Serine protease selectively degrading mucin and uses thereof

Abstract

The present application relates to a serine protease that selectively degrades mucin and uses thereof. The serine protease is serine protease EatA, and the protein contains a passenger domain with serine protease EatA activity, and the amino acid sequence of the passenger domain is at least 90% identical to SEQ ID NO: 2, preferably the amino acid sequence shown in SEQ ID NO: 2. The serine protease EatA can selectively degrade mucin in disease-related mucus and is highly stable over a long period of time.

Description

[Technical Field]

[0001] This application relates to the field of biomedical technology, and in particular to serine proteases that selectively degrade mucins and their use in degrading mucins. [Background technology]

[0002] Mucins are highly glycosylated macromolecular proteins expressed by secretory cells in the human body, which are generally distributed in epithelial and glandular tissues of the digestive and respiratory tracts, lungs, kidneys, ovaries, breasts, and pancreas. Under normal physiological conditions, mucins lubricate and protect the surfaces of human tissues. Mucin expression and composition are strictly regulated. However, in some pathological conditions, such as cancer and inflammation, uncontrolled changes in mucin expression and composition occur, resulting in the accumulation of these mucins, which can have a significant adverse effect on patients. Abnormal mucin expression can result in serious clinical and pathological features in both cancer and non-cancerous diseases, such as pseudomyxoma peritonei, chronic obstructive pulmonary disease, colon mucinous carcinoma, and ovarian mucinous carcinoma.

[0003] Pseudomyxoma peritonei is a type of cancer whose etiology remains unclear, but which is often clinically diagnosed as originating from organs and tissues such as the ovaries, appendix, and intestinal mucous glands. Cancer cells eventually colonize the peritoneum and the peritoneal surfaces of abdominal organs. As the tumor grows, it secretes large amounts of mucins, particularly mucin MUC2, the predominant gel-forming mucin in the small and large intestine. Gel-forming mucins form gel-like, jelly-like polymers at specific pH, salt concentration, and concentrations of mucins from the same family. The core region of the MUC2 molecule is highly glycosylated, making it highly resistant to degradation by common proteases such as proteinase K, pepsin, and trypsin. Covalent interactions at the C- and N-termini of the protein chain favor the formation of MUC2 dimers and polymers, respectively. This gel-like mucin forms and envelops lesions such as cancer cell clusters, gradually progressing to diffuse metastatic lesions and leading to pseudomyxoma peritonei, resulting in ubiquitous mucus distribution within the peritoneum. Because this type of cancer cell is encapsulated in a large amount of gel-like mucin, it is difficult for drugs to reach the target using conventional delivery methods, resulting in poor therapeutic effects. In addition, the large amount of intraperitoneal mucin distributed throughout the patient's body can have adverse effects on the patient, and in severe cases, the large amount of mucin can compress organs, causing intestinal obstruction as a complication and threatening the patient's life.

[0004] Chronic obstructive pulmonary disease (COPD) is a chronic lung disease that restricts airflow in and out of the lungs due to abnormalities in the small airways. It is the third leading cause of death worldwide (2019, WTO). The etiology of COPD includes long-term exposure to harmful gases and particles, as well as individual factors such as childhood events affecting lung development and genetic factors. COPD causes ongoing, progressive respiratory symptoms, including shortness of breath, cough, and phlegm production. The airway narrowing process can also occur. In some cases, the lungs can be destroyed. Patients with COPD experience abnormal mucus secretion, which can block the airways and cause inflammation and swelling of the airway lining. COPD is also sometimes referred to as "emphysema" or "chronic bronchitis." Emphysema generally refers to damage to the small alveoli at the end of the lung's airways. Chronic bronchitis is chronic coughing caused by airway inflammation accompanied by phlegm production.

[0005] The etiology of ovarian mucinous carcinoma is still unknown, but since it is mostly derived from mucinous epithelial cells, both ovaries of the patient are affected, and the ovarian surface is affected. Mucin is present in extracellular spaces, destructive stromal infiltration, nodular growth, ovarian hilar invasion, vascular invasion, signet ring cells, and extensive necrosis, and more than 90% of the cells in ovarian mucinous tumors contