Conjugate of fusion protein of GLP-1 and FGF21

Abstract

The present disclosure provides a conjugate comprising a GLP-1, a polypeptide linker, and a polypeptide of FGF21. Also provided are pharmaceutical compositions comprising the conjugate and methods for treating diseases.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application number 202180002753.4, application date January 8, 2021, and invention name “Conjugate of fusion protein of GLP-1 and FGF21”. The original application is the Chinese national phase application of the PCT international application with international application number PCT / CN2021 / 070766, which claims priority of application serial numbers PCT / CN2020 / 071566 and PCT / CN2020 / 130515 filed on January 11, 2020 and November 20, 2020, respectively. Field of the Invention

[0002] The present invention relates to fusion protein conjugates, pharmaceutical compositions thereof, and methods of preventing and / or treating diseases using such substances. Background Art

[0003] 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) derived from post-translational processing of the proglucagon peptide. The original GLP-1 product, GLP-1, stimulates insulin synthesis and secretion and has been shown to prevent hyperglycemia in diabetes, particularly type 2 diabetes. However, endogenous GLP-1 has a half-life of only approximately 2 minutes, which results in fasting plasma levels of GLP-1 of only 0-15 pmol / L.

[0004] Fibroblast growth factor 21 (FGF21), a member of the fibroblast growth factor (FGF) family, is a hormone synthesized in several metabolically active organs and regulates glucose and lipid homeostasis. Due to its diverse metabolic functions in multiple target organs, the biology of FGF21 is inherently complex. FGF21 has been reported to play a role in organs such as the liver, adipocytes, pancreas, hypothalamus, and muscle tissue (Fisher FM, Annu Rev Physiol, 2016, 78:223).

[0005] Metabolic disorders, often associated with insulin resistance, visceral obesity, and atherogenic dyslipidemia, pose a significant and escalating public health and clinical challenge worldwide. However, existing treatments for metabolic diseases suffer from issues such as short half-lives and / or low efficacy.

[0006] Therefore, there is a need for improved therapeutic regimens for treating metabolic diseases. Summary of the Invention

[0007] Provided herein are conjugates of the fusion proteins, as well as pharmaceutical compositions and methods of use for treating / preventing metabolic disorders.

[0008] In one aspect, the present disclosure provides a Polypeptide Conjugate comprising a fusion polypeptide conjugated to at least one clearance reducing moiety (CRM),

[0009] wherein the fusion polypeptide comprises or consists essentially of GLP-1, a polypeptide linker, and FGF21 from N-terminus to C-terminus;

[0010] wherein the fusion polypeptide further comprises at least one conjugable residue conjugated to a CRM, and wherein the first conjugable residue is in FGF21; and

[0011] The length of the polypeptide linker is at least 0-120 amino acid residues.

[0012] In certain embodiments, the polypeptide linker is at least 4, 5, 8, 10, 20, 30, 40, 48, 50, 60, 70, 80, 90, 100, 110, or 120 amino acid residues in length.

[0013] In certain embodiments, the Polypeptide Conjugate does not comprise an antibody fragment (eg, Fc).

[0014] In certain embodiments, the fusion polypeptide further comprises a second conjugatable residue.

[0015] In certain embodiments, the fusion polypeptide comprises at most one conjugable residue or at most two conjugable residues.

[0016] In certain embodiments, the second conjugatable residue is present in the GLP-1 or polypeptide linker.

[0017] In certain embodiments, FGF21 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:36 while retaining the basic biological activity of SEQ ID NO:36, and / or GLP-1 comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:50 while retaining the basic biological activity of SEQ ID NO:50.

[0018] In certain embodiments, GLP-1 contains no more than 9, 8, 7, 6, 5, 4, or 3 mutations relative to SEQ ID NO: 50 while retaining the basic biological activity of SEQ ID NO: 50, and / or FGF21 contains no more than 12, 11, 10, 9, 8, or 7 mutations relative to SEQ ID NO: 36 while retaining the basic biological activity of SEQ ID NO: 36.

[0019] In certain embodiments, the first conjugable residue in FGF21 is at a position selected from positions 170, 171, 172, 173, 174, 180, 181, and 182 relative to SEQ ID NO:36.

[0020] In certain embodiments, the first conjugable residue in FGF21 comprises a residue that was introduced, optionally by substitution or by insertion.

[0021] In certain embodiments, the conjugable residue is selected from the group consisting of lysine, cysteine, and an unnatural amino acid.

[0022] In certain embodiments, the first conjugable residue in FGF21 is lysine.

[0023] In certain embodiments, the conjugable residue in FGF21 comprises a substitution relative to SEQ ID NO: 36 selected from the group consisting of G170K, P171K, S172K, Q173K, G174K, A180K, S181K, and ins182K.

[0024] In certain embodiments, FGF21 further comprises one or more mutations at positions selected from K56, K59, K69, and K122 relative to SEQ ID NO: 36, wherein the mutations are mutations to non-conjugatable residues.

[0025] In certain embodiments, the non-conjugable residue is selected from the group consisting of arginine (R), glutamine (Q), alanine (A), glycine (G), histidine (H), serine (S), and threonine (T).

[0026] In certain embodiments, FGF21 comprises mutations of K56R, K59R, K69R, and K122R.

[0027] In certain embodiments, FGF21 further comprises one or more additional mutations at a position selected from positions 121, 168, 171, 180, and 181 relative to SEQ ID NO: 36, with the proviso that the first conjugatable residue is not further mutated.

[0028] In certain embodiments, the one or more additional mutations in FGF21 are selected from N121Q, M168L, P171G, A180E, and del181S.

[0029] In certain embodiments, FGF21 comprises at most one conjugatable residue.

[0030] In certain embodiments, FGF21 comprises or consists of a combination of mutations relative to SEQ ID NO: 36 selected from the group consisting of:

[0031] 1) 121Q, 168L, 180K, 171G, 56R, 59R, 69R and 122R;

[0032] 2) 121Q, 168L, 171K, 56R, 59R, 69R and 122R;

[0033] 3) 121Q, 168L, 174K, 171G, 56R, 59R, 69R and 122R;

[0034] 4) 121Q, 168L, 171K, 56R, 59R, 69R, 122R and 180E;

[0035] 5) 121Q, 168L, 174K, 171G, 56R, 59R, 69R, 122R and 180E;

[0036] 6) 121Q, 168L, 180K, 56R, 59R, 69R and 122R;

[0037] 7)121Q, 168L, 180K, 171G, 56R, 59R, 69R, 122R and del181S;

[0038] 8) 121Q, 168L, 170K, 56R, 59R, 69R and 122R;

[0039] 9) 121Q, 168L, 170K, 56R, 59R, 69R, 122R and 180E;

[0040] 10) 121Q, 168L, 170K, 171G, 56R, 59R, 69R, 122R and 180E;

[0041] 11) 121Q, 168L, 180E, 171G, 56R, 59R, 69R, 122R, and 181K; and

[0042] 12)121Q, 168L, 180E, 171G, 56R, 59R, 69R, 122R and ins182K.

[0043] In certain embodiments, FGF21 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-46 and 118-119.

[0044] In certain embodiments, the GLP-1 comprises no conjugatable residues or comprises at most one conjugatable residue.

[0045] In certain embodiments, the second conjugatable residue is in GLP-1.

[0046] In certain embodiments, the second conjugable residue in GLP-1 is lysine.

[0047] In certain embodiments, the conjugatable residue in GLP-1 comprises or consists of K26 or K34 relative to SEQ ID NO:50.

[0048] In certain embodiments, the conjugatable residue in GLP-1 comprises a residue that has been introduced, optionally by substitution or by insertion.

[0049] In certain embodiments, the conjugatable residue in GLP-1 is introduced by a substitution selected from the group consisting of E27K and R36K relative to SEQ ID NO:50.

[0050] In certain embodiments, the GLP-1 further comprises substitution of K26 and / or K34 to a non-conjugatable residue, with the proviso that if K26 or K34 is a conjugatable residue, it is not substituted.

[0051] In certain embodiments, the non-conjugable residue is selected from the group consisting of arginine (R), glutamine (Q), alanine (A), glycine (G), histidine (H), serine (S), and threonine (T).

[0052] In certain embodiments, the substitution at K26 is selected from K26R and K26Q, and / or the substitution at K34 is selected from K34R and K34Q.

[0053] In certain embodiments, the conjugatable residue in GLP-1 comprises or consists of cysteine ​​or an unnatural amino acid.

[0054] In certain embodiments, the conjugatable residue in GLP-1 is introduced by substitution at a position selected from the group consisting of K26, K34, E27, and R36 relative to SEQ ID NO:50.

[0055] In certain embodiments, the GLP-1 further comprises one or more additional mutations at a position selected from the group consisting of A8, G22, K34, R36, and H7 relative to SEQ ID NO: 50, or any combination thereof, with the proviso that if K34 is a conjugatable residue, it is not further mutated.

[0056] In certain embodiments, the one or more additional mutations in GLP-1 are selected from the group consisting of H7IA, H7IPA, A8G, G22E, K34R, and R36G, or any combination thereof.

[0057] In certain embodiments, GLP-1 comprises the amino acid sequence X7X8EGTFTSDVSSYLEX 22 QAAX 26X 27 FIAWLVX 34 GX 36 G (SEQ ID NO: 51),

[0058] Wherein: X7 is H, imidazole-4-acetic acid (IA) or imidazole propionic acid (IPA); X8 is A, G, S, V, Aib, T, I or L; X 22 G or E; X 26 K, R or C; X 27 E, K or C; X 34 is R, K or C, and X 36 is K, R, G or C, with the proviso that GLP-1 contains at most one conjugatable residue or no conjugatable residue.

[0059] In certain embodiments, X7 is H, X8 is G, X 22 E; X 26 K or R; X 27 E; X 34 is R, and X 36 For G.

[0060] In certain embodiments, the GLP-1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 52, 53, and 115.

[0061] In certain embodiments, a polypeptide linker comprises one or more units of a repeating sequence.

[0062] In certain embodiments, the repetitive sequence consists of no more than 4, 5, or 6 types of amino acid residues selected from the group consisting of G, Q, A, E, P, and S.

[0063] In certain embodiments, the repeat sequence comprises or consists of an amino acid sequence selected from the group consisting of: G a S b, SEQ ID NO: 65 (GAQP), SEQ ID NO: 92 (GQEP), SEQ ID NO: 93 (GEQP), SEQ ID NO: 94 (GPQE), SEQ ID NO: 95 (GPEQ), SEQ ID NO: 96 (GSEP), SEQ ID NO: 97 (GESP), SEQ ID NO: 98 (GPSE), SEQ ID NO: 99 (GPES), SEQ ID NO: 100 (GQAP), SEQ ID NO: 101 (GPAQ), SEQ ID NO: 102 (GPQA), SEQ ID NO: 103 (GSQP), SEQ ID NO: 104 (GASP), SEQ ID NO: 105 (GPAS), SEQ ID NO: 106 (GPSA), SEQ ID NO: 107 (GGGS), SEQ ID NO: 108 (GSGS), SEQ ID NO: 57 (GGGGS) and GS, wherein a and b are independently an integer selected from 1 to 5.

[0064] In certain embodiments, the polypeptide linker comprises no conjugatable residues or comprises at most one conjugatable residue.

[0065] In certain embodiments, the polypeptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 54-65, 109-113, 3, and 47-49.

[0066] In certain embodiments, the second conjugatable residue is in a polypeptide linker.

[0067] In certain embodiments, the conjugatable residue in the polypeptide linker comprises a residue that has been introduced, optionally by substitution or by insertion.

[0068] In certain embodiments, the conjugable residue in the polypeptide linker is the C-terminal residue of the polypeptide linker, or is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 residues (including the conjugable residue) away from the N-terminal residue of FGF21.

[0069] In certain embodiments, the polypeptide linker comprising a second conjugatable residue comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-75 and 89-91.

[0070] In certain embodiments, the fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4-22, 24, 26-35, 77-88, and 116-117.

[0071] In another aspect, the present disclosure provides a Polypeptide Conjugate comprising a fusion polypeptide conjugated to at least one clearance reducing moiety (CRM),

[0072] wherein the fusion polypeptide comprises or consists essentially of GLP-1, a polypeptide linker, and FGF21 from N-terminus to C-terminus;

[0073] wherein the at least one CRM is conjugated to at least one conjugatable residue in the fusion polypeptide; and

[0074] The length of the polypeptide linker is at least 0-120 amino acid residues.

[0075] In certain embodiments, the polypeptide linker is at least 4, 5, 8, 10, 20, 24, 28, 30, 40, 48, 50, 60, 70, 80, 90, 100, 110, or 120 amino acid residues in length.

[0076] In certain embodiments, the Polypeptide Conjugate does not comprise an antibody fragment (eg, Fc).

[0077] In certain embodiments, the fusion polypeptide comprises at most one conjugable residue or at most two conjugable residues.

[0078] In certain embodiments, the fusion polypeptide comprises a first conjugable residue conjugated to a CRM, and wherein the first conjugable residue is in FGF21, in a polypeptide linker, or in GLP-1. In certain embodiments, the first conjugable residue is in FGF21.

[0079] In certain embodiments, the fusion polypeptide comprises a first conjugable residue and a second conjugable residue, wherein:

[0080] 1) Both conjugable residues are present in the polypeptide linker,

[0081] 2) Both conjugable residues are present in FGF21,

[0082] 3) Both conjugated residues are present in GLP-1,

[0083] 4) the first conjugable residue is present in FGF21 and the second conjugable residue is present in the polypeptide linker,

[0084] 5) the first conjugable residue is present in GLP-1 and the second conjugable residue is present in FGF21, or

[0085] 6) The first conjugable residue is present in GLP-1 and the second conjugable residue is present in the polypeptide linker.

[0086] In certain embodiments, the first conjugable residue is lysine, cysteine, or an unnatural amino acid residue, or both the first conjugable residue and the second conjugable residue are lysine, cysteine, or an unnatural amino acid residue.

[0087] In certain embodiments, FGF21 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:36 while retaining the basic biological activity of SEQ ID NO:36, and / or GLP-1 comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:50 while retaining the basic biological activity of SEQ ID NO:50.

[0088] In certain embodiments, GLP-1 contains no more than 9, 8, 7, 6, 5, 4, or 3 mutations relative to SEQ ID NO: 50 while retaining the basic biological activity of SEQ ID NO: 50, and / or FGF21 contains no more than 12, 11, 10, 9, 8, or 7 mutations relative to SEQ ID NO: 36 while retaining the basic biological activity of SEQ ID NO: 36.

[0089] In certain embodiments, FGF21 comprises one or more mutations at a position selected from positions 121, 168, 171, 180, and 181 relative to SEQ ID NO:36.

[0090] In certain embodiments, the one or more additional mutations in FGF21 are selected from N121Q, M168L, P171G, A180E, and del181S, or any combination thereof.

[0091] In certain embodiments, FGF21 comprises no conjugatable residues or comprises at most one conjugatable residue.

[0092] In certain embodiments, the conjugatable residue in FGF21 is at a position selected from positions 71, 170, 171, 172, 173, 174, 180, 181, and 182 relative to SEQ ID NO:36.

[0093] In certain embodiments, the conjugatable residue in FGF21 comprises a residue that has been introduced, optionally by substitution or by insertion.

[0094] In certain embodiments, the conjugable residue in FGF21 is lysine.

[0095] In certain embodiments, the conjugable residue in FGF21 comprises a substitution relative to SEQ ID NO: 36 selected from the group consisting of G170K, P171K, S172K, Q173K, G174K, A180K, S181K, S71K, and ins182K.

[0096] In certain embodiments, FGF21 further comprises one or more mutations at positions selected from K56, K59, K69, and K122 relative to SEQ ID NO: 36, wherein the mutations are mutations to non-conjugatable residues.

[0097] In certain embodiments, the non-conjugable residue is selected from the group consisting of arginine (R), glutamine (Q), alanine (A), glycine (G), histidine (H), serine (S), and threonine (T).

[0098] In certain embodiments, FGF21 comprises mutations of K56R, K59R, K69R, and K122R.

[0099] In certain embodiments, FGF21 comprises or consists of a combination of mutations relative to SEQ ID NO: 36 selected from the group consisting of:

[0100] 1) 121Q, 168L, 180K, 171G, 56R, 59R, 69R and 122R;

[0101] 2) 121Q, 168L, 171K, 56R, 59R, 69R and 122R;

[0102] 3) 121Q, 168L, 174K, 171G, 56R, 59R, 69R and 122R;

[0103] 4) 121Q, 168L, 171K, 56R, 59R, 69R, 122R and 180E;

[0104] 5) 121Q, 168L, 174K, 171G, 56R, 59R, 69R, 122R and 180E;

[0105] 6) 121Q, 168L, 180K, 56R, 59R, 69R and 122R;

[0106] 7)121Q, 168L, 180K, 171G, 56R, 59R, 69R, 122R and del181S;

[0107] 8) 121Q, 168L, 170K, 56R, 59R, 69R and 122R;

[0108] 9) 121Q, 168L, 170K, 56R, 59R, 69R, 122R and 180E;

[0109] 10) 121Q, 168L, 170K, 171G, 56R, 59R, 69R, 122R and 180E;

[0110] 11) 121Q, 168L, 180E, 171G, 56R, 59R, 69R, 122R, and 181K;

[0111] 12) 121Q, 168L, 180E, 171G, 56R, 59R, 69R, 122R and ins182K;

[0112] 13) 71K, 121Q, 168L, 56R, 59R, 69R and 122R;

[0113] 14) 71K, 121Q, 168L, 171G, 56R, 59R, 69R, and 122R; and

[0114] 15)71K, 121Q, 168L, 171G, 56R, 59R, 69R, 122R, 180E.

[0115] In certain embodiments, FGF21 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-46 and 118-119.

[0116] In certain embodiments, the conjugatable residue in FGF21 is cysteine.

[0117] In certain embodiments, the conjugable residue in FGF21 comprises a substitution relative to SEQ ID NO: 36 selected from the group consisting of G170C, P171C, S172C, Q173C, G174C, A180C, S181C, S71C, and ins182C.

[0118] In certain embodiments, FGF21 comprises or consists of a combination of mutations relative to SEQ ID NO: 36 selected from the group consisting of:

[0119] 1)121Q, 168L, 171C, 180E;

[0120] 2)121Q, 168L, 171G, 174C, 180E;

[0121] 3)121Q, 168L, 171G, 180C;

[0122] 4)121Q, 168L, 171G, 180E;

[0123] 5)71C, 121Q, 168L, 171G, 180E and

[0124] 6)121Q, 168L, 180E, 171G, ins 182C.

[0125] In certain embodiments, FGF21 comprises an amino acid sequence selected from SEQ ID NOs: 136-140 and 171.

[0126] In certain embodiments, the GLP-1 comprises one or more mutations at positions selected from the group consisting of A8, G22, K34, R36, and H7 relative to SEQ ID NO: 50, or any combination thereof.

[0127] In certain embodiments, the one or more mutations are selected from the group consisting of H7IA, H7IPA, A8G, G22E, K34R, and R36G, or any combination thereof.

[0128] In certain embodiments, the GLP-1 comprises no conjugatable residues or comprises at most one conjugatable residue.

[0129] In certain embodiments, the conjugable residue in GLP-1 is lysine.

[0130] In certain embodiments, the conjugatable residue in GLP-1 comprises or consists of K26 or K34 relative to SEQ ID NO:50.

[0131] In certain embodiments, the conjugatable residue in GLP-1 comprises a residue that has been introduced, optionally by substitution or by insertion.

[0132] In certain embodiments, the conjugatable residue in GLP-1 is introduced by a substitution selected from the group consisting of E27K and R36K relative to SEQ ID NO: 50. In such embodiments, the GLP-1 further comprises substitution of K26 and / or K34 to a non-conjugatable residue.

[0133] In certain embodiments, the non-conjugable residue is selected from the group consisting of arginine (R), glutamine (Q), alanine (A), glycine (G), histidine (H), serine (S), and threonine (T).

[0134] In certain embodiments, the substitution at K26 is selected from K26R and K26Q, and / or the substitution at K34 is selected from K34R and K34Q.

[0135] In certain embodiments, the conjugatable residue in GLP-1 comprises or consists of cysteine ​​or an unnatural amino acid.

[0136] In certain embodiments, the conjugatable residue in GLP-1 is introduced by substitution at a position selected from the group consisting of K26, K34, E27, and R36 relative to SEQ ID NO:50.

[0137] In certain embodiments, the conjugable residue in GLP-1 comprises or consists of cysteine ​​and is introduced by substitution at a position selected from the group consisting of K26C, K34C, E27C, and R36C relative to SEQ ID NO:50.

[0138] In certain embodiments, GLP-1 comprises the amino acid sequence X7X8EGTFTSDVSSYLEX22QAAX26X27FIAWLVX34GX36G (SEQ ID NO: 51),

[0139] wherein: X7 is H, imidazole-4-acetic acid (IA) or imidazole propionic acid (IPA); X8 is A, G, S, V, Aib, T, I or L; X22 is G or E; X26 is K, R or C; X27 is E, K or C; X34 is R, K or C, and X36 is K, R, G or C.

[0140] In certain embodiments, X7 is H, X8 is G, X22 is E; X26 is K or R; X27 is E; X34 is R, and X36 is G.

[0141] In certain embodiments, the GLP-1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 52, 53, and 115.

[0142] In certain embodiments, the GLP-1 comprises the amino acid sequence of SEQ ID NO: 120 or 52.

[0143] In certain embodiments, the polypeptide linker comprises one or more units (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) of a repeat sequence. In certain embodiments, the polypeptide linker comprises two or more different repeat sequences.

[0144] In certain embodiments, the repetitive sequence consists of no more than 4, 5, or 6 types of amino acid residues selected from the group consisting of G, Q, A, E, P, T, and S.

[0145] In certain embodiments, the repetitive sequence comprises or consists of an amino acid sequence selected from the group consisting of GaSb, SEQ ID NO: 65 (GAQP), SEQ ID NO: 92 (GQEP), SEQ ID NO: 93 (GEQP), SEQ ID NO: 94 (GPQE), SEQ ID NO: 95 (GPEQ), SEQ ID NO: 96 (GSEP), SEQ ID NO: 97 (GESP), SEQ ID NO: 98 (GPSE), SEQ ID NO: 99 (GPES), SEQ ID NO: 100 (GQAP), SEQ ID NO: 101 (GPAQ), SEQ ID NO: 102 (GPQA), SEQ ID NO: 103 (GSQP), SEQ ID NO: 104 (GASP), SEQ ID NO: 105 (GPAS), SEQ ID NO: 106 (GPSA), SEQ ID NO: 107 (GGGS), SEQ ID NO: 108 (GSGS), SEQ ID NO: 57 (GGGGS), SEQ ID NO: 58 (GGGGS), SEQ ID NO: 59 (GGGGS), SEQ ID NO: 61 (GGGGS), SEQ ID NO: 62 (GGGGS), SEQ ID NO: 63 (GGGGS), SEQ ID NO: 64 (GGGGS), SEQ ID NO: 65 (GGGGS), SEQ ID NO: 66 (GGGGS), SEQ ID NO: 67 (GGGGS), SEQ ID NO: 68 (GGGGS), SEQ ID NO: 69 (GGGGS), SEQ ID NO: 70 (GGGGS), SEQ ID NO: 71 (GGGGS), SEQ ID NO: 72 (GGGGS), SEQ ID NO: 73 (GGGGS), SEQ ID NO: 74 (GGGGS), SEQ ID NO: 75 (GGG NO:143(GSAPGSPAGSPTGSAPGSPA) and GS, wherein a and b are independently an integer selected from 1 to 5.

[0146] In certain embodiments, the polypeptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 54-65, 109-113, 3, 47-49, and 141-147.

[0147] In certain embodiments, the polypeptide linker comprises no conjugatable residues, at most one conjugatable residue, or at most two conjugatable residues.

[0148] In certain embodiments, the conjugatable residue in the polypeptide linker comprises a residue that has been introduced, optionally by substitution or by insertion.

[0149] In certain embodiments, the conjugable residue in the polypeptide linker is the C-terminal residue of the polypeptide linker, or is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70 residues (including the conjugable residue) away from the N-terminal residue of FGF21.

[0150] In certain embodiments, the conjugable residue in the polypeptide linker is lysine, and the polypeptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-75 and 89-91.

[0151] In certain embodiments, the conjugable residue in the polypeptide linker is cysteine, and the polypeptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 122-132.

[0152] In certain embodiments, the polypeptide linker comprises two conjugable residues and comprises the amino acid sequence of SEQ ID NO: 133 or 134.

[0153] In certain embodiments, the conjugable residue is lysine and the fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4-22, 24, 26-35, 77-88, 116-117.

[0154] In certain embodiments, the conjugable residue is cysteine ​​and the fusion polypeptide comprises an amino acid sequence selected from the group consisting of 148-165, 168-170, 1, 23, 25, and 76.

[0155] In certain embodiments of any aspect disclosed herein, the CRM comprises a structure of *-ABCDE, wherein A, B, C, D, and E are interconnected by amide bonds, and the * end of A is linked to a conjugable residue on a polypeptide complex, and wherein:

[0156] A is selected from the group consisting of a bond, a, b, c and d are independently integers from 0 to 4, R 1 is hydrogen or -COOH;

[0157] B is selected from the group consisting of a bond, e is an integer from 1 to 4,

[0158] C is a bond or R 2 is -CH2SO3H or -COOH, f is an integer from 1 to 4, and n is an integer from 1 to 25;

[0159] D is selected from the group consisting of a bond, g and h are independently 0 or 1, and R 3 is H or -CH2COOH;

[0160] E is an acidic group having the formula:

[0161]

[0162] Where W represents -(CR 4 R 5 ) l -,

[0163] R 4 and R 5independently selected from the group consisting of hydrogen, halogen, cyano, hydroxy, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, hydroxyalkyl, amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy, and carboxamide,

[0164] R 6 Selected from hydroxyl or NR 7 R 8 ;

[0165] R 7 and R 8 Independently selected from the group consisting of hydrogen, alkyl, hydroxyl and and

[0166] l is an integer from 10 to 20,

[0167] or a pharmaceutically acceptable salt thereof.

[0168] In certain embodiments, the CRM is conjugated to at least one lysine residue in the fusion polypeptide.

[0169] In certain embodiments, A is a bond, B is a bond, and C is a bond.

[0170] In certain embodiments, A is And B is

[0171] In certain embodiments, A is And B is a bond.

[0172] In certain embodiments, the CRM comprises the following structure:

[0173]

[0174] In certain embodiments, the CRM is conjugated to at least one cysteine ​​residue in the fusion polypeptide.

[0175] In certain embodiments, A is And B is

[0176] In certain embodiments, the CRM comprises the following structure:

[0177]

[0178] In another aspect, the present disclosure provides a pharmaceutical composition comprising the Polypeptide Conjugate disclosed herein and a pharmaceutically acceptable carrier.

[0179] In another aspect, the present disclosure provides a method of preventing or treating a metabolic disorder in a subject in need thereof, comprising administering a therapeutically effective amount of a Polypeptide Conjugate disclosed herein or a pharmaceutical composition disclosed herein.

[0180] In some embodiments, the metabolic disorder is diabetes, obesity, nonalcoholic steatohepatitis (NASH), cardiovascular such as dyslipidemia, arteriosclerosis, alcoholic steatohepatitis (ASH), diabetic nephropathy, gestational diabetes, metabolic syndrome such as metabolic syndrome X, nonalcoholic fatty liver disease (NAFLD), end-stage liver disease, hepatic steatosis (fatty liver), cirrhosis, or primary biliary cirrhosis (PBC).

[0181] In another aspect, the present disclosure provides a polynucleotide encoding a fusion polypeptide as described herein.

[0182] In another aspect, the present disclosure provides a vector comprising the polynucleotide disclosed herein.

[0183] In another aspect, the present disclosure provides a host cell comprising a vector disclosed herein.

[0184] In another aspect, the present disclosure provides a method for producing a fusion polypeptide as described herein, comprising culturing a host cell disclosed herein under conditions that allow expression of a polynucleotide disclosed herein.

[0185] In another aspect, the present disclosure provides a method for producing a Polypeptide Conjugate disclosed herein, comprising conjugating a clearance reducing moiety to a fusion polypeptide as described herein.

[0186] 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, "fusion polypeptide" means one fusion polypeptide or more than one fusion polypeptide.

[0187] In all cases where a series of numerical values ​​are described in this application, it should be understood that any numerical value described can be the upper or lower limit of a numerical range. It should be further understood that the present invention encompasses all such numerical ranges, that is, the range with a combination of a numerical upper limit and a numerical lower limit, wherein the numerical value of each of the upper and lower limits can be any numerical value described herein. The scope provided herein is understood to include all values ​​within the scope. For example, 1-10 is understood to include all values ​​1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, as well as appropriate time-divided numerical values. Similarly, the range limited by "at least" is understood to include the lower limit provided and all higher numerals.

[0188] As used herein, "about" is understood to include within three standard deviations of the mean or within the standard tolerance range in the particular art. In certain embodiments, about is understood to mean a variation of no more than 0.5.

[0189] The articles "a" and "an" are used herein to refer to one or to more than one (ie, to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0190] The term "including" is used herein to mean and is interchangeable with the phrase "including but not limited to". Similarly, "such as" is used herein to mean and is interchangeable with the phrase "such as but not limited to".

[0191] The term "or" is used herein inclusively to mean, and interchangeably with, the terms "and / or," unless the context clearly dictates otherwise.

[0192] Terms like "comprising" are to be construed as open-ended terms (ie, meaning "including, but not limited to,") unless otherwise indicated.

[0193] The term "consisting essentially of" implies the exclusion of all, most, or all but negligible amounts of other elements, or it allows for elements not explicitly recited, but excludes elements found in the prior art or that affect the basic or novel properties. When a polypeptide is referred to as "consisting essentially of" an amino acid sequence, this means that the polypeptide is primarily composed of the amino acid sequence, with no more than 10 (e.g., no more than 6, 5, 4, 3, 2, or 1) additional amino acid residues at one or both ends of the polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0194] Figures 1A-1F In vivo activity of the fusion protein conjugates was demonstrated. To evaluate the effect of the fusion protein conjugates on body weight, 10-week-old male C57BL / 6J mice were subcutaneously administered 30 nmol / kg of the fusion protein conjugates (as indicated in the figure legends), semaglutide, or vehicle control daily for 6, 10, or 14 days. Body weight was measured daily. Figure 1A Shown are body weight losses from day 1 to day 11 of treatment. Figure 1B Shown is the body weight loss on day 11. Compared with vehicle control: *, p<0.05; ***, p<0.001, compared with semaglutide (JP51144): ##, p<0.01; ###, p<0.001. Figure 1C Shown are weight losses following protein treatment from day 1 to day 15. Figure 1DShown is body weight loss on day 15. Compared with vehicle control: ***, p < 0.001; ns, not statistically significant; compared with semaglutide (JP51144): #, p < 0.05; ##, p < 0.01; ###, p < 0.001. Figure 1E Shown are weight losses following protein treatment from day 1 to day 11. Figure 1F Shown is body weight loss on day 11. Compared to vehicle control: ***, p < 0.001. All data are expressed as mean and standard error (SEM).

[0195] Figure 2 Pharmacokinetic measurements of the fusion protein conjugates are shown. Each fusion protein conjugate was injected subcutaneously into C57BL / 6 mice (n=3 / group) once, and plasma samples were collected at the designated time points and analyzed by ELISA. Plasma concentrations are indicated as mean and standard error (SEM).

[0196] Figures 3A-3B Demonstration of in vivo efficacy in DIO mice To evaluate the effect of the fusion proteins on body weight, 17-week-old DIO mice (C57BL / 6 mice on a high-fat diet for 13 weeks) were subcutaneously dosed with different concentrations of the compounds daily for 21 days. Figure 3A Shown is body weight loss at 10 nmol / kg. Figure 3B The effect on body weight at 30 nmol / kg is shown. Data are presented as mean and standard error (SEM). For each treatment group, n=5.

[0197] Figures 4A-4F Demonstration of in vivo efficacy in DIO mice To evaluate the in vivo effects of compounds on body weight, blood glucose, plasma insulin, and plasma triglycerides, 17-week-old DIO mice (C57BL / 6 mice on a high-fat diet for 13 weeks) were subcutaneously dosed with different concentrations of compound daily for 21 days. Figure 4A Body weight loss after protein treatment is shown. Compared with vehicle control: *, p < 0.05, **, p < 0.01 ***, p < 0.001, ****, p < 0.0001; compared with semaglutide (JP51144): #, p < 0.05. Figure 4B Shown is the weight change on day 22. Figure 4C Shown are the changes in fasting blood glucose after treatment. Figure 4D The terminal plasma insulin concentration on day 22 is shown. Figure 4E Plasma triglyceride concentrations in terminal plasma on day 22 are shown. Figure 4F Hepatic triglyceride levels are shown on day 22. Data are presented as mean and standard error (SEM). For each treatment group, n=5.

[0198] Figure 5 All sequences disclosed in this disclosure are shown. DETAILED DESCRIPTION

[0199] The following description of the present invention is intended only to illustrate various embodiments of the present invention. As such, the specific modifications discussed should not be construed as limiting the scope of the present invention. It will be apparent to those skilled in the art that various equivalents, variations, and modifications may be made without departing from the scope of the present invention, and it will be understood that such equivalent embodiments are intended to be included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0200] definition

[0201] "Percentage (%) sequence identity" is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum number of identical amino acids (or nucleic acids). In other words, the percentage (%) sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of identical amino acid residues (or bases) relative to the reference sequence to which it is compared by the total number of amino acid residues (or bases) in the candidate sequence or the reference sequence, whichever is shorter. Conservative substitutions of amino acid residues are not considered identical residues. Alignment for the purpose of determining percent amino acid (or nucleic acid) sequence identity can be performed, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of the US 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 Bioinformatics Institute, see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin et al., Methods in Enzymology, 267:383-402 (1997)). MA et al., Bioinformatics (Oxford, England), 23(21):2947-8 (2007) and ALIGN or Megalign (DNASTAR) software. One skilled in the art can use the default parameters provided by the tools, or can customize parameters suitable for alignment, for example, by selecting an appropriate algorithm.

[0202] "Conservative substitutions" with respect to amino acid sequences refer to replacing an amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties. For example, conservative substitutions can be made between amino acid residues having hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between residues having neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between residues having acidic side chains (e.g., Asp, Glu), between amino acids having basic side chains (e.g., His, Lys, and Arg), or between residues having aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative substitutions generally do not cause significant changes in the conformational structure of the protein, and therefore, the biological activity of the protein can be retained.

[0203] As used herein, the term "functional form" refers to different forms (such as variants, fragments, fusions, derivatives and mimetics) of a parent molecule that, despite differences in amino acid sequence or chemical structure, retain the basic biological activity of the parent molecule. As used herein, the expression "retaining basic biological activity" means exhibiting at least a portion (e.g., not less than about 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%) or all of the biological activity of the parent molecule. The functional form of a parent polypeptide can include naturally occurring variant forms and non-naturally occurring forms, such as those obtained by recombinant methods or chemical synthesis. The functional form can contain non-natural amino acid residues.

[0204] As used herein, the term "variant" refers to a polypeptide that has at least 70% sequence identity with a parent polypeptide and retains at least some of the functionality of the parent polypeptide. A variant may differ from the parent polypeptide in one or more amino acid residues. For example, a variant may have a substitution, addition, deletion, insertion, or truncation of one or more amino acid residues of the parent polypeptide.

[0205] As used herein, the term "fragment" refers to a partial sequence of a parent polypeptide of any length. The fragment may still retain at least some of the functions of the parent polypeptide.

[0206] As used herein, the term "derivative" refers to a chemically modified polypeptide or fusion polypeptide in which one or more clearly defined substituents have been covalently attached to one or more specific amino acid residues of the polypeptide or fusion polypeptide. Exemplary chemical modifications can be, for example, alkylation, acylation, esterification, amidation, phosphorylation, glycosylation, labeling, methylation, or conjugation to one or more moieties of one or more amino acids.

[0207] 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, as used herein, an amino acid mimetic can be a synthetic structure (known or unknown) that may or may not be an amino acid, but retains the functional characteristics of the parent amino acid while differing in structure from the parent amino acid. Examples include methacryloyl or acryloyl derivatives of amides, β-, γ-, and δ-imino acids (e.g., piperidine-4-carboxylic acid), and the like.

[0208] As used herein, "treating" or "treatment" of a condition includes preventing or alleviating the condition, slowing the onset or rate of development of the condition, reducing the risk of developing the condition, preventing or delaying the development 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 some combination thereof.

[0209] As used herein, the term "vector" refers to a medium into which a polynucleotide encoding a protein can be operably inserted to cause expression of the protein. A vector can be used to transform, transduce, or transfect a host cell so that the genetic elements it carries are expressed in the host cell. Examples of vectors include plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phages or M13 phages; and animal viruses. The categories of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40). A vector can contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, a vector can contain an origin of replication. A vector can also include materials that facilitate its entry into cells, including but not limited to viral particles, liposomes, or protein envelopes. A vector can be an expression vector or a cloning vector. The present disclosure provides vectors (e.g., expression vectors) containing a nucleic acid sequence encoding a fusion polypeptide as provided herein, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (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, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p1 5TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0210] As used herein, the phrase "host cell" refers to a cell into which an exogenous polynucleotide and / or vector has been introduced.

[0211] The term "pharmaceutically acceptable" indicates that the specified carrier, vehicle, diluent, excipient and / or salt is generally chemically and / or physically compatible with the other ingredients making up the formulation and physiologically compatible with the recipient thereof.

[0212] As used herein, the term "subject" or "individual" or "animal" or "patient" refers to a human or non-human animal, including a mammal or primate, for whom diagnosis, prognosis, amelioration, prevention and / or treatment of a disease or condition is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cattle, bears, and the like.

[0213] In one aspect, the present disclosure provides a Polypeptide Conjugate comprising a fusion polypeptide conjugated to at least one clearance reducing moiety (CRM), wherein the fusion polypeptide comprises or consists essentially of GLP-1, a polypeptide linker, and FGF21 from N-terminus to C-terminus, wherein at least one CRM is conjugated to at least one conjugable residue in the fusion polypeptide. In certain embodiments, the polypeptide linker is at least 0-120 amino acid residues in length.

[0214] In another aspect, the present disclosure provides a polypeptide conjugate comprising a fusion polypeptide conjugated to at least one clearance reducing moiety (CRM), wherein the fusion polypeptide comprises or consists essentially of GLP-1, a polypeptide linker, and FGF21 from N-terminus to C-terminus; wherein the fusion polypeptide further comprises at least one conjugable residue conjugated to the CRM, and wherein the first conjugable residue is in FGF21; and wherein the polypeptide linker is at least 0-120 amino acid residues in length.

[0215] Fusion peptide

[0216] When applied to amino acid sequences (e.g., peptides, polypeptides, or proteins), the term "fusion" or "fused" refers to the combination of two or more amino acid sequences into a single amino acid sequence that does not occur naturally, for example, by chemical bonding or recombinant means. A fused amino acid sequence can be produced by genetic recombination of two encoding polynucleotide sequences and can be expressed by introducing a construct containing the recombinant polynucleotide into a host cell.

[0217] The terms "protein," "peptide," and "polypeptide" are used interchangeably herein and refer to polymers of amino acid residues linked by covalent bonds such as peptide bonds. Proteins or polypeptides as provided herein can comprise naturally occurring amino acid residues or non-natural amino acid residues, or both. Polypeptides, peptides, and proteins provided herein can comprise amino acid residues of any suitable length, for example, at least 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more amino acid residues.

[0218] As used herein, the term "naturally occurring" amino acid residue refers to an amino acid residue found in a naturally occurring protein or peptide in both D and L stereoisomeric forms if its structure allows such stereoisomeric forms. Examples of naturally occurring amino acid residues include, but are not limited to, the 20 standard amino acids, including glycine (Gly or G), alanine (Ala or A), valine (Val or V), leucine (Leu or L), isoleucine (Ile or I), serine (Ser or S), cysteine ​​(Cys or C), threonine (Thr or T), methionine (Met or M), proline (Pro or P), phenylalanine (Phe or F), tyrosine (Tyr or Y), tryptophan (Trp or W), histidine (His or H), lysine (Lys 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, pyrrolysine (PYL), selenocysteine, pyrroline-carboxy-lysine (PCL), sarcosine, β-alanine, phosphoserine, γ-carboxyglutamate, and ornithine. Examples of naturally occurring amino acid residues that are the D stereoisomer include, for example, D-aspartic acid, D-serine, D-cysteine, D-alanine, D-glutamic acid, and the like.

[0219] "Amino acid analogs" refers to compounds that have the same basic chemical structure as a naturally occurring amino acid (i.e., an alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but will retain the same basic chemical structure as a naturally occurring amino acid.

[0220] As used herein, "non-natural" amino acid residues refer to any amino acid residues not found in nature, including but not limited to modified amino acid residues and / or amino acid mimetics, which are not one of the known naturally occurring amino acids, but act in a manner similar to naturally occurring amino acids. Modified amino acid residues or mimetics can be produced by adding chemical entities (such as carbohydrate groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, and linkers for conjugation), functionalization or other modifications. Non-natural amino acids can also refer to amino acids manufactured by chemical synthesis. Exemplary non-natural amino acids include but are not limited to 2-aminoisobutyric acid (Aib), imidazole-4-acetic acid (IA), imidazole propionic acid (IPA), α-aminobutyric acid (Abu), tert-butylglycine (Tle), 3-aminomethylbenzoic acid, anthranilic acid, deaminohistidine (abbreviated as DesaminoHis, alias imidazole propionic acid, abbreviated as lmpr), amino acid β analogs such as β-alanine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, Nα -acetyl-histidine, α-fluoro-methyl-histidine, α-methyl-histidine, α,α-dimethyl-glutamate, 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.

[0221] The introduction of non-natural amino acids into fusion polypeptides, polypeptide fragments and / or polypeptide complexes can be achieved by the 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. Pat. No. 7,083,970. Briefly, some of these expression systems involve site-directed mutagenesis to introduce stop codons (e.g., amber (UAG), ochre (UAA), and opal (UGA) codons) into the open reading frame encoding the fusion polypeptides of the present disclosure. Other codons can also be introduced into the expression system for unnatural amino acids, such as four-base codons (e.g., AGGA, AGGU, CGGU, CGCU, CGAU, CCCU, CUCU, CUAU, and GGGU), five-base codons, six-base codons, etc. Such expression vectors are then introduced into a host that can utilize a tRNA specific for the introduced stop codon or other codon and that carries the selected unnatural amino acid.

[0222] GLP-1

[0223] As used herein, the term "glucagon-like peptide-1" or "GLP-1" is intended to broadly encompass native GLP-1 peptide and all functional forms thereof, such as functional variants, fragments, fusions, derivatives and mimetics thereof.

[0224] 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: 50. The residue numbering in GLP-1 is with reference to the sequence of SEQ ID NO: 50, which begins with His at position 7 and ends with Gly at position 37.

[0225] The functional form of the native GLP-1 peptide is capable of activating the GLP-1 receptor at a level comparable to, or not less than about 20% (or not less than 30%, 40%, 50%, 60%, 70%, 80%, 90%) of, the level of the native GLP-1 peptide. Activation of the GLP-1 receptor typically initiates a signal transduction pathway, resulting in an insulinotropic effect or other physiological effects as known in the art. Many functional forms of GLP-1 peptides are known in the art, such as, but not limited to, liraglutide, semaglutide, dulaglutide, albiglutide, and those disclosed in WO2000055203A1, WO 98 / 08871, WO 2006 / 097537, WO2007139589A1, WO1998019698A1, WO2001098331A2, WO2003040309A2, WO2005000892A2, WO2015000942A1, WO2016083499A1, the disclosures of which are incorporated herein in their entireties.

[0226] In certain embodiments, the GLP-1 provided herein comprises an amino acid sequence having at least 70% (e.g., at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) sequence identity to SEQ ID NO: 50 while retaining substantial biological activity of SEQ ID NO: 50.

[0227] In certain embodiments, the GLP-1 comprises no more than 9, 8, 7, 6, 5, 4, 3, or 2 substitutions relative to SEQ ID NO: 50 while retaining the substantial biological activity of SEQ ID NO: 50. In certain embodiments, the GLP-1 comprises at least 2, 3, 4, 5, 6, 7, 8, or 9 substitutions relative to SEQ ID NO: 50 while retaining the substantial biological activity of SEQ ID NO: 50.

[0228] In certain embodiments, GLP-1 further comprises one or more mutations. Those skilled in the art will appreciate that various amino acid substitutions, such as conservative amino acid substitutions, can be made in the sequence of any of the polypeptides described herein without necessarily reducing their activity. Examples of amino acid substitutions include replacing an L-amino acid with its corresponding D-amino acid, replacing cysteine ​​with homocysteine ​​or other unnatural amino acids with thiol-containing side chains, replacing lysine with homolysine, diaminobutyric acid, diaminopropionic acid, ornithine, or other unnatural amino acids with amino-containing side chains, or replacing alanine with norvaline, etc.

[0229] Various substitutions have been introduced into native GLP-1 peptides and have been shown to retain or even improve their biological activity. In certain embodiments, the GLP-1 comprises one or more mutations at positions selected from the group consisting of A8, G22, K34, R36, and H7 relative to SEQ ID NO: 50, and any combination thereof. For example, substitutions at A8 are believed to be useful for preventing DPP4 enzymatic cleavage at that residue, substitutions at G22 are desirable for improving activity and solubility, and substitutions at R36 are useful for reducing immunogenicity. Examples of substitutions at these positions include, but are not limited to, H7IA, H71IPA, A8G, A8S, A8V, A8Aib, A8T, A8I, A8L, G22E, K34R, R36G, or any combination thereof, as well as the substitutions described in U.S. Patent No. 8,273,854, which is incorporated herein in its entirety. In certain embodiments, one or more additional substitutions comprise conservative substitutions.

[0230] In certain embodiments, GLP-1 comprises a substitution at A8 selected from the group consisting of A8G, A8S, A8V, A8Aib, A8T, and A8L. In certain embodiments, GLP-1 comprises a substitution at G22E. In certain embodiments, GLP-1 comprises a substitution at R36G. In certain embodiments, GLP-1 comprises a substitution at H7 such as H7IA or H7IPA. In certain embodiments, GLP-1 comprises a substitution at K34 such as K34R.

[0231] In certain embodiments, GLP-1 comprises or consists of one or more substitutions selected from the group consisting of A8G, K26R, K34R, G22E, and R36G. In certain embodiments, GLP-1 comprises or consists of one or more substitutions selected from the group consisting of A8G, K34R, G22E, and R36G.

[0232] FGF21

[0233] As used herein, the term "fibroblast growth factor 21" or "FGF21" is intended to broadly encompass native human FGF21 and its functional variants, fragments, fusions, derivatives or mimetics. Native human FGF21 consists of 209 amino acid residues (Uniprot database, accession number Q9NSA1), of which amino acid residues 1-28 are signal polypeptides and amino acid residues 29-209 are mature polypeptides of 181 residues. The FGF21 provided in the present disclosure may be a mature polypeptide function or a functional variant, fragment, fusion, derivative or mimetics thereof. The mature polypeptide of FGF21 is included herein with SEQ ID NO: 36. As used herein, the residue numbering in FGF21 refers to the sequence of SEQ ID NO: 36, which starts with His at position 1 and ends with Ser at position 181.

[0234] The functional form of the mature polypeptide of FGF21 is capable of activating the FGF21 receptor at a level comparable to, or not less than about 20% (or not less than 30%, 40%, 50%, 60%, 70%, 80%, 90%) of the mature polypeptide of native human FGF21. Activation of the FGF21 receptor can result in biological activities, such as the ability to activate glucose uptake in adipocytes, the ability to lower blood sugar and triglyceride levels, or the ability to reduce body weight (Tezze C et al., Front Physiol. 2019, 10:419). Many functional forms of mature polypeptides of FGF21 are known in the art, such as, but not limited to, WO2019043457A2, WO2018088838A1, WO2018039081A1, WO2017220706A1, WO2017180988A2, WO2017116207A1, WO2017093465, WO2017059371A1, WO2016102562A1, WO2016065326A, WO2013173158A1, WO2013052311 A1, WO2013033452A2, WO2012066075A1, WO2012059873A2, WO2012010553A1, WO2011140086A2, WO2010084169A2, WO2010065439A1, WO2008121563A2, WO2006028595A2, WO2006028714A1, WO2005113606A2, WO2016102562A, the disclosures of which are incorporated herein in their entirety.

[0235] In certain embodiments, the FGF21 provided herein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 36 while retaining substantial biological activity of SEQ ID NO: 36.

[0236] In certain embodiments, FGF21 comprises no more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 substitutions relative to SEQ ID NO: 36 while retaining the substantial biological activity of SEQ ID NO: 36. In certain embodiments, FGF21 comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 substitutions relative to SEQ ID NO: 36 while retaining the substantial biological activity of SEQ ID NO: 36.

[0237] In certain embodiments, FGF21 comprises one or more mutations. In certain embodiments, the one or more mutations comprise conservative substitutions. In certain embodiments, the one or more mutations are at a position selected from positions 121, 168, 171, 180, and 181 relative to SEQ ID NO:36. In certain embodiments, the one or more mutations in FGF21 are selected from N121Q, M168L, P171G, A180E, and del181S. As used herein, "del181S" refers to the deletion of the serine at position 181 of FGF21.

[0238] connector

[0239] As used herein, the term "polypeptide linker" can be any suitable polypeptide that is capable of reacting with at least two entities to be connected (e.g., polypeptides) to connect the entities to form a molecule, or to maintain the entities tightly enough to maintain an associated state. The polypeptide linker can be integrated into the molecule or structure obtained by the connection. In certain embodiments, the polypeptide linker separates GLP-1 and FGF21 without substantially interfering with the respective biological activities of GLP-1 and FGF21. The polypeptide linker can be composed of amino acid residues linked together by peptide bonds. The polypeptide linker may further contain one or more non-natural amino acids.

[0240] In certain embodiments, the length of the polypeptide linker is at least 4, 8, 10, 20, 24, 28, 30, 40, 48, 50, 60, 70, 80, 90, 100, 110, 120 or more amino acid residues. In certain embodiments, the length of the polypeptide linker is at most 4, 8, 10, 20, 24, 28, 30, 40, 48, 50, 60, 70, 80, 90, 100, 110 or 120 amino acid residues. In certain embodiments, the length of the polypeptide linker is 0-120, 1-120, 4-120, 8-120, 10-120, 20-120, 20-110, 20-100, 20-90 or 20-80 amino acid residues. Without wishing to be bound by any theory, it is believed that the appropriate length of the polypeptide linker can further improve the biological activity or stability or pharmacokinetic parameters of the connected polypeptide molecule.

[0241] Any suitable polypeptide linker can be used. For example, a polypeptide linker can comprise or consist of amino acid residues selected from the group consisting of glycine (G), serine (S), alanine (A), methionine (M), asparagine (N), glutamine (Q), cysteine ​​(C), proline (P), glutamic acid (E), threonine (T), and lysine (K). In some embodiments, a polypeptide linker can be composed of a majority of non-sterically hindered amino acids, such as glycine and alanine.

[0242] In some embodiments, the linker is polyglycine, polyalanine, a combination of glycine and alanine (such as poly(Gly-Ala)), or a combination of glycine and serine (such as poly(Gly-Ser)).

[0243] In certain embodiments, the polypeptide linker comprises or consists of one or more repetitions of a repetitive sequence. In certain embodiments, the polypeptide linker comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 repetitions of a repetitive sequence, or within any numerical range limited by any two of the above listed numbers.

[0244] In certain embodiments, the repetitive sequence comprises or consists of no more than 4, 5, or 6 types of amino acid residues selected from the group consisting of G, Q, A, E, P, T, and S. In certain embodiments, the repetitive sequence comprises Q. In certain embodiments, the repetitive sequence consists of no more than 3, 4, or 5 types of amino acid residues selected from the group consisting of G, A, S, Q, and P. In certain embodiments, the repetitive sequence consists of G, A, Q, and P. In certain embodiments, the repetitive sequence consists of G, A, E, and P. In certain embodiments, the repetitive sequence consists of G, A, S, and P. In certain embodiments, the repetitive sequence does not comprise negatively charged amino acid residues.

[0245] In certain embodiments, the repeat sequence comprises or consists essentially of the amino acid residues G and S. In certain embodiments, the repeat sequence comprises or consists essentially of an amino acid sequence selected from the group consisting of: GS, GGSG, G a S b 、S a G b , wherein a and b are independently selected from integers from 1 to 5. In certain embodiments, the polypeptide linker comprises or consists essentially of: (GS)n, (GGSG)n, (G a S b )n、(S a G b )n, wherein a and b are independently selected from integers from 1 to 5, and n is selected from integers from 1 to 60.

[0246] In certain embodiments, the repeat sequence comprises or consists of an amino acid sequence selected from the group consisting of: G a S b, SEQ ID NO: 65 (GAQP), SEQ ID NO: 92 (GQEP), SEQ ID NO: 93 (GEQP), SEQ ID NO: 94 (GPQE), SEQ ID NO: 95 (GPEQ), SEQ ID NO: 96 (GSEP), SEQ ID NO: 97 (GESP), SEQ ID NO: 98 (GPSE), SEQ ID NO: 99 (GPES), SEQ ID NO:100(GQAP), SEQ ID NO:101(GPAQ), SEQ ID NO:102(GPQA), SEQ ID NO:103(GSQP), SEQ ID NO:104(GASP), SEQ ID NO:105(GPAS), SEQ ID NO:106(GPSA), SEQ ID NO:107(GGGS), SEQ ID NO:108(GSGS)、SEQ ID NO:57(GGGGS)、SEQ ID NO:143(GSAPGSPAGSPTGSAPGSPA) and GS, wherein a and b are independently an integer selected from 1 to 5.

[0247] In certain embodiments, the polypeptide linker comprises or consists of a sequence selected from the group consisting of SEQ ID NOs: 54-65, 109-113, 3, 47-49, and 141-147.

[0248] In certain embodiments, the polypeptide linker comprises or consists of more than one repeat sequence. For example, the polypeptide linker comprises or consists of 2, 3, or 4 different repeat sequences. In certain embodiments, the polypeptide linker comprises or consists of sequential or tandem repeats of different repeat sequences. The number of repeats of each repeat sequence can be independently selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more.

[0249] In certain embodiments, the fusion polypeptide comprises GLP-1, a polypeptide linker, and FGF21 from the N-terminus to the C-terminus. In certain embodiments, the fusion polypeptide is a single-chain polypeptide. A single-chain polypeptide may still have intrachain bonds within the molecule, but does not include complexes formed by one or more interchain bonds.

[0250] In certain embodiments, the fusion polypeptide consists essentially of GLP-1, a polypeptide linker, and FGF21 from N-terminus to C-terminus.

[0251] In certain embodiments, the Polypeptide Conjugate does not comprise an antibody fragment (e.g., Fc). Antibodies are immunoglobulin molecules that have the ability to specifically bind to a particular antigen. Antibodies contain two functionally independent parts, a variable domain called "Fab" that binds to the antigen, and a constant domain called "Fc," which comprises the non-antigen binding fragment and is associated with effector functions such as complement activation and attack by phagocytes. The Fc domain has a long serum half-life.

[0252] Conjugable residues

[0253] The fusion polypeptides provided herein further comprise at least one conjugable residue conjugated to a CRM.

[0254] As used herein, the term "conjugable residue" refers to an amino acid residue at a specific position within a fusion polypeptide that not only possesses a functional group capable of chemically or enzymatically conjugating to a chemical moiety, but also is positioned within the fusion polypeptide such that it readily undergoes such conjugation in a conjugation reaction of the specific functional group. As used herein, "conjugation" refers to a reaction that joins two molecules together to form a physical entity. For example, a covalent bond connecting two molecules can be formed in conjugation.

[0255] The skilled person will understand that whether a residue is a conjugable residue will depend on the given conjugation reaction and / or the functional group to be conjugated. If the functional group to be conjugated is an amino group and / or the conjugation reaction is specific or selective for amino groups, then a cysteine ​​residue (i.e., not having an amino functional group in its side chain) will not be a conjugable group, regardless of its position in the polypeptide, while a lysine residue at a specific position may be a conjugable group. Similarly, if the functional group to be conjugated is a thiol group and / or the conjugation reaction is specific or selective for thiol groups, then a lysine residue (i.e., not having a thiol functional group in its side chain) will not be a conjugable residue, regardless of its position, while an unpaired cysteine ​​residue that does not form a disulfide bond (whether intrachain or interchain) may be a conjugable residue.

[0256] In certain embodiments, the fusion polypeptide comprises a conjugated residue that has a conjugation reaction that is specific or selective for amino groups or amines (e.g., an acylation reaction). In certain embodiments, the fusion polypeptide comprises a conjugated residue that has a conjugation reaction that is specific or selective for thiol groups (e.g., a maleimide reaction or a reaction for disulfide bond formation).

[0257] When referring to an amino acid residue, the expression "conjugable" is intended to mean that the residue at a particular position in the fusion polypeptide is sufficiently accessible for conjugation. For example, a cysteine ​​residue that is part of a disulfide bridge is not a conjugable residue in the present disclosure.

[0258] The conjugated residue can be a natural amino acid residue, a non-natural amino acid residue, a modified amino acid residue or an amino acid mimetic. Examples of conjugated residues include, but are not limited to, lysine, cysteine ​​or a non-natural amino acid residue.

[0259] The fusion polypeptides provided herein comprise at least one conjugated residue conjugated to a CRM. In certain embodiments, the fusion polypeptide comprises at most one conjugated residue. In certain embodiments, the fusion polypeptide comprises at most two conjugated residues.

[0260] In certain embodiments, the fusion polypeptide comprises a first conjugable residue conjugated to a CRM. In certain embodiments, the first conjugable residue is in FGF21, in a polypeptide linker, or in GLP-1. In certain embodiments, the first conjugable residue is in FGF21.

[0261] In certain embodiments, the fusion polypeptide comprises a first conjugable residue and a second conjugable residue. In certain embodiments, the first and second conjugable residues are both present in the same constituent component of the fusion polypeptide. For example, the first and second conjugable residues are both present in FGF21, in a polypeptide linker, or in GLP-1. In certain embodiments, the first and second conjugable residues are present in different constituent components of the fusion polypeptide. For example, the first conjugable residue is present in FGF21 and the second conjugable residue is present in a polypeptide linker, the first conjugable residue is present in GLP-1 and the second conjugable residue is present in FGF21, or the first conjugable residue is present in GLP-1 and the second conjugable residue is present in a polypeptide linker. As used herein, the terms "first" and "second" are used only to designate one of the two conjugable residues and do not in any way imply that conjugation is performed in any order between the first and second conjugable residues.

[0262] In certain embodiments, the conjugable residue may be a naturally occurring residue found in the native FGF21 sequence, or a naturally occurring residue found in the native GLP-1 sequence. In certain embodiments, such a naturally occurring residue is a naturally occurring lysine residue.

[0263] In certain embodiments, when native GLP-1 or native FGF21 has more than one naturally occurring conjugable residue, one or more of such naturally occurring conjugable residues can be removed, for example, by mutation. For example, native GLP-1 has two naturally occurring lysine residues and native FGF21 has four naturally occurring lysine residues, at least some of which, or all of which, can be mutated without significantly reducing the biological activity of the fusion polypeptide.

[0264] In certain embodiments, a conjugable residue may alternatively be an introduced residue, for example, by substituting a non-conjugable residue for a conjugable residue, or by inserting a conjugable residue. The term "non-conjugable residue" refers to an amino acid residue in a fusion polypeptide that is not a conjugable residue for a given functional group and / or a given conjugation reaction. For example, when lysine is a conjugable residue, any amino acid residue may be considered a non-conjugable residue if it does not contain an amino group and / or does not react in a conjugation reaction that is specific or selective for an amino group.

[0265] In certain embodiments, such introduced conjugable residues are lysine residues. In certain embodiments, such introduced conjugable residues are cysteine ​​residues.

[0266] In certain embodiments, the first and second conjugated residues have the same chemical identity. For example, the first and second conjugated residues are both lysine, both cysteine, or both unnatural amino acid residues.

[0267] Conjugatable residues in FGF21

[0268] In certain embodiments, FGF21 comprises a first conjugable residue. In certain embodiments, FGF21 comprises no conjugable residues or comprises at most one conjugable residue.

[0269] In certain embodiments, the conjugated residue in FGF21 is an introduced residue, optionally by substitution or insertion. Such introduced conjugated residues can be introduced at a position that does not significantly reduce the biological activity of FGF21. In certain embodiments, the introduced conjugated residue in FGF21 is in the C-terminal region of FGF21, for example, at a position no more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue away from the most C-terminal residue, or at a position relative to the most C-terminal residue in SEQ ID NO:36, or at a position extending from the most C-terminal residue in SEQ ID NO:36. The C-terminal extension of FGF21 can be described with reference to SEQ ID NO:36 by numbering from residue 181, which is the most C-terminal residue in SEQ ID NO:36. Position 182 relative to SEQ ID NO:36 refers to the first residue extending from residue 181 in SEQ ID NO:36, i.e., from the C-terminus. Similarly, position 183 refers to the second residue extending from the C-terminus, and so on. For example, "ins182K" relative to SEQ ID NO: 36 refers to the lysine residue attached or inserted at position 182 relative to SEQ ID NO: 36. In certain embodiments, the conjugated residue introduced into FGF21 is selected from positions 170, 171, 172, 173, 174, 180, 181, 71, and 182 relative to SEQ ID NO: 36.

[0270] In certain embodiments, the conjugated residue in FGF21 comprises or consists of lysine. In certain embodiments, the conjugated residue in FGF21 comprises a substitution selected from the group consisting of G170K, P171K, S172K, Q173K, G174K, A180K, S181K, S71K, and ins182K relative to SEQ ID NO: 36.

[0271] In certain embodiments, naturally occurring lysine residues in FGF21 have been removed, for example, by substitution of naturally occurring lysine residues with non-conjugatable residues or deletion of naturally occurring lysine residues, or any other means that prevents the side chains of naturally occurring lysine residues from reacting in a given conjugation reaction.

[0272] In certain embodiments, FGF21 comprises one or more mutations at positions selected from K56, K59, K69, and K122 relative to SEQ ID NO: 36, wherein the mutation is a mutation to a non-conjugable residue. In certain embodiments, the non-conjugable residue is selected from the group consisting of arginine (R), glutamine (Q), alanine (A), glycine (G), histidine (H), serine (S), and threonine (T). In certain embodiments, FGF21 comprises a substitution of K56 selected from the group consisting of K56R, K56Q, K56A, K56G, K56H, K56S, and K56T. In certain embodiments, FGF21 comprises or further comprises a substitution of K59 selected from the group consisting of K59R, K59Q, K59A, K59G, K59H, K59S, and K59T. In certain embodiments, FGF21 comprises or further comprises a substitution of K69 selected from the group consisting of K69R, K69Q, K69A, K69G, K69H, K69S, and K69T. In certain embodiments, FGF21 comprises or further comprises a substitution of K122 selected from the group consisting of K122R, K122Q, K122A, K122G, K122H, K122S, and K122T. In certain embodiments, FGF21 comprises mutations of K56R, K59R, K69R, and K122R.

[0273] In certain embodiments, FGF21 comprises or consists of a combination of mutations relative to SEQ ID NO: 36 selected from the group consisting of:

[0274] 1) N121Q, M168L, A180K, P171G, K56R, K59R, K69R, and K122R;

[0275] 2) N121Q, M168L, P171K, K56R, K59R, K69R, and K122R;

[0276] 3) N121Q, M168L, G174K, P171G, K56R, K59R, K69R, and K122R;

[0277] 4) N121Q, M168L, P171K, K56R, K59R, K69R, K122R, and A180E;

[0278] 5) N121Q, M168L, G174K, P171G, K56R, K59R, K69R, K122R, and A180E;

[0279] 6) N121Q, M168L, A180K, K56R, K59R, K69R, and K122R;

[0280] 7) N121Q, M168L, A180K, P171G, K56R, K59R, K69R, K122R, and del181S;

[0281] 8) N121Q, M168L, G170K, K56R, K59R, K69R, and K122R;

[0282] 9) N121Q, M168L, G170K, K56R, K59R, K69R, K122R, and A180E;

[0283] 10) N121Q, M168L, G170K, P171G, K56R, K59R, K69R, K122R, and A180E;

[0284] 11) N121Q, M168L, A180E, P171G, K56R, K59R, K69R, K122R, and S181K;

[0285] 12) N121Q, M168L, A180E, P171G, K56R, K59R, K69R, K122R, and ins182K;

[0286] 13) 71K, 121Q, 168L, 56R, 59R, 69R and 122R;

[0287] 14) 71K, 121Q, 168L, 171G, 56R, 59R, 69R, and 122R; and

[0288] 15)71K, 121Q, 168L, 171G, 56R, 59R, 69R, 122R, 180E.

[0289] In certain embodiments, FGF21 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-46 and 118-119.

[0290] In certain embodiments, the conjugatable residue in FGF21 comprises or consists of cysteine.

[0291] Without wishing to be bound by any theory, it is believed that the cysteine ​​residues at positions 75 and 93 in FGF21 relative to SEQ ID NO: 36 are non-conjugable residues. The two cysteine ​​residues form an intramolecular disulfide bond in mature FGF21 and are therefore inaccessible to the conjugating agent in the conjugation reaction. Therefore, FGF21, which only has cysteine ​​residues at positions 75 and 93, is considered to contain no conjugable residues for thiol conjugation reactions.

[0292] In certain embodiments, the conjugable residue in FGF21 comprises a substitution relative to SEQ ID NO: 36 selected from the group consisting of G170C, P171C, S172C, Q173C, G174C, A180C, S181C, S71C, and ins182C.

[0293] In certain embodiments, the conjugable residues in FGF21 comprise or consist of non-natural amino acids. Non-natural amino acids can contain a variety of functional or reactive groups that provide additional functionality and / or reactivity. Particular non-natural amino acids useful for conjugating moieties to the fusion polypeptides of the present disclosure include those with side chains containing azide, alkyne, alkene, cycloalkyne, or halide groups.

[0294] In certain embodiments, FGF21 comprises or consists of a combination of mutations relative to SEQ ID NO: 36 selected from the group consisting of:

[0295] 1)121Q, 168L, 171C, 180E;

[0296] 2)121Q, 168L, 171G, 174C, 180E;

[0297] 3)121Q, 168L, 171G, 180C;

[0298] 4)121Q, 168L, 171G, 180E;

[0299] 5) S71C, 121Q, 168L, 171G, 180E; and

[0300] 6)121Q, 168L, 180E, 171G, ins 182C.

[0301] In certain embodiments, FGF21 does not comprise a conjugatable residue. In certain embodiments, FGF21 comprises mutations 121Q, 168L, 171G, and 180E relative to SEQ ID NO: 36.

[0302] In certain embodiments, FGF21 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 136-140 and 171.

[0303] Conjugable residues in GLP-1

[0304] In certain embodiments, GLP-1 comprises no conjugable residue or comprises at most one conjugable residue.In certain embodiments, FGF21 comprises a first conjugable residue and GLP-1 comprises a second conjugable residue.

[0305] In certain embodiments, the conjugable residue in GLP-1 is a naturally occurring amino acid residue. In certain embodiments, the conjugable residue in GLP-1 comprises or consists of lysine.

[0306] The native GLP-1 peptide as set forth in SEQ ID NO: 50 has two naturally occurring lysine (K) residues, K26 and K34. In certain embodiments, the conjugable residue in GLP-1 is a naturally occurring residue found in the native GLP-1 peptide, such as K26 or K34.

[0307] In certain embodiments, the conjugable residue in GLP-1 comprises an introduced residue, for example, by substituting a non-conjugable residue for a conjugable residue, or by inserting a conjugable residue (e.g., at the N-terminus or C-terminus of GLP-1 or within the GLP-1 sequence). Any non-conjugable residue in GLP-1 can be substituted with a conjugable residue, as long as such substitution does not substantially reduce the biological activity of GLP-1 or the fusion polypeptide.

[0308] In certain embodiments, the conjugable residues in GLP-1 are introduced by substitutions at E27 and R36 relative to the GLP-1 of SEQ ID NO: 50. In certain embodiments, the conjugable residues in GLP-1 are introduced by substitutions selected from the group consisting of E27K and R36K. In such embodiments, the GLP-1 further comprises substitutions of K26 and / or K34 to non-conjugable residues.

[0309] In certain embodiments, one or both of the naturally occurring lysine residues in GLP-1 have been removed, for example, by substitution of a non-conjugatable residue for the naturally occurring lysine residue or deletion of the naturally occurring lysine residue, or any other means that prevents the side chain of the naturally occurring lysine residue from reacting in a given conjugation reaction.

[0310] In certain embodiments, GLP-1 further comprises substitution of K26 and / or K34 to a non-conjugable residue. In certain embodiments, for lysine conjugation reactions, the non-conjugable residue is optionally selected from the group consisting of arginine (R), glutamine (Q), alanine (A), glycine (G), histidine (H), serine (S), and threonine (T).

[0311] In certain embodiments, the GLP-1 comprises a substitution at K26 selected from the group consisting of K26R, K26Q, K26A, K26G, K26H, K26S, and K26T. In certain embodiments, the GLP-1 comprises or further comprises a substitution at K34 selected from the group consisting of K34R, K34Q, K34A, K34G, K34H, K34S, and K34T. In certain embodiments, the substitution at K26 is selected from the group consisting of K26R and K26Q, and / or the substitution at K34 is selected from the group consisting of K34R and K34Q. In certain embodiments, the GLP-1 comprises K26R and / or K34R relative to SEQ ID NO: 50.

[0312] In certain embodiments, the conjugatable residue in GLP-1 comprises a cysteine ​​residue.

[0313] In certain embodiments, the conjugable residue in GLP-1 is a non-natural amino acid residue (NNAA).

[0314] In certain embodiments, the cysteine ​​residue or NNAA in GLP-1 is introduced by substitution at a position selected from the group consisting of K26, K34, E27, and R36 relative to SEQ ID NO: 50. In certain embodiments, the cysteine ​​residue or NNAA in GLP-1 is introduced by substitution at a position selected from the group consisting of K26, E27, and R36 relative to SEQ ID NO: 50.

[0315] In certain embodiments, GLP-1 comprises the amino acid sequence X7X8EGTFTSDVSSYLEX 22 QAAX 26 X 27 FIAWLVX 34 GX 36 G (SEQ ID NO: 51), wherein: X7 is H, IA or IPA; X8 is A, G, S, V, Aib, T, I or L; X 22 G or E; X 26 K, R or C; X 27 E, K or C; X 34 is R, K or C, and X 36 is K, R, G or C, with the proviso that GLP-1 has at most one lysine residue (K) or at most one cysteine ​​(C) in the amino acid sequence.

[0316] In certain embodiments, X7 is H, X8 is G, X 22 E; X 26 K, R or C; X 27 E, K or C; X 34 is R, K or C, and X36 is K, R, C or G, provided that GLP-1 has at most one lysine residue (K) or at most one cysteine ​​residue (C) in the amino acid sequence.

[0317] In certain embodiments, X7 is H, X8 is G, X 22 E; X 26 K or R; X 27 E; X 34 is R, and X 36 is K or G, with the proviso that GLP-1 has at most one lysine residue (K) in the amino acid sequence.

[0318] In certain embodiments, the GLP-1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 52, 53, and 115.

[0319] In certain embodiments, the GLP-1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 120 or 52.

[0320] In certain embodiments, the GLP-1 comprises no conjugatable residues. In certain embodiments, the GLP-1 comprises no lysine, no cysteine, and / or no unnatural amino acid residues.

[0321] Conjugable residues in the linker

[0322] In certain embodiments, the polypeptide linker comprises at most one or at most two conjugable residues, and FGF21 or GLP-1 comprises no conjugable residues. In certain embodiments, FGF21 comprises a first conjugable residue, and the polypeptide linker comprises a second conjugable residue.

[0323] A conjugated residue can be introduced into the linker sequence by, for example, substitution or insertion. In certain embodiments, a non-conjugated amino acid residue in a polypeptide linker is replaced with a conjugated residue. In certain embodiments, a conjugated residue is inserted into a polypeptide linker. For example, a second conjugated residue can be inserted within a repeat of a repetitive sequence, between two repeats of a repetitive sequence, or at the N-terminus or C-terminus of a polypeptide linker.

[0324] In certain embodiments, a conjugated residue is introduced into a polypeptide linker at any suitable position. In certain embodiments, a conjugated residue is introduced into a polypeptide linker at a position that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70 residues (including conjugated residues) away from the most N-terminal residue of FGF21. In certain embodiments, a conjugated residue is introduced into the polypeptide linker at a position that is at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70 residues (including conjugated residues) from the N-terminal residue of FGF21. In certain embodiments, a conjugated residue is introduced into the polypeptide linker at the position of the C-terminal residue of the polypeptide linker. In certain embodiments, a conjugable residue is introduced into the polypeptide linker at a position that is at least 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 residues (including conjugable residues) away from the most C-terminal residue of GLP-1. In certain embodiments, a conjugable residue is introduced into the polypeptide linker at a position that is at most 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 residues (including conjugable residues) away from the most C-terminal residue of GLP-1.

[0325] In one embodiment, the polypeptide linker comprises a substitution with a conjugated residue that is a lysine. Alternatively, the polypeptide linker comprises a substitution with a conjugated residue that is a cysteine ​​or non-natural amino acid residue. In one embodiment, the conjugated residue in the polypeptide linker is a lysine residue introduced by substitution at the position of the most C-terminal residue of the polypeptide linker.

[0326] In certain embodiments, the conjugated residue in the polypeptide linker is lysine, and the polypeptide linker comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-75 and 89-91. Alternatively, the lysine residue in each of these exemplary polypeptide linkers (i.e., SEQ ID NOs: 66-75 and 89-91) can be substituted with cysteine ​​or a non-natural amino acid residue.

[0327] In certain embodiments, the conjugable residue in the polypeptide linker is cysteine, and the polypeptide linker comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 121-134. In certain embodiments, the polypeptide linker comprises two conjugable residues, and the polypeptide linker comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 133 or 134. Alternatively, the cysteine ​​residue in each of these exemplary polypeptide linkers (i.e., SEQ ID NOs: 121-134) can be substituted with lysine or a non-natural amino acid residue.

[0328] In certain embodiments, the fusion polypeptide comprises at most one conjugable residue. In some embodiments, the single conjugable residue is selected from positions 170, 171, 172, 173, 174, 180, 181, 71, and 182 of FGF21 relative to SEQ ID NO: 36. In some embodiments, the single conjugable residue is in the polypeptide linker, for example, at a position that is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 residues (including the conjugable residue) away from the most C-terminal residue of GLP-1.

[0329] In certain embodiments, the fusion polypeptide comprises at most two conjugable residues. In such embodiments, the first conjugable residue is at a position selected from positions 170, 171, 172, 173, 174, 180, 181, 71, and 182 in FGF21 relative to SEQ ID NO: 36, and the second conjugable residue is at the position of the most C-terminal residue of the polypeptide linker in the polypeptide linker, or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 residues (including the conjugable residue) away from the most C-terminal residue of GLP-1, or at a position selected from positions 26, 34, 27, and 36 in GLP-1 relative to SEQ ID NO: 50. In some embodiments, the first conjugable residue is in a polypeptide linker and is at the C-terminus of the polypeptide linker, and the second conjugable residue is also in a polypeptide linker at a position that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70 residues (including conjugable residues) away from the N-terminal residue of FGF21. In some embodiments, the first conjugable residue is in a polypeptide linker and is at the C-terminus of the polypeptide linker, and the second conjugable residue is also in a polypeptide linker at a position that is at or near the center of the polypeptide linker. In some embodiments, the first and second conjugable residues are both in the polypeptide linker and are independently at a position that is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 residues (including the conjugable residue) away from the most C-terminal residue of GLP-1.

[0330] In certain embodiments, the fusion polypeptides provided herein comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4-22, 24, 26-35, 77-88, and 116-117, wherein the conjugated residue is lysine. Table 1A below shows exemplary fusion polypeptide sequences by SEQ ID NO, as well as the SEQ ID NOs of the FGF21, polypeptide linker, and GLP-1 sequences contained in the fusion polypeptide. Table 1A below also shows mutations in GLP-1 and FGF21, as well as the sequence and number of repeats in the polypeptide linker sequence.

[0331] Table 1A. Exemplary fusion polypeptide sequences

[0332]

[0333]

[0334]

[0335]

[0336] **: Mutations in GLP-1 refer to mutations relative to SEQ ID NO: 50;

[0337] # : Mutation in FGF21 means mutation relative to SEQ ID NO: 36;

[0338] In certain embodiments, the fusion polypeptides provided herein comprise an amino acid sequence selected from the group consisting of 148-165, 168-170, 1, 23, 25, and 76, wherein the conjugable residue is cysteine. Table 1B below shows exemplary fusion polypeptide sequences by SEQ ID NO, as well as the SEQ ID NOs of the FGF21, polypeptide linker, and GLP-1 sequences contained in the fusion polypeptide. Table 1B below also shows mutations in GLP-1 and FGF21, as well as the sequence and number of repeats in the polypeptide linker sequence.

