A multi-domain active protein for treating metabolic diseases

Abstract

The present invention belongs to the field of biopharmaceuticals, and particularly relates to a multi-domain active protein for treating metabolic diseases. The multi-domain active protein has a structural formula as shown in Formula I: A-L a -F-L b -B. Compared with the prior art, the multi-domain active protein of the present invention has a long half-life and supports a once-weekly dosing frequency; the GLP-1R agonist activity of the multi-domain active protein is increased by more than 200 times at most; the multi-domain active protein has good stability in vivo and in vitro and low immunogenicity; since no unnatural amino acids need to be introduced and no chemical synthesis and cross-linking steps are involved, it can be prepared by a recombinant method, greatly simplifying the preparation process.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceuticals, and particularly relates to a multi-domain active protein for treating metabolic diseases. Background Art

[0002] Diabetes can be classified into type 1 diabetes and type 2 diabetes according to pathological characteristics. Type 1 diabetes is mainly characterized by insufficient insulin secretion and requires daily insulin injection; while type 2 diabetes is caused by the body's inability to effectively utilize insulin. The majority of diabetes patients are type 2 diabetes patients. It is estimated that about 80-90% of type 2 diabetes patients are significantly obese (Center for disease control and prevention (CDC) National Diabetes Fact Sheet, 2014).

[0003] Conventional chemical drugs for treating type 2 diabetes, such as sulfonylureas and thiazolidinediones, have obvious hypoglycemic effects, but the main drawback is that they can cause weight gain (Kahn SE, Haffner SM, Heise MA, Herman WH, Holman RR, Jones NP, et al. Glycemic durability of rosiglitazone, metformin, or glyburidemonotherapy. N Engl J Med 2006; 355(23): 2427-43.). The protein drugs for type 2 diabetes are mainly GLP-1R (GLP-1 receptor) agonists, such as Dulaglutide (trade name: ), Albiglutide (trade name ), Liraglutide (trade name and are respectively used for treating obesity and diabetes), Exenatide (trade name ), Lixisenatide (trade name ) and Semaglutide, etc. GLP-1R agonists have significant blood glucose-lowering effects. Moreover, different from insulin, the blood glucose-lowering effect of GLP-1R agonists is strictly blood glucose-dependent, not easily causing hypoglycemia, and also having the effect of weight loss. For example, the weight loss of Dulaglutide is approximately 2.9 kg, while Liraglutide (once a day, dose 3 mg) approved for weight loss has a weight loss of about 8 kg. The weight loss of these drugs is mainly through appetite control, and most of them do not exceed 10% of the average weight. Although bariatric surgery can significantly improve obesity and treat diabetes, its application is not extensive because most patients are not willing to accept this surgery due to surgical risks and long-term sequelae considerations (Obesity and Diabetes, New Surgical and Nonsurgical Approaches, Springer Publishing House, 2015).

[0004] It is reported that the secretion of incretin in patients undergoing surgical weight loss surgery will surge (Obesity and Diabetes, New Surgical and Nonsurgical Approaches, Springer Publishing House, 2015). Therefore, currently, the new generation of diabetes drugs mainly focuses on the research of dual-acting or multi-acting incretin receptor agonists, such as GLP-1R / GIPR and GLP-1R / GCGR dual agonists, and even GLP-1R / GIPR / GCGR triple agonists.

[0005] Among them, the receptors of glucagon and GLP-1 (glucagon-like peptide-1) are structurally related, but these two hormones show diametrically opposite effects in controlling glucose. Clinically, GLP-1 and its analogs are mainly used for blood glucose control in diabetic patients, while glucagon is used for acute hypoglycemia. In recent years, more and more studies have shown that although glucagon has the risk of increasing blood glucose, it can effectively reduce weight; more importantly, GLP-1 and glucagon seem to have positive additive or synergistic physiological effects. For example, the dual agonist of glucagon receptor (GCGR) and GLP-1 receptor (GLP-1R) can lose weight more effectively than the single agonist of GLP-1R. Although GCGR activation may lead to an increase in blood glucose level, this risk can be appropriately offset by GLP-1R activation.

[0006] Currently, dual agonists of GLP-1R and GCGR are generally based on oxyntomodulin or glucagon, and are modified (oxyntomodulin analogs or glucagon analogs) to improve their short-acting and enzymatic degradation defects. Most of these analogs mutate the serine (Ser) at the second position into the unnatural amino acid Aib to resist the enzymatic degradation of DPP-IV. This is because natural glucagon and oxyntomodulin are similar to natural GLP-1 and are extremely vulnerable to hydrolysis by DPP-IV protease in serum, resulting in inactivation (Victor A. Gault et al., A novel GLP-1 / glucagon hybrid peptide with triple-acting agonist activity at GIP, GLP-1 and glucagon receptors and therapeutic potential in high-fat fed mice, J Biol Chem., 288(49):35581-91. 2013; Bhat VK et al., A DPP-IV-resistant triple-acting agonist of GIP, GLP-1 and glucagon receptors with potent glucose-lowering and insulinotropic actions in high-fat-fed mice, Diabetologia, 56(6):1417-24. 2013; John A. Pospisilik et al.; Metabolism of glucagon by dipeptidyl peptidase IV(CD26), Regulatory Peptides 96:133–141, 2001; Hinke SA et al., Dipeptidyl peptidase IV(DPIV / CD26) degradation of glucagon. Characterization of glucagon degradation products and DPIV-resistant analogs, J Biol Chem 275:3827–3834, 2000; Alessia Santoprete et al., DPP-IV-resistant, long-acting oxyntomodulin derivatives, J.Pept.Sci., 17:270–280, 2011).

[0007] FGF21 belongs to the members of the polypeptide family that is widely expressed in developmental and adult tissues and plays important roles in various physiological functions. FGF21 is mainly expressed in pancreatic β-cells, liver, WAT, and skeletal muscle. Recently, it has been found that it is also expressed at low levels in thymus, vascular endothelium, kidney, and testicular tissues, with obvious tissue specificity, and belongs to the "endocrine" hormones of the FGF family together with FGF15 / 19 and FGF23.

[0008] As an important metabolic regulator, FGF21 has been proven to improve various metabolic abnormalities in preclinical models of type II diabetes (T2DM). FGF21 has potential effects as a therapeutic drug for diabetic patients in terms of improving insulin sensitivity, improving blood glucose control, reducing body weight, lowering low-density lipoprotein cholesterol (LDL-C) and triglyceride levels, while increasing high-density lipoprotein cholesterol level (HDL-C). In diabetic mice and monkeys, human FGF21 can reduce fasting serum glucose concentration, reduce fasting serum triglyceride, insulin, and glucagon concentrations. In addition, in a rodent model of diet-induced obesity, administration of FGF21 results in dose-dependent overall body weight loss. Therefore, FGF21 has the potential to treat diseases such as diabetes, obesity, dyslipidemia, and metabolic syndrome.

[0009] However, FGF21 has a very short serum half-life: 30 minutes in mice and 2 hours in monkeys. Therefore, daily injection or continuous infusion of the corresponding FGF21 protein is required to maintain in vivo biological activity. In human studies, circulating FGF21 levels tend to increase in patients with obesity, dyslipidemia, TDM2, and other insulin resistance-related diseases. Studies have shown that an increase in FGF21 concentration is associated with an increased risk of CVD, and it can also lead to osteoporosis and affect reproduction (by promoting metabolism and thus causing energy deficiency), etc. (Wei W, Dutchak PA, Wang X, Ding X, Wang X, Bookout AL, et al. Fibroblast growth factor 21 promotes bone loss by potentiating the effects of peroxisome proliferator-activated receptor gamma. Proc Natl Acad Sci USA. 2012;109(8):3143–8; Fibroblast growth factor 21 has no direct role in regulating fertility in female mice, Mol Metab, 5(8):690-8, 2016). The homology of the FGF family sequence and the widespread distribution of the FGFR1 receptor also raise concerns about the potential safety issues associated with the clinical use of high doses of FGF21 (Kharitonenkov A & DiMarchi R: Fibroblast growth factor 21 night watch: advances and uncertainties in the field. J Intern Med. 2017 Mar;281(3):233-246.).

[0010] Both GCGR / GLP-1R dual agonists and FGF21 analogs are used to treat diabetes and weight loss respectively. In addition, there are reports on the preparation of a dual-active protein by fusing GLP-1 analog and FGF21 through Fc (YH25723, a Novel Long-Acting GLP-1 / FGF21 Dual Agonist Provides More Potent and Sustained Glycemic Control and Greater Weight Loss Compared with Single Agonists in Animal Models, The American Diabetes Association, 2016). As mentioned above, currently, dual- or even triple-effect polypeptides based on the modification of Glucagon or gastrin-releasing peptide generally require the substitution of some amino acids with unnatural amino acids to improve stability and activity, and even need to be modified by fatty acids or PEG. It is extremely difficult technically to fuse and express with FGF21 analogs to prepare a single molecule. Currently, there is no report on the combination use of dual- or multi-effect agonist polypeptides and FGF21 analogs. Summary of the Invention

[0011] In order to overcome the problems existing in the prior art, the purpose of the present invention is to provide a multi-domain active protein for treating metabolic and related diseases, its preparation and application. The multi-active protein of the present invention has a significant weight loss effect and can be clinically used to treat related diseases such as diabetes, weight loss, non-alcoholic fatty liver, and hyperlipidemia.

[0012] In order to achieve the above purpose and other related purposes, the present invention adopts the following technical solutions:

[0013] In the first aspect of the present invention, there is provided a multi-domain active protein, the structure of which includes a structure shown in Formula I, and the structure shown in Formula I is: A-L a -F-L b -B, wherein A is a GCGR / GLP-1R dual agonist active peptide, F is a long-acting protein unit, B is natural FGF21 or an FGF21 analog, and L a does not exist or is a linker, and L b does not exist or is a linker.

[0014] The multi-domain active protein has at least the triple activities of GLP-1, GCG and FGF21.

[0015] Further, in Formula I, the A includes a structure shown in Formula II, and the structure shown in Formula II is:

[0016] HSQGTFTSD-X 10 -S-X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -F-X 23 -X 24 -WL-X 27 -X 28 -X 29 -X 30 -X z 。

[0017] Wherein, said X 10 is selected from any one of V, L or Y; said X 12 is selected from any one of S, E or K; said X 13 is selected from any one of Y or Q; said X 14 is selected from any one of L or M; said X 15 is selected from any one of D or E; said X 16 is selected from any one of S, E or G; said X 17 is selected from any one of R, E or Q; said X 18 is selected from any one of R, E or A; said X 19 is selected from any one of A or V; said X 20 is selected from any one of Q, R or K; said X 21 is selected from any one of D, L or E; said X 23 is selected from any one of V or I; said X 24 is selected from any one of Q, A or E; said X 27 is selected from any one of M, K or V; said X 28 is selected from any one of N or K; said X 29 is selected from any one of G or T; said X 30 is G or absent; said X z is absent or selected from any one of GPSSGAPPPS (SEQ ID NO.3), PSSGAPPPS (SEQ ID NO.4),

[0018] SSGAPPPS (SEQ ID NOIL-11.5), GPSSGAPPS (SEQ ID NO.6), PSSGAPPS (SEQ IDNO.7) or KRNRNN IA (SEQ ID NO.8).

