Tri-target polypeptide and fusion protein thereof

Abstract

A tri-target polypeptide exerts the effects of lowering blood glucose, lowering blood lipids and promoting weight loss, and long-acting advantages are conferred to the triple-target polypeptide. The excellent effects above against specific indications are retained, and the long-acting advantages are achieved, such that the administration frequency can be reduced. Experimental results show that the polypeptide and a fusion protein can significantly lower blood glucose, lower blood lipids and / or promote weight loss, and can effectively prevent, treat or ameliorate diabetes, obesity, obesity-related diseases or metabolic disorders.

Description

A three-target polypeptide and fusion protein thereof

[0001] This application claims priority to the Chinese patent application No. 202410783583.3, filed on June 17, 2024, and entitled "A three-target polypeptide and fusion protein thereof", the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application relates to the field of polypeptides and fusion protein drugs, in particular to a three-target polypeptide, fusion protein and application thereof. BACKGROUND

[0003] Obesity, lipid-lowering

[0004] (1) Disease introduction:

[0005] More than 1 billion people worldwide, or 1 in 8, suffer from obesity. It is estimated that the number of overweight and obese people (BMI≥25kg / m2) worldwide could exceed 4 billion by 2035. In 2020, this number was more than 260 million. The proportion of overweight and obese people increased from 14% in 1990 to 24% in 2020. Data over the past 35 years shows a significant upward trend in global obesity rates. According to the provided information, the following key points about the epidemiology of obesity and overweight in China and the United States can be summarized:

[0006] The problem of overweight and obesity in China is becoming increasingly serious. The latest data shows that more than half of Chinese adults (50.7%) are overweight or obese, 6.8% of children under 6 years old are overweight, and 3.6% are obese, 11.1% of adolescents aged 6-17 are overweight, and 7.9% are obese. The prevalence of overweight and obesity in China shows a clear regional difference, with the north being higher than the south overall. The prevalence is higher in men and older people. In recent years, the rate of overweight and obesity in rural areas has risen faster than in urban areas, and the urban-rural gap is gradually narrowing.

[0007] In the United States, the problem of overweight and obesity is also serious. Data from 2015-2016 shows that 39.8% of adults are obese, and 31.8% of adolescents are overweight or obese. It is estimated that nearly half of American adults will be obese by 2030. Obesity rates vary among different races, regions, genders, and ages in the United States. The obesity rate among African Americans and Hispanics is higher than that among whites and Asians, and the obesity rate is generally higher in the southern states.

[0008] Overweight and obesity are associated with multiple health problems and are risk factors for many chronic diseases, such as diabetes and heart disease. In China and the United States, overweight and obesity have become one of the major causes of death and disability. The pathogenesis of obesity is complex and related to multiple factors, including genetics, lifestyle, and socioeconomic status. The increase in fast food and the decrease in physical activity are important reasons. China and the United States need to take effective public health policies, strengthen health education, and promote healthy lifestyles to curb the trend of obesity and reduce the disease burden caused by obesity.

[0009] (2) Mainstream treatment options and pipeline progress:

[0010] The Expert Consensus on Obesity Prevention and Control in Chinese Residents states that when lifestyle interventions are ineffective, patients with BMI ≥ 30 kg / m 2 or BMI ≥ 27 kg / m 2 with obesity-related diseases (such as hypertension and type 2 diabetes) may consider drug therapy. The Chinese Guide to Medical Nutrition Therapy for Overweight / Obesity (2021) recommends that adult patients with BMI ≥ 27 kg / m 2 and obesity-related complications use drug weight loss on the basis of lifestyle intervention. Currently, only orlistat is approved for marketing in China, which can be used for long-term treatment of obesity, and the recommended dose is 120 mg, taken 3 times a day. Some drugs for treating diabetes, such as GLP-1 receptor agonists, are also recommended for use in obese or overweight diabetic patients, but the selection of drugs is very limited.

[0011] The 2022 American Gastroenterological Association (AGA) guidelines recommend that for obese or overweight adults with weight-related complications, lifestyle interventions should be added to drug therapy rather than using continuous lifestyle interventions alone. The FDA-approved weight loss drugs in the United States include orlistat, lorcaserin, phentermine / topiramate extended-release, liraglutide, semaglutide, and naltrexone / bupropion extended-release. The 2022 AGA guidelines recommend semaglutide, phentermine / topiramate extended-release, liraglutide, and naltrexone / bupropion extended-release for obese or overweight adults with complications based on weight loss efficacy. The 2015 American Endocrine Society guidelines state that patients with BMI ≥ 30 kg / m 2 or BMI ≥ 27 kg / m 2 with complications such as hypertension, hyperlipidemia, and type 2 diabetes may consider drug therapy. The use of weight loss drugs requires evaluation of efficacy and safety, and generally it is recommended to continue medication only when weight loss is more than 5%.

[0012] In summary, both Chinese and American guidelines recommend the use of medication in addition to lifestyle interventions for obese patients who do not respond well to them. Currently, only orlistat is approved for weight loss in China, while the United States has a variety of weight loss drugs available and recommends drugs with novel mechanisms such as GLP-1 receptor agonists. Clinical use of weight loss drugs requires a careful balance of benefits and risks, and regular assessments of efficacy and safety are necessary.

[0013] GLP-1 receptor agonists are currently a hot target in the development of weight-loss drugs. GLP-1 drugs already approved for marketing in China include Novo Nordisk's liraglutide (indications include overweight / obesity) and semaglutide (approved only for type 2 diabetes). Several domestic pharmaceutical companies are developing GLP-1 drugs for weight-loss indications, such as Huadong Medicine, Livzon Pharmaceutical Group, Hanyu Pharmaceutical, and Tonghua Dongbao. Among them, Huadong Medicine's liraglutide injection received the first GLP-1-based weight-loss indication approval in China in 2023. Oral and long-acting formulations, as well as GLP-1 / GIP dual agonists, are expected to further enrich the selection of weight-loss drugs. Examples include Novo Nordisk's oral semaglutide and Eli Lilly's Tirzepatide. With the rising incidence of obesity and the approval of new drugs, the Chinese weight-loss drug market is expected to grow rapidly, potentially reaching 38.3 billion yuan by 2030.

[0014] Both Chinese and American guidelines recommend drug intervention as an important means of obesity treatment. GLP-1 receptor agonists are currently the most popular target for weight loss drug development, and oral and long-acting formulations are expected to improve patient compliance. Domestic pharmaceutical companies are actively investing in this area, and the market prospects are broad. However, attention should be paid to the selection of drug indications and their rational use to avoid blind or inappropriate use.

[0015] (3) Unmet clinical needs:

[0016] The following unmet clinical needs exist in the treatment of obesity in the United States regarding drug efficacy:

[0017] In the United States, the number of approved weight-loss drugs is limited. Currently, the FDA has approved fewer than 10 drugs for the long-term treatment of obesity, including orlistat and semaglutide. Compared to the vast obese population, the existing drug options are limited and cannot meet diverse clinical needs. Some weight-loss drugs have poor efficacy. Early weight-loss drugs, such as amphetamines, while effective, were highly addictive and had significant side effects, and most have been banned. The weight-loss efficacy of existing approved drugs also needs further improvement to better control weight. Taking semaglutide as an example, according to the provided information, although semaglutide, as a new type of weight-loss drug, has significant efficacy, it also has some major drawbacks:

[0018] 1. Increased risk of gastrointestinal adverse reactions

[0019] A recent study in JAMA showed that GLP-1 agonists, including semaglutide, may increase the risk of gastrointestinal diseases such as pancreatitis, gastroparesis, and intestinal obstruction by 4-9 times.

[0020] Common adverse reactions of oral semaglutide tablets include nausea, vomiting, diarrhea, constipation, stomach pain, etc. The larger the dose, the more obvious the side effects.

[0021] 2. Injection is not portable

[0022] Although it has the advantage of one injection per week, injection is still less portable than oral administration.

[0023] 3. Potential risk of abuse

[0024] There is a risk of non-obese people abusing it for non-medical weight loss purposes.

[0025] 4. Long-term safety needs to be observed

[0026] As a new drug, the long-term safety of semaglutide needs to be further evaluated.

[0027] Therefore, although semaglutide performs well in terms of weight loss efficacy, its gastrointestinal adverse reactions and other issues still need to be addressed to better meet clinical needs. At the same time, we need to be vigilant about its potential risk of abuse.

[0028] In addition, due to the huge market demand for weight loss drugs (estimated at $200 billion in 2035), drug supply shortages affect patient use. The demand for new weight loss drugs such as semaglutide is high, and the supply is insufficient, causing shortages and affecting normal medication for patients. It takes time to increase production capacity. The drug is expensive, and patients have a heavy burden. New weight loss drugs such as semaglutide cost up to $10,000 per year, with limited coverage by medical insurance, and patients have a heavy economic burden, affecting drug accessibility and compliance. Finally, new weight loss drugs still need to be developed. For different types of obesity and patient characteristics, new weight loss drugs with better efficacy, higher safety, and more convenient use still need to be developed to meet the diverse needs of patients.

