CN116143939A

CN116143939A - Multiple active protein for treating metabolic diseases

Info

Publication number: CN116143939A
Application number: CN202210991405.0A
Authority: CN
Inventors: 黄岩山
Original assignee: Zhejiang Doer Biologics Co Ltd
Current assignee: Zhejiang Doer Biologics Co Ltd
Priority date: 2017-11-24
Filing date: 2017-11-24
Publication date: 2023-05-23
Also published as: CN109836503A; WO2019101036A1; CN109836503B

Abstract

The invention belongs to the field of biological medicines, and particularly relates to a multi-active protein for treating metabolic diseases. The structural formula of the multiple active protein of the inventionThe method comprises the following steps: A-L-F or A-L ₁ ‑F‑L ₂ -B. Compared with the prior art, the multi-active protein has the following beneficial effects: the multiple active proteins have long half-lives and support the frequency of administration once a week; the GLP-1R agonistic activity of the multi-activity protein is improved to more than 200 times, and the proportion of GCG to GLP-1 is only 1: about 1; the multiple active protein has good stability and low immunogenicity.

Description

Multiple active protein for treating metabolic diseases

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a multi-active protein for treating metabolic diseases.

Background

Diabetes mellitus is classified into type one diabetes mellitus and type two diabetes mellitus according to pathological characteristics. Type one diabetes is mainly manifested by insulin hyposecretion, requiring daily insulin injections; and type II diabetes is caused by the inability of the human body to effectively utilize insulin. Of which type II diabetics account for the vast majority. It is estimated that approximately 80-90% of patients with type II diabetes are significantly obese (Center for disease control and prevention (CDC) National Diabetes Fact Sheet, 2014).

Conventional agents for the treatment of type II diabetes, such as sulfonylureas, thiazolidinediones, etc., have remarkable hypoglycemic effects, but have the major disadvantage of causing weight gain (Kahn SE, haffner SM, heise MA, herman WH, holman RR, jones NP, et al Glycmic durability of rosiglitazone, metformin, or glyburide monotherapy.N Engl J Med 2006;355 (23): 2427-43.). Whereas the protein drugs for type two diabetes are mainly GLP-1R (GLP-1 receptor) agonists, such as dolapride (trade name:

) Abirubu peptide (Albiglutide, trade name->

) Liraglutide (trade name +.>

And

For the treatment of obesity and diabetes, respectively), exenatide (Exenatide, trade name +.>

) Lixisenatide (trade name>

) And a cable Ma Lutai (semaglute) which may be about to be marketed. The GLP-1R agonist has remarkable hypoglycemic effect, and unlike insulin, the hypoglycemic effect of the GLP-1R agonist is strictly blood sugar-dependent, is not easy to cause hypoglycemia, and has the effect of reducing body weight. For example, the reduced weight of dolapride is about 2.9 kgWhile Liraglutide (dose 3 mg) approved for weight loss weighs about 8 kg once a day. The weight loss of these drugs is mainly controlled by appetite and most of them do not exceed 10% of the average body weight. Bariatric surgery (Bariatric surgery), while significantly improving obesity and treating diabetes, is not widely used because most patients are not willing to accept such surgery due to the risk of surgery and long-term sequelae (Obesity and Diabetes, new Surgical and Nonsurgical Approaches, springer press, 2015).

Incretin (Incretin) secretion has been reported to proliferate in patients undergoing surgical bariatric surgery (Obesity and Diabetes, new Surgical and Nonsurgical Approaches, springer press, 2015). Thus, the current new generation of diabetic drugs is mainly focused on the study of dual or multiple effect Incretin (Incretin) receptor agonists, such as GLP-1R/GIPR and GLP-1R/GCGR dual agonists, even GLP-1R/GIPR/GCGR triple agonists.

Among them, the receptors for Glucagon (Glucagon) and GLP-1 (Glucagon-like peptide-1) are structurally related, but these two hormones exhibit diametrically opposite effects in controlling glucose. Clinically, GLP-1 and its analogs are mainly used for glycemic control in diabetics, while Glucagon (Glucago) is used for acute hypoglycemia. In recent years, more and more studies have demonstrated that Glucagon (Glucagon), while at risk of increasing blood glucose, is effective in reducing body weight; more importantly, GLP-1 appears to have a positive additive or electrophysiological effect with Glucoago, e.g., a GLP-1 receptor (GLP-1R) dual agonist can reduce weight more effectively than a GLP-1R single agonist. While GCGR agonism may lead to increased blood glucose levels, this risk may be appropriately offset by GLP-1R agonism.

Currently dual agonists of GLP-1R and GCGR are commonly based on Oxyntomodulin (Oxyntomodulin) or Glucagon and are engineered to ameliorate their shortness of potency and enzymatic breakdown (Oxyntomodulin analogs or Glucagon analogs). Most of these analogues mutate serine (Ser) at the second position to the unnatural amino acid Aib to resist DPP-IV enzymatic cleavage. This is because both natural Glucoago and oxyntomodulin are similar to natural GLP-1and are highly susceptible to inactivation by hydrolysis by DPP-IV protease in serum (Victor A. Gault et al, A novel GLP-1/Glucagon hybrid peptide with triple-acting agonist activity at GIP, GLP-1and Glucagon receptors and therapeutic potential in high-fast fed mie, J Biol Chem 288 (49): 35581-91.2013; bhat VK et al, A DPP-IV-resistance test-acting agonist of GIP, GLP-1and Glucagon receptors with potent glucose-lowering and insulinotropic actions in high-fast-fed mie, diabetes 56 (6): 1417-24.2013;John A.Pospisilik et al; metabolism of Glucagon by dipeptidyl peptidase IV (CD 26), regulatory Peptides96:133-141,2001;Hinke SA et al, dipeptidyl peptidase IV (DPI V/CD 26) degradation of glago. Characterization of Glucagon degradation products and DPIV-resistant analogs, J Biol Chem 275:3827-3834,2000;Alessia Santoprete et al, DPP-IV-Pestat, J acting oxyntomodulin derivatives, scuted et al, 5417-280,2011:5417. The risk of immunogenicity by mutation is extremely high. There are also few Glucago analogs of crosslinked fatty acids that retain the natural Ser at the second position (Henderson SJ et al, robust anti-obesity and metabolic effects of a dual GLP-1/Glucagon receptor peptide agonist in rodents and non-human matrices, diabetes Obes Metab, 2016). The analogue (MEDI 0382) comprises 30 amino acids, mutated by 9 amino acids compared to the natural Glucagon. Meanwhile, although the second position of MEDI0382 also retains the natural Ser amino acid, only once-a-day dosing frequency can be supported.

Oxyntomodulin analogues, although exhibiting a preliminary hypoglycemic and hypolipidemic effect, have a still uncertain mechanism of action: the oxyntomodulin receptor has not been found, and it is only through a mouse or cellular assay with GCGR or GLP-1R knockdown that verifies that oxyntomodulin can bind to these 2 receptors to act. In addition, oxyntomodulin is capable of agonizing GLP-1R and GCGR, but has quite low activity (about one tenth and one hundredth of that of natural GLP-1 and Glucoago, respectively), and oxyntomodulin analogues used in general studies have been designed to have about 1:1 agonizing activity of GLP-1R and GCGR, and most studies consider that the hypoglycemic effect and lipid-lowering effect are best when the activity is about 1:1 (Peptide-based GLP-1/Glucoago co-agonists: a double-edged sword to combat diabesity, hitesh Soni,95:5-9, 2016). In addition, methods based on Glucago sequences have been developed, and most of them are introduced in the form of unnatural amino acids. Because of the extremely high safety requirements of hypoglycemic drugs, one of the difficulties in the development of such receptor agonists is the need to comprehensively consider the influence of immunogenicity, and generally has higher homology with human sequences, the lower the risk of immunogenicity in humans. The production rate of antibody reaches 49% by Taspoglutide (introducing unnatural amino acid Aib), a GLP-1R agonist hypoglycemic agent developed by the cooperation of Roche and Proprietary, finally all phase III clinical studies are stopped (Julio Rosenstock et al, the Fate of Taspoglutide, a Weekly GLP-1Receptor Agonist,Versus wice-Daily, exenatide for Type 2,Diabetes Care,36:498-504,2013).

Another problem relates to the half-life of GLP-1R/GCGR dual agonist. Most of the research at present adopts a way of cross-linking fatty acid or PEG. The fatty acid crosslinking can reduce the activity loss to the greatest extent, however, the half life can only be maintained for about 12 hours, so that the administration mode can only be adopted every day; PEG cross-linking, while more effective in extending half-life relative to fatty acids, causes significant problems with activity impairment. More importantly, there is no fusion Glucoago analog or GLP-1R/GCGR bispecific agonist reported to be effective against DPP-IV degradation. According to the prior art, glucago analogs are combined with F _C Or human serum albumin, and introducing the unnatural amino acid Aib at the second position is extremely difficult to achieve. Whereas, if the natural GLP-1 is mimicked, mutation of the second Ser of the natural Glucoago to other natural amino acids would result in a significant decrease in GCGR agonistic activity (Alessia Santoprete et al, DPP-IV-resistance, long-acting oxyntomodulin derivatives, J.Pept.Sci.,17:270-280,2011). The common method is to chemically synthesize small peptide with unnatural amino acid and then F _C Crosslinking is performed by PEG or fatty acid, etc. Such as HM12525A Crosslinking of human F based on GLP-1R/GCGR bispecific small peptide _C Is prepared by the method. (Jahoon Kang et al, the ultra-long acting LAPSGLP/GCG dual ag, HM12525A, demonstrated safety and prolonged pharmacokinetics in healthy volunteers: alpha phase 1first-in-human study,51st European Association for The Study of Diabetes (EASD), stock holm, sweden; september 14-18,2015).

Disclosure of Invention

In order to overcome the problems in the prior art, the invention aims to provide a multi-domain active protein for treating metabolism and related diseases, and preparation and application thereof. The multiple active protein has obvious weight reducing effect, and can be clinically used for treating related diseases such as diabetes, weight loss, non-alcoholic fatty liver, hyperlipidemia and the like.

In order to achieve the above and other related objects, the present invention adopts the following technical scheme:

the first aspect of the present invention provides a multiple active protein, wherein the structure of the multiple active protein comprises a structure shown in formula I, and the structure shown in formula I is: A-L-F formula I

In the formula I, A is a GCGR/GLP-1R double-effect agonist, F is a long-acting protein unit, and L is a connecting chain for connecting the A and the F.

Further, the GCGR/GLP-1R dual agonist is selected from an analog of native Glucago (SEQ ID NO. 44) or other polypeptide or protein having GCGR/GLP-1R dual agonistic activity. In an embodiment of the present invention, the structure of a includes a structure shown in formula II, where the structure shown in formula II is:

HSQGTFTSDYSKYLD ₁₆ ₁₇ ₁₈ XXXAQDFVQWLMN ₂₉ XX ^z (SEQ ID NO. 141) formula II, wherein X ₁₆ Any one selected from amino acids other than Y, N, W, and H; x is X ₁₇ Any one selected from amino acids other than P, L, T, F and H; x is X ₁₈ Any one of amino acids other than those selected from P, F, H and W; in addition X ₁₇ And X is ₁₈ Can not be R, X at the same time ₂₉ Is T or deletion, X ^z Selected from GGPSSGAPPPS (SEQ ID NO. 3), GPSSGAPPPS (SEQ ID NO. 4), PSSGAPPPS (SEQ ID No. 5), SSGAPPPS (SEQ ID No. 6), GGPSSGAPPS (SEQ ID No. 7), GPSSGAPPS (SEQ ID No. 8), PSSGAPPS (SEQ ID No. 9), or SSGAPPS (SEQ ID No. 10).

The A can resist in vivo protease hydrolysis.

The amino acid sequence of A can be shown as any one of SEQ ID NO.46, SEQ ID NO.55, SEQ ID NO.59, SEQ ID NO.68 and SEQ ID NO. 74.

Currently, most of the hybrid peptides, whether based on Glucoagon or oxyntomodulin, have been engineered at the second position to mutate Ser to the unnatural amino acid Aib or D-form amino acid (D-Ser) (see for details review the literature Peptide-based GLP-1/Glucoagon co-agoniss: a double-edged sword to combat diabesity, hitesh Soni,95:5-9, 2016) to protect against hydrolysis by serum DPP-IV enzyme. Similar to native GLP-1 (SEQ ID NO.1, HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG), native Glucoagon (SEQ ID NO. 44) is highly susceptible to hydrolysis of DPP-IV in serum resulting in inactivation (Victor A. Gault et al, A-level GLP-1/Glucagon hybrid peptide with triple-acting agonist activity at GIP, GLP-1and Glucagon receptors and therapeutic potential in high-fat fed micro, J Biol Chem.,288 (49): 35581-91.2013; bhat VK et al, ADPP-IV-resistance triple-acting agonist of GIP, GLP-1and Glucagon receptors with potent glucose-lowering and insulinotropic actions in high-fat-fed micro, diabetes, 56 (6): 1417-24.2013;John A.Pospisilik et al; metabolism of Glucagon by dipeptidyl peptidase IV (CD 26), regulatory Peptides 96:133-141,2001;Hinke SA et al, dipeptidyl peptidase IV (DPIV/CD 26) degradation of glucose. Characterization of Glucagon degradation products and DPIV-resistant analogs, J Biol Chem 275:3827-3834,2000). However, the inventors have found that the GCG analogs obtained by the screening of the present invention retain Ser in the natural second position, in combination with F _C After fusion, stability was also sufficient to support once-a-week dosing frequency, reducing the potential risk of immunogenicity. Modification at positions 16, 17 and 18, in addition to attenuating degradation of the GCG analog by endopeptidases, also better maintains GCGR agonistic activity. In one embodiment of the invention, multipleA in the structure of the active protein A-L-F is a GCG analogue, and the multiple active protein of the structure A-L-F has extremely high resistance to DPP-IV hydrolysis when compared with the corresponding small peptide without the L-F part. In contrast, as reported, the second Ser of the small peptide, which does not contain an L-F moiety, is highly susceptible to inactivation by DPP-IV attack. Furthermore, the stability of the multiple active proteins of the A-L-F structure in serum is comparable to the analogues of the second introduced unnatural amino acid Aib or D-Ser. There is no prior art at present that discloses fusion F _C To increase the DPP-IV enzyme resistance of the GCG analog. As is well known, the second position of GLP-1 is A, even with F _C After fusion, GLP-1 is still very susceptible to degradation by DPP-IV to hydrolyze the first two amino acids HA. Thus, long acting GLP-1 analogs currently marketed and clinically administered at once a week frequency must have the second amino acid mutated to glycine Gly (e.g., dolapride and Abirudin) or Aib (e.g., soxh Ma Lutai) to maintain N-terminal stability. Similarly, for Glucago analog polypeptides, numerous reports have shown above that GCG analogs with the second amino acid being the natural Ser are highly susceptible to inactivation by DPP-IV attack, and that degradation must be avoided by mutating the second amino acid to an unnatural amino acid while retaining GCGR agonistic activity. According to the prior art, GCG analogues are combined with F _C Fusion and modification of the second amino acid is not possible. Whereas if the natural GLP-1 is mimicked, mutation of the second amino acid Ser of natural Glucago to other natural amino acids would likely result in a significant decrease in GCGR agonistic activity (Alessia Santoprete et al, DPP-IV-resistance, long-acting oxyntomodulin derivatives, J.Pept.Sci.,17:270-280,2011). The only method for realizing the administration period (long half-life) once a week and keeping high activity is to replace the Ser at the second position with the unnatural amino acid and simultaneously use PEG or F _C Crosslinking of The macromolecules (Jahoon Kang et al, the ultra-long acting) ^LAPS GLP/GCG dual agonist,HM12525A,demonstrated safety and prolonged pharmacokinetics in healthy volunteers:a phase 1first-in-human study，51 ^st European Association for the Study of Diabetes (EASD), stockholm, sweden; september 14-18,2015). The inventors have surprisingly found that,preferred GCG analogs and F provided by the present invention _C After fusion, an extremely high DPP-IV resistance is obtained, and pharmacodynamic experiments show that the stable performance supports the administration frequency once a week. This effect is achieved by means of fusion, which is not currently visible. The second Ser is reserved, so that the immunogenicity risk is reduced, the agonistic activity of the GCGR is reserved to the greatest extent, and the weight reduction effect can be effectively achieved.

Ritzel U et al reported that a GLP-1 analog polypeptide with a second mutation from Ala to Ser (Ritzel U et al A synthetic glucagon-like peptide-1analog with improved plasma stability,J Endocrinol, 159 (1): 93-102, 1998) has an effect against DPP-IV. Based on this, pich KM et al (Pich KM et al, protein engineering strategies for sustained glucagon-like peptide-1receptor-dependent control of glucose homeostasis, diabetes,57 (7): 1926-34, 2008) also reported a GLP-1 with HS at the N-terminal and antibody F _C Fusion active protein (CNTO 736). However, as noted above, numerous reports indicate that the N-terminus of a Glucago analog is very unstable when it retains the native HS sequence, and even if the fatty acid is crosslinked, the half-life is difficult to last for more than 12 hours; furthermore, the inventors have found that the natural Glucago with HS sequence is directly related to F _C Fusion (SEQ ID NO. 75), or Glucoagon-cex sequence reported by Joseph R.Chabenne et al (Joseph R.Chabenne et al Optimization of the Native Glucagon Sequence for Medicinal Purposes, J Diabetes Sci technology.4 (6): 1322-1331, 2010) with F _C Fusion (SEQ ID NO. 76) still does not possess significant properties against DPP-IV enzyme. This suggests that although GLP-1 and Glucago belong to members of the Incretin (Incretin) family, the different sequences themselves have large conformational differences and thus differ in their resistance to proteases.

In addition, it has been reported that the 16-18 positions of Glucoago are also sites subject to degradation, so that most Glucoago analogs are engineered or modified at this site (PEG or fatty acid, etc.), while more mutations are introduced at additional sites to balance GCGR and GLP-1R agonistic activity, to obtainA polypeptide having high activity against both receptors. Such as MEDI0382 (Henderson SJ et al, robust anti-obesity and metabolic effects of a dual GLP-1/Glucagon receptor peptide agonist in rodents and non-human matrices, diabetes Obes Metab, 2016) currently in clinical study, comprises 30 amino acids. In comparison to the natural Glucago, 9 mutations were introduced. Meanwhile, although the second position of MEDI0382 also retains the natural Ser amino acid, only once-a-day dosing frequency can be supported. The GCG analog in the invention retains the natural S amino acid at the second position of the N end, only makes mutation of not more than 3 amino acids, and then is matched with a long-acting unit (such as F _C ) Once fused, it is sufficient to support once-a-week dosing frequency. In certain embodiments of the invention, the C-terminal T is further deleted in order to reduce the agonistic activity of the GCGR to balance the ratio of activity of the GCGR to GLP-1R. The deletion of T at the C-terminus has no effect on the stability of the Glucagon analog. Therefore, the invention has the greatest advantages that fewer site mutations are adopted to achieve the aim of optimal receptor agonistic activity and stability, unnatural amino acids are not introduced during mutation, and the potential immunogenicity is reduced and the product is conveniently and directly prepared by utilizing a recombination technology.

The study found that Glucago-cex sequence reported by Joseph R.Chabenne et al was compared with F _C The fusion was made to give an active protein (C002G 12S3A1F4, SEQ ID NO. 76) with no significant DPP-IV resistance (example 4). In ICR mice IPGTT test and DIO mice weight loss test (examples 8 and 9) showed that C002G12S3A1F4 also had no significant and sustained hypoglycemic effect. At the same time, it can be seen in example 4 that, although it is likewise a mutant F at positions 16-18 _C The stability of the fused GCG analog, different dimeric double-acting proteins, is greatly different. The spatial conformation of the protein is extremely complex, so that the mutation according to formula II not only improves the stability of the peptide chain interior, but also makes it possible to alter the GCG analog with F _C The interaction conformation between chains further improves the stability of the N-terminus of the fusion protein. More importantly, compared with the existing GCG and mutants, the A-L-F structural protein provided by the invention has the advantage of obviously improved GLP-1R agonistic activity. Although the following are providedHowever Joseph R.Chabenne et al and Richard D.DiMarchi et al reported that adding a C-terminal small peptide cex of Exendin-4 (SEQ ID NO.4, GPSSGAPPPS) to the C-terminus of Glumagon increased the GLP-1R agonistic activity from 0.7% to 1.6% by about 2-fold (Optimization of the Native Glucagon Sequence for Medicinal Purposes, J Diabetes Sci technology.4 (6): 1322-1331, 2010 and patent US 9018164B 2), but the ratio of GCGR agonistic activity to GLP-1R agonistic activity was only 35: about 1. Further, evers A et al report (Evers A et al, design of Novel Exendin-Based Dual Glucagon-like Peptide1 (GLP-1)/Glucagon Receptor Agonists, J Med chem.;60 (10): 4293-4303.2017) that GLP-1R agonistic activity was reduced by about 3-fold and GCG activity was reduced by about 14-fold after addition of cex sequence to the C-terminus of the GCG analog (Table 2, peptides 7 and 8).

That is, simply adding the C-terminal peptide cex sequence of Exendin-4 (e.g., GPSSGAPPPS) to the C-terminus of native Glucago does not significantly increase or even further attenuate the agonistic activity of GLP-1R. On the other hand, when small peptides such as Glucoago and GLP-1 are expressed in fusion with carrier fusion proteins such as Fc and albumin, the activity tends to be significantly reduced due to steric hindrance (YAN-SHAN HUANG et al Preparation and characterization of a novel exendin-4human serum albumin fusion protein expressed in Pichia pastoris,J.Pept.Sci.2008;14:588-595), and this change in activity is unpredictable. The inventors have unexpectedly found that the effect on GLP-1R activity is quite different from that of GCGR after fusion of a GCG analog to Fc. Additional cex or analogous sequence (SEQ ID NO. 3-10) is added to the C-terminus of the GCG analog and fused further to F _C Chain-time, the analog of GCG is directly connected with F _C Compared with chain fusion, the GLP-1R agonistic activity retention rate of the structure containing the cex sequence is obviously improved by more than 200 times, but the GCG activity retention rate is basically unchanged and even slightly reduced.

In addition, a well-known concept for those skilled in the art, especially those skilled in the art of recombinant protein medicine, is: the mutation at any one site on the protein sequence is not expected accurately, and especially for small peptides with the number of amino acids of GLP-1, exendin-4 or Glucago being only 30-40, the effect of single site mutation or simultaneous mutation at a plurality of sites is more difficult to predict. For example, it was reported by Joseph Chabenne et al (Joseph Chabenne et al, A Glucagon analog chemically stabilized for immediate treatment of life-threatening hypoglycemia, molecular Metabolism,3:293-300, 2014) that alanine scanning (Ala scan) was performed on Glucoago (SEQ ID NO. 44), that the relative residual activity remained spanning from 0.2% to 100% after each site of Glucoago was independently replaced with alanine, and that mutations at

positions

1, 2, 3, 4, 6-12, 14, 15, 22, 23, 25-27, 29 of Glucoago would significantly attenuate GCGR agonistic activity (Table 4 in the article). However, it can be seen in other reports that the simultaneous mutation of several of these sites with other amino acid substitutions does not always correspond to the results of alanine scanning. As reported by Jonathan W Day et al (Jonathan W Day et al, A new Glucagon and GLP-1co-agonist eliminates obesity in rodents, nature Chemical Biology,5:749-757, 2009) hybrid peptide (Chimera 2 herein), mutation of Val at position 23 to Ile slightly increased the agonistic activity of GCGR. However, alanine scanning showed that substitution with alanine at position 23 resulted in essentially complete loss of GCGR agonistic activity (down to only 1.1% remaining).

Most importantly, the invention provides a multi-active protein with GCGR/GLP-1R double-effect agonism activity. Currently dual agonists of GLP-1R and GCGR are commonly engineered based on the native Oxynomodulin (SEQ ID NO.2, HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNN IA) or Glucago (SEQ ID NO. 44) sequences. The agonistic activity of the analogues used in the general studies on GLP-1R and GCGR was generally about 1:1, and most studies considered that the 1:1 activity was the best in reducing blood glucose and lipid (Peptide-based GLP-1/Glucago co-agonists: a double-edged sword to combat diabesity, hitesh Soni,95:5-9, 2016).

Oxynomodulin (SEQ ID NO. 2) has a relatively low agonistic activity per se for both receptors, whereas Glucago (SEQ ID NO. 44) has an increased agonistic activity for GLP-1R after sequence mutation, but the activity is inevitably decreased after addition of PEG or fatty acid, especially in the case of PEG modification. However, the dual active proteins of the present invention retain most of the full GLP-1R and GCGR agonistic activity. The increase in activity predicts a decrease in dose, and lower doses allow for smoother glycemic control, thereby improving ease of administration well and reducing the risk of potential immunogenicity. It is well known that side effects of GLP-1 analogs and fusion proteins thereof, including dizziness, nausea, etc., are dose dependent, and that lowering the dose can reduce gastrointestinal side effects. Meanwhile, glucago can improve the metabolism rate, increase the fat consumption, play a more remarkable role in reducing weight, reduce the risk of hypoglycemia, and is suitable for being combined with other hypoglycemic agents such as insulin and the like.

GLP-1R and GCGR agonism and downstream signaling and their physiological effects are extremely complex and as yet not completely understood. At present, the coincidence is considered to be approximately: glucose enters pancreatic beta cells through GLUT2 and glycolysis occurs and pyruvate is produced. Pyruvic acid enters mitochondria for oxidative metabolism and ATP production. Intracellular ATP increases will shut off K _ATP (ATP-sensitive potassium ion, ATP-sensitive potassium) channel, depolarizing the envelope, opening up the calcium channel, increasing extracellular calcium influx, a series of changes that lead to insulin exocrine. GLP-1 increases insulin exocrine secretion through a range of mechanisms: GLP-1R binds to G.alpha.s, activates adenylate cyclase, converts ATP to cAMP, and mobilizes PKA and Epac signaling factors downstream. This results in a series of cellular reactions, including the turning off of K _ATP The channel promotes fusion of insulin secretory granules with the envelope (Chris de Graaf et al, glucago-Like Peptide-1and Its Class B G Protein-Coupled Receptors: A Long March to Therapeutic Successes, pharmacological Reviews,68 (4) 954-1013, 2016). GCGR is similar to GLP-1R, and intracellular cAMP up-regulation occurs upon binding to Glucago. GLP-1R and GCGR both belong to the GPCR family, with 7 transmembrane regions. After binding to the respective ligand, the C-terminal end of the receptor is phosphorylated, and beta-arrestin (beta-arestin) is enriched and bound to the receptor, most Eventually leading to endocytosis of the receptor (Jorgensen, R et al, oxyntomodulin differentially affects Glucagon-like peptide-1receptor-arrestin recruitment and signaling through G α, J.Pharmacol. Exp. Ther.322,148-154, 2007).