[0019] Further, the amino acid sequence of A is as shown in any one of SEQ ID NOs. 44 to 92.

[0020] In Formula I, B is natural FGF21 (SEQ ID NO. 136) or an FGF21 analog. B includes the following structure:

[0021] HPIPDSSPLLQFGGQVRQ X 19 YLYTDDAQQTE X 31 HLEI X 36 EDGTVG X 43 A X 45 DQSPESLL QL X 56 ALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRE X 98 LLEDGYNVYQSEAH GLPLH X 118 PGN X 122 SPHRDPAPRGP X 134 RFLPLPGLPPALPEPPGILAPQPPDVGSSDPL X 167 MV X 170 X 171 SQ X 174 RSPS X 179 X 18 0X 181 , wherein the N-terminal HPIPDSS may be absent or partially absent; X 19 is selected from R, Y, V, E or C; X 31 is selected from A or C; X 36 is selected from R or K; X 43 is selected from G or C; X 45 is selected from A, K, E or V; X 56 is selected from K, R, V or I; X 98 is selected from L, R or D; X 118 is selected from L or C; X 122 is selected from K or R; X 134 is selected from A or C; X 167Selected from S, A or R; X 170 Selected from G or E; X 171 Selected from P or G; X 174 Selected from G, A or L; X 179 Selected from Y, A or F; X 180 Selected from A or E; X 181 Selected from S, K or absent.

[0022] The FGF21 analog is an active protein having the same or similar biological functions as natural FGF21 (SEQ ID NO. 136) and having a homology of more than 80% with natural FGF21 (SEQ ID NO. 136). Preferably, the FGF21 analog has a homology of more than 85% with natural FGF21 (SEQ ID NO. 136); more preferably, the FGF21 analog has a homology of more than 90% with natural FGF21 (SEQ ID NO. 136); more preferably, the FGF21 analog has a homology of more than 95% with natural FGF21 (SEQ ID NO. 136). Exemplarily, the FGF21 analog may be selected from FGF21 analogs or mutants described in patents or patent applications such as US20140213512, US8188040, US9493530, WO 2016114633, US 20150291677, US 9422353, US 8541369, US7622445, US7576190, US20070142278, US9006400 or US 20130252884.

[0023] Preferably, the FGF21 analog is as shown in SEQ ID NO. 137-148.

[0024] Furthermore, the F is the F derived from mammalian immunoglobulin C portion. The immunoglobulin is a polypeptide chain molecule containing disulfide bonds and generally has two light chains and two heavy chains. The F of the immunoglobulin used here C portion has the common meaning of the term in the field of immunology. Specifically, this term refers to an antibody fragment obtained by removing two antigen-binding regions (Fab fragments) from an antibody. The F C portion may include a hinge region and extend through the CH2 and CH3 domains to the C-terminus of the antibody. The F C portion may further include one or more glycosylation sites. There are 5 human immunoglobulins in the human body with different effector characteristics and pharmacokinetic properties: IgG, IgA, IgM, IgD and IgE. IgG is the immunoglobulin with the highest content in serum. IgG also has the longest serum half-life (about 23 days) among all immunoglobulins.

[0025] Further, F can be selected from the complete F of immunoglobulins, C the F C fragments of parts, or mutants of the F C parts of immunoglobulins.

[0026] The immunoglobulin F used in the present invention C part is derived from the F C region of mammalian IgG1, IgG2 or IgG4 or mutants thereof; preferably, it can be the F C region of human IgG1, IgG2 or IgG4 or mutants thereof; more preferably, it can be the F C region of human IgG1 or IgG4 or mutants thereof. In a preferred embodiment, the 297th position of the F C domain is replaced by glycine or alanine. The above content is in accordance with the EU index number of Kabat (Kabat, E.A. et al., sequences of proteins of immunological interest, 5th edition, public health service, National Institutes of Health, Bethesda, MD (1991)).

[0027] In a preferred embodiment, the F C domain is from human IgG4 and is as shown in SEQ ID NO. 16. In a preferred embodiment, the F C domain is from human IgG1 and is as shown in SEQ ID NO. 12. The K at the end of the F C chain can be removed to facilitate the homogeneity of the expression product.

[0028] In some embodiments, the amino acid sequence of the F can be as shown in any one of SEQ ID NOs. 9 - 18.

[0029] In the above multi - domain active protein of the present invention, the GCGR / GLP - 1R dual - acting agonist peptide A is fused with natural FGF21 or FGF21 analog B through the long - acting protein unit F to form a recombinant fusion protein with at least GLP - 1, GCG and FGF21 triple - acting activities. A and F can be connected by a linker chain, such as L a , or can be directly fused without adding a linker chain. F and B can be connected by a linker chain, such as L b , or can be directly fused without adding a linker chain.

[0030] When L a and Lb When it is a linking chain, the linking chain is a flexible polypeptide of appropriate length composed of glycine (G), serine (S) and / or alanine (A), so that adjacent protein domains can move freely relative to each other. When it is necessary to ensure that the two adjacent domains do not interfere with each other spatially, a longer linking chain can be used.

[0031] The preferred linking chains of the present invention include units rich in G, S and / or A, such as (GS)n, (GGS)n, (GGSG)n, (GGGS)nA, (GGGGS)nA, (GGGGA)nA, etc., where n is an integer from 1 to 10. In a preferred embodiment, the amino acid length of the linking chain is 5 - 26. The exemplary linking chains are each independently selected from Table 2.

[0032] Furthermore, the amino acid sequence of the linking chain can be as shown in any one of SEQ ID NO.19 - 41.

[0033] Furthermore, the amino acid sequence of the multi - domain active protein is as shown in any one of SEQ ID NO.150 - 208.

[0034] The multi - domain active protein provided by the present invention is an F C fusion protein, retaining the conventional properties of F C , such as binding to FcRn to extend the in - vivo half - life, and the property of achieving efficient purification by binding to Protein A or G - type fillers with high affinity and high specificity during the separation and purification process. In addition, these multi - domain active proteins can not only effectively resist the degradation of proteases in serum inside the protein, but also effectively prevent the degradation at the N - terminus. For Incretin - like polypeptides such as Glucagon or GLP - 1, the integrity of the N - terminus is crucial for determining their biological activity. The in - vivo half - lives of native Glucagon and GLP - 1 are short. Besides the reason of small molecular weight, more importantly, it is due to hydrolysis by DPP - IV enzyme in the body and hydrolysis inside the polypeptide. In an embodiment of the present invention, after native Glucagon is fused with F C , it will still be rapidly degraded by DPP - IV and inactivated; while the corresponding Glucagon analog can significantly resist the attack of DPP - IV.

[0035] In the second aspect of the present invention, there is provided an isolated polynucleotide encoding the aforementioned multi - domain active protein.

[0036] In the third aspect of the present invention, there is provided a recombinant expression vector containing the aforementioned isolated polynucleotide.

[0037] In a fourth aspect of the present invention, there is provided a host cell which contains the aforementioned recombinant expression vector or has the aforementioned isolated polynucleotide integrated into its genome.

[0038] In a fifth aspect of the present invention, there is provided a method for preparing the aforementioned multi-domain active protein, which includes culturing the aforementioned host cell under suitable conditions to express the multi-domain active protein, and then separating and purifying to obtain the multi-domain active protein.

[0039] In a sixth aspect of the present invention, there is provided the use of the aforementioned multi-domain active protein in the preparation of a medicament for treating diabetes metabolic-related diseases.

[0040] The multi-domain active protein provided by the present invention can be used to treat metabolic syndrome. Metabolic syndrome is typically characterized by clustering of at least three or more of the following risk factors: (1) abdominal obesity (excessive adipose tissue inside or around the abdomen), (2) atherogenic dyslipidemia, dyslipidemia, including high triglycerides, low HDL cholesterol and high LDL cholesterol, which enhances the accumulation of plaques in the arterial wall, (3) elevated blood pressure, (4) insulin resistance or glucose intolerance, (5) a thrombotic state, such as high fibrin or plasminogen activator inhibitor-1 in the blood, and (6) a pro-inflammatory state, such as elevated C-reactive protein in the blood. Other risk factors may include aging, hormonal imbalance and genetic factors.

[0041] In addition, the multi-domain active protein of the present invention can also be used to treat obesity. In some aspects, the multi-domain active protein of the present invention treats obesity through mechanisms such as reducing appetite, decreasing food intake, lowering the body fat level of patients, and increasing energy consumption.

[0042] In a seventh aspect of the present invention, there is provided a method for treating metabolic-related diseases, which includes administering the aforementioned multi-domain active protein to a subject.

[0043] The present invention further provides a method for promoting weight loss or preventing weight gain, which includes administering the aforementioned multi-domain active protein to a subject.

[0044] In an eighth aspect of the present invention, there is provided a composition which contains the aforementioned multi-domain active protein or a culture of the aforementioned host cell, and a pharmaceutically acceptable carrier.

[0045] In a ninth aspect of the present invention, there is provided the use of the aforementioned multi-domain active protein in the preparation of a fusion protein.

[0046] In a tenth aspect of the present invention, there is provided a fusion protein which contains the aforementioned multi-domain active protein in its structure.

[0047] In the eleventh aspect of the present invention, there is provided another composition for treating metabolic and related diseases, comprising a GCGR / GLP-1R dual-agonist active protein and a long-acting FGF21 analog.

[0048] The structure of the GCGR / GLP-1R dual-agonist active protein includes: A-L a -F, and the structure of A includes the structure shown in Formula II, and the structure shown in Formula II is:

[0049] HSQGTFTSD-X 10 -S-X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -F-X 23 -X 24 -WL-X 27 -X 28 -X 29 -X 30 -X z .

[0050] Wherein, the X 10 is selected from any one of V, L or Y; the X 12 is selected from any one of S, E or K; the X 13 is selected from any one of Y or Q; the X 14 is selected from any one of L or M; the X 15 is selected from any one of D or E; the X 16 is selected from any one of S, E or G; the X 17 is selected from any one of R, E or Q; the X 18 is selected from any one of R, E or A; the X 19 is selected from any one of A or V; the X 20 is selected from any one of Q, R or K; the X 21 is selected from any one of D, L or E; the X 23 is selected from any one of V or I; the X 24 is selected from any one of Q, A or E; the X 27 is selected from any one of M, K or V; the X 28 is selected from any one of N or K; the X 29 is selected from any one of G or T; the X 30 is G or absent; the X zAbsent or selected from any one of GPSSGAPPPS (SEQ ID NO.3), PSSGAPPPS (SEQ ID NO.4), SSGAPPPS (SEQ ID NO.5), GPSSGAPPS (SEQ ID NO.6), PSSGAPPS (SEQ ID NO.7) or KRNRNN IA (SEQ ID NO.8).

