increase in terminal phase half-life compared to comparative polypeptide conjugates having a CRM conjugated on GLP-1 and lacking a peptide linker, such as semaglutide, as determined under equivalent in vivo investigation conditions and in the same type of experimental animals.

[0021] In certain embodiments, the double-conjugated polypeptide conjugates provided herein have blood, 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 comparative polypeptide conjugates having a CRM without a peptide linker and conjugated on GLP-1, such as semaglutide, as determined under equivalent in vivo study conditions and in the same type of experimental animals.

[0022] In certain embodiments, the double-conjugated polypeptide conjugates provided herein offer an extended duration of therapeutic efficacy compared to comparative polypeptide conjugates having a CRM conjugated on GLP-1 and lacking a peptide linker, such as semaglutide, as determined under equivalent in vivo investigation conditions and in the same type of experimental animals. In certain embodiments, the extended duration of therapeutic efficacy may be characterized by the area under the curve (AUC) of the time response curve and / or the duration of the therapeutic response.

[0023] In certain embodiments, the therapeutic response includes a decrease in body weight, a decrease in food intake, or a decrease in glucose levels (fasting glucose levels or non-fasting glucose levels). In certain embodiments, the double-conjugated polypeptide conjugates provided herein have an increase in AUC to the time response curve of at least 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 180%, and 200% compared to comparative polypeptide conjugates having a CRM without a peptide linker and conjugated on GLP-1, such as semaglutide, as determined under equivalent in vivo investigation conditions and in the same type of experimental animals. In certain embodiments, the double-conjugated polypeptide conjugates provided herein have an increase in the duration of the therapeutic response of at least 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 180%, 200%, 300%, or 400% compared to comparative polypeptide conjugates having a CRM conjugated on GLP-1 and lacking a peptide linker, such as semaglutide, as determined under equivalent in vivo study conditions and in the same type of experimental animals.

[0024] In some embodiments, GLP-1 comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 1, while retaining substantial biological activity of SEQ ID NO: 1.

[0025] In some embodiments, GLP-1 contains at most 9, 8, 7, 6, 5, or 4 mutations compared to SEQ ID NO: 1, while retaining the substantial biological activity of SEQ ID NO: 1.

[0026] In some embodiments, GLP-1 comprises or consists of one or more mutations at positions selected from the group consisting of A8, G22, Q23, E27, K26, A30, K34, R36, and H7, or any combination thereof, compared to SEQ ID NO: 1.

[0027] In some embodiments, GLP-1 includes or consists of one or more substitutions selected from the group consisting of A8Aib, G22E, K26R, K34R, and R36G, or any combination thereof.

[0028] In some embodiments, the first CRM residue is a lysine residue, and the polypeptide conjugate contains only one lysine residue.

[0029] In some embodiments, both the first and second CRM residues are lysine residues, and the polypeptide conjugate contains only two lysine residues.

[0030] In some embodiments, the second lysine residue is located within GLP-1 and is arbitrarily selected from the group consisting of K23, K26, K27, K30, and K34.

[0031] In some embodiments, all lysine residues other than the CRM residue in GLP-1 are replaced with non-lysine residues optionally selected from the group consisting of arginine (R), glutamine (Q), alanine (A), glycine (G), histidine (H), serine (S), and threonine (T).

[0032] In some embodiments, the first CRM residue is a cysteine ​​residue, and the polypeptide conjugate contains only one cysteine ​​residue.

[0033] In some embodiments, the first and second CRM residues are both cysteine residues, and the polypeptide conjugate comprises only two cysteine residues.

[0034] In some embodiments, the second cysteine residue is within GLP-1, and is optionally selected from the group consisting of C23, C26, C27, C30, and C34.

[0035] In some 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 the CRM residue.

[0036] In some embodiments, the first and second amino acid residues are non-natural amino acid residues, and the polypeptide conjugate comprises only two non-natural amino acid residues as CRM residues.

[0037] In some embodiments, GLP-1 comprises the amino acid sequence of X7X8EGTFTSDVSSYLEX 22 X 23 AAX 26 X 27 FIX 30 WLVX 34 GX 36 G (SEQ ID NO: 2), wherein: X7 is H, imidazole-4-acetate (IA), or imidazole propionic acid (IPA); X8 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, X 36 is R or G.

[0038] In some embodiments, X7 is H, X8 is G or Aib, X 22 is G or E; X 23is Q, C, or K, and X 26 is K, R, or C; X 27 is E, K, or C; X 30 is A, C, or K, and X 34 is K, R, or C, and X 36 It is R or G.

[0039] In some embodiments, GLP-1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 8, 10, 12, 13, 15, 17, 18, 20, 22, 23, 25, 27, 28, 30, 32, 33, 35, 36, 38, and 40-44.

[0040] In some embodiments, GLP-1 contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-6, 8-11, 13-16, 18-21, 23-26, 28-31, 33, 34, 36-39, and 184.

[0041] In some embodiments, the polypeptide linker has a length of at least 10 amino acid residues (for example, at least 12, 24, 32, 40, 48, 60, or 80 amino acid residues).

[0042] In some embodiments, the polypeptide linker has a length of 12 to 80 amino acid residues.

[0043] 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, excluding the CRM residue.

[0044] In some embodiments, the polypeptide linker comprises or consists of one or more repeats of a repeat sequence, excluding the CRM residue.

[0045] In some embodiments, the repeat sequence is sequence number 45 (GQEPGAQP), sequence number 46 (GAQPGAQP), sequence number 47 (GQEP), sequence number 48 (GAQP), sequence number 49 (GAQPGQEPGAQP), sequence number 50 (GAQPGQEP), sequence number 51 (GEQP), sequence number 52 (GPQE), sequence number 53 (GPEQ), sequence number 54 (GSEP), sequence number 55 (GESP), sequence number 56 (GPSE), sequence number 57 (GPES), sequence number 58 (GQAP), sequence number 59 (GPAQ), sequence number 60 (GPQA), sequence number 61 (GS The sequence consists of sequences selected from the group comprising QP, sequence numbers 62 (GASP), 63 (GPAS), 64 (GPSA), 65 (GGGS), 66 (GSGS), 67 (GGGGS), 68 (GQEPGQAP), 69 (GQAPGQEP), 70 (SEPATSGSETPGTSESATPESGPGTSTEPSEG), 71 (SEPATS), 72 (GSETPG), 73 (TSESAT), 74 (PESGPG), 75 (TSTEPS), and GS.

[0046] In some embodiments, the polypeptide linker comprises (repeat 1)r(repeat 2)s(repeat 3)x(repeat 4)y, where repeat 1, repeat 2, repeat 3, and repeat 4 are linked via peptide bonds or via one or more amino acid residues, and repeat 1, repeat 2, repeat 3, and repeat 4 independently correspond to SEQ ID NOs. 45 (GQEPGAQP), 46 (GAQPGAQP), 47 (GQEP), 48 (GAQP), 49 (GAQPGQEPGAQP), 50 (GAQPGQEP), 51 (GEQP), 52 (GPQE), 53 (GPEQ), 54 (GSEP), 55 (GESP), 56 (GPSE), 57 (GPES), 58 (GQAP), The array contains or consists of an array selected from the group consisting of sequence number 59 (GPAQ), sequence number 60 (GPQA), sequence number 61 (GSQP), sequence number 62 (GASP), sequence number 63 (GPAS), sequence number 64 (GPSA), sequence number 65 (GGGS), sequence number 66 (GSGS), sequence number 67 (GGGGS), sequence number 68 (GQEPGQAP), sequence number 69 (GQAPGQEP), sequence number 70 (SEPATSGSETPGTSESATPESGPGTSTEPSEG), sequence number 71 (SEPATS), sequence number 72 (GSETPG), sequence number 73 (TSESAT), sequence number 74 (PESGPG), sequence number 75 (TSTEPS), and GS, where r, s, x, and y are integers independently selected from 0 to 30 or 0 to 20, provided that r, s, x, and y are not simultaneously 0.

[0047] In some embodiments, x and y are 0, r and s are integers independently selected from 1 to 30, and repeat 1 and repeat 2 are a) Repeat 1 contains or consists of the sequence of sequence number 45, and Repeat 2 contains or consists of the sequence of sequence number 48; b) Repeat 1 contains or consists of the sequence of sequence number 47, and Repeat 2 contains or consists of the sequence of sequence number 48; c) Repeat 1 contains or consists of the sequence of sequence number 48, and Repeat 2 contains or consists of the sequence of sequence number 47; d) Repeat 1 contains or consists of the sequence of sequence number 48, and Repeat 2 contains or consists of the sequence of sequence number 45; e) Repeat 1 contains or consists of the sequence of sequence number 49, and Repeat 2 contains or consists of the sequence of sequence number 48; f) Repeat 1 contains or consists of the sequence of sequence number 70, and Repeat 2 contains or consists of the sequence of sequence number 48; g) Repeat 1 contains or consists of the sequence of sequence number 48, and Repeat 2 contains or consists of the sequence of sequence number 68; and h) Repeat 1 contains or consists of the sequence of sequence number 70, and Repeat 2 contains or consists of the sequence of sequence number 47. This is a combination selected from a group consisting of [the specified elements].

[0048] In some embodiments, r, x, and y are 0, s is an integer selected from 1 to 30 or 0 to 20, and Repeat 1 includes or consists of an array of sequence number 45.

[0049] In some embodiments, y is 0, r, s, and x are integers independently selected from 1 to 30, and repeat 1, repeat 2, and repeat 3 are a) Repeat 1 contains or consists of the sequence of sequence number 48; Repeat 2 contains or consists of the sequence of sequence number 49; Repeat 3 contains or consists of the sequence of sequence number 48; b) Repeat 1 contains or consists of the sequence of sequence number 48; Repeat 2 contains or consists of the sequence of sequence number 47; Repeat 3 contains or consists of the sequence of sequence number 48; c) Repeat 1 contains or consists of the sequence of sequence number 70; Repeat 2 contains or consists of the sequence of sequence number 47; Repeat 3 contains or consists of the sequence of sequence number 48; d) Repeat 1 contains or consists of the sequence of sequence number 70; Repeat 2 contains or consists of the sequence of sequence number 48; Repeat 3 contains or consists of the sequence of sequence number 47. e) Repeat 1 contains or consists of the sequence of sequence number 45, Repeat 2 contains or consists of the sequence of sequence number 58; Repeat 3 contains or consists of the sequence of sequence number 48, f) Repeat 1 contains or consists of the sequence of sequence number 48; Repeat 2 contains or consists of the sequence of sequence number 68; Repeat 3 contains or consists of the sequence of sequence number 45. g) Repeat 1 contains or consists of the sequence of sequence number 48, Repeat 2 contains or consists of the sequence of sequence number 45; Repeat 3 contains or consists of the sequence of sequence number 48, and h) Repeat 1 contains or consists of the sequence of sequence number 48; Repeat 2 contains or consists of the sequence of sequence number 45; Repeat 3 contains or consists of the sequence of sequence number 68. This is a combination selected from a group consisting of [the specified elements].

[0050] In some embodiments, r, s, x, and y are integers independently selected from 1 to 30 or 0 to 20, and repeat 1, repeat 2, repeat 3, and repeat 4 are a) Repeat 1 contains or consists of the sequence of sequence number 48; Repeat 2 contains or consists of the sequence of sequence number 50; Repeat 3 contains or consists of the sequence of sequence number 47; Repeat 4 contains or consists of the sequence of sequence number 48. b) Repeat 1 contains or consists of the sequence of sequence number 45, Repeat 2 contains or consists of the sequence of sequence number 68; Repeat 3 contains or consists of the sequence of sequence number 45, Repeat 4 contains or consists of the sequence of sequence number 48, c) Repeat 1 contains or consists of the sequence of sequence number 48; Repeat 2 contains or consists of the sequence of sequence number 68; Repeat 3 contains or consists of the sequence of sequence number 45; Repeat 4 contains or consists of the sequence of sequence number 48. d) Repeat 1 contains or consists of the sequence of sequence number 48, Repeat 2 contains or consists of the sequence of sequence number 58; Repeat 3 contains or consists of the sequence of sequence number 48, Repeat 4 contains or consists of the sequence of sequence number 45, and e) Repeat 1 contains or consists of the sequence of sequence number 48; Repeat 2 contains or consists of the sequence of sequence number 45; Repeat 3 contains or consists of the sequence of sequence number 69; Repeat 4 contains or consists of the sequence of sequence number 68. This is a combination selected from a group consisting of [the specified elements].

[0051] In some embodiments, the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 76-89, except that one residue is substituted by a CRM residue.

[0052] In some embodiments, the peptide linker includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 90-117, 175, and 176.

[0053] In some embodiments, the polypeptide conjugate comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 118-174 and 177-183.

[0054] In some embodiments, the CRM comprises a plasma protein binding moiety, a polymer, Fc, HSA (albumin), an Xten sequence, or a PAS sequence.

[0055] In some embodiments, the CRM includes an albumin-binding moiety.

[0056] In some embodiments, the albumin-binding moiety comprises the structure *-ABCDE, or a pharmaceutically acceptable salt thereof, where A, B, C, D, and E are interconnected via amide bonds, and the * terminus of A is connected to the reactive group of the CRM residue in the polypeptide complex, in the formula: A is a combination, [ka] Selected from, where a, b, c, and d are independent integers from 0 to 4, R 1 is hydrogen or -COOH; B is a combination, [ka] Selected from, where the α-th order is concatenated to the α'-th order, and e is an integer from 1 to 4. C is a combination or [ka] And R 2 is -CH2SO3H or -COOH, f is an integer from 1 to 4, n is an integer from 1 to 25, and if B is not associative, the β' position is linked to the β position, or if B is associative, the β' position is linked to the α' position; D is bond, [ka] Selected from, where g and h are independently 0 or 1, R 3 is H or -CH2COOH, 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, the γ' position is linked to the γ position; If B is a bond and C is a bond, then the γ' position is linked to the α position; E is the formula: [ka] It is an acidic group having, In the formula, W is -(CR4 R 5 ) l - represents, R 4 and R 5 These are independently selected from the group consisting of hydrogen, halogen, cyano, hydroxyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, hydroxyalkyl, amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy, and carboxamide. R 6 is hydroxyl or NR 7 R 8 Selected from; R 7 and R 8 These are independently hydrogen, alkyl, hydroxyl, and [ka] Selected from the group consisting of, l is an integer between 10 and 20, 10 and 18, or 10 and 16, for example, 12, 14, 16, or 18. If D is not a bond, then the δ position is linked to the δ' position. If C is not a bond and D is a bond, then the δ position is linked to the γ position. If B is not a bond, C is a bond, and D is a bond, then the δ position is linked to the β position. If A is not a bond, and B, C, and D are all bonds, then the δ position is linked to the α' position.

[0057] In some embodiments, the CRM is conjugated to a lysine residue.

[0058] In some embodiments, A is a coupling.

[0059] In some embodiments, A is a bond, and B is a bond or [ka] And in the formula, e is 1, 2, or 3.

[0060] In some embodiments, A is a coupling and B is [ka] And C is, [ka] In the formula, the β' position is linked to the β position, where e is 1, 2, or 3, f is 1, 2, or 3, and n is 1 or 2. In a particular embodiment, D is a bond, and E is in the formula: [ka] It is an acidic group having R 2 is -COOH, and R 6 is hydroxyl. In certain embodiments, W is -(CR 4 R 5 ) l - represents R 4 and R 5 l is independently a hydrogen atom, and l is an integer between 10 and 20, 10 and 18, or 10 and 16, for example, 12, 14, 16, or 18.