It has been shown that GCGR has a different endocytosis efficiency than GLP-1R, and that endocytosis of the Receptor affects downstream signal transduction (Functional Consequences of Glucagon-like Peptide-1Receptor Cross-talk and Trafficking, J Biol Chem, sarah Noerklit Roed, etc. 290 (2): 1233-1243, 2015), ultimately possibly affecting tissue cell physiological function. As indicated by Kuna, r.s. Et al, blocking GLP-1R endocytosis reduces insulin release from pancreatic cells (Glucagon-like peptide-1receptor-mediated endosomal cAMP generation promotes glucose-stimulated insulin secretion in pancreatic-cells, kuna, r.s. Et al, am.j. Physiol. Endocrinol Metab,305, E161-E170, 2013). In addition, there is a difference in endocytosis efficiency between the two types of receptors, e.g., the endocytosis efficiency of GCGR is lower than that of GLP-1R. In order to avoid that dual-effect agonists of GLP-1R and GCGR are simultaneously combined with GLP-1R and GCGR, the existing dual-effect agonists of GLP-1R and GCGR are mostly based on one peptide chain structure. However, if at F _C The double-effect agonist of GLP-1R and GCGR is constructed on the basis of bivalent protein, and can be combined with two receptors of GLP-1R and GCGR simultaneously, so that heterodimerization and crosslinking of the two receptors are caused to influence endocytosis of the respective receptors, and intracellular signal transmission is influenced to influence normal physiological functions of the receptors. There have been few reports of co-expression of GCGR and GLP-1R on the same tissue cell surface (Dominik Schelshorn et al, lateral Allosterism in the Glucagon Receptor Family: glucoago-Like Peptide 1 derivatives G-Protein-Coupled Receptor Heteromer Formation, molecular Pharmacology,81 (3) 309-318, 2012). It was also found during the course of the present invention that the ability of GCGR/GLP-1R dual agonist active proteins of different structures to induce insulin in rat BRIN-BD11 cells was completely different. A number of factors influence the effects of heterodimerization. Therefore, how to obtain GLP-1R and GCGR dual-effect agonists with normal physiological effects is an extremely difficult task. Such as GLP-1R/GCGR receptor agonist and F _C Length and structure of connecting peptide betweenNot only affects the activity of the protein, but also is related to GLP-1R and GCGR crosslinking. In a preferred embodiment, the linker is a flexible polypeptide of glycine (G), serine (S) and/or alanine (a) of suitable length, and preferably of suitable sequence and length to reduce potential receptor heterodimerization such that the bivalent dual agonist still has the best GLP-1R and GCGR agonistic activity.

Preferred connecting chains of the invention include those containing units enriched in G, S and/or A, such as exemplified by (GS) n, (GGS) n, (GGSG) n, (GGGS) nA, (GGGGS) nA, (GGGGA) nA, etc., n being an integer from 1 to 10, in a preferred embodiment the amino acid length of the connecting chain is from 5 to 26. Exemplary connecting chains are each independently selected from table 2.

Further, the amino acid sequence of the connecting chain L in the formula I can be shown as any one of SEQ ID NO. 21-43.

Further, the F is F derived from a mammalian immunoglobulin _C Part(s). The immunoglobulin is a disulfide-bond containing polypeptide chain molecule, typically having two light chains and two heavy chains. Immunoglobulin F as used herein _C Part has the usual meaning of terms in the field of immunology. In particular, the term refers to antibody fragments obtained by removing two antigen binding regions (Fab fragments) from an antibody. F (F) _C The portion may include a hinge region and extend through the CH ₂ And CH (CH) ₃ The domain reaches the C-terminus of the antibody. F (F) _C The moiety may further comprise one or more glycosylation sites. The human body has 5 human immunoglobulins with different effector characteristics and pharmacokinetic properties: igG, igA, igM, igD and IgE. IgG is the highest content of immunoglobulins in serum. IgG also has the longest serum half-life (about 23 days) among all immunoglobulins.

Further, F may be selected from the group consisting of complete F of immunoglobulin _C Part of immunoglobulin F _C Fragments of parts or F of immunoglobulins _C Partial mutants.

Immunoglobulin F for use in the invention _C F derived in part from mammalian IgG1, igG2 or IgG4 _C A region or mutant thereof; preferably, it may be derived from human IgG1, igG2 or IgG 4F _C A region or mutant thereof; more preferably, F may be derived from human IgG1 or IgG4 _C A region or a mutant thereof. In a preferred embodiment, F _C The position 297 of the domain is replaced with glycine or alanine. The above is numbered according to the EU index of kabat (kabat, E.A. et al, sequences of proteins of immunological interest, fifth edition, public health service, national Institutes of Health, bethesda, MD (1991)).

In a preferred embodiment, F _C The domain is from human IgG1 and is shown in SEQ ID NO. 12. In a preferred embodiment, said F _C The domain is from human IgG4 as shown in SEQ ID NO. 16. The F is _C The removal of K at the chain end facilitates improved homogeneity of the expression product.

The amino acid sequence of F can be shown in any one of SEQ ID NO. 11-20.

The multiple active protein provided by the invention is F _C Fusion protein, reserve F _C Such as binding to FcRn to extend in vivo half-life. In addition, the multiple active proteins can effectively resist the degradation of protease in serum to the inside of the protein and also can effectively prevent the degradation of N-terminal. For an Incretin-type polypeptide, such as Glucago or GLP-1, the N-terminal integrity is critical in determining its biological activity. Natural Glucagon and GLP-1 have a short half-life in vivo, and in addition to this reason of small molecular weight, it is more important to be due to DPP-IV enzymatic hydrolysis in the receptor. In one embodiment of the invention, natural Glucago and F _C After fusion, the fusion is still rapidly degraded by DPP-IV to be inactivated; whereas the corresponding Glucago analogues are significantly resistant to DPP-IV attack. Both liraglutide and MEDI0382 described above retain the second natural amino acid, but the dosing cycle can only support once a day. The multiple active proteins of the present invention, however, retain the second natural amino acid, with a significant increase in stability sufficient to support once-a-week dosing frequency.

Further, a second aspect of the present invention provides a multi-stageThe heavy active protein has triple-effect agonistic activity, and the structural formula of the heavy active protein comprises a structure shown in a formula III, wherein the structure shown in the formula III is as follows: A-L ₁ -F-L ₂ -B, wherein a is a GCGR/GLP-1R dual agonist, F is a long acting protein unit, B is native FGF21 (SEQ ID No. 143) or FGF21 analogue, L ₁ For the connecting strand, the sequence is selected from any one of SEQ ID NOS.21-43; l (L) ₂ Absent or selected from any one of SEQ ID NOS.21-43.

Further, the structure of A comprises a structure shown in a formula II, and the structure shown in the formula II is as follows:

HSQGTFTSDYSKYLD ₁₆ ₁₇ ₁₈ XXXAQDFVQWLMN ₂₉ XX ^z (SEQ ID NO. 141) formula II, wherein X ₁₆ Any one selected from amino acids other than Y, N, W, and H; x is X ₁₇ Any one selected from amino acids other than P, L, T, F and H; x is X ₁₈ Any one selected from amino acids other than P, F, H and W; in addition X ₁₇ And X is ₁₈ Can not be R, X at the same time ₂₉ Is T or deletion, X ^z Selected from any one of GGPSSGAPPPS (SEQ ID NO. 3), GPSSGAPPPS (SEQ ID NO. 4), PSSGAPPPS (SEQ ID NO. 5), SSGAPPPS (SEQ ID NO. 6), GGPSSGAPPS (SEQ ID NO. 7), GPSSGAPPS (SEQ ID NO. 8), PSSGAPPS (SEQ ID NO. 9) or SSGAPPS (SEQ ID NO. 10).

The amino acid sequence of A can be shown as any one of SEQ ID NO.46, SEQ ID NO.54, SEQ ID NO.55 and SEQ ID NO. 68.

The FGF21 analogue described by formula III may be selected from FGF21 analogues or mutants as described in patents or patent applications such as US20140213512, US8188040, US9493530, WO 2016114633, US 20150291677, US 9422353, US 8541369, US7622445, US7576190, US20070142278, US 9006400 or US 20130252884. Further, the amino acid sequence of the FGF21 analogue is shown as SEQ ID NO.144, SEQ ID NO.145 or SEQ ID NO. 146. In another in vivo efficacy embodiment of the invention, the triple active protein group has a more pronounced weight-reducing effect than the same dose of the double active protein + FGF21 analogue co-administered group. But not so much on appetite. It was demonstrated that the side effects of the tri-active protein could be lower and the safety was improved (example 12).

The B of formula III may also be natural leptin (SEQ ID NO. 155) and analogues thereof, selected from the variants, derivatives or analogues described in U.S. Pat. No. 7307142, U.S. Pat. No. 7423113 or U.S. Pat. No. 3,084, etc. patents or patent applications;

the B described in formula III may also be amyl and its analogues.

In a second aspect of the invention, there is provided an isolated polynucleotide encoding the aforementioned multiple active protein.

In a third aspect of the invention there is provided a recombinant expression vector comprising the aforementioned isolated polynucleotide.

In a fourth aspect of the invention, there is provided a host cell comprising the recombinant expression vector described above or the isolated polynucleotide described above having an exogenous source integrated into the genome.

In a fifth aspect of the present invention, there is provided a method for producing the aforementioned multi-active protein, comprising culturing the aforementioned host cell under appropriate conditions to express the multi-active protein, and then isolating and purifying the multi-active protein.

In a sixth aspect of the invention, there is provided the use of a multi-active protein as hereinbefore described in the manufacture of a medicament for the treatment of a disease associated with diabetes metabolism.

The multi-active protein provided by the invention can be used for treating metabolic syndrome. Metabolic syndrome is generally characterized by clustering at least three or more of the following risk factors: (1) abdominal obesity (excessive adipose tissue in or around the abdomen), (2) atherogenic dyslipidemia, including high triglycerides, low HDL cholesterol and high LDL cholesterol, which enhance plaque accumulation in the arterial wall, (3) elevated blood pressure, (4) insulin resistance or glucose intolerance, (5) thrombotic conditions such as high fibrin or plasminogen activator inhibitor-1 in the blood, and (6) pro-inflammatory conditions such as elevated C-reactive protein in the blood. Other risk factors may include aging, hormonal imbalance, and genetic factors.

In addition, the multiple active proteins of the invention are also useful in the treatment of obesity. In some aspects, the multiple active proteins of the invention treat obesity by mechanisms such as decreased appetite, decreased food intake, decreased fat levels in a patient, increased energy expenditure, and the like.

In a seventh aspect of the invention, there is provided a method of treating a metabolic-related disorder comprising administering to a subject the aforementioned multi-active protein.

The invention further provides a method of promoting weight loss or preventing weight gain comprising administering said multiple active protein to a subject.

In an eighth aspect of the invention, there is provided a composition comprising a culture of the aforementioned multi-active protein or the aforementioned host cell, and a pharmaceutically acceptable carrier.

In a ninth aspect of the invention there is provided the use of a multiplex active protein as hereinbefore described in the preparation of a fusion protein.

In a tenth aspect of the present invention there is provided a multidomain protein comprising the aforementioned multiple active protein in its structure.

In summary, compared with the prior art, the invention has the following beneficial effects:

(1) The multiple active proteins of the invention have long half-lives and support the frequency of administration once a week;

(2) The GLP-1R agonism activity of the multi-activity protein is improved to more than 200 times at most;

(3) The multi-activity protein has good in-vivo and in-vitro stability and low immunogenicity.

(4) The non-natural amino acid is not required to be introduced, and chemical synthesis and crosslinking steps are not required to be involved, so that the preparation can be realized through a recombination method, and the preparation process is greatly simplified.

Drawings

FIG. 1 shows the electrophoresis pattern (10% SDS-PAGE) of purified partial recombinant proteins, lanes 1-6 are C240G12S3A1F4, C368G12S3A1F4, C225G12S3A1F4, C495G12S3A1F4, C382G12S3A1F4 and C462G12S3A1F4 non-reduced samples, respectively; 7-12 are samples subjected to reduction treatment of C240G12S3A1F4, C368G12S3A1F4, C225G12S3A1F4, C495G12S3A1F4, C382G12S3A1F4 and C462G12S3A1F4, respectively; m is a protein standard substance: 97.2, 66.4, 44.3, 29, 20.1, 14.3KD.

FIG. 2A is a graph of GCGR survival.

FIG. 2B is a graph of GLP-1R activity results.

Fig. 3A: results of serum stability over time.

Fig. 3B: results of serum stability over time.

Fig. 3C: results of serum stability over time.

Fig. 3D: results of serum stability over time.

Fig. 4: stimulation of islet cells by dimeric recombinant proteins obtained in examples 6 and 7 from fusion of GCG analogs with different length connecting chains and F (SEQ ID No. 12).

Fig. 5: figure of the hypoglycemic effect of the dimeric recombinant protein of example 8 in normal ICR mice.

Fig. 6: effect of the dimeric recombinant protein in example 9 on DIO mouse body weight.

Fig. 7: example 11 electrophoresis pattern of fusion proteins obtained by purification (10% SDS-PAGE), lanes 1-6 are non-reducing treated samples of C382F4FGF1, C382F4FGF2, C382F4FGF3, C495F4FGF1, C495F4FGF2 and C495F4FGF3, respectively; 7-12 are samples reduced by C382F4FGF1, C382F4FGF2, C382F4FGF3, C495F4FGF1, C495F4FGF2 and C495F4FGF3, respectively; m is a self-made protein standard substance: 140. 97.2, 66.4, 44.3, 29, 20.1, 14.3KD.

Fig. 8: effect of the three active proteins in example 12 on DIO mouse body weight.

Fig. 9: effect of the three active proteins in example 12 on diet of DIO mice. The food intake of the DIO mice in the PBS group was 100%, and the ordinate represents the percentage of food intake in the other groups.

Fig. 10: effect of the three active proteins in example 14 on DIO mouse body weight.

Fig. 11: effect of the three active proteins in example 14 on diet of DIO mice. The food intake of the DIO mice in the PBS group was 100%, and the ordinate represents the percentage of food intake in the other groups.

Detailed Description

Term interpretation:

the term "diabetes" includes type one diabetes, type two diabetes, gestational diabetes, and other symptoms that cause hyperglycemia. The term is used for metabolic disorders in which the pancreas does not produce enough insulin, or cells of the body fail to respond appropriately to insulin, so that the efficiency of glucose uptake by tissue cells decreases resulting in glucose accumulation in the blood.