[0051] Said F is a long-acting protein unit, and F can be selected from the complete F of immunoglobulin C portion, the F of immunoglobulin C fragment of the portion or the mutant of the F of immunoglobulin C Further, the amino acid sequence of said F is shown in SEQ ID NO.9-18.

[0052] L a Absent or is a linker chain. When it is a linker chain, the linker chain is a flexible polypeptide of a suitable length composed of glycine (G), serine (S) and / or alanine (A), so that adjacent protein domains can move freely relative to each other. When it is necessary to ensure that the two adjacent domains do not interfere with each other spatially, a longer linker chain can be used. Exemplary linker chains are such as (GS)n, (GGS)n, (GGSG)n, (GGGS)nA, (GGGGS)nA, (GGGGA)nA, etc., where n is an integer from 1 to 10. Exemplary linker chains can be independently selected from Table 2.

[0053] Further, the amino acid sequence of said GCGR / GLP-1R dual-acting agonist protein is any one of SEQ ID NO.96, SEQ ID NO.98, SEQ ID NO.100, SEQ ID NO.102, SEQ ID NO.104, SEQ ID NO.106, SEQ ID NO.108, SEQ ID NO.110, SEQ ID NO.112, SEQ ID NO.114-133.

[0054] The structure of said long-acting FGF21 analog includes: F-L b -B, and the structure of said B includes:[[]]

[0055] HPIPDSSPLLQFGGQVRQ X 19 YLYTDDAQQTE X 31 HLEI X 36 EDGTVG X 43 AX 45 DQSPESLLQL X 56 ALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRE X 98 LLEDGYNVYQSEAHGLPLH X 118 PGN X 122 SPHRDPAPRGP X 134 RFLPLPGLPPALPEPPGILAPQPPDVGSSDPL X 167 MV X 170 X 171 SQ X 174 RSPS X 179 X 18 0X 181 , wherein the N-terminal HPIPDSS may be absent or partially absent; X 19 is selected from R, Y, V, E or C; X 31 is selected from A or C; X 36 is selected from R or K; X 43 is selected from G or C; X 45 is selected from A, K, E or V; X 56 is selected from K, R, V or I; X 98 is selected from L, R or D; X 118 is selected from L or C; X 122 is selected from K or R; X 134 is selected from A or C; X 167 is selected from S, A or R; X 170 is selected from G or E; X 171 is selected from P or G; X 174 is selected from G, A or L; X 179 is selected from Y, A or F; X 180 is selected from A or E; X 181 is selected from S, K or absent.

[0056] The FGF21 analog described above is an active protein that has the same or similar biological functions as natural FGF21 (SEQ ID NO. 136) and has a homology of more than 80% with natural FGF21 (SEQ ID NO. 136). Preferably, the FGF21 analog has a homology of more than 85% with natural FGF21 (SEQ ID NO. 136); more preferably, the FGF21 analog has a homology of more than 90% with natural FGF21 (SEQ ID NO. 136); more preferably, the FGF21 analog has a homology of more than 95% with natural FGF21 (SEQ ID NO. 136). Exemplarily, the FGF21 analog described above can be selected from FGF21 analogs or mutants described in patents or patent applications such as US20140213512, US8188040, US9493530, WO 2016114633, US 20150291677, US 9422353, US 8541369, US7622445, US7576190, US20070142278, US9006400 or US 20130252884. The FGF21 analogs are shown as SEQ ID NO. 137-148.

[0057] The F can be selected from the complete F of an immunoglobulin C portion, a fragment of the F of an immunoglobulin C portion or a mutant of the F of an immunoglobulin C portion. The amino acid sequence of the F can be shown as any one of SEQ ID NO. 9-18. L b is absent or is a linker chain. When L b is a linker chain, the linker chain includes units containing G, S, and / or A, such as (GS)n, (GGS)n, (GGSG)n, (GGGS)nA, (GGGGS)nA, (GGGGA)nA, etc., where n is an integer from 1 to 10. In a preferred embodiment, the amino acid length of the linker chain is 5-26. Exemplary linker chains are each independently selected from Table 2. Further, the amino acid sequence of the linker chain can be shown as any one of SEQ ID NO. 19-41.

[0058] Further, the amino acid sequence of the long-acting FGF21 analog is shown as any one of SEQ ID NO: 210-221.

[0059] In a twelfth aspect of the present invention, there is provided the use of the aforementioned composition in the preparation of a medicament for treating metabolic-related diseases.

[0060] In a thirteenth aspect of the present invention, there is provided another method for treating metabolic-related diseases, which includes administering to a subject the aforementioned composition comprising a GCGR / GLP-1R dual-agonistic active protein and a long-acting FGF21 analog.

[0061] The present invention further provides a method for promoting weight loss or preventing weight gain, which includes administering to a subject the aforementioned composition comprising a GCGR / GLP-1R dual-agonistic active protein and a long-acting FGF21 analog.

[0062] It is well known that incretin-like hormone proteins such as GLP-1 analogs and Exendin-4 can cause side effects such as nausea and vomiting in patients, and these are dose-related. Therefore, when an ideal blood glucose level can be maintained, minimizing the dosage theoretically alleviates the uncomfortable side effects of patients. In addition, there have also been reports on the effects of FGF21 on osteoporosis and reproduction (Fibroblast growth factor-21 concentration in serum and synovial fluid is associated with radiographic bone loss of knee osteoarthritis. Scand J Clin Lab Invest. 2015 Apr;75(2):121-5; Fibroblast growth factor 21 has no direct role in regulating fertility in female mice. Mol Metab. 5(8):690-8, 2016). Theoretically, the risk of drug side effects is proportional to the dosage. In the field of diabetes drugs, the safety of drugs is extremely high. The inventors have found that the GCG / GLP-1 / FGF21 triple-active protein and the above-mentioned composition have good effects on controlling blood glucose and body weight at very low dosages, have little impact on the gastrointestinal tract, greatly reduce the dosage of FGF21, and thus significantly reduce the potential side effect risk.

[0063] Compared with the prior art, the present invention has the following beneficial effects:

[0064] (1) The multi-domain active protein of the present invention has a long half-life and supports a once-weekly dosing frequency;

[0065] (2) The GLP-1R agonistic activity of the multi-domain active protein of the present invention is increased by up to more than 200 times;

[0066] (3) The multi-domain active protein of the present invention has good stability in vivo and in vitro and low immunogenicity;

[0067] (4) Since there is no need to introduce non-natural amino acids and no chemical synthesis or cross-linking steps are involved, it can be prepared by recombinant methods, greatly simplifying the preparation process. Description of the Drawings

[0068] Figure 1 Reduction electrophoresis diagram (10% SDS-PAGE) of the partially purified triple-effect active protein obtained. Lanes 1-13 are C002L 13 F4L 10 W, C240L 12 F8L 12 M1, C495L 13 F8L 10 M1, C266L 13 F7L 13 M4, C462L9F2L9M 10 , C611L 11 F4L 11 M 11 , C563L 14 F8L9M3, C382L 13 F8L 10 M2, C623L9F3L 10 M1, C731L5F2L9M9, C353L 13 F3L 10 M4, C227L 12 F5L 14 M4, C137L 10 F8L9M5; M is the protein standard: 97.2, 66.4, 44.3, 29, 20.1, 14.3 KD.

[0069] Figure 2A : Graph showing the results of serum stability over time.

[0070] Figure 2B : Graph showing the results of serum stability over time.

[0071] Figure 2C : Graph showing the results of serum stability over time.

[0072] Figure 3 : Graph showing the blood sugar lowering effect of the active protein in Example 8 in normal ICR mice.

[0073] Figure 4 : Graph showing the effect of the active protein in Example 9 on the body weight of DIO mice.

[0074] Figure 5: Effect of the active protein in Example 9 on the appetite of DIO mice; taking the food intake of DIO mice in the PBS group as 100%, and the vertical axis is the percentage of the food intake of other groups compared thereto.

[0075] Figure 6 : Effect of the active protein in Example 10 on the body weight of DIO mice.

[0076] Figure 7 : Effect of the active protein in Example 10 on the appetite of DIO mice; taking the food intake of DIO mice in the PBS group as 100%, and the vertical axis is the percentage of the food intake of other groups compared thereto. Detailed implementation manners

[0077] Term explanations:

[0078] The term "diabetes" includes type 1 diabetes, type 2 diabetes, gestational diabetes, and other conditions that cause hyperglycemia. This term is used for metabolic disorders in which the pancreas does not produce enough insulin, or the body's cells do not respond properly to insulin, so that the efficiency of tissue cells to absorb glucose decreases, resulting in the accumulation of glucose in the blood.

[0079] Type 1 diabetes is also known as insulin-dependent diabetes and juvenile-onset diabetes, which is caused by the destruction of β cells and usually results in absolute insulin deficiency.

[0080] Type 2 diabetes is also known as non-insulin-dependent diabetes and adult-onset diabetes, which is generally associated with insulin resistance.

[0081] The term "obesity" means an excess of adipose tissue. When energy intake exceeds energy expenditure, excess calories are stored in fat, resulting in obesity. In this article, individuals with a body mass index (BMI = weight (kg) divided by the square of height (m)) exceeding 25 are considered obese.

[0082] Incretin: Incretin is a gastrointestinal hormone that regulates blood glucose by enhancing glucose-stimulated insulin secretion (also known as glucose-dependent insulin secretion, GSIS) (Drucker.D J, Nauck, MA, Lancet 368: 1696-705, 2006). Incretin can also slow down the rate of nutrient absorption by delaying gastric emptying and directly reduce food absorption. At the same time, incretin also inhibits the secretion of glucagon by intestinal α cells. So far, there are two known incretins: glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP).

[0083] PreproGlucagon: It is a precursor polypeptide composed of 158 amino acids, which is differentially processed in tissues to form a variety of structurally related proglucagon-derived peptides, including glucagon, glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), and oxyntomodulin (OXM).

[0084] GIP: It is a 42-amino acid peptide obtained by proteolytic processing of a 133-amino acid precursor (pre-pro-GIP). These molecules are involved in a variety of biological functions, including glucose homeostasis, insulin secretion, gastric emptying, intestinal growth, and regulation of food intake.

[0085] Glucagon-like peptide (GLP-1): The sequence is as shown in SEQ ID NO: 1; it is an incretin hormone polypeptide of 30 or 31 amino acids secreted from intestinal L-cells, and has two active forms, 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 insulin secretion, inhibition of glucagon production, delayed gastric emptying, and appetite suppression (Tharakan G, Tan T, Bloom S. Emerging therapies in the treatment of ‘diabesity’: beyond GLP-1. Trends Pharmacol Sci 2011;32(1):8-15.). The therapeutic potential of native GLP-1 is limited because it can be rapidly degraded by dipeptidyl peptidase-4 (DPP-4), neutral endopeptidase (NEP), plasma kallikrein, or plasmin. Since native GLP-1 has an ultra-short half-life of only about 2 minutes in vivo, methods have emerged to improve efficacy for the treatment of diabetes and obesity by using chemical modifications and / or formulation forms (Lorenz M, Evers A, Wagner M. Recent progress and future options in the development of GLP-1 receptor 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-1 receptor agonists for type 2 diabetes. Expert Opin Investig Drugs 2016;25(2):145-58).

[0086] Oxyntomodulin is a 37-amino acid small peptide, and its sequence is shown in SEQ ID NO: 2; it contains the complete 29-amino acid sequence of Glucagon (SEQ ID NO: 42). Oxyntomodulin is a dual agonist of GLP-1R and GCGR and is secreted together with GLP-1 by intestinal L-cells after a meal. Similar to Glucagon, oxyntomodulin produces significant weight loss in humans and rodents. The weight loss activity of oxyntomodulin has been compared with equimolar doses of selective GLP-1 agonists in obese mice. It has been found that compared with selective GLP-1R agonists, oxyntomodulin has an antihyperglycemic effect, can significantly reduce body weight and has lipid-lowering activity (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 native oxyntomodulin reduces 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, randomized, 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: a 11andomized controlled trial; Wynne K et al., Int J Obes (Lond), 30:1729-36, 2006). However, due to its small molecular weight and degradation by DPP-IV, oxyntomodulin has a short half-life.At present, dual-acting agonists of GLP-1 receptor (GLP-1R) and glucagon receptor (GCGR) are generally based on oxyntomodulin, and mutations (oxyntomodulin analogs) are made to improve the short-acting and enzymatic defects of oxyntomodulin, and most of them adopt the method of mutating the second serine Ser to α-aminoisobutyric acid (Aib), and introduce non-natural amino acids to resist DPP-IV enzymatic hydrolysis. Although oxyntomodulin analogs have shown preliminary hypoglycemic and fat-reducing effects, their mechanism of action is still unclear, and oxyntomodulin receptors have not been discovered. At present, only GCGR or GLP-1R knockout mice or cell experiments have been used to verify that oxyntomodulin can bind to these two receptors and work.

[0087] Glucagon is a 29 amino acid peptide corresponding to amino acids 53-81 of preproglucagon, and the sequence is shown in SEQ ID NO: 42 (CGFanelli 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 (Habegger KM 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 increasing blood glucose levels under hypoglycemic conditions, regulating hepatic ketone production, regulating bile acid metabolism and satiety effects through the vagus nerve by stimulating glycogenolysis and gluconeogenesis. Glucagon has been used therapeutically for acute hypoglycemia, and glucagon receptor activation reduces food intake and promotes lipolysis and weight loss in animals and humans.

[0088] The term "receptor agonist" can be defined as a polypeptide, protein or other small molecule that binds to a receptor and elicits the usual response of the natural ligand.

[0089] "GLP-1 receptor (GLP-1R) agonists" can be defined as peptides, proteins or other small molecules that bind to GLP-1R and can trigger characteristic reactions identical or similar to those of natural GLP-1. GLP-1R agonists fully or partially activate GLP-1R, which in turn triggers a series of downstream signaling pathways in cells, resulting in corresponding cell activities, such as insulin secretion from β cells; typical GLP-1R agonists include natural GLP-1 and its mutants and analogs, such as exenatide and liraglutide.

[0090] GLP-1 Analogue: In this text, "GLP-1 analogue" or "GLP-1 mutant" both refer to GLP-1R agonists and can be used interchangeably.

[0091] Glucagon Receptor (GCGR) Agonist: That is, Glucagon receptor agonist, which can be defined as a polypeptide, protein or other small molecule that binds to GCGR and can trigger the same or similar characteristic responses as native Glucagon. GCGR agonists activate GCGR completely or partially, and then cause a series of downstream signal pathway reactions within cells, generating corresponding cellular activities: such as glycogenolysis, gluconeogenesis, fatty acid oxidation and ketogenesis in hepatocytes, etc.

[0092] Glucagon Analogue: In this text, "Glucagon analogue", "GCG analogue", "Glucagon mutant" and "GCG mutant" all refer to Glucagon receptor agonists and can be used interchangeably.

[0093] GCGR / GLP-1R Dual-Acting Peptide: The GCGR / GLP-1R dual-acting peptide of the present invention includes proteins or polypeptides that can simultaneously activate GLP-1R and GCGR. Such as the dual agonist based on Oxyntomodulin reported by Alessandro Pocai et al. (Glucagon-Like Peptide 1 / Glucagon Receptor Dual Agonism Reverses Obesity in Mice, Diabetes; 58(10):2258-2266, 2009), or the dual agonist based on Glucagon reported by Richard D. DiMarchi et al. (US9018164 B2). In this text, "dual agonist", "bispecific active protein" or "dual-acting active protein" are synonymous.

[0094] FGF21 (Fibroblast growth factor 21), together with FGF15 / 19 and FGF23, belongs to the "endocrine" hormones of the FGF family. FGF21 is an important hormone that regulates glucose and lipid metabolism. Different from the mechanism regulated by insulin, FGF21 promotes glucose uptake in adipocytes by upregulating the expression of GLUT1. The binding of FGF21 to its receptor requires the assistance of the transmembrane protein β-Klotho. By binding to the FGFR / β-Klotho receptor complex, it stimulates signal transduction and elicits biological effects in the liver, adipose tissue, and pancreas. β-Klotho is selectively expressed in the pancreas, liver, and adipose tissue, which also explains the specificity of the action of FGF21 on these tissues (Kurosu H et al., Tissue-specific expression of betaKlotho and fibroblast growth factor (FGF) receptor isoforms determines metabolic activity of FGF19 and FGF21. J Biol Chem 282:26687-26695, 2007; Kharitonenkov A et al., (2008b) FGF-21 / FGF-21 receptor interaction and activation is determined by betaKlotho. J Cell Physiol 215:1-7). In the presence of the co-receptor β-Klotho, FGF21 can bind to and activate three FGFR subtypes (1c, 2c, and 3c). Other FGFR subtypes, such as FGFR1b, 2b, and 3b, do not form a complex with β-Klotho and thus do not serve as FGF21 receptors. Evidence shows that among the FGFR receptors that bind FGF21, FGFR1 plays a major role in regulating FGF21 activity. The N- and C-termini of FGF21 are very important for its biological activity. The N-terminus binds to FGFR, and the C-terminus binds to β-Klotho (Micanovic R, et al (2009) Different roles of N- and C-termini in the functional activity of FGF21. J Cell Physiol 219:227-234). The mouse FGF21 protein consists of 210 amino acids with a signal peptide composed of 30 amino acids at the amino terminus. The human FGF21 protein consists of 209 amino acids with a signal peptide composed of 28 amino acids at the amino terminus. The human FGF21 protein has approximately 75% homology with the mouse.FGF21 is mainly expressed in pancreatic β-cells, the liver, WAT, and skeletal muscle, showing obvious tissue specificity. Human FGF21 is easily degraded by prolyl peptidase (FAP, a serine protease) in vivo, with a half-life of 30 minutes in mice and 2 hours in monkeys.

[0095] Multi-domain: A domain is a region with specific structure and independent function in a biological macromolecule, especially in a protein. In a globular protein, a domain has its own specific tertiary structure, and its function does not depend on the rest of the protein molecule. However, different domains in the same protein are often connected by short sequences without secondary structure. Different domains in a protein form a multi-domain. In the present invention, the multi-domain refers to a fusion protein containing a GCG analog, FGF21 and its analogs, and FC, which has GCGR agonist activity, GLP-1R agonist activity, and FGF21 activity.

[0096] Dimer: The dimer referred to in the present invention is formed through the natural non-covalent and covalent interactions of the constant region (F C ) of immunoglobulin. If not otherwise specified, the dimers formed by F C are all homodimers, as described in the dimer provided by the present invention. The active protein described in Formula I of the present invention will form dimers due to the presence of F C .

[0097] Triple-active protein: In this article, "triple-agonist active protein", "triple-active agonist active protein", "triple-specific dimer active protein", etc. are all synonymous and can be used interchangeably.

[0098] EC 50 (concentration for 50% of maximal effect) refers to the concentration required for a certain drug or substance to stimulate 50% of its corresponding biological response. The lower the EC 50 value, the stronger the stimulating or agonistic ability of the drug or substance. For example, more intuitively, it can be manifested as a stronger intracellular signal caused, and thus a better ability to induce the production of a certain hormone.

[0099] Cell biological activity

[0100] For the in vitro cell activity assays of the GLP-1R and GCGR agonist activities of the present invention, the luciferase reporter gene detection method is used. This method is based on the principle that after GLP-1R and GCGR are activated, they can activate the downstream cAMP pathway. The activity assay of FGF21 and its analogs is obtained by co-transfecting FGF21R and β-klotho into the same CHO cells and detecting the fluorescence change caused by the signal.

[0101] Joseph R. Chabenne et al. and Richard D. DiMarchi et al. have reported that adding a small peptide cex (GPSSGAPPPS) at the C-terminus of Exendin-4 to the C-terminus of Glucagon can increase the agonistic activity of GLP-1R by about 2 times (Optimization of the Native Glucagon Sequence for Medicinal Purposes, JDiabetes Sci Technol. 4(6):1322–1331, 2010 and Patent US9018164 B2), but the ratio of GCGR agonistic activity to GLP-1R agonistic activity only reaches about 35:1. In addition, Evers A et al. reported (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 after adding the cex sequence to the C-terminus of the GCG analog, the GLP-1R agonistic activity decreased by about 3 times the original, and the activity of GCG decreased by about 14 times (Table 2, Peptides 7 and 8 in the article).

[0102] In one embodiment of the present invention, when the GCG analog containing GPSSGAPPPS or a similar sequence is further fused to the F C chain, the GLP-1R agonistic activity is increased by more than an astonishing 200 times (EC 50 ~1.1 nM). However, for the GCG analogs calculated based on the data disclosed in US9018164 B2 and by Joseph R. Chabenne et al. in the corresponding patents and literature, the change in the GLP-1R agonistic activity ratio before and after adding GPSSGAPPPS or a similar sequence is only about 2 times (for example, the percentage of GLP-1R agonistic activity of native Glucagon in the article is 0.7%, and it increases to 1.6% after adding the GPSSGAPPPS sequence). That is to say, adding the GPSSGAPPPS sequence to the C-terminus of the Glucagon polypeptide does not significantly increase its GLP-1R agonistic activity.

[0103] Stability of multi-domain active proteins

[0104] Native Glucagon has multiple sensitive degradation sites, including the DPP-IV degradation site at the second position and the SRR site at positions 16-18. Although it has been reported that F CIt can improve the chemical stability and serum stability of active proteins. However, for GLP-1 or Glucagon analogs whose N-terminus must be exposed, the effect of F C seems to be not generalizable. After natural GLP-1 or Glucagon is fused with F C , obvious degradation of it under the condition of 37-degree serum can still be observed. In the present invention, mutations resistant to protease hydrolysis are introduced on the basis of natural Glucagon to increase its stability. After these mutants are fused with F C , the stability is further improved.