[0061] In some embodiments, A is a bond, B is a bond, and C is a bond.

[0062] In some embodiments, A is a bond, B is a bond, and C is [ka] In the equation, f is 1, 2, or 3, and n is 1 or 2.

[0063] In some embodiments, A is [ka] In the equation, a is 1, 2, or 3, b is 1, 2, or 3, and c is 1 or 2.

[0064] In some embodiments, A is [ka] And B is [ka] In the formula, the α-position is linked to the α'-position, where a is 1, 2, or 3, b is 1, 2, or 3, c is 1 or 2, and e is 1, 2, or 3.

[0065] In some embodiments, A is [ka] And B is [ka] And C is [ka] In the formula, the α position is linked to the α' position, the β' position is linked to the β position, and in the formula, a is 1, 2, or 3, b is 1, 2, or 3, c is 1 or 2, e is 1, 2, or 3, f is 1, 2, or 3, and n is 1 or 2.

[0066] In some embodiments, A is [ka] And B is [ka] C is a bond, where the α-position is linked to the α'-position, where a is 1, 2, or 3, b is 1, 2, or 3, c is 1 or 2, and e is 1, 2, or 3.

[0067] In some embodiments, A is [ka] B is a combination, where a is 1, 2, or 3, b is 1, 2, or 3, and c is 1 or 2.

[0068] In some embodiments, A is [ka] B is a bond, and C is [ka] In the formula, the β' position is linked to the α' position, where a is 1, 2, or 3, b is 1, 2, or 3, c is 1 or 2, f is 1, 2, or 3, and n is 1 or 2.

[0069] In some embodiments, A is [ka] B is a bond, C is a bond, where a is 1, 2, or 3, b is 1, 2, or 3, and c is 1 or 2.

[0070] In some embodiments, D is a bond.

[0071] In some embodiments, A is [ka] And B is [ka] And C is [ka] D is a bond, where the α-position is linked to the α'-position, the β'-position is linked to the β-position, where a is 1, 2, or 3, b is 1, 2, or 3, c is 1 or 2, e is 1, 2, or 3, f is 1, 2, or 3, and n is 1 or 2.

[0072] In some embodiments, D is [ka] In the equation, g is either 0 or 1, and h is either 0 or 1.

[0073] In some embodiments, A is [ka] And B is [ka] or bond, C is bond, D is [ka] In the formula, a is 1, 2, or 3, b is 1, 2, or 3, c is 1 or 2, e is 1, 2, or 3, g is 0 or 1, h is 0 or 1, if B is not a bond, the α position is linked to the α' position and the γ' position is linked to the β position, and if B is a bond, the γ' position is linked to the α' position.

[0074] In some embodiments, D is [ka] That is the case.

[0075] In some embodiments, A is [ka] And B is [ka] And C is bonded or [ka] And D is [ka] If C is a bond, the α position is linked to the α' position and the γ' position is linked to the β position, or if C is not a bond, the α position is linked to the α' position, the β' position is linked to the β position and the γ' position is linked to the γ position, where a is 1, 2, or 3, b is 1, 2, or 3, c is 1 or 2, e is 1, 2, or 3, f is 1, 2, or 3, and n is 1 or 2.

[0076] In some embodiments, the CRM is given by the following formula: [ka] It includes the structure.

[0077] In some embodiments, the CRM is conjugated to a cysteine ​​residue.

[0078] In some embodiments, A is [ka] And B is [ka] In this equation, the α-position is connected to the α'-position.

[0079] In some embodiments, A is [ka] And B is [ka] And C is [ka] In the formula, the α position is linked to the α' position, the β' position is linked to the β position, and in the formula, a is 1, 2, or 3, b is 1, 2, or 3, c is 1 or 2, d is 1, 2, or 3, f is 1, 2, or 3, and n is 1 or 2. In a particular embodiment, D is a bond, and E is in the formula: [ka] It is an acidic group having R 2 is -COOH, and R 6 is hydroxyl. In certain embodiments, W is -(CR 4 R 5 ) l - represents R 4 and R 5 l is independently a hydrogen atom, and l is an integer between 10 and 20, 10 and 18, or 10 and 16, for example, 12, 14, 16, or 18.

[0080] In some embodiments, A is [ka] And B is [ka] And C is [ka] D is a bond, where the α position is linked to the α' position, the β' position is linked to the β position, where a is 1, 2, or 3, b is 1, 2, or 3, c is 1 or 2, d is 1, 2, or 3, f is 1, 2, or 3, and n is 1 or 2.

[0081] In some embodiments, A is [ka] And B is [ka] In the formula, the α-position is linked to the α'-position, and in the formula, d is 1, 2, or 3, and e is 1, 2, or 3.

[0082] In some embodiments, A is [ka] And B is [ka] C is a bond, in the formula the α-position is linked to the α'-position, in the formula d is 1, 2, or 3, and e is 1, 2, or 3.

[0083] In some embodiments, A is [ka] And B is [ka] C is a bond, and D is [ka] In the formula, the α position is linked to the α' position, the γ' position is linked to the β position, d is 1, 2, or 3, e is 1, 2, or 3, g is 0 or 1, and h is 0 or 1.

[0084] In some embodiments, the CRM is given by the following formula: [ka] It includes the structure.

[0085] In a second embodiment, the disclosure provides a polynucleotide encoding the polypeptide portion (or fragment thereof) of the polypeptide conjugate of the first embodiment.

[0086] In a third embodiment, the disclosure provides a vector comprising a polynucleotide according to the second embodiment.

[0087] In a fourth embodiment, the disclosure provides a host cell comprising a vector according to the third embodiment.

[0088] In some embodiments, the host cell is a prokaryotic or eukaryotic cell.

[0089] In a fifth embodiment, the disclosure provides a method for producing a polypeptide conjugate of the first embodiment, comprising culturing a host cell of the fourth embodiment under conditions that enable the expression of a polynucleotide of the second embodiment to obtain the polypeptide portion of the polypeptide conjugate.

[0090] In some embodiments, the host cell is a prokaryotic or eukaryotic cell.

[0091] In some embodiments, the polypeptide portion is expressed as a soluble protein.

[0092] In some embodiments, the method further includes conjugating the polypeptide portion with CRM.

[0093] In a sixth embodiment, the disclosure provides a pharmaceutical composition comprising a polypeptide conjugate according to the first embodiment and a pharmaceutically acceptable salt.

[0094] In a seventh aspect, the Disclosure provides a method for preventing or treating a metabolic disorder in a subject requiring such treatment, comprising administering a therapeutically effective dose of a polypeptide conjugate according to the first aspect.

[0095] In an eighth aspect, the Disclosure provides a method for managing the weight of a subject in need, comprising administering a therapeutically effective dose of a polypeptide conjugate provided herein and thereby managing the subject's weight.

[0096] In a ninth aspect, the Disclosure provides a method for reducing food intake in a subject in need, comprising administering a therapeutically effective dose of a polypeptide conjugate provided herein, thereby reducing the subject's food intake.

[0097] In a tenth aspect, the Disclosure provides a method for reducing body weight in a subject in need, comprising administering a therapeutically effective dose of a polypeptide conjugate provided herein, thereby reducing the subject's body weight.

[0098] In certain embodiments, metabolic disorders include diabetes mellitus, obesity, overweight, non-alcoholic steatohepatitis (NASH), cardiovascular-like dyslipidemia, arteriosclerosis, alcoholic steatohepatitis (ASH), diabetic nephropathy, gestational diabetes, metabolic syndromes such as metabolic syndrome X, non-alcoholic fatty liver disease (NAFLD), end-stage liver disease, fatty liver disease, cirrhosis, or primary biliary cirrhosis (PBC).

[0099] In certain embodiments, diabetes may 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 (young-onset adult-onset diabetes), gestational diabetes, and elevated HbA1c levels.

[0100] In a particular embodiment, the subject is a human being.

[0101] In a particular embodiment, the subject has a fasting blood glucose level of 125 mg / dL or higher.

[0102] In a particular embodiment, the subject has a body mass index (BMI) of at least 25 or higher than 25.

[0103] In certain embodiments, the polypeptide conjugate is administered in a dosage regimen less frequently than once daily, once every three days, once a week, once every two weeks, once every three weeks, or once a month.

[0104] In certain embodiments, the polypeptide conjugate is administered twice a week, once a week, once every two weeks, once every three weeks, once a month, or once every two months.

[0105] In a particular embodiment, the medication regimen has a dosing interval ranging from approximately every three days to approximately once a month, or from approximately once a week to approximately once a month.

[0106] In certain embodiments, the polypeptide conjugate is administered subcutaneously, intravenously, or orally.

[0107] 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) grammatical object of the article. For example, “a fusion polypeptide” means one or more fusion polypeptides.

[0108] In all occurrences in this application where a series of enumerated numbers are present, it should be understood that any of the enumerated numbers may be the upper or lower limit of a range of numbers. It should be further understood that the invention encompasses all such ranges of numbers, i.e., domains having combinations of numerical upper and lower limits, where the numerical values ​​for each of the upper and lower limits may be any of the numerical values ​​enumerated herein. A domain provided herein is understood to include all values ​​within the domain. For example, 1-10 is understood to include all the values ​​1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, as well as any decimal values. Similarly, a domain delimited by “at least” is understood to include the lower and all higher numbers provided.

[0109] As used herein, “approximately” is understood to include within three standard deviations of the mean or within the standard range of acceptance in the particular art. In certain embodiments, “approximately” is understood to mean a variation of at most 0.5.

[0110] The articles “a” and “an” are used herein to refer to one or more than one (i.e., at least one) grammatical object of the article. For example, “an element” means one type of element or more than one type of element.

[0111] The term "including" is used herein to mean "including, but not limited to," and is interchangeable with it. Similarly, "etc." is used herein to mean "equals, but not limited to," and is interchangeable with it.

[0112] The term "or" is used herein inclusively and interchangeably to mean the term "and / or" unless otherwise clearly indicated in the context. [Brief explanation of the drawing]

[0113] [Figure 1] Figure 1 (Figures 1A-1D) shows the in vivo activity of molecules 001, 002, and 012 in C57BL / 6 mice. To assess body weight, 10-week-old male C57BL / 6 mice were administered the test drug substance once subcutaneously. Figure 1A shows body weight loss after treatment with molecule 001 from day 1 to day 8. Figure 1B shows body weight loss after treatment with molecule 002 from day 1 to day 8. Figure 1C shows body weight loss after treatment with molecule 012 or molecule 001 from day 1 to day 8. Figure 1D shows body 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 control. [Figure 2] Figure 2 (Figures 2A, 2B, and 2C) shows the in vivo activity of molecules 001, 002, and 012 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 mean and standard error (SEM). Figure 2C shows non-fasting glucose after a single dose of molecule 012. To assess glucose, 10-week-old male db / db mice were subcutaneously administered the specified GLP-1 polypeptide conjugate at the indicated doses. Fasting glucose (Figures 2A and 2B) or non-fasting glucose (Figure 2C) was measured at various time points, with 5 animals used in each group. Delta blood glucose is glucose minus the baseline level. Data are shown as mean and standard error (SEM). [Figure 3]Figure 3 (Figures 3A and 3B) shows the in vivo efficacy in diet-induced obesity (DIO) mice. To evaluate the effects of each GLP-1 polypeptide conjugate on body weight, food intake, and glucose levels, 22-week-old DIO mice (C57BL / 6 mice fed a high-fat diet for 13 weeks) were subcutaneously administered various concentrations of specified GLP-1 polypeptide conjugates every other day for 25 days. Figure 3A shows the changes in body weight using dose titration of 10, 30, and 100 nmol / kg of molecular weight 012. Figure 3B shows the changes in fasting glucose immediately after treatment. Data are expressed as mean and standard error (SEM). [Figure 4] Figure 4 (Figures 4A-4L) shows all sequences disclosed herein. [Modes for carrying out the invention]

[0114] Detailed explanation The following description of the present invention is intended to illustrate various embodiments of the invention. Therefore, specific modifications discussed should not be construed as limitations on the scope of the invention. It will be apparent to those skilled in the art that various equivalents, variations, and modifications can be realized without departing from the scope of the invention, and it will be understood that such equivalent embodiments should be incorporated herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0115] definition As used herein, the term "amino acid" refers to an organic compound containing an amine (-NH2) and a carboxyl (-COOH) functional group, along with a side chain specific to each amino acid.

[0116] As used herein, the term “naturally occurring” amino acid residue refers to an amino acid residue found in a natural protein or peptide, including all of its D and L stereoisomers, where 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), and lysine (L This includes 20 standard amino acids, including ys 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 their natural analogs, such as canavanine, pyrrolidine (PYL), selenocysteine, pyrroline-carboxylysine (PCL), sarcosine, beta-alanine, phosphoserine, γ-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, and D-glutamic acid.

[0117] "Amino acid analogs" are compounds that have the same basic chemical structure as naturally occurring amino acids, namely, a carbon atom bonded to hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have a modified R group (e.g., norleucine) or a modified peptide skeleton, but will retain the same basic chemical structure as naturally occurring amino acids.

[0118] As used herein, “unnatural” amino acid residues refer to any amino acid residue not found in nature, including, but not limited to, modified amino acid residues and / or amino acid mimetic compounds that are not one of the known naturally occurring amino acids but function in a similar manner to naturally occurring amino acids. Modified amino acids or mimetic compounds may be created by the addition of chemical entities such as carbohydrate groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers for conjugation, functionalization, or other modifications. Unnatural amino acids may also refer to amino acids produced by chemical synthesis. Examples of non-natural amino acids include 2-aminoisobutyric acid (Aib), imidazole-4-acetate (IA), imidazolepropionic acid (IPA), α-aminobutyric acid (Abu), tert-butylglycine (Tle), β-alanine, 3-aminomethylbenzoic acid, anthranilic acid, des-amino-histidine (abbreviated as DesaminoHis, alternative name imidazopropionic acid, abbreviated as lmpr), beta analogs of amino acids such as β-alanine, 2-amino-histidine, β-hydroxyhistidine, homohistidine, Nα-acetylhistidine, α-fluoromethyl This includes, but is not limited to, ethyl-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.

[0119] The introduction of non-natural amino acids into polypeptides can be achieved by techniques described in Wang et al., Science 292:498-500, 2001; Deiters et al., J Am Chem Soc 125:11782-11783, 2003; Wang and Schultz, Science 301:964-967, 2003; Zhang et al., Science 303:371-373, 2004, or U.S. Patent No. 7,083,970. Briefly, some of these expression systems involve site-directed mutagenesis, which introduces stop codons such as amber (UAG), ochre (UAA), and opal (UGA) codons into an open reading frame encoding the fusion polypeptide of this disclosure. Other codons, such as 4-base codons (e.g., AGGA, AGGU, CGGU, CGCU, CGAU, CCCU, CUCU, CUAU, and GGGU), 5-base codons, and 6-base codons, can also be introduced into expression systems for non-natural amino acids. Such expression vectors are then introduced into a host that can utilize tRNA specific to the introduced stop codon or other codons and carrying the selected non-natural amino acid. As another example, non-natural amino acids may be chemically synthesized and inserted into or attached to polypeptides by chemical reactions such as acylation.