[0029] Compared with the United States, the unmet clinical needs gap for weight loss indications in China is even larger. The number of obese people in China increased from 191 million in 2017 to 230 million in 2021, and is expected to reach 329 million in 2030. Currently, China has more obese people than the United States, ranking first in the world. At the same time, China currently only has orlistat approved for the treatment of obesity, with a treatment penetration rate of only about 0.25%.

[0030] In addition, there are differences in lifestyle interventions for obesity between China and the United States. The United States has established health insurance covering the whole life cycle and special groups, while China still lacks a recognized lifestyle intervention program for obesity management. The United States started earlier in obesity drug clinical trials, with more drugs approved for marketing. In recent years, the number of related clinical trials in China has increased, but it still needs time to be approved for marketing. In summary, there are significant differences in obesity incidence, treatment availability, and medical insurance support between China and the United States, leading to different clinical needs for obesity treatment. China needs to accelerate the development and approval of new drugs and expand patient accessibility to meet the treatment needs of the large obese population.

[0031] (4) Obesity-related diseases

[0032] Obesity and obstructive sleep apnea (OSA)

[0033] Obesity is the most important risk factor for OSA. There is a bidirectional causal relationship between obesity and OSA. Obesity leads to OSA, while OSA can also lead to further weight gain through various mechanisms such as appetite hormone imbalance, fatigue, and low mood. Obesity and OSA often coexist and have similar complications such as diabetes, hypertension, and cardiovascular disease. Weight loss can improve OSA, and OSA treatment can also help control weight and metabolic disorders.

[0034] In China, overweight and obesity can increase the risk of OSA by 1.5-2.4 times. In the United States, about 70% of adult OSA patients are obese. The higher the BMI, the higher the prevalence of OSA. Weight gain can exacerbate OSA. Studies have shown that a 10% increase in body weight can increase the risk of OSA by 30% and AHI by 32%. Conversely, a 10% weight loss can reduce AHI by 26%. In children, obesity is also a major risk factor for OSA. Obese children have a 46% higher risk of OSA than normal-weight children. Obesity in adolescence has a more significant impact on OSA.

[0035] In China, the overall prevalence of OSA is about 4%, with a higher prevalence in obese populations. A survey in Shanghai showed that the prevalence of OSA in people over 30 years old was 3.62%. In the United States, OSA affects about 25% of adults, with a prevalence of up to 45% in obese populations. With the prevalence of obesity, the prevalence of OSA is expected to further increase. In addition, abdominal obesity is more closely related to OSA. Fat deposition in the abdominal cavity and around the pharynx makes the upper airway space smaller, and the airway more prone to collapse, leading to OSA.

[0036] In summary, obesity is the most important risk factor for OSA, and the two are mutually causal, forming a vicious cycle that poses a serious burden on individual health and public health. Screening and treatment of OSA while preventing and treating obesity are crucial for controlling the prevalence of both diseases.

[0037] Obesity and CVD

[0038] Obesity is one of the major risk factors for CVD. In China, the risk of CVD in overweight and obese people is significantly higher than that in normal weight people. Obesity can increase the risk of CVD by 1.5-2.4 times. With the continuous rise in obesity rates in China and the United States, the burden of CVD will also increase. In 2019, there were 549,500 CVD deaths in China attributable to high BMI. Obesity leads to CVD through a variety of direct and indirect pathophysiological mechanisms. Abdominal obesity is more closely related to CVD. Weight loss can improve metabolic indicators and reduce the risk of CVD.

[0039] Diabetes, glucose-lowering

[0040] (1) Disease introduction:

[0041] The prevalence of diabetes in China continues to rise. In 2007, it was 9.7%, in 2013, it was 10.4%, and in 2017, it reached 11.2%. It is estimated that by 2045, the number of people with diabetes in China will reach 174.4 million. The national survey from 2015 to 2017 showed that the prevalence of diabetes among people aged 18 and over in China was 12.8%, and the prevalence of prediabetes was 35.2%. It is estimated that the total number of people with diabetes in China is 129.8 million. Diabetes prevalence is higher in men, the elderly, and urban populations. It is higher in southern regions than in northern regions. Obesity is a major risk factor. In the United States, the overall prevalence of diabetes in 2019 was 11.3%, or 37.3 million people. Among them, the diagnosed accounted for 8.7%, and the undiagnosed accounted for 23%.

[0042] Among people aged 65 and over in the United States, 29.2% have diabetes. Among different races, American Indians / Alaska Natives (14.7%) and Hispanics (12.5%) have the highest prevalence. In 2014, the global prevalence of diabetes among adults aged 18 and over was 8.5%. From 2000 to 2019, the standardized mortality rate due to diabetes worldwide increased by 3%.

[0043] Diabetes control in patients in China and the United States is not ideal, and diabetes and its complications have placed a huge burden on the health care system, and prevention and control need to be strengthened.

[0044] (2) Mainstream treatment options and pipeline progress

[0045] Current diabetes treatment drugs include oral drugs and injectable preparations.

[0046] Oral hypoglycemic agents include insulin secretagogues, non-insulin secretagogues, dipeptidyl peptidase-4 inhibitors (DPP-4 inhibitors), and sodium-glucose co-transporter 2 inhibitors (SGLT-2 inhibitors).

[0047] Insulin and insulin analogues, glucagon-like polypeptide-1 receptor agonists (GLP-1 receptor agonists) are injection preparations.

[0048] GLP-1 receptor agonists are initially used for the treatment of type 2 diabetes, and the indications are later extended to obesity, cardiovascular disease, non-alcoholic steatohepatitis, Alzheimer's disease, chronic kidney disease, etc. The GLP-1 drug penetration rate in Chinese patients with type 2 diabetes is low, and there is a large growth space in the future. In addition to GLP-1 single-target drugs, GLP-1 and GIP, GCG, etc. Multi-target combination is also a research hotspot. GLP-1 / GIP dual-target drug tirzepatide has been approved for listing, and the efficacy in reducing blood sugar and weight is better. GLP-1 / GCG, GLP-1 / GIP / GCG triple-target drugs are also expected to further improve the efficacy. GLP-1 / GIP / GCG triple-target drug Retatrutide of Eli Lilly has outstanding clinical efficacy, and can reduce the average body weight by 24.2% after 48 weeks of treatment. Many domestic enterprises have also laid out GLP-1 multi-target drugs, such as UBT251 of Union Pharmaceutical and DR10624 of East Pharmaceutical. The Chinese insulin market is growing rapidly, and diabetes drugs have become the second largest drug in the world. The risk of diabetes in obese people is 5 times that of non-obese people. GLP-1 drugs are expected to maintain strong growth in the field of diabetes and obesity. SUMMARY

[0049] Therefore, the present application provides polypeptides, fusion proteins and applications thereof. The polypeptides and fusion proteins provided by the present application can significantly reduce blood sugar, reduce lipids and / or reduce weight, and can effectively prevent, treat or improve diabetes, obesity, obesity-related diseases or metabolic abnormalities.

[0050] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0051] In a first aspect, the present application provides a triple-target polypeptide or a pharmaceutically acceptable salt, solvate, hydrate, complex, complex, chelate, derivative, variant, analog thereof, wherein the polypeptide structure is shown as formula I: X1-X2-Q-G-T-F-T-S-D-X 10 -S-X 12 -Y-L-D-X 16 -X 17 -X 18 -A-X 20 -X 21 -F-X 23 -X 24 -W-L-X 27 -X 28 -X 29 -X 30 -X 31 -X32 -X 33 -X 34 -X 35 -X 36 -X 37 -D-R-V-Y-I-H-P

[0052] Formula I

[0053] wherein:

[0054] X1is selected from H or Y;

[0055] X2is selected from S or A;

[0056] X 10 is selected from T, U or Y;

[0057] X 12 is selected from E or K;

[0058] X 16 is selected from E or S;

[0059] X 17 is selected from A, E, K, Q or R;

[0060] X 18 is selected from A or R;

[0061] X 20 is selected from D, K, Q or R;

[0062] X 21 is selected from D or E;

[0063] X 23 is selected from I or V;

[0064] X 24 is selected from A, C, D or Q;

[0065] X 27 is selected from E, L or M;

[0066] X 28 is selected from A, D or N;

[0067] X 29 is selected from G or T;

[0068] X 30 is selected from K, L, G, R or absent;

[0069] X 31 is selected from G, R or absent;

[0070] X 32 is selected from N or absent;

[0071] X 33 is selected from R or absent;

[0072] X 34 is selected from N or absent;

[0073] X 35 is selected from N or absent;

[0074] X 36 is selected from I or absent;

[0075] X 37 is selected from A or absent.