[0105] Currently, almost all GCGR / GLP-1R dual agonists designed and developed based on Oxyntomodulin and Glucagon introduce mutations resistant to DPP-IV at the second position, such as the mutation of L-type to D-type amino acids (L-Ser mutated to D-Ser), or the introduction of unnatural amino acids such as Aib (Matthias H. et al., Unimolecular Polypharmacy forTreatment of Diabetes and Obesity, 24:51–62,2016). However, in the examples of the present invention, the dual active protein conforming to Formula II and retaining natural L-Ser at the second position exhibits very high serum stability, and there is still no significant sign of DPP-IV degradation after 24 hours, while the corresponding polypeptide without fusion with F C is rapidly hydrolyzed by DPP-IV (Table 9). The inventors of the present invention prepared the active protein C001L C F8 (SEQ ID NO.93) fused by natural Glucagon and F 13 , and the active protein C002 L C F8 (SEQ ID NO.94) fused by Glucagon-cex reported by JosephR.Chabenne et al. and F 13 as a control to verify whether the fusion with F C improves the stability. However, C001L 13 F8 (SEQ ID NO.93) and C002L 13Neither F8 (SEQ ID NO. 94) showed any obvious sign of resistance to DPP-IV. Although it has been reported that binding to serum albumin (such as HSA) may help improve the stability of proteins (such as liraglutide), however, without mutation at the second position, it is simply impossible to make the half-life last more than 12 hours, that is, it is impossible to support once-weekly dosing frequency. The pharmacokinetic and pharmacodynamic tests of the GCG analogs provided by the present invention show that it is sufficient to support once-weekly dosing frequency, rather than once-daily (such as albumin-bound liraglutide) as generally reported. The retention of natural amino acids further reduces the risk of immunogenicity, and avoiding chemical cross-linking also makes the preparation process simpler and more convenient.

[0106] Intraperitoneal glucose tolerance test (IPGTT)

[0107] In one of the embodiments, an IPGTT experiment was conducted. After injecting glucose into the mice administered with the multi-domain active protein, extremely stable blood glucose fluctuations were presented.

[0108] Weight loss, appetite control, and pharmacokinetic experiments in DIO mice

[0109] The potential weight loss effect of GCGR agonists has been reported in many papers. However, due to the easy degradation and extremely small molecular weight of natural Glucagon, its potential for drug development is extremely small. Currently, Glucagon analogs are mainly used for acute hypoglycemic symptoms. Clinical reports on the use of long-acting GCG analogs for weight loss in diabetic patients are also emerging continuously. As is well known, obesity is one of the causes of insulin resistance in diabetic patients, and the amount of weight loss is an important indicator for evaluating an antidiabetic drug. In addition, after administration of the multi-domain active protein of the present invention to DIO mice, a significant reduction in body weight was induced. The roles of GCGR / GLP-1R dual agonists, FGF21 and its analogs in reducing blood glucose and lipid metabolism are well known. Stanislaus S et al. reported (Stanislaus S et al., A novel Fc FGF21 with improved resistance to proteolysis, increased affinity towards β-Klotho and enhanced efficacy in mice and cynomolgus monkeys Endocrinology. 2017 May 1; 158(5):1314-1327.) F C- At a dose of 3 mg / kg (about 30 nM / kg), after continuous administration for 4 weeks, the body weight loss is more than 15%. In Example 9 of the present invention, the triple-active protein at a dose of 30 nM / kg achieved a weight loss of about 30%, while the appetite showed basically no obvious change. Similarly, at a dose of 10 nM / kg, the weight loss was also close to 15%. In addition, the composition of the dual-active protein and the long-acting FGF21 analog in Example 10 (A-L a -F+F-L b -B) also showed a synergistic effect: the combined administration composition could reduce the body weight by 35% or more, while the dual-active protein at the same dose could only reduce the body weight by no more than 13%, and the long-acting FGF21 analog had a body weight loss of less than 10%.

[0110] Clinical application prospects

[0111] Clinically, the multi-active protein of the present invention has potential pharmacokinetic properties suitable for administration once a week or more. The dose depends on the administration frequency and mode, the age, sex, weight and general condition of the treated subject, the condition and severity of the treatment, any concomitant diseases to be treated, and other factors obvious to those skilled in the art. At the same time, according to the situation of the treated person and other pathological conditions, the multi-active protein of the present invention can be administered or applied in combination with one or more other therapeutic active compounds or substances. For example, other selectable therapeutic active compounds include, but are not limited to, anti-diabetic drugs, anti-hyperlipidemic drugs, anti-obesity drugs, anti-hypertensive drugs, and reagents for treating diabetes or diabetes-related complications.

[0112] Metabolic syndrome is associated with an increased risk of other conditions related to coronary heart disease and vascular plaque accumulation, such as stroke and peripheral vascular disease, and becomes atherosclerotic cardiovascular disease (ASCVD). Patients with metabolic syndrome can develop from an early insulin resistance state to fully developed type 2 diabetes, and the risk of ASCVD further increases. Without being limited to any specific theory, the relationship between insulin resistance, metabolic syndrome, and vascular diseases may involve one or more common pathogenic mechanisms, including impaired insulin-stimulated vasodilation, reduced insulin resistance-related availability caused by enhanced oxidative stress, and abnormalities of adipocyte-derived hormones (such as adiponectin) (Lteif, Mather, Can. J. Cardiol. 20 (Suppl B): 66B-76B, 2004)

[0113] The active protein of the present invention can also be used to treat obesity. In some aspects, the active protein of the present invention treats obesity through mechanisms such as reducing appetite, decreasing food intake, reducing the body fat level of patients, and increasing energy consumption.

[0114] In some potential embodiments, the active protein of the present invention can be used to treat non-alcoholic fatty liver disease (NAFLD). NAFLD refers to a broad spectrum of liver diseases, ranging from simple fatty liver (steatosis) to non-alcoholic steatohepatitis (NASH) to cirrhosis (irreversible end-stage scarring of the liver). All stages of NAFLD are characterized by fat accumulation in liver cells. Simple fatty liver is a type of abnormal accumulation of certain types of fat, triglycerides, in liver cells without inflammation or scarring. In NASH, fat accumulation is associated with varying degrees of liver inflammation (hepatitis) and scarring (fibrosis). Inflammatory cells can damage liver cells (hepatocyte necrosis). In the terms "steatohepatitis" and "steatonecrosis", steatosis refers to fat infiltration, hepatitis refers to inflammation in the liver, and necrosis refers to damaged liver cells. NASH can ultimately lead to liver scarring (fibrosis) and then to irreversible end-stage scarring (cirrhosis), and cirrhosis caused by NASH is the last and most severe stage within the NAFLD spectrum.

[0115] Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific embodiments; it should also be understood that the terms used in the embodiments of the present invention are for describing specific embodiments, rather than for limiting the protection scope of the present invention. The test methods without specific conditions noted in the following examples are generally carried out under conventional conditions or according to the conditions recommended by each manufacturer.

[0116] When an embodiment gives a numerical range, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of this technology. In addition to the specific methods, devices, and materials used in the embodiments, according to the knowledge of those skilled in the art of this technology and the description of the present invention, any methods, devices, and materials similar or equivalent to the methods, devices, and materials described in the embodiments of the present invention can also be used to implement the present invention.

[0117] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the fields of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in this technical field. These techniques have been well described in the existing literature. For details, see Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and 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. Wassarman 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.

[0118] Example 1 Screening of GCG Analogs (Screening of Glucagon Analogs)

[0119] The amino acid sequence of natural GLP-1 is shown in SEQ ID NO.1, specifically:

[0120] HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG.

[0121] The amino acid sequence of natural Oxyntomodulin is shown in SEQ ID NO.2, specifically:

[0122] HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNN IA.

[0123] The GCG analog of the present invention is denoted as A, and A is a GCGR / GLP-1R dual-agonist active peptide, selected from all peptide chains capable of achieving dual-agonist activity for GCGR and GLP-1R.

[0124] The structural formula of A is shown in Formula II:

[0125] HSQGTFTSD-X 10 -S-X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -F-X 23 -X 24 -WL-X 27 -X 28 -X 29 -X 30 -X z 。

[0126] Wherein, the X 10 is selected from any one of V, L or Y; the X 12 is selected from any one of S, E or K; the X 13 is selected from any one of Y or Q; the X 14 is selected from any one of L or M; the X 15 is selected from any one of D or E; the X 16 is selected from any one of S, E or G; the X 17 is selected from any one of R, E or Q; the X 18 is selected from any one of R, E or A; the X 19 is selected from any one of A or V; the X 20 is selected from any one of Q, R or K; the X 21 is selected from any one of D, L or E;

[0127] The X 23 is selected from any one of V or I; the X 24 is selected from any one of Q, A or E; the X 27 is selected from any one of M, K or V; the X 28 is selected from any one of N or K; the X 29 is selected from any one of G or T; the X 30 is G or absent; the X zAbsent or selected from any one of GPSSGAPPPS (SEQ ID NO.3), PSSGAPPPS (SEQ ID NO.4), SSGAPPPS (SEQ ID NO.5), GPSSGAPPS (SEQ ID NO.6), PSSGAPPS (SEQ ID NO.7), or KRNRNNIA (SEQ ID NO.8).

[0128] Table 1 lists the amino acid sequences of some GCG analogs of the present invention:

[0129] Table 1

[0130]

[0131]

[0132] Preparation of dual - effect active protein A - La - F in Example 2

[0133] In this example, a dimer dual - effect active protein A - L a -F is obtained by fusing a GCG analog with a linker chain L a -F. Among them, A is the same as A in Example 1. Specifically, the structural formula of the said A is shown in Formula II:

[0134] HSQGTFTSD - X 10 -S - X 12 -X 13 -X 14 -X 15 -X 16 -X 17 -X 18 -X 19 -X 20 -X 21 -F - X 23 -X 24 -WL - X 27 -X 28 -X 29 -X 30 -X z .

[0135] Among them, the said X 10 is selected from any one of V, L, or Y; the said X 12 is selected from any one of S, E, or K; the said X 13 is selected from any one of Y or Q; the said X 14 is selected from any one of L or M; the said X 15 is selected from any one of D or E; the said X 16 is selected from any one of S, E, or G; the said X 17 is selected from any one of R, E, or Q; the said X18 Any one selected from R, E or A; said X 19 Any one selected from A or V; said X 20 Any one selected from Q, R or K; said X 21 Any one selected from D, L or E; said X 23 Any one selected from V or I; said X 24 Any one selected from Q, A or E; said X 27 Any one selected from M, K or V; said X 28 Any one selected from N or K; said X 29 Any one selected from G or T; said X 30 Is G or absent; said X z Absent or any one selected from GPSSGAPPPS (SEQ ID NO.3), PSSGAPPPS (SEQ ID NO.4), SSGAPPPS (SEQ ID NO.5), GPSSGAPPS (SEQ ID NO.6), PSSGAPPS (SEQ ID NO.7) or KRNRNN IA (SEQ ID NO.8).

[0136] Said F is a long-acting protein unit, and F can be selected from the complete F of an immunoglobulin C portion, the F of an immunoglobulin C fragment of the portion or mutant of the F of an immunoglobulin C portion. The amino acid sequence of said F is shown in SEQ ID NO.9-18.

