WO2024027553A1

WO2024027553A1 - Bifunctional fusion protein and use thereof

Info

Publication number: WO2024027553A1
Application number: PCT/CN2023/109575
Authority: WO
Inventors: 龚大为; 黄俭; 徐军; 朱大龙
Original assignee: 无锡市华盛康肽生物技术有限公司
Priority date: 2022-08-03
Filing date: 2023-07-27
Publication date: 2024-02-08
Also published as: CN117510646A

Abstract

The present invention relates to the field of biomedicine, and particularly to a bifunctional fusion protein and the use thereof. The bifunctional fusion protein comprises an Fc fragment, wherein one end of the Fc fragment is connected with a GLP1 polypeptide or an analog thereof, the other end of the Fc fragment is connected with a ligand of an APJ receptor, the ligand of the APJ receptor is an Elabela polypeptide fragment, and the GLP1 polypeptide or the analog thereof and the ligand of the APJ receptor are both connected with the Fc fragment via a connecting peptide. It is confirmed that in animal models of disease, the bifunctional fusion protein can effectively reduce the blood glucose level and weight of diabetic mice, and can improve the heart function of rats with heart failure. Therefore, the bifunctional fusion protein has significant anti-diabetic and anti-heart failure effects, and can treat diabetic cardiopathy while controlling blood glucose.

Description

A bifunctional fusion protein and its use

Technical field

The present invention relates to the field of biomedicine, and in particular to a bifunctional fusion protein and its use.

Background technique

The prevalence of diabetes is gradually increasing due to an aging population and changing lifestyles. Diabetes is divided into type 1 (T1DM) and type 2 (T2DM) diabetes, and most patients have T2DM. According to recent estimates from the International Diabetes Federation, the number of adults living with diabetes exceeded 537 million in 2021 and is expected to reach 783 million by 2045 (1). .The diabetes epidemic places a severe economic burden on health systems and society. The global cost of diabetes treatment will be US$966 billion in 2021 and is expected to reach more than US$1 trillion by 2045. Diabetes greatly increases the risk of diabetic heart disease. Diabetic heart disease includes coronary artery disease (CAD), cardiac autonomic neuropathy (CAN), and diabetic cardiomyopathy (DCM), which are characterized by molecular, structural, and functional changes in the myocardium and cardiac dysfunction that ultimately lead to cardiac dysfunction. Failure (HF). In patients with DM, the prevalence of HF ranges from 9% to 22%, which is 4 times higher than in the general population, and is even higher in patients with DM ≥60 years of age. In patients with diabetes, cardiac dysfunction is often clinically asymptomatic and is often not detected until late in the disease. Even among asymptomatic, normotensive patients with well-controlled diabetes, 50% are thought to exhibit some degree of cardiac dysfunction. It is generally accepted that one of the hallmarks of the diabetic heart is left ventricular (LV) diastolic dysfunction, which also includes cardiac fibrosis, cardiac hypertrophy, and coronary microvascular perfusion impairment. The mechanisms leading to diabetic heart disease are multifactorial, including metabolic disorders, inflammation, ROS/oxidative stress,

On the other hand, HF is a major risk factor for the pathogenesis of new-onset T2DM. In a cohort study including HF with reduced ejection fraction (HFrEF) and HF with preserved ejection fraction (HFpEF), the incidence of diabetes ranged from 10% to 47%. Additionally, up to 60% of HF patients have insulin resistance, and HF patients The incidence of diabetes among middle-aged and elderly people is more than twice that of people of similar age. Risk factors for developing diabetes in patients with HF include elevated body mass index and waist circumference, smoking history, and blood glucose or HbA1c Elevated systolic blood pressure, longer duration of HF, diuretic therapy, and degree of heart failure. Mortality in HF patients with diabetic cardiomyopathy is higher than in patients with diabetes or HF alone. Therefore, patients with diabetic heart disease are a unique and large patient group that require specialized treatment strategies and medical care.

Glucagon-like peptides, including glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2), both derived from glucagon Proglucagon is composed of 158 amino acids and can be cleaved into different peptide chains. Because GLP-1 has the pharmacological effects of promoting insulin secretion, protecting pancreatic beta cells, inhibiting glucagon secretion, inhibiting gastric emptying, and reducing appetite, it can be used clinically for the treatment of type 2 diabetes and obesity. However, GLP1 cannot be used directly as a drug because it is cleaved by DPP4 and rapidly degraded in the body. The half-life of native GLP-1 is very short, approximately 1-2 minutes, and is caused by two reasons: (a) degradation by dipeptidyl peptidase-4 (DPP-4) and (b) renal elimination. Intact as well as degraded and inactivated GLP-1 metabolites are rapidly cleared from the circulation by the kidneys. Pharmaceutical companies make GLP1 analogs by changing the amino acid sequence and structure of GLP1. This analog retains the physiological function of GLP1, but has a significantly extended half-life and can be used for clinical purposes. There are currently several GLP1 analogues on the market. GLP1 analogues act against diabetes by activating GLP1 receptors.