[0076] In some embodiments of the present application, the polypeptide has:

[0077] (I) an amino acid sequence as shown in any one of SEQ ID No. 1 to 10;

[0078] (II) an amino acid sequence obtained by substituting, deleting or adding 1 or 2 or 3 amino acids to the amino acid sequence as described in (I), and which is functionally identical to the amino acid sequence as described in (I); or

[0079] (III) an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more homology to the amino acid sequence as described in (I) or (II).

[0080] In some embodiments of the present application, the polypeptide has a free or chemically modified peptide chain end.

[0081] Preferably, the chemical modification includes a chemical modification of the amino terminal end or a chemical modification of the carboxyl terminal end.

[0082] Preferably, the chemical modification of the amino terminal end includes acylation, sulfonylation, alkylation and PEG modification; and the chemical modification of the carboxyl terminal end includes amidation, sulfonylation and PEG modification.

[0083] Preferably, the chemical modification of the amino terminal end is acetylation, benzoylation or sulfonylation of the amino group; the alkylation of the amino terminal end is C1-6alkylation or aralkylation; and the chemical modification of the carboxyl terminal end is substitution of OH in the carboxyl group with NH2or sulfonylamide or functionalization of OH in the carboxyl group with a PEG molecule.

[0084] In a second aspect, the present application also provides a fusion protein comprising the polypeptide or its pharmaceutically acceptable salt, solvate, hydrate, complex, compound, chelate, derivative, variant or analogue thereof as the active part, and a long-acting part, which is connected by the above two parts.

[0085] Preferably, the long-acting moiety is selected from the group consisting of Fc region of immunoglobulin, albumin, antibody and / or antigen-binding fragment.

[0086] In some embodiments of the present application, the fusion protein has:

[0087] (I) an amino acid sequence as shown in any one of SEQ ID No. 11 to 20;

[0088] (II) an amino acid sequence obtained by substituting, deleting or adding 1 or 2 or 3 amino acids to the amino acid sequence as described in (I), and which has the same function as the amino acid sequence as described in (I); or

[0089] (III) an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more homology to the amino acid sequence as described in (I) or (II).

[0090] In a third aspect, the present application further provides use of any one of the following in the preparation of a drug for reducing blood sugar, reducing blood lipid and / or reducing weight, or a drug for preventing, treating and / or improving diabetes, obesity, obesity-related diseases or metabolic abnormalities:

[0091] (I) the polypeptide or a pharmaceutically acceptable salt, solvate, hydrate, complex, compound, chelate, derivative, variant or analogue thereof;

[0092] (II) the fusion protein.

[0093] In a fourth aspect, the present application further provides a drug, which comprises any one of (I) or (II) and at least one of pharmaceutically acceptable adjuvant, excipient, carrier and solvent:

[0094] (I) the polypeptide or a pharmaceutically acceptable salt, solvate, hydrate, complex, compound, chelate, derivative, variant or analogue thereof;

[0095] (II) the fusion protein;

[0096] Preferably, it can also contain other effective ingredients.

[0097] In a fifth aspect, the present application further provides a method for reducing blood sugar, reducing blood lipid and / or reducing weight, or a method for preventing, treating and / or improving diabetes, obesity, obesity-related diseases or metabolic abnormalities, which administers, takes or uses any one of the following:

[0098] (I) the polypeptide or a pharmaceutically acceptable salt, solvate, hydrate, complex, compound, chelate, derivative, variant or analogue thereof;

[0099] (II) the fusion protein;

[0100] (III) the medicament.

[0101] The polypeptide provided by the present application realizes the synergistic effect of three targets GLP-1R, GCGR and MASR, and plays a role in reducing blood sugar, reducing fat and losing weight. The mechanism is as follows:

[0102] After GLP-1R is activated, insulin secretion is enhanced in a glucose concentration-dependent manner, glucagon secretion is inhibited, gastric emptying is delayed, satiety is increased, food intake is reduced through central appetite suppression, and energy consumption is increased through central and peripheral mechanisms, thereby achieving the effects of reducing blood sugar and losing weight. After GCGR is activated, the hyperglycemic effect can be counteracted by the insulin secretion and glucagon secretion inhibition effects of GLP-1R, and the hyperglycemic effect of GCGR activation can also alleviate the side effect of hypoglycemia caused by excessive GLP-1R effect. The effects of GCGR activation, such as increasing fatty acid oxidation and heat generation, reducing fat deposition and promoting energy consumption, can synergize with GLP-1R to reduce weight. GLP-1R and GCGR synergize to increase insulin secretion, enhance insulin sensitivity, improve insulin resistance and beta cell dysfunction, suppress appetite, reduce weight, and greatly reduce the occurrence of hypoglycemia. Meanwhile, MasR is activated to enhance insulin-induced glucose uptake in skeletal muscle, improve insulin sensitivity, synergize with the GLP-1R-promoted insulin secretion effect, and enhance the hypoglycemic effect.

[0103] On the basis of the above research, the three-target polypeptide provided by the present application is long-acting, which retains the excellent effects of the foregoing specific indications while having the advantages of long-acting, can reduce the frequency of administration, improve the convenience of medication, and improve patient compliance.

[0104] ①Improve patient compliance: Traditional polypeptide drugs need to be administered frequently, which is inconvenient for patients. Long-acting can prolong the administration interval and improve patient compliance.

[0105] ②Maintain effective blood drug concentration: Long-acting can maintain a relatively stable effective blood drug concentration in the body, avoiding large fluctuations in blood drug concentration.

[0106] ③Reduce adverse reactions: Long-acting preparations can avoid adverse reactions caused by excessively high peak blood drug concentration.

[0107] ④Improve efficacy: Compared with intermittent administration, continuous exposure to an effective concentration range can improve efficacy.

[0108] ⑤Reduce systemic exposure: Long-acting can reduce systemic exposure and reduce the risk of systemic toxicity.

[0109] Advantages of long-acting technology

[0110] Common long-acting modification methods include sustained-release preparations, chemical modification, and fusion proteins. Different long-acting technologies have different advantages and disadvantages, and the appropriate technology route needs to be selected according to the physicochemical properties of the specific drug, the route of administration, and other factors. The molecule of the present application uses a fusion protein approach, in which a polypeptide is fused to a long half-life protein (such as albumin, antibody, etc.), and the long half-life characteristics of the fusion protein are used to achieve long-acting.

[0111] The experimental results show that:

[0112] Compared with the Vehicle group, polypeptides 9G014, 8, and 16 mg / kg significantly reduced the body fat weight in a dose-dependent manner (P<0.001), with a decrease rate of 30.19%, 64.69%, and 65.87%, respectively. Compared with the Vehicle group, polypeptide 9G01 significantly reduced the fat index in a dose-dependent manner (P<0.001), with a decrease rate of 14.86%, 37.38%, and 47.19%, respectively. Compared with the Vehicle group, polypeptide 9G01 significantly reduced the Lee's index in a dose-dependent manner (P<0.001), with a decrease rate of 6.76%, 12.32%, and 14.25%, respectively.

[0113] Compared with the Vehicle group, polypeptide 9G01 significantly reduced serum TG, TC, HDL, and LDL in a dose-dependent manner (P<0.05, P<0.01, P<0.001).

[0114] Compared with the Vehicle group, polypeptide 9G01 significantly reduced serum Glu (P<0.001) by 62.08%, 76.26%, and 74.04%.

[0115] Compared with the Vehicle group, polypeptide 9G01 significantly reduced AUC0-30min, AUC0-60min, and AUC0-120min (P<0.001).

[0116] The above experimental results show that polypeptide 9G01 has a therapeutic effect on obesity and metabolic abnormalities in mice, and can dose-dependently reduce the body weight, food intake, body fat weight, fat index, Lee's index of obese mice, with an effect better than LY3437943 and comparable to dulaglutide. Polypeptide 9G01 can dose-dependently reduce serum TG, TC, LDL, and HDL, with an effect better than the positive control. Polypeptide 9G01 can improve glucose intolerance and reduce blood glucose, with an effect better than the positive control drug. The minimum effective dose of polypeptide 9G01 is 4 mg / kg, and the best effective dose is 8 mg / kg.

[0117] In conclusion, the polypeptide and fusion protein provided by the present application can significantly reduce blood sugar, blood lipid and / or weight, and can effectively prevent, treat or improve diabetes, obesity, obesity-related diseases or metabolic abnormalities. BRIEF DESCRIPTION OF DRAWINGS

[0118] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below.