[0137] The present invention abbreviates the sequence shown in SEQ ID NO.9 as F1, specifically:

[0138] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0139] The present invention abbreviates the sequence shown in SEQ ID NO.10 as F2, specifically:

[0140] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

[0141] The present invention abbreviates the sequence shown in SEQ ID NO.11 as F3, specifically:

[0142] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0143] The present invention abbreviates the sequence shown in SEQ ID NO.12 as F4, specifically:

[0144] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

[0145] The present invention abbreviates the sequence shown in SEQ ID NO.13 as F5, specifically:

[0146] ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

[0147] The present invention abbreviates the sequence shown in SEQ ID NO.14 as F6, specifically:

[0148] ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

[0149] The present invention abbreviates the sequence shown in SEQ ID NO.15 as F7, specifically:

[0150] ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。

[0151] The present invention abbreviates the sequence shown in SEQ ID NO.16 as F8, specifically:

[0152] ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

[0153] The present invention abbreviates the sequence shown in SEQ ID NO.17 as F9, specifically:

[0154] ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

[0155] The present invention abbreviates the sequence shown in SEQ ID NO.18 as F10, specifically:

[0156] ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG。

[0157] L aIt is a linking chain, which is a flexible polypeptide composed of glycine (G), serine (S) and / or alanine (A) with an appropriate length, so that adjacent protein domains can move freely relative to each other. When it is necessary to ensure that the two adjacent domains do not interfere with each other spatially, a longer linking chain can be used. Exemplary linking chains are, for example, (GS)n, (GGS)n, (GGSG)n, (GGGS)nA, (GGGGS)nA, (GGGGA)nA, etc., where n is an integer from 1 to 10. Exemplary linking chains can be independently selected from Table 2 respectively.

[0158] Table 2

[0159]

[0160]

[0161] Partial bifunctional active protein A-L a -F has the amino acid sequence as shown in Table 3:

[0162] Table 3

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169] Based on knowing the amino acid sequence of A-L a -F, those skilled in the art can prepare it using the prior art: due to the presence of the F C sequence, protein purification can be carried out by Protein A resin chromatography with high affinity and high specificity. Here, only one feasible preparation method is exemplarily given.

[0170] The preparation process is as follows:

[0171] (1) According to the protein sequence and the amino acid codon table, design its DNA sequence. Prepare the polynucleotide DNA fragments corresponding to A, L a , and F in the recombinant protein respectively. Each DNA fragment can be synthesized and spliced by conventional solid-phase synthesis techniques;

[0172] (2) Design primers for nested PCR amplification, and splice A, L a, the DNA fragments corresponding to F respectively, to obtain the target gene. The PCR splicing technology (including primer design, PCR introduction of mutations, and enzymatic digestion, etc.) is a well-known public technology in the art. Those skilled in the art should be aware that the PCR splicing process in this example is not the only method. For example, the target gene can also be obtained by gene synthesis. After successfully obtaining the target gene, the target gene is cloned into the mammalian cell expression vector pTT5 (Yves Durocher), and transformed into Escherichia coli Top10F'; after positive clone identification, it is inoculated into 500 ml of LB medium, cultured overnight, the bacteria are collected by centrifugation, and Omega Endo-Free Plasmid Maxi Kit is used to extract the plasmid;

[0173] (3) Transfecting Hek293F cells and cell expression: Take 1.0 mg of plasmid and dilute it to 25 ml with Freestyle 293 expression medium (Thermofisher); take 3.0 mg of PEI (linear, 25KD) and dilute it to 25 ml with Freestyle 293 expression medium, add it to the plasmid solution, mix well, and incubate at room temperature for 30 minutes; meanwhile, take Hek293F cells in the logarithmic growth phase (viability > 95%), count; centrifuge at 1100 RPM for 10 minutes, discard the supernatant; resuspend the cells with 450 ml of Freestyle293 expression medium; after the incubation of the PEI-plasmid mixture is completed, add it to the cell suspension, and culture it at 37 °C with 5% CO2 and shaking at 140 RPM; after 7 hours, replace the Freestyle 293 expression medium with 1000 ml of 293SFM II medium (Thermofisher) and continue to culture for 7 days;

[0174] (4) Purification of the recombinant protein: Centrifuge the cell culture solution at 8000 rpm at high speed for 10 min to obtain the supernatant and load it onto a Protein A column (Bogelong (Shanghai) Biotechnology Co., Ltd.) pre-equilibrated with equilibration buffer (20 mM PB, 0.5 M NaCl, pH 7), and elute with 100% (the eluent is 0.1 M Gly-HCl, pH 3.0); neutralization solution (1 M Tris-HCl, pH 8.0) is pre-added to the collection tube, and the eluted samples are collected; finally, add neutralization solution to 1 / 10 of the volume of the eluted samples, and measure the protein concentration using the conventional Bradford method;

[0175] (5) Identification of the physicochemical properties of the recombinant protein: SDS-PAGE electrophoresis or amino acid sequence verification of the purified recombinant protein is consistent with the expectation.

[0176] Example 3 Preparation of the triple-effect active protein

[0177] The triple - effect active protein of the present invention contains multiple domains and has triple - effect agonist activity. The structural formula of the triple - effect active protein is shown as Formula I: A - L a - F - L b - B, where A is a GCGR / GLP - 1R dual - effect agonist peptide, F is a long - acting protein unit, B is natural FGF21 or an FGF21 analog, and L a is a linker chain, L b is a linker chain.

[0178] In Formula I, the structural formula of A is shown as Formula II:

[0179] HSQGTFTSD - X 10 - S - X 12 - X 13 - X 14 - X 15 - X 16 - X 17 - X 18 - X 19 - X 20 - X 21 - F - X 23 - X 24 - WL - X 27 - X 28 - X 29 - X 30 - X z 。

[0180] Among them, the X 10 is selected from any one of V, L or Y; the X 12 is selected from any one of S, E or K; the X 13 is selected from any one of Y or Q; the X 14 is selected from any one of L or M; the X 15 is selected from any one of D or E; the X 16 is selected from any one of S, E or G; the X 17 is selected from any one of R, E or Q; the X 18 is selected from any one of R, E or A; the X 19 is selected from any one of A or V; the X 20 is selected from any one of Q, R or K; the X 21 is selected from any one of D, L or E; the X 23 is selected from any one of V or I; the X 24 is selected from any one of Q, A or E; the X 27 is selected from any one of M, K or V; the X 28 is selected from any one of N or K; the X 29 is selected from any one of G or T; the X 30is G or absent; said X z is absent or selected from any one of GPSSGAPPPS (SEQ ID NO.3), PSSGAPPPS (SEQ ID NO.4), SSGAPPPS (SEQ ID NO.5), GPSSGAPPS (SEQ ID NO.6), PSSGAPPS (SEQ ID NO.7) or KRNRNN IA (SEQ ID NO.8).

[0181] In formula I, said F is optionally selected from the complete F of an immunoglobulin C portion, the F of an immunoglobulin C fragment of the portion or a mutant of the F of an immunoglobulin C portion, as shown in SEQ ID NO.9 - 18.

[0182] In formula I, said B is natural FGF21 (SEQ ID NO.136) or an FGF21 analog. The structural formula of said B is:

[0183] HPIPDSSPLLQFGGQVRQ X 19 YLYTDDAQQTE X 31 HLEI X 36 EDGTVG X 43 A X 45 DQSPESLL QL X 56 ALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRE X 98 LLEDGYNVYQSEAH GLPLH X 118 PGN X 122 SPHRDPAPRGP X 134 RFLPLPGLPPALPEPPGILAPQPPDVGSSDPL X 167 MV X 170 X 171 SQ X 174 RSPS X 179 X 18 0X181 .

[0184] Among them, the N-terminal HPIPDSS can be absent or partially absent; X 19 is selected from R, Y, V, E or C; X 31 is selected from A or C;

[0185] X 36 is selected from R or K; X 43 is selected from G or C; X 45 is selected from A, K, E or V; X 56 is selected from K, R, V or I; X 98 is selected from L, R or D; X 118 is selected from L or C; X 122 is selected from K or R; X 134 is selected from A or C; X 167 is selected from S, A or R; X 170 is selected from G or E; X 171 is selected from P or G; X 174 is selected from G, A or L; X 179 is selected from Y, A or F;

[0186] X 180 is selected from A or E; X 181 is selected from S, K or absent.

[0187] The FGF21 analog described above is an active protein having the same or similar biological function as natural FGF21 (SEQ ID NO. 136) and having a homology of more than 80% with natural FGF21 (SEQ ID NO. 136). Preferably, the FGF21 analog has a homology of more than 85% with natural FGF21 (SEQ ID NO. 136); more preferably, the FGF21 analog has a homology of more than 90% with natural FGF21 (SEQ ID NO. 136); more preferably, the FGF21 analog has a homology of more than 95% with natural FGF21 (SEQ ID NO. 136). Preferably, the FGF21 analog is as shown in SEQ ID NOs. 137-148.

[0188] The amino acid sequences of natural FGF21 and some analogs are shown in Table 4 in detail:

[0189] Table 4

[0190]

[0191] L a is absent or is a linking chain, L b is absent or is a linking chain. When L a and L bWhen it is a linking chain, the linking chain is a flexible polypeptide of a suitable length composed of glycine (G), serine (S) and / or alanine (A), so that adjacent protein domains can move freely relative to each other. When it is necessary to ensure that the two adjacent domains do not interfere with each other spatially, a longer linking chain can be used. Exemplary linking chains are, for example, (GS)n, (GGS)n, (GGSG)n, (GGGS)nA, (GGGGS)nA, (GGGGA)nA, etc., where n is an integer from 1 to 10. Exemplary linking chains can each independently be selected from Table 2.

[0192] Based on the GCGR / GLP-1R dual-activity protein, at the C-terminus of F C FGF21 or an FGF21 analogue is fused through a linking peptide chain to prepare an exemplary triple-activity protein as described in Table 5.

[0193] Table 5

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209] After knowing A-L a -F-L bBased on the amino acid sequence of -B, those skilled in the art can prepare it using existing technologies. Since it has the F C sequence, protein purification can be carried out by Protein A resin chromatography with high affinity and high specificity. The specific method can refer to the preparation method in Example 2. The recombinant protein obtained by purification was verified by SDS-PAGE electrophoresis or amino acid sequence, and both were consistent with the expectation. Among them, Figure 1 is the SDS-PAGE electrophoresis diagram of the partially purified sample.