[0339] Table 1B. Exemplary fusion polypeptide sequences

[0340]

[0341]

[0342]

[0343] **: Mutations in GLP-1 refer to mutations relative to SEQ ID NO: 50;

[0344] # : Mutation in FGF21 means mutation relative to SEQ ID NO: 36;

[0345] § The fusion polypeptides having the amino acid sequences of SEQ ID NOs: 166 and 167 contained inactive GLP-1 or inactive FGF21 and served as negative controls.

[0346] Clearance Modifier

[0347] The present disclosure provides a Polypeptide Conjugate comprising a fusion polypeptide provided herein conjugated to at least one clearance reducing moiety (CRM).

[0348] As used herein, the term "conjugate" refers to a compound that is the result of two or more molecules being joined together to form a physical entity. For example, a polypeptide conjugate of the present disclosure refers to a compound that is the result of a fusion polypeptide joined together with one or more clearance-modifying moieties. The molecules can be linked together by covalent bonds, non-covalent bonds, linkers, chemical modifications, or protein fusions, or by any means known to those skilled in the art. Preferably, the molecules are linked together by covalent bonds. The connection can be permanent or reversible. In some embodiments, certain cleavable or non-cleavable linkers may be included.

[0349] As used herein, the term "clearance modifying moiety" or "CRM" refers to a moiety that can alter one or more pharmacokinetic (PK) properties (e.g., extend in vivo half-life). Examples of CRMs can include, but are not limited to, polyethylene glycol (PEG), glucuronic acid, or other sugar-based linkers, polar, positively or negatively charged groups that can increase the hydrolysis rate of the succinimidyl ring and reduce or minimize the rate of the reverse Michael reaction, and thus reduce or minimize the rate of loss of the fusion polypeptide.

[0350] In certain embodiments, the CRM comprises a plasma protein binding portion, a polymer, human serum albumin (HSA) and functional fragments thereof, an Xten sequence or a PAS sequence. In certain embodiments, the Xten sequence is an extended recombinant polypeptide sequence having an amino acid sequence described in WO2007103515, WO2009023270, WO2010091122, WO2011123813, WO2013130683, WO2017146979, WO2011084808, WO2013040093, WO2013122617, WO2014011819, WO2013184216, WO2014164568, WO2015023891, WO2016077505 and WO2017040344, the disclosures of which are incorporated herein by reference in their entirety. In certain embodiments, the term "PAS" (also used interchangeably with the term "APS") refers to amino acid repeats consisting of Ala, Ser, and Pro residues, as described in US Pat. No. 8,563,521 B2, the disclosure of which is incorporated herein in its entirety.

[0351] In certain embodiments, the CRM comprises an albumin binding moiety. The term "albumin binding moiety" refers to any functional moiety that is capable of binding to albumin (e.g., human serum albumin) or any functional fragment thereof with sufficient specificity, preferably non-covalently. The albumin binding moiety attached to the therapeutic fusion polypeptide, polypeptide, or polypeptide complex typically has an affinity for human serum albumin of less than 10 μM, and preferably less than 1 pM. The albumin binding moiety may include, but is not limited to, an albumin binding domain, an albumin binding sequence from a synthetic peptide, and an albumin binding chemical moiety. For example, the albumin binding moiety is selected from the albumin binding domain from Streptococcus protein G, the albumin binding domain from the Peptostreptococcus magnus protein PAB, and an albumin binding peptide having the core sequence DICLPRWGCLW (SEQ ID NO: 114). Many small peptides that serve as albumin binding moieties have been described in J. Biol Chem., 277, 38 (2002), 35035-35043. For another example, the albumin binding moiety is selected from a linear and branched lipophilic moiety containing 4-40 carbon atoms, a compound having a cyclopentaphenanthrene skeleton, etc. For example, the albumin binding moiety is of the formula CH3(CH2) v CO-NHCH(COOH)(CH2)2CO-, wherein v is an integer from 10 to 24.

[0352] In certain embodiments, the albumin binding moiety comprises the following structure: *-ABCDE, wherein A, B, C, D and E are interconnected by amide bonds and the * end of A is linked to a conjugable residue on the polypeptide complex, and wherein:

[0353] A is selected from the group consisting of a bond, a, b, c and d are independently integers from 0 to 4, R 1 is hydrogen or -COOH;

[0354] B is selected from the group consisting of a bond, e is an integer from 1 to 4,

[0355] C is a bond or R 2 is -CH2SO3H or -COOH, f is an integer from 1 to 4, and n is an integer from 1 to 25;

[0356] D is selected from the group consisting of a bond, g and h are independently 0 or 1, and R 3 is H or -CH2COOH;

[0357] E is an acidic group having the formula:

[0358]

[0359] Where W represents -(CR 4 R 5 ) l -,

[0360] R 4 and R 5 independently selected from the group consisting of hydrogen, halogen, cyano, hydroxy, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, hydroxyalkyl, amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy, and carboxamide,

[0361] R 6 Selected from hydroxyl or NR 7 R 8 ;

[0362] R 7 and R 8 Independently selected from the group consisting of hydrogen, alkyl, hydroxyl and And l is an integer from 10 to 20.

[0363] In certain embodiments, the CRM is conjugated to a lysine residue, optionally in a polypeptide linker or in GLP-1 or in FGF21.

[0364] In certain embodiments, A is a bond.

[0365] In certain embodiments, A is a bond, and B is a bond or where e is 1, 2, or 3.

[0366] In certain embodiments, A is a bond and B is And C is wherein e is 1, 2, or 3, f is 1, 2, or 3, and n is 1 or 2. In certain embodiments, D is a bond, and E is an acidic group having the formula: In certain embodiments, R 2 is -COOH, and R 6 In certain embodiments, W represents -(CR 4 R 5 ) l -, R 4 and R 5 are independently hydrogen, and l is an integer from 10 to 20.

[0367] In certain embodiments, A is a bond, B is a bond, and C is a bond.

[0368] In certain embodiments, A is a bond, B is a bond, and C is Wherein f is 1, 2 or 3, and n is 1 or 2.

[0369] In certain embodiments, A is wherein a is 1, 2, or 3, b is 1, 2, or 3, and c is 1 or 2.

[0370] In certain embodiments, A is And B is wherein a is 1, 2, or 3, b is 1, 2, or 3, c is 1 or 2, and e is 1, 2, or 3.

[0371] In certain embodiments, A is B is And C is wherein a is 1, 2, or 3, b is 1, 2, or 3, c is 1, 2, or 3, e is 1, 2, or 3, f is 1, 2, or 3, and n is 1 or 2.

[0372] In certain embodiments, A is B is and C is a bond, wherein a is 1, 2, or 3, b is 1, 2, or 3, c is 1 or 2, and e is 1, 2, or 3.

[0373] In certain embodiments, A is and B is a bond, wherein a is 1, 2, or 3, b is 1, 2, or 3, and c is 1 or 2.

[0374] In certain embodiments, A is B is a bond, and C is wherein a is 1, 2, or 3, b is 1, 2, or 3, c is 1 or 2, f is 1, 2, or 3, and n is 1 or 2.

[0375] In certain embodiments, A is B is a bond, and C is a bond, wherein a is 1, 2, or 3, b is 1, 2, or 3, and c is 1 or 2.

[0376] In certain embodiments, D is a bond.

[0377] In certain embodiments, A is B is C is and D is a bond, wherein a is 1, 2, or 3, b is 1, 2, or 3, c is 1 or 2, e is 1, 2, or 3, f is 1, 2, or 3, and n is 1 or 2.

[0378] In certain embodiments, D is Where g is 0 or 1, and h is 0 or 1.

[0379] In certain embodiments, A is B is or bond, C is a bond, and D is wherein a is 1, 2, or 3, b is 1, 2, or 3, c is 1 or 2, e is 1, 2, or 3, g is 0 or 1, and h is 0 or 1.

[0380] In certain embodiments, D is

[0381] In certain embodiments, A is B is C is a bond or And D is wherein a is 1, 2, or 3, b is 1, 2, or 3, c is 1, 2, or 3, e is 1, 2, or 3, f is 1, 2, or 3, and n is 1 or 2.

[0382] In certain embodiments, the CRM comprises the following structure (also referred to as -OOC-(CH2)16-CO-gGlu-2XADO, where 2XADO refers to two consecutive ADO moieties and ADO is an abbreviation for 8-amino-3,6-dioxaoctanoic acid):

[0383]

[0384] In certain embodiments, the CRM is conjugated to at least one cysteine ​​residue in the fusion polypeptide.

[0385] In certain embodiments, A is And B is

[0386] In certain embodiments, A is B is And C is wherein a is 1, 2, or 3, b is 1, 2, or 3, c is 1 or 2, d is 1, 2, or 3, f is 1, 2, or 3, and n is 1 or 2. In certain embodiments, D is a bond, and E is an acidic group having the formula: In certain embodiments, R 2 is -COOH, and R 6 In certain embodiments, W represents -(CR 4 R 5 ) l -, R 4 and R 5 are independently hydrogen, and l is an integer from 10 to 20.

[0387] In certain embodiments, A is B is C is and D is a bond, wherein a is 1, 2, or 3, b is 1, 2, or 3, c is 1 or 2, d is 1, 2, or 3, f is 1, 2, or 3, and n is 1 or 2.

[0388] In certain embodiments, A is And B is wherein d is 1, 2, or 3, and e is 1, 2, or 3.

[0389] In certain embodiments, A is B is and C is a bond, wherein d is 1, 2, or 3, and e is 1, 2, or 3.

[0390] In certain embodiments, A is B is C is a bond, and D is wherein d is 1, 2, or 3, e is 1, 2, or 3, g is 0 or 1, and h is 0 or 1.

[0391] In certain embodiments, the CRM comprises the following structure:

[0392]

[0393]

[0394] Exemplary fusion polypeptide conjugates via lysine residues are shown in Table 2A and Table 2B below.

[0395] Table 2A Exemplary Fusion Polypeptide Conjugates

[0396]

[0397]

[0398]

[0399] **A group refers to HOOC-(CH2)16-CO-gGlu-2XADO or

[0400] Table 2B Exemplary Fusion Polypeptide Conjugates

[0401]

[0402]

[0403] **B group refers to HOOC-(CH2)20-CO-gGlu-2XADO-EDA-CO-CH2 or

[0404] § Fusion polypeptides having the amino acid sequences of SEQ ID NOs: 166 and 167, comprising inactive GLP-1 or inactive FGF21 served as negative controls.

[0405] In certain embodiments, the conjugates provided herein comprise a fusion polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 6-15, and a CRM is linked to a conjugated lysine residue in the fusion polypeptide. Alternatively, the conjugated lysine residue in the fusion polypeptide selected from SEQ ID NOs: 6-15 can be substituted with cysteine ​​or a non-natural amino acid, and the CRM is conjugated to such cysteine ​​or non-natural amino acid.

[0406] In certain embodiments, the conjugates provided herein comprise a fusion polypeptide comprising an amino acid sequence selected from SEQ ID NO: 25, and one CRM is linked to a conjugatable cysteine ​​residue in the fusion polypeptide.

[0407] In certain embodiments, the conjugates provided herein comprise a fusion polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4-5, 16-22, 24, 26-35, 77-88, and 116-117, and two CRMs, each linked to a conjugated lysine residue in the fusion polypeptide. Alternatively, the conjugated lysine residue in the fusion polypeptide selected from the group consisting of SEQ ID NOs: 2, 4-5, 16-22, 24, 26-35, 77-88, and 116-117 can be substituted with cysteine ​​or a non-natural amino acid, and the two CRMs are conjugated to such cysteine ​​or non-natural amino acid residues.

[0408] In certain embodiments, the conjugates provided herein comprise a fusion polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4-5, 16-22, 24, 26-35, 77-88, and 116-117, and two CRMs, each of which is linked to one of the conjugated lysine residues in the fusion polypeptide. In certain embodiments, the first CRM is conjugated to a conjugated lysine residue in FGF21, and the second CRM is conjugated to a conjugated lysine residue in GLP-1 or a polypeptide linker.

[0409] In certain embodiments, the conjugates provided herein comprise a fusion polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 148-165, 168-170, 1, 23, and 76, and two CRMs, each linked to a conjugated cysteine ​​residue in the fusion polypeptide. In certain embodiments, the first CRM is conjugated to a conjugated cysteine ​​residue in FGF21, and the second CRM is conjugated to a conjugated cysteine ​​residue in the polypeptide linker. In certain embodiments, both the first and second CRMs are conjugated to a conjugated cysteine ​​residue in the polypeptide linker.

[0410] In certain embodiments, the present disclosure provides a Polypeptide Conjugate comprising a fusion polypeptide conjugated to a CRM or a pharmaceutically acceptable salt thereof, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: N, and the CRM covalently linked to the mth residue (which is lysine, i.e., mK) in the fusion polypeptide, counting from the N-terminus to the C-terminus, wherein:

[0411] 1) N is 6, m is 222, and mK is 222K (this type of conjugate molecule is also referred to as compound 6),

[0412] 2) N is 7, m is 221, and mK is 221K (this type of conjugate molecule is also referred to as compound 7),

[0413] 3) N is 8, m is 225, and mK is 225K (this type of conjugate molecule is also referred to as compound 8),

[0414] 4) N ​​is 9, m is 231, and mK is 231K (this type of conjugate molecule is also referred to as compound 9),

[0415] 5) N is 10, m is 222, and mK is 222K (this type of conjugate molecule is also referred to as compound 10),

[0416] 6) N is 11, m is 221, and mK is 221K (this type of conjugate molecule is also referred to as compound 11),

[0417] 7) N is 12, m is 225, and mK is 225K (this type of conjugate molecule is also referred to as compound 12),

[0418] 8) N is 13, m is 222, and mK is 222K (this type of conjugate molecule is also referred to as compound 13),

[0419] 9) N is 14, m is 222, and mK is 222K (such conjugate molecules are also referred to as compound 14), or

[0420] 10) N is 15, m is 231, and mK is 231K (such conjugate molecules are also referred to as compound 15), and

[0421] The CRM has the following structure:

[0422]

[0423] In certain embodiments, the present disclosure provides a polypeptide conjugate comprising a fusion polypeptide conjugated to a CRM or a pharmaceutically acceptable salt thereof,

[0424] wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: N, and a CRM covalently linked to the mth residue (which is cysteine, i.e., mC) in the fusion polypeptide, counting from the N-terminus to the C-terminus, and wherein:

[0425] 1) N is 25, m is 51, and mC is 51C (this type of conjugate molecule is also referred to as compound 76),

[0426] The CRM has the following structure:

[0427]

[0428] or a pharmaceutically acceptable salt thereof.

[0429] In certain embodiments, the present disclosure provides a polypeptide conjugate comprising a fusion polypeptide conjugated to a first CRM and a second CRM, or a pharmaceutically acceptable salt thereof,

[0430] wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: X, and the first CRM and the second CRM are covalently linked to the pth residue and the qth residue (both lysine, i.e., pK or qK) of the fusion polypeptide, respectively, as counted from the N-terminus to the C-terminus, wherein one CRM is linked to one residue, wherein:

[0431] 1) X is 2, p is 20, pK is 20K, q is 231, and qK is 231K (this type of conjugate molecule is also referred to as Compound 2),

[0432] 2) X is 4, p is 51, pK is 51K, q is 231, and qK is 231K (this type of conjugate molecule is also referred to as compound 4),

[0433] 3) X is 5, p is 51, pK is 51K, q is 222, and qK is 222K (this type of conjugate molecule is also referred to as compound 5),

[0434] 4) X is 16, p is 20, pK is 20K, q is 222, and qK is 222K (this type of conjugate molecule is also referred to as Compound 16),

[0435] 5) X is 17, p is 20, pK is 20K, q is 221, and qK is 221K (this type of conjugate molecule is also referred to as Compound 17),

[0436] 6) X is 18, p is 20, pK is 20K, q is 225, and qK is 225K (this type of conjugate molecule is also referred to as Compound 18),

[0437] 7) X is 19, p is 51, pK is 51K, q is 222, and qK is 222K (this type of conjugate molecule is also referred to as Compound 19),

[0438] 8) X is 20, p is 51, pK is 51K, q is 221, and qK is 221K (this type of conjugate molecule is also referred to as compound 20),

[0439] 9) X is 21, p is 51, pK is 51K, q is 225, and qK is 225K (this type of conjugate molecule is also referred to as compound 21),

[0440] 10) X is 22, p is 51, pK is 51K, q is 231, and qK is 231K (this type of conjugate molecule is also referred to as compound 22),

[0441] 11) X is 24, p is 51, pK is 51K, q is 221, and qK is 221K (this type of conjugate molecule is also referred to as compound 24),

[0442] 12) X is 26, p is 36, pK is 36K, q is 210, and qK is 210K (this type of conjugate molecule is also referred to as compound 26),

[0443] 13) X is 27, p is 41, pK is 41K, q is 215, and qK is 215K (this type of conjugate molecule is also referred to as compound 27),

[0444] 14) X is 28, p is 71, pK is 71K, q is 245, and qK is 245K (this type of conjugate molecule is also referred to as compound 28),

[0445] 15) X is 29, p is 111, pK is 111K, q is 285, and qK is 285K (this type of conjugate molecule is also referred to as compound 29),

[0446] 16) X is 30, p is 51, pK is 51K, q is 225, and qK is 225K (this type of conjugate molecule is also referred to as compound 30),

[0447] 17) X is 31, p is 79, pK is 79K, q is 253, and qK is 253K (this type of conjugate molecule is also referred to as compound 31),

[0448] 18) X is 32, p is 71, pK is 71K, q is 245, and qK is 245K (this type of conjugate molecule is also referred to as compound 32),

[0449] 19) X is 33, p is 39, pK is 39K, q is 213, and qK is 213K (this type of conjugate molecule is also referred to as compound 33),

[0450] 20) X is 34, p is 35, pK is 35K, q is 209, and qK is 209K (this type of conjugate molecule is also referred to as compound 34),

[0451] 21) X is 35, p is 20, pK is 20K, q is 221, and qK is 221K (this type of conjugate molecule is also referred to as compound 35),

[0452] 22) X is 77, p is 36, pK is 36K, q is 207, and qK is 207K (this type of conjugate molecule is also referred to as compound 37),

[0453] 23) X is 78, p is 41, pK is 41K, q is 212, and qK is 212K (this type of conjugate molecule is also referred to as compound 38),

[0454] 24) X is 79, p is 71, pK is 71K, q is 242, and qK is 242K (this type of conjugate molecule is also referred to as compound 39),

[0455] 25) X is 80, p is 111, pK is 111K, q is 282, and qK is 282K (this type of conjugate molecule is also referred to as compound 40),

[0456] 26) X is 81, p is 79, pK is 79K, q is 250, and qK is 250K (this type of conjugate molecule is also referred to as compound 41),

[0457] 27) X is 82, p is 71, pK is 71K, q is 242, and qK is 242K (this type of conjugate molecule is also referred to as compound 42),

[0458] 28) X is 83, p is 39, pK is 39K, q is 210, and qK is 210K (this type of conjugate molecule is also referred to as compound 43),

[0459] 29) X is 84, p is 35, pK is 35K, q is 206, and qK is 206K (this type of conjugate molecule is also referred to as compound 44),

[0460] 30) X is 85, p is 46, pK is 46K, q is 222, and qK is 222K (this type of conjugate molecule is also referred to as compound 45,

[0461] 31) X is 86, p is 41, pK is 41K, q is 222, and qK is 222K (this type of conjugate molecule is also referred to as compound 46),

[0462] 32) X is 87, p is 36, pK is 36K, q is 222, and qK is 222K (this type of conjugate molecule is also referred to as compound 47),

[0463] 33) X is 88, p is 51, pK is 51K, q is 222, and qK is 222K (this type of conjugate molecule is also referred to as compound 48),

[0464] 34) X is 116, p is 51, pK is 51K, q is 232, and qK is 232K (this type of conjugate molecule is also referred to as compound 49), or

[0465] 35) X is 117, p is 51, pK is 51K, q is 233, and qK is 233K (this conjugate molecule is also referred to as compound 50), and

[0466] The CRM has the following structure:

[0467]

[0468] In certain embodiments, the present disclosure provides a polypeptide conjugate comprising a fusion polypeptide conjugated to a first CRM and a second CRM, or a pharmaceutically acceptable salt thereof,

[0469] wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: X, and the first CRM and the second CRM are covalently linked to the pth residue and the qth residue (both cysteine, i.e., pC or qC) of the fusion polypeptide, respectively, counted from the N-terminus to the C-terminus, wherein one CRM is linked to one residue, wherein:

[0470] 1) X is 148, p is 51, pC is 51C, q is 222, and qC is 222C (this type of conjugate molecule is also referred to as compound 51),

[0471] 2) X is 149, p is 51, pC is 51C, q is 225, and qC is 225C (this type of conjugate molecule is also referred to as compound 52),

[0472] 3) X is 150, p is 51, pC is 51C, q is 233, and qC is 233C (this type of conjugate molecule is also referred to as compound 53),

[0473] 4) X is 151, p is 51, pC is 51C, q is 231, and qC is 231C (this type of conjugate molecule is also referred to as compound 54),

[0474] 5) X is 152, p is 51, pC is 51C, q is 222, and qC is 222C (this type of conjugate molecule is also referred to as compound 55),

[0475] 6) X is 153, p is 51, pC is 51C, q is 225, and qC is 225C (this type of conjugate molecule is also referred to as compound 56),

[0476] 7) X is 154, p is 51, pC is 51C, q is 231, and qC is 231C (this type of conjugate molecule is also referred to as compound 57),

[0477] 8) X is 155, p is 55, pC is 55C, q is 229, and qC is 229C (this type of conjugate molecule is also referred to as compound 58),

[0478] 9) X is 156, p is 59, pC is 59C, q is 233, and qC is 233C (this type of conjugate molecule is also referred to as compound 59),

[0479] 10) X is 157, p is 51, pC is 51C, q is 225, and qC is 225C (this type of conjugate molecule is also referred to as compound 60),

[0480] 11) X is 158, p is 46, pC is 46C, q is 225, and qC is 225C (this type of conjugate molecule is also referred to as compound 61)

[0481] 12) X is 159, p is 41, pC is 41C, q is 225, and qC is 225C (this type of conjugate molecule is also referred to as compound 62),

[0482] 13) X is 160, p is 51, pC is 51C, q is 222, and qC is 222C (this type of conjugate molecule is also referred to as compound 63),

[0483] 14) X is 161, p is 46, pC is 46C, q is 222, and qC is 222C (this type of conjugate molecule is also referred to as compound 64),

[0484] 15) X is 162, p is 41, pC is 41C, q is 222, and qC is 222C (this type of conjugate molecule is also referred to as compound 65),

[0485] 16) X is 163, p is 51, pC is 51C, q is 225, and qC is 225C (this type of conjugate molecule is also referred to as compound 66),

[0486] 17) X is 164, p is 46, pC is 46C, q is 235, and qC is 235C (this type of conjugate molecule is also referred to as compound 67),

[0487] 18) X is 165, p is 51, pC is 51C, q is 222, and qC is 222C (this type of conjugate molecule is also referred to as compound 68),

[0488] 19) X is 168, p is 52, pC is 52C, q is 282, and qC is 282C (this type of conjugate molecule is also referred to as Compound 71),

[0489] 20) X is 169, p is 62, pC is 62C, q is 282, and qC is 282C (this type of conjugate molecule is also referred to as Compound 72),

[0490] 21) X is 170, p is 42, pC is 42C, q is 282, and qC is 282C (this type of conjugate molecule is also referred to as Compound 73),

[0491] 22) X is 1, p is 51, pC is 51C, q is 122, and qC is 122C (this type of conjugate molecule is also referred to as Compound 74),

[0492] 23) X is 23, p is 40, pC is 40C, q is 51, and qC is 51C (this type of conjugate molecule is also referred to as Compound 75), or

[0493] 24) X is 76, p is 50, pC is 50C, q is 71, and qC is 71C (this conjugate molecule is also referred to as compound 77), and

[0494] The CRM has the following structure:

[0495]

[0496] or a pharmaceutically acceptable salt thereof.

[0497] In another aspect, the present disclosure provides a method of producing a conjugate provided herein, comprising conjugating a clearance reducing moiety to a fusion polypeptide provided herein.

[0498] The CRM can be linked to the fusion polypeptide or polypeptide complex by covalent binding, affinity binding, embedding, coordination binding, complexing, association, blending or addition.

[0499] In certain embodiments, the fusion polypeptide can be linked to the CRM directly or indirectly, such as through another moiety or through a coupling agent.

[0500] The fusion polypeptide can be conjugated at, for example, a lysine residue, a cysteine ​​residue or an unnatural amino acid by a suitable conjugation reaction.

[0501] For example, a fusion polypeptide with a conjugated residue (such as lysine) can react with an amino-reactive reagent. In certain embodiments, CRM is conjugated to a conjugated lysine residue via an acyl group in an acylation reaction. Exemplary methods of acylation reactions are described in, for example, WO2009083549 and WO2010029159, the contents of which are incorporated herein in their entirety. The CRM to be conjugated in the acylation reaction can contain a carboxylic acid group, an α, ω-fatty diacid residue, an activated ester, or an activated N-hydroxyimide ester. Examples of activated esters include O-succinimide reagents, such as N-hydroxysuccinimide (NHS) or sulfo-NHS esters and imidoester compounds, such as Traut's reagent, which can react with the ε-amino group of a conjugated lysine residue to form an amide or amidine bond. Additional examples of suitable amino-reactive reagents include O-acylisoureas, N-hydroxytriazole esters, acid anhydrides, phenyl-activated esters, P-hydroxamate-activated esters, acyl imidazoles, acylbenzotriazoles, acyl azides, acyl halides, phosphonium salts, and ammonium / uronium salts.

[0502] For another example, a fusion polypeptide having a conjugated residue (such as cysteine) can be linked to a thiol-reactive reagent. In certain embodiments, the CRM is conjugated to a conjugated cysteine ​​residue via maleimide or iodoacetamide to form a carbon-sulfur bond. In certain embodiments, the CRM is conjugated to a conjugated cysteine ​​residue via a disulfide to form a disulfide bond. Additional examples of suitable thiol-reactive groups include dienyl sulfones, α-haloacyl groups, or other thiol-reactive conjugation partners. For details, see Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, 1997, Non-Radioactive Labelling: A Practical Approach, Academic Press, London; Means (1990) Bioconjugate Chemistry 1:2; Hermanson, G. in Bioconjugate Techniques (1996) Academic Press, San Diego, pp. 40-55, 643-671.

[0503] For example, a fusion polypeptide having a conjugable residue (such as an NNAA) can be conjugated to a CRM such that a stable linkage can be formed between the NNAA of the fusion polypeptide and the CRM. For example, an NNAA containing a keto or aldehyde or β-diketone moiety can react with a reagent containing a hydrazide or O-alkylhydroxylamine, hydroxylamine to form a hydrazone or O-alkylated oxime bond. For another example, an NNAA containing an azide group can react with an alkyne derivative to form a stable triazole linker (and vice versa) via a copper (I)-catalyzed [3+2] cycloaddition. For another example, an NNAA containing an azide group can be linked to an appropriate water-soluble phosphine-containing reagent to form an amide bond via a Staudinger ligation. In addition, the thioester portion of the NNAA can react with an amine-containing reagent to form an amide bond. Fusion polypeptides provided herein incorporating NNAA can be conjugated to reagents by cycloaddition reactions, such as the (4+2) cycloaddition between a diene and a dienophile (Diels-Alder reaction), the (3+2) cycloaddition by a 1,3-dipolar Huisgen cycloaddition, and the (3+2) cycloaddition by a nitrone-olefin cycloaddition. Cycloaddition methods suitable for antibody conjugation have been described, for example, in WO05003294, US20120004183, WO06009901, WO07130453, and U.S. Patent No. 6,737,236.

[0504] For another example, the fusion polypeptide can be conjugated to biotin, which is then indirectly conjugated to a CRM, which is conjugated to avidin. For another example, the fusion polypeptide can be linked to a coupling agent, which is further linked to a CRM. Examples of coupling agents include bifunctional moieties such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of iminoesters (such as dimethyl diimidoadipate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as 2,6-toluene diisocyanate), and bis-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) to provide disulfide bonds.

[0505] Additional methods for conjugating CRMs to fusion polypeptides are found in, for example, U.S. Pat. No. 5,208,020; U.S. Pat. No. 6,4411,163; WO2005037992; WO2005081711; and WO2006 / 034488, which are incorporated herein by reference in their entireties. Specific examples of methods for preparing the conjugates of the present disclosure are also included in the experimental section of this disclosure.

[0506] The fusion polypeptides and conjugates thereof provided herein have the combined benefits of two mechanisms of action and have synergistic effects against diabetes / NASH as well as additional benefits. The fusion polypeptides and conjugates provided herein provide better efficacy than semaglutide and FGF21 alone at much less systemic exposure, indicating a potentially better safety profile and a potentially improved therapeutic index.

[0507] Pharmaceutical composition

[0508] In another aspect, the present disclosure also provides a pharmaceutical composition comprising the fusion polypeptide conjugate provided herein and a pharmaceutically acceptable carrier.

[0509] The term "pharmaceutically acceptable" indicates that the specified carrier, vehicle, diluent, excipient and / or salt is generally chemically and / or physically compatible with the other ingredients making up the formulation and physiologically compatible with the recipient thereof.

[0510] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical formulation other than the active ingredient that is biologically active and non-toxic to the subject. Pharmaceutically acceptable carriers for 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, sequestering / chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0511] 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, one or more antioxidants (such as methionine) are included in the pharmaceutical compositions provided herein to reduce oxidation of the polypeptide complex or bispecific polypeptide complex. This reduction in oxidation prevents or reduces the loss of binding affinity, thereby improving the stability of the protein and extending the shelf life to the greatest extent. Therefore, in certain embodiments, a composition comprising a fusion polypeptide, polypeptide complex or conjugate disclosed herein and one or more antioxidants (such as methionine) is provided.

[0512] For further illustration, pharmaceutically acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, injection), isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection; non-aqueous vehicles, such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents at bacteriostatic or fungistatic concentrations; isotonic agents, such as sodium chloride or dextrose; buffers, such as phosphate or citrate buffers; antioxidants, such as sodium bisulfate; local anesthetics, such as procaine hydrochloride; suspending and dispersing agents, such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone; emulsifiers, such as polysorbate 80 (TWEEN-80); chelating agents, such as ethylenediaminetetraacetic acid (EDTA) or ethylene glycol tetraacetic acid (EGTA), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents used as carriers can be added to pharmaceutical compositions in multidose containers and include phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients can include, for example, water, normal saline, dextrose, glycerol, or ethanol. Suitable nontoxic auxiliary substances can include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers, or reagents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.

[0513] The pharmaceutical composition can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation or powder. Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, polyvinyl pyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0514] In an embodiment, the pharmaceutical composition is formulated as an injectable composition. Injectable pharmaceutical compositions can 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. Injectable formulations may include sterile and / or pyrogen-free solutions that can be used immediately for injection; sterile dry soluble products that are combined with a solvent just before use, such as lyophilized powders, including subcutaneous tablets; sterile suspensions that can be used immediately for injection; sterile dry insoluble products that are combined with a vehicle just before use; and sterile and / or pyrogen-free emulsions. Solutions can be aqueous or non-aqueous.

[0515] In certain embodiments, unit dose parenteral formulations are packaged in ampoules, vials, or syringes with needles.All preparations for parenteral administration should be sterile and pyrogen-free, as is known and practiced in the art.

[0516] In certain embodiments, a sterile lyophilized powder is prepared by dissolving a fusion polypeptide, polypeptide complex, or conjugate as disclosed herein in a suitable solvent. The solvent may contain excipients that enhance the stability of the powder or a 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, glycerol, glucose, sucrose, or other suitable agents. The solvent may contain a buffer, such as citrate, sodium phosphate, or potassium phosphate, or other such buffers known to those skilled in the art. In one embodiment, the buffer is at approximately neutral pH. The solution is then sterile filtered and lyophilized under standard conditions known to those skilled in the art to provide the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial may contain a single or multiple doses of a fusion polypeptide, polypeptide complex, or conjugate, or combination thereof, as provided herein. Overfilling the vial by a small amount (e.g., about 10%) beyond that required for a single dose or a set of doses is acceptable to facilitate accurate sampling and dosing. The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature.

[0517] Reconstitution of the lyophilized powder with water for injection provides a formulation for parenteral administration. In one embodiment, for reconstitution, sterile and / or pyrogen-free water or other liquid suitable carrier is added to the lyophilized powder. The exact amount depends on the given selected therapy and can be determined empirically.

[0518] Administration of the pharmaceutical compositions as described herein can be carried out by any route known to a physician of ordinary skill in the art. One embodiment is peripheral parenteral administration via a sterile syringe or some other mechanical device (such as an infusion pump). In certain embodiments, the peripheral parenteral route is an intravenous, intramuscular, subcutaneous, or intraperitoneal route of administration.

[0519] In certain embodiments, the fusion polypeptides, polypeptide complexes, or conjugates described herein are formulated in a form suitable for non-parenteral administration, such as oral, rectal, nasal, or lower respiratory tract administration.

[0520] In certain embodiments, the fusion polypeptides, polypeptide complexes, or conjugates described herein are formulated in solid formulations, such as freeze-dried or spray-dried, which are then reconstituted in a suitable diluent solution prior to administration. Standard pharmaceutical formulation techniques, such as those described in Remington: The Science and Practice of Pharmacy (D.B. Troy, ed., 21st ed., Lippincott, Williams & Wilkins, 2006), can be used. Alternatively, the fusion polypeptides, polypeptide complexes, or conjugates described herein can be formulated for administration by the following routes: tongue, sublingual, buccal, oral, oral, gastric and intestinal, nasal, pulmonary (e.g., via bronchioles and alveoli or a combination thereof), epidermal, dermal, transdermal, vaginal, rectal, ocular (e.g., via conjunctiva), ureteral, transdermal, or pulmonary. As yet another option, the fusion polypeptides, polypeptide complexes or conjugates described herein can be formulated for administration transdermally, for example, by needle-free injection or by a patch (optionally an iontophoretic patch), or transmucosally, for example, buccally.

[0521] Treatment

[0522] In another aspect, the disclosure provides a method of preventing or treating a metabolic disorder in a subject in need thereof, comprising administering a therapeutically effective amount of a Polypeptide Conjugate or pharmaceutical composition provided herein.

[0523] Also provided are methods of treatment comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide conjugate or pharmaceutical composition as provided herein, thereby treating or preventing a condition or disorder. In certain embodiments, the subject has been identified as having a disorder or condition that may be responsive to a polypeptide conjugate or pharmaceutical composition as provided herein.

[0524] In certain embodiments, the metabolic disorder is diabetes, obesity, nonalcoholic steatohepatitis (NASH), cardiovascular such as dyslipidemia, arteriosclerosis, alcoholic steatohepatitis (ASH), diabetic nephropathy, gestational diabetes, metabolic syndrome such as metabolic syndrome X, nonalcoholic fatty liver disease (NAFLD), end-stage liver disease, hepatic steatosis (fatty liver), cirrhosis, or primary biliary cirrhosis (PBC).

[0525] For example, metabolic conditions or disorders that can be treated or improved using the Polypeptide Conjugates or pharmaceutical compositions provided herein include conditions in which a human subject has a fasting blood glucose level of 125 mg / dL or greater, e.g., 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, or greater than 200 mg / dL. Blood glucose levels can be determined in the fed or fasted state or randomly. Metabolic conditions or disorders can also include conditions in which a subject is at increased risk of developing a metabolic condition. For human subjects, such conditions include a fasting blood glucose level of 100 mg / dL.

[0526] The therapeutically effective amount of the Polypeptide Conjugates or pharmaceutical compositions provided herein will depend on various factors known in the art, such as the subject's weight, age, past medical history, current medication, health status and the possibility of cross-reactions, allergies, sensitivities and adverse side effects, as well as the route of administration and the extent of disease progression. A person of ordinary skill in the art (e.g., a physician or veterinarian) may proportionally reduce or increase the dosage as indicated by these and other circumstances or requirements. A therapeutically effective amount may be the amount of the Polypeptide Conjugates or pharmaceutical compositions provided herein that elicits a biological or medical response in a tissue system, animal, or human that is being sought by a researcher, physician, or other clinician, including mitigating or ameliorating the symptoms of the disease or condition being treated, i.e., supporting observable levels of one or more desired biological or medical responses, such as reducing blood sugar, insulin, triglycerides, or cholesterol levels; reducing weight; or improving glucose tolerance, energy expenditure, or insulin sensitivity.

[0527] In certain embodiments, the Polypeptide Conjugates or pharmaceutical compositions provided herein can be administered at a therapeutically effective dose of about 0.01 mg / kg to about 100 mg / kg (e.g., about 0.01 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, or about 100 mg / kg). In some of these embodiments, the polypeptide conjugates or pharmaceutical compositions provided herein are administered at a dosage of about 50 mg / kg or less, and in some of these embodiments, the dosage is 10 mg / kg or less, 5 mg / kg or less, 1 mg / kg or less, 0.5 mg / kg or less, or 0.1 mg / kg or less. In certain embodiments, the dosage can be changed during treatment. For example, in certain embodiments, the initial dosage can be higher than the subsequent dosage. In certain embodiments, the dosage can be changed during treatment, depending on the experimenter's response.

[0528] Dosage regimens may be adjusted to provide the optimal desired response (eg, a therapeutic response). For example, a single dose may be administered, or several divided doses may be administered over time.

[0529] The Polypeptide Conjugates or pharmaceutical compositions provided herein can be administered by any route known in the art, such as parenteral (e.g., subcutaneous, intraperitoneal, intravenous (including intravenous infusion), intramuscular or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal or topical) routes.

[0530] The polypeptide conjugate or pharmaceutical composition may be administered alone or in combination with one or more additional therapeutic means or agents.

[0531] In certain embodiments, when used to treat metabolic diseases, the polypeptide conjugates or pharmaceutical compositions provided herein can be administered in combination with any other therapeutic agent used to treat metabolic diseases or any related medical conditions. As used herein, "administered in combination" includes simultaneous administration as part of the same pharmaceutical composition, simultaneous administration as independent compositions, or administration at different times as independent compositions. As the phrase "in combination" is used herein, a composition administered before or after another agent is considered to be administered "in combination" with the agent, even if the composition and the second agent are administered by different routes. Where possible, the additional therapeutic agent administered in combination with the fusion polypeptide, polypeptide complex, or conjugate provided herein is administered according to the schedule listed in the product information sheet for the additional therapeutic agent, or according to the Physicians' Desk Reference (Physicians' Desk Reference, 70th Edition (2016)) or a regimen well known in the art.

[0532] Preparation method

[0533] The present disclosure provides isolated nucleic acids or polynucleotides encoding fusion polypeptides as described herein.

[0534] As used herein, the term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless explicitly limited, the term encompasses polynucleotides containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a specific polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (see Batzer et al., Nucleic Acids 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)).

[0535] Nucleic acids or polynucleotides encoding the fusion polypeptides described herein can be constructed using recombinant techniques. To this end, DNA encoding a GLP-1 polypeptide and DNA encoding an FGF21 polypeptide can be obtained and operably linked to allow transcription and expression in a host cell to produce the fusion polypeptide. A polynucleotide sequence encoding a polypeptide linker can also be operably linked to allow expression of the desired product.

[0536] The encoding polynucleotide sequence may further be operably linked to one or more regulatory sequences, optionally in an expression vector, so that expression or production of the fusion polypeptide is enabled and under appropriate control.

[0537] Recombinant techniques known in the art can be used to insert the encoding polynucleotide sequence into a vector for further cloning (DNA amplification) or expression. 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., a prokaryotic promoter such as T7, T7lac, Sp6, araBAD, trp, lac, tac, pLm, A3, lac, lpp, npr, pac, syn, trc, and T3, or a eukaryotic promoter such as SV40, CMV, and EF-1α) and a transcription termination sequence.

[0538] Vectors and host cells

[0539] In another aspect, the present disclosure provides a vector comprising a polynucleotide provided herein.

[0540] The vector comprising the polynucleotide sequence provided herein can be introduced into a host cell for cloning or gene expression. As used herein, the phrase "host cell" refers to a cell into which an exogenous polynucleotide and / or vector has been introduced. In other embodiments, the vector is extrachromosomal. If necessary, the host cell can be isolated. In certain embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[0541] Suitable host cells for cloning or expressing the DNA in the vectors herein are primarily prokaryotes. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia, for example Escherichia coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, for example Salmonella typhimurium; Serratia, for example Serratia marcescens. marcescans); and Shigella, and Bacilli, such as Bacillus subtilis and Bacillus licheniformis; Pseudomonas, such as Pseudomonas aeruginosa; and Streptomyces. In some embodiments, the host cell is a eukaryotic organism, such as yeast and mammalian cells (e.g., immortalized mammalian cells).

[0542] Any suitable method known to those skilled in the art, such as transformation, transfection or transduction, can be used to introduce a vector comprising a polynucleotide sequence as provided herein into a host cell. In one embodiment, the polynucleotide sequence encoding the fusion polypeptide can be subcloned into an expression vector, which is expressed as inclusion bodies in the host cell. The vector can be a viral vector, and any suitable viral vector can be used in this capacity.

[0543] In another aspect, the present disclosure provides a host cell comprising a vector as provided herein. The host cell is a prokaryotic cell or a eukaryotic cell. Host cells transformed with the above-mentioned expression or cloning vectors can be cultured in a conventional nutrient medium, which may be optionally modified to induce promoters, select transformants, or amplify cloning vectors.

[0544] In another aspect, the present disclosure provides a method of producing a fusion polypeptide as described herein, comprising culturing a host cell as provided herein under conditions that allow expression of the fusion polypeptide as described herein.

[0545] In order to produce fusion polypeptide as described herein, host cells transformed with expression vectors can be cultured in a variety of culture media. Commercially available bacterial growth media, such as Terrific broth, LB broth, LB agar, M9 minimal culture medium, MagiaMedia culture medium and ImMedia culture medium (ThermoFisher) are suitable for culturing bacterial host cells. Commercially available culture media, such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle's Medium (Dulbecco's Modified Eagle's Medium, DMEM, Sigma) are suitable for culturing eukaryotic host cells. Any of these culture media can optionally supplement hormones and / or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium salts, magnesium salts and phosphates), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN TM Drugs), trace elements (defined as inorganic compounds typically present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions (such as temperature, pH, etc.) are those previously used with the host cell selected for expression and will be apparent to those of ordinary skill.

[0546] In one aspect, the present disclosure provides a method of expressing a fusion polypeptide as described herein, comprising culturing a host cell provided herein under conditions whereby the fusion polypeptide as described herein is expressed.

[0547] In certain embodiments, the fusion polypeptide is expressed as inclusion bodies. In certain embodiments, the method further comprises renaturing the fusion polypeptide from the inclusion bodies.

[0548] When using recombinant technology, fusion polypeptides as described herein can be produced in the intracellular, periplasmic space, or directly secreted into the culture medium. If the product is produced in the cell, then as a first step, the microparticle debris of the host cell or cleavage fragments is removed, for example, by centrifugation or ultrafiltration. Carter et al., " Biotechnology (Bio / Technology) " 10:163-167 (1992) describes a program for separating proteins secreted into the periplasmic space of Escherichia coli. In simple terms, the cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) through about 30min. Cell debris can be removed by centrifugation. In the case where the product is secreted into the culture medium, generally first a commercially available protein concentration filter is used, such as Amicon (Amicon) or Millipore (Millipore) Pellicon ultrafiltration unit is concentrated from the supernatant of this type of expression system. Protease inhibitors, such as PMSF, can be included in any of the aforementioned steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants.

[0549] In certain embodiments, the method further comprises isolating the fusion polypeptide.

[0550] Fusion polypeptides as described herein produced by cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography.

[0551] Other techniques for protein purification, such as ion exchange column fractionation, ethanol precipitation, reversed-phase HPLC, silica gel chromatography, heparin SEPHAROSE TM Chromatography, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatographing, SDS-PAGE, and ammonium sulfate precipitation are also useful, depending on the protein to be recovered.

[0552] Reagent test kit

[0553] Also provided are kits for practicing the disclosed methods. Such kits may include a pharmaceutical composition as described herein, which may be provided in a sterile container. Optionally, instructions for using the provided pharmaceutical composition to treat metabolic disorders may also be included or made available to a patient or healthcare provider.

[0554] In one aspect, the kit comprises (a) a pharmaceutical composition comprising a therapeutically effective amount of a fusion polypeptide conjugate; and (b) one or more containers for the pharmaceutical composition. Such kits may also include instructions for their use; the instructions may be customized according to the precise metabolic disorder being treated. The instructions may describe the uses and properties of the materials provided in the kit. In certain embodiments, the kit includes instructions for use by a patient to treat metabolic disorders such as elevated glucose levels, elevated insulin levels, diabetes, obesity, non-alcoholic steatohepatitis (NASH), cardiovascular such as dyslipidemia, arteriosclerosis, alcoholic steatohepatitis (ASH), diabetic nephropathy, gestational diabetes, metabolic syndromes such as metabolic syndrome X, non-alcoholic fatty liver disease (NAFLD), end-stage liver disease, hepatic steatosis (fatty liver), cirrhosis, or primary biliary cirrhosis (PBC).

[0555] Instructions can be printed on base material (such as paper or plastics etc.), and can be present in test kit as package insert, be present in the label of the container of test kit or its component (being associated with packaging) etc.In other embodiments, instructions exist as the electronic storage data file present in suitable computer readable storage medium (such as CD-ROM, floppy disk etc.).In another other embodiment, actual instructions are not present in test kit, but are provided for as the means of obtaining instructions from remote source by the Internet.An embodiment of this embodiment is the test kit comprising a website, can check instructions and / or can download instructions from the website on the website.Usually wish that some or all components of test kit are packaged in suitable packaging to maintain sterile.The component of test kit can be packaged in test kit accommodating element to make single unit that is easy to dispose, wherein test kit accommodating element (such as box or similar structure) can be or can not be airtight container, for example to further keep the sterility of some or all components of test kit.

[0556] Exemplary embodiments

[0557] The following numbered embodiments, while not limiting, are illustrative of certain aspects of the present disclosure:

[0558] 1. A polypeptide conjugate comprising a fusion polypeptide conjugated to at least one clearance reducing moiety (CRM),

[0559] wherein the fusion polypeptide comprises or consists essentially of GLP-1, a polypeptide linker, and FGF21 from N-terminus to C-terminus;

[0560] wherein the at least one CRM is conjugated to at least one conjugatable residue in the fusion polypeptide; and

[0561] The length of the polypeptide linker is at least 0-120 amino acid residues.

[0562] 2. The Polypeptide Conjugate of embodiment 1, wherein the polypeptide linker is at least 4, 5, 8, 10, 20, 24, 28, 30, 40, 48, 50, 60, 70, 80, 90, 100, 110, or 120 amino acid residues in length.

[0563] 3. The polypeptide conjugate of embodiment 1, wherein the polypeptide conjugate does not comprise an antibody fragment (eg, Fc).

[0564] 4. The Polypeptide Conjugate of any one of embodiments 1 to 3, wherein the fusion polypeptide comprises at most one conjugatable residue or at most two conjugatable residues.

[0565] 5. The polypeptide conjugate of any one of embodiments 1 to 4, wherein the fusion polypeptide comprises a first conjugable residue conjugated to a CRM, and wherein the first conjugable residue is in the FGF21, in the polypeptide linker, or in the GLP-1.

[0566] 6. The Polypeptide Conjugate of embodiment 4, wherein the fusion polypeptide comprises a first conjugable residue and a second conjugable residue, wherein:

[0567] 1) Both conjugable residues are present in the polypeptide linker,

[0568] 2) Both conjugable residues are present in the FGF21,

[0569] 3) both conjugable residues are present in the GLP-1,

[0570] 4) the first conjugable residue is present in the FGF21 and the second conjugable residue is present in the polypeptide linker,

[0571] 5) the first conjugable residue is present in the GLP-1 and the second conjugable residue is present in the FGF2, or

[0572] 6) The first conjugable residue is present in the GLP-1 and the second conjugable residue is present in the polypeptide linker.

[0573] 7. The polypeptide conjugate of any one of embodiments 1 to 6, wherein the first conjugable residue is lysine, cysteine, or a non-natural amino acid residue, or both the first conjugable residue and the second conjugable residue are lysine, cysteine, or a non-natural amino acid residue.

[0574] 8. A polypeptide conjugate according to any preceding embodiment, wherein the FGF21 comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 36 while retaining the essential biological activity of SEQ ID NO: 36, and / or the GLP-1 comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 50 while retaining the essential biological activity of SEQ ID NO: 50.

[0575] 9. A Polypeptide Conjugate according to any preceding embodiment, wherein the GLP-1 comprises no more than 9, 8, 7, 6, 5, 4, or 3 mutations relative to SEQ ID NO: 50 while retaining the substantial biological activity of SEQ ID NO: 50, and / or the FGF21 comprises no more than 12, 11, 10, 9, 8, or 7 mutations relative to SEQ ID NO: 36 while retaining the substantial biological activity of SEQ ID NO: 36.

[0576] 10. The Polypeptide Conjugate of any preceding embodiment wherein the FGF21 comprises one or more mutations at a position selected from 121, 168, 171, 180 and 181 relative to SEQ ID NO: 36.

[0577] 11. The polypeptide conjugate of embodiment 10, wherein the one or more mutations in FGF21 are selected from N121Q, M168L, P171G, A180E and del181S, or any combination thereof.

[0578] 12. The Polypeptide Conjugate of any preceding embodiment wherein the FGF21 comprises no conjugatable residues or at most one conjugatable residue.

[0579] 13. The Polypeptide Conjugate of any preceding embodiment, wherein the conjugatable residue in FGF21 is located at a position selected from positions 170, 171, 172, 173, 174, 180, 181, 71, and 182 relative to SEQ ID NO: 36.

[0580] 14. The Polypeptide Conjugate of any preceding embodiment wherein the conjugatable residue in FGF21 comprises an introduced residue, optionally by substitution or by insertion.

[0581] 15. The polypeptide conjugate of embodiment 14, wherein the conjugable residue in FGF21 is lysine.

[0582] 16. The polypeptide conjugate of embodiment 15, wherein the conjugable residue in the FGF21 comprises a substitution relative to SEQ ID NO: 36 selected from the group consisting of G170K, P171K, S172K, Q173K, G174K, A180K, S181K, S71K, and ins182K.

[0583] 17. The polypeptide conjugate of embodiment 15 or 16, wherein the FGF21 further comprises one or more mutations at positions selected from K56, K59, K69 and K122 relative to SEQ ID NO: 36, wherein the mutations are mutations to non-conjugatable residues.

[0584] 18. The polypeptide conjugate of embodiment 17, wherein the non-conjugable residue is selected from the group consisting of arginine (R), glutamine (Q), alanine (A), glycine (G), histidine (H), serine (S), and threonine (T).

[0585] 19. The polypeptide conjugate of embodiment 18, wherein the FGF21 comprises mutations of K56R, K59R, K69R and K122R.

[0586] 20. The Polypeptide Conjugate of any preceding embodiment wherein the FGF21 comprises or consists of a combination of mutations relative to SEQ ID NO: 36 selected from the group consisting of:

[0587] 1) 121Q, 168L, 180K, 171G, 56R, 59R, 69R and 122R;

[0588] 2) 121Q, 168L, 171K, 56R, 59R, 69R and 122R;

[0589] 3) 121Q, 168L, 174K, 171G, 56R, 59R, 69R and 122R;

[0590] 4) 121Q, 168L, 171K, 56R, 59R, 69R, 122R and 180E;

[0591] 5) 121Q, 168L, 174K, 171G, 56R, 59R, 69R, 122R and 180E;

[0592] 6) 121Q, 168L, 180K, 56R, 59R, 69R and 122R;

[0593] 7)121Q, 168L, 180K, 171G, 56R, 59R, 69R, 122R and del181S;

[0594] 8) 121Q, 168L, 170K, 56R, 59R, 69R and 122R;

[0595] 9) 121Q, 168L, 170K, 56R, 59R, 69R, 122R and 180E;

[0596] 10) 121Q, 168L, 170K, 171G, 56R, 59R, 69R, 122R, and 180E

[0597] 11) 121Q, 168L, 180E, 171G, 56R, 59R, 69R, 122R, and 181K;

[0598] 12) 121Q, 168L, 180E, 171G, 56R, 59R, 69R, 122R, and ins182K

[0599] 13) 71K, 121Q, 168L, 56R, 59R, 69R and 122R;

[0600] 14) 71K, 121Q, 168L, 171G, 56R, 59R, 69R, and 122R; and

[0601] 15)71K, 121Q, 168L, 171G, 56R, 59R, 69R, 122R, 180E.

[0602] 21. The Polypeptide Conjugate of any preceding embodiment, wherein the FGF21 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-46 and 118-119.

[0603] 22. The polypeptide conjugate of embodiment 14, wherein the conjugatable residue in FGF21 is cysteine.

[0604] 23. The polypeptide conjugate of embodiment 22, wherein the conjugatable residue in FGF21 comprises a substitution relative to SEQ ID NO: 36 selected from the group consisting of G170C, P171C, S172C, Q173C, G174C, A180C, S181C, S71C, and ins182C.

[0605] 24. The Polypeptide Conjugate of any one of embodiments 12, 22 and 23 wherein the FGF21 comprises or consists of a combination of mutations relative to SEQ ID NO: 36 selected from the group consisting of:

[0606] 1)121Q, 168L, 171C, 180E;

[0607] 2)121Q, 168L, 171G, 174C, 180E;

[0608] 3)121Q, 168L, 171G, 180C;

[0609] 4)121Q, 168L, 171G, 180E;

[0610] 5) 71C, 121Q, 168L, 171G, 180E; and

[0611] 6)121Q, 168L, 180E, 171G, ins 182C.

[0612] 25. The Polypeptide Conjugate of any one of embodiments 12 and 22 to 24, wherein the FGF21 comprises an amino acid sequence selected from SEQ ID NOs: 136-140 and 171.

[0613] 26. The Polypeptide Conjugate of any preceding embodiment wherein the GLP-1 comprises one or more mutations at a position selected from the group consisting of A8, G22, K34, R36 and H7, or any combination thereof, relative to SEQ ID NO: 50.

[0614] 27. The polypeptide conjugate of embodiment 26, wherein the one or more mutations are selected from the group consisting of H7IA, H7IPA, A8G, G22E, K34R and R36G, or any combination thereof.

[0615] 28. The Polypeptide Conjugate of any preceding embodiment wherein the GLP-1 comprises no conjugatable residues or at most one conjugatable residue.

[0616] 29. The Polypeptide Conjugate of embodiment 28, wherein the conjugatable residue in the GLP-1 is lysine.

[0617] 30. The Polypeptide Conjugate of embodiment 29 wherein the conjugatable residue in the GLP-1 comprises or consists of K26 or K34 relative to SEQ ID NO: 50.

[0618] 31. The Polypeptide Conjugate of embodiment 29 wherein the conjugatable residue in the GLP-1 comprises an introduced residue, optionally by substitution or by insertion.

[0619] 32. The Polypeptide Conjugate of embodiment 26 wherein the conjugatable residue in the GLP-1 is introduced by a substitution selected from the group consisting of E27K and R36K relative to SEQ ID NO: 50.

[0620] 33. The Polypeptide Conjugate of any one of embodiments 30 to 32 wherein the GLP-1 further comprises substitution of K26 and / or K34 to a non-conjugatable residue, provided that if K26 or K34 is a conjugatable residue, it is not substituted.

[0621] 34. The polypeptide conjugate of embodiment 33, wherein the non-conjugable residue is selected from the group consisting of arginine (R), glutamine (Q), alanine (A), glycine (G), histidine (H), serine (S), and threonine (T).

[0622] 35. The Polypeptide Conjugate of embodiment 33 or 34 wherein the substitution at K26 is selected from K26R and K26Q, and / or the substitution at K34 is selected from K34R and K34Q.

[0623] 36. The Polypeptide Conjugate of embodiment 28, wherein the conjugatable residue in the GLP-1 comprises or consists of cysteine ​​or an unnatural amino acid.

[0624] 37. The Polypeptide Conjugate of embodiment 36, wherein the conjugatable residue in the GLP-1 is introduced by substitution at a position selected from the group consisting of K26, K34, E27 and R36 relative to SEQ ID NO: 50.

[0625] 38. A fusion polypeptide according to embodiment 37, wherein the conjugable residue in the GLP-1 comprises or consists of cysteine ​​and is introduced by substitution at a position selected from the group consisting of K26C, K34C, E27C and R36C relative to SEQ ID NO: 50.

[0626] 39. The Polypeptide Conjugate of any one of embodiments 26 to 38 wherein the GLP-1 comprises the amino acid sequence X7X8EGTFTSDVSSYLEX22QAAX26X27FIAWLVX34GX36G (SEQ ID NO:51) wherein: X7 is H, imidazole-4-acetic acid (IA) or imidazole propionic acid (IPA); X8 is A, G, S, V, Aib, T, I or L; X22 is G or E; X26 is K, R or C; X27 is E, K or C; X34 is R, K or C, and X36 is K, R, G or C.

[0627] 40. The polypeptide conjugate of embodiment 39 wherein X7 is H, X8 is G, X22 is E; X26 is K or R; X27 is E; X34 is R, and X36 is G.

[0628] 41. The Polypeptide Conjugate of any one of embodiments 26 to 35, 39 and 40 wherein the GLP-1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 52, 53 and 115.

[0629] 42. The Polypeptide Conjugate of any one of embodiments 36 to 40 wherein the GLP-1 comprises the amino acid sequence of SEQ ID NO: 120 or 52.

[0630] 43. The Polypeptide Conjugate of any preceding embodiment, wherein the polypeptide linker comprises one or more units of a repeat sequence.

[0631] 44. The Polypeptide Conjugate of embodiment 43, wherein the repeat sequence consists of no more than 4, 5 or 6 types of amino acid residues selected from the group consisting of G, Q, A, E, P, T and S.

[0632] 45. The Polypeptide Conjugate of embodiment 43, wherein the repeat sequence comprises or consists of an amino acid sequence selected from the group consisting of: G a S b , SEQ ID NO:65(GAQP), SEQ ID NO:92(GQEP), SEQ ID NO:93(GEQP), SEQ ID NO:94(GPQE), SEQ ID NO:95(GPEQ), SEQ ID NO:96(GSEP), SEQ ID NO:97(GESP), SEQ ID NO:98(GPSE), SEQ ID NO:99(GPES), SEQ ID NO:100(GQAP), SEQ ID NO:101(GPAQ), SEQ ID NO:102(GPQA), SEQ ID NO:103(GSQP), SEQ ID NO:104(GASP), SEQ ID NO:105(GPAS), SEQ ID NO:106(GPSA), SEQ ID NO:107(GGGS), SEQ ID NO:108(GSGS)、SEQID NO:57(GGGGS)、SEQ ID NO:143(GSAPGSPAGSPTGSAPGSPA) and GS, wherein a and b are independently an integer selected from 1 to 5.

[0633] 46. ​​The Polypeptide Conjugate of any one of embodiments 43 to 45, wherein the polypeptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 54-65, 109-113, 3, 47-49, and 141-147.

[0634] 47. The Polypeptide Conjugate of any preceding embodiment wherein the Polypeptide Linker comprises no conjugatable residues or comprises at most one conjugatable residue, or at most two conjugatable residues.

[0635] 48. The Polypeptide Conjugate of embodiment 47, wherein the conjugatable residue in the Polypeptide Linker comprises an introduced residue, optionally by substitution or by insertion.

[0636] 49. The polypeptide conjugate of embodiment 48, wherein the conjugable residue in the polypeptide linker is the C-terminal most residue of the polypeptide linker, or is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 residues (including the conjugable residue) away from the C-terminal most residue of the GLP-1.

[0637] 50. The Polypeptide Conjugate of embodiment 48 or 49, wherein the conjugatable residue in the polypeptide linker is lysine and the polypeptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-75 and 89-91.

[0638] 51. The polypeptide conjugate of embodiment 48 or 49, wherein the conjugatable residue in the polypeptide linker is cysteine, and the polypeptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 122-132.

[0639] 52. The polypeptide conjugate of embodiment 51, wherein the polypeptide linker comprises two conjugable residues and comprises the amino acid sequence of SEQ ID NO: 133 or 134.

[0640] 53. The Polypeptide Conjugate of any one of embodiments 1 to 21, 26 to 35, 39 to 41, 43 to 50, wherein the conjugatable residue is lysine and the fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4-22, 24, 26-35, 77-88, 116-117.

[0641] 54. The polypeptide conjugate of any one of embodiments 1 to 14, 22 to 28, 36 to 40, 42 to 49, 51 and 52, wherein the conjugatable residue is cysteine ​​and the fusion polypeptide comprises an amino acid sequence selected from the group consisting of 148-165, 168-170, 1, 23, 25 and 76.

[0642] 55. The Polypeptide Conjugate of any one of the preceding embodiments,

[0643] wherein the CRM comprises a structure of *-ABCDE, and

[0644] in:

[0645] A is selected from the group consisting of a bond, a, b, c and d are independently integers from 0 to 4, R 1 is hydrogen or -COOH;

[0646] B is selected from the group consisting of a bond, e is an integer from 1 to 4,

[0647] C is a bond or R 2 is -CH2SO3H or -COOH, f is an integer from 1 to 4, and n is an integer from 1 to 25;

[0648] D is selected from the group consisting of a bond, g and h are independently 0 or 1, and R 3 is H or -CH2COOH;

[0649] E is an acidic group having the formula:

[0650]

[0651] Where W represents -(CR 4 R 5 ) l -,

[0652] R 4 and R 5 independently selected from the group consisting of hydrogen, halogen, cyano, hydroxy, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, hydroxyalkyl, amino, aminoalkyl, carboxyl, carboxylalkyl, alkoxy, aryloxy, and carboxamide,

[0653] R 6 Selected from hydroxyl or NR 7 R 8 ;

[0654] R 7 and R 8 Independently selected from the group consisting of hydrogen, alkyl, hydroxyl and and

[0655] l is an integer from 10 to 20,

[0656] or a pharmaceutically acceptable salt thereof.

[0657] 56. The conjugate of embodiment 55, wherein the CRM is conjugated to at least one lysine residue in the fusion polypeptide.

[0658] 57. The Polypeptide Conjugate of embodiment 56, wherein A is a bond, B is C is D is a bond, and E is an acidic group having the formula:

[0659] wherein e is 1, 2, or 3, f is 1, 2, or 3, and n is 1 or 2, and R 6 、R 2 and W are as defined in embodiment 48.

[0660] 58. The Polypeptide Conjugate of embodiment 57, wherein R 2 is -COOH, and R 6 is hydroxy and W is as defined in embodiment 48.

[0661] 59. The Polypeptide Conjugate of embodiment 58, wherein W represents -(CR 4 R 5 ) l -, R 4 and R 5 are independently hydrogen, and l is an integer from 10 to 20.

[0662] 60. The Polypeptide Conjugate of embodiment 56 wherein the CRM comprises the following structure:

[0663]

[0664] 61. The polypeptide conjugate of embodiment 55, wherein the CRM is conjugated to at least one cysteine ​​residue in the fusion polypeptide.

[0665] 62. The Polypeptide Conjugate of embodiment 61, wherein A is And B is

[0666] 63. The Polypeptide Conjugate of embodiment 62 wherein the CRM comprises the following structure:

[0667]

[0668] 64. A Polypeptide Conjugate comprising a fusion polypeptide conjugated to a CRM, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: N, and the CRM covalently linked to the mth residue (which is lysine, i.e., mK) in the fusion polypeptide, counting in the N-terminal to C-terminal direction, and wherein:

[0669] 1) N is 6, m is 222, and mK is 222K (this type of conjugate molecule is also referred to as compound 6),

[0670] 2) N is 7, m is 221, and mK is 221K (this type of conjugate molecule is also referred to as compound 7),

[0671] 3) N is 8, m is 225, and mK is 225K (this type of conjugate molecule is also referred to as compound 8),

[0672] 4) N ​​is 9, m is 231, and mK is 231K (this type of conjugate molecule is also referred to as compound 9),

[0673] 5) N is 10, m is 222, and mK is 222K (this type of conjugate molecule is also referred to as compound 10),

[0674] 6) N is 11, m is 221, and mK is 221K (this type of conjugate molecule is also referred to as compound 11),

[0675] 7) N is 12, m is 225, and mK is 225K (this type of conjugate molecule is also referred to as compound 12),

[0676] 8) N is 13, m is 222, and mK is 222K (this type of conjugate molecule is also referred to as compound 13),

[0677] 9) N is 14, m is 222, and mK is 222K (such conjugate molecules are also referred to as compound 14), or

[0678] 10) N is 15, m is 231, and mK is 231K (such conjugate molecules are also referred to as compound 15), wherein the CRM has the following structure:

[0679]

[0680] or a pharmaceutically acceptable salt thereof.

[0681] 65. A Polypeptide Conjugate comprising a fusion polypeptide conjugated to a CRM, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: N, and the CRM covalently linked to the mth residue (which is cysteine, i.e., mC) in the fusion polypeptide, counted in the N-terminal to C-terminal direction, and wherein:

[0682] 1) N is 25, m is 51, and mC is 51C (this type of conjugate molecule is also referred to as compound 76), wherein the CRM has the following structure:

[0683]

[0684] or a pharmaceutically acceptable salt thereof.

[0685] 66. A polypeptide conjugate comprising a fusion polypeptide conjugated to a first CRM and a second CRM

[0686] wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: X, and the first CRM and the second CRM are covalently linked to the pth residue and the qth residue (both lysine, i.e., pK or qK) of the fusion polypeptide, respectively, as counted from the N-terminus to the C-terminus, wherein one CRM is linked to one residue, wherein:

[0687] 1) X is 2, p is 20, pK is 20K, q is 231, and qK is 231K (this type of conjugate molecule is also referred to as Compound 2),

[0688] 2) X is 4, p is 51, pK is 51K, q is 231, and qK is 231K (this type of conjugate molecule is also referred to as compound 4),

[0689] 3) X is 5, p is 51, pK is 51K, q is 222, and qK is 222K (this type of conjugate molecule is also referred to as compound 5),

[0690] 4) X is 16, p is 20, pK is 20K, q is 222, and qK is 222K (this type of conjugate molecule is also referred to as Compound 16),

[0691] 5) X is 17, p is 20, pK is 20K, q is 221, and qK is 221K (this type of conjugate molecule is also referred to as Compound 17),

[0692] 6) X is 18, p is 20, pK is 20K, q is 225, and qK is 225K (this type of conjugate molecule is also referred to as Compound 18),

[0693] 7) X is 19, p is 51, pK is 51K, q is 222, and qK is 222K (this type of conjugate molecule is also referred to as Compound 19),

[0694] 8) X is 20, p is 51, pK is 51K, q is 221, and qK is 221K (this type of conjugate molecule is also referred to as compound 20),

[0695] 9) X is 21, p is 51, pK is 51K, q is 225, and qK is 225K (this type of conjugate molecule is also referred to as compound 21),

[0696] 10) X is 22, p is 51, pK is 51K, q is 231, and qK is 231K (this type of conjugate molecule is also referred to as compound 22),

[0697] 11) X is 24, p is 51, pK is 51K, q is 221, and qK is 221K (this type of conjugate molecule is also referred to as compound 24),

[0698] 12) X is 26, p is 36, pK is 36K, q is 210, and qK is 210K (this type of conjugate molecule is also referred to as compound 26),

[0699] 13) X is 27, p is 41, pK is 41K, q is 215, and qK is 215K (this type of conjugate molecule is also referred to as compound 27),

[0700] 14) X is 28, p is 71, pK is 71K, q is 245, and qK is 245K (this type of conjugate molecule is also referred to as compound 28),

[0701] 15) X is 29, p is 111, pK is 111K, q is 285, and qK is 285K (this type of conjugate molecule is also referred to as compound 29),

[0702] 16) X is 30, p is 51, pK is 51K, q is 225, and qK is 225K (this type of conjugate molecule is also referred to as compound 30),

[0703] 17) X is 31, p is 79, pK is 79K, q is 253, and qK is 253K (this type of conjugate molecule is also referred to as compound 31),

[0704] 18) X is 32, p is 71, pK is 71K, q is 245, and qK is 245K (this type of conjugate molecule is also referred to as compound 32),

[0705] 19) X is 33, p is 39, pK is 39K, q is 213, and qK is 213K (this type of conjugate molecule is also referred to as compound 33),

[0706] 20) X is 34, p is 35, pK is 35K, q is 209, and qK is 209K (this type of conjugate molecule is also referred to as compound 34),

[0707] 21) X is 35, p is 20, pK is 20K, q is 221, and qK is 221K (this type of conjugate molecule is also referred to as compound 35),

[0708] 22) X is 77, p is 36, pK is 36K, q is 207, and qK is 207K (this type of conjugate molecule is also referred to as compound 37),

[0709] 23) X is 78, p is 41, pK is 41K, q is 212, and qK is 212K (this type of conjugate molecule is also referred to as compound 38),

[0710] 24) X is 79, p is 71, pK is 71K, q is 242, and qK is 242K (this type of conjugate molecule is also referred to as compound 39),

[0711] 25) X is 80, p is 111, pK is 111K, q is 282, and qK is 282K (this type of conjugate molecule is also referred to as compound 40),

[0712] 26) X is 81, p is 79, pK is 79K, q is 250, and qK is 250K (this type of conjugate molecule is also referred to as compound 41),

[0713] 27) X is 82, p is 71, pK is 71K, q is 242, and qK is 242K (this type of conjugate molecule is also referred to as compound 42),

[0714] 28) X is 83, p is 39, pK is 39K, q is 210, and qK is 210K (this type of conjugate molecule is also referred to as compound 43),

[0715] 29) X is 84, p is 35, pK is 35K, q is 206, and qK is 206K (this type of conjugate molecule is also referred to as compound 44),

[0716] 30) X is 85, p is 46, pK is 46K, q is 222, and qK is 222K (this type of conjugate molecule is also referred to as compound 45,

[0717] 31) X is 86, p is 41, pK is 41K, q is 222, and qK is 222K (this type of conjugate molecule is also referred to as compound 46),

[0718] 32) X is 87, p is 36, pK is 36K, q is 222, and qK is 222K (this type of conjugate molecule is also referred to as compound 47),

[0719] 33) X is 88, p is 51, pK is 51K, q is 222, and qK is 222K (this type of conjugate molecule is also referred to as compound 48),

[0720] 34) X is 116, p is 51, pK is 51K, q is 232, and qK is 232K (this type of conjugate molecule is also referred to as compound 49), or

[0721] 35) X is 117, p is 51, pK is 51K, q is 233, and qK is 233K (this conjugate molecule is also referred to as compound 50), and

[0722] The CRM has the following structure:

[0723]

[0724] or a pharmaceutically acceptable salt thereof.

[0725] 67. A polypeptide conjugate comprising a fusion polypeptide conjugated to a first CRM and a second CRM

[0726] wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: X, and the first CRM and the second CRM are covalently linked to the pth residue and the qth residue (both cysteine, i.e., pC or qC) of the fusion polypeptide, respectively, counted from the N-terminus to the C-terminus, wherein one CRM is linked to one residue, wherein:

[0727] 1) X is 148, p is 51, pC is 51C, q is 222, and qC is 222C (this type of conjugate molecule is also referred to as compound 51),

[0728] 2) X is 149, p is 51, pC is 51C, q is 225, and qC is 225C (this type of conjugate molecule is also referred to as compound 52),

[0729] 3) X is 150, p is 51, pC is 51C, q is 233, and qC is 233C (this type of conjugate molecule is also referred to as compound 53),

[0730] 4) X is 151, p is 51, pC is 51C, q is 231, and qC is 231C (this type of conjugate molecule is also referred to as compound 54),

[0731] 5) X is 152, p is 51, pC is 51C, q is 222, and qC is 222C (this type of conjugate molecule is also referred to as compound 55),

[0732] 6) X is 153, p is 51, pC is 51C, q is 225, and qC is 225C (this type of conjugate molecule is also referred to as compound 56),

[0733] 7) X is 154, p is 51, pC is 51C, q is 231, and qC is 231C (this type of conjugate molecule is also referred to as compound 57),

[0734] 8) X is 155, p is 55, pC is 55C, q is 229, and qC is 229C (this type of conjugate molecule is also referred to as compound 58),

[0735] 9) X is 156, p is 59, pC is 59C, q is 233, and qC is 233C (this type of conjugate molecule is also referred to as compound 59),

[0736] 10) X is 157, p is 51, pC is 51C, q is 225, and qC is 225C (this type of conjugate molecule is also referred to as compound 60),

[0737] 11) X is 158, p is 46, pC is 46C, q is 225, and qC is 225C (this type of conjugate molecule is also referred to as compound 61)

[0738] 12) X is 159, p is 41, pC is 41C, q is 225, and qC is 225C (this type of conjugate molecule is also referred to as compound 62),

[0739] 13) X is 160, p is 51, pC is 51C, q is 222, and qC is 222C (this type of conjugate molecule is also referred to as compound 63),

[0740] 14) X is 161, p is 46, pC is 46C, q is 222, and qC is 222C (this type of conjugate molecule is also referred to as compound 64),

[0741] 15) X is 162, p is 41, pC is 41C, q is 222, and qC is 222C (this type of conjugate molecule is also referred to as compound 65),

[0742] 16) X is 163, p is 51, pC is 51C, q is 225, and qC is 225C (this type of conjugate molecule is also referred to as compound 66),

[0743] 17) X is 164, p is 46, pC is 46C, q is 235, and qC is 235C (this type of conjugate molecule is also referred to as compound 67),

[0744] 18) X is 165, p is 51, pC is 51C, q is 222, and qC is 222C (this type of conjugate molecule is also referred to as compound 68),

[0745] 19) X is 168, p is 52, pC is 52C, q is 282, and qC is 282C (this type of conjugate molecule is also referred to as Compound 71),

[0746] 20) X is 169, p is 62, pC is 62C, q is 282, and qC is 282C (this type of conjugate molecule is also referred to as Compound 72),

[0747] 21) X is 170, p is 42, pC is 42C, q is 282, and qC is 282C (this type of conjugate molecule is also referred to as Compound 73),

[0748] 22) X is 1, p is 51, pC is 51C, q is 122, and qC is 122C (this type of conjugate molecule is also referred to as Compound 74),

[0749] 23) X is 23, p is 40, pC is 40C, q is 51, and qC is 51C (this type of conjugate molecule is also referred to as Compound 75), or

[0750] 24) X is 76, p is 50, pC is 50C, q is 71, and qC is 71C (this conjugate molecule is also referred to as compound 77), and

[0751] The CRM has the following structure:

[0752]

[0753] or a pharmaceutically acceptable salt thereof.

[0754] 68. A pharmaceutical composition comprising the Polypeptide Conjugate according to any one of the preceding embodiments and a pharmaceutically acceptable carrier.

[0755] 69. A method of preventing or treating a metabolic disorder in a subject in need thereof, comprising administering a therapeutically effective amount of the Polypeptide Conjugate of any one of Embodiments 1 to 67 or the pharmaceutical composition of Embodiment 68.

[0756] 70. The method of embodiment 69, wherein the metabolic disorder is diabetes, obesity, non-alcoholic steatohepatitis (NASH), cardiovascular such as dyslipidemia, arteriosclerosis, 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).

[0757] 71. A polynucleotide encoding a fusion polypeptide as defined in any one of embodiments 1 to 67.

[0758] 72. A vector comprising the polynucleotide according to embodiment 71.

[0759] 73. A host cell comprising the vector of embodiment 72.

[0760] 74. A method for producing a fusion polypeptide as defined in any one of embodiments 1 to 67, comprising culturing the host cell according to embodiment 73 under conditions allowing expression of the polynucleotide according to embodiment 71.

[0761] 75. A method for producing a Polypeptide Conjugate as defined in any one of embodiments 1 to 67, comprising conjugating a clearance reducing moiety to the fusion polypeptide of any one of embodiments 1 to 67.

[0762] Example

[0763] Example 1: Recombinant expression of fusion protein

[0764] The GLP-1 / FGF21 fusion proteins listed in Table 1 were produced in a bacterial E. coli expression system using a BL21(DE3) derivative strain. DNA encoding the GLP-1 / FGF21 fusion precursors was codon-optimized for E. coli expression, synthesized de novo, and subcloned into a pET-derived expression vector (Novagen). Amino acid substitutions were made by modifying the corresponding genetic code. When the cell density reached an OD600 of 2.0 in Terrific Broth (TB) medium, overexpression of the GLP-1 / FGF21 precursors as inclusion bodies was induced with 0.5 mM isopropyl bd-thiogalactopyranoside (IPTG). Cells were harvested after 20-22 hours of protein induction at 37°C. The quality of the inclusion bodies was analyzed by microscopic imaging.

[0765] Example 2: Refolding and purification

[0766] Cells are collected and lysed by a cell crusher (900 bar, twice). The insoluble portion containing the fusion protein is collected and washed twice by centrifugation (8,000 × g, 30 min). 6M urea and 10mM DTT buffer are then used to dissolve the inclusion bodies. After 0.5 hour, the solution is diluted with refolding buffer and stirred at room temperature for 12 hours. After removing the label by protease, the protein is loaded onto an anion exchange chromatography using Q Sepharose fast flow resin (GE Healthcare), and further refined by anion exchange chromatography using SuperQ-5PW resin (TOSOH). The sample in each step is characterized by LC / MS to confirm the correct molecular weight.

[0767] Example 3: Preparation of fusion protein conjugates

[0768] To a solution of GLP1 / FGF21 fusion protein (75 mg) in NaOH or Tris buffer, CRM reagent (i.e., HOOC-(CH2)16-CO-gGlu-2XADO, HOOC-(CH2)16-CO-gGlu-2XADO-EDA-CO-CH2, or HOOC-(CH2)20-CO-gGlu-2XADO-EDA-CO-CH2) (6 mg or 12 mg) in an organic solvent was added dropwise. The reaction was stirred at room temperature for 1 h. The product was then subjected to anion exchange chromatography using Source 30Q resin (GE Healthcare).

[0769] The conjugated fusion proteins were detected and characterized by LC-MS using a Waters BioAccord LC-MS system or by UPLC using a Waters Acquity UPLC system, following the supplier's manual and using conditions optimized for the different conjugates (as shown in Table 2).

[0770] Example 4: In vitro activity of exemplary fusion protein conjugates.

[0771] method:

[0772] The in vitro GLP-1 activity of some exemplary fusion protein conjugates was measured using a BHK cell line overexpressing the human GLP-1 receptor and a CRE luciferase reporter. The test fusion protein conjugates were measured in 3-fold serial dilutions at a maximum concentration of 1 nM. After treating the cells with the fusion protein conjugate for 4 hours, luciferase activity was measured by the Steadylite plus kit (PerkinElmer, 6066751). Semaglutide was tested as a control under parallel conditions. Semaglutide was purchased from Novo Nordisk, batch JP51144. According to the product label, semaglutide is a human GLP-1 analog conjugated to HOOC-(CH2)16-CO-gGlu-2XADO at residue 26 (lysine).

[0773] The in vitro FGF21 activity of the exemplary fusion protein conjugates was assessed using a HEK293 cell line overexpressing human β-Klotho. The test fusion protein conjugates were measured in 3-fold serial dilutions at a maximum concentration of 200 nM. After treating the cells with the fusion protein conjugates for 5 min, p-ERK levels were measured using a p-ERK kit (Cisbio, 64ERKPEH).

[0774] The activity of each exemplary fusion protein conjugate was determined by the EC derived from nonlinear regression analysis. 50 express.

[0775] in conclusion:

[0776] The exemplary fusion protein conjugates showed GLP-1 activity that was 3-9 times less potent than semaglutide, and FGF21 activity of similar or equal magnitude compared to native FGF21 (Table 3).

[0777] Table 3: In vitro activities of exemplary fusion protein conjugates.

[0778]

[0779]

[0780] Example 5: In vivo activity of exemplary fusion protein conjugates.

[0781] method:

[0782] 10 week old male C57BL / 6 mice were injected subcutaneously with 30nmol / kg exemplary fusion protein conjugate every day for 10 or 14 days (n=5). The body weight of each individual animal was monitored every day. Day 1 and day 10 (or day 14) were the first and last day of the molecule dose. BW reduction %=100*(BW of the nth day - BW of the 1st day) / (BW of the 1st day). Data are indicated as mean and standard error (SEM) or combined values. Statistical analysis was performed by one-way ANOVA. The weight loss on day 11 in Table 5 was calculated by -1*(BW reduction %-BW reduction % of vehicle group).

[0783] Table 5 Weight loss of C57BL / 6 mice on day 11

[0784]

[0785]

[0786] in conclusion:

[0787] All tested exemplary fusion protein conjugates (Compounds 4, 5, and 10) exhibited more enhanced and sustained weight loss compared to semaglutide during the 10-day dosing period ( Figures 1A-1B During the 14-day dosing period, all tested exemplary fusion protein conjugates (Compounds 2 and 9-15) exhibited more enhanced and sustained weight loss compared to semaglutide ( Figures 1C-1D All tested exemplary fusion protein conjugates (Compounds 19-22) exhibited sustained weight loss compared to vehicle control ( Figures 1E-1F All tested molecules in Table 5 showed better weight loss efficacy than semaglutide. However, compared with compounds 10 and 13, compound 14 exhibited lower weight loss efficacy, indicating that negatively charged linkers may have an adverse effect on fusion protein activity.

[0788] Example 6: Pharmacokinetic Measurements of Exemplary Fusion Protein Conjugates

[0789] method:

[0790] 6-8 week old male C57BL / 6 mice were administered a single subcutaneous dose of 30 nmol / kg of exemplary fusion protein conjugates (compounds 4, 9, 10, 19, 63) or non-acylated fusion protein (i.e., compound 3, which has the same amino acid sequence as compound 2 but without any conjugation) (n=3 / group). Plasma samples were collected before (-5 min), 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after injection. For compound 3, plasma collection time points were -5 min, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, and 6 h. The concentration of fusion protein conjugates or non-acylated fusion proteins in plasma was measured by ELISA analysis with immunoreactivity against both the C-terminal FGF21 and the intact N-terminus of GLP-1. Pharmacokinetic parameters were calculated by WinNonlin based on plots showing plasma concentrations of each protein versus time after subcutaneous injection.

[0791] in conclusion:

[0792] Acylated fusion protein conjugates including compounds 4, 9, 10, 19, and 63 showed longer T than the non-acylated fusion protein (i.e., compound 3). 1 / 2 and greater AUC, indicating that CRM conjugation improved the pharmacokinetic profile. In addition, diacylation significantly prolonged the half-life of the protein conjugate compared to the monoacylated molecule (diacylated compounds 4 and 19 compared to monoacylated compounds 9 and 10). Compounds 4 and 19 also showed better pharmacokinetic properties than semaglutide ( Figure 2 and Table 4 ).

[0793] Table 4: Pharmacokinetic parameters.

[0794]

[0795] Pharmacokinetic data were analyzed by WinNonlin software. Tmax, Cmax, Tmax and Tmax of each molecule were calculated. 1 / 2 , AUC and MRT.

[0796] Example 7. Comparison of GLP-1 / FGF21 fusion protein conjugate with GLP1 alone and FGF21 alone.

[0797] method:

[0798] 17-week-old DIO male C57BL / 6 mice (approximately 45 g) were subcutaneously injected with the designated protein (i.e., compounds 63, 69, and 70) daily for 21 days. Body weight was measured daily, and five animals were used per treatment group. Day 1 and day 21 were the first and last days of molecule dosing. BW reduction % = 100 * (BW on day n - BW on day 1) / (BW on day 1). Data are presented as mean and standard error (SEM).

[0799] in conclusion:

[0800] like Figures 3A-3B As shown in , compound 63 at both 10 nmol / kg and 30 nmol / kg showed better body weight control than GLP1 alone (compound 70) and FGF21 alone (compound 69), indicating that there is an additive or synergistic effect when combining GLP1 and FGF21.

[0801] Example 8 Efficacy studies in disease models

[0802] The selected fusion protein conjugates were evaluated in disease animal models (DIO mice, db / db mice, NASH models, etc.) to determine body weight, food intake, glucose efficiency, and dose response. Several biomarkers were also measured, including fasting insulin, plasma triglycerides, and liver triglycerides.

[0803] method:

[0804] 17-week-old DIO male C57BL / 6 mice (about 45g) were subcutaneously injected with the specified protein (i.e., compound 63) every day for 21 days. Body weight was measured every day, and five animals were used for each treatment group. Fasting blood glucose was also monitored on the specified day. The body weight and glucose of each individual animal were monitored. Day 1 and day 21 were the first and last days of the molecular dose. BW reduction %=100*(BW on the nth day - BW on the 1st day) / (BW on the 1st day). Terminal blood was collected, and EDTA-2k plasma was prepared and frozen at -80°C for biomarker measurement. Liver was also collected and frozen in liquid nitrogen and stored at -80°C.

[0805] Data are presented as mean and standard error (SEM). Statistical analysis was performed by one-way ANOVA.

[0806] in conclusion:

[0807] Figures 4A-4F Compound 63 at 30 nmol / kg was shown to provide better body weight and glycemic control and lipid profiles than semaglutide at 30 nmol / kg. Dose-dependent efficacy was also observed when comparing the 10 nmol / kg and 30 nmol / kg results.

[0808] Example 9: PK studies in non-human primates.

[0809] The pharmacokinetics of selected fusion protein conjugates were evaluated in monkeys by subcutaneous and intravenous injections, respectively.

[0810] Example 10: Stability study.

[0811] To test stability, different conjugate fusion proteins were formulated in buffers with different compositions (pH 6, 7, 7.4, and 8.0) and stored at 4° C. for 2-4 weeks. HMWP% and LMW% were analyzed by size exclusion chromatography (SEC)-HPLC. Concentration and modification were analyzed by reverse phase (RP)-UPLC and LC / MS.

[0812] Example 11: Immunogenicity assessment.