[0120] Sequence identity percentage (%) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those in a reference sequence, after the sequences have been aligned and gaps introduced where necessary to achieve the maximum number of identical amino acids (or nucleic acids). In other words, the sequence identity percentage (%) of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of amino acid residues (or bases) that are identical to those in the reference sequence being compared by the total number of amino acid residues (or bases) in either the candidate sequence or the reference sequence, whichever is shorter. Conservative substitutions of amino acid residues are not considered identical. Alignment for the purpose of determining amino acid (or nucleic acid) sequence identity percentage can be achieved using publicly available tools such as BLASTN, BLASTp (available on the website of the National Center for Biotechnology Information (NCBI), 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 on the website of the European Institute for Bioinformatics, see also Higgins DGet 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. Those skilled in the art can use the initial parameters provided by the tool, or customize the parameters for alignment as needed, for example, by selecting an appropriate algorithm.

[0121] A "conservative substitution" in relation to amino acid sequences refers to replacing an amino acid residue with a different amino acid residue that has a side chain with similar physiological and chemical properties. For example, conservative substitutions can occur 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 substitutions usually do not cause significant changes in the protein's three-dimensional structure and therefore can preserve the protein's biological activity.

[0122] As used herein, the term “functional form” refers to various forms of a parent molecule (such as variants, fragments, fusions, derivatives, and mimics) that, despite having differences in amino acid sequence or chemical structure, still retain substantial biological activity of the parent molecule. As used herein, the expression “retain substantial biological activity” means exhibiting at least some (e.g., at least 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 may include both naturally occurring variant forms and non-natural forms, such as those obtained by recombinant or chemical synthesis. Functional forms may contain non-natural amino acid residues.

[0123] As used herein, the term “variant” refers to a polypeptide having at least 70% sequence identity with the parent polypeptide. A variant may differ from the parent peptide by one or more amino acid residues. For example, a variant may have one or more amino acid residues of the parent polypeptide substituted, added, deleted, inserted, or excised.

[0124] As used herein, the term “fragment” refers to a partial sequence of a parent polypeptide of any length. A fragment may still retain at least some of the functionality of the parent polypeptide.

[0125] As used herein, the term “derivative” refers to a chemically modified polypeptide or fusion polypeptide in which one or more clearly defined substituents are covalently attached to one or more specific amino acid residues of the polypeptide or fusion polypeptide. Exemplary chemical modifications may include, for example, alkylation, acylation, esterification, amidation, phosphorylation, glycosylation, labeling, methylation of one or more amino acids, or conjugation with one or more moieties.

[0126] As used herein, the term “mimetic” refers to a molecular structure that acts as a substitute for an amino acid, peptide, polypeptide, or fusion polypeptide. For example, an amino acid mimetic as used herein may be an amino acid or not, but may be a synthetic structure (known or unknown) that retains the functional characteristics of the parent amino acid, while the structure of the amino acid mimetic differs from that of the parent amino acid. Examples include methacryloyl or acryloyl derivatives of amides, β-, γ-, and δ-imino acids (such as piperidine-4-carboxylic acid).

[0127] As used herein, “treating” or “treating” a medical condition includes preventing or alleviating the condition, slowing the rate of 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 ending symptoms associated with the condition, causing complete or partial regression of the condition, curing the condition, or any combination thereof.

[0128] As used herein, the term “vector” refers to a vehicle into which a protein-coding polynucleotide can be operably inserted to result in the expression of that protein. A vector can be used to transform, transduce, or transfect a host cell to result in the expression of a genetic element it carries within the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phages or M13 phages, and animal viruses. Categories of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription start sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain a replication origin. The vector may also contain materials that assist its entry into cells, including but not limited to viral particles, liposomes, or protein coatings. The vector may be an expression vector or a cloning vector. This disclosure provides a vector (e.g., an expression vector) containing a nucleic acid sequence provided herein encoding a fusion polypeptide, at least one promoter operably linked to the nucleic acid sequence (e.g., SV40, CMV, EF-1α), and at least one select marker.Examples of vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40), lambda phages, and M13 phages, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, p This includes, but is 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, etc.

[0129] As used herein, the term “host cell” refers to a cell into which an exogenous polynucleotide and / or vector has been introduced.

[0130] The term "pharmaceutically acceptable" indicates that a specified carrier, vehicle, diluent, excipient, and / or salt is substantially chemically and / or physically compatible with the other components of the formulation and physiologically compatible with its recipient.

[0131] As used herein, the terms “subject,” “individual,” “animal,” or “patient” refer to human or non-human animals, including mammals or primates, that require diagnosis, prognosis, improvement, prevention, and / or treatment for a disease or disorder. Mammal subjects include humans, domesticated animals, agricultural animals, and animals kept for zoos, exercise, or as pets, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cattle, bears, etc.

[0132] Polypeptide conjugate In one embodiment, the present disclosure provides a polypeptide conjugate comprising a single bioactive peptide attached to the N-terminus of a peptide linker, and a first CRM conjugated to a first clearance reduction (CRM) residue in the peptide linker, wherein the bioactive peptide comprises a GLP-1 receptor agonist, and the first CRM residue is located at least 5 amino acid residues (excluding the CRM residue) away from the C-terminal amino acid residue of the GLP-1 receptor agonist.

[0133] The terms “peptide” and “polypeptide” are used interchangeably herein and refer to polymers of amino acid residues linked by covalent bonds, such as peptide bonds. The peptides or polypeptides provided herein may contain naturally occurring or unnatural amino acid residues, or both. The polypeptides and peptides provided herein may contain amino acid residues of any suitable length, 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.

[0134] A polypeptide conjugate contains a single, i.e., only one, bioactive peptide. The term "single" in relation to a bioactive peptide is intended to mean that the polypeptide conjugate does not contain two or more different bioactive peptides each attached to a peptide linker. However, a single bioactive peptide may contain fragments or portions derived from different bioactive peptides fused together, for example, as a hybrid or chimera, as long as these portions are not integrated as a single peptide and each attached to a linker. However, if 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 this disclosure.

[0135] As used herein, the term “bioactive peptide” means a peptide having a biological function or activity, such as a physiological function or therapeutic function. In certain embodiments, a bioactive peptide is therapeutically active. A peptide that does not have a biological function when used alone is not a bioactive peptide. For example, a peptide linker is not a bioactive peptide unless, when used alone, it has its own biological function or activity.

[0136] Bioactive peptides include glucagon-like peptide-1 (GLP-1) receptor agonists.

[0137] The term "glucagon-like peptide-1 (GLP-1) receptor" (also known as GLP1R) refers to a receptor protein found on beta cells in the pancreas and neurons in the brain, containing 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. The GLP-1 receptor is involved in regulating blood glucose levels by enhancing insulin secretion. When expressed in the brain, the GLP-1 receptor may also be involved in regulating appetite.

[0138] As used herein, the terms “glucagon-like peptide-1 (GLP-1) receptor agonist” or “GLP-1 receptor agonist” refer to molecules that can bind to and activate the GLP-1 receptor. GLP-1 receptor agonists can elicit a GLP-1 receptor response that is comparable to, or partially comparable to, that of the native ligand.

[0139] As used herein, the terms “clearance-modifying portion” or “CRM” refer to a portion that can modify one or more pharmacokinetic (PK) properties (e.g., increasing the half-life in vivo). Examples of CRMs, without limitation, may include 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, decrease or minimize the rate of the reverse Michael reaction, and thus decrease or minimize the rate of drug loss, as well as the rate of loss of linker groups from bioactive peptides to other thiol-containing proteins and small molecules.

[0140] As used herein, "CRM residue" refers to an amino acid residue that is conjugated to CRM.

[0141] As used herein, the term “conjugate” refers to a compound resulting from two or more molecules joined together to form a single physical entity. For example, the conjugate in this disclosure means a compound resulting from a polypeptide and one or more clearance modification moieties joined together. The molecules may be joined together by covalent, non-covalent, linker, chemical modification, or protein fusion, or by any means known to those skilled in the art. Preferably, the molecules may be joined together by covalent bonds. The conjugate may be permanent or reversible. In some embodiments, certain cleavable or incleavable linkages may be included.

[0142] While CRM conjugation to GLP-1 receptor agonists can extend the half-life of GLP-1 receptor agonists, it can also negatively affect the biological activity of GLP-1 receptor agonists, making them less active than their unconjugated counterparts. For example, the conjugated GLP-1 derivative semaglutide has a significantly extended half-life, but its GLP-1 receptor binding is reduced to 1 / 939th of that of its unconjugated counterpart in the presence of human serum albumin (HSA) (J.Med.Chem.2015,58,7370-7380).

[0143] However, the inventors have unexpectedly discovered that certain CRM-conjugated polypeptides, including GLP-1 receptor agonists, can possess both an extended half-life and retained biological activity when the 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. When the CRM conjugation on the peptide linker is located close to the C-terminus of the GLP-1 receptor agonist, it is thought to significantly reduce biological activity, which can be much lower than when the conjugation is on the GLP-1 receptor agonist itself. However, when the conjugation is located sufficiently far from the C-terminus of the GLP-1 receptor agonist, the CRM-conjugated GLP-1 receptor agonist can possess both an extended half-life and retained biological activity. Such unexpected effects can be observed using certain GLP-1 receptor agonists and certain CRMs.

[0144] In a particular embodiment, the first CRM residue is located at least 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 35, 38, 40, 45, 46, 50, 55, 58, 60, 65, 70, 75, or 78 residues (excluding the CRM residue) away from the C-terminal amino acid residue of the GLP-1 receptor agonist. In a particular embodiment, the distance between the first CRM residue and the C-terminal amino acid of the GLP-1 receptor agonist is 10-120, 15-120, 20-120, 25-120, 30-120, 31-120, 32-120, 33-120, 34-120, 35-120, 36-120, 37-120, 38-120, 39-120, 40-120, 30-80, 31-80, 32-80, 33-80, 34-80, 35-80, 36-80, 37-80, 38-80, 39-80, or 40-80.

[0145] In certain embodiments, the GLP-1 receptor agonist in the polypeptide conjugate has a length of at most 70, 60, or 50 amino acid residues. In certain embodiments, the GLP-1 receptor agonist in the polypeptide conjugate has a length of at most 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, or 30 amino acid residues.

[0146] In certain embodiments, the GLP-1 receptor agonist in the polypeptide conjugate includes or is GLP-1 as provided in this disclosure. In some embodiments, the GLP-1 in the polypeptide conjugate provided herein includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 8, 10, 12, 13, 15, 17, 18, 20, 22, 23, 25, 27, 28, 30, 32, 33, 35, 36, 38, 40-44, and 184. In certain embodiments, the GLP-1 in the polypeptide conjugate includes or consists of the amino acid sequence of SEQ ID NO: 3 (8Aib, 34R, 36G) or SEQ ID NO: 8 (8Aib, 22E, 34R, 36G).

[0147] Without wishing to be constrained by any theory, it appears that CRM (or fatty acid) conjugation to GLP-1 may reduce its activity in the presence of HSA. For example, a decrease in GLP-1 activity has been reported with respect to semaglutide, which has a fatty acid conjugated to K26 of GLP-1 (8Aib, 36R) and showed a significant decrease in activity in the presence of HSA compared to its unconjugated counterpart. Since HSA is present in human blood and unavoidable under physiological conditions, a decrease in GLP-1 receptor agonist activity in the presence of HSA would likely impair the therapeutic activity of the protein conjugate. However, some polypeptide conjugates provided herein may retain most of the GLP-1 activity in the presence of HSA despite CRM conjugation. In such embodiments, the polypeptide conjugates provided herein are monoconjugated and have a CRM conjugated to a peptide linker rather than to a bioactive peptide (e.g., GLP-1).

[0148] In some of these embodiments, such monoconjugated polypeptide conjugates provided herein have increased GLP-1 receptor agonist activity in the presence of HSA compared to a comparative polypeptide conjugate that lacks a peptide linker and has a CRM conjugated on GLP-1. In certain embodiments, the GLP-1 receptor agonist activity in the presence of HSA 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 comparative polypeptide conjugate is semaglutide. In certain embodiments, the monoconjugated polypeptide conjugates provided herein activate the GLP-1 receptor in the presence of human serum albumin (HSA) with an EC50 of 50% or less (or 40%, 30%, 20%, 10%, 5%, or 3% or less) than that of semaglutide in the presence of HSA, as 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 HSA, with an EC50 comparable to (e.g., 20% to 300% of) that of semaglutide in the absence of HSA, as determined in the same or equivalent assay. In certain embodiments, the amount of human serum albumin (HSA) allows for assessment of the effect of HSA 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% HSA.

[0149] In certain embodiments, the monoconjugated polypeptide conjugates provided herein bind to HSA with binding affinity (KD) comparable to that of semaglutide (e.g., about 70% to about 500%), as determined in the same or equivalent assay.

[0150] In certain embodiments, the monoconjugated polypeptide conjugates provided herein have at least equivalent terminal phase half-lives to comparative polypeptide conjugates having a CRM conjugated on GLP-1 and lacking a peptide linker, such as semaglutide, as determined under equivalent in vivo investigation conditions and in the same type of experimental animal. Suitable animals for determining terminal phase half-lives include, for example, mice, rats, miniature pigs, or monkeys. In certain embodiments, the terminal phase half-life is determined in a suitable animal after a single intravenous, subcutaneous, or oral administration of a dose appropriate to provide therapeutic efficacy.

[0151] In certain embodiments, the polypeptide conjugate further comprises a second CRM conjugated to a second CRM residue. In other words, the disclosure also provides a double-conjugated polypeptide conjugate. It has been unexpectedly discovered by the inventors that the second CRM conjugation can further extend the half-life of the polypeptide conjugate. In certain embodiments, a double-conjugated polypeptide conjugate having both a first and a second CRM conjugation may have a half-life that is at least 1, 2, 3, 4, 5, 6 times, or even longer, than the corresponding polypeptide conjugate having only one CRM conjugation (e.g., semaglutide).

[0152] The second CRM residue can be located at any suitable position. In certain embodiments, the second CRM residue is located within the GLP-1 receptor agonist or within the peptide linker.

[0153] In a particular embodiment, the second CRM residue is located at K26 of the GLP-1 peptide. It has been reported that conjugation with two fatty acid moieties on the GLP-1 peptide can significantly reduce its activity or further inactivate the conjugated GLP-1 peptide. Therefore, it has been unexpectedly found by the inventors that conjugation both on GLP-1 (e.g., K26) and on the peptide linker at a position sufficiently far from the C-terminus of the GLP-1 peptide can minimize the negative effects of fatty acid conjugation on the biological activity of the GLP-1 polypeptide in the presence of HSA.

[0154] In some of these embodiments, such double-conjugated polypeptide conjugates provided herein have equivalent or acceptable GLP-1 receptor agonist activity in the presence of HSA compared to comparative polypeptide conjugates that lack a peptide linker and have a CRM conjugated on GLP-1. In certain embodiments, the double-conjugated polypeptide conjugates provided herein activate the GLP-1 receptor in the presence of HSA with an EC50 of 2000% or less (or 1500%, 1000%, 900%, 800%, 700%, 600%, or 500% or less) of comparative polypeptide conjugates that lack a peptide linker and have a CRM conjugated on GLP-1, such as semaglutide, as determined in the same or equivalent assay. In certain embodiments, the double-conjugated polypeptide conjugates provided herein activate the GLP-1 receptor in the absence of HSA at an EC50 comparable to (e.g., 500%, 400%, 300%, or 200% or less) that of a comparative polypeptide conjugate having a CRM without a peptide linker and conjugated on GLP-1, such as semaglutide, as determined in the same or equivalent assay. In certain embodiments, the double-conjugated polypeptide conjugate provided herein activates the GLP-1 receptor at a first EC50 in the presence of HSA and at a second EC50 in the absence of HSA, where the first EC50 is higher than the second EC50 but no more than 1000 times that of the second EC50, for example, the first EC50 (in the presence of HSA) is 900 times, 800 times, 700 times, 600 times, or 500 times less than the second EC50 (in the absence of HSA). In certain embodiments, HSA is present in an in vitro assay for GLP-1 receptor activation in an appropriate amount that allows for assessment of the effect of HSA binding on GLP-1 receptor activation.In certain embodiments, the in vitro assay for GLP-1 receptor activation is performed in the presence of an appropriate amount of human serum albumin (HSA) that allows for assessment of the effect of HSA 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% HSA.