[0119] Figure 1 shows the effect on body weight;

[0120] Figure 2 shows the effect on body weight loss rate (vs. Day 1);

[0121] Figure 3 shows the effect on body weight loss rate (vs. Vehicle);

[0122] Figure 4 shows the change of food intake per week during administration;

[0123] Figure 5 shows the change of food intake at 4W of administration;

[0124] Figure 6 shows the effect on fat weight; *** P<0.001 vs Control; ### P<0.001 vs Vehicle;

[0125] Figure 7 shows the effect on fat index; *** P<0.001 vs Control; ### P<0.001 vs Vehicle;

[0126] Figure 8 shows the effect on Lee's index; *** P<0.001 vs Control; ### P<0.001 vs Vehicle;

[0127] Figure 9 shows the effect on serum TG; # P<0.05, ## P<0.01 vs Vehicle;

[0128] Figure 10 shows the effect on serum TC; *** P<0.001 vs Control; ### P<0.001 vs Vehicle;

[0129] Figure 11 shows the effect on serum LDL; *** P<0.001 vs Control; ## P<0.01, ### P<0.001 vs Vehicle;

[0130] Figure 12 shows the effect on serum HDL; *** P < 0.001 vs Control; ### P < 0.001 vs Vehicle;

[0131] Figure 13 shows the effect on glycemia; * P < 0.05, ** P < 0.01 vs Control; ## P < 0.01, ### P < 0.001 vs Vehicle;

[0132] Figure 14 shows the effect on fructosamine; * P < 0.05, ** P < 0.01 vs Control; ## P < 0.01, ### P < 0.001 vs Vehicle;

[0133] Figure 15 shows the effect on 30 min glucose tolerance; * P < 0.05, ** P < 0.01 vs Control; ## P < 0.01, ### P < 0.001 vs Vehicle;

[0134] Figure 16 shows the effect on 60 min glucose tolerance; * P < 0.05, ** P < 0.01 vs Control; ## P < 0.01, ### P < 0.001 vs Vehicle;

[0135] Figure 17 shows the effect on 120 min glucose tolerance; * P < 0.05, ** P < 0.01 vs Control; ## P < 0.01, ### P < 0.001 vs Vehicle. DETAILED DESCRIPTION

[0136] The present application discloses polypeptides, fusion proteins and applications thereof, and those skilled in the art can refer to the content herein, and appropriately improve process parameters to achieve. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The methods and applications of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0137] Terminology

[0138] “Polypeptide” is used herein in its broadest form to refer to a compound of two or more subunit amino acids, amino acid analogs or other peptidomimetics. Thus, the term “polypeptide” includes short peptide sequences as well as longer polypeptides and proteins. As used herein, the term “amino acid” refers to natural and / or unnatural or synthetic amino acids, including both D or L optical isomers, as well as amino acid analogs and peptidomimetics.

[0139] As used herein, the term “amino acid” includes the standard twenty genetically-encoded amino acids and their corresponding “D” stereoisomers (as compared to the natural “L” form), omega-amino acids, other naturally occurring amino acids, nonstandard amino acids (e.g., alpha, alpha-disubstituted amino acids, N-alkyl amino acids, etc.), and chemically derivatized amino acids (see below).

[0140] When a specific amino acid is recited, such as “alanine” or “Ala” or “A”, unless otherwise explicitly stated, the term refers to both L-a alanine and D-alanine. Other nonstandard amino acids can also be suitable components of the polypeptides of the present application, so long as the polypeptide retains the desired functional properties. For the illustrated peptides, each encoded amino acid residue is represented by a one-letter designation corresponding to the conventional amino acid trivial name, where appropriate.

[0141] Those skilled in the art will appreciate that the polypeptides of the present application can include or consist of one or more amino acids that have been modified or derivatized.

[0142] Chemical derivatives of one or more amino acids can be achieved by reaction with functional side groups. Such derivative molecules include, for example, molecules in which the free amino group has been derivatized to form an amine hydrochloride, a p-toluenesulfonyl group, a carboxyphenoxy group, a t-butoxy carbonyl group, a chloroacetyl group, or a formyl group. The free carboxyl group can be derivatized to form a salt, a methyl and ethyl ester or other type of ester, and a hydrazide. The free hydroxyl group can be derivatized to form an O-acyl or O-alkyl derivative. Peptides containing naturally occurring amino acid derivatives of the twenty standard amino acids are also included as chemical derivatives. For example: 4-hydroxyproline can be substituted for proline; 5- hydroxylysine can be substituted for lysine; 3-methylhistidine can be substituted for histidine; homoserine can be substituted for serine and ornithine can be substituted for lysine. Derivatives also include peptides containing one or more additions or deletions, as long as the necessary activity is maintained. Other included modifications are amidation, amino-terminal acylation (e.g., acetylation or mercaptoacetic acid amidation), carboxyl-terminal amidation (e.g., with ammonia or methylamine), and similar terminal modifications.

[0143] It will further be appreciated by those skilled in the art that peptidomimetic compounds can also be useful. The term 'peptidomimetic' refers to a compound that mimics the conformation and desired characteristics of a particular peptide as a therapeutic agent. For example, the polypeptide includes not only molecules in which the amino acid residues are linked by peptide (-CO-NH-) bonds, but also molecules in which the peptide bonds are reversed. For example, such retro-inverso peptide mimetics can be prepared using methods known in the art, such as the methods described in Meziere et al. (1997), which is incorporated herein by reference. Such methods involve making pseudo-peptides that contain changes in the backbone that do not involve the orientation of the side chain. Retro-inverso peptides, which contain NH-CO bonds instead of CO-NH peptide bonds, are more resistant to proteolysis. Alternatively, the polypeptide can be a peptidomimetic compound in which one or more of the amino acid residues are linked by -y(CH2 NH)- bonds instead of the conventional amide bond. In a further alternative, the peptide bond can be omitted altogether, with the proviso that a suitable linker moiety is used that preserves the spacing between the carbon atoms of the amino acid residues; it can be advantageous for the linker moiety to have substantially the same charge distribution and substantially the same planarity as a peptide bond.

[0144] It will also be appreciated that the polypeptide can be conveniently blocked at its N- or C-terminus to help reduce susceptibility to extracellular proteolytic digestion. A variety of uncoded or modified amino acids, such as D-amino acids and N-methyl amino acids, have also been used to modify mammalian peptides. In addition, the putative biologically active conformation can be stabilized by covalent modification, such as cyclization or by the introduction of a lactam or other type of bridge, for example, see Veber et al. (1978) and Thursell et al. (1983), both of which are incorporated herein by reference.

[0145] A "derivative" or "variant" of any polypeptide described herein can be a substitution, deletion, or addition variant of the sequence. A variant can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,... 30... 40 or more amino acid substitutions and / or deletions from the sequence. A "deletion" variant can include a deletion of a single amino acid, a deletion of a small group of amino acids (such as 2, 3, 4 5 or more amino acids), or a deletion of a larger region of amino acids, such as a deletion of a particular amino acid domain or other feature. A "substitution" variant preferably involves replacing one or more amino acids with the same number of amino acids and making conservative amino acid substitutions. For example, an amino acid can be replaced with an alternative amino acid having similar properties, e.g., another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid, or another aliphatic amino acid. Some properties of the 20 primary amino acids that can be used to select suitable substituents are as follows:

[0146] Table 1

[0147] Amino acids herein can refer to the full name, three letter code, or one letter code.

[0148] Preferred "derivatives" or "variants" include derivatives or variants in which the amino acids present in the sequence are structural analogs of the naturally occurring amino acids. The amino acids used in the sequence can also be derivatized or modified.

[0149] Preferably, the amino acid sequence of the variant has more than 60% or more than 70% (e.g., 75 or 80%), preferably more than 85%, e.g., more than 90% or 95% amino acid identity to the sequence shown in the sequences disclosed herein. This level of amino acid identity can be seen over the full length of the relevant SEQ ID NO sequence or over a portion of the sequence, such as over 20, 30, 50, 75, 100, 150, 200 or more amino acids, depending on the size of the full length polypeptide.

[0150] "Antibodies or antigen-binding fragments thereof" include essentially intact antibody molecules, as well as chimeric antibodies, humanized antibodies, isolated human antibodies, single-chain antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy and / or light chains, and antigen-binding fragments and derivatives thereof. Suitable antigen-binding fragments and derivatives include Fv fragments (e.g., single-chain Fv and disulfide-bonded Fv), Fab-like fragments (e.g., Fab fragments, Fab' fragments, and F(ab)2 fragments), single variable domains (e.g., VH domains and VL domains), and single-domain antibodies (dAbs, including single and dual formats [i.e., dAb-linker-dAb] as well as nanobodies). Potential advantages of using antibody fragments rather than whole antibodies are numerous. Smaller size fragments can lead to improved pharmacological properties, such as better penetration of solid tissues. Furthermore, antigen-binding fragments (such as Fab, Fv, ScFv, and dAb antibody fragments) can be expressed and secreted in E. coli, allowing for easy production of large quantities of the fragments.