Apelin receptor (APJ) is a G protein-coupled receptor. Its endogenous ligands include Elabela (ELA) and Apelin. Elabela is a new APJ ligand peptide discovered in recent years. It has been widely used in cardiovascular diseases. Plays a key role in development and cardiovascular disease. As a new hormone peptide, Elabela exhibits cardioprotective activity by activating APJ receptors. Elabela replacement therapy may become an effective method for the treatment of heart disease. In addition, Elabela can promote the differentiation of human embryonic stem cells, enhance myocardial contractility, promote the formation of cardiomyocytes, and increase cardiac contractility. Elabela is widely expressed in kidney and vascular endothelial cells and significantly improves various pulmonary hypertension models, suggesting that Elabela replacement therapy may have certain prospects in the treatment of hypertension. Elabela is a short peptide of 32 amino acids that can be degraded into ELA21, ELA14, and ELA11. These degradation fragments are physiologically active. The in vivo half-life of ELA 21 is 13 minutes, but the in vivo half-life after fusion with IgG-Fc is 44 hours. The fusion protein has significant anti-heart failure, renal protection, and anti-inflammatory effects.

Contents of the invention

There is currently no molecule that can simultaneously activate GLP1 receptors and APJ receptors for the treatment of diabetic heart disease. In view of the above shortcomings of the prior art, the purpose of the present invention is to provide a bifunctional fusion protein and its use to solve the problems in the prior art.

In order to achieve the above objectives and other related objectives, the present invention provides a bifunctional fusion protein, which includes an Fc fragment. One end of the Fc fragment is connected to a GLP1 polypeptide or an analog thereof, and the other end is connected to a ligand of an APJ receptor. The ligand of the APJ receptor is an Elabela polypeptide fragment. The bifunctional fusion protein can activate GLP1R and APJ receptors, and the GLP1-Elabela bifunctional fusion protein was named ACF210.

In certain embodiments of the invention, the GLP1 polypeptide or analog thereof and the ligand of the APJ receptor are connected to the Fc fragment through a linker.

In certain embodiments of the invention, the Fc fragment is selected from the group consisting of Fc fragments of IgA, IgD, IgE, IgG or IgM.

The GLP1 analog is selected from benaglutide, exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide or semaglutide.

In certain embodiments of the invention, the Elabela polypeptide fragment is selected from Elabela-21, Elabela-14, or Elabela-11.

The bifunctional fusion protein also includes a signal peptide connected to a GLP1 polypeptide or an analog thereof.

The present invention also provides a nucleic acid molecule encoding the bifunctional fusion protein.

The present invention also provides an expression vector containing the above-mentioned nucleic acid molecule.

The present invention also provides a host cell containing the above-mentioned expression vector.

The invention also provides a method for preparing a bifunctional fusion protein, which method includes the following steps:

a) Under expression conditions, culture the host cells as described above to express the bifunctional fusion protein;

b) Isolate and purify the bifunctional fusion protein described in step a).

The present invention also provides a pharmaceutical composition, which contains an effective amount of the above-mentioned bifunctional fusion protein and one or more pharmaceutically acceptable carriers or excipients.

The present invention also provides the use of the above-mentioned bifunctional fusion protein and pharmaceutical composition in preparing drugs for preventing and treating diabetes, obesity, renal function damage and/or cardiovascular disease.

The present invention also provides the use of the bifunctional fusion protein or pharmaceutical composition in preparing products with any one or more of the following functions:

1) Activate GLP1 receptor;

2) Activate APJ receptors;

3) Lower blood sugar;

4) Reduce urine output;

5) Reduce urinary glucose;

6) Reduce urinary protein;

7) Reduce food intake;

8) Reduce body weight;

9) Improve cardiac ejection fraction or cardiac fractional shortening;

10) Reduce the BNP or TnT content in the blood.

As mentioned above, the bifunctional fusion protein of the present invention and its use have the following beneficial effects: ACF210 fusion protein has been confirmed in animal disease models to effectively reduce the blood sugar level and body weight of diabetic mice, and can improve the heart function of heart failure rats. Function, reduce the size of cardiac infarction, so it has significant anti-diabetic and anti-heart failure effects, and can treat diabetic heart disease while controlling blood sugar.

Description of the drawings

Figure 1 shows a schematic structural diagram of the ACF210 fusion protein of the present invention.