Type one diabetes, also known as insulin dependent diabetes mellitus and juvenile onset diabetes mellitus, is caused by beta cell destruction, often resulting in absolute insulin deficiency.

Type II diabetes, also known as non-insulin dependent diabetes mellitus and adult onset diabetes mellitus, is commonly associated with insulin resistance.

The term "obesity" means an excess of adipose tissue, which is stored in fat when energy intake exceeds energy expenditure, resulting in obesity. Obesity is herein optimally regarded as the formation of excess adipose tissue of any extent that is dangerous to health. Individuals having a body mass index (BMI = weight (kilograms) divided by height (meters) squared) of greater than 25 are considered obese herein.

Incretin (Incretin): incretins are gastrointestinal hormones that regulate blood glucose by enhancing glucose-stimulated insulin secretion (also known as glucose-dependent insulin secretion, GSIS) (Dracker. D J, nauck, mass., lancet368:1696-705, 2006). Incretins can also slow down the rate of nutrient absorption and directly reduce food absorption by delaying gastric emptying. At the same time, incretins also inhibit the secretion of Glucagon (Glucagon) by intestinal alpha cells. There are two known incretins to date: glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP).

preproGlucagon (preproGlucagon): is a precursor polypeptide of 158 amino acids that is differentially processed in tissue to form a variety of structurally related pro-Glucagon derived peptides, including Glucagon (Glucagon), glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), and Oxyntomodulin (OXM).

GIP: is a 42 amino acid peptide derived from a 133 amino acid precursor (pre-pro-GIP) by proteolytic processing, and these molecules are involved in a variety of biological functions including glucose homeostasis, insulin secretion, gastric emptying and intestinal growth, and regulation of food intake.

Glucagon-like peptide (GLP-1): is a 30 or 31 amino acid polypeptide incretin hormone secreted from intestinal L-cells, and has two active forms of GLP-1 (7-36) and GLP-1 (7-37). GLP-1 is released into the circulation after a meal and exerts its biological activity by activating the GLP-1 receptor. GLP-1 has many biological effects including glucose-dependent insulinotropic secretion, inhibition of glucagon production, delay of gastric emptying, and appetite suppression (Tharakan G, tan T, bloom S.Emerging therapies in the treatment of 'diabedity': before-b-on GLP-1.Trends Pharmacol Sci2011;32 (1): 8-15), among others. Natural GLP-1 has limited therapeutic potential due to its ability to be rapidly degraded by dipeptidyl peptidase-4 (DPP-4), neutral Endopeptidase (NEP), plasma kallikrein, or plasmin, etc. Because natural GLP-1 has an ultrashort half-life of only about 2 minutes in vivo, methods for treating diabetes and obesity by improving efficacy using chemical modifications and/or formulation formats have emerged (Lorenz M, evers A, wagner M. Recent progress and future options in the development of GLP-1receptor agonists for the treatment of diabesity.Bioorg Med Chem Lett 2013;23 (14): 4011-8.Tomlinson B,Hu M,Zhang Y,Chan P,Liu ZM.An overview of new GLP-1receptor agonists for type 2diabetes.Expert Opin Investig Drugs 2016;25 (2): 145-58).

Oxyntomodulin (Oxyntomodulin) is a small peptide of 37 amino acids, the sequence of which is shown in SEQ ID NO:2 is shown in the figure; it comprises the complete 29 amino acid sequence of Glucagon Glucago (SEQ ID NO. 44). Oxyntomodulin is a dual agonist of GLP-1R and GCGR, secreted together with GLP-1 by intestinal L-cells after meal. Similar to Glucagon glucon, oxyntomodulin produces significant weight loss in humans and rodents. The weight loss activity of oxyntomodulin has been compared in obese mice with equimolar doses of a selective GLP-1 agonist. Oxyntomodulin has been found to have an antihyperglycemic effect, a significant weight loss and a lipid lowering activity compared to the selective GLP-1R agonists (The Glucagon receptor is involved in mediating the body weight-lowering effects of oxyntomodulin, kosinski JR et al, obesity (Silver Spring), 20): 1566-71, 2012). In overweight and obese patients, subcutaneous administration of natural oxyntomodulin reduced body weight by 1.7 kg within four weeks. Oxyntomodulin has also been shown to reduce food intake and increase energy expenditure in humans (Subcutaneous oxyntomodulin reduces body weight in overweight and obese subjects: a double-blind, randomised, controlled trial, wynne K et al, diabetes,54:2390-5, 2005;Oxyntomodulin increases energy expenditure in addition to decreasing energy intake in overweight and obese humans:aandomized controlled trial;Wynne K et al, int J Obes (Lond), 30:1729-36, 2006). However, oxyntomodulin has a short half-life, also due to the small molecular weight and degradation of DPP-IV. Currently dual-effect agonists of the GLP-1 receptor (GLP-1R) and the glucagon receptor (GCGR) are commonly based on oxyntomodulin, and are mutated (oxyntomodulin analogues) in order to improve the shortness of action and enzymatic hydrolysis of oxyntomodulin, and the method of mutating the serine Ser at the second position into alpha-aminoisobutyric acid (Aib) is mostly adopted, and the enzymatic hydrolysis of DPP-IV is resisted by introducing unnatural amino acids. Oxyntomodulin analogues, although exhibiting preliminary hypoglycemic and hypolipidemic effects, have a still unknown mechanism of action, and oxyntomodulin receptors have not been found, and oxyntomodulin has been shown to act by binding to these 2 receptors only in mice or cell assays in which GCGR or GLP-1R is knocked out.

Glucagon (Glucagon) is a 29 amino acid peptide corresponding to amino acids 53-81 of preproglucagon, the sequence of which is shown in SEQ ID NO.44 (C.G.Fanelli et al, nutrition, metabolism & Cardiovascular Diseases (2006) 16, S28-S34). Glucagon receptor activation has been shown to increase energy expenditure and reduce food intake in both rodents and humans (habeger k.m. et al the metabolic actions of Glucagon revisited, nat.rev. Endocrinol.2010,6, 689-697) and these effects are stable and sustained in rodents. Glucagon has many physiological effects, such as by stimulating glycogenolysis and gluconeogenesis, increasing blood glucose levels in hypoglycemic conditions, regulating liver ketone production, regulating bile acid metabolism, and satiety effects through the vagus nerve. Therapeutically, glucagon has been used in acute hypoglycemia, with glucagon receptor activation reducing food intake and promoting lipolysis and weight loss in animals and humans.

The term "receptor agonist" may be defined as a polypeptide, protein, or other small molecule that binds to a receptor and elicits the usual response of a natural ligand.

A "GLP-1 receptor (GLP-1R) agonist" may be defined as a polypeptide, protein, or other small molecule that binds to GLP-1R and is capable of eliciting a characteristic response that is the same or similar to native GLP-1. GLP-1R agonists produce the corresponding cellular activity by activating GLP-1R either completely or partially, which in turn causes a series of intracellular downstream signaling pathway responses: such as insulin secretion by beta cells; typical GLP-1R agonists include native GLP-1 and mutants, analogs thereof, such as exenatide, liraglutide (Liraglutide), and the like.

GLP-1R analogs: as used herein, "GLP-1 analog" or "GLP-1 mutant" are both GLP-1R agonists and are mutually generic.

Glucagon Receptor (GCGR) agonists, i.e., glucogen receptor agonists, may be defined as polypeptides, proteins, or other small molecules that bind to GCGR and are capable of eliciting the same or similar characteristic responses as native Glucagon (glucogen). GCGR agonists produce corresponding cellular activity by activating GCGR either fully or partially, which in turn causes a series of intracellular downstream signaling pathway responses: such as glycogenolysis, glycogenesis, fatty acid oxidation, ketogenesis, etc.

Glucago analogs: as used herein, "Glucago analog", "GCG analog", "Glucago mutant" and "GCG mutant" are intended to mean Glucago receptor agonists, which are mutually generic.

GCGR/GLP-1R dual agonist: the GCGR/GLP-1R dual-effect agonists of the present invention include proteins or polypeptides that are capable of agonizing GLP-1R and GCGR simultaneously. Oxynodomodulin-based dual-effect agonists as reported by Alessandro Pocai et al (Glucago-Like Peptide 1/Glucagon Receptor Dual Agonism Reverses Obesity in Mice, diabetes;58 (10): 2258-2266, 2009), or Glucago-based dual-effect agonists as reported by Richard D.DiMarchi et al (US 9018164B 2). As used herein, a "dual agonist" or "dual active protein" is synonymous.

FGF21: fibroblast growth factor (fibroblast growth factor, FGF), also known as heparin binding growth factor (heparin binding growth factor), is a class of polypeptide substances secreted primarily by the pituitary and hypothalamus. FGF has various effects such as promotion of fibroblast mitosis, growth of mesodermal cells, stimulation of angiogenesis, etc. FGF21 is an important member of the FGF family, and this hormone is currently being developed as a drug for use as a weight-loss drug and for the treatment of diabetes, and has entered the clinical trial stage. FGF21 plays a physiological role through the FGF21 receptor and the co-receptor β -klotho.

Leptin (leptin): mainly produced by white adipose tissue. The precursor consists of 167 amino acid residues, and comprises a signal peptide of 21 amino acids at the N-terminal, and the signal peptide of the precursor is cut off in blood to become leptin mature peptide (leptin) of 146 amino acids. Leptin has a wide range of biological effects such as acting on the metabolic regulation center of the hypothalamus, exerting an effect of suppressing appetite, reducing energy intake, increasing energy consumption, and suppressing fat synthesis.

Dimer: the dimer referred to in the present invention is a dimer formed by the constant region (F _C ) Natural non-covalent and covalent interactions. F unless otherwise specifically indicated _C The dimers formed are all homodimers, as described for the dimers provided herein.

Dimer double-effect active protein: herein, "dual active protein," "dimer dual active," and "dual agonistic active protein" are synonymous and are used interchangeably. Meaning ofRefers to a fusion protein with GCGR and GLP-1R agonistic activity, and the fusion protein has F _C The moiety, thus, has two peptide chains, and forms a dimeric structure by non-covalent and covalent interactions between the two peptide chains.

Dimeric three-way active protein: as used herein, "triple-effect active protein", "triple-effect agonistic active protein", "triple-active agonistic active protein", "dimeric triple-effect active protein" are synonymous and are used interchangeably. Meaning fusion proteins having both GCGR, GLP-1R agonistic activity and FGF21 activity (or leptin activity), which fusion proteins are due to the presence of F _C The moiety, thus, has two peptide chains, and forms a dimeric structure by non-covalent and covalent interactions between the two peptide chains.

IC ₅₀ (half maximal inhibitory concentration) refers to the half-inhibitory concentration of the antagonist being measured. It indicates the concentration of a drug or substance (inhibitor) required to inhibit its corresponding 50% biological response (or some substance contained in the response, such as an enzyme, cellular receptor or microorganism). The lower the IC50 value, the more inhibitory the drug or substance, e.g., more intuitively, the better the binding affinity to the receptor. Is a measure of the effectiveness of a substance in inhibiting a particular biological or biochemical function.

EC ₅₀ (concentration for 50%of maximal effect) refers to the concentration of a drug or substance required to stimulate 50% of its corresponding biological response. The lower the EC50 value, the more potent the stimulation or agonism of the drug or substance, e.g., more intuitively, the more potent the intracellular signal that can be seen, and thus the better the ability to induce the production of a hormone.

Low Density Lipoprotein (LDL): one of the plasma lipoproteins is the main carrier of cholesterol in the blood, which tends to deposit cholesterol on the arterial wall. Leukocytes attempt to digest low density lipoproteins, but in the process they become toxins. More and more leukocytes are attracted to the areas where changes occur, so that the arterial wall may become inflamed. Over time, these plaque deposits can accumulate on the arterial wall as the process continues, such that the passageway becomes very narrow and lacks toughness. If too much plaque builds up, the artery may be completely occluded. When the complex of LDL and cholesterol (LDL-C) creates too much plaque on the arterial wall, blood will not flow freely through the artery. Plaque may collapse suddenly in the artery at any time, resulting in blockage of the blood vessels and eventual heart disease.

High density protein (HDL): helps to clear LDL from the artery, acts as a scavenger, and cleans LDL from the artery and back to the liver.

Triglyceride (TG): is another type of fat used to store the energy of overeating. High levels of triglycerides in the blood are associated with atherosclerosis. High triglycerides can be caused by overweight and obesity, lack of physical movement, smoking, excessive alcohol consumption and high carbohydrate intake (over 60% of total calories). Sometimes time-based or genetic diseases are the cause of high triglycerides. People with high triglycerides typically have high total cholesterol levels, including high LDL cholesterol and low HDL cholesterol, as well as many people with heart disease or diabetes.

Cell biological Activity

In-vitro cell activity measurement of GLP-1R and GCGR agonistic activity of the invention adopts a luciferase reporter gene detection method. This approach is based on the principle that GLP-1R and GCGR activate the downstream cAMP pathway after agonism. The activity of FGF21 and analogues thereof was determined by co-transfecting FGF21R with β -klotho in the same CHO cell and detecting the change in fluorescence caused by the signal.

Joseph R.Chabenne et al and Richard D.DiMarchi et al have reported that adding a C-terminal small peptide cex (GPSSGAPPPS) from Exendin-4 to the C-terminus of Glucago can increase GLP-1R agonistic activity by about 2-fold (Optimization of the Native Glucagon Sequence for Medicinal Purposes, J Diabetes Sci technology.4 (6): 1322-1331, 2010 and patent US 9018164B 2). Further, evers A et al report (Evers A et al, design of Novel Exendin-Based Dual Glucagon-like Peptide 1 (GLP-1)/Glucagon Receptor Agonists, J Med chem.;60 (10): 4293-4303.2017) that GLP-1R agonistic activity was decreased by about 3-fold and GCG activity was decreased by about 14-fold after addition of cex sequence to the C-terminus of the GCG analog (Table 2, peptides 7 and 8).