[0813] The immunogenicity of selected fusion protein conjugates was also assessed by in silico (iTope and TCED methods) and ex vivo (EpiScreen) methods. Sequence Listing <110> Beijing Zhipeptide Biopharmaceutical Technology Co., Ltd. (BEIJING QL BIOPHARMACEUTICAL CO., LTD.) <120> Conjugate of fusion protein of GLP-1 and FGF21 <130> 074585-8002CN02 <150> PCT / CN2020 / 130515 <151> 2020-11-20 <150> PCT / CN2020 / 071566 <151> 2020-01-11 <160> 171 <170> PatentIn version 3.5 <210> 1 <211> 232 <212> PRT <213> Artificial sequence <220> <223> composite <400> 1 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Cys His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Lys Thr Cys Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Lys Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Glu Ser 225 230 <210> 2 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> Composite <400> 2 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu<​​​​​​​​​​​​​​​​​ Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Lys Ser 225 230 <210> 3 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Composite <400> 3 Gly Pro Ala Ser Gly Pro Ala Ser Gly Pro Ala Ser Gly Pro Ala Ser 1 5 10 15 Gly Pro Ala Ser 20 <210> 4 <211> 232 <212> PRT <213> Artificial sequence <220> <223> Composite <400> 4 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Lys His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Lys Ser 225 230 <210> 5 <211> 232 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 5 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Lys His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Lys Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Ala Ser 225 230 <210> 6 <211> 232 <212> PRT [[ID=​​​​​​​​​​​​​​​​​​​​​​ Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Lys Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Ala Ser 225 230 <210> 7 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 7 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Ser His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60<00021​​​​​​​​​​​​​​​ Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Lys Pro Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Ala Ser 225 230 <210> 8 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 8 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Ser His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln 210 215 220 Lys Arg Ser Pro Ser Tyr Ala Ser 225 230 <210> 九 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> Composite <400> 九 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 二十五 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45<00022(...)Gly Gly Ser His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Lys Ser 225 230 <210> 10 <211> 232 <212> PRT <213> artificial sequence <220> <223> Compound <400> 10 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Ser His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Lys Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Glu Ser 225 230 <210> 11 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 11 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Ser His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Lys Pro Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Glu Ser 225 230 <210> 12 <211> 232 <212> PRT <213> Artificial sequence <220> <223> Synthetic substance <400> 12 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Ser His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln 210 215 220 Lys Arg Ser Pro Ser Tyr Glu Ser 225 230 <210> 13 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Substance <400> 13 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly 35 40 45 Ala Gln Pro His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Lys Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Glu Ser 225 230 <210> 14 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Substance <400> 14 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Gln Glu Pro Gly Gln Glu Pro Gly Gln Glu Pro Gly Gln Glu Pro Gly 35 40 45 Gln Glu Pro His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Lys Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Glu Ser 225 230 <210> 15 <211> 232 <212> PRT <213> Artificial sequence <220> <223> Composite <400> 15<00​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Pro Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Lys Ser 225 230 <210> 16 <211> 232 <212> PRT <213> Artificial sequence <220> <223> Synthetic substance <400> 16 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Ser His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Lys Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Glu Ser 225 230 <210> 17 <211> 232 <212> PRT <213> Artificial sequence <220> <223> Synthetic compound <400> 17 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Ser His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Lys Pro Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Glu Ser 225 230 <210> 18 <211> 232 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 18 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Ser His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln 210 215 220 Lys Arg Ser Pro Ser Tyr Glu Ser ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Lys Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Glu Ser 225 230 <210> 20 <211> 232 <212> PRT <213> Artificial sequence <220> <223> Synthetic compound <400> 20 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Lys His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Lys Pro Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Glu Ser 225 230 <210> 21 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Substance <400> 21 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Lys His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln 210 215 220 Lys Arg Ser Pro Ser Tyr Glu Ser 225 230 <210> 22 <211> 231 <212> PRT <213> Artificial Sequence <220> <223> Composite <400> 22 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Lys His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Lys 225 230 <210> 23 <211> 232 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 23 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Cys Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Cys His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Lys Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Glu Ser 225 230 <210> 24 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 24 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Lys His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Lys Gly Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Glu Ser 225 230 <210> 25 <211> 232 <212> PRT <213> Artificial sequence <220> <223> Synthetic compound <400> 25 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Cys His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Lys Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Glu Ser ​​​​​​​​​​​​​​​​​​​​​​20 25 30 Gly Gly Gly Lys His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe 35 40 45 Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln 50 55 60 Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala 65 70 75 80 Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro 85 90 95 Gly Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln 100 105 110 Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala 115 120 125 Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln 130 135 140 Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro 145 150 155 160 His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro 165 170 175 Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln 180 185 190 Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser 195 200 205 Gln Lys Arg Ser Pro Ser Tyr Glu Ser 210 215 <210> 27 <211> 222 <212> PRT <213> Artificial Sequence <220> <223> Composite <400> 27 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Lys His Pro Ile Pro Asp Ser Ser 35 40 45 Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr 50 55 60 Asp Asp Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly 65 70 75 80 Thr Val Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu 85 90 95 Arg Ala Leu Arg Pro Gly Val Ile Gln Ile Leu Gly Val Arg Thr Ser 100 105 110 Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His 115 120 125 Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly 130 135 140 Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro 145 150 155 160 Gly Gln Arg Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg 165 170 175 Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly 180 185 190 Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser 195 200 205 Leu Val Gly Gly Ser Gln Lys Arg Ser Pro Ser Tyr Glu Ser 210 215 220 <210> 28 <211> 252 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Substance <400> 28 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 50 55 60 Gly Ser Gly Gly Gly Gly Lys His Pro Ile Pro Asp Ser Ser Pro Leu 65 70 75 80 Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp 85 90 95 Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val 100 105 110 Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala 115 120 125 Leu Arg Pro Gly Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe 130 135 140 Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp 145 150 155 160 Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn 165 170 175 Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln 180 185 190 Arg Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu 195 200 205 Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu 210 215 220 Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val 225 230 235 240 Gly Gly Ser Gln Lys Arg Ser Pro Ser Tyr Glu Ser[[ID=I7]] 245 250 <210> 29 <211> 292 <21~> PRT <213> Artificial Sequence <220> <223> Synthetic Substance <400> 29 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 [ Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly 35 40 45 Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly 50 55 60 Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly 65 70 75 80 Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly 85 90 95 Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Lys His 100 105 110 Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg 115 120 125 Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu 130 135 140 Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro 145 150 155 160 Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly Val Ile Gln Ile 165 170 175 Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala 180 185 190 Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu 195 200 205 Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly 210 215 220 Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His Arg Asp Pro Ala 225 230 235 240 Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala 245 250 255 Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly 260 265 270 Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln Lys Arg Ser Pro 275 280 285 Ser Tyr Glu Ser 290 <210> 30 <211> 232 <212> PRT <213> Artificial sequence <220> <223> Synthetic compound <(400)> 30 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly 35 40 45 Ala Gln Lys His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln 210 215 220 Lys Arg Ser Pro Ser Tyr Glu Ser 225 230 <210> 31 <211> 260 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 31 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly 35 40 45 Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly 50 55 60 Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Lys His 65 70 75 80 Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg 85 90 95 Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu 100 105 110 Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro 115 120 125 Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly Val Ile Gln Ile 130 135 140 Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala 145 150 155 160 Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu 165 170 175 Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly 180 185 190 Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His Arg Asp Pro Ala 195 200 205 Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala 210 215 220 Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly 225 230 235 240 Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln Lys Arg Ser Pro 245 250 255 Serum Tyr Glu Serum 260 <210> 32 <211> 252 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 32 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly 35 40 45 Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly 50 55 60 Ala Gln Pro Gly Ala Gln Lys His Pro Ile Pro Asp Ser Ser Pro Leu 65 70 75 80 Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp 85 90 95 Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val 100 105 110 Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala 115 120 125 Leu Arg Pro Gly Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe 130 135 140 Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp 145 150 155 160 Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn 165 170 175 Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln 180 185 190 Arg Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu 195 200 205 Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu 210 215 220 Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val 225 230 235 240 Gly Gly Ser Gln Lys Arg Ser Pro Ser Tyr Glu Ser 245 250 <210> 33 <211> 220 <212> PRT <213> Artificial sequence <220> <223> Synthetic compound <400> 33 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Ala Gln Pro Gly Ala Gln Lys His Pro Ile Pro Asp Ser Ser Pro Leu 35 40 45 Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp 50 55 60 Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val 65 70 75 80 Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala 85 90 95 Leu Arg Pro Gly Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe 100 105 110 Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp 115 120 125 Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn 130 135 140 Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln 145 150 155 160 Arg Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu 165 170 175 Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu 180 185 190 Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val 195 200 205 Gly Gly Ser Gln Lys Arg Ser Pro Ser Tyr Glu Ser 210 215 220 <210> 34 <211> 216 <212> PRT <213> Artificial sequence <220> <223> Composite <400> 34 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Ala Gln Lys His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 35 40 45 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 50 55 60 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 65 70 75 80 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly 85 90 95 Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg 100 105 110 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 115 120 125 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 130 135 140 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 145 150 155 160 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 165 170 175 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 180 185 190 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln 195 200 205 Lys Arg Ser Pro Ser Tyr Glu Ser 210 215 <210> 35 <211> 232 <212> PRT <213> Artificial Sequence <220><( <223> Synthetic Substance <400> 35 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Ser His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Lys Gly Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Glu Ser 225 230 <210> 36 <211> 181 <212> PRT <213> Artificial Sequence <220> <223> Composite <400> 36 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val 1 5 10 15 Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30 Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45 Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln 50 55 60 Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly 65 70 75 80 Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg 85 90 95 Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105 110 Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro 115 120 125 Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140 Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val 145 150 155 160 Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser 165 170 175 Pro Ser Tyr Ala Ser 180 <210> 37 <211> 181 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 37 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val 1 5 10 15 Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30 Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45 Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly Val Ile Gln 50 55 60 Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly 65 70 75 80 Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg 85 90 95 Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105 110 Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His Arg Asp Pro 115 120 125 Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140 Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val 145 150 155 160 Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln Gly Arg Ser 165 170 175 Pro Ser Tyr Lys Ser 180 <210> 38 <211> 181 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 38 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val 1 5 10 15 Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30 Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45 Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly Val Ile Gln 50 55 60 Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly 65 70 75 80 Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg 85 90 95 Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105 110 Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His Arg Asp Pro 115 120 125 Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140 Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val 145 150 155 160 Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Lys Ser Gln Gly Arg Ser 165 170 175 Pro Ser Tyr Ala Ser 180 <210> 39 <211> 181 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Substance <400> 39 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val 1 5 10 15 Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30 Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45 Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly Val Ile Gln 50 55 60 Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly 65 70 75 80 Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg 85 90 95 Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105 110 Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His Arg Asp Pro 115 120 125 Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140 Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val 145 150 155 160 Gly Ser Ser Asp Pro Leu Ser Leu Val Lys Pro Ser Gln Gly Arg Ser 165 170 175 Pro Ser Tyr Ala Ser 180 <210> 40 <211> 181 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 40 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val 1 5 10 15 Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30 Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45 Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly Val Ile Gln 50 55 60 Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly 65 70 75 80 Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg 85 90 95 Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105 110 Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His Arg Asp Pro 115 120 125 Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140 Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val 145 150 155 160 Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln Lys Arg Ser 165 170 175 Pro Ser Tyr Ala Ser 180 <210> 41 <211> 181 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 41 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val 1 5 10 15 Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30 Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45 Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly Val Ile Gln 50 55 60 Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly 65 70 75 80 Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg 85 90 95 Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105 110 Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His Arg Asp Pro 115 120 125 Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140 Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val 145 150 155 160 Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Lys Ser Gln Gly Arg Ser 165 170 175 Pro Ser Tyr Glu Ser 180 <210> 42 <211> 181 <212> PRT <213> Artificial sequence <220> <223> Synthetic substance <400> 42 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val 1 5 10 15 Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30 Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45 Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly Val Ile Gln 50 55 60 Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly 65 70 75 80 Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg 85 90 95 Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105 110 Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His Arg Asp Pro 115 120 125 Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140 Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val 145 150 155 160 Gly Ser Ser Asp Pro Leu Ser Leu Val Lys Pro Ser Gln Gly Arg Ser 165 170 175 Pro Ser Tyr Glu Ser 180 <210> 43 <211> 181 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 43 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val 1 5 10 15 Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30 Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45 Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly Val Ile Gln 50 55 60 Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly 65 70 75 80 Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg 85 90 95 Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105 110 Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His Arg Asp Pro 115 120 125 Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140 Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val 145 150 155 160 Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln Lys Arg Ser 165 170 175 Pro Ser Tyr Glu Ser 180 <210> 44 <211> 181 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 44 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val 1 5 10 15 Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30 Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45 Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly Val Ile Gln 50 55 60 Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly 65 70 75 80 Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg 85 90 95 Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105 110 Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His Arg Asp Pro 115 120 125 Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140 Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val 145 150 155 160 Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Pro Ser Gln Gly Arg Ser 165 170 175 Pro Ser Tyr Lys Ser 180 <210> 45 <211> 180 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 45 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val 1 5 10 15 Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30 Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45 Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly Val Ile Gln 50 55 60 Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly 65 70 75 80 Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg 85 90 95 Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105 110 Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His Arg Asp Pro 115 120 125 Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140 Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val 145 150 155 160 Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln Gly Arg Ser 165 170 175 Pro Ser Tyr Lys 180 <210> 46 <211> 181 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 46 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val 1 5 10 15 Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30 Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45 Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly Val Ile Gln 50 55 60 Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly 65 70 75 80 Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg 85 90 95 Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105 110 Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His Arg Asp Pro 115 120 125 Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140 Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val 145 150 155 160 Gly Ser Ser Asp Pro Leu Ser Leu Val Lys Gly Ser Gln Gly Arg Ser 165 170 175 Pro Ser Tyr Glu Ser 180 <210> 47 <211> 20 <212> PRT <213> Synthetic Sequence <220> <223> Synthetic <400> 47 Gly Pro Ser Ala Gly Pro Ser Ala Gly Pro Ser Ala Gly Pro Ser Ala 1 5 10 15 Gly Pro Ser Ala 20 <210> 48 <211> 20 <212> PRT <213> Synthetic Sequence <220> <223> Synthetic <400> 48 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser 1 5 10 15 Gly Gly Gly Ser 20 <210> 49 <211> 20 <212> PRT <213> artificial hierarchy <220> <223> compound <400> 49 Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 1 5 10 15 Gly Ser Gly Ser 20 <210> 50 <211> 31 <212> PRT <213> artificial hierarchy <220> <223> compound <400> 50 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30 <210> 51 <211> 31 <212> PRT <213> artificial hierarchy <220> <223> compound <220> <221> MISC_FEATURE <222> (1)..(1) <223> Xaa is His, imidazole-4-acetic acid (IA), or imidazole propionic acid (IPA). <220> <221> MISC_FEATURE <222> (2)..(2) <223> Xaa is Ala, Gly, Ser, Val, Aib, Thr, He or Leu. <220> <221> MISC_FEATURE <222> (16) <223> Xaa is Gly or Glu <220> <221> MISC_FEATURE <222> (20)..(20) <223> Xaa is Lys, Arg or Cys. <220> <221> MISC_FEATURE <222> (21)..(21) <223> Xaa is Glu, Lys or Cys. <220> <221> MISC_FEATURE <222> (28)..(28) <223> Xaa is Arg, Lys or Cys. <220> <221> MISC_FEATURE <222> (30)..(30) <223> Xaa is Lys, Arg, Gly or Cys. <400> 51 Xaa Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa 1 5 10 15 Gln Ala Ala Xaa Xaa Phe Ile Ala Trp Leu Val Xaa Gly Xaa Gly 20 25 30 <210> 52 <211> 31 <212> PRT <213> Artificial sequence <220> <223> composite <400> 52 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly 20 25 30 <210> 53 <211> 31 <212> PRT <213> Artificial sequence <220> <223> composite <400> 53 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly 20 25 30 <210> 54 <211> 20 <212> PRT <213> Artificial sequence <220> <223> composite <400> 54 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 55 <211> 20 <212> PRT <213> Artificial sequence <220> <223> compound <400> 55 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 1 5 10 15 Gly Ala Gln Pro 20 <210> 56 <211> 20 <212> PRT <213> artificial hierarchy <220> <223> compound <400> 56 Gly Gln Glu Pro Gly Gln Glu Pro Gly Gln Glu Pro Gly Gln Glu Pro 1 5 10 15 Gly Gln Glu Pro 20 <210> 57 <211> 5 <212> PRT <213> artificial hierarchy <220> <223> compound <400> 57 Gly Gly Gly Gly Ser 1 5 <210> 58 <211> 10 <212> PRT <213> artificial hierarchy <220> <223> compound <400> 58 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 59 <211> 40 <212> PRT <213> artificial hierarchy <220> <223> Composite <400> 59 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Ser 35 40 <210> 60 <211> 80 <212> PRT <213> Artificial sequence <220> <223> Composite <400> 60 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 1 5 10 15 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 20 25 30 Gly Ala Glq Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 35 40 45 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 50 55 60 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 65 70 75 80 <210> 61 <211> 25 It should be noted that in line 40, "Glq" might be a typo and is likely meant to be "Gln".<212> PRT <213> artificial hierarchy <220> <223> compound <400> 61 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 <210> 62 <211> 48 <212> PRT <213> artificial hierarchy <220> <223> compound <400> 62 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 1 5 10 15 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 20 25 30 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 35 40 45 <210> 63 <211> 40 <212> PRT <213> artificial hierarchy <220> <223> compound <400> 63 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 1 5 10 15 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 20 25 30 Gly Ala Gln Pro Gly Ala Gln Pro 35 40 <210> 64 <211> 8 <212> PRT <213> Artificial sequence <220> <223> composite <400> 64 Gly Ala Gln Pro Gly Ala Gln Pro 1 5 <210> 65 <211> 4 <212> PRT <213> Artificial sequence <220> <223> composite <400> 65 Gly Ala Gln Pro 1 <210> 66 <211> 20 <212> PRT <213> Artificial sequence <220> <223> composite <400> 66 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Lys 20 <210> 67 <211> 5 <212> PRT <213> Artificial sequence <220> <223> composite <400> 67 Gly Gly Gly Gly Lys 1 5 <210> 68 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Composite <400> 68 Gly Gly Gly Gly Ser Gly Gly Gly Gly Lys 1 5 10 <210> 69 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> Composite <400> 69 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Lys 35 40 <210> 70 <211> 80 <212> PRT <213> Artificial Sequence <220> <223> Composite<000^3831><400> 70 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 1 5 10 15 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 20 25 30 Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro 35 40 45 Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro 50 55 60 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Lys 65 70 75 80 <210> 71 <211> 20 <212> PRT <213> artificial sequence <220> <223> Synthesis <400> 71 Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro 1 5 10 15 Gly Ala Gln Lys 20 <210> 72 <211> 48 <212> PRT <213> artificial sequence <220> <223> Synthesis <400> 72 Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro 1 5 10 15 Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro 20 25 30 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Lys 35 40 45 <210> 73 <211> 40 <212> PRT <213> Artificial sequence <220> <223> composite <400> 73 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 1 5 10 15 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 20 25 30 Gly Ala Gln Pro Gly Ala Gln Lys 35 40 <210> 74 <211> 8 <212> PRT <213> Artificial sequence <220> <223> composite <400> 74 Gly Ala Gln Pro Gly Ala Gln Lys 1 5 <210> 75 <211> 4 <212> PRT <213> Artificial sequence <220> <223> composite <400> 75 Gly Ala Gln Lys 1 <210> 76 <211> 252 <212> PRT <213> Artificial sequence <220> <223> Composite <400> 76 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 50 55 60 Gly Ser Gly Gly Gly Gly Cys His Pro Ile Pro Asp Ser Ser Pro Leu 65 70 75 80 Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp 85 90 95 Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val 100 105 110 Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala 115 120 125 Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe 130 135 140 It should be noted that there seems to be a misspelling in the tag "0003912" in the original text, which should probably be "0003912". And in the translation, the "37" in "Leu Lys Pro Gly Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe" should be "37" for consistency.Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp 145 150 155 160 Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn 165 170 175 Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln 180 185 190 Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu 195 200 205 Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu 210 215 220 Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val 225 230 235 240 Gly Gly Ser Gln Gly Arg Ser Pro Ser Tyr Glu Ser 245 250 <210> 77 <211> 217 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 77 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Gly Gly Gly Lys His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe 35 40 45 Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln 50 55 60 Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala 65 70 75 80 Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro 85 90 95 Gly Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln 100 105 110 Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala 115 120 125 Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln 130 135 140 Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro 145 150 155 160 His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro 165 170 175 Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln 180 185 190 Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Lys Ser 195 200 205 Gln Gly Arg Ser Pro Ser Tyr Glu Ser 210 215 <210> 78 <211> 222 <212> PRT <213> Artificial Sequence <220> <223> Composition <400> 78 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly[[ID=​​​​​​​​​​​​​​​​​​​​100 105 110 Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His 115 120 125 Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly 130 135 140 Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro 145 150 155 160 Gly Gln Arg Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg 165 170 175 Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly 180 185 190 Ile Leu Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser 195 200 205 Leu Val Gly Lys Ser Gln Gly Arg Ser Pro Ser Tyr Glu Ser 210 215 220 <210> 79 <211> 252[[ID=-33]] <212> PRT <213> Artificial Sequence <220> <223> Synthetic Substance <400> 79 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 50 55 60 Gly Ser Gly Gly Gly Gly Lys His Pro Ile Pro Asp Ser Ser Pro Leu 65 70 75 80 Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp 85 90 95 Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val 100 105 110 Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala 115 120 125 Leu Arg Pro Gly Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe 130 135 140 Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp 145 150 155 160 Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn 165 170 175 Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln 180 185 190 Arg Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu 195 200 205 Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu 210 215 220 Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val 225 230 235 240 Gly Lys Ser Gln Gly Arg Ser Pro Ser Tyr Glu Ser 245 250 <210> 80 <211> 292 <212> PRT <213> Artificial Sequence <220> <223> Composite <400> 80 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly 35 40 45 Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly 50 55 60 Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly 65 70 75 80 Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly 85 90 95 Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Lys His 100 105 110 Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg 115 120 125 Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu 130 135 140 Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro 145 150 155 160 Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly Val Ile Gln Ile 165 170 175 Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala 180 185 190 Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu 195 200 205 Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly 210 215 220 Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His Arg Asp Pro Ala 225 230 235 240 Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala 245 250 255 Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly 260 265 270 Ser Ser Asp Pro Leu Ser Leu Val Gly Lys Ser Gln Gly Arg Ser Pro 275 280 285 Ser Tyr Glu Ser 290 [[ID=2二十二]]<210> 81 <211> 260 <二十一二> PRT<二十一二><213> Artificial sequence [ <220> <223> Synthetic compound <400> 81 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu[[ID=3七十六]]<四十五>1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30<00041五零一>Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly 35 40 45 Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly 50 55 60 It should be noted that there seems to be an error in the original text where the tag is repeated as [[ID=2二十二]] and is repeated as [[ID=二十一二]]. I've translated it as accurately as possible based on the given text. Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Lys His 65 70 75 80 Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg 85 90 95 Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu 100 105 110 Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser Pro 115 120 125 Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly Val Ile Gln Ile 130 135 140 Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly Ala 145 150 155 160 Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg Glu 165 170 175 Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His Gly 180 185 190 Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His Arg Asp Pro Ala 195 200 205 Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala 210 215 220 Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly 225 230 235 240 Ser Ser Asp Pro Leu Ser Leu Val Gly Lys Ser Gln Gly Arg Ser Pro 245 250 255 Ser Tyr Glu Ser 260 <210> 82 <211> 252 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 82 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly 35 40 45 Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly 50 55 60 Ala Gln Pro Gly Ala Gln Lys His Pro Ile Pro Asp Ser Ser Pro Leu 65 70 75 80 Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp 85 90 95 Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val 100 105 110 Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala 115 120 125 Leu Arg Pro Gly Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe 130 135 140 Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp 145 150 155 160 Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn 165 170 175 Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln 180 185 190 Arg Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu 195 200 205 Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu 210 215 220 Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val 225 230 235 240 Gly Lys Ser Gln Gly Arg Ser Pro Ser Tyr Glu Ser 245 250 <210> 83 <211> 220 <212> PRT <213> Artificial sequence <220> <223> Composition <400> 83 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu<D 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Ala Gln Pro Gly Ala Gln Lys His Pro Ile Pro Asp Ser Ser Pro Leu[[ID=2,]] 35 40 45 Leu Gln Phe Gly Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp 50 55 60 Ala Gln Gln Thr Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val 65 70 75 80 Gly Gly Ala Ala Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala 85 90 95 Leu Arg Pro Gly Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe 100 105 110 Leu Cys Gln Arg Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp 115 120 125 Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn 130 135 140 Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln 145 150 155 160 Arg Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu 165 170 175 Pro Leu Pro Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu 180 185 190 Ala Pro Gln Pro Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val 195 200 205 Gly Lys Ser Gln Gly Arg Ser Pro Ser Tyr Glu Ser 210 215 220 <210> 84 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Substance <400> 84 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Ala Gln Lys His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 35 40 45 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 50 55 60 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 65 70 75 80 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly 85 90 95 Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg 100 105 110 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 115 120 125 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 130 135 140 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 145 150 155 160 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 165 170 175 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 180 185 190 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Lys Ser Gln 195 200 205 Gly Arg Ser Pro Ser Tyr Glu Ser 210 215 <210> 85 <211> 232 <212> PRT <213> Artificial sequence <220> <223> Synthetic compound <400> 85 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Lys Gly Gly 35 40 45 Gly Gly Ser His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr<00042​​​​​​​​​​​​​​Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Lys Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Glu Ser 225 230 <210> 86 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 86 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly<00​ Gly Gly Gly Ser Gly Gly Gly Gly Lys Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Ser His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Lys Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Glu Ser 225 230 <210> 87 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> Composite <400> 87 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Gly Gly Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Ser His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Lys Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Glu Ser 225 230 <210> 88 <211> 232 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 88 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly 35 40 45 Ala Gln Lys His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Lys Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Glu Ser 225 230 <210> 89 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Composite <400> 89 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Lys Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 90 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Composite <400> 90 Gly Gly Gly Gly Ser Gly Gly Gly Gly Lys Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 91 <211> 20 <212> PRT <213> Artificial sequence <220> <223> composite <400> 91 Gly Gly Gly Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 92 <211> 4 <212> PRT <213> Artificial sequence <220> <223> composite <400> 92 Gly Gln Glu Pro 1 <210> 93 <211> 4 <212> PRT <213> Artificial sequence <220> <223> composite <400> 93 Gly Glu Gln Pro 1 <210> 94 <211> 4 <212> PRT <213> Artificial sequence <220> <223> composite <400> 94 Gly Pro Gln Glu 1 <210> 95 <211> 4 <212> PRT <213> Artificial sequence <220> <223> composite <400> 95 Gly Pro Glu Gln 1 <210> 96 <211> 4 <212> PRT <213> Artificial sequence <220> <223> composite <400> 96 Gly Ser Glu Pro 1 <210> 97 <211> 4 <212> PRT <213> Artificial sequence <220> <223> composite <400> 97 Gly Glu Ser Pro 1 <210> 98 <211> 4 <212> PRT <213> Artificial sequence <220> <223> composite <400> 98 Gly Pro Ser Glu 1 <210> 99 <211> 4 <212> PRT <213> Artificial sequence <220> <223> composite <400> 99 Gly Pro Glu Ser 1 <210> 100 <211> 4 <212> PRT <213> Artificial sequence <220> <223> composite <400> 100 Gly Gln Ala Pro 1 <210> 101 <211> 4 <212> PRT <213> Artificial sequence <220> <223> composite <400> 101 Gly Pro Ala Gln 1 <210> 102 <211> 4 <212> PRT <213> Artificial sequence <220> <223> composite <400> 102 Gly Pro Gln Ala 1 <210> 103 <211> 4 <212> PRT <213> Artificial sequence <220> <223> composite <400> 103 Gly Ser Gln Pro 1 <210> 104 <211> 4 <212> PRT <213> Artificial sequence <220> <223> composite <400> 104 Gly Ala Ser Pro 1 <210> 105 <211> 4 <212> PRT <213> Artificial sequence <220> <223> composite <400> 105 Gly Pro Ala Ser 1 <210> 106 <211> 4 <212> PRT <213> Artificial sequence <220> <223> composite <400> 106 Gly Pro Ser Ala 1 <210> 107 <211> 4 <212> PRT <213> Artificial sequence <220> <223> composite <400> 107 Gly Gly Gly Ser 1 <210> 108 <211> 4 <212> PRT <213> Artificial sequence <220> <223> composite <400> 108 Gly Ser Gly Ser 1 <210> 109 <211> 20 <212> PRT <213> Artificial sequence <220> <223> composite <400> 109 Gly Gln Ala Pro Gly Gln Ala Pro Gly Gln Ala Pro Gly Gln Ala Pro 1 5 10 15 Gly Gln Ala Pro 20 <210> 110 <211> 20 <212> PRT <213> Artificial sequence <220> <223> compound <400> 110 Gly Ser Gln Pro Gly Ser Gln Pro Gly Ser Gln Pro Gly Ser Gln Pro 1 5 10 15 Gly Ser Gln Pro 20 <210> 111 <211> 20 <212> PRT <213> artificial hierarchy <220> <223> compound <400> 111 Gly Pro Ala Gln Gly Pro Ala Gln Gly Pro Ala Gln Gly Pro Ala Gln 1 5 10 15 Gly Pro Ala Gln 20 <210> 112 <211> 20 <212> PRT <213> artificial hierarchy <220> <223> compound <400> 112 Gly Pro Gln Ala Gly Pro Gln Ala Gly Pro Gln Ala Gly Pro Gln Ala 1 5 10 15 Gly Pro Gln Ala 20 <210> 113 <211> 20 <212> PRT <213> artificial hierarchy <220> <223> compound <400> 113 Gly Ala Ser Pro Gly Ala Ser Pro Gly Ala Ser Pro Gly Ala Ser Pro 1 5 10 15 Gly Ala Ser Pro 20 <210> 114 <211> 11 <212> PRT <213> Artificial sequence <220> <223> composite <400> 114 Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp 1 5 10 <210> 115 <211> 31 <212> PRT <213> Artificial sequence <220> <223> composite <400> 115 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Lys Gly 20 25 30 <210> 116 <211> 232 <212> PRT <213> Artificial sequence <220> <223> composite <400> 116 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Lys His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Glu Lys 225 230 <210> 117 <211> 233 <212> PRT <213> Artificial Sequence <220> <223> Composite <400> 117 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Lys His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Glu Ser Lys 225 230 <210> 118 <211> 181 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 118 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val 1 5 10 15 Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30 Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45 Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly Val Ile Gln 50 55 60 Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly 65 70 75 80 Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg 85 90 95 Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105 110 Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His Arg Asp Pro 115 120 125 Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140 Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val 145 150 155 160 Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln Gly Arg Ser 165 170 175 Pro Ser Tyr Glu Lys 180 <210> 119 <211> 182 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 119 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val 1 5 10 15 Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30 Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45 Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Arg Pro Gly Val Ile Gln 50 55 60 Ile Leu Gly Val Arg Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly 65 70 75 80 Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg 85 90 95 Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105 110 Gly Leu Pro Leu His Leu Pro Gly Gln Arg Ser Pro His Arg Asp Pro 115 120 125 Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140 Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val 145 150 155 160 Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln Gly Arg Ser 165 170 175 Pro Ser Tyr Glu Ser Light 180 <210> 120 <211> 31 <212> PRT <213> artificial sequence <220> <223> Synthesis <400> 120 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly 20 25 30 <210> 121 <211> 29 <212> PRT <213> artificial sequence <220> <223> Synthesis <400> 121 Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu Gln Ala 1 5 10 15 Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Gly Gly 20 25 <210> 122 <211> 20 <212> PRT <213> artificial hierarchy <220> <223> compound <400> 122 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Cys 20 <210> one two three <211> 20 <212> PRT <213> artificial hierarchy <220> <223> compound <400> one two three Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 1 5 10 15 Gly Ala Gln Cys 20 <210> 124 <211> twenty four <212> PRT <213> artificial hierarchy <220> <223> compound <400> 124 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 1 5 10 15 Gly Ala Gln Pro Gly Ala Gln Cys 20 <210> 125 <211> 28 <212> PRT <213> Artificial sequence <220> <223> Composite <400> 125 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 1 5 10 15 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Cys 20 25 <210> 126 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Composite <400> 126 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Cys Gly 1 5 10 15 Gly Gly Gly Ser 20 [[ID=四十八]] [[ID=四十九]]<210> 127[[ID=五十]] [[ID=五十一]]<211> 20[[ID=五十二]] [[ID=五十三]]<212> PRT[[ID=五十四]] [[ID=五十五]]<213> Artificial sequence[[ID=五十六]] [[ID=五十七]]<二百二十>[[ID=五十八]] [[ID=五十九]]<223> Composite[[ID=六十]] [[ID=六十一]]<400> 127[[ID=六十二]] [[ID=六十三]]Gly Gly Gly Gly Ser Gly Gly Gly Gly Cys Gly Gly Gly Gly Ser Gly[[ID=六十四]] [[ID=六十五]]1 5 10 15[[ID=六十六]] [[ID=六十七]]Gly Gly Gly Ser[[ID=六十八]] [[ID=六十九]]20[[ID=七十]] [[ID=七十一]]<210> 128 [[ID=七十二]] <211> 20 <212> PRT <213> artificial hierarchy <220> <223> compound <400> 128 Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr Gly Ser Ala Pro 1 5 10 15 Gly Ser Pro Cys 20 <210> 129 <211> 30 <212> PRT <213> artificial hierarchy <220> <223> compound <400> 129 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Cys Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 30 <210> 130 <211> 80 <212> PRT <213> artificial hierarchy <220> <223> compound <400> 130 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 1 5 10 15 Gly Ala Gln Pro Cys Gln Ala Pro Gly Ala Gln Pro Gly Ala Gln Pro 20 25 30 Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro 35 40 45 Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro 50 55 60 Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro 65 70 75 80 <210> 131 <211> 80 <212> PRT <213> artificial sequence <220> <223> Synthesis <400> 131 Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro 1 5 10 15 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Gln Cys Pro 20 25 30 Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro 35 40 45 Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro 50 55 60 Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro 65 70 75 80 <210> 132 <211> 80 <212> PRT <213> Artificial Sequence <220> <223> Composite <400> 132 Gly Ala Gln Pro Gly Ala Gln Pro Gly Gln Cys Pro Gly Ala Gln Pro 1 5 10 15 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 20 25 30 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 35 40 45 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 50 55 60 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 65 70 75 80 <210> 133 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Composite <400> 133 Gly Gly Gly Gly Ser Gly Gly Gly Cys Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Cys 20 <210> 134 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> Composite <400> 134 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Cys 35 40 <210> 135 <211> 168 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 135 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val 1 5 10 15 Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His[[ID=​​​​​​​​​​​​​​​​85 90 95 Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105 110 Gly Leu Pro Leu His Leu Pro Gly Gln Lys Ser Pro His Arg Asp Pro 115 120 125 Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140 Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val 145 150 155 160 Gly Ser Ser Asp Pro Leu Ser Cys 165 <210> 136 <211> 181 <212> PRT <213> artificial sequence <220> <223> synthesis <400> 136 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val 1 5 10 15 Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30 Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45 Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln 50 55 60 Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly 65 70 75 80 Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg 85 90 95 Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105 110 Gly Leu Pro Leu His Leu Pro Gly Gln Lys Ser Pro His Arg Asp Pro 115 120 125 Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140 Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val 145 150 155 160 Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Cys Ser Gln Gly Arg Ser 165 170 175 Pro Ser Tyr Glu Ser 180 <210> 137 <211> 181 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 137 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val 1 5 10 15 Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30 Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45 Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln 50 55 60 Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly 65 70 75 80 Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg 85 90 95 Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105 110 Gly Leu Pro Leu His Leu Pro Gly Gln Lys Ser Pro His Arg Asp Pro 115 120 125 Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140 Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val 145 150 155 160 Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln Cys Arg Ser 165 170 175 Pro Ser Tyr Glu Ser 180 <210> 138 <211> 181 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Substance <400> 138 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val 1 5 10 15 Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30 Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45 Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln 50 55 60 Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly 65 70 75 80 Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg 85 90 95 Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105 110 Gly Leu Pro Leu His Leu Pro Gly Gln Lys Ser Pro His Arg Asp Pro 115 120 125 Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140 Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val 145 150 155 160 Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln Gly Arg Ser 165 170 175 Pro Ser Tyr Cys Ser 180 <210> 139 <211> 181 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 139 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val 1 5 10 15 Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30 Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45 Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln 50 55 60 Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly 65 70 75 80 Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg 85 90 95 Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105 110 Gly Leu Pro Leu His Leu Pro Gly Gln Lys Ser Pro His Arg Asp Pro 115 120 125 Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140 Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val 145 150 155 160 Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln Gly Arg Ser 165 170 175 Pro Ser Tyr Glu Ser 180 <210> 140 <211> 182 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 140 His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val 1 5 10 15 Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25 30 Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser 35 40 45 Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile Gln 50 55 60 Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly 65 70 75 80 Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg 85 90 95 Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His 100 105 110 Gly Leu Pro Leu His Leu Pro Gly Gln Lys Ser Pro His Arg Asp Pro 115 120 125 Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140 Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val 145 150 155 160 Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln Gly Arg Ser 165 170 175 Pro Ser Tyr Glu Ser Cys 180 <210> 141 <211> 24 <212> PRT <213> Artificial sequence <220> <223> composite <400> 141 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 1 5 10 15 Gly Ala Gln Pro Gly Ala Gln Pro 20 <210> 142 <211> 28 <212> PRT <213> Artificial sequence <220> <223> composite <400> 142 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 1 5 10 15 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 20 25 <210> 143 <211> 20 <212> PRT <213> Artificial sequence <220> <223> composite <400> 143 Gly Ser Ala Pro Gly Ser Pro Ala Gly Ser Pro Thr Gly Ser Ala Pro 1 5 10 15 Gly Ser Pro Ala 20 <210> 144 <211> 30 <212> PRT <213> Artificial sequence <220> <223> composite <400> 144 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 30 <210> 145 <211> 80 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Substance <400> 145 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 1 5 10 15 Gly Ala Gln Pro Gly Gln Ala Pro Gly Ala Gln Pro Gly Ala Gln Pro 20 25 30[[ID=3 1]] Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 35 40 45 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 50 55 60 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 65 70 75 80 <210> 146 <211> 80 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Substance <400> 146 Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro 1 5 10 15 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Gln Ala Pro 20 25 30 Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro 35 40 45 Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro 50 55 60 Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro 65 70 75 80 <210> 147 <211> 80 <212> PRT <213> artificial sequence <220> <223> Synthesis <400> 147 Gly Ala Gln Pro Gly Ala Gln Pro Gly Gln Ala Pro Gly Ala Gln Pro 1 5 10 15 Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro 20 25 30 Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro 35 40 45 Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro Gly Path Gln Pro 50 55 60 Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro 65 70 75 80 <210> 148 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> Composite <400> 148 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Cys His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Lys Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Cys Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Glu Ser 225 230 <210> 149 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Substance <400> 149 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Cys His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Lys Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln 210 215 220 Cys Arg Ser Pro Ser Tyr Glu Ser 225 230 <210> 150 <211> 233 <212> PRT <213> Synthetic sequence <220> <223> Synthetic compound <400> 150 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Cys His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Lys Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Glu Ser Cys 225 230 <210> 151 <211> 232 <212> PRT <213> Artificial sequence <220> <223> Composite <400> 151 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Cys His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Lys Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Cys Ser 225 230 <210> 152 <211> 232 [[ID=​​​​​​​​​​​​​​​​​Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly 35 40 45 Ala Gln Cys His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Lys Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Cys Ser Gln 210 215 220 Gly Arg Ser Pro Ser Tyr Glu Ser 225 230 <210> 153 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Substance <400> 153 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Gly Gly Gly 20 25 30 Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly Ala Gln Pro Gly 35 40 45 Ala Gln Cys His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly 50 55 60 Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr 65 70 75 80 Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala 85 90 95 Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly 100 105 110 Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg 115 120 125 Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys 130 135 140 Ser Phe Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser 145 150 155 160 Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Gln Lys Ser Pro His 165 170 175 Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly 180 185 190 Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro 195 200 205 Pro Asp Val Gly Ser Ser Asp Pro Leu Ser Leu Val Gly Gly ...

Claims

1. A polypeptide conjugate comprising a fusion polypeptide and a clearance reducing moiety (CRM), wherein the CRM is conjugated to a conjugable residue in the fusion polypeptide, wherein: 1) The amino acid sequence of the fusion polypeptide is SEQ ID NO: 9, wherein the conjugated residue is a lysine residue at position 231; 2) the amino acid sequence of the fusion polypeptide is SEQ ID NO: 10, wherein the conjugated residue is a lysine residue at position 222; 3) the amino acid sequence of the fusion polypeptide is SEQ ID NO: 11, wherein the conjugated residue is a lysine residue at position 221; 4) the amino acid sequence of the fusion polypeptide is SEQ ID NO: 12, wherein the conjugated residue is a lysine residue at position 225; 5) the amino acid sequence of the fusion polypeptide is SEQ ID NO: 13, wherein the conjugated residue is a lysine residue at position 222; 6) the amino acid sequence of the fusion polypeptide is SEQ ID NO: 14, and the conjugated residue is a lysine residue at position 222; or 7) The amino acid sequence of the fusion polypeptide is SEQ ID NO: 15, wherein the conjugated residue is a lysine residue at position 231; The CRM comprises the following structure:

2. A pharmaceutical composition comprising the polypeptide conjugate according to claim 1 and a pharmaceutically acceptable carrier.

3. Use of the polypeptide conjugate of claim 1 or the pharmaceutical composition of claim 2 in the preparation of a medicament for preventing or treating a metabolic disorder in a subject in need thereof, wherein the metabolic disorder is diabetes, obesity, non-alcoholic steatohepatitis (NASH), arteriosclerosis, diabetic nephropathy, metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), or hepatic steatosis. The use according to claim 3 , wherein the metabolic disorder is fatty liver.

5. A polynucleotide encoding the fusion polypeptide as defined in claim 1. A vector comprising the polynucleotide according to claim 5 . A host cell comprising the vector according to claim 6 .

8. A method for producing a fusion polypeptide as defined in claim 1, comprising culturing the host cell according to claim 7 under conditions which allow expression of the polynucleotide according to claim 5.

9. A method for producing a polypeptide conjugate as defined in claim 1, comprising conjugating a clearance reducing moiety to the fusion polypeptide of claim 1.

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