[0155] In certain embodiments, the double-conjugated polypeptide conjugates provided herein bind to HSA with a higher binding affinity, represented by a significantly lower KD value (e.g., 50%, 40%, 30%, or 20% or less) than comparative polypeptide conjugates having a CRM without a peptide linker and conjugated on GLP-1, such as semaglutide, as determined in the same or equivalent assay.

[0156] In certain embodiments, the double-conjugated polypeptide conjugates provided herein have enhanced pharmacokinetic properties compared to comparative polypeptide conjugates having a CRM conjugated on a bioactive peptide (e.g., GLP-1) and lacking a peptide linker, the pharmacokinetic properties being determined by measuring the blood concentration of the polypeptide conjugate after administration of a therapeutically effective dose to the subject.

[0157] In certain embodiments, the double-conjugated polypeptide conjugates provided herein have at least a 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 180%, 200%, 300%, or 400% increase in terminal phase half-life compared to comparative polypeptide conjugates having a CRM conjugated on GLP-1 and lacking a peptide linker, such as semaglutide, as determined under equivalent in vivo investigation conditions and in the same type of experimental animal. Suitable animals for determining terminal phase half-life include, for example, mice, rats, miniature pigs, or monkeys. In certain embodiments, the terminal phase half-life is determined in a suitable animal after a single intravenous or subcutaneous administration of a dose appropriate to provide therapeutic efficacy.

[0158] In certain embodiments, the double-conjugated polypeptide conjugates provided herein have blood, plasma, or serum concentrations that remain within a 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 comparative polypeptide conjugates having a CRM without a peptide linker and conjugated on GLP-1, such as semaglutide, as determined under equivalent in vivo study conditions and in the same type of experimental animals. As used herein, the term “therapeutic window” means a range of concentration levels of the polypeptide conjugate in the blood, plasma, or serum that provides therapeutic benefit or efficacy to the target condition being treated without having unacceptable toxicity. The range of concentration levels may range from the minimum concentration that elicits a therapeutic response to the maximum concentration that provides a therapeutic response without inducing unacceptable toxicity.

[0159] In certain embodiments, the double-conjugated polypeptide conjugates provided herein offer an extended duration of therapeutic efficacy compared to comparative polypeptide conjugates having a CRM conjugated on GLP-1 and lacking a peptide linker, such as semaglutide, as determined under equivalent in vivo investigation conditions and in the same type of experimental animals. Such extended therapeutic efficacy may be characterized by the area under the curve (AUC) for a time response curve, which may be plotted after a single dose or after repeated doses, appropriate for providing the intended therapeutic effect in subjects with metabolic pathologies (e.g., disease model animals). The extended duration of therapeutic efficacy may also be characterized by the duration of the therapeutic response.

[0160] In certain embodiments, the therapeutic response includes a decrease in body weight, a decrease in food intake, or a decrease in glucose levels (fasting glucose levels or non-fasting glucose levels). In certain embodiments, the double-conjugated polypeptide conjugates provided herein have an increase in AUC to the time response curve of at least 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 180%, and 200% compared to comparative polypeptide conjugates having a CRM without a peptide linker and conjugated on GLP-1, such as semaglutide, as determined under equivalent in vivo investigation conditions and in the same type of experimental animals. In certain embodiments, the double-conjugated polypeptide conjugates provided herein have an increase in the duration of the therapeutic response of at least 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 180%, 200%, 300%, or 400% compared to comparative polypeptide conjugates having a CRM conjugated on GLP-1 and lacking a peptide linker, such as semaglutide, as determined under equivalent in vivo study conditions and in the same type of experimental animals. In certain embodiments, the experimental animals for the therapeutic response are disease model animals such as db / db mice or diet-induced obesity (DIO) animals.

[0161] GLP-1 receptor agonists A GLP-1 receptor agonist may include both the natural ligand of the receptor, and artificially designed or modified molecules that exhibit agonist activity comparable to that of the natural ligand, or exhibit at least 30%, 40% or 50% of the agonist activity of the natural ligand. In certain embodiments, the GLP-1 receptor agonist comprises the amino acid sequence of native GLP-1, oxyntomodulin, exendin-4, exenatide, beinaglutide, efpeglenatide, langlenatide, semaglutide, taspoglutide, pegapamotide, liraglutide, albiglutide, dulaglutide, or lixisenatide.

[0162] In certain embodiments, the GLP-1 receptor agonist comprises GLP-1.

[0163] As used herein, the term "glucagon-like peptide-1" or "GLP-1" is intended to broadly encompass native GLP-1 peptides, and all functional forms thereof, such as functional variants, fragments, fusions, derivatives, and mimetics thereof.

[0164] As used herein, the term "native GLP-1 peptide" refers to native human glucagon-like peptide-1 (GLP-1(7-37)), the sequence of which is set forth in SEQ ID NO: 1. As used herein, when referring to a specific amino acid residue 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), thus the first residue in SEQ ID NO: 1, which is histidine (H), is referred to as 7H, meaning that it corresponds to the 7th residue in GLP(1-37); the 31st residue in SEQ ID NO: 1, which is glycine (G), is referred to as 37G, meaning that it corresponds to the 37th residue in GLP(1-37).

[0165] Functional forms of the natural GLP-1 peptide can activate the GLP-1 receptor at levels comparable to or at least 20% (or at least 30%, 40%, 50%, 60%, 70%, 80%, 90%) that of the natural GLP-1 peptide. Activation of the GLP-1 receptor typically initiates signaling pathways that result in insulin secretion stimuli or other physiological effects, as is well known in the art. Functional forms of the natural GLP-1 peptide may contain one or more substitutions, additions, or deletions compared to SEQ ID NO: 1. Many functional forms of the natural GLP-1 peptide, such as, without limitation, liraglutide, semaglutide, dulaglutide, albiglutide, and those disclosed in WO2000055203A1, WO98 / 08871, and WO2006 / 097537, the entire disclosure of which is incorporated herein, are well known in the art.

[0166] In certain embodiments, the GLP-1 provided herein comprises an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 1 (for example, 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%), while retaining substantial biological activity of SEQ ID NO: 1.

[0167] In certain embodiments, GLP-1 contains at least 9, 8, 7, 6, 5, 4, 3, or 2 mutations (e.g., additions, deletions, substitutions) compared to SEQ ID NO: 1, while retaining the substantial biological activity of SEQ ID NO: 1. In certain embodiments, GLP-1 contains 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 the substantial biological activity of SEQ ID NO: 1.

[0168] Those skilled in the art will understand that various amino acid substitutions, such as conservative amino acid substitutions, can be made in the sequence of any of the polypeptide fragments described herein without necessarily reducing their activity. Examples of amino acid substitutions include substituting an L-amino acid for the corresponding D-amino acid, substituting cysteine ​​for homocysteine ​​or other unnatural amino acids having a thiol-containing side chain, substituting lysine for homolysine, diaminobutyric acid, diaminopropionic acid, ornithine, or other unnatural amino acids having an amino acid-containing side chain, or substituting alanine for norvaline or the like.

[0169] Various substitutions have been introduced into the natural GLP-1 peptide and have been shown to maintain or further enhance its biological activity. In certain embodiments, GLP-1 comprises or consists of one or more mutations at a position selected from the group consisting of A8, G22, Q23, E27, K26, A30, K34, R36, and H7, and / or any combination thereof. For example, a substitution at A8 is useful for inhibiting DPP4 enzymatic cleavage at that residue, a substitution at G22 is desirable for improving activity and solubility, and a substitution at R36 appears useful for reducing immunogenicity. Examples of substitutions at these positions include, but are not limited to, H7IA, H7IPA, A8G, A8S, A8V, A8Aib, A8T, A8I, A8L, G22E, K34R, R36G, and the substitutions described in U.S. Patent No. 8,273,854, which is incorporated herein by its entirety. In certain embodiments, one or more substitutions include conservative substitutions. As used herein, the residue numbering in GLP-1 refers to the 31-amino acid sequence specified in Sequence ID No. 1, also known as GLP-1(7-37), where residue 7 is histidine (H7, i.e., the first residue in Sequence ID No. 1) and residue 37 is glycine (G37, i.e., the last residue in Sequence ID No. 1).

[0170] In certain embodiments, GLP-1 includes substitution of A8 selected from the group consisting of A8G, A8S, A8V, A8Aib, A8T, A8I, and A8L. In certain embodiments, GLP-1 includes substitution of G22E. In certain embodiments, GLP-1 includes substitution of R36G. In certain embodiments, GLP-1 includes substitution of H7 which is H7IA or H7IPA. In certain embodiments, GLP-1 includes substitution of K34 which is K34R.

[0171] In a particular embodiment, GLP-1 includes or consists of one or more substitutions at positions selected from the group consisting of H7, A8, G22, K34, and R36, or any combination thereof. In a particular embodiment, GLP-1 includes or consists of one or more substitutions at positions selected from the group consisting of H7IA, H7IPA, A8G, A8Aib, K34R, G22E, and R36G, or any combination thereof.

[0172] In a particular embodiment, GLP-1 includes or consists of one or more substitutions selected from the group consisting of A8Aib, K26R, G22E, K34R, and R36G, or any combination thereof.

[0173] In a particular embodiment, GLP-1 is X7X8EGTFTSDVSSYLEX 22 X 23 AAX 26 X 27 FIX 30 WLVX 34 GX 36 The amino acid sequence G (SEQ ID NO: 2) is included in the formula, where: X7 is H, imidazole-4-acetate (IA), or imidazole propionic acid (IPA); X8 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, and X 36 It is R or G.

[0174] In a particular embodiment, X7 is H, X8 is G or Aib, and 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 K, R, or C, and X 36 It is R or G.

[0175] In a particular embodiment, GLP-1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3-44, and 184.

[0176] In certain embodiments, GLP-1 contains at most one lysine residue. In certain embodiments, GLP-1 contains at most one cysteine ​​residue.

[0177] In a particular embodiment, GLP-1 is Sequence ID 1 (WT GLP-1) Sequence ID 3 (8Aib, 34R, 36G), Sequence ID 5 (8Aib, 26R, 27K, 34R, 36G), Sequence ID 7 (8Aib, 26R 34R, 36G), Sequence ID 8 (8Aib, 22E, 34R, 36G), Sequence ID 10 (8Aib, 22E, 26R, 27K, 34R, 36G), Sequence ID 12 (8Aib, 22E, 26R, 34R, 36G), Sequence ID 13 (8Aib, 34R), Sequence ID 15 (8Aib, 26R, 27K, 34R), Sequence ID 17 (8Aib, 26R, 34R), SEQ ID NO: 18 (8Aib, 22E, 34R), SEQ ID NO: 20 (8Aib, 22E, 26R, 27K, 34R), SEQ ID NO: 22 (8Aib, 22E, 26R, 34R), SEQ ID NO: 23 (8G, 34R, 36G), SEQ ID NO: 25 (8G, 26R, 27K, 34R, 36G), SEQ ID NO: 27 (8G, 26R, 34R, 36G), SEQ ID NO: 28 (8G, 22E, 34R, 36G), SEQ ID NO: 30 (8G, 22E, 26R, 27K, 34R, 36G), SEQ ID NO: 32 (8G, 22E, 26R, 34R, 36G), SEQ ID NO: 33 (8G, 34R), SEQ ID NO: 35 (8G, 26R, 34R), SEQ ID NO: 36 (8G, 22E, 34R), SEQ ID NO: 38 (8G, 22E, 26R, 27K, 34R), SEQ ID NO: 40 (8G, 22E, 26R, 34R), SEQ ID NO: 41 (8G, 22E, 36G), SEQ ID NO: 42 (8G, 36G) SEQ ID NO: 43 (8G, 22E), and SEQ ID NO: 44 (8G) comprises an amino acid sequence selected from the group consisting of:

[0178] In a specific embodiment, GLP-1 is SEQ ID NO: 3 (8Aib, 34R, 36G), SEQ ID NO: 4 (8Aib, 26C, 34R, 36G), SEQ ID NO: 5 (8Aib, 26R, 27K, 34R, 36G), SEQ ID NO: 6 (8Aib, 27C, 34R, 36G), SEQ ID NO: 8 (8Aib, 22E, 34R, 36G) SEQ ID NO: 9 (8Aib, 22E, 26C, 34R, 36G), SEQ ID NO: 10 (8Aib, 22E, 26R, 27K, 34R, 36G), Sequence ID 11 (8Aib, 22E, 27C, 34R, 36G), Sequence ID 13 (8Aib, 34R), Sequence ID 14 (8Aib, 26C, 34R), Sequence ID 15 (8Aib, 26R, 27K, 34R), Sequence ID 16 (8Aib, 27C, 34R), Sequence ID 18 (8Aib, 22E, 34R), Sequence ID 19 (8Aib, 22E, 26C, 34R), Sequence ID 20 (8Aib, 22E, 26R, 27K, 34R), Sequence ID 21 (8Aib, 22E, 27C, 34R), Sequence ID 23 (8G, 34R, 36G), Sequence ID 24 (8G, 26C, 34R, 36G), Sequence ID 25 (8G, 26R, 27K, 34R, 36G), Sequence ID 26 (8G, 27C, 34R, 36G), Sequence ID 28 (8G, 22E, 34R, 36G), Sequence ID 29 (8G, 22E, 26C, 34R, 36G), Sequence ID 30 (8G, 22E, 26R, 27K, 34R, 36G), Sequence ID 31 (8G, 22E, 27C, 34R, 36G), Sequence ID 33 (8G, 34R) Sequence ID 34 (8G, 26C, 34R), Sequence ID 36 (8G, 22E, 34R), Sequence ID 37 (8G, 22E, 26C, 34R), Sequence ID 38 (8G, 22E, 26R, 27K, 34R), Sequence ID 39 (8G, 22E, 27C, 34R), and Sequence ID 184 (8Aib, 22E, 26C, 34R, 36G) It contains an amino acid sequence selected from the group consisting of the following.

[0179] In a particular embodiment, GLP-1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 8, 12, 32, and 184.

[0180] In a particular embodiment, GLP-1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 8, 28, 29, and 184.

[0181] In a particular embodiment, GLP-1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 8, and 28.

[0182] In a particular embodiment, GLP-1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 12, and 32.

[0183] In a particular embodiment, GLP-1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 29, and 184.

[0184] In a particular embodiment, GLP-1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 32.

[0185] Peptide linker In polypeptide conjugates, the GLP-1 receptor agonist is attached to the N-terminus of the peptide linker. The GLP-1 receptor agonist can also be attached to the peptide linker via direct linkage, such as a covalent bond like a peptide bond.

[0186] A peptide linker may consist of amino acid residues linked together by peptide bonds. The peptide linker may further contain one or more non-natural amino acids.

[0187] In a particular embodiment, the peptide linker has a length of at least 10 amino acid residues (e.g., at least 15, 20, 24, 40, 50, 60, 70, or 80 amino acid residues). In a particular embodiment, the polypeptide linker has a length of 10-120, 15-120, 20-120, 25-120, 30-120, 31-120, 32-120, 33-120, 34-120, 35-120, 36-120, 37-120, 38-120, 39-120, 40-120, 20-80, 30-80, 31-80, 32-80, 33-80, 34-80, 35-80, 36-80, 37-80, 38-80, 39-80, or 40-80 amino acid residues. In certain embodiments, the polypeptide linker has a length of 12-80, 20-60, 30-60, 30-50, 30-40, 40-50, 30-45, or 35-45 amino acid residues.

[0188] In a particular embodiment, the peptide linker has a length of 12 to 80 amino acid residues.

[0189] Any suitable polypeptide linker may be used. For example, the polypeptide linker may contain 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 consist of a majority of sterically unhinged amino acids, such as glycine and alanine. In some embodiments, the linker is polyglycine, polyalanine, a combination of glycine and alanine (e.g., poly(Gly-Ala)), or a combination of glycine and serine (e.g., poly(Gly-Ser)).

[0190] In a particular embodiment, the peptide linker consists of amino acid residues selected from the group consisting of G, Q, A, E, P, S, and T, excluding the CRM residue.

[0191] In certain embodiments, the peptide linker comprises or consists of one or more repeats of the repeat sequence, excluding the CRM residue. In certain embodiments, the peptide linker comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 repeats of the repeat sequence, or any number within the range defined by any two of the numbers listed above.

[0192] In a particular embodiment, the repeat sequence contains or consists of at least four, five, or six types of amino acid residues selected from the group consisting of G, Q, A, E, P, T, and S. In a particular embodiment, the repeat sequence contains or consists of at least four, five, or six types of amino acid residues selected from the group consisting of G, Q, A, E, P, and S. In a particular embodiment, the repeat sequence contains Q. In a particular embodiment, the repeat sequence consists of Q and at least three, four, or five types of amino acid residues selected from the group consisting of G, A, E, P, and S.

[0193] In a particular embodiment, the repeat sequence consists of G, Q, A, E, and P.

[0194] In a particular embodiment, the repeat sequence includes or consists of sequences selected from the group consisting of SEQ ID NOs. 45 to 75 and GS.

[0195] In certain embodiments, the polypeptide linker comprises or consists 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.

[0196] In a particular embodiment, the polypeptide linker comprises or consists of (repeat 1)r(repeat 2)s(repeat 3)x(repeat 4)y, in the formula: Repeats 1, 2, 3, and 4 are linked via peptide bonds or via one or more amino acid residues; Repeat 1, Repeat 2, Repeat 3, and Repeat 4 independently contain or consist of sequences selected from the group consisting of SEQ ID NOs. 45-75 and GS. Given that r, s, x, and y are not all 0 at the same time, r, s, x, and y are integers independently selected from 0 to 30 (e.g., 0 to 29, 0 to 28, 0 to 27, 0 to 26, 0 to 25, 0 to 24, 0 to 23, 0 to 22, 0 to 21, 0 to 20, 0 to 19, 0 to 18, 0 to 17, 0 to 16, 0 to 15, 0 to 14, 0 to 13, 0 to 12, 0 to 11, 0 to 10, 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, 0 to 4, 0 to 3, 0 to 2, or 0 to 1).

[0197] In a particular embodiment, x and y are 0, r and s are integers independently selected from 1 to 30 (e.g., 1 to 29, 1 to 28, 1 to 27, 1 to 26, 1 to 25, 1 to 24, 1 to 23, 1 to 22, 1 to 21, 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2), and repeat 1 and repeat 2 are a) Repeat 1 contains or consists of the sequence of sequence number 45, and Repeat 2 contains or consists of the sequence of sequence number 48; b) Repeat 1 contains or consists of the sequence of sequence number 47, and Repeat 2 contains or consists of the sequence of sequence number 48; c) Repeat 1 contains or consists of the sequence of sequence number 48, and Repeat 2 contains or consists of the sequence of sequence number 47; d) Repeat 1 contains or consists of the sequence of sequence number 48, and Repeat 2 contains or consists of the sequence of sequence number 45; e) Repeat 1 contains or consists of the sequence of sequence number 49, and Repeat 2 contains or consists of the sequence of sequence number 48; f) Repeat 1 contains or consists of the sequence of sequence number 70, and Repeat 2 contains or consists of the sequence of sequence number 48; g) Repeat 1 contains or consists of the sequence of sequence number 48, and Repeat 2 contains or consists of the sequence of sequence number 68; and h) Repeat 1 contains or consists of the sequence of sequence number 70, and Repeat 2 contains or consists of the sequence of sequence number 47. This is a combination selected from a group consisting of [the specified elements].

[0198] In a particular embodiment, r, x, and y are 0, s is an integer selected from 1 to 30 (e.g., 1 to 29, 1 to 28, 1 to 27, 1 to 26, 1 to 25, 1 to 24, 1 to 23, 1 to 22, 1 to 21, 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2), and repeat 1 contains or consists of an array of sequence number 45.

[0199] In a particular embodiment, y is 0, r, s, and x are integers independently selected from 1 to 30 (e.g., 1 to 29, 1 to 28, 1 to 27, 1 to 26, 1 to 25, 1 to 24, 1 to 23, 1 to 22, 1 to 21, 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2), and repeat 1, repeat 2, and repeat 3 are, a) Repeat 1 contains or consists of the sequence of sequence number 48; Repeat 2 contains or consists of the sequence of sequence number 49; Repeat 3 contains or consists of the sequence of sequence number 48; b) Repeat 1 contains or consists of the sequence of sequence number 48; Repeat 2 contains or consists of the sequence of sequence number 47; Repeat 3 contains or consists of the sequence of sequence number 48; c) Repeat 1 contains or consists of the sequence of sequence number 70; Repeat 2 contains or consists of the sequence of sequence number 47; Repeat 3 contains or consists of the sequence of sequence number 48; d) Repeat 1 contains or consists of the sequence of sequence number 70; Repeat 2 contains or consists of the sequence of sequence number 48; Repeat 3 contains or consists of the sequence of sequence number 47. e) Repeat 1 contains or consists of the sequence of sequence number 45, Repeat 2 contains or consists of the sequence of sequence number 58; Repeat 3 contains or consists of the sequence of sequence number 48, f) Repeat 1 contains or consists of the sequence of sequence number 48; Repeat 2 contains or consists of the sequence of sequence number 68; Repeat 3 contains or consists of the sequence of sequence number 45. g) Repeat 1 contains or consists of the sequence of sequence number 48, Repeat 2 contains or consists of the sequence of sequence number 45; Repeat 3 contains or consists of the sequence of sequence number 48, and h) Repeat 1 contains or consists of the sequence of sequence number 48; Repeat 2 contains or consists of the sequence of sequence number 45; Repeat 3 contains or consists of the sequence of sequence number 68. This is a combination selected from a group consisting of [the specified elements].

[0200] In a particular embodiment, r, s, x, and y are integers independently selected from 1 to 30 (e.g., 1 to 29, 1 to 28, 1 to 27, 1 to 26, 1 to 25, 1 to 24, 1 to 23, 1 to 22, 1 to 21, 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2), and repeat 1, repeat 2, repeat 3, and repeat 4 are, a) Repeat 1 contains or consists of the sequence of sequence number 48; Repeat 2 contains or consists of the sequence of sequence number 50; Repeat 3 contains or consists of the sequence of sequence number 47; Repeat 4 contains or consists of the sequence of sequence number 48. b) Repeat 1 contains or consists of the sequence of sequence number 45, Repeat 2 contains or consists of the sequence of sequence number 68; Repeat 3 contains or consists of the sequence of sequence number 45, Repeat 4 contains or consists of the sequence of sequence number 48, c) Repeat 1 contains or consists of the sequence of sequence number 48; Repeat 2 contains or consists of the sequence of sequence number 68; Repeat 3 contains or consists of the sequence of sequence number 45; Repeat 4 contains or consists of the sequence of sequence number 48. d) Repeat 1 contains or consists of the sequence of sequence number 48, Repeat 2 contains or consists of the sequence of sequence number 58; Repeat 3 contains or consists of the sequence of sequence number 48, Repeat 4 contains or consists of the sequence of sequence number 45, and e) Repeat 1 contains or consists of the sequence of sequence number 48; Repeat 2 contains or consists of the sequence of sequence number 45; Repeat 3 contains or consists of the sequence of sequence number 69; Repeat 4 contains or consists of the sequence of sequence number 68. This is a combination selected from a group consisting of [the specified elements].

[0201] In a particular embodiment, the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 76-89, except that one residue is substituted by a CRM residue.

[0202] In a particular embodiment, the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 90-117, 175, and 176.

[0203] CRM residue In certain embodiments, the polypeptide conjugate may be monoconjugated with a CRM. In certain embodiments, the first CRM residue is a lysine residue, and the polypeptide conjugate contains only one lysine residue. In such embodiments, the peptide linker contains only one lysine residue, and the GLP-1 receptor agonist does not contain a lysine residue.

[0204] In certain embodiments, the single bioactive peptide in the polypeptide conjugate comprises GLP-1, and the naturally occurring residues found in the native GLP-1 sequence, namely K26 and K34, may be substituted with non-lysine residues. In certain embodiments, GLP-1 includes substitutions of K26 selected from the group consisting of K26R, K26Q, K26A, K26G, K26H, K26S, and K26T. In certain embodiments, GLP-1 includes, or further includes, substitutions of K34 selected from the group consisting of K34R, K34Q, K34A, K34G, K34H, K34S, and K34T. In certain embodiments, the K26 substitution is selected from K26R and K26Q, and / or the K34 substitution is selected from K34R and K34Q. Throughout this specification, when a K residue is present in GLP-1, the position of K is identified by reference to the amino acid sequence of GLP-1(1-37). For example, K26 indicates that the 26th position, which references the amino acid sequence of GLP-1(1-37), corresponds to the 20th position, which references the amino acid sequence of GLP-1(7-37), i.e., sequence number 1. Similarly, K34 indicates that the 34th position, which references the amino acid sequence of GLP-1(1-37), corresponds to the 28th position, which references the amino acid sequence of GLP-1(7-37), i.e., sequence number 1.

[0205] In certain embodiments, GLP-1 may further include one or more additional mutations at positions selected from the group consisting of A8, G22, E27, R36, and H7, and / or any combination thereof. In certain embodiments, GLP-1 includes K26R and K34R. In certain embodiments, GLP-1 is Sequence ID 7 (8Aib, 26R, 34R, 36G), Sequence ID 12 (8Aib, 22E, 26R, 34R, 36G), Sequence ID 17 (8Aib, 26R, 34R), Sequence ID 22 (8Aib, 22E, 26R, 34R), Sequence ID 27 (8G, 26R, 34R, 36G), Sequence ID 32 (8G, 22E, 26R, 34R, 36G), Sequence ID 35 (8G, 26R, 34R), and Sequence ID 40 (8G, 22E, 26R, 34R) It contains an amino acid sequence selected from the group consisting of the following.

[0206] In certain embodiments, the first CRM residue 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 ​​residues, and therefore any GLP-1 derivative (including those provided herein) can be used as long as it does not contain a cysteine ​​residue.

[0207] In certain embodiments, the first CRM residue is a non-natural amino acid residue, and the polypeptide conjugate contains only one non-natural amino acid residue as the CRM residue. Non-natural amino acids may contain a variety of functional groups or reactive groups that can provide additional function and / or reactivity. Certain non-natural amino acids that are useful for the purpose of conjugating a portion into the fusion polypeptide of this disclosure include those having side chains with azides, alkynes, alkenes, cycloalkynes, or halides.

[0208] If the first CRM residue is cysteine ​​or a non-natural amino acid residue, GLP-1 may contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 8, 10, 12, 13, 15, 17, 18, 20, 22, 23, 25, 27, 28, 30, 32, 33, 35, 36, 38, and 40-44.

[0209] In certain embodiments, the polypeptide conjugate further comprises a second CRM residue. In certain embodiments, the second CRM residue is located within the peptide linker. In such embodiments, the second CRM residue may be at any suitable distance from the first CRM residue, insofar as both CRM residues can be properly conjugated. In certain embodiments, the second CRM residue is located within a GLP-1 receptor agonist, e.g., GLP-1. In certain embodiments, the second CRM residue is a naturally occurring residue found in the natural GLP-1 sequence, or an introduced residue, e.g., by substitution of a naturally occurring residue or insertion of a new residue.

[0210] In certain embodiments, both the first and second CRM residues are lysine residues, and the polypeptide conjugate contains only two lysine residues. In certain embodiments, the second lysine residue is present 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 natural GLP-1 sequence, e.g., 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, as long as such substitution does not substantially reduce the GLP-1R agonist activity of GLP-1. The second CRM residue can be introduced by substitution of, for example, Q23K, E27K, or A30K. In certain embodiments, all lysine residues other than the CRM residue in GLP-1 are replaced with non-lysine residues, so that the polypeptide conjugate does not contain additional lysine residues other than the CRM residue. For example, if K26 is the second CRM residue, then K34 is substituted with a non-lysine residue, and vice versa. Non-lysine residues can be selected by those skilled in the art, and examples include arginine (R), glutamine (Q), alanine (A), glycine (G), histidine (H), serine (S), or threonine (T).

[0211] If both the first and second CRM residues are lysine residues, GLP-1 may contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 5, 8, 10, 13, 15, 18, 20, 23, 25, 28, 30, 33, 36, and 38.

[0212] In certain embodiments, both the first and second CRM residues are cysteine ​​residues, and the polypeptide conjugate contains only two cysteine ​​residues. In certain embodiments, the second cysteine ​​residue is located within GLP-1. In certain embodiments, the second cysteine ​​residue is located within the peptide linker.

[0213] In a particular embodiment, both the first and second CRM residues are unnatural amino acid residues, and the polypeptide conjugate contains only two unnatural amino acid residues as CRM residues.

[0214] In certain embodiments, a cysteine ​​residue or a 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 compared to SEQ ID NO: 1. In certain embodiments, a cysteine ​​residue or NNAA in GLP-1 is introduced by substitution at the position of K26 or E27 compared to SEQ ID NO: 1. In certain embodiments, the second CRM residue in GLP-1 is cysteine ​​and is introduced by substitution at a position selected from the group consisting of Q23C, K26C, E27C, A30C, and K34C.

[0215] If both the first and second CRM residues are cysteine ​​residues, GLP-1 may contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, 9, 11, 14, 16, 19, 21, 24, 26, 29, 31, 34, 37, 39, and 184.

[0216] CRM In certain embodiments, the CRM comprises a plasma protein binding moiety, a polymer, Fc, human serum albumin (HSA) and its functional fragment, an Xten sequence, or a PAS sequence. In certain embodiments, the Xten sequence is an extended recombinant polypeptide sequence having the amino acid sequences described in WO2007103515, WO2009023270, WO2010091122, WO2011123813, WO2013130683, WO2017146979, WO2011084808, WO2013040093, WO2013122617, WO2014011819, WO2013184216, WO2014164568, WO2015023891, WO2016077505, and WO2017040344, the disclosure of which is incorporated in their entirety. 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, in which its disclosure is incorporated in its entirety.