In one embodiment of the invention, the GCG analog containing the C-terminal extension peptide of Exendin-4 is further fused to F _C GLP-1R agonistic activity is surprisingly increased by more than 200-fold (EC 50 of about 1.1 nM) when the chain is present. The proportion of GLP-1R agonistic activity of GCG analogs calculated from the data disclosed in U.S. Pat. No. 3,979,B 2 and Joseph R.Chabenne et al in the corresponding patents and literature was only about 2-fold change before and after increasing the sequence of the C-terminal extension peptide cex of Exendin-4 (e.g., the percentage of GLP-1R agonistic activity of native Gluagon in the text was 0.7% and increased to 1.6% after increasing the sequence of GPSSGAPPPS). That is, adding a GPSSGAPPPS sequence to the C-terminus of a Glucago polypeptide does not significantly increase the agonistic activity of its GLP-1R.

Stability of multiple active proteins

Natural Glucago has multiple susceptible degradation sites, including the second DPP-IV degradation site, and the SRR site at positions 16-18. Although there are reports that F _C Can improve the chemical stability and serum stability of the active protein, however, for GLP-1 or Glumagon analogues with which the N-terminus must be exposed, F _C The effect of (2) appears to be indiscriminate. Native GLP-1 or Glucago with F _C After fusion, significant degradation was still observed under 37 degrees serum conditions. The invention introduces mutation resisting 16-18 bit protease hydrolysis based on natural Glucago to increase its stability. These mutants and F _C After fusion, stability is further improved. In the examples of the present invention, about 50% of GCGR agonistic activity could still be detected at 72 hours. In this case, the native Glucago and F containing the cex sequence _C The dimer formed by fusion (SEQ ID NO. 76) has hardly detected any activity.

At present, almost all GCGR/GLP-1R double-effect agonists developed based on Oxytomodulin and Glucago designs introduce a mutation resisting DPP-IV at the second position, such as L-type mutation into D-type amino acid (L-Ser mutation into D-Ser), or unnatural amino acid Aib and the like (Matthias H.

Etc., unimolecular Polypharmacy for Treatment of Diabetes and Obesity,24:51-62,2016). However, in the examples of the invention, the second dimeric active protein preserving native L-Ser exhibits very high serum stability, and there is no sign of significant DPP-IV degradation at 24 hours, without fusion F _C The corresponding polypeptide of (B) was rapidly hydrolyzed by DPP-IV (Table 5). The present inventors prepared natural Glucago and F _C Fused active protein C001G12S3A1F4 (SEQ ID NO. 75) and Glucago-cex and F reported by Joseph R.Chabenne et al _C Fused active protein C002G12S3A1F4 (SEQ ID NO. 76) was used as a control to verify if F _C Fusion improves stability. However, neither C001G12S 3A1F4 (SEQ ID NO. 75) nor C002G12S3A1F4 (SEQ ID NO. 76) showed obvious signs of resistance to DPP-IV. Although binding to serum albumin (e.g., HSA) has been reported to potentially help to improve protein stability (e.g., liraglutide), if the second site is not mutated, half-life cannot last for more than 12 hours at all, i.e., it is impossible to support dosing frequency once a week. The GCG analog drug generation and efficacy test provided by the present invention showed sufficient frequency of administration to support once a week, rather than once a day (e.g., albumin-binding liraglutide) as commonly reported. The retention of natural amino acids further reduces the risk of immunogenicity, avoids chemical cross-linking and also makes the preparation process easier and more convenient.

Glucose stimulated insulin secretion assay (GSIS)

GLP-1 or an analogue thereof is known to act on islet beta cells by agonizing GLP-1R, promoting transcription of insulin genes, synthesis and secretion of insulin. Clinically, GLP-1 analogs are often used in combination with insulin. Although the detailed mechanism of GLP-1R agonism is still not completely uncovered, the fact that cAMP signaling after receptor agonism and rapid endocytosis of the receptor is clear. Several studies have shown that cAMP signals are a different signaling pathway than GLP-1R receptor endocytosis, but both affect insulin secretion (Agonist-induced internalization of the Glucagon-like peptide-1receptor is mediated by the Gaq pathway,Aiysha Thompson, et al, biochemical Pharmacology,93:72-84, 2015;Molecular Characterisation of Small Molecule Agonists Effect on the Human Glucagon Like Peptide-1Receptor Internalisation,Aiysha Thompson, et al, PLOS ONE). Thus, attenuation of cAMP signaling and endocytosis would, in theory, disrupt insulin secretion and thus affect the physiological efficacy of GLP-1 analogs. After GLP-1R cross-links with GCGR to form heterodimers, endocytosis is significantly reduced, affecting insulin secretion.

In one embodiment of the invention, the partial GCGR/GLP-1R agonist induced insulin secretion is significantly reduced. There have been studies showing the phenomenon that various human cell surfaces express various intestinal hormone receptors simultaneously (Dominik Schelshorn et al, lateral Allosterism in the Glucagon Receptor Family: glucose-Like Peptide 1 index G-Protein-Coupled Receptor Heteromer Formation, molecular pharmacology,81 (3) 309-318, 2012). Therefore, if the GCGR/GLP-1R dual-effect agonist is combined with the GLP-1R and GCGR receptor, the original physiological effect of the dual-effect agonist is possibly reduced, and some potential unknown effects are more difficult to predict. In addition to the reduction of insulin secretion, unexpected toxic side effects may also occur. The safety requirements for diabetic drugs are extremely high, and therefore, the inventors believe that dimers that do not crosslink the receptor should be superior.

Abdominal glucose tolerance test (IPGTT)

In one embodiment, an IPGTT experiment was performed. Mice administered with bispecific active protein exhibited very smooth blood glucose fluctuations after glucose injection.

Weight loss experiments in DIO mice

There have been many reports of potential weight loss effects of GCGR agonists. However, natural Glucagon has little potential for patent drugs because of its susceptibility to degradation and minimal molecular weight. Glucago analogs are currently used primarily for acute hypoglycemic symptoms. Clinical reports of long-acting GCG analogs for weight loss in diabetics are also emerging. It is known that obesity is one of the causes of insulin resistance in diabetics, and weight loss is an important index for evaluating a hypoglycemic agent. In addition, the multiple active proteins of the invention induced a significant decrease in body weight following DIO mouse administration. Rat pharmacokinetic studies the bispecific active proteins of the invention have improved pharmacokinetic properties, i.e. they have an extended half-life in vivo. In one embodiment, various bispecific active proteins are administered subcutaneously in rats and serum is taken at various time points to determine blood concentration to evaluate their pharmacokinetic properties.

Clinical application prospect

Clinically, the multiple active proteins of the invention have potentially pharmacokinetic properties suitable for once weekly or more administration. The dosage will depend on the frequency and mode of administration, the age, sex, weight and general condition of the subject being treated, the condition and severity of the treatment, any concomitant diseases to be treated, and other factors apparent to those skilled in the art. Meanwhile, depending on the condition of the subject and other pathological conditions, the multi-active proteins of the present invention may be administered or applied in combination with one or more other therapeutically active compounds or substances, such as other therapeutically active compounds that may be selected include, but are not limited to, antidiabetics, antihyperlipidemic agents, antiobesity agents, antihypertensive agents and agents for treating complications resulting from or associated with diabetes.

Metabolic syndrome is associated with increased risk of coronary heart disease and other conditions associated with the accumulation of vascular plaques, such as stroke and peripheral vascular disease, becoming atherosclerotic cardiovascular disease (ASCVD). Patients with metabolic syndrome may develop fully mature type two diabetes from an early insulin resistance state, and the risk of ASCVD is further increased. Without being bound to any particular theory, the relationship between insulin resistance, metabolic syndrome and vascular disease may involve one or more co-morbidity mechanisms including insulin-stimulated vasodilation disorders, reduced availability of insulin resistance-related events due to oxidative stress enhancement, and abnormalities in adipocyte-derived hormones such as adiponectin (Lteif, mather, can.J. cardiol.20 (journal B): 66B-76B, 2004)

The active proteins of the invention are also useful for the treatment of obesity. In some aspects, the active proteins of the invention treat obesity by mechanisms such as decreased appetite, decreased food intake, decreased fat levels in a patient, increased energy expenditure, and the like.

In some potential embodiments, the active proteins of the invention are useful for treating non-alcoholic fatty liver disease (NAFLD). NAFLD refers to a broad spectrum of liver diseases ranging from simple fatty liver (steatosis) to nonalcoholic steatohepatitis (NASH) to cirrhosis (irreversible advanced scarring of the liver). All disease phases of NAFLD have fat accumulation in liver cells. Fatty liver alone is an abnormal accumulation of certain types of fat, triglycerides in liver cells, but without inflammation or scarring. In NASH, fat accumulation is associated with varying degrees of liver inflammation (hepatitis) and scarring (fibrosis). Inflammatory cells can destroy liver cells (hepatocyte necrosis). In the terms "steatohepatitis" and "steatonecrosis", steatosis refers to steatoinfiltration, hepatitis refers to inflammation in the liver, and necrosis refers to disrupted liver cells. NASH can ultimately lead to scarring of the liver (fibrosis) and then to irreversible advanced scarring (cirrhosis), with NASH resulting cirrhosis being the last and most severe stage within the NAFLD spectrum.

Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention. The test methods in the following examples, in which specific conditions are not noted, are generally conducted under conventional conditions or under conditions recommended by the respective manufacturers.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present invention employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related arts. These techniques are well described in the prior art literature and see, in particular, sambrook et al MOLECULAR CLONING: a LABORATORY MANUAL, second edition, cold Spring Harbor Laboratory Press,1989and Third edition,2001; ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, john Wiley & Sons, new York,1987and periodic updates; the series METHODS IN ENZYMOLOGY, academic Press, san Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, third edition, academic Press, san Diego,1998; METHODS IN ENZYMOLOGY, vol.304, chromatin (p.m. wassman and a.p. wolffe, eds.), academic Press, san Diego,1999; and METHODS IN MOLECULAR BIOLOGY, vol.119, chromatin Protocols (p.b. becker, ed.) Humana Press, totowa,1999, etc.

Example 1 screening of GCG analog (screening of glucagon analog)

The GCG analogue is marked as A, and the structural formula of A is shown as a formula II (SEQ ID NO. 141):

HSQGTFTSDYSKYLD ₁₆ ₁₇ ₁₈ XXXAQDFVQWLMN ₂₉ XX ^z (SEQ ID NO. 141). Wherein X is ₁₆ Any one selected from amino acids other than Y, N, W, and H; x is X ₁₇ Any one selected from amino acids other than P, L, T, F and H; x is X ₁₈ Any one selected from amino acids other than P, F, H and W; in addition X ₁₇ And X is ₁₈ Can not be R, X at the same time ₂₉ Is T or deletion, X ^z Selected from GGPSSGAPPPS (SEQ ID NO. 3), GPSSGAPPPS (SE)Q ID No. 4), PSSGAPPPS (SEQ ID No. 5), SSGAPPPS (SEQ ID No. 6), GGPSSGAPPS (SEQ ID No. 7), GPSSGAPPS (SEQ ID No. 8), PSSGAPPS (SEQ ID No. 9) or SSGAPPS (SEQ ID No. 10).

The amino acid sequences of some of the GCG analogs of the invention are listed in table 1:

TABLE 1

Further, the structural formula of the recombinant protein is shown as formula I: A-L-F is represented by the formula I,

in the formula I, the structural formula of A is shown as the formula II: HSQGTFTSDYSKYLD ₁₆ ₁₇ ₁₈ XXXAQDFVQWLMN ₂₉ XX ^z (SEQ ID NO. 141) wherein X ₁₆ Any one selected from amino acids other than Y, N, W, and H; x is X ₁₇ Any one selected from amino acids other than P, L, T, F and H; x is X ₁₈ Any one selected from amino acids other than P, F, H and W; in addition X ₁₇ And X is ₁₈ Can not be R, X at the same time ₂₉ Is T or deletion, X ^z Selected from any one of GGPSSGAPPPS (SEQ ID NO. 3), GPSSGAPPPS (SEQ ID NO. 4), PSSGAPPPS (SEQ ID NO. 5), SSGAPPPS (SEQ ID NO. 6), GGPSSGAPPS (SEQ ID NO. 7), GPSSGAPPS (SEQ ID NO. 8), PSSGAPPS (SEQ ID NO. 9) or SSGAPPS (SEQ ID NO. 10).

In formula I, F is a long-acting protein unit, and F can be selected from the complete F of immunoglobulin _C Part of immunoglobulin F _C A partial fragment or mutant of the FC portion of an immunoglobulin as shown in SEQ ID nos. 11-20.

For example, F may be the complete FC portion of natural IgG1, the amino acid sequence of which is shown in SEQ ID NO.11, specifically:

EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

f can be a mutant of the FC portion of natural IgG1, the amino acid sequence of which is shown in SEQ ID NO.12, specifically:

EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

f can be the complete FC portion of natural IgG2, the amino acid sequence of which is shown in SEQ ID NO.13, specifically:

ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

f can be the complete FC portion of natural IgG4, and the amino acid sequence is shown in SEQ ID NO.14, specifically:

ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

f can be a mutant of the FC portion of natural IgG4, the amino acid sequence of which is shown in SEQ ID NO.15, specifically:

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

f can be a mutant of the FC portion of natural IgG4, the amino acid sequence of which is shown in SEQ ID NO.16, specifically:

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

f can be a mutant of the FC portion of natural IgG4, and the amino acid sequence can be shown as SEQ ID NO.17, specifically:

ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

F can be a mutant of the FC portion of natural IgG4, and the amino acid sequence can be shown as SEQ ID NO.18, specifically:

ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

f can be a mutant of the FC portion of natural IgG1, and the amino acid sequence can be shown as SEQ ID NO.19, specifically:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

f can be a mutant of the FC portion of natural IgG1, and the amino acid sequence can be shown as SEQ ID NO.20, specifically:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

in formula I, L is a linker chain which is a flexible polypeptide of glycine (G), serine (S) and/or alanine (A) of suitable length such that adjacent protein domains can move freely relative to each other. Longer connecting chains can be used when it is necessary to ensure that the two adjacent domains do not spatially interfere with each other. The connecting chain is exemplified by (GS) n, (GGS) n, (GGSG) n, (GGGS) nA, (GGGGS) nA, (GGGGA) nA, etc., n is an integer of 1 to 10, and in a preferred embodiment, the amino acid length of the connecting chain is 5 to 26. Exemplary connecting chains are each independently selected from table 2.