[0151] The phrase "antibody or antigen-binding fragment thereof" is also intended to encompass antibody mimetics (e.g., non-antibody scaffold structures that have a high degree of stability but allow for variability to be introduced at certain positions). Those skilled in the biochemistry art will be familiar with many such molecules, as discussed in Gebauer and Skerra, 2009, the disclosure of which is incorporated herein by reference. Exemplary antibody mimetics include: affibodies (also known as Trinectins; Nygren, 2008, FEBS J, 275, 2668-2676); CTLDs (also known as Tetranectins; Innovations Pharmac. Technol. (2006), 27-30); adnectins (also known as monobodies; Meth. Mol. Biol., 352 (2007), 95-109); anticalins (Drug Discovery Today (2005), 10, 23-33); DARPins (ankyrins; Nat. Biotechnol. (2004), 22, 575-582); avimers (Nat. Biotechnol. (2005), 23, 1556-1561); minibodies (FEBS J, (2007), 274, 86-95); peptide aptamers (Expert. Opin. Biol. Ther. (2005), 5, 783-797); Kunitz domains (J. Pharmacol. Exp. Ther. (2006) 318, 803-809); affilins (Trends. Biotechnol. (2005), 23, 514-522); affimers (Avacta Life Sciences, Wetherby, UK). Also included within the scope of the present invention are chimeric T cell receptors (also known as chimeric immunoreceptors and chimeric antigen receptors or CARs) (see Pule et al., 2003, the disclosure of which is incorporated herein by reference). These are engineered receptors that graft arbitrary specificities onto immune effector cells. Typically, CARs are used to graft the specificity of a monoclonal antibody onto a T cell; transfer of their coding sequences is facilitated by retroviral vectors. The most common form of such molecules is a fusion that includes a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to a CD3-zeta transmembrane and intracellular domain. When T cells express this fusion molecule, they recognize and kill target cells expressing the specificity of the transferred monoclonal antibody.

[0152] A polynucleotide of the application can encode any of the polypeptides described herein. The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. Non-limiting examples of polynucleotides include a gene, a gene fragment, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide of the application can be provided in isolated or substantially isolated form. Substantially isolated means that the polypeptide is essentially separated from any surrounding medium, but not necessarily completely isolated. A polynucleotide can be mixed with a carrier or diluent that does not interfere with the intended use of the polynucleotide and is still considered to be substantially isolated.

[0153] A nucleic acid sequence which "encodes" a selected polypeptide is a nucleic acid molecule, which when expressed in vivo, transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. For purposes of the application, such nucleic acid sequences can include, but are not limited to, cDNA from a viral, prokaryotic or eukaryotic mRNA, genomic sequences from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. A transcription termination sequence can be located 3' to the coding sequence.

[0154] A suitable polynucleotide sequence can also be a variant of one of these particular polynucleotide sequences. For example, the variant can be a substitution, deletion or addition variant of any of the above nucleic acid sequences. The variant polynucleotide can include 1, 2, 3, 4, 5, up to 10, up to 20, up to 30, up to 40, up to 50, up to 75 or more nucleic acid substitutions and / or deletions from the sequences given in the sequence listing. Suitable variants can have at least 70% homology, preferably at least 80% or 90% (and more preferably at least 95%, 97% or 99%) homology with the polynucleotide of any one of the nucleic acid sequences disclosed herein. Preferably, these levels of homology and identity exist at least in respect of the coding regions of the polynucleotide. Methods of measuring homology are well known in the art, and the skilled person will understand that in the present context, homology is calculated on the basis of nucleic acid identity. Such homology can exist within a region of at least 15, preferably at least 30, for example at least 40, 60, 100, 200 or more contiguous nucleotides. Such homology can exist over the entire length of the unmodified polynucleotide sequence.

[0155] The difference between a homolog and the sequence in the associated polynucleotide can lie in having fewer than 3, 5, 10, 15, 20, or more mutations (each mutation can be a substitution, deletion, or insertion). These mutations can be measured in regions of at least 30, for example, at least 40, 60, or 100 or more consecutive nucleotides in the homolog. In one embodiment, the variant sequence may differ from the specific sequence given in the sequence listing due to redundancy in the genetic code. The DNA code has four major nucleic acid residues (A, T, C, and G) and uses these residues to “spell” three-letter codons, which represent amino acids and proteins encoded in the genes of an organism. The linear sequence of codons along the DNA molecule is translated into a linear sequence of amino acids in one or more proteins encoded by these genes. The code is highly degenerate, with 61 codons encoding 20 natural amino acids, and 3 codons representing a “stop” signal. Therefore, most amino acids are encoded by more than one codon—in fact, several amino acids are encoded by four or more different codons. Therefore, the variant polynucleotides of the present invention can encode the same polypeptide sequence as another polynucleotide of the present invention, but can have different nucleic acid sequences due to the use of different codons to encode the same amino acid. Therefore, the polypeptides of the present invention can be produced or delivered in the form of a polynucleotide encoding and capable of expressing it. The polynucleotides of the present invention can be synthesized according to methods well known in the art, as described by way of example in Green and Sambrook (2012, *Molecular Cloning: A Lab Manual*, 4th ed.; Cold Spring Harbor Publishing; the disclosure of which is incorporated herein by reference). The nucleic acid molecules of the present invention can be provided in the form of expression cassettes containing a control sequence operatively linked to an insert sequence, thereby allowing the polypeptides of the present invention to be expressed in vivo. These expression cassettes are then typically provided in vectors (e.g., plasmids or recombinant viral vectors). Such expression cassettes can be administered directly to a host subject. Alternatively, vectors comprising the polynucleotides of the present invention can be administered to a host subject. Preferably, a genetic vector is used to prepare and / or administer the polynucleotide. A suitable vector can be any vector capable of carrying a sufficient amount of genetic information and allowing expression of the polypeptides of the present invention.

[0156] Accordingly, the present application includes expression vectors comprising such polynucleotide sequences. Such expression vectors are routinely constructed in the art of molecular biology, and can for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers, and other elements, such as for example polyadenylation signals, which can be necessary, and positioned in the correct orientation to allow expression of the peptides of the present application. Other suitable vectors will be apparent to those skilled in the art (see Green and Sambrook, supra). The present application also includes cells that have been modified to express the polypeptides of the present application. Such cells include transient or, preferably, stable higher eukaryotic cell lines (such as mammalian cells or insect cells), lower eukaryotic cells (such as yeast), or prokaryotic cells (such as bacterial cells). Particular examples of cells that can be modified by insertion of a vector or expression cassette encoding a polypeptide of the present application include mammalian HEK293T, CHO, HeLa, NS0, and COS cells. Preferably, the cell line chosen is not only stable, but also allows for mature glycosylation and cell surface expression of the polypeptide. Such cell lines of the present application can be cultured using routine methods to produce the polypeptides of the present application, or can be used to deliver the antibodies of the present application to a subject for therapeutic or prophylactic purposes. Alternatively, the polynucleotides, expression cassettes, or vectors of the present application can be administered to cells from a subject ex vivo, which are then returned to the body of the subject.

[0157] A "nucleic acid molecule" includes DNA (e.g., genomic DNA or complementary DNA) and mRNA molecules, which can be single-stranded or double-stranded. "Isolated" means that the nucleic acid molecule is not located in or otherwise provided in a cell.

[0158] Those skilled in the art will appreciate that a nucleic acid molecule can be codon-optimized for expression of a polypeptide in a particular host cell, for example for expression in human cells (e.g., see Angov, 2011, the disclosure of which is incorporated herein by reference).

[0159] It will be appreciated by persons skilled in the art that additional compounds can also be included in the pharmaceutical composition, including chelating agents such as EDTA, citrate, EGTA, or glutathione. The pharmaceutical composition can be prepared in a manner known in the art, in a manner that has sufficient storage stability and is suitable for administration to humans and animals. For example, the pharmaceutical composition can be lyophilized, for example by freeze-drying, spray-drying, spray-chilling, or by particle formation using supercritical particles. "Pharmaceutically acceptable" means a nontoxic material that does not reduce the effectiveness of the dendritic cell and tumor cell related antigen binding activity of the polypeptides of the application. Such pharmaceutically acceptable buffers, carriers, or excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th Ed., Editor: A. R Gennaro, Mack Publishing Company (1990) and the handbook of Pharmaceutical Excipients, 3rd Ed., Editor: A. Kibbe, Pharmaceutical Press (2000), the disclosures of which are incorporated herein by reference).