[0217] In certain embodiments, the CRM includes an albumin-binding moiety. The term “albumin-binding moiety” refers to any functional moiety capable of binding with sufficient specificity, preferably non-covalently, to albumin (e.g., human serum albumin) or any functional fragment thereof. Albumin-binding moieties attached to therapeutic fusion polypeptides, polypeptides, or polypeptide complexes typically have an affinity of less than 10 μM, preferably less than 1 pM, to human serum albumin. The albumin-binding moiety may, without limitation, include 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 may be selected from an albumin-binding domain derived from streptococcal protein G, an albumin-binding domain derived from Peptostreptococcus magnus protein PAB, or an albumin-binding peptide having the core sequence DICLPRWGCLW (SEQ ID NO: 173). Several small peptides that constitute the albumin-binding moiety are described in J. Biol Chem. 277, 38 (2002) 35035-35043. As another example, the albumin-binding moiety can be selected from linear and branched lipohophilic moies containing 4 to 40 carbon atoms, compounds having a cyclopentanophenanthrene skeleton, etc. For example, the albumin-binding moiety is of the formula CH3(CH2) v It is the base of CO-NHCH(COOH)(CH2)2CO-, where v is an integer between 10 and 24.

[0218] In a particular embodiment, the albumin-binding moiety comprises the structure *-ABCDE, or a pharmaceutically acceptable salt thereof, where A, B, C, D, and E are interconnected via amide bonds, and the * terminus of A is connected to the reactive group of a conjugateable residue on the polypeptide complex, in the formula: A is a combination, [ka] Selected from, where a, b, c, and d are independent integers from 0 to 4, R 1is hydrogen or -COOH; B is a combination, [ka] Selected from, where e is an integer from 1 to 4, and in the expression, the α-th order is concatenated to the α'-th order. C is a combination or [ka] And R 2 is -CH2SO3H or -COOH, f is an integer from 1 to 4, n is an integer from 1 to 25, and if B is not associative, the β' position is linked to the β position, or if B is associative, the β' position is linked to the α' position; D is bond, [ka] Selected from, where g and h are independently 0 or 1, R 3 is H or -CH2COOH, 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, the γ' position is linked to the γ position; If B is a bond and C is a bond, then the γ' position is linked to the α' position; E is the formula: [ka] It is an acidic group having, In the formula, W is -(CR 4 R 5 ) l - represents, R 4 and R 5 These are independently selected from the group consisting of hydrogen, halogen, cyano, hydroxyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, hydroxyalkyl, amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy, and carboxamide. R 6is hydroxyl or NR 7 R 8 Selected from; R 7 and R 8 These are independently hydrogen, alkyl, hydroxyl, and [ka] Selected from the group consisting of, l is an integer between 10 and 20. If D is not a bond, then the δ position is linked to the δ' position. If C is not a bond and D is a bond, then the δ position is linked to the γ position. If B is not a bond, C is a bond, and D is a bond, then the δ position is linked to the β position. If A is not a bond, and B, C, and D are all bonds, then the δ position is linked to the α' position.

[0219] In certain embodiments, the CRM is conjugated to a lysine residue, which optionally is located within the peptide linker or GLP-1.

[0220] In a particular embodiment, A is a coupling.

[0221] In a particular embodiment, A is a bond, and B is a bond or [ka] In the formula, e is 1, 2, or 3. In a particular embodiment, A is a bond and B is [ka] And C is, [ka] In the formula, the β' position is linked to the β position, where e is 1, 2, or 3, f is 1, 2, or 3, and n is 1 or 2. In a particular embodiment, D is a bond, and E is in the formula:

Chem.

[0222] In a specific embodiment, A is a bond, B is a bond, and C is a bond.

[0223] In a specific embodiment, A is a bond, B is a bond, and C is

Chem.

[0224] In a specific embodiment, A is

Chem.

[0225] In a specific embodiment, A is

Chem.

Chem.

[0226] In a particular embodiment, A is [ka] And B is [ka] And C is [ka] In the formula, the α position is linked to the α' position, the β' position is linked to the β position, and in the formula, a is 1, 2, or 3, b is 1, 2, or 3, c is 1 or 2, e is 1, 2, or 3, f is 1, 2, or 3, and n is 1 or 2.

[0227] In a particular embodiment, A is [ka] And B is [ka] C is a bond, where the α-position is linked to the α'-position, where a is 1, 2, or 3, b is 1, 2, or 3, c is 1 or 2, and e is 1, 2, or 3.

[0228] In a particular embodiment, A is [ka] B is a combination, where a is 1, 2, or 3, b is 1, 2, or 3, and c is 1 or 2.

[0229] In a particular embodiment, A is [ka] B is a bond, and C is [ka] In the formula, the β' position is linked to the α' position, where a is 1, 2, or 3, b is 1, 2, or 3, c is 1 or 2, f is 1, 2, or 3, and n is 1 or 2.

[0230] In a particular embodiment, A is [ka] B is a bond, C is a bond, where a is 1, 2, or 3, b is 1, 2, or 3, and c is 1 or 2.

[0231] In a particular embodiment, D is a bond.

[0232] In a particular embodiment, A is [ka] And B is [ka] And C is [ka] D is a bond, where the α-position is linked to the α'-position, the β'-position is linked to the β-position, where a is 1, 2, or 3, b is 1, 2, or 3, c is 1 or 2, e is 1, 2, or 3, f is 1, 2, or 3, and n is 1 or 2.

[0233] In a particular embodiment, D is [ka] In the equation, g is either 0 or 1, and h is either 0 or 1.

[0234] In a particular embodiment, A is [ka] And B is [ka] or bond, C is bond, D is [ka] If B is not a bond, the α position is linked to the α' position and the γ' position is linked to the β position, or if B is a bond, the γ' position is linked to the α' position, where a is 1, 2, or 3, b is 1, 2, or 3, c is 1 or 2, e is 1, 2, or 3, g is 0 or 1, and h is 0 or 1.

[0235] In a particular embodiment, D is [ka] That is the case.

[0236] In a particular embodiment, A is [ka] And B is [ka] And C is bonded or [ka] And D is [ka] In the formula, the α position is linked to the α' position, if C is a bond, the γ' position is linked to the β position, or if C is not a bond, the β' position is linked to the β position and the γ' position is linked to the γ position, in the formula, a is 1, 2, or 3, b is 1, 2, or 3, c is 1 or 2, e is 1, 2, or 3, f is 1, 2, or 3, and n is 1 or 2.

[0237] In such an embodiment, the CRM is given by the following formula: [ka] It contains the structure (also known as -HOOC-(CH2)16-CO-gGlu-2XADO, where 2XADO means two consecutive ADO moieties, and ADO is an abbreviation for 8-amino-3,6-dioxaoctanoic acid).

[0238] In certain embodiments, the CRM is conjugated to a cysteine ​​residue, which optionally is located within the peptide linker or GLP-1.

[0239] In a particular embodiment, A is [ka] And B is [ka] In this equation, the α-position is connected to the α'-position.

[0240] In a particular embodiment, A is [ka] And B is [ka] And C is [ka] In the formula, the α position is linked to the α' position, the β' position is linked to the β position, and in the formula, a is 1, 2, or 3, b is 1, 2, or 3, c is 1 or 2, d is 1, 2, or 3, f is 1, 2, or 3, and n is 1 or 2. In a particular embodiment, D is a bond, and E is in the formula: [ka] It is an acidic group having the δ position linked to the γ position. In certain embodiments, R2 is -COOH and R6 is hydroxyl. In certain embodiments, W represents -(CR4R5)l-, where R4 and R5 are independently hydrogen and l is an integer between 10 and 20.

[0241] In a particular embodiment, A is [ka] And B is [ka] And C is [ka] D is a bond, where the α position is linked to the α' position, the β' position is linked to the β position, where a is 1, 2, or 3, b is 1, 2, or 3, c is 1 or 2, d is 1, 2, or 3, f is 1, 2, or 3, and n is 1 or 2.

[0242] In a particular embodiment, A is [ka] And B is [ka] In the formula, the α-position is linked to the α'-position, and in the formula, d is 1, 2, or 3, and e is 1, 2, or 3.

[0243] In a particular embodiment, A is [ka] And B is [ka] C is a bond, in the formula the α-position is linked to the α'-position, in the formula d is 1, 2, or 3, and e is 1, 2, or 3.

[0244] In a particular embodiment, A is [ka] And B is [ka] C is a bond, and D is [ka] In the formula, the α position is linked to the α' position, the γ' position is linked to the β position, d is 1, 2, or 3, e is 1, 2, or 3, g is 0 or 1, and h is 0 or 1.

[0245] In such an embodiment, the CRM is given by the following formula: [ka] It contains the structure (also known as HOOC-(CH2)16-CO-gGlu-2XADO-EDA-CO-CH2 or HOOC-(CH2)20-CO-gGlu-2XADO-EDA-CO-CH2).

[0246] Conjugate In a particular embodiment, the polypeptide conjugate comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 118-174 and 177-183.

[0247] In certain embodiments, the polypeptide conjugates provided herein include an amino acid sequence selected from the group consisting of SEQ ID NOs: 119, 126-128, 136-139, 147-150, 158-161, 169-174, and 177-183, as well as a CRM attached to a lysine or cysteine ​​residue. In certain embodiments, the CRM includes the structure -HOOC-(CH2)16-CO-gGlu-2XADO (with respect to lysine conjugation) or HOOC-(CH2)16-CO-gGlu-2XADO-EDA-CO-CH2 (with respect to cysteine ​​conjugation).

[0248] In certain embodiments, the polypeptide conjugates provided herein include an amino acid sequence selected from the group consisting of SEQ ID NOs: 118, 120-125, 129-135, 140-146, 151-157, 162-168, 173, 174, and 177-183, as well as two CRMs attached to two lysine residues or two cysteine ​​residues, respectively. In certain embodiments, the CRM includes the structure -HOOC-(CH2)16-CO-gGlu-2XADO (with respect to lysine conjugation) or HOOC-(CH2)16-CO-gGlu-2XADO-EDA-CO-CH2 (with respect to cysteine ​​conjugation).

[0249] In certain embodiments, the polypeptide conjugate provided herein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 118, 120, 121, 122, 123, 124, 125, 129, 130, 131, 132, 133, 134, 135, 140, 141, 142, 143, 144, 145, 146, 173, 174, and 177-183, as well as two CRMs attached to two lysine residues, respectively. In certain embodiments, the CRM comprises the structure -HOOC-(CH2)16-CO-gGlu-2XADO.

[0250] In certain embodiments, the polypeptide conjugate provided herein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 119, 126, 127, 128, 136, 137, 138, 139, 147, 148, 149, and 150, as well as a CRM attached to a lysine residue. In certain embodiments, the CRM comprises the structure -HOOC-(CH2)16-CO-gGlu-2XADO.

[0251] In certain embodiments, the polypeptide conjugate provided herein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 151, 152, 153, 154, 155, 156, 157, 162, 163, 164, 165, 166, 167, 168, and 177-183, and two CRMs attached to two cysteine ​​residues, respectively. In certain embodiments, the CRM comprises the structure HOOC-(CH2)16-CO-gGlu-2XADO-EDA-CO-CH2.

[0252] In certain embodiments, the polypeptide conjugate provided herein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 159, 160, 161, 169, 170, 171, and 172, and a CRM attached to a cysteine ​​residue. In certain embodiments, the CRM comprises the structure HOOC-(CH2)16-CO-gGlu-2XADO-EDA-CO-CH2.

[0253] In certain embodiments, CRM is conjugated to a non-natural amino acid residue in the fusion polypeptide.

[0254] Table 1 below provides detailed information on each of the exemplary polypeptide conjugates, including the sequence numbers of the polypeptide portion, GLP-1, and peptide linker, as well as the CRM residues. Mutations in GLP-1, as well as the repeat sequences and number of repeats in the peptide linker sequence, are also shown.

[0255] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0256] In a particular embodiment, the polypeptide conjugate includes the structure shown below, in which amino acid residues are represented as single-letter abbreviations in gray circles. Molecule 001: [ka] Molecule 002: [ka] Molecule 012: [ka]

[0257] Preparation method This disclosure provides isolated nucleic acids or polynucleotides that encode the polypeptide portion (or fragment thereof) of the polypeptide conjugate provided herein.

[0258] As used herein, the terms “nucleic acid” or “polynucleotide” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and their polymers, either in single-stranded or double-stranded form. Unless specifically limited, the terms include polynucleotides containing known analogs of natural nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a similar manner to naturally occurring nucleotides. Unless otherwise indicated, a particular polynucleotide sequence implicitly includes its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as explicitly indicated sequences. Specifically, degenerate codon substitution can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or a deoxyinosine residue (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)).

[0259] The nucleic acids or polynucleotides encoding polypeptides (or fragments thereof) provided herein can be constructed using recombinant techniques. For this purpose, DNA encoding a GLP-1 receptor agonist (such as GLP-1) and DNA encoding a peptide linker can be obtained and operably linked to enable transcription and expression in host cells to produce a fusion polypeptide. If necessary, polynucleotide sequences encoding one or more linkers can also be operably linked to enable expression of the desired product.

[0260] The coding polynucleotide sequence may optionally be further operably ligated to one or more regulatory sequences in the expression vector so that the expression or production of the fusion polypeptide or polypeptide complex is feasible and under proper control.

[0261] The coding polynucleotide sequence can be inserted into a vector for further cloning (DNA amplification) or expression using recombination 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.

[0262] Vectors and host cells In another embodiment, this disclosure provides a vector comprising a polynucleotide provided herein.

[0263] Vectors comprising polynucleotide sequences 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 the exogenous polynucleotide and / or vector has been introduced. In other embodiments, the vector is extrachromosomal. Host cells may be isolated if desired. In certain embodiments, the host cell is a prokaryotic or eukaryotic cell.

[0264] Suitable host cells for cloning or expressing DNA in vectors as described herein are primarily prokaryotes. Suitable prokaryotes for this purpose include eubacteria such as Gram-negative or Gram-positive organisms, e.g., Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces. In some embodiments, the host cells are eukaryotes such as yeast and mammalian cells (e.g., immortalized mammalian cells).

[0265] Vectors comprising polynucleotide sequences 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, a polynucleotide sequence encoding a GLP-1 polypeptide may be subcloned within an expression vector, which is then expressed as an inclusion body in the host cell. The vector may be a viral vector, and any suitable viral vector can be used in this capacity.

[0266] In another embodiment, the present disclosure provides host cells containing vectors provided herein. The host cells are prokaryotic or eukaryotic cells. Host cells transformed with the expression or cloning vectors described above can be cultured in conventional nutrient media, which may be modified as needed, for inducing promoters, selecting transformants, or amplifying cloning vectors.

[0267] In another embodiment, the Disclosure provides a method for producing a polypeptide conjugate provided herein, comprising culturing a host cell provided herein under conditions that enable the expression of a polynucleotide provided herein to obtain the polypeptide portion of the polypeptide conjugate.

[0268] For the production of the polypeptide portion provided herein, host cells transformed with the expression vector can be cultured in a variety of media. Commercial 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. Commercial media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium (DMEM) (Sigma) are suitable for culturing eukaryotic host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphates), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN® drugs), trace elements (defined as inorganic compounds normally present at final concentrations within the micromolar range), and glucose or equivalent energy sources. Any other necessary supplements may also be included in appropriate concentrations known to those skilled in the art. Culture conditions such as temperature and pH are those previously used with the host cells selected for expression and will be obvious to those skilled in the art.

[0269] In one embodiment, the disclosure provides a method for 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 a particular embodiment, the polypeptide portion is expressed as a soluble polypeptide.