Figure 2 shows the dose-effect response curves for ACF210, Fc-ELA21 and Trulicity.

Figure 3 shows the fasting blood glucose data after administration of ACF210 to the db/db mouse model of the present invention. Vehicle is the solvent control, and ACF210 is the ELA21-Fc-GLP1 fusion protein. The data is the mean ± s.e.

Figure 4 shows the urinary volume, urinary glucose and protein excretion results of diabetic mice after administration of ACF210 of the present invention.

Figure 5 shows the effect of ACF210 of the present invention on food intake and body weight of diabetic mice.

Figure 6 shows the effect of ACF210 of the present invention on cardiac ejection fraction (EF) and cardiac fractional shortening (FS) in rat myocardial infarction model.

Figure 7 shows the effect of ACF210 of the present invention on serum markers of cardiac injury and heart failure in rat myocardial infarction model.

Detailed ways

The invention provides a bifunctional fusion protein. The bifunctional fusion protein includes an Fc fragment. One end of the Fc fragment is connected to a GLP1 polypeptide or an analog thereof, and the other end is connected to a ligand of an APJ receptor. The ligand of the APJ receptor is connected to the Fc fragment. The body is an Elabela polypeptide fragment. The bifunctional fusion protein can activate GLP1R and APJ receptors. In the present invention, the GLP1-Elabela bifunctional fusion protein in which the Fc fragment is connected to one end of the GLP1 polypeptide or its analogue and the other end of the Fc fragment is connected to the Elabela polypeptide fragment is named for ACF210.

In certain embodiments of the present invention, the N-terminus of the Fc fragment is connected to a GLP1 polypeptide or an analog thereof, and the C-terminus is connected to a ligand of the APJ receptor.

In certain embodiments of the invention, the GLP1 polypeptide or analog thereof and the ligand of the APJ receptor are connected to the Fc fragment through a linker. The term "linker peptide (linker)" refers to a short peptide that can connect two polypeptide sequences, generally a peptide with a length of 2-30 amino acids. The linking peptide can be any linking peptide in the art that is suitable for forming polypeptides. For example, the linking peptide can be (G4S)3 linker. In one embodiment, the GLP1 polypeptide or analog thereof is connected to the Fc fragment through a first linker peptide (linker1), and the amino acid sequence of the first linker peptide is as shown in SEQ ID NO.1: GGGGGGGSGGGGSGGGGSA in another implementation In this method, the ligand of the APJ receptor is connected to the Fc fragment through a second linker peptide (linker2), and the amino acid sequence of the second linker peptide is as shown in SEQ ID NO. 2: GGGGGSGGGGSGGGGS.

In certain embodiments of the invention, the Fc fragment is selected from the group consisting of Fc fragments of IgA, IgD, IgE, IgG or IgM. The IgG is selected from IgG1, IgG2, IgG3 or IgG4. In one embodiment, the Fc fragment is selected from the Fc fragment of IgG4, and the amino acid sequence of the Fc fragment is shown in SEQ ID NO.3:

In one embodiment, the GLP1 analog is selected from dulaglutide, and the amino acid sequence is as shown in SEQ ID NO. 4: HGEGTFTSDVSSYLEEQAAKEFIAWLVK.

The human Elabela gene is located on chromosome 4, GRCh38.p12, and contains 3 exons. The precursor protein encoded by this gene is composed of 54 amino acids, which is then cleaved by the Golgi apparatus to form a mature polypeptide of 32 amino acids. Elabela protein contains two double arginine sequences, which can be recognized and cleaved by furin to cleave the Elabela-32 protein into Elabela polypeptide fragments of different sizes. Polypeptide fragments of different lengths are named according to the number of amino acids: Elabela-32 (Ela32), Elabela-21 (Ela21), Elabela-14 (Ela14), Elabela-11 (Ela11).

In certain embodiments of the invention, the Elabela polypeptide fragment is selected from Elabela-21, Elabela-14 or Elabela-11. In one embodiment, the Elabela polypeptide fragment is selected from Elabela-21, and the amino acid sequence is as shown in SEQ ID NO. 5: LRKHNCLQRRCMPLHSRVPFP.

As used herein, the term "signal peptide" refers to a peptide present at one end of a protein that targets the protein to the secretory pathway. Following translation of the mRNA, the signal peptide is cleaved into the endoplasmic reticulum following translocation. Signal peptides are also called signal sequences, leader sequences, or leader peptides. Typically, signal peptides are shorter (eg, 5-30, 5-25, 5-20, 5-15, or 5-10 amino acids long) peptides. The signal peptide can be present at the N-terminus of the protein. Incorporation of a signal peptide onto a fusion protein can promote secretion and/or production of the protein.

Suitable signal peptides for use in the present invention may be heterogeneous sequences derived from different eukaryotic and prokaryotic proteins, in particular secreted proteins. In some embodiments, a suitable signal peptide is a leucine-rich sequence. Suitable signal peptides may be derived from human growth hormone (hGH), immunoglobulin heavy chain, serum albumin preproprotein, Igκ light chain precursor, azurocidin preproprotein, cystatin S prepro body, trypsinogen 2 precursor, potassium channel blocker, α-conotoxin lp1.3, α-conotoxin, α-galactosidase, cellulose, aspartic acid protease Nepenthes ( nepenthesin)-1, acid chitinase, K28 prepro-toxin, killer toxin zygocin precursor, and cholera toxin.

In one embodiment, the sequence of the signal peptide is shown in SEQ ID NO. 6: MGWSCIILFLVATATGVHS.

In one embodiment, the amino acid sequence of the bifunctional fusion protein is shown in SEQ ID NO.7, and the nucleotide sequence is shown in SEQ ID NO.8.

In animal models of diabetes and heart failure, the bifunctional fusion protein exhibits significant anti-diabetic activity and cardioprotective activity.

The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 8.

Those skilled in the art know that the nucleic acid molecule encoding the above-mentioned fusion protein amino acid sequence can appropriately introduce substitutions, deletions, changes, insertions or additions to provide a homolog of the nucleic acid molecule.

In the present invention, the term "expression vector" refers to a conventional expression vector in the art that contains appropriate regulatory sequences, such as promoters, terminators, enhancers, etc., and the expression vector can be a virus or a plasmid. The expression vector is, for example, pcDNA3.1(+), pDHFR or pTT5.

In the present invention, the term "host cell" refers to various conventional host cells in this field, as long as the vector can stably replicate itself and the nucleic acid molecules carried can be effectively expressed. The host cell is selected from prokaryotic expression cells or eukaryotic expression cells. The host cell is, for example, selected from: COS, CHO, CHO-K1, NSO, sf9, sf21, DH5α, BL21(DE3), TG1, BL21(DE3), 293F cells or 293E cells.

b) Isolate and purify the bifunctional fusion protein described in step a).

In the present invention, the term "effective amount" refers to the amount or dosage that produces the desired effect in the treated individual after the pharmaceutical composition of the present invention is administered to a patient, and the expected effect includes the improvement of the individual's condition.

"Pharmaceutically acceptable" means that the molecular entities or pharmaceutical compositions do not produce adverse, allergic or other adverse reactions when properly administered to animals or humans.

The "pharmaceutically acceptable carrier or excipient" should be compatible with the active ingredient, that is, it can be blended with it without significantly reducing the effect of the drug under normal circumstances. Specific examples of substances that can be used as pharmaceutically acceptable carriers or excipients are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, Ethylcellulose and methylcellulose; tragacanth powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive Oils, corn oil, and cocoa butter; polyols, such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; colorants; Flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; and phosphate buffers, etc. These substances are used as needed to aid the stability of the formulation or to help enhance the activity or its bioavailability or to produce an acceptable mouthfeel or odor in the case of oral administration.

In the present invention, unless otherwise specified, the dosage form of the pharmaceutical composition is not particularly limited. It can be made into dosage forms such as injections, oral liquids, tablets, capsules, dropping pills, sprays, etc., and can be prepared by conventional methods. The choice of drug dosage form should match the mode of administration.

The cardiovascular disease is selected from heart failure, cardiomyopathy, coronary atherosclerotic heart disease (coronary heart disease), and arrhythmia. The heart failure includes acute or chronic heart failure, heart failure with preserved ejection fraction (HFpEF), and heart failure with reduced ejection fraction (HFrEF).

In a preferred embodiment, the cardiovascular disease is diabetic heart disease, which is a heart disease complicated or associated with diabetes. The diabetic heart disease includes coronary atherosclerotic heart disease, diabetic cardiomyopathy, arrhythmia, and diabetic heart failure.

1) Activate GLP1 receptor;

2) Activate APJ receptors;

3) Lower blood sugar;

4) Reduce urine output;

5) Reduce urinary glucose;

6) Reduce urinary protein;

7) Reduce food intake;

8) Reduce body weight;

9) Improve cardiac ejection fraction or cardiac fractional shortening;

10) Reduce the BNP or TnT content in the blood.

The present invention also provides a method for treating diabetes, obesity, renal impairment and/or cardiovascular disease, the method comprising administering a therapeutically effective amount of the bifunctional fusion protein or pharmaceutical composition to a subject.