[0210] Example 4 Preparation of FGF21 Analogue with Fused FC

[0211] In this example, F with the code name F9 C was fused with natural FGF21 and FGF21 analogues with the code names M1 - M 12 to obtain long-acting FGF21 analogues. The structural formula of the long-acting FGF21 analogue is F-L b -B. The F can be selected from the complete F C portion of an immunoglobulin, a fragment of the F C portion of an immunoglobulin, or a mutant of the F C portion of an immunoglobulin. The amino acid sequence of the F can be as shown in any one of SEQ ID NO.9 - 18. L b does not exist or is a linker chain. When L b is a linker chain, the linker chain includes units rich in G, S, and / or A, such as (GS)n, (GGS)n, (GGSG)n, (GGGS)nA, (GGGGS)nA, (GGGGA)nA, etc., where n is an integer from 1 to 10. In a preferred embodiment, the amino acid length of the linker chain is 5 - 26. Exemplary linker chains are each independently selected from Table 2. Further, the amino acid sequence of the linker chain can be as shown in any one of SEQ ID NO.19 - 41. The B is natural FGF21 (SEQ ID NO.136) or an FGF21 analogue. The structural formula of the B is:

[0212] HPIPDSSPLLQFGGQVRQ X 19 YLYTDDAQQTE X 31 HLEI X 36 EDGTVG X 43 A X 45 DQSPESLLQL X 56ALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRE X 98 LLEDGYNVYQSEAHGLPLH X 118 PGN X 122 SPHRDPAPRGP X 134 RFLPLPGLPPALPEPPGILAPQPPDVGSSDPL X 167 MV X 170 X 171 SQ X 174 RSPS X 179 X 18 0X 181 wherein, the N-terminal HPIPDSS may be absent or partially absent; X 19 is selected from R, Y, V, E or C; X 31 is selected from A or C; X 36 is selected from R or K; X 43 is selected from G or C; X 45 is selected from A, K, E or V; X 56 is selected from K, R, V or I; X 98 is selected from L, R or D; X 118 is selected from L or C; X 122 is selected from K or R; X 134 is selected from A or C; X 167 is selected from S, A or R; X 170 is selected from G or E; X 171 is selected from P or G; X 174 is selected from G, A or L; X 179 is selected from Y, A or F; X 180 is selected from A or E; X 181 is selected from S, K or absent.

[0213] The FGF21 analog is an active protein that has the same or similar biological functions as natural FGF21 (SEQ ID NO. 136) and has a homology of more than 80% with natural FGF21 (SEQ ID NO. 136). Preferably, the FGF21 analog has a homology of more than 85% with natural FGF21 (SEQ ID NO. 136); more preferably, the FGF21 analog has a homology of more than 90% with natural FGF21 (SEQ ID NO. 136); more preferably, the FGF21 analog has a homology of more than 95% with natural FGF21 (SEQ ID NO. 136). Exemplarily, the FGF21 analog may be selected from FGF21 analogs or mutants 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. The FGF21 analogs are shown in SEQ ID NOs. 137 - 148.

[0214] The amino acid sequence of the long - acting FGF21 analog can be shown as SEQ ID NOs. 209 - SEQ ID NO. 221. Synthesizing DNA according to the protein sequence and sub - cloning it into a recombinant expression vector is a conventional method in the technical field. Similar to Example 2, transfect Hek293F cells and cell expression. Similarly, due to the presence of the F C sequence, the process of separation and purification can also refer to Example 2.

[0215] F9L 10 The W sequence is shown in SEQ ID NO. 209:

[0216] ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS。

[0217] F9L 10 The M1 sequence is shown in SEQ ID NO.210:

[0218] ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES。

[0219] F9L 10The M2 sequence is shown in SEQ ID NO. 211:

[0220] ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHCPGNKSPHRDPAPRGPCRFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES。

[0221] F9L 10 The M3 sequence is shown in SEQ ID NO. 212:

[0222] ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGADSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHCPGNKSPHRDPAPRGPCRFLPLPGLPPALPEPPGILAPQPPDVGSSDPLAMVGPSQGRSPSYAS。

[0223] F9L 10 The M4 sequence is shown in SEQ ID NO.213:

[0224] ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHCPGNKSPHRDPAPRGPCRFLPLPGLPPALPEPPGILAPQPPDVGSSDPLAMVGGSQGRSPSYES。

[0225] F9L 10 The M5 sequence is shown in SEQ ID NO.214:

[0226] ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAEDQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHCPGNKSPHRDPAPRGPCRFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES。

[0227] F9L 10 The M6 sequence is shown in SEQ ID NO.215:

[0228] ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHCPGNKSPHRDPAPRGPCRFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVEPSQGRSPSYAS。

[0229] F9L 10The M7 sequence is shown in SEQ ID NO. 216:

[0230] ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGAHPIPDSSPLLQFGGQVRQEYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSYES。

[0231] F9L 10 The M8 sequence is shown in SEQ ID NO. 217:

[0232] ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGGSQGRSPSAAS。

[0233] F9L 10 The M9 sequence is shown in SEQ ID NO. 218:

[0234] ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHCPGNKSPHRDPAPRGPCRFLPLPGLPPALPEPPGILAPQPPDVGSSDPLAMVGGSQGRSPSYAS。

[0235] F9L 10 M 10 The sequence is shown in SEQ ID NO. 219:

[0236] ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGADSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHCPGNKSPHRDPAPRGPCRFLPLPGLPPALPEPPGILAPQPPDVGSSDPLAMVGGSQGRSPSYAS。

[0237] F9L 10 M 11 The sequence is shown in SEQ ID NO.220:

[0238] ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLAMVGGSQGRSPSYES。

[0239] F9L 10 M 12The sequence is shown as SEQ ID NO.221:

[0240] ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAGGGGAGGGGAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRERLLEDGYNVYQSEAHGLPLHCPGNKSPHRDPAPRGPCRFLPLPGLPPALPEPPGILAPQPPDVGSSDPLAMVGGSQGRSPSYAS。

[0241] Example 5 In vitro cell activity detection

[0242] The dual - effect active protein obtained in Example 2 was subjected to in vitro activity determination, including GLP - 1R agonist activity detection and GCGR agonist activity detection.

[0243] GLP - 1R agonist activity detection:

[0244] The GLP - 1R agonist activity was detected by the luciferase reporter gene assay (Jonathan W Day et al.: Nat Chem Biol. 2009 Oct; 5(10):749 - 57). The human GLP - 1R gene was cloned into the mammalian cell expression plasmid pCDNA3.1 to construct the recombinant expression plasmid pCDNA3.1 - GLP - 1R. At the same time, the full - length gene of luciferase was cloned onto the pCRE plasmid to obtain the pCRE - Luc recombinant plasmid. The pCDNA3.1 - GLP - 1R and pCRE - Luc plasmids were transfected into CHO cells at a molar ratio of 1:10, and stable transfected expression strains were screened to obtain the recombinant CHO / GLP - 1R stable cell line.

[0245] Cells were cultured in DMEM / F12 medium containing 10% FBS and 300 μg / ml G418 in a 9-cm cell culture dish. When the confluence reached about 90%, the culture supernatant was discarded. After adding 2 ml of trypsin and digesting for 3 min, 2 ml of DMEM / F12 medium containing 10% FBS and 300 μg / ml G418 was added for neutralization. The cells were transferred to a 15-ml centrifuge tube, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 2 ml of DMEM / F12 medium containing 10% FBS and 300 μg / ml G418 for counting. The cells were diluted with DMEM / F12 medium containing 10% FBS to 3×10 5 , and 100 μl was seeded into each well of a 96-well plate, that is, 5×10 4 / well. After attachment, the medium was changed to DMEM / F12 medium containing 0.2% FBS for culture. After discarding the supernatant of the cells seeded in the 96-well plate, the purified recombinant proteins (Table 6, Table 7) or native Glucagon (Hangzhou Zhongtai Biochemical Co., Ltd., GLUC-004) and native GLP-1 (Hangzhou Zhongtai Biochemical Co., Ltd., GLUC-016B) were used as controls and diluted to a series of specified concentrations with DMEM / F12 medium containing 0.1% FBS, and then added to the cell culture wells, 100 μl / well. After stimulation for 6 h, the detection was carried out. The detection was performed according to the instructions of the Luciferase reporter kit (Ray Biotech, Cat: 68-LuciR-S200).

[0246] Detection method for GCGR agonist activity:

[0247] The detection of GCGR agonist activity also adopted a similar luciferase reporter gene detection method. The GCGR gene was cloned into the mammalian cell expression plasmid pCDNA3.1 to construct the recombinant expression plasmid pCDNA3.1-GCGR. The construction of the stable transfected cell line HEK 293T / GCGR by co-transfecting the HEK 293T cells with the pCRE-Luc recombinant plasmid was the same as above.

[0248] Detection method for FGF21 activity:

[0249] The FGF21 activity assay was performed using a method similar to that in the literature with appropriate modifications (Xu J et al., Polyethylene glycolmodified FGF21 engineered to maximize potency and minimize vacuole formation, Bioconjug Chem.; 24(6):915-25, 2013). The puromycin resistance gene pac was amplified by PCR, cloned into pcDNA3.1(+), and replaced the original G418 resistance gene. The GAL4DBD-ELK1, IRES, and KLB (β-klotho) genes were amplified by PCR and cloned into the pcDNA-Puro plasmid in sequence to construct the plasmid pcDNA-GAL4DBD-ELK1-IRES-KLB-Puro for cell transfection and screening. The plasmid was extracted and reserved using the Omega Endo-Free Plasmid Midi Kit. The cell transfection process was as follows: Hek293T cells were seeded in a 6-well plate at 3x10 5 cells per well and cultured overnight.

[0250] After washing the cells twice with Opti-MEM medium, 2 ml of Opti-MEM medium was added. The cell transfection reagent was prepared in the following ratio: Lipofectamine 2000 (6 μl): pFR-Luc (4.6 μg): pcDNA-GAL4DBD-ELK1-IRES-KLB-Puro (1 μg). After standing for 20 min, it was slowly added to the 6-well plate and mixed while adding. After culturing for 6 h, the medium was changed to DMEM + 10% FBS medium and continued to be cultured at 37 °C and 5% CO2. A stable transfected cell line with FGF21 activity response was obtained. After the cells grew confluent in the culture dish, they were digested with trypsin to prepare a cell suspension (1x105 cells / ml, DMEM + 5% FBS + 1 μg / ml puromycin), seeded in a 96-well plate at 100 μl per well, and cultured overnight. Gradient concentrations of the test sample were added and allowed to act for 6 h, and fluorescence detection was performed using the Luciferase Reporter Assay Kit (68-LucifR-S200).

[0251] The activity assay results of some dual-effect active proteins are shown in Tables 6 and 7:

[0252] Table 6

[0253]

[0254]

[0255] Description: The protein numbers in the table follow the following rules: polypeptide code + linker code + FC code, e.g., C240L 13 F4, indicating that the C240 polypeptide is fused with the IgG FC with the code F4 through the linker with the code L 13 of the linker.

[0256] a. The ratio of GLP-1R agonist activity before and after inserting GPSSGAPPPS or a similar sequence (also known as the Cex sequence, any one of SEQ ID NOs. 3-8 in the present invention) between the GCG analog and the Fc chain.

[0257] b. The ratio calculated based on the GLP-1R agonist activity data of native Glucagon and Glucagon Cex disclosed in Table 2 of US9018164 B2.

[0258] c. The ratio calculated based on the GLP-1R agonist activity data of native Glucagon and Glucagon Cex disclosed in Table 1 of the article by Joseph R. Chabenne et al. (Joseph R. Chabenne et al., Optimization of the Native Glucagon Sequence for Medicinal Purposes, J Diabetes Sci Technol. 4(6):1322–1331, 2010).

[0259] As shown in Tables 6 - 8, when the sequence containing the CEX extension peptide is fused with F (SEQ ID NO. 16) through (GGGGS)3A (SEQ ID NO. 31) to prepare a dimer, the agonist activity against GLP-1R is increased by more than 200 times, while there is no significant difference in the agonist activity against GCGR.

[0260] Table 7

[0261]

[0262] Activity detection of the triple-activity protein

[0263] The activity results of the triple-activity protein prepared in Example 3 are shown in Table 8:[[]]END]]

[0264] Table 8

[0265]

[0266]

[0267]

[0268]

[0269] The protein numbers in the table follow the following rules: polypeptide code + linker code + F C code + linker code + FGF21 mutant code, such as C209L 13 F4L 13 M9, indicating that the C209 polypeptide is fused with IgG F with the code F4 C through the linker with the code L 13 and then further fused with the FGF21 mutant with the code M9 through the linker with the code L 13 of the linker.

[0270] Example 6 DPP-IV Enzyme Stability of the Triple-Active Protein

[0271] Dissolve 5 μM of the purified triple-active protein in 10 mM HEPES buffer (containing 0.05 mg / ml BSA), add recombinant DPP-IV enzyme at a final concentration of 10 nM, incubate at 37 °C for 24 hours, and then detect the activity of GCGR cells in vitro. Activity retention rate = (activity after DPP-IV enzyme treatment / activity before treatment) × 100%.

[0272] In this example, GCG analogs with the unnatural amino acids Aib or D-Ser introduced at the second position were used as controls:

[0273] GD Ser GS: H-D-Ser-QGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPS (SEQ ID NO.134);

[0274] G Aib GS: H-Aib-QGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPS (SEQ ID NO.135);

[0275] C364 (SEQ ID NO.70), C382 (SEQ ID NO.57), C495 (SEQ ID NO.53), C462 (SEQ ID NO.66), C225 (SEQ ID NO.47), and C209 (SEQ ID NO.71) were used as controls for the stability experiment in this example.

[0276] The results are shown in Table 9:

[0277] Table 9

[0278]

[0279]

[0280] Example 7 Serum Stability Test of Tri-Effect Active Protein

[0281] In vitro cell detection method:

[0282] (1) Take the tri-effect active protein, first ultrafilter and concentrate it, then dilute it to 1.6 mg / ml with 20 mM PB pH 7.4. After sterile filtration, dilute the serum (FBS, GEMINI 900-108, A97E00G) 10-fold, mix well, and dispense it into sterile centrifuge tubes;

[0283] (2) Take Glucagon (SEQ ID NO: 42, Hangzhou Zhongtai Biochemical Co., Ltd., GLUC-004) separately, dilute it to 0.2 mg / ml, after sterile filtration, dilute the serum 10-fold, mix well, and dispense it into sterile centrifuge tubes;

[0284] (3) Freeze 1-2 tubes of the above samples at -20 °C as controls, and place several other tubes in a 37 °C incubator, and sample and detect the GCGR agonist activity at different time points;

[0285] (4) After subculturing HEK 293T / GCGR cells twice, seed them in 96-well plates and detect the activity of the samples. Residual activity: Take the activity value at 0 hour as 100%, and the values measured at subsequent time points are compared with it. Except for C002L 13 F4L 13 W, the results are similar to those in Table 9, and there is no significant difference in the serum stability of each tri-effect active protein. The relative activity of the exemplary tri-effect active protein changes over time as shown in Figure 2A -C.

[0286] Example 8 Glucose Tolerance Test (IPGTT) in Normal ICR Mice

[0287] Group normal ICR mice, with 8 mice in each group. Fast overnight, collect blood from the tail (recorded as the blood glucose sample at t = 0 minute), and subcutaneously inject the tri-effect active protein (40 nmol / kg, acetate buffer), the composition (combination administration group) or physiological saline PBS. The combination administration group was pre-mixed before administration (each 40 nmol / kg, acetate buffer). After 15 minutes, intraperitoneally inject glucose (2 g / kg body weight), and measure the blood glucose level at t = 30 minutes, t = 60 minutes, t = 120 minutes and t = 240 minutes. The animals remained fasting during the experiment to prevent interference from food intake. The results are as shown in Figure 3 shown.

[0288] Example 9 Pharmacodynamic Study of Continuous Administration of Tri-Effect Active Protein in Diet-Induced Obesity (DIO) Mice

[0289] Male C57BL / 6J mice at 7 weeks of age were fed a high-fat diet (60% kcal from fat) and continued to be raised for 16 weeks (23 weeks in total). When their body weight reached approximately 55 g, the experiment was conducted. Breeding conditions: 12 h light / 12 h dark, ad libitum feeding, single-cage breeding. One day before administration, the mice were grouped according to their body weight and body weight growth curve (8 mice / group), and subcutaneous administration was performed the next day. As shown in Table 10, the active protein was administered at a dose of 10 nmol / kg body weight or 30 nmol / kg body weight, once every 4 days; the negative control group was injected with physiological saline (PBS) at 5 μl / g body weight; the positive control group was injected with liraglutide (30 nmol / kg body weight), once a day for 28 consecutive days. The body weight and food intake of the mice were measured every day. They were sacrificed on the 5th day after the last administration. Blood was collected from the orbital cavity. The plasma specimens were stored at -80 °C. The average body weight changes of each group of animals before and at the time of sacrifice were calculated. The results of body weight changes are as Figure 4 shown; the changes in total food intake are as Figure 5 shown.

[0290] Table 10

[0291] Sample SEQ ID NO. Dose (nM) <![CDATA[C002L 13 F8L 10 W]]> 149 10 <![CDATA[C495L 13 F8L 10 M2]]> 160 10 <![CDATA[C382L 13 F8L 10 M2]]> 167 10 <![CDATA[C462L 13 F8L 10 M2]]> 180 10 <![CDATA[C495L 13 F8L 10 M2]]> 160 30 <![CDATA[C382L 13 F8L 10 M2]]> 167 30 <![CDATA[C462L 13 F8L 10 M2]]> 180 30

[0292] Pharmacodynamic study of combination administration in diet-induced obesity (DIO) mice in Example 10

[0293] The difference between this example and Example 9 is that the dual-effect active protein and the long-acting FGF21 analog were administered in combination in the form of a compound. Male C57BL / 6J mice at 7 weeks of age were fed a high-fat diet (60% kcal from fat) and continued to be raised for 16 weeks (23 weeks in total). When their body weight reached approximately 55 g, the experiment was conducted. Breeding conditions: 12 h light / 12 h dark, ad libitum feeding, single-cage breeding. One day before administration, the mice were grouped according to their body weight and body weight growth curve (8 mice / group), and subcutaneous administration was performed the next day. The dual-effect active protein and the long-acting FGF21 analog were mixed according to the doses shown in Table 11 before administration and administered at a dose of 15 nmol / kg body weight or 30 nmol / kg body weight, once every 4 days; the negative control group was injected with physiological saline (PBS) at 5 μl / g body weight; the positive control group was injected with liraglutide (30 nmol / kg body weight), once a day for 28 consecutive days. The body weight and food intake of the mice were measured every day. They were sacrificed on the 5th day after the last administration. Blood was collected from the orbital cavity. The plasma specimens were stored at -80 °C. The average body weight changes of each group of animals before and at the time of sacrifice were calculated. The results of body weight changes are as Figure 6 shown; the changes in total food intake are as Figure 7 shown.

[0294] Table 11

[0295]

[0296]

[0297] As described above, it is only a preferred embodiment of the present invention, and does not impose any formal or substantial restrictions on the present invention. It should be pointed out that for those of ordinary skill in the art of this technology, without departing from the method of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as within the protection scope of the present invention. Any equivalent changes such as slight modifications, decorations, and evolutions made by those skilled in the art who are familiar with this specialty without departing from the spirit and scope of the present invention by using the technical content disclosed above are equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications, and evolutions made to the above embodiments based on the substantial technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A multi-domain active protein, the structure of which includes the structure shown in Formula I, and the structure shown in Formula I is: A-L a -F-L b -B, wherein, A is a GCGR / GLP-1R dual-acting agonist peptide, F is the Fc of an immunoglobulin, B is natural FGF21 or an FGF21 mutant having the same or similar biological function as natural FGF21, and L a is a linker chain, and L b is a linker chain; the amino acid sequence of the said A is shown as any one of SEQ ID NO.44, SEQ ID NO.46-47, SEQ ID NO.49, SEQ ID NO.51-55, SEQ ID NO.57-62, SEQ ID NO.65-68 or SEQ ID NO.70-92.

2. The multi-domain active protein according to claim 1, wherein The amino acid sequence of said B is any one of SEQ ID NOs. 136 to 148.

3. The multi-domain active protein according to claim 1, characterized in that, It also includes any one or more of the following features: (1) the amino acid sequence of said F is as shown in any one of SEQ ID NOs. 9 to 18; (2) said L a has an amino acid sequence as shown in any one of SEQ ID NOs. 19 to 41; (3) said L b has an amino acid sequence as shown in any one of SEQ ID NOs. 19 to 41.

4. The multi-domain active protein according to claim 1, wherein The amino acid sequence of said multi-domain active protein is any one of SEQ ID NOs. 150 to 159, SEQ ID NOs. 161 to 166, SEQ ID NOs. 168 to 179, SEQ ID NOs. 181 to 208.

5. A composition for treating metabolic and related diseases, comprising a GCGR / GLP-1R dual-acting agonist active protein with an amino acid sequence of any one of SEQ ID NOs. 98, 106, and 114, and a long-acting FGF21 analog with an amino acid sequence of any one of SEQ ID NOs. 210 to 212, wherein the metabolic and related diseases are selected from at least one of diabetes, obesity, non-alcoholic fatty liver, and hyperlipidemia.

6. An isolated polynucleotide encoding the multi-domain active protein according to any one of claims 1-4 or the composition for treating metabolic and related diseases according to claim 5.

7. A recombinant expression vector comprising the isolated polynucleotide according to claim 6.

8. A host cell containing the recombinant expression vector according to claim 7 or having an exogenous isolated polynucleotide according to claim 6 integrated into its genome.

9. The preparation method of the multi-domain active protein according to any one of claims 1-4 or the composition for treating metabolic and related diseases according to claim 5, characterized in that, Culturing the host cell according to claim 8 under suitable conditions to express the multi-domain active protein or the composition for treating metabolic and related diseases, and then isolating and purifying to obtain the multi-domain active protein or the composition for treating metabolic and related diseases.

10. Use of the multi-domain active protein according to any one of claims 1-4 or the composition for treating metabolic and related diseases according to claim 5 in the preparation of a drug for treating metabolic-related diseases, wherein the metabolic-related diseases are selected from at least one of diabetes, obesity, non-alcoholic fatty liver, and hyperlipidemia.

11. A composition containing the multi-domain active protein according to any one of claims 1-4 or the composition for treating metabolic and related diseases according to claim 5 or the culture of the host cell according to claim 8, and a pharmaceutically acceptable carrier.

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