TABLE 2

SEQ ID NO.	(Code)	Connecting chain
			21	G ₄ S ₁	GGGGS
22	G ₄ A ₁	GGGGA
			23	G ₆ S ₂	GGGSGGGS
24	G ₄ S ₄	GSGSGSGS
			25	G ₈ S ₂	(GGGGS) ₂
26	G ₈ A ₂	(GGGGA) ₂
			27	G ₉ S ₃	GGSGGGSGGGGS
28	G ₉ S ₂ A ₁	GGSGGGAGGGGS
			29	G ₁₂ S ₃	(GGGGS) ₃
30	G ₁₂ A ₃	(GGGGA) ₃
			31	G ₁₂ A ₁ S ₃	(GGSGG) ₃ A
32	G ₁₂ A ₄	(GGGGA) ₃ A
			33	G ₁₂ S ₃ A ₁	(GGGGS) ₃ A
34	G ₁₂ S ₁ A ₃	(GGGGA) ₃ S
			35	G ₁₃ S ₄	GS(GGGGS) ₃
36	G ₁₆ A ₄	(GGGGA) ₄
			37	G ₁₆ S ₄	(GGGGS) ₄
38	G ₁₇ S ₅	GS(GGGGS) ₄
			39	G ₂₀ S ₅	(GGGGS) ₅
40	G ₂₀ S ₅ A ₁	(GGGGS) ₅ A
			41	G ₂₄ S ₆	(GGGGS) ₆
42	G ₂₈ S ₇	(GGGGS) ₇
			43	G ₃₂ S ₈	(GGGGS) ₈

Example 2 preparation of dimer double active protein

In example 1, the amino acid sequence of the dimeric double-acting protein (structural formula shown in formula I) obtained by fusing the GCG analog with the connecting chain and F is as follows:

The amino acid sequence of C001G12S3A1F4 is shown as SEQ ID NO.75, specifically:

HSQGTFTSDYSKYLDSRRAQDFVQWLMNTGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C002G12S3A1F4 is shown in SEQ ID NO.76, specifically:

HSQGTFTSDYSKYLDSRRAQDFVQWLMNTGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C240G12S3A1F4 is shown in SEQ ID NO.77, specifically:

HSQGTFTSDYSKYLDERAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C320G12S3A1F4 is shown as SEQ ID NO.78, specifically:

HSQGTFTSDYSKYLDYQAAQDFVQWLMNTGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C276G12S3A1F4 is shown in SEQ ID NO.79, and specifically comprises:

HSQGTFTSDYSKYLDSRAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C368G12S3A1F4 is shown as SEQ ID NO.80, and specifically comprises the following steps:

HSQGTFTSDYSKYLDEQAAQDFVQWLMNTGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C225G12S3A1F4 is shown as SEQ ID NO.81, and specifically comprises the following steps:

HSQGTFTSDYSKYLDERAAQDFVQWLMNTGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C302G12S3A1F4 is shown as SEQ ID NO.82, specifically:

HSQGTFTSDYSKYLDEPAAQDFVQWLMNTGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of the C163G12S3A1F4 is shown as SEQ ID NO.83, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSRAAQDFVQWLMNTGPSSGAPPPS GGGGS GGGGS GGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C350G12S3A1F4 is shown as SEQ ID NO.84, and specifically comprises the following steps:

HSQGTFTSDYSKYLDNQEAQDFVQWLMNTGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C271G12S3A1F4 is shown in SEQ ID NO.85, and specifically comprises:

HSQGTFTSDYSKYLDGRAAQDFVQWLMNTGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C232G12S3A1F4 is shown as SEQ ID NO.86, specifically:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNTGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C495G12S3A1F4 is shown as SEQ ID NO.87, and specifically comprises the following steps:

HSQGTFTSDYSKYLDEQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C307G12S3A1F4 is shown in SEQ ID NO.88, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSLAAQDFVQWLMNTGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C382G12S3A1F4 is shown in SEQ ID NO.89, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C227G12S3A1F4 is shown as SEQ ID NO.90, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNTGPSSGAPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

The amino acid sequence of C266G12S3A1F4 is shown as SEQ ID NO.91, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C137G12S3A1F4 is shown as SEQ ID NO.92, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSERAQDFVQWLMNTGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C399G12S3A1F4 is shown as SEQ ID NO.93, specifically:

HSQGTFTSDYSKYLDGERAQDFVQWLMNTGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C395G12S3A1F4 is shown in SEQ ID NO.94, and is specifically as follows:

HSQGTFTSDYSKYLDEQSAQDFVQWLMNTPSSGAPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C394G12S3A1F4 is shown in SEQ ID NO.95, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQPAQDFVQWLMNTSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C392G12S3A1F4 is shown as SEQ ID NO.96, and specifically comprises the following steps:

HSQGTFTSDYSKYLDEEAAQDFVQWLMNTGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C353G12S3A1F4 is shown as SEQ ID NO.97, specifically:

HSQGTFTSDYSKYLDEQAAQDFVQWLMNGPSSGAPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C352G12S3A1F4 is shown in SEQ ID NO.98, and specifically comprises the following steps:

HSQGTFTSDYSKYLDEQAAQDFVQWLMNGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C228G12S3A1F4 is shown as SEQ ID NO.99, and specifically comprises the following steps:

HSQGTFTSDYSKYLDEERAQDFVQWLMNTGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C462G12S3A1F4 is shown as SEQ ID NO.100, and specifically comprises the following steps:

HSQGTFTSDYSKYLDEEAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C187G12S3A1F4 is shown as SEQ ID NO.101, specifically:

HSQGTFTSDYSKYLDSQRAQDFVQWLMNTGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C334G12S3A1F4 is shown as SEQ ID NO.102, specifically:

HSQGTFTSDYSKYLDSTAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C364G12S3A1F4 is shown as SEQ ID NO.103, specifically:

HSQGTFTSDYSKYLDGQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C209G12S3A1F4 is shown as SEQ ID NO.104, specifically:

HSQGTFTSDYSKYLDSEAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C289G12S3A1F4 is shown as SEQ ID NO.105, and is specifically as follows:

HSQGTFTSDYSKYLDGEAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

The amino acid sequence of C0382G12S3A1F4 is shown as SEQ ID NO.142, specifically:

HSQGTFTSDYSEYLDSERARDFVAWLEAGGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

on the basis of knowledge of the amino acid sequence of the dimeric double-acting protein, the person skilled in the art can prepare it using the prior art: due to the F bearing _C The sequences, therefore, can be purified by high affinity and high specificity Protein A resin chromatography, only one possible preparation being given here by way of example.

The preparation process is as follows:

(1) The DNA sequence is designed according to the protein sequence and the amino acid codon table. Respectively preparing polynucleotide DNA fragments corresponding to A, L, F in the recombinant protein, wherein each DNA fragment can be synthesized and spliced by a conventional solid-phase synthesis technology;

(2) Primers are designed for nested PCR amplification, corresponding DNA fragments of A, L, F are spliced to obtain target genes, and PCR splicing technology (including primer design, PCR introduction mutation, enzyme digestion and the like) is a well-known technology known to those skilled in the art. It will be appreciated by those skilled in the art that the PCR splicing process of the present embodiment is not the only method, and that the desired gene can be obtained by, for example, gene synthesis. After successfully obtaining the target gene, cloning the target gene into a mammalian cell expression vector pTT5 (Yves Durocher) to transform escherichia coli Top10F'; after positive clone identification, the cells were inoculated into 500ml of LB medium, cultured overnight, and collected by centrifugation, using Omega E.Z.N.A.

Endo-Free Plasmid Maxi Kit plasmid was extracted.

(3) Transfection of Hek293F cells and cell expression: 1.0mg of plasmid was diluted to 25ml using Freestyle293 expression medium (thermosusher); 3.0mg PEI (linear, 25 KD) was diluted to 25ml using Freestyle293 expression medium, added to plasmid solution, mixed well and incubated for 30 min at room temperature; at the same time, hek293F cells grown in log phase (viability>95%), count; 1100RPM, centrifuging for 10 minutes, and discarding the supernatant; cells were resuspended using 450ml Freestyle293 expression medium;after incubation of PEI plasmid mixture was completed, it was added to the cell suspension at 37℃with 5% CO ₂ Shake culturing at 140 RPM; after 7 hours, the Freestyle293 expression medium was replaced with 1000ml of 293sfm II medium (thermoshifier) and cultivation was continued for 7 days.

(4) Purification of recombinant proteins: centrifuging the cell culture solution at 8000rpm for 10min at high speed to obtain supernatant, loading onto Protein A column (Bogurone (Shanghai) Biotechnology Co., ltd.) balanced with balancing solution (20mM PB,0.5M NaCl,pH7), and eluting with 100% (eluent 0.1M Gly-HCl, pH 3.0); adding neutralization solution (1M Tris-HCl, pH 8.0) into a collecting pipe in advance, and collecting an eluted sample; finally, the neutralization solution is added to 1/10 of the volume of the eluted sample, and the protein concentration is determined by the conventional Bradford method.

(5) Physical and chemical property identification of recombinant proteins: SDS-PAGE electrophoresis of recombinant proteins obtained by purification is performed, and FIG. 1 is an SDS-PAGE electrophoresis of an exemplary purified sample.

Example 3 in vitro cell Activity assay

The double-effect active protein obtained in the example 2 is subjected to in vitro activity measurement, including GLP-1R agonist activity detection and GCGR agonist activity detection.

GLP-1R agonism activity assay:

GLP-1R agonistic activity is detected by adopting a luciferase reporter gene detection method. Cloning the human GLP-1R gene into a mammalian cell expression plasmid pCDNA3.1 to construct a recombinant expression plasmid pCDNA3.1-GLP-1R, and cloning a full-length luciferase (luciferase) gene onto a pCRE-EGFP plasmid to replace the EGFP gene to obtain the pCRE-Luc recombinant plasmid. The pCDNA3.1-GLP-1R and pCRE-Luc plasmids are transfected into CHO cells according to the mol ratio of 1:10, and stable transgenic expression strains are screened to obtain recombinant CHO/GLP-1R stable transgenic cell strains.

Culturing cells in 9-cm cell culture dish with DMEM/F12 medium containing 10% FBS and 300 μg/ml G418, discarding culture supernatant when confluence reaches about 90%, adding 2ml pancreatin for 3min, adding 2ml DMEM/F12 medium containing 10% FBS and 300 μg/ml G418 for neutralization, transferring to 15ml centrifuge tube, centrifuging at 1000rpm for 5min, discarding supernatant, adding 2ml DMEM/F12 medium containing 10% FBS and 300 min Mu.g/ml of G418 in DMEM/F12 medium was resuspended and counted. The cells were diluted to 3X 10 with DMEM/F12 medium containing 10% FBS ⁵ 100 μl, 5×10, of each well is plated in a 96-well plate ⁴ After attachment, the wells were replaced with DMEM/F12 medium containing 0.2% FBS. After cell removal of the supernatant from 96-well plates, purified recombinant protein (Table 3) or native Glucoagon (GLUC-004, hangzhou peptide Biochemical Co., ltd.) was diluted to a series of indicated concentrations with DMEM/F12 medium containing 0.1% FBS as a control with native GLP-1 (GLUC-016B, hangzhou peptide Biochemical Co., ltd.) and added to the cell culture wells, and the wells were stimulated for 6 hours for detection. Detection was performed according to the instructions of the luciferase reporter assay kit (Lucifersae reporter kit, ray Biotech, cat: 68-LucIR-S200).

GCGR agonist activity assay:

the GCGR agonistic activity assay was also performed using the luciferase reporter assay. The GCGR gene is cloned into a mammalian cell expression plasmid pCDNA3.1 to construct recombinant expression plasmid pCDNA3.1-GCGR, and HEK 293T cells are transfected and HEK 293T/GCGR is stably transformed. FIG. 2 is a graph of results of the measurement of active EC50 of a portion of a dimeric dual active protein.

The results are shown in Table 3:

TABLE 3 Table 3

In addition, other dimeric active proteins without Exendin-4C-terminal sequence were also shown: in vitro activity assays were performed for CG283G12S3A1F4 (SEQ ID NO. 106), CG214G12S3A1F4 (SEQ ID NO. 107), CG267G12S3A1F4 (SEQ ID NO. 108), C308G12S3A1F4 (SEQ ID NO. 109), C224G12S3A1F4 (SEQ ID NO. 110), CG308G12S3A1F4 (SEQ ID NO. 111), C319G12S3A1F4 (SEQ ID NO. 112), C214G12S3A1F4 (SEQ ID NO. 113), C303G12S3A1F4 (SEQ ID NO. 114), CG303G12S3A1F4 (SEQ ID NO. 115), including GLP-1R agonist activity assays and GCGR agonist activity assays.