"Subjects" include but are not limited to animals, preferably mammals; the mammals are preferably rodents, artiodactyls, perissodactyls, lagomorphs, primates, etc. The mammals include, for example, humans, non-human primates (such as monkeys), mice, pigs, cattle, goats, rabbits, rats, guinea pigs, hamsters, horses, monkeys, sheep or other non-human mammals; non-human mammals; Mammals include, for example, non-mammalian vertebrates, such as birds (eg, chickens or ducks) or fish, as well as non-mammalian invertebrates.

"Treatment" or "therapy" for a condition includes preventing or alleviating a condition, reducing the rate at which a condition occurs or develops, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition , reduce or terminate symptoms associated with a condition, produce a complete or partial reversal of a condition, cure a condition, or a combination of the above.

The "therapeutically effective dose" or "effective dose" in the present invention refers to the dose or concentration of a certain drug that effectively treats related diseases or alleviates disease states. For example, for the bifunctional fusion protein disclosed in the present invention, the therapeutically effective dose is at this dose or concentration. The bifunctional fusion protein can activate GLP1 receptor, activate APJ receptor, lower blood sugar, reduce urine output, Reduce urinary glucose, reduce urinary protein, reduce food intake, reduce body weight, improve cardiac ejection fraction or cardiac shortening fraction, reduce BNP or TnT levels in the blood, or reduce any symptoms related to diabetes, obesity, renal function damage, cardiovascular disease Symptoms or markers associated with preventing or delaying the development of diabetes, obesity, renal impairment, and/or cardiovascular disease, or some combination of the above.

Specifically, when administered to subjects, the dosage varies depending on the age and weight of the patient, the characteristics and severity of the disease, and the route of administration. You can refer to the results of animal experiments and various situations. The total dosage cannot exceed A certain range.

In some embodiments, the methods described herein can be further administered in combination with one or more additional therapies (eg, one or more additional therapeutic agents and/or one or more treatment regimens).

In some embodiments, the administration method is, for example, subcutaneous injection, intravenous injection, oral administration, respiratory inhalation and other routes.

In some embodiments, the frequency of administration may be once a day, once every 2 days, once a week, once a month, or once every few months, etc.

The following describes the embodiments of the present invention through specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. Various details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

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 specific embodiments; it should also be understood that the terms used in the embodiments of the present invention are for describing specific specific embodiments, They are not intended to limit the scope of the invention; in the specification and claims of the invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

When the examples give numerical ranges, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and materials used in the embodiments, those skilled in the art can also use methods, equipment, and materials described in the embodiments of the present invention based on their understanding of the prior art and the description of the present invention. Any methods, equipment and materials similar or equivalent to those in the prior art may be used to implement the present invention.

Example 1 Design and preparation of GLP1-Elabela bifunctional fusion protein ACF210

1.Design of ACF210

Figure 1 is a schematic structural diagram of ACF210. As shown in the figure, the Fc fragment of immunoglobulin IgG is the structural skeleton. The GLP1 polypeptide analog and the Elabela (ELA) polypeptide fragment are located at the N-terminus and C-terminus of Fc respectively. GLP1 or A linker is designed between ELA and Fc skeleton, and the secretion signal peptide is located at the N-terminus of the entire fusion protein. The composition of the entire ACF210 fusion protein, starting from the N-terminus, is the secretion signal peptide, GLP1 analog, the first linking peptide (GGGGGGGSGGGGSGGGGSA), the IgG4-Fc fragment, the second linking peptide (GGGGGSGGGGSGGGGS), and the ELA21 peptide fragment, among which the ELA21 peptide The fragment is a polypeptide of the Elabela family. The amino acid sequence of the fusion protein ACF210 is shown in SEQ ID NO. 7. Different underlines represent the secretion signal peptide, GLP1 analog, first linking peptide, IgG4-Fc fragment, second linking peptide, and ELA21 peptide in the fusion protein. The amino acid sequence of the fragment; the nucleotide sequence of the fusion protein ACF210 is shown in SEQ ID NO. 8.

2. Preparation of fusion protein ACF210

The preparation of fusion protein ACF210 is an existing technology. In this application, the fusion protein ACF210 is entrusted to WuXi Biologics, a third-party organization, to complete. The example preparation process is as follows: The DNA sequence of the ACF210 fusion protein molecule is cloned into pcDNA ^TM 3.1 ( +) (V79020, ThermoFisher) mammalian gene expression plasmid, the accuracy of the sequence is verified by sequencing after cloning. After the sequence is correct, large-scale expression plasmid preparation is performed to obtain sufficient plasmid DNA. The plasmid was transfected into CHO-K1 cells with polyethylenimine (PEI), and the fusion protein was expressed in large quantities in the CHO-K1 cells. The recombinant protein was transiently expressed after expanding the culture to 3L. The culture supernatant was collected on the 11th day of culture, and the protein concentration in the supernatant was 1.2g/L. The supernatant was clarified by centrifugation at 10,000 × g for 40 min and sterile filtered through 0.45 μm and 0.22 μm filters. The filtrate was purified using Protein A affinity chromatography (MabSelect SuRe resin, GE Healthcare Life Science) and SEC-HPLC. The SEC eluate was buffer exchanged with 50mM His, 125mM Arg, pH5.5 buffer to obtain the final product, with an assay purity of over 98%. The obtained fusion proteins are used for in vitro and in vivo experiments or treatments.