[0160] In one embodiment, the pharmaceutical composition of the present application can be in the form of a liposome, in which the polypeptide is combined with amphiphilic agents, such as lipids, in addition to other pharmaceutically acceptable carriers, which exist in aggregated form as micelles, insoluble monolayers, and liquid crystals. Suitable lipids for liposome formulation include, but are not limited to, monoglycerides, diglycerides, sulpholipids, lyso- lecithins, phospholipids, saponins, bile acids, and the like. Suitable lipids also include lipids modified in the polar head group with poly(ethylene glycol) in order to prolong the circulation time in the bloodstream. The preparation of such liposome formulations can be found in, for example, US 4,235,871, the disclosure of which is incorporated herein by reference. The pharmaceutical composition of the present application can also be in the form of biodegradable microspheres. Aliphatic polyesters, such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), copolymers of PLA and PGA (PLGA), or poly(caprolactone) (PCL), and polyanhydrides have been widely used as biodegradable polymers in microsphere production. The preparation of such microspheres can be found in US 5,851,451 and EP 0213303, the disclosures of which are incorporated herein by reference. In further embodiments, the pharmaceutical composition of the present application is provided in the form of a polymer gel, in which a polymer (such as starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, ethylhydroxyethylcellulose, alginates, carrageenans, hyaluronic acid and its derivatives, polyacrylic acid, polyvinylimidazole, polysulphonates, polyethylene glycol / polyethylene oxide, polyethylene oxide / polypropylene oxide copolymers, polyvinyl alcohol / polyvinyl acetate of varying degrees of hydrolysis, and polyvinylpyrrolidone) is used for the thickening of a solution containing a medicament. The polymer can also include gelatin or collagen. Alternatively, the polypeptide can simply be dissolved in saline, water, polyethylene glycol, propylene glycol, ethanol, or oils such as safflower oil, corn oil, peanut oil, cottonseed oil, or sesame oil, tragacanth gum, and / or various buffers. It will be appreciated that the pharmaceutical composition of the present application can contain ions and a defined pH for enhancing the action of the active polypeptide. Additionally, the composition can be subjected to conventional pharmaceutical operations such as sterilization, and / or can contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, fillers, and the like. The pharmaceutical composition of the present application can be administered by any suitable route known to those skilled in the art. Thus, possible routes of administration include parenteral (intravenous, subcutaneous and intramuscular), topical, ocular, nasal, pulmonary, buccal, oral, parenteral, vaginal and rectal. Likewise, administration from an implant is also possible. In a preferred embodiment, the pharmaceutical composition is administered parenterally, e.g., intravenously, intracerebroventricularly, intraarticularly, intraarterially, intraperitoneally, intrathecally, intraventricularly, sternal, intracranially, intramuscularly or subcutaneously, or the pharmaceutical composition can be administered by infusion technology. The pharmaceutical composition can conveniently be in the form of a sterile aqueous solution, which can contain other substances, for example enough salts or dextrose, to make the solution isotonic with the blood.If necessary, the aqueous solution should be appropriately buffered (preferably, buffered to a pH of 3 to 9). The preparation of suitable parenteral formulations under aseptic conditions can be readily achieved by standard pharmaceutical techniques well known to those skilled in the art.

[0161] The polypeptides, fusion proteins and applications thereof provided by the present application use raw materials and reagents that can be purchased from the market.

[0162] The present application is further described below in conjunction with examples:

[0163] Example 1 Target design and screening verification

[0164] The Fc fusion proteins designed for the GLP1R / GCGR / MasR three targets design a total of 10 lead molecules for the three targets.

[0165] (1) The polypeptide sequences generated in the design process of the present application are

[0166] Polypeptide 1: HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIADRVYIHP (SEQ ID NO: 01)

[0167] Polypeptide 2: YAQGTFTSDTSKYLDSARADDFVDWLLNTLRNRNNIADRVYIHP (SEQ ID NO: 02)

[0168] Polypeptide 3: HSQGTFTSDYSKYLDSARAQDFVDWLLNTGRNRNNIADRVYIHP (SEQ ID NO: 03)

[0169] Polypeptide 4: YAQGTFTSDYSKYLDEQAAKEFIAWLMNTDRVYIHP (SEQ ID NO: 04)

[0170] Polypeptide 5: YAQGTFTSDUSKYLDEQAAKEFIAWLLDTDRVYIHP (SEQ ID NO: 05)

[0171] Polypeptide 6: YAQGTFTSDUSKYLDERAAQDFVQWLLDGRGDRVYIHP (SEQ ID NO: 06)

[0172] Polypeptide 7: HSQGTFTSDYSKYLDEKRAKEFVCWLMNTDRVYIHP (SEQ ID NO: 07)

[0173] Polypeptide 8: HSQGTFTSDYSEYLDSERARDFVQWLEAGGDRVYIHP (SEQ ID NO: 08)

[0174] Polypeptide 9: HSQGTFTSDYSKYLDERAAQDFVQWLLDTDRVYIHP (SEQ ID NO: 09)

[0175] Polypeptide 10: HSQGTFTSDYSKYLDEQAAKEFIAWLMNTDRVYIHP (SEQ ID NO: 10)

[0176] (2) The related protein sequences generated in the design process of the present application are

[0177] Polypeptide 1 G01

[0178] Polypeptide 2 G01:

[0179] Polypeptide 3 G01:

[0180] Polypeptide 4 G01:

[0181] Polypeptide 5 G01:

[0182] Polypeptide 6 G01:

[0183] Polypeptide 7 G01:

[0184] Polypeptide 8 G01:

[0185] Polypeptide 9 G01:

[0186] Polypeptide 10 G03:

[0187] Example 2 Preparation method of polypeptide

[0188] 1. Polypeptides 1-10 are prepared by conventional solid-phase polypeptide synthesis method.

[0189] The polypeptides are detected by conventional mass spectrometry detection method, and the results are shown in the following table.

[0190] Table 2. Polypeptide mass spectrometry results

[0191] 2. Taking polypeptide 9G01 as an example, the preparation method of the fusion protein is as follows:

[0192] Step 1 Construction of vector of polypeptide 9G01

[0193] The polypeptide gene of the present application is obtained by gene synthesis to obtain the target gene fragment, and then the target gene is connected to the pcDNA3.4 vector by molecular cloning technology. After sequencing, the expression vector plasmid with the correct target gene sequence is obtained. The expression vector plasmid with correct sequencing is transformed into competent cells, and then a large amount of plasmid for transient transfection and expression is obtained by using a large amount of extraction kit. The specific steps are as follows:

[0194] First, prepare the expression vector with correct sequencing, and take the competent cells (DH-5α) from the-80℃ refrigerator, mark in the ice box, and add 30-50ng of the expression vector plasmid to the competent cells (DH-5α) in the clean bench, mix well, ice bath for 30min, then heat shock in the 42℃ water bath for 90s, immediately put into the ice box, then add LB medium in the clean bench, cultivate at 37℃ for 1h, then take part of the supernatant and coat on the LB plate containing antibiotics, pick single colonies on the second day, and put into 50ml of LB medium containing antibiotics for overnight culture. On the third day, use OMEGE plasmid extraction kit for plasmid extraction, and follow the instructions for extraction steps to obtain a large amount of plasmid for transient transfection.

[0195] Step 2 Expression and production of polypeptide 9G01

[0196] The expression plasmid is mixed with the transfection reagent by lipofection, and then added into Expi CHO cells. After 5-7 days of culture in a constant temperature incubator, the cell culture containing polypeptide 9G01 is harvested. The cell culture is centrifuged to obtain the supernatant, and the affinity chromatography column is used to capture the target protein. The polypeptide 9G01 content is determined by A280 method. The specific steps are as follows:

[0197] First, prepare the plasmid and transfection reagent in a clean bench, add Expi CHO cell culture medium (volume is 1 / 10 of the transfection volume) into a 50 ml centrifuge tube, add the expression vector plasmid (100 μg / 100 ml), and add the filtered transfection reagent PEI (1 g / L Polysciences) into the centrifuge tube according to the mass ratio (plasmid: PEI = 1:3). Mix and stand for 10 min, pour the plasmid / PEI mixture into the Expi CHO cells to be transfected, mix, and put the shake flask into a 37°C, 270 rpm, 8% CO2 incubator for culture. After 24 h, add the feed medium for culture, and after 5 d, the culture is completed, and the sample is sent for titer detection. Centrifuge at 13000 rpm for 20 min to harvest the culture supernatant, and purify the supernatant by Protein A affinity chromatography (affinity filler: HiTrap MabSelect SuRe GE). The loading flow rate is 1 ml / min. After loading, wash with phosphate buffer, and then elute with sodium acetate buffer at pH 3.4. Collect each tube of about 500 μl, read the 280 nm absorbance value using a NanoDrop instrument, replace the buffer system of the high-concentration protein to neutral phosphate buffer by using a dialysis bag, and detect the purity by SEC-HPLC, which is not less than 90%.

[0198] Other fusion proteins disclosed in the present application can be prepared by referring to the above method, which is not described herein.

[0199] The polypeptide 9G01 was detected, and the results are shown in the following table:

[0200] (1) Polypeptide 9G01 N / C terminal sequencing results

[0201] Table 3

[0202] (2) Polypeptide 9G01 complete molecular weight detection results

[0203] Table 4

[0204] The polypeptide 10G03 was detected by using ultra-high performance liquid chromatography and high-resolution / high-precision mass spectrometry (LC-MS / MS method), and the results are shown in the following table:

[0205] Table 5. Polypeptide 10G03 N / C terminal sequence results

[0206] Other fusion proteins were determined by using the above method, and the amino acid sequence was consistent with the designed sequence, which is not described herein.