[0270] When recombinant techniques are used, the polypeptides provided herein may be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If the product is produced intracellularly, the first step is to remove particulate debris, whether from the host cell or lysed fragments, by, for example, centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describe a procedure for isolating proteins secreted into the periplasmic space of E. coli. Briefly, the cell paste is thawed for about 30 minutes in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation. If the product is secreted into the culture medium, the supernatant from such an expression system is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. To inhibit protein degradation, protease inhibitors such as PMSF may be included in one of the aforementioned steps, and antibiotics may be included to prevent the growth of accidental contaminants.

[0271] In a particular embodiment, the method further includes isolating the polypeptide.

[0272] Polypeptides provided herein, prepared from cells, can be purified using, for example, hydroxyl apatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography.

[0273] Other techniques for protein purification, such as fractionation with ion exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography with silica, chromatography with heparin SEPHAROSE® chromatography on anion or cation exchange resins (e.g., polyaspartate columns), chromatographic focusing, SDS-PAGE, and ammonium phosphate precipitation, can also be used depending on the protein to be recovered.

[0274] In certain embodiments, the method further comprises conjugating a polypeptide with CRM. The polypeptide may be conjugated by a suitable conjugation reaction at, for example, a lysine residue, a cysteine ​​residue, or a non-natural amino acid.

[0275] For example, polypeptides having one or more CRM residues, such as lysine, can react with amino-reactive substances. 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 entire contents of which are incorporated herein. The CRM to be conjugated in the acylation reaction may include, among many 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-succinimidyl reagents such as N-hydroxysuccinimidyl (NHS) or sulfo-NHS esters, and imide ester compounds such as Traut's reagent, which can react with the ε-amino group of the conjugateable lysine residue to form an amide or amidine bond. Additional examples of suitable amino-reactive substances include O-acyl isourea, N-hydroxytriazole esters, anhydrides, phenyl-reactive esters, P-hydroxamic acid-reactive esters, acylimidazoles, acylbenzotriazoles, acyl azides, acid halides, phosphonium salts, and aminonium / uronium salts.

[0276] As another example, polypeptides having one or more CRM residues, such as cysteine, can be conjugated to thiol-reactive substances. In certain embodiments, the CRM is conjugated to a cysteine ​​residue in an alkylation reaction. In certain embodiments, the CRM is conjugated to a conjugable cysteine ​​residue via maleimide or iodoacetamide to form a carbon-sulfur bond. In certain embodiments, the CRM is conjugated to a conjugable cysteine ​​residue via a disulfide to form a disulfide bond. Additional examples of suitable thiol-reactive groups include dienyl sulfones, α-haloacyls, or other thiol-reactive conjugation partners. For further details, please refer to 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.

[0277] For example, polypeptides having one or more CRM residues, such as non-natural amino acid residues (NNAAs), can be conjugated to CRMs, thereby forming a stable link between the NNAA and CRM of the fusion polypeptide. For instance, NNAAs containing a keto group, an aldehyde, or a β-diketo moiety can react with hydrazide- or O-alkylhydroxylamine-, or hydroxylamine-containing active materials to form hydrazone or O-alkylated oxime links. As another example, NNAAs containing an azide group can react with alkyne derivatives to form a stable triazole linker by copper(I)-catalyzed [3+2] cycloaddition (and vice versa). As yet another example, NNAAs containing an azide group can ligate with a suitable water-soluble phosphine-containing active material to form an amide linkage by Staudinger ligation. Furthermore, thioester moieties in NNAAs can react with amine-containing active materials to form amide links. Fusion polypeptides provided herein, incorporating NNAA, can be conjugated with active ingredients by cyclization reactions, such as (4+2) cyclization between diene and dienophile (Diels-Alder reaction), (3+2) cyclization by 1,3-dipole hysgene cyclization, and (3+2) cyclization by nitrone-olefin cyclization. Suitable cyclization methods for antibody conjugation are described, for example, in WO05003294, US20120004183, WO06009901, WO07130453, and U.S. Patent No. 6,737,236.

[0278] As another example, a polypeptide may be conjugated to biotin, and then indirectly conjugated to a CRM which is conjugated to avidin. Furthermore, as yet another example, a fusion polypeptide or polypeptide complex may be linked to a coupling agent, which then further links to a CRM. Examples of coupling agents include difunctional moieties such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and iminothiolane (IT), difunctional derivatives of imide esters (such as dimethylHCl adipimidoate), 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 disulfide linkages.

[0279] Additional methods for conjugating CRM to polypeptides can be found, for example, in U.S. Patent Nos. 5,208,020; 6,4411,163; WO2005037992; WO2005081711; and WO2006 / 034488, which are incorporated herein by reference in their entirety. Specific examples of methods for preparing the conjugates of this disclosure are also included in the experimental portion of this disclosure.

[0280] Pharmaceutical composition In another embodiment, the Disclosure also provides pharmaceutical compositions comprising polypeptide conjugates and pharmaceutically acceptable carriers provided herein.

[0281] The term "pharmaceutically acceptable" indicates that a specified carrier, vehicle, diluent, excipient, and / or salt is substantially chemically and / or physically compatible with the other components of the formulation and physiologically compatible with its recipient.

[0282] "Pharmacologically acceptable carrier" refers to a component in a pharmaceutical formulation other than the active ingredient that is biologically acceptable and non-toxic to the target. Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating or sealing agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0283] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated hydroxytoluene, and / or propyl gallate. As disclosed herein, the encapsulation of one or more antioxidants, such as methionine, in the pharmaceutical compositions provided herein reduces the oxidation of polypeptide complexes or bispecific polypeptide complexes. This reduction in oxidation prevents or reduces the decrease in binding affinity, thereby improving protein stability and maximizing shelf life. Accordingly, in certain embodiments, compositions are provided that include a fusion polypeptide, polypeptide complex, or conjugate disclosed herein, and one or more antioxidants such as methionine.

[0284] To further illustrate, pharmaceutically acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose, and Ringer's lactate injection; non-aqueous vehicles such as plant-derived fixative oils, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents at bacteriostatic or fungiostatic concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphoric acid or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone; emulsifiers such as polysorbate 80 (TWEEN-80); and 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 used as carriers, including phenol or cresol, mercury compounds, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoate esters, thimerosal, benzalkonium chloride, and benzethonium chloride, may be added to pharmaceutical compositions in multi-dose containers. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or active ingredients such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

[0285] Pharmaceutical compositions may be liquid solutions, suspensions, emulsions, pills, capsules, tablets, sustained-release formulations, or powders. Oral formulations may contain standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, and magnesium carbonate.

[0286] In embodiments, the pharmaceutical composition is formulated into an injectable composition. The injectable pharmaceutical composition may be prepared in any conventional form, such as a liquid solution, suspension, emulsion, or a solid form suitable for producing a liquid solution, suspension, or emulsion. Preparations for injection may include sterile and / or nonpyrogenic solutions ready for injection, sterile dry soluble products such as lyophilized powders ready to be combined with a solvent immediately before use, including subcutaneous injection tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle immediately before use, and sterile and / or nonpyrogenic emulsions. The solutions may be either aqueous or non-aqueous.

[0287] In certain embodiments, a unit dose of parenteral preparation is packaged in an ampoule, vial, or syringe with a needle. All preparations for parenteral administration must be sterile and nonpyrogenic, as is known and practiced in the art.

[0288] In certain embodiments, a sterile lyophilized powder is prepared by dissolving a polypeptide conjugate disclosed herein in a suitable solvent. The solvent may contain excipients that improve the stability of the powder or the reconstituted solution prepared from the powder, or other pharmacological components. Excipients that may be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable active ingredients. In one embodiment, the solvent may contain a buffer such as citrate, sodium phosphate, or potassium, or other such buffers known to those skilled in the art, at approximately a neutral pH. Subsequent sterile filtration of the solution, followed by lyophilization under standard conditions known to those skilled in the art, provides the desired formulation. In one embodiment, the resulting solution will be dispensed into vials for lyophilization. Each vial may contain a single-dose or multiple-dose amount of the fusion polypeptide, polypeptide complex, or conjugate, or composition thereof, provided herein. Overfilling the vial with a small amount (e.g., about 10%) more than what is needed for a single dose or a series of doses is acceptable to facilitate accurate sample extraction and accurate dosing. The lyophilized powder can be stored under suitable conditions, such as about 4°C to room temperature.

[0289] The reconstitution of lyophilized powder with water for injection provides a formulation for use in parenteral administration. In one embodiment, for reconstitution, a suitable carrier of sterile and / or non-pyrogenic water or other liquid is added to the lyophilized powder. The exact amount may be determined empirically, depending on the selected therapy being given.

[0290] The administration of the pharmaceutical compositions described herein may 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 any other mechanical device such as an infusion pump. In certain embodiments, a peripheral venous route is a route of administration that is intravenous, intramuscular, subcutaneous, or intraperitoneal.

[0291] In certain embodiments, the polypeptide conjugates described herein are formulated into a form suitable for parenteral administration, such as oral, rectal, nasal, or lower respiratory tract administration.

[0292] In certain embodiments, the polypeptide conjugate described herein may be formulated into a solid formulation, such as by lyophilization or spray drying, which is then restored in an appropriate dilution solution before administration. Standard pharmaceutical formulation techniques, such as those described in Remington: The Science and Practice of Pharmacy (DB Troy, Editor, 21st Edition, Lippincott, Williams & Wilkins, 2006), may be employed. Alternatively, the polypeptide conjugate described herein may be formulated for administration via the tongue, sublingual, buccal, oral, gastric and intestinal, nasal, intrapulmonary, e.g., bronchioles and alveoli or a combination thereof, epidermal, cutaneous, transdermal, vaginal, rectal, ocular, e.g., conjunctiva, ureter, transdermal, or intrapulmonary routes. Further options include formulation for administration via needleless injection or patch, optionally iontophoresis patch, or transmucosal, e.g., buccal administration.

[0293] Treatment method In another embodiment, the Disclosure provides a method for preventing or treating a metabolic disorder in a subject requiring such treatment, comprising administering a therapeutically effective dose of a polypeptide conjugate provided herein.

[0294] Therapies are also provided, which include administering a therapeutically effective dose of the polypeptide conjugate provided herein to a subject in need thereof, thereby treating or preventing a disease or disorder. In certain embodiments, a subject is identified as having a disorder or disease that may respond to the polypeptide conjugate provided herein.

[0295] In certain embodiments, metabolic disorders include diabetes mellitus, obesity, overweight, non-alcoholic steatohepatitis (NASH), cardiovascular 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).

[0296] In certain embodiments, the condition of diabetes mellitus includes all forms of diabetes, such as hyperglycemia, type 2 diabetes mellitus, impaired glucose tolerance, type 1 diabetes mellitus, non-insulin-dependent diabetes mellitus, MODY (young-onset adult-onset diabetes mellitus), gestational diabetes mellitus, and / or elevated HbA1c levels.

[0297] In certain embodiments, the condition of diabetes includes diabetic complications such as vascular damage.

[0298] In another embodiment, the present disclosure provides a method for managing body weight in a subject requiring such management, comprising administering a therapeutically effective dose of a polypeptide conjugate provided herein.

[0299] In another embodiment, the present disclosure provides a method for reducing food intake in a subject requiring such reduction, comprising administering a therapeutically effective dose of a polypeptide conjugate provided herein.

[0300] In another embodiment, the Disclosure provides a method for reducing body weight in a subject in need, comprising administering a therapeutically effective dose of a polypeptide conjugate provided herein.

[0301] In certain embodiments, conditions or metabolic disorders that can be treated or improved using polypeptide conjugates provided herein include conditions in which a human subject has a fasting blood glucose level of 125 mg / dL or higher, for example, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, or 200 mg / dL. Blood glucose levels may be determined by diet, fasting, or randomly. Metabolic conditions or disorders may also include conditions in which a subject has an increased risk of developing a metabolic condition. With respect to human subjects, such conditions include a fasting blood glucose level of 100 mg / dL.

[0302] In certain embodiments, conditions or metabolic disorders that can be treated or improved using 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 higher 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 to 30, 26 to 30, 27 to 30, 28 to 30, 25 to 29, or 25 to 28.

[0303] The therapeutically effective dose of polypeptide conjugates provided herein will depend on various factors known in the art, such as the subject's body weight, age, medical history, current medications, health status, and potential for cross-reactivity, allergies, sensitivities, and adverse side effects, as well as the route of administration and the degree of disease progression. The dosage may be proportionally reduced or increased by those skilled in the art (e.g., physicians or veterinarians) as indicated by these and other circumstances or requirements. The therapeutically effective dose may be the amount of fusion polypeptides, polypeptide complexes, and conjugates provided herein that elicit a biological or pharmacokinetic response in a tissue system, animal, or human, including the alleviation or improvement of symptoms of the disease or disorder being treated, as sought by researchers, physicians, or other clinicians; i.e., an amount that supports an observable level of one or more desired biological or pharmacokinetic responses, such as a decrease in blood glucose, insulin, triglyceride, or cholesterol levels; a decrease in body weight; or an improvement in glucose tolerance, energy expenditure, or insulin sensitivity.

[0304] In certain embodiments, the polypeptide conjugates provided herein may be administered in therapeutically effective doses ranging from 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 embodiments of these procedures, the polypeptide conjugate provided herein is administered in a dose of about 50 mg / kg or less, and in certain embodiments of these procedures, the dose is 10 mg / kg or less, 5 mg / kg or less, 1 mg / kg or less, 0.5 mg / kg or less, or 0.1 mg / kg or less. In certain embodiments, the administered dose may vary over the course of treatment. For example, in certain embodiments, the initial dose may be higher than subsequent doses. In certain embodiments, the administered dose may fluctuate over the course of treatment depending on the subject's response.

[0305] In certain embodiments, the polypeptide conjugates provided herein may be administered to subjects (e.g., humans) in dosing regimens less frequent than once daily, once every three days, once a week, once every two weeks, once every three weeks, or once a month. In certain embodiments, the polypeptide conjugates provided herein may be administered to subjects (e.g., humans) at dosing intervals of once a week, once every two weeks, once every three weeks, or once a month. Therapeutic efficacy with lower dosing frequencies has the potential to improve patient compliance and long-term treatment success. Semaglutide, a currently available treatment, is administered once a week. Without wishing to be constrained by any theory, certain polypeptide conjugates provided herein have a significantly extended half-life and appear suitable for treating metabolic conditions with lower dosing frequencies than semaglutide, for example, less 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 a month). In certain embodiments, the dosing regimen is a continuous dosing regimen selected from dosing twice a week, once a week, once every two weeks, once every three weeks, once a month, or once every two months. In a particular embodiment, the medication regimen has a dosing interval ranging from approximately every three days to approximately once a month, or from approximately once a week to approximately once a month.

[0306] The medication regimen may be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single dose may be administered, or several divided doses may be administered over time.

[0307] Polypeptide conjugates provided herein may be administered by any route known in the art, such as parenteral (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, or intradermal injection, including subcutaneous, intraperitoneal, intravenous injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or local) routes. In certain embodiments, polypeptide conjugates provided herein are administered subcutaneously, intravenously, intramuscularly, or intradermally.

[0308] Polypeptide conjugates can be administered alone or in combination with one or more additional therapeutic agents or therapeutic devices.