Example 2 In vitro GLP-1R receptor and APJ receptor activation experiments verify that ACF210 fusion protein has GLP1 and Elabela dual function

In order to verify that the ACF210 fusion protein has the dual activity of activating GLP1R and APJR, the in vitro biological activities of ACF210 (GLP1-Fc-ELA21) and single-drug fusion proteins Fc-ELA21 and Trulicity (GLP1-Fc) were first compared in vitro. Trulicity is a fusion protein drug already on the market from Eli Lilly Pharmaceutical Company. It is a fusion protein of a GLP-1 peptide analog and an Fc fragment. Trulicity is an agonist of GLP1R. Trulicity causes the activation of adenylyl cyclase by binding to the GLP1R receptor. The activation of this enzyme increases intracellular cAMP content. Fc-ELA21 is also a fusion protein, which is a fusion protein of ELA21 polypeptide and Fc fragment. ELA21 polypeptide belongs to the Elabela family and is an agonist of APJ receptors. After activation of APJ receptors, it can inhibit the activity of adenylate cyclase, thereby reducing cAMP levels. Therefore, the activation of different receptors can be analyzed by detecting intracellular cAMP concentration. status.

The ACTOne endpoint assay (BD Biosciences) method is a commonly used detection method to detect intracellular cAMP content. HEK-293 cells, the tool cells used for detection, stably express cyclic nucleotide gated (CNG) channel proteins and simultaneously express GLP1R receptors or APJ receptors. Under the action of different fusion proteins, HEK-293 cells The cAMP content in the body will change accordingly.

HEK293 cells were first cultured in modified Eagle medium (DMEM) containing 10% heat-inactivated fetal calf serum and 1 μg/mL puromycin Dulbecco at 37°C, 5% CO ₂ . Cells were harvested by mild trypsinization and seeded at a density of 50,000 cells per well in poly-D-lysine-coated 96-well cell culture plates (BD Biocoating) and cultured overnight. The next day, add BD ACTOne Membrane Potential Dye to each well and store the plate at room temperature for 2 hours. During the experiment, cells were first incubated with DPBS or different concentrations of ligands for 60 minutes. Dose-response curves were drawn using the ratio of Ft/F0 (Ft represents the fluorescence intensity 60 minutes after the addition of the agonist, F0 represents the fluorescence intensity before the addition of the compound). Then the EC50 value, which is the concentration value that causes 50% of the maximum effect, is calculated through the dose response curve to represent the receptor activating activity of GLP1-Fc, Fc-ELA and ACF210. The dose-effect response curves of various reagents are shown in Figure 2 . The results show that the activation of APJ receptors by ACF210 is similar to that of the positive control Fc-ELA21, and the activity of ACF210 in activating GLP1R is equivalent to that of the positive control Trulicity. The specific values are shown in Table 1. It can be seen that Fc-ELA can only activate APJ receptors, and Trulicity can only Activates GLP1 receptors, and ACF210 has dual activity, activating both GLP1R and APJ receptors.

Table 1 The activity of ACF210 fusion protein in activating GLP-1R receptor and APJ receptor

Example 3 In vivo pharmacodynamic verification of ACF210 in db/db mouse model

The animal experimental protocol was approved by the IACUC committee. db/db mice are commonly used animal models in diabetes research and were therefore used to conduct in vivo pharmacodynamic studies of ACF210. The animal experiment steps are as follows:

(1) db/db mice can start the experiment after they are 5 weeks old and have adapted to the environment for one week, and the average non-fasting blood sugar exceeds 16.7mmol/L.

(2) Day-6 to Day-5: 20 animals were placed in the metabolic cage. The mice could move freely in the cage to obtain food and water. 24-hour urine was collected to detect urine sugar and urine microalbumin.

(3) Day-3: Determine the body weight of 20 animals, and measure the blood glucose of the animals after fasting for 6 hours (when the animals are fasting, change cages at the same time), collect 110 μL of blood from the mandible, and collect plasma (EDTA anticoagulation, 7000rpm, 4℃, 10min) , store at -80°C, and use a blood glucose meter to detect blood sugar (blood sugar will be tested twice in a row. If the difference between the two test results is greater than 10%, a third test will be performed, and the final result will be the average value of the tests).