[0207] Example 3: In vitro activity detection method

[0208] 1. Cell culture and reagent preparation

[0209] 1) Cell lines: Flpin-CHO-GCGR, Flpin-293-GLP1R

[0210] 2) Complete medium: F12K + 10% fetal bovine serum + 1* penicillin streptomycin + 800 μg / ml hygromycin and DMEM + 10% fetal bovine serum + 1* penicillin streptomycin + 200 μg / ml hygromycin.

[0211] 3) Assay buffer: 1* HBSS + 20 mM HEPES + 0.1% BSA + 500 μΜ IBMX.

[0212] 2. Assay of the tested compound on GLP-1R / GCGR receptor activity

[0213] 1) Digest Flpin-CHO-GCGR, Flpin-293-GLP1R stable pool, resuspend in assay buffer, seed in 384 cell culture plates, seeding density is 8000, 20000 per well respectively, seeding volume is 15 μL per well.

[0214] 2) Dilute the compound with assay buffer.

[0215] 3) Add 5 μL of compound per well, incubate at 37°C for 30 minutes.

[0216] 4) Freeze-thaw Eu-cAMP tracer and Ulight-anti-cAMP, dilute with lysis buffer.

[0217] 5) Add 10 μl Eu-cAMP tracer to the assay well, then add 10 μl Ulight-anti-cAMP to the assay well.

[0218] 6) Centrifuge the reaction plate at 200g for 30s at room temperature, stand for 1h at 25°C, then collect data using Envision.

[0219] 3. Assay of the tested compound on MASR receptor activity

[0220] 1) Preparation of KH solution and high potassium solution

[0221] Krebs-Henseleit (KH) solution (mM): NaCl 120, KCl 4.5, CaCl21.5, KH2PO41.2, MgSO41.2, NaHCO315, glucose 10, pH 7.4.

[0222] K + - Krebs-Henseleit (KH) solution (mM): NaCl 120, KCl 4.5, CaCl21.5, KH2PO41.2, MgSO41.2, NaHCO315, glucose 10, pH 7.4. +K-H solution (mM): NaCl 60, KCl 63.5, CaCl2 1.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 15, glucose 10, pH 7.4.

[0223] 2) Preparation of vascular ring samples: After anesthesia, the mesenteric artery blood vessels of rats were quickly removed and immersed in pre-cooled K-H solution (filled with 95% air and 5% CO2 mixed gas, pH = 7.4), and the surrounding adherent tissues were carefully peeled off under a stereomicroscope and cut into 2 mm long segments for use. The arterial rings were hung on two L-shaped metal needles, one connected to a tension transducer and the other connected to a vascular fine adjustment device (adjusting the load tension), and the vascular tension was recorded by the BL-420N biological signal measuring system. The arterial rings were immersed in a 1 mL constant temperature tissue bath containing K-H solution, maintained at 37°C and continuously supplied with a mixture of 95% air and 5% CO2.

[0224] 3) Vascular function recording: Before the experiment, the vascular rings were given a pre-tension of 0.65g and balanced for about 90min, during which fresh K-H solution was replaced every 15min. After the balance was completed, the vascular rings were contracted with high-K+-KH solution (containing K+ 60mM) to detect the contractility of the arterial rings, and those with a difference in contraction amplitude of <10% in two times were selected for the experiment.

[0225] 4) Preparation of vascular concentration gradient relaxation curve: After the vascular rings were balanced, 80μL of a vascular contractile agent was added to an 8mL constant temperature tissue bath, and after the vascular contraction entered the plateau phase, Ach or the test drug was added in turn from low concentration to high concentration to draw the concentration gradient contraction curve.

[0226] Results of in vitro activity detection of Example 4

[0227] A Flpin-293-GLP1R, Flpin-CHO-GCGR stable cell line reporter system was constructed for the GLP1R / GCGR target point, and GLP-1 and Glucagon were used as controls, respectively. The lead molecules with higher affinity to GLP1R / GCGR receptors were screened through the reporter system, and the designed lead molecules were screened and optimized. For MasR receptor molecules, the functional effects of the lead molecules were observed by observing the degree of vascular concentration gradient relaxation.

[0228] In the first round of design and target activity screening, polypeptide 6, polypeptide 7, polypeptide 9, and polypeptide 10, four compounds had GLP1R / GCGR target activity, so the functions of MasR were further observed, as shown in Table 6, and polypeptide 6, polypeptide 7, polypeptide 9, and polypeptide 10 were screened.

[0229] Further, the C-terminus of the four compounds is connected to IgG4 FC through a GS connector to form a fusion protein, which is the second round of Fc long-acting design, marked as polypeptide 6G01, polypeptide 7G01, polypeptide 9G01, polypeptide 10G03. The second round is based on the compounds screened in the first round, which have reached the required activity. GLP-1 and Glucagon are used as controls for target activity screening. Polypeptide 9G01 is the best candidate compound, as shown in Table 7.

[0230] The third round is further pharmacological optimization. The positive controls of GLP-1R and GCGR are GLP-1(7-37) and Glucagon, respectively, and the control of MASR is Ach. According to the experimental results in Tables 8 and 9, polypeptide 9G01 is the best candidate compound.

[0231] Table 6. First round design and screening EC 50 (nM)

[0232] Note: NA represents no results of target verification, and ND represents undetected

[0233] Table 7. Second round design (long-acting design) EC 50 (nM)

[0234] Table 8. CMC screening and further verification EC 50 (nM)

[0235] Table 9. CMC screening and further verification EC 50 (nM)

[0236] Example 5 Therapeutic effect of subcutaneous injection of polypeptide 9G01 on high-fat combined high-fructose diet-induced obesity and metabolic abnormalities in mice

[0237] Methods: Mice were fed high-fat feed combined with 25% high-fructose drinking water for 16 weeks to induce an obesity metabolic abnormality model, and the body weight was measured once a week. After 16 weeks of modeling, according to the body weight, 8 model mice and 6 blank mice were randomly selected, and serum was taken for biochemical detection. Compared with the blank control mice, the model mice had significant differences in body weight, which was considered to be a successful modeling test. The successfully modeled mice were randomly divided into 6 groups according to the body weight, namely the model control group (Vehicle), the positive drug control group Dulaglutide, the positive drug control group LY3437943, the polypeptide 9G014, 8, 16 mg / kg dose groups, each group of 12; another blank control group (Control) of 12. The test substances and positive control groups were given subcutaneous injection of drugs, and the model control group and the blank control group were given subcutaneous injection of normal saline, twice a week, for 4 weeks of continuous administration. After the experiment, blood was collected and biochemical indexes were detected; fat tissue was removed for determination.

[0238] Results are as follows:

[0239] 1.1 Effect on body weight

[0240] After 4W of administration, compared with the Vehicle group, polypeptide 9G01 can significantly reduce the body weight of mice in a dose-dependent manner (vs. Vehicle P<0.001); compared with the Control group, there was no statistical difference in the body weight of mice in the polypeptide 9G01 8, 16 mg / kg dose groups. The results are shown in Table 10, Table 10, and Figure 1.

[0241] Compared with Day 1, polypeptide 9G01 can dose-dependently increase the body weight loss rate; the body weight loss rate of mice in the polypeptide 9G01 8, 16 mg / kg groups is comparable to that in the Dulaglutide group. The results are shown in Table 10, Figure 2.

[0242] Compared with Vehicle, polypeptide 9G01 can dose-dependently increase the body weight loss rate; the body weight loss rate of mice in the polypeptide 9G01 8, 16 mg / kg groups is comparable to that in the Dulaglutide group. The results are shown in Table 10, Figure 3.

[0243] Table 10. Effect on body weight (g) Notes: * P<0.05, ** P<0.01, *** P<0.001 vs. Control; # P<0.05, ## P<0.01, ### P<0.001 vs. Vehicle

[0244] Table 10. Effect on body weight (g) Notes: *P<0.05, ** P<0.01, *** P<0.001 vs. Control; # P<0.05, ## P<0.01, ### P<0.001 vs. Vehicle

[0245] 1.2 Effect on food intake

[0246] During the administration period, the food intake of polypeptide 9G01 remained stable every week, and the food intake of LY3437943 and Dulaglutide groups was inhibited in the early stage and gradually recovered in the later stage. The results are shown in Figure 4.

[0247] After 4 weeks of administration, compared with Vehicle, polypeptide 9G01 could reduce food intake in a dose-dependent manner, but there was no statistically significant difference. The results are shown in Figure 5.