The amino acid sequence of CG283G12S3A1F4 is shown as SEQ ID NO.106, specifically:

HSQGTFTSDYSKYLDERAAQDFVQWLMNGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of CG214G12S3A1F4 is shown as SEQ ID NO.107, specifically:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of CG267G12S3A1F4 is shown as SEQ ID NO.108, specifically:

HSQGTFTSDYSKYLDSRAAQDFVQWLMNGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C308G12S3A1F4 is shown as SEQ ID NO.109, specifically:

HSQGTFTSDYSKYLDEQAAQDFVQWLMNTGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C224G12S3A1F4 is shown as SEQ ID NO.110, and specifically comprises the following steps:

HSQGTFTSDYSKYLDERAAQDFVQWLMNTGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of CG308G12S3A1F4 is shown as SEQ ID NO.111, specifically:

HSQGTFTSDYSKYLDEQAAQDFVQWLMNGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C319G12S3A1F4 is shown as SEQ ID NO.112, specifically:

HSQGTFTSDYSKYLDGQAAQDFVQWLMNGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of C214G12S3A1F4 is shown as SEQ ID NO.113, specifically:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNTGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

The amino acid sequence of C303G12S3A1F4 is shown as SEQ ID NO.114, and specifically comprises the following steps:

HSQGTFTSDYSKYLDEEAAQDFVQWLMNTGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the amino acid sequence of CG303G12S3A1F4 is shown as SEQ ID NO.115, specifically:

HSQGTFTSDYSKYLDEEAAQDFVQWLMNGGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

the results of the measurement are shown in Table 4, compared with the recombinant protein of the present invention:

TABLE 4 Table 4

Description: a. to add the GLP-1R agonistic activity ratio of Exendin-4 before and after the C-terminal extension peptide Cex (in the present invention, any one of SEQ ID NO. 3-10).

b. The ratios obtained were calculated from GLP-1R agonistic activity data of native Glucoago to Glucoago Cex as disclosed in Table 2 of US 9018164B 2.

c. The ratios obtained were calculated from GLP-1R agonist activity data of the native Glucoago and Glucoago Cex disclosed in Table 1 of Joseph R.Chabenne et al (Joseph R.Chabenne et al Optimization of the Native Glucagon Sequence for Medicinal Purposes, J Diabetes Sci technology.4 (6): 1322-1331, 2010).

As shown in tables 3 and 4, when the sequence containing the extended peptide was passed through (GGGGS) ₃ After A (SEQ ID NO. 33) and F (SEQ ID NO. 16) are fused to prepare a dimer, the agonistic activity to GLP-1R is improved by more than 200 times, and the agonistic activity to GCGR is not obviously different.

EXAMPLE 4 DPP-IV enzyme resistance stability

Purified dimeric double-acting protein 5uM was dissolved in 10mM HEPES buffer (containing 0.05mg/ml BSA, and 10nM final concentration of recombinant DPP-IV enzyme was added thereto), and after 24 hours incubation at 37℃the in vitro GCGR cell activity assay was examined.

In this example, GCG analogs with the second unnatural amino acid Aib or D-Ser introduced were used as controls:

GDSerGS：H-D-Ser-QGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPS(SEQ ID NO.116)；

GAibGS：H-Aib-QGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPS(SEQ ID NO.117)；

c364 (SEQ ID NO. 72), C382 (SEQ ID NO. 59), C495 (SEQ ID NO. 55), C462 (SEQ ID NO. 68), C225 (SEQ ID NO. 49) and C209 (SEQ ID NO. 73) were used as controls for the stability test in this example.

The results are shown in Table 5:

TABLE 5

EXAMPLE 5 serum stability test

In vitro cell assay:

(1) Taking dimer double-effect active protein, ultrafiltering and concentrating, diluting to 1.6mg/ml with 20mM PB pH7.4, sterilizing and filtering, diluting serum (FBS, GEMINI 900-108, A97E 00G) 10 times, mixing well, and packaging into sterile centrifuge tube;

(2) In addition, glucago (SEQ ID NO.44, GLUC-004, hangzhou peptide Biochemical Co., ltd.) is diluted to 0.2mg/ml, sterilized and filtered, and serum is diluted 10 times, mixed uniformly and packaged in a sterile centrifuge tube;

(3) The sample 1-2 is frozen at-20 ℃ to be used as a control, and the other samples are placed in a 37 ℃ incubator to be sampled and detected for GCGR agonistic activity at different time points;

(4) HEK 293T/GCGR cells were passaged twice and then plated in 96-well plates to examine the activity of the samples. The relative activity over time is shown in figures 3A-D. From the results of FIG. 3, it can be seen that C240G12S3A1F4, C276G12S3A1F4, C368G12S3A1F4, C225G12S3A1F4, C163G12S3A1F4, C232G12S3A1F4, C495G12S3A1F4, C382G12S3A1F4, C271G12S3A1F4, C227G12S3A1F4, C266G12S3A1F4, C399G12S3A1F4, C392G12S3A1F4, C353G12S3A1F4, C137G12S3A1F4, C289G12S3A1F4, C209G12S3A1F4, C352G12S3A1F4, C228G12S3A1F4, C462G12S3A1F4, C187G12S 1A 1F4, C364G12S3A1F4 are significantly more stable than the other serum dimers.

Residual activity: the activity value at 0 hours was taken as 100%, and the values measured at the subsequent time points were compared with the activity value at 0 hours.

Example 6 Activity assay of dimeric double-acting proteins of different Link lengths

Fusion of the GCG analog of example 1 to human IgG 1F via different lengths of the connecting chain _C (SEQ ID NO. 12) N-terminus. That is, the amino acid sequence of the dimeric double-acting protein obtained by fusing the GCG analog with the different length connecting chain and F (SEQ ID NO. 12) is as follows:

the amino acid sequence of the C382G4A1F1 is shown as SEQ ID NO.118, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

the amino acid sequence of C382G4S1F1 is shown as SEQ ID NO.119, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

the amino acid sequence of the C382G6S2F1 is shown as SEQ ID NO.120, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGSGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

the amino acid sequence of C382G4S4F1 is shown as SEQ ID NO.121, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGSGSGSGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

the amino acid sequence of the C382G8S2F1 is shown as SEQ ID NO.122, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

the amino acid sequence of C382G8A2F1 is shown as SEQ ID NO.123, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGAGGGGAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

the amino acid sequence of the C382G9S3F1 is shown as SEQ ID NO.124, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGSGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

the amino acid sequence of C382G9S2A1F1 is shown as SEQ ID NO.125, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGSGGGAGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

the amino acid sequence of the C382G12S3F1 is shown as SEQ ID NO.126, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

the amino acid sequence of the C382G12A3F1 is shown as SEQ ID NO.127, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGAGGGGAGGGGAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

The amino acid sequence of C382G12A1S3F1 is shown as SEQ ID NO.128, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGSGGGGSGGGGSGGAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

the amino acid sequence of C382G12S3A1F1 is shown as SEQ ID NO.129, specifically:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

the amino acid sequence of the C382G12A4F1 is shown as SEQ ID NO.130, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGAGGGGAGGGGAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

the amino acid sequence of C382G12S1A3F1 is shown as SEQ ID NO.131, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGAGGGGAGGGGASEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

the amino acid sequence of the C382G13S4F1 is shown as SEQ ID NO.132, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

the amino acid sequence of the C382G16A4F1 is shown as SEQ ID NO.133, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGAGGGGAGGGGAGGGGAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

the amino acid sequence of the C382G16S4F1 is shown as SEQ ID NO.134, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

the amino acid sequence of C382G17S5F1 is shown as SEQ ID NO.135, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGSGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

the amino acid sequence of the C382G20S5F1 is shown as SEQ ID NO.136, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

the amino acid sequence of C382G20S5A1F1 is shown as SEQ ID NO.137, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSGGGGSGGGGSAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

the amino acid sequence of the C382G24S6F1 is shown as SEQ ID NO.138, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

the amino acid sequence of the C382G28S7F1 is shown as SEQ ID NO.139, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

the amino acid sequence of the C382G32S8F1 is shown as SEQ ID NO.140, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

the dimeric double-acting protein can be prepared by the person skilled in the art using the prior art, knowing its amino acid sequence.

The preparation method used in this example is the same as in example 2.

The dimer double-effect active protein obtained in the example was subjected to in vitro activity assays, including GLP-1R agonist activity assays and GCGR agonist activity assays. The detection method was the same as in example 3.

The test results are shown in Table 6.

TABLE 6

EXAMPLE 7 glucose stimulated insulin secretion assay (GSIS)

This example refers to the method of Aisliding M.Lynch et al (A novel DPP IV-resistance C-terminally extended Glucagon analogue exhibits weight-lowering and diabetes-protective effects in high-fat-fed mice mediated through Glucagon and GLP-1receptor activation,Aisling M.Lynch et al, diabetes, 57:1927-1936, 2014) using rat BRIN-BD11 cells for measuring insulin release induced by active protein stimulation, with the modification that 1.0X10 per well was added in a 24 well plate (Orange Scientific, brainel' Alleud, belgium) ⁶ The cells were incubated overnight at 37℃and the supernatant was removed by centrifugation, and 1.0ml KRB (115 mM NaCl, 4.7mM KCl, 1.28mM CaCl) was added to each well ₂ 、1.2mM MgSO ₄ 、1.2mM KH ₂ PO ₄ 、25mM HEPES、10mM NaHCO ₃ NaOH adjusts pH to 7.4), 0.1% (wt/vol.) BSA, and 1.1mM glucose. After cells were incubated at 37℃for 40 min, the supernatant was centrifuged off and replaced with 1.0ml of fresh KRB solution with gradient concentrations of active protein in Table 6. After incubation at 37℃for 20 minutes, the buffer was removed by centrifugation and stored overnight at-20℃before immunoradiometric detection of insulin content. The results are shown in FIG. 4.

EXAMPLE 8 glucose tolerance test (IPGTT) in normal ICR mice

Normal ICR mice were grouped into 8 groups. Overnight fast, tail blood sampling (noted as t=0 min blood glucose) and subcutaneous injection of dimer double-acting protein (40 nmol/kg acetate buffer) or physiological saline PBS, wherein C002G12S3A1F4 was administered at a dose of 250nmol/kg (acetate buffer). Glucose (2 g/kg body weight) was injected intraperitoneally after 15 minutes and blood glucose levels were measured at t=30 minutes, t=60 minutes, t=120 minutes, and t=240 minutes. Animals are still fasted during the experiment to prevent interference with food intake. The results are shown in FIG. 5.

EXAMPLE 9 study of efficacy of continuous administration in obesity (DIO) induced mice

The purpose of this example was to investigate the effect of different dimeric double-activity proteins on DIO mouse body weight. Male C57BL/6J mice of 7 weeks old were fed high fat diet (60% kcal from fat) for a further 16 weeks (total 23 weeks) and tested at a weight of about 55 g. Feeding conditions: the mice were grouped (8 mice/group) according to body weight and body weight growth curve one day prior to dosing, and were treated subcutaneously the next day, with 12h light/12 h darkness, free feeding, single cage feeding. Dosing was performed at a dose of 50nmol/kg body weight, once every 4 days, sham PBS injection on other days, and dosing was continued for 28 days; the negative control group was injected with physiological saline (PBS) at a rate of 5 ul/g body weight; positive control groups were injected with liraglutide (40 nmol/kg body weight), and the body weight of the mice was measured once a day. The 5 th day after the last dose was sacrificed. The orbit is bled. The plasma samples were stored at-80 ℃. The average body weight change before and at the time of sacrifice was calculated for each group of animals. The results are shown in FIG. 6.

Example 10 pharmacokinetic Parameter (PK) determination of rats

Male SD rats were grouped around 6 weeks, 8 per group. Dosing or physiological saline was according to the regimen of table 7. All rats began to drink and eat freely after dosing was completed; the time point at which the administration was completed was set to 0 and served as a time reference for the subsequent experiments. The drug generation detection of the dimer double-effect active protein adopts a sandwich ELISA method. Namely, mouse anti-human IgG4 FC mab (9002-01, I2013-NG 56,0.25 mg/ml); plates (96 well plates, corning, 42592), 200 ng/well, overnight at 4 ℃; after PBST plates are washed for 4 times, the plates are sealed by 5% milk powder solution, and the room temperature is 1h; then PBST washes the plate for 4 times; after dilution of rat serum with PBST, incubation was carried out for 2h at 37 ℃; PBST washing the plate for 6 times; then, the rabbit anti-Glucoagon antibody (self-made: immunity to natural Glucoagon polypeptide (SEQ ID NO. 44) is diluted by 1% BSA solution, and the anti-Glucoagon antibody (Hangzhou Hua' an Biotechnology Co., ltd.) is obtained by purifying Protein G, and the mixture is subjected to biotin labeling, 200X, 0.2 mg/ml) and 1:2000 incubation for 2 hours at 37 ℃; PBST washing the plate for 6 times; diluting strep-HRP with 1% BSA solution, incubating for 1.5h at 37 ℃ and keeping the concentration of strep-HRP at 1:5 ten thousand; PBST washing the plate for 6 times; finally TMB developed, OD450 read.

TABLE 7

The results are shown in Table 8:

TABLE 8

Dimer double-effect active protein	T _1/2 (hours)	T _max (hours)
			C240G12S3A1F4(SEQ ID NO.77)	41.2	24
C382G12S3A1F4(SEQ ID NO.89)	42.5	24
			C495G12S3A1F4(SEQ ID NO.87)	43.7	24
C364G12S3A1F4(SEQ ID NO.103)	42.6	24
			C462G12S3A1F4(SEQ ID NO.100)	39.5	24
C227G12S3A1F4(SEQ ID NO.90)	42.2	24
			C368G12S3A1F4(SEQ ID NO.80)	37.8	24
C266G12S3A1F4(SEQ ID NO.91)	38.4	24
			C002G12S3A1F4(SEQ ID NO.76)	8.5	6

EXAMPLE 11 construction and Activity determination of dimeric triple-effect active proteins fused with FGF21 analog

Further fusion of FGF21 analogues at the C-terminus of the aforementioned dimeric double-acting protein was performed to construct fusion proteins (triple-acting proteins fused with FGF21 analogues) as shown in table 9. The amino acid sequence of each fusion protein in table 9 is as follows:

the amino acid sequence of the natural FGF21 is shown as SEQ ID NO.143, and specifically comprises the following steps:

HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS。

the amino acid sequence of FGF21 analogue 1 is shown as SEQ ID NO.144, and specifically comprises the following steps:

HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES。

the amino acid sequence of FGF21 analogue 2 is shown as SEQ ID NO.145, and specifically comprises the following steps:

HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHCPGNKSPHRDPAPRGPCRFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES。

the amino acid sequence of FGF21 analogue 3 is shown as SEQ ID NO.146, and specifically comprises the following steps:

DSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHCPGNKSPHRDPAPRGPCRFLPLPGLPPALPEPPGILAPQPPDVGSSDPLAMVGPSQGRSPSYAS。

c382 The amino acid sequence of F4FGF is shown as SEQ ID NO.147, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS。

c382 The amino acid sequence of F4FGF1 is shown as SEQ ID NO.148, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES。

the amino acid sequence of the C382F4FGF2 is shown as SEQ ID NO.149, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHCPGNKSPHRDPAPRGPCRFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES。

c382 The amino acid sequence of F4FGF3 is shown as SEQ ID NO.150, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGADSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHCPGNKSPHRDPAPRGPCRFLPLPGLPPALPEPPGILAPQPPDVGSSDPLAMVGPSQGRSPSYAS。

c495 The amino acid sequence of F4FGF is shown as SEQ ID NO.151, and specifically comprises the following steps:

HSQGTFTSDYSKYLDEQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS。

c495 The amino acid sequence of F4FGF1 is shown as SEQ ID NO.152, and specifically comprises the following steps:

HSQGTFTSDYSKYLDEQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES。

C495 The amino acid sequence of F4FGF2 is shown as SEQ ID NO.153, and specifically comprises the following steps:

HSQGTFTSDYSKYLDEQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHCPGNKSPHRDPAPRGPCRFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES。

c495 The amino acid sequence of F4FGF3 is shown as SEQ ID NO.154, and specifically comprises the following steps:

HSQGTFTSDYSKYLDEQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGADSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHCPGNKSPHRDPAPRGPCRFLPLPGLPPALPEPPGILAPQPPDVGSSDPLAMVGPSQGRSPSYAS。

the preparation method of the fusion protein belongs to the prior art: due to the F bearing _C Sequences, thusProtein purification can be performed by high affinity and high specificity Protein A resin chromatography, see the procedure in example 2. The electrophoretogram of the fusion protein obtained by purification is shown in FIG. 7. Preparation of native FGF21, FGF21 analogs reference reports of Xu J et al (Xu J et al Polyethylene glycol modified FGF21 engineered to maximize potency and minimize vacuole formation, bioconjug chem.;24 (6): 915-25, 2013) and were modified as follows: the expression vector is PET30, and the host bacterium is BL21 (DE 3) (merck China). The inclusion bodies were washed four times with washing solution (50mM Tris,150mM NaCl,2M urea, pH 8.0), and weighed; adding 1ml of denaturing liquid (50mM Tris,150mM NaCl,8M urea,10mM DTT,pH 8.5) into each 0.1g of inclusion body according to the mass volume ratio of (1:10), and gently mixing and dissolving for more than 5 hours at room temperature by a shaking table; diluting and renaturating according to the proportion of 1:100-200. Slowly dripping the denatured liquid into the renaturation liquid, and continuously stirring in the process; after the dripping is completed, the renaturation solution containing the protein is placed at 4 ℃ for 24 hours; after the completion, the mixture was filtered with a 0.45um filter membrane for chromatographic purification.

Construction of living cells:

PCR amplified puromycin resistance gene pac, cloned to pcDNA3.1 (+), replaces the original G418 resistance gene. GAL4DBD-ELK1, IRES and KLB (beta-klotho) genes are amplified by PCR and cloned on pcDNA-Puro plasmids in sequence, and plasmids pcDNA-GAL4DBD-ELK1-IRES-KLB-Puro are constructed for cell transfection screening. The plasmid used was Omega E.Z.N.A.

Endo-Free Plasmid Midi Kit is extracted for later use. The cell transfection procedure was as follows: hek293T cells were plated in 6 well plates, 3X 10 per well ⁵ Cells were cultured overnight.

After washing the cells twice with Opti-MEM medium, 2ml of Opti-MEM medium was added. Cell transfection reagents were prepared in the following proportions: lipofectamine 2000 (6 μl): pFR-Luc (4.6 μg): pcDNA-GAL4DBD-ELK1-IRES-KLB-Puro (1. Mu.g). Standing for 20min, slowly adding into 6-hole plate, and mixing. After culturing for 6h, DMEM+10% FBS culture medium is changed, 37 ℃ and 5% CO ₂ Culturing was continued. And screening to obtain stable transgenic cell strain with FGF21 activity response.

Cell viability:

after the cells had grown up in the dishes, they were trypsinized, cell suspensions (1X 105 cells/ml, DMEM+5% FBS+1. Mu.g/ml puromycin) were prepared, 96-well plates were plated, 100. Mu.l per well, and cultured overnight. The sample to be tested was added in gradient concentration and allowed to act for 6 hours, and fluorescence detection was performed using Luciferase Reporter Assay Kit (68-LucifR-S200). The results are shown in Table 9:

TABLE 9

EXAMPLE 12 study of the efficacy of the three active proteins fused to FGF21 and analogues thereof in DIO mice

Male C57BL/6J mice of 7 weeks old were fed high fat diet (60% kcal from fat) for a further 16 weeks (total 23 weeks) and tested at a weight of about 55 g. Feeding conditions: the mice were grouped according to body weight and body weight growth curve (8 mice/group) one day prior to dosing, followed by subcutaneous dosing treatment (table 10) at 12h light/12 h dark, free feeding, single cage feeding. The double-effect active protein is dosed at 30 nmol/kg body weight or 90 nmol/kg body weight once in 4 days; FGF21 analogues were administered twice daily at 30 nmol/kg body weight; the negative control group was injected with physiological saline (PBS) at a rate of 5 ul/g body weight; the positive control group was injected with liraglutide (30 nmol/kg body weight), once daily, for 28 consecutive days, and the body weight and feeding amount of the mice were measured daily. The 5 th day after the last dose was sacrificed. The orbit is bled. The plasma samples were stored at-80 ℃. The average body weight change before and at the time of sacrifice was calculated for each group of animals. The weight change results are shown in fig. 8; the change in total intake is shown in fig. 9.

Table 10

EXAMPLE 13 construction and Activity determination of Tri-active protein fused to leptin

At the C-terminal of the aforementioned dimeric double-acting protein, natural leptin (SEQ ID NO. 155) and its analogues may be further fused, and the fusion protein (triple-acting protein fused with leptin) in Table 11 is constructed and obtained. The amino acid sequence of each fusion protein in table 11 is as follows:

TABLE 11

The amino acid sequence of the natural leptin is shown as SEQ ID NO.155, and specifically comprises the following steps:

VPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC。

c382 The amino acid sequence of F4Lep is shown as SEQ ID NO.156, and specifically comprises the following steps:

HSQGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSAVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC。

c495 The amino acid sequence of F4Lep is shown as SEQ ID NO.157, and specifically comprises the following steps:

HSQGTFTSDYSKYLDEQAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSAVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC。

c462 The amino acid sequence of F4Lep is shown as SEQ ID NO.158, and specifically comprises the following steps:

HSQGTFTSDYSKYLDEEAAQDFVQWLMNGGPSSGAPPPSGGGGS GGGGS GGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSAVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC。

c364 The amino acid sequence of F4Lep is shown as SEQ ID NO.159, and specifically comprises the following steps:

HSQGTFTSDYSKYLDGQAAQDFVQWLMNGGPSSGAPPPSGGGGS GGGGS GGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSAVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC。

c289 The amino acid sequence of F4Lep is shown as SEQ ID NO.160, and is specifically as follows:

HSQGTFTSDYSKYLDGEAAQDFVQWLMNGGPSSGAPPPSGGGGSGGGGSGGGGSAESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSGGGGSGGGGSAVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC。

EXAMPLE 14 study of the efficacy of leptin-fused triple-active proteins in DIO mice

Male C57BL/6J mice of 7 weeks old were fed high fat diet (60% kcal from fat) for a further 16 weeks (total 23 weeks) and tested at a weight of about 55 g. Feeding conditions: the mice were grouped according to body weight and body weight growth curve (8 mice/group) one day prior to dosing, followed by subcutaneous dosing treatment (table 12) at 12h light/12 h darkness, free feeding, single cage feeding. The negative control group was dosed at 30 nmol/kg body weight and injected with physiological saline (PBS) at 5 ul/g body weight; double-effect active protein was given once a day for 4 days, sham PBS for other days, and leptin was administered twice daily at 30 nmol/kg body weight for 28 days, and the body weight and feeding amount of the mice were measured daily. The 5 th day after the last dose was sacrificed. The orbit is bled. The plasma samples were stored at-80 ℃. The average body weight change before and at the time of sacrifice was calculated for each group of animals. The weight change results are shown in fig. 10; the change in total intake is shown in fig. 11.

While the invention has been described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and additions may be made without departing from the scope of the invention. Equivalent embodiments of the present invention will be apparent to those skilled in the art having the benefit of the teachings disclosed herein, when considered in the light of the foregoing disclosure, and without departing from the spirit and scope of the invention; meanwhile, any equivalent changes, modifications and evolution of the above embodiments according to the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. The structure of the multiple active protein comprises a structure shown in a formula I, wherein the structure shown in the formula I is as follows: A-L-F, wherein A is GCGR/GLP-1R double-effect agonist, F is long-acting protein unit, L is a connecting chain for connecting A and F;

the structure of A comprises a structure shown in a formula II, and the structure shown in the formula II is as follows: HSQGTFTSDYSKYLDX ₁₆ X ₁₇ X ₁₈ AQDFVQWLMNX ₂₉ Xz formula II, wherein X ₁₆ Any one selected from amino acids other than Y, N, W, and H; x is X ₁₇ Any one selected from amino acids other than P, L, T, F and H; x is X ₁₈ Any one selected from amino acids other than P, F, H and W; in addition X ₁₇ And X is ₁₈ Can not be R, X at the same time ₂₉ For T or deletion, xz is selected from GGPSSGAPPPS, GPSSGAPPPS, PSSGAPPPS, SSGAPPPSAny of GGPSSGAPPS, GPSSGAPPS, PSSGAPPS or SSGAPPS;

the F is an FC moiety derived from a mammalian immunoglobulin;

the L is a G, S and/or A-rich connecting chain;

preferably, the amino acid sequence of A is shown as any one of SEQ ID NO.46, SEQ ID NO.54, SEQ ID NO.55, SEQ ID NO.59, SEQ ID NO.68 and SEQ ID NO. 74.

2. The multiple active protein of claim 1, further comprising any one or more of the following features:

(1) The amino acid sequence of F is shown in any one of SEQ ID NO. 11-20; (2) The amino acid sequence of L is shown in any one of SEQ ID NO. 21-43.

3. The multiple active protein of claim 1, wherein the amino acid sequence of the multiple active protein is as shown in any one of SEQ ID No.77, SEQ ID No.87, and SEQ ID No. 89.

4. The structure of the multiple active protein comprises a structure shown in a formula III, wherein the structure shown in the formula III is as follows: A-L1-F-L2-B, wherein A is a GCGR/GLP-1R dual-effect agonist, F is a long-acting protein unit, B is natural FGF21 or FGF21 analogue, L1 is a connecting chain connecting A and F, and L2 is absent or a connecting chain connecting B and F;

The structure of A comprises a structure shown in a formula II, and the structure shown in the formula II is as follows: HSQGTFTSDYSKYLDX ₁₆ X ₁₇ X ₁₈ AQDFVQWLMNX ₂₉ Xz formula II, wherein X ₁₆ Any one selected from amino acids other than Y, N, W, and H; x is X ₁₇ Any one selected from amino acids other than P, L, T, F and H; x is X ₁₈ Any one selected from amino acids other than P, F, H and W; in addition X ₁₇ And X is ₁₈ Can not be R, X at the same time ₂₉ Is T or deleted, xz is selected from any one of GGPSSGAPPPS, GPSSGAPPPS, PSSGAPPPS, SSGAPPPS, GGPSSGAPPS, GPSSGAPPS, PSSGAPPS or SSGAPPS;

the F is an FC moiety derived from a mammalian immunoglobulin;

the L2 is a G, S and/or A-rich connecting chain;

5. The multi-active protein of claim 4, further comprising any one or more of the following features:

(1) The amino acid sequence of F is shown in any one of SEQ ID NO. 11-20; (2) The amino acid sequence of the L1 is shown in any one of SEQ ID NO. 21-43; (3) The amino acid sequence of the L2 is shown in any one of SEQ ID NO. 21-43; (4) The amino acid sequence of the B is shown as any one of SEQ ID NO.143-146 and SEQ ID NO. 155.

6. The multiple active protein of claim 4, wherein the amino acid sequence of the multiple active protein is as shown in any one of SEQ ID nos. 147-154 or 156-160.

7. An isolated polynucleotide encoding the multi-active protein of any one of claims 1-6.

8. A recombinant expression vector comprising the isolated polynucleotide of claim 7.

9. A host cell comprising the recombinant expression vector of claim 8 or the isolated polynucleotide of claim 7 integrated into the genome with an exogenous source.

10. The method for producing a multi-active protein according to claim 1 to 6,

culturing the host cell of claim 9 under suitable conditions to express the multi-active protein, and then isolating and purifying to obtain the multi-active protein.

11. Use of a multiple active protein according to any one of claims 1-6 in the manufacture of a medicament for the treatment of diabetes and obesity related diseases.

12. A composition comprising a culture of the multi-active protein of any one of claims 1-6 or the host cell of claim 9, and a pharmaceutically acceptable carrier.