[0309] In certain embodiments, when used to treat metabolic disorders, the polypeptide conjugates provided herein may be administered in combination with any other therapeutic agents for use in the treatment of metabolic disorders or any related medical disorders. 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 administered before or after another active agent is considered to be administered “in combination” with that active agent, as the phrase is used herein, even if the composition and the second active agent are administered via different routes. Where possible, additional therapeutic agents administered in combination with the fusion polypeptides, polypeptide complexes, or conjugates provided herein are administered according to the schedule listed on the product information sheet for 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 compounds that may be administered in combination with pharmaceutical compositions containing polypeptide conjugates provided herein includes rosiglitizone, pioglitizone, repaglinide, nateglitinide, metformin, exenatide, sitagliptin, pramulintide, glipizide, glimepriride, acarbose, and miglitol.

[0310] kit Kits for implementing the disclosed methods are also provided. Such kits may include pharmaceutical compositions such as those described herein, including nucleic acids encoding polypeptide conjugates provided herein, vectors and cells containing such nucleic acids, and pharmaceutical compositions containing such nucleic acid-containing compounds, which may be provided in sterile containers. Optionally, instructions for use on how to employ the provided pharmaceutical compositions in the treatment of metabolic disorders may also be included or made available for use by patients or healthcare providers.

[0311] In one embodiment, the kit comprises (a) a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide conjugate or a variant form thereof provided herein; and (b) one or more containers for the pharmaceutical composition. Such a kit may also include instructions for use; the instructions may be tailored to the specific metabolic disorder being treated. The instructions may describe the use and properties of the materials provided in the kit. In certain embodiments, the kit includes instructions for use to a patient for administering an administration to treat metabolic disorders such as elevated glucose levels, elevated insulin levels, diabetes mellitus, obesity, non-alcoholic steatohepatitis (NASH), cardiovascular dyslipidemia, atherosclerosis, alcoholic steatohepatitis (ASH), diabetic nephropathy, metabolic syndromes such as metabolic syndrome X, non-alcoholic fatty liver disease (NAFLD), end-stage liver disease, hepatic steatosis (fatty liver), cirrhosis, or primary biliary cirrhosis (PBC).

[0312] Instructions for use may be printed on a substrate such as paper or plastic, and may be present as an accompanying document within the kit, on the kit container or on a label of its components (e.g., associated with the packaging). In another embodiment, instructions for use may exist as an electronic data file on a suitable computer-readable storage medium, such as a CD-ROM or diskette. In yet another embodiment, the actual instructions for use are not present in the kit, but means are provided for obtaining the instructions from a remote supplier, such as via the internet. An example of this embodiment is a kit that includes a web address from which the instructions can be viewed and / or downloaded. Often, it would be desirable that some or all components of the kit be packaged in appropriate packaging that maintains sterility. The components of the kit may be packaged in a kit storage element that makes a single, easily handled unit, and the kit storage element, such as a box or similar structure, may or may not be a sealed container, for example, to further maintain sterility for some or all of the components of the kit. [Examples]

[0313] [Example 1] Recombinant expression and purification of GLP-1 protein The GLP-1 proteins listed in Table 1 were produced from a bacterial E. coli expression system using the BL21(DE3) derivative strain. The DNA encoding the GLP-1 precursor was codon-optimized for E. coli expression, de novo-synthesized, and subcloned into a PET derivative expression vector (Novagen). Amino acid substitutions were performed by modifying the corresponding gene code. When the cell density reached OD600 of 2.0 in Terrific Broth (TB) medium, GLP-1 precursor overexpression was induced with 0.5 mM isopropyl bd-thiogalactoside (IPTG). After protein induction at 37°C for 20-22 hours, the cells were collected. The collected cells were dissolved in 20 mM Tris pH 8.0, 0.15 M NaCl buffer using a cell grinder (900 bar, twice). The soluble fraction containing the GLP-1 protein was recovered by centrifugation (8,000 × g, 30 minutes). After removing the tag with protease, the protein was purified by reverse-phase chromatography. Samples from each step were characterized by LC / MS to confirm the correct molecular weight.

[0314] [Example 2] Incorporation of non-proteinogenic amino acids into 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 GLP-1 protein in the organic solvent. The reaction mixture was stirred at room temperature for 3 hours. Then, piperidine was added to the reaction solution to remove the Fmoc protecting group.

[0315] [Example 3] Preparation of GLP-1 compounds using CRM A solution of GLP-1 protein in NaOH was to be added in droplet form to a CRM reagent (i.e., HOOC-(CH2)16-CO-gGlu-2XADO) in an organic solvent. The reaction mixture was stirred at room temperature for 1 hour. The product was then subjected to reverse-phase chromatography, which yielded the compounds listed in Table 1 above.

[0316] The conjugated GLP-1 proteins were detected and characterized by LC-MS using the Waters BioAccord LC-MS system or by UPLC using the Waters Acquity UPLC system, using conditions optimized for various conjugates according to the supplier's manual.

[0317] [Example 4] In vitro activity Methods: In vitro GLP-1 activity was measured using BHK cell lines overexpressing the human GLP-1 receptor and CRE luciferase reporter, with and without 1% human serum albumin (HSA). Test fusion proteins were measured at 1 nM or 100 nM as the highest concentration, in the absence or presence of 1% HSA, using 3-fold serial dilutions. After treating cells with the molecule for 4 hours, luciferase activity was measured using the Steadylite plus kit (Perkin Elmer, 6066751).

[0318] The activity of each protein was represented by EC50, which was derived from nonlinear regression analysis.

[0319] Conclusion: Almost all molecules exhibit efficacy comparable to or even better than semaglutide in assays without HSA supplementation. However, various molecules exhibit varying degrees of reduction in GLP-1 activity in the presence of 1% HSA (i.e., EC 50 This showed an increase in linker length. The data in Table 2 suggested that linker length, fatty acid position, and the number of conjugated fatty acid moieties may be related to GLP-1 activity. As shown in Table 2, molecules with short linker lengths (i.e., 12 amino acid residues) (whether monoacylated or biacylated) had significantly lower GLP-1 activity compared to such molecules with longer linker lengths. As the distance between the C-terminal residue of GLP-1 and the CRM residue (i.e., lysine in the linker) increased, the GLP-1 activity of the molecule in the presence of HSA appeared to steadily improve.

[0320] As shown in Table 2, monoacylated molecules 002, 010, and 011 showed significantly higher activity than semaglutide in the presence of 1% HSA. For the biacylated molecules 012, 016, 004, 006, 007, 005, 001, and 061, all showed relatively lower GLP-1 activity than semaglutide in the presence of 1% HSA, but were still within acceptable limits.

[0321] [Table 2]

[0322] [Example 5] In vivo activity in C57 lean mice Methods: Ten-week-old male C57BL / 6 mice were subcutaneously injected 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. Body weight loss % = 100 * (BW on day n - BW on day 1) / (BW on day 1). Data are presented as mean and standard error (SEM). The area under the curve (AUC ΔBW 0~8d) for body weight loss (%) from day 0 to day 8 was calculated.

[0323] Conclusion: Molecules 001, 002, and 012 showed dose-dependent efficacy for weight control (Figures 1A, 1B, 1C, 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 in vitro activity data. Molecules 001, 016, and 012, which contain bifatty acids, exhibited more sustained efficacy than semaglutide, suggesting that molecules 001, 016, and 012 may have longer half-lives (Figures 1A, 1C, and 1D).

[0324] [Table 3]

[0325] [Example 6] In vivo activity in db / db mice Methods: Ten-week-old male db / db mice were given a single subcutaneous injection of protein. Fasting glucose was measured at various time points, with three animals used in each group. Delta serum glucose is glucose minus the baseline level. Data are presented as mean and standard error (SEM).

[0326] Conclusion: Both molecules 001 and 002 demonstrated dose-dependent efficacy for glucose control (Figures 2A and 2B). Molecule 001, containing a bifatty acid, exhibited more sustained efficacy than semaglutide, suggesting that molecule 002 may have a longer half-life.

[0327] Methods: Ten-week-old male db / db mice were given a single subcutaneous injection of protein. Non-fasting glucose was measured at various time points, with five animals used in each group. Delta blood glucose is glucose minus the baseline level. Data are presented as mean and standard error (SEM). The area under the curve (AUC ΔBG 0~192h) for delta blood glucose from 0 to 192 hours was calculated.

[0328] Conclusion: Molecule 012 demonstrated dose-dependent efficacy for glucose regulation (Figure 2C, Table 4). Molecule 012, containing a bifatty acid, exhibited more sustained efficacy than semaglutide.

[0329] [Table 4]

[0330] [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 before administration (-5 minutes), and at 0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72, 96, 120, 144, 168, and 192 hours after injection. Plasma protein concentrations were measured by ELISA assay. Pharmacokinetic parameters were calculated using WinNonlin based on graphs showing plasma concentrations of each protein versus time after subcutaneous injection.

[0331] Conclusion: Molecules 001 and 012 showed longer half-lives in mice than semaglutide and molecule 002 (Table 5), consistent with in vivo efficacy.

[0332] [Table 5]

[0333] Methods: Male SD rats aged 6–8 weeks were administered a single subcutaneous dose of 15 nmol / kg protein (n=3 / group) and a single intravenous dose of 15 nmol / kg protein (n=3 / group). Plasma samples were collected before administration (-5 minutes), and at 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 24 hours, 32 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, 144 hours, 168 hours, 192 hours, 216 hours, and 240 hours after subcutaneous administration. Plasma samples were also collected before administration (-5 minutes), and at 0.083 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 24 hours, 32 hours, 48 ​​hours, 72 hours, 96 hours, and 120 hours after intravenous administration. The concentrations of polypeptide conjugates in plasma were measured by LC-MS / MS. Pharmacokinetic parameters were calculated using WinNonlin based on graphs showing the plasma concentration versus time of each polypeptide conjugate after subcutaneous injection.

[0334] Conclusion: As shown in Table 6, molecules 001 and 012 exhibited longer half-lives than semaglutide, with a 12-hour half-life in rats. 1 / 2 It has been reported to have [specific characteristic] (reported in sc administration and in non-clinical review by the FDA).

[0335] [Table 6]

[0336] Methods: Ten-month-old male Bama miniature pigs 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 from the molecular 012 and semaglutide groups were collected before administration (-5 minutes) and at 0.5 hours, 1 hour, 3 hours, 8 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 168 hours, 336 hours, 504 hours, and 672 hours after subcutaneous administration. Plasma samples from the molecular 012 and semaglutide groups were also collected before administration (-5 minutes) and at 0.083 hours, 0.5 hours, 1 hour, 3 hours, 8 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 168 hours, 336 hours, and 504 hours after intravenous administration. For the semaglutide group, the protein concentration in plasma was measured by ELISA assay. For the molecular 012 group, the protein concentration in plasma was measured by LC-MS / MS. Based on graphs showing the plasma concentration versus time of each GLP-1 polypeptide conjugate after administration, pharmacokinetic parameters were calculated using WinNonlin.

[0337] Conclusion: Molecule 012 showed a longer half-life than semaglutide in miniature pigs (Table 7).

[0338] [Table 7]

[0339] [Example 8] Efficacy studies in disease models Selected molecules are assessed in disease animal models (e.g., db / db mice) to determine body weight, food intake, and glucose potency in relation to 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).

[0340] method: 22-week-old male DIO C57BL / 6 mice (approximately 50g) were subcutaneously injected with a specified GLP-1 polypeptide conjugate (i.e., molecule 012) every other day (Q2D) 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 in each treatment group. Body weight and fasting blood glucose were monitored for each individual animal, but food intake for each group of animals was measured together. Days 1 and 25 were the first and last days of molecular administration. Data are presented as mean and standard error (SEM) or pooled values. Statistical analysis was performed by one-way ANOVA. Weight loss on day 25 is calculated as -1 * (BW loss % - BW loss %) in the vehicle group; cumulative decrease in food intake is calculated as -100 * (cumulative food intake - cumulative food intake in the vehicle) / cumulative food intake in the vehicle.

[0341] Conclusion: In the DIO study, as shown in Figures 3A, 3B, and Table 8, molecule 012 demonstrated dose-dependent efficacy in weight loss, reduced food intake, and control of blood glucose levels.

[0342] [Table 8]

[0343] [Example 9] PK research in non-human primates The pharmacokinetics of the selected molecules will be assessed in monkeys. Both subcutaneous and intravenous injections will be administered.

[0344] Methods: Male cynomolgus monkeys aged 3-5 years were administered a single subcutaneous dose of 5 nmol / kg protein (n=2 / group). Plasma samples from the semaglutide group were collected before administration (-5 minutes), and at 2, 4, 6, 8, 24, 48, 72, 96, 120, 144, 216, 288, 360, 432, and 504 hours after injection. Plasma samples from the 012 group were collected before administration (-5 minutes), and at 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 protein concentration in plasma was measured by ELISA assay. For group 012, the plasma protein concentration was measured by LC-MS / MS. Based on graphs showing the plasma concentration of each protein versus time after subcutaneous injection, pharmacokinetic parameters were calculated using WinNonlin.

[0345] Conclusion: Molecule 012 showed a longer half-life than semaglutide and molecule 002 in monkeys (Table 9).

[0346] [Table 9]

[0347] [Example 10] Immunogenicity assessment The selected GLP-1 polypeptide conjugates are also assessed for immunogenicity using in silico (iTope and TCED methods) and ex vivo (EpiScreen) methods. [Example 11]

[0348] Human serum albumin binding Methods: Molecular binding to serum albumin was characterized by surface plasmon resonance in a Biacore 8K instrument. Serum albumin from different species was covalently bound to the surface of a CM5 sensor chip until it reached 4000 RU. The chip was blocked with 1 M ethanolamine at a flow rate of 10 μL / min for 420 seconds. Each molecular 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. A binding buffer without molecules was delivered onto the chip at a flow rate of 20 seconds to allow spontaneous dissociation of the bound molecules for 30 seconds.

[0349] Conclusion: Molecules 004, 001, 006, and 012 showed higher binding affinity to human serum albumin than semaglutide and molecule 002 (see Table 10), which is consistent with PK data.

[0350] [Table 10]

Claims

1. A polypeptide conjugate comprising a polypeptide portion and two clearance reduction portions (CRMs) conjugated to lysine (K) residues of the polypeptide portion, The polypeptide portion comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 118, 129, and 133. The CRM is given by the following formula: 【Chemistry 1】 A polypeptide conjugate containing the structure.

2. Structure shown below: 【Chemistry 2】 (Amino acid residues are represented by a single-letter abbreviation inside a circle.) A polypeptide conjugate according to claim 1, comprising:

3. A pharmaceutical composition comprising the polypeptide conjugate described in claim 1 and a pharmaceutically acceptable carrier.

4. A pharmaceutical composition according to claim 3 for use in a method of preventing or treating a metabolic disorder in a subject requiring the same, wherein the method comprises administering a therapeutically effective amount of the polypeptide conjugate according to claim 1.

5. The pharmaceutical composition according to claim 4, wherein the metabolic disorder is diabetes mellitus, obesity, weight gain, 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, or primary biliary cirrhosis (PBC).

6. The pharmaceutical composition according to claim 4, wherein the polypeptide conjugate 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.

7. A pharmaceutical composition according to claim 3 for use in a method for reducing food intake in a subject requiring it, wherein the method comprises administering a therapeutically effective amount of the polypeptide conjugate according to claim 1.

8. A pharmaceutical composition according to claim 3 for use in a method for reducing body weight in a subject requiring such reduction, wherein the method comprises administering a therapeutically effective amount of the polypeptide conjugate according to claim 1.

Images