(4) Day-1: The animals were randomly divided into 4 groups according to body weight, fasting blood sugar, urine sugar, and urine microalbumin, with 10 animals in each group. Animals with relatively high or low values will not participate in subsequent experiments.

(5) Administration: From Day 1 to Day 29, the drug is administered every other day. The first administration is on Day 1 and the last administration is on Day 29. Subcutaneous administration. The administration volume is 5 mL/kg. The administration location is from the neck From the skin on the back to the skin on the buttocks, the locations of two consecutive administrations are different.

(6) Day 1 and Day 29: Animals change cages in the morning and fast. After fasting for 6 hours, the blood glucose of the animals 0min before administration, and blood glucose at 30min, 1hr, 2hr, 3hr and 6hr after administration are measured with a blood glucose meter. blood sugar.

(7) Day1,8,16,24: Detect the animal’s weight and food consumption.

(8) Day 8, 16, 24: Animals fast in the morning and change cages. After fasting for 6 hours, blood glucose will be measured with a blood glucose meter (all blood glucose testing environments will be conducted under the same conditions).

(9) Day 26 to Day 27: After weighing and measuring food consumption, the animals are placed in metabolic cages. The mice can move freely in the cages to obtain food and water. 24-hour urine collection is performed to detect urine sugar and trace amounts of urine. protein. No obvious toxic or side effects of the drug were found during the experiment. No animal deaths occurred.

The experimental results are as follows:

1. ACF210 reduces blood sugar in the in vivo disease animal model db/db mice.

As shown in Figure 3, the initial average blood glucose concentration of all experimental animals was 440 mg/dL. During the 29 days of experimental administration, the blood glucose of mice treated with ACF210 was relatively maintained at the initial level of the experiment, which was better than that of the untreated mice. There was a significant difference in the blood glucose levels of the control mice, indicating that ACF210 has a significant anti-hyperglycemic effect in the db/db mouse model.

2.ACF210 improves renal function in diabetic mice

As shown in Figure 4, ACF210 has the effect of reducing urine output, urinary glucose, and urinary protein in db/db mice. This shows that while ACF210 maintains the glomerular filtration rate, it also significantly reduces urine sugar and protein, and the glomerular filtration function is significantly protected.

3.ACF210 suppresses appetite and reduces body weight.

Under normal circumstances, GLP1 can act on the gastrointestinal tract to control energy intake, prolong the retention time of food in the stomach, suppress appetite, and thereby reduce weight.

As shown in Figure 5, AFC210 significantly inhibited food intake (left) and reduced body weight (right). These effects indicate that the role of GLP1 in ACF210 is fully exerted.

Example 4 In vivo pharmacodynamic verification of ACF210 in myocardial infarction rat model

A myocardial infarction model was created by ligation of the left anterior descending coronary artery. The specific steps were as follows: 6-week-old SD rats were subjected to ligation of the left anterior descending coronary artery under anesthesia to create a myocardial infarction model. The rat was placed on the operating table and anesthetized with isoflurane (2% inhalation). After the chest was opened, the heart was taken out of the chest, the left anterior descending LAD coronary artery was found, ligated with 5/0 silk suture, and then put back into the heart. The chest cavity is closed after removing air and blood. The success of the operation was confirmed by the rising T wave inversion of the ST segment of the electrocardiogram.

1. Detection of cardiac output fraction and cardiac contraction fraction

One week after surgery, rats were randomly divided into ACF210 (1 mg/kg/day) and vehicle groups for treatment. Echocardiographic Ultrasound Measurements Animals underwent echocardiographic measurements before treatment and weekly after treatment. Direct measurement of left ventricular internal diameter diastole (LVIDd) and systole using M lead intraperitoneal diameters (LDIDs) and left ventricular ejection fraction (LVEF). The systolic fraction (FS) is calculated by the following formula: FS=(LVIDd-LVIDs)/LVIDd×100%.

The results are shown in Figure 6. The LVEF and FS index of the control animals decreased significantly after surgery and remained at low levels in the following weeks, indicating heart failure in the animals. The cardiac functions of rats treated with ACF210 were maintained at close to normal levels, indicating that ACF210 can reduce the degree of heart failure in the rat myocardial infarction model and that ACF210 has a significant anti-heart failure effect.

2. Detection of cardiac and heart failure markers

One week after surgery, rats were randomly divided into ACF210 (1 mg/kg/day) and vehicle groups for treatment. Rat serum was collected before ACF210 or vehicle treatment and weekly during treatment. The heart and heart failure marker BNP (rat BNP ELISA kit, MSKbio, China) and the damage marker TnT (Rat Tn-T ELISA kt, mlbio, China) were measured using ELISA.