[0248] 1.3 Effect on fat weight and Lee's index

[0249] Compared with the Control group, the fat weight, fat index, and Lee's index of the Vehicle group were significantly increased (P<0.001); compared with the Vehicle group, polypeptide 9G01 at 4, 8, and 16 mg / kg could significantly reduce the fat weight in a dose-dependent manner (P<0.001), with a decrease rate of 30.19%, 64.69%, and 65.87%, respectively; compared with the Vehicle group, polypeptide 9G01 could significantly reduce the fat index in a dose-dependent manner (P<0.001), with a decrease rate of 14.86%, 37.38%, and 47.19%, respectively; compared with the Vehicle group, polypeptide 9G01 could significantly reduce the Lee's index in a dose-dependent manner (P<0.001), with a decrease rate of 6.76%, 12.32%, and 14.25%, respectively; compared with the Vehicle group, LY3437943 and Dulaglutide could significantly reduce the fat weight, fat index, and Lee's index (P<0.001). The results are shown in Table 11, Figures 6, 7, and 8.

[0250] Table 11. Effect on fat weight and Lee's index Note: *** P<0.001 vs. Control; # P<0.05, ## P<0.01, ### P<0.001 vs. Vehicle

[0251] 1.4 Effect on blood lipids

[0252] At 4W, compared with the Control group, the serum TC, HDL and LDL of the Vehicle group were significantly increased (P<0.001); compared with the Vehicle group, the polypeptide 9G01 could significantly reduce the serum TG, TC, HDL and LDL in a dose-dependent manner (P<0.05, P<0.01, P<0.001). Compared with the Vehicle group, LY3437943 and Dulaglutide significantly reduced (P<0.001) serum TC, HDL and LDL. The results are shown in Table 12, Table 12-2, Figure 9, Figure 10, Figure 11 and Figure 12.

[0253] Table 12. Effects on blood lipids Note: *** P<0.001 vs. Control; # P<0.05, ## P<0.01, ### P<0.001 vs. Vehicle

[0254] Table 12-2. Effects on blood lipids Note: *** P<0.001 vs. Control; # P<0.05, ## P<0.01, ### P<0.001 vs. Vehicle

[0255] 1.5 Effects on blood glucose, glycosylated hemoglobin and fructosamine

[0256] At 4W, after 16h fasting, compared with the Control group, the serum Glu of the Vehicle group was significantly increased (P<0.001); compared with the Vehicle group, the polypeptide 9G01 significantly (P<0.001) reduced serum Glu, with a reduction rate of 62.08%, 76.26% and 74.04%; compared with the Vehicle group, the polypeptide 9G01 had no difference in reducing serum HbA1c and FUN. Compared with the Vehicle group, LY3437943 and Dulaglutide significantly reduced (P<0.05, P<0.001) serum Glu and FUN. The results are shown in Table 13, Figure 13 and Figure 14.

[0257] Table 13. Effects on blood glucose, glycosylated hemoglobin and fructosamine Note: *** P<0.001 vs. Control; # P<0.05, ## P<0.01, ### P<0.001 vs. Vehicle

[0258] 1.6 Effect on glucose tolerance

[0259] Mice were fasted overnight without water for 16 h, and the glucose tolerance test was performed after the end of the 8thdose in the fourth week. Compared with the Control group, the Vehicle group significantly increased AUC 0-30min , AUC 0-60min , AUC 0-120min ; compared with the Vehicle group, polypeptide 9G01 significantly (P<0.001) reduced AUC 0-30min , AUC 0-60min , AUC 0-120min . Compared with the Vehicle group, LY3437943 and Dulaglutide significantly (P<0.01, P<0.001) reduced AUC 0-30min , AUC 0-60min , AUC 0-120min . The results are shown in Table 14, Figures 15-17.

[0260] Table 14. Effect on glucose tolerance Note: *** P<0.001 vs. Control; # P<0.05, ## P<0.01, ### P<0.001 vs. Vehicle

[0261] Conclusion: Polypeptide 9G01 has a therapeutic effect on obesity combined with metabolic abnormalities in mice, and can dose-dependently reduce the body weight, food intake, body fat weight, fat index, Lee's index of obese mice, and the effect is better than that of LY3437943 and comparable to that of Dulaglutide; can dose-dependently reduce serum TG, TC, LDL, HDL, and the effect is better than that of the positive control; improves glucose intolerance and reduces blood glucose in mice, and the effect of reducing blood glucose is better than that of the positive control drug; the minimum effective dose of polypeptide 9G01 is 4 mg / kg, and the best effective dose is 8 mg / kg.

[0262] The above describes in detail a three-target polypeptide and a fusion protein thereof provided by the present application. The principles and implementation modes of the present application are described by using specific examples, and the above description of the examples is only used to help understand the method and core idea of the present application. It should be noted that those skilled in the art can make some improvements and modifications to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

Claims

1. A three-target polypeptide or a pharmaceutically acceptable salt, solvate, hydrate, complex, chelate, derivative, variant, or analog thereof, characterized in that, The polypeptide structure is shown in Formula I: X1-X2-QGTFTSDX 10 -SX 12 -YLDX 16 -X 17 -X 18 -AX 20 -X 21 -FX 23 -X 24 -WLX 27 -X 28 - X 29 -X 30 -X 31 -X 32 -X 33 -X 34 -X 35 -X 36 -X 37 -DRVYIHP Formula I in: X1 is selected from H or Y; X2 is selected from S or A; X 10 Selected from T, U, or Y; X 12 Selected from E or K; X 16 Selected from E or S; X 17 Choose from A, E, K, Q, or R; X 18 Selected from A or R; X 20 Selected from D, K, Q, or R; X 21 Choose from D or E; X 23 Selected from I or V; X 24 Choose from A, C, D, or Q; X 27 Selected from E, L, or M; X 28 Choose from A, D, or N; X 29 Selected from G or T; X 30 Selected from K, L, G, R, or not present; X 31 Selected from G, R, or not present; X 32 Selected from N or not present; X 33 Selected from R or does not exist; X 34 Selected from N or not present; X 35 Selected from N or not present; X 36 Selected from I or not present; X 37 Selected from A or does not exist.

2. The polypeptide or its pharmaceutically acceptable salt, solvate, hydrate, complex, chelate, derivative, variant, or analogue as described in claim 1, characterized in that, The polypeptide has the following characteristics: (I) An amino acid sequence as shown in any one of SEQ ID No. 1 to 10; (II) An amino acid sequence that is functionally identical to the amino acid sequence described in (I) obtained by substituting, deleting, or adding one, two, or three amino acids to the amino acid sequence described in (I); or (III) An amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology with the amino acid sequence described in (I) or (II).

3. The polypeptide or its pharmaceutically acceptable salt, solvate, hydrate, complex, chelate, derivative, variant, or analogue as described in claim 1 or 2, characterized in that, The peptide chain ends of the polypeptide are either free or chemically modified; Preferably, the chemical modification includes chemical modification at the amino terminus or chemical modification at the carboxyl terminus; Preferably, the chemical modification of the amino terminus includes acylation, sulfonation, alkylation, and PEG modification; the chemical modification of the carboxyl terminus includes amidation, sulfonation, and PEG modification. Preferably, the chemical modification of the amino terminus is acetylation, benzoylation, or sulfonation of the amino group; the alkylation of the amino terminus is C1-6 alkylation or aralkylation; and the chemical modification of the carboxyl terminus is that the OH group in the carboxyl group is replaced by NH2 or by sulfonamide, or the OH group in the carboxyl group is connected to a functionalized PEG molecule.

4. A fusion protein, characterized in that, The fusion protein comprises, as an active portion, a polypeptide as described in any one of claims 1 to 3 or a pharmaceutically acceptable salt, solvate, hydrate, complex, chelate, derivative, variant or analog thereof, and a long-acting portion, wherein the fusion protein is formed by linking the two portions described above. Preferably, the extended-acting portion is selected from the Fc region of immunoglobulins, albumin, antibodies, and / or antigen-binding fragments.

5. The fusion protein as described in claim 4, characterized in that, The fusion protein has the following characteristics: (I) An amino acid sequence as shown in any one of SEQ ID No. 11 to 20; (II) An amino acid sequence that is functionally identical to the amino acid sequence described in (I) obtained by substituting, deleting, or adding one, two, or three amino acids to the amino acid sequence described in (I); or (III) An amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology with the amino acid sequence described in (I) or (II).

6. A drug, characterized in that, The drug comprises any one of (I) or (II) below, and at least one of pharmaceutically acceptable excipients, excipients, carriers and solvents; (I) The polypeptide as described in any one of claims 1-3, or a pharmaceutically acceptable salt, solvate, hydrate, complex, chelate, derivative, variant, or analog thereof; (II) The fusion protein as described in claim 4 or 5 Preferably, it may also contain other active ingredients.

7. A method for lowering blood sugar, lowering blood lipids, and / or losing weight, or a method for preventing, treating, and / or improving diabetes, obesity, obesity-related diseases, or metabolic abnormalities, characterized in that, Administer, take or use any of the following: (I) The polypeptide as described in any one of claims 1-3, or a pharmaceutically acceptable salt, solvate, hydrate, complex, chelate, derivative, variant, or analog thereof; (II) The fusion protein as described in claim 4 or 5; or (III) The drug as described in claim 6.

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