The results are shown in Figure 7. After myocardial infarction, the BNP and TnT concentrations of rats in the control group gradually increased. Although the concentrations of BNP and TnT in the ACF210 treatment group also increased, the increase was significantly reduced, indicating that ACF210 has the effect of treating heart failure and protecting heart damage. .

The above examples are for illustrating the disclosed embodiments of the present invention and are not to be construed as limitations of the present invention. In addition, various modifications and variations in methods of the invention set forth herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included in the scope of the present invention.

Claims

A bifunctional fusion protein, characterized in that the bifunctional fusion protein includes an Fc fragment. One end of the Fc fragment is connected to a GLP1 polypeptide or an analog thereof, and the other end is connected to a ligand of an APJ receptor. The Fc fragment is The ligand is an Elabela polypeptide fragment.
The bifunctional fusion protein according to claim 1, characterized in that the ligand of the APJ receptor is apelin, and/or the N-terminus of the Fc fragment is connected with GLP1 polypeptide or its analogue, and the C-terminus is connected with Ligands for APJ receptors.
The bifunctional fusion protein according to claim 1, characterized in that, the GLP1 polypeptide or its analog and the ligand of the APJ receptor are connected to the Fc fragment through a connecting peptide; preferably, the GLP1 polypeptide or its analog is connected through The first connecting peptide is connected to the Fc fragment, and the amino acid sequence of the first connecting peptide is as shown in SEQ ID NO. 1; and/or the ligand of the APJ receptor is connected to the Fc fragment through the second connecting peptide, and the second connecting peptide is connected to the Fc fragment. The amino acid sequence of the peptide is shown in SEQ ID NO.2.
The bifunctional fusion protein according to claim 1, characterized in that the Fc fragment is selected from the Fc fragment of IgA, IgD, IgE, IgG or IgM; preferably, the Fc fragment is selected from the Fc fragment of IgG4; preferably , the amino acid sequence of the Fc fragment is shown in SEQ ID NO.3.
The bifunctional fusion protein according to claim 1, characterized in that the GLP1 analog is selected from the group consisting of benaglutide, exenatide, liraglutide, lixisenatide, albiglutide, duraglutide Lutide or semaglutide; preferably, the GLP1 analog is selected from dulaglutide, and the amino acid sequence is as shown in SEQ ID NO. 4.
The bifunctional fusion protein according to claim 1, wherein the Elabela polypeptide fragment is selected from Elabela-21, Elabela-14 or Elabela-11; preferably, the Elabela polypeptide fragment is selected from Elabela-21, and the amino acid The sequence is shown as SEQ ID NO.5.
The bifunctional fusion protein according to claim 1, characterized in that the bifunctional fusion protein also includes a signal peptide, and the signal peptide is connected to a GLP1 polypeptide or an analog thereof; preferably, the sequence of the signal peptide is as follows SEQ ID NO.6 is shown.
The bifunctional fusion protein according to claim 1, wherein the amino acid sequence of the bifunctional fusion protein is shown in SEQ ID NO. 7.
A nucleic acid molecule, characterized in that the nucleic acid molecule encodes the bifunctional fusion protein of any one of claims 1-8.
The nucleic acid molecule according to claim 9, wherein the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO. 8.
An expression vector, characterized in that the expression vector contains the nucleic acid molecule of claim 9 or 10.
A host cell, characterized in that the host cell contains the expression vector of claim 11.
The preparation method of the bifunctional fusion protein according to any one of claims 1 to 8, characterized in that the preparation method includes the following steps:

a) Under expression conditions, culture the host cell as claimed in claim 12 to express the bifunctional fusion protein;

b) Isolate and purify the bifunctional fusion protein described in step a).
A pharmaceutical composition comprising the bifunctional fusion protein according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier or excipient.
The use of the bifunctional fusion protein according to any one of claims 1 to 8 and the pharmaceutical composition according to claim 14 in the preparation of drugs for preventing and treating diabetes, obesity, renal dysfunction and/or cardiovascular disease.
The use according to claim 15, characterized in that the cardiovascular disease is selected from the group consisting of heart failure, coronary heart disease, cardiomyopathy and arrhythmia.
The use of the bifunctional fusion protein of any one of claims 1 to 8 or the pharmaceutical composition of claim 14 in the preparation of products with any one or more of the following functions:

1) Activate GLP1 receptor;

2) Activate APJ receptors;

3) Lower blood sugar;

4) Reduce urine output;

5) Reduce urinary glucose;

6) Reduce urinary protein;

7) Reduce food intake;

8) Reduce body weight;

9) Improve cardiac ejection fraction or cardiac fractional shortening;

10) Reduce the BNP or TnT content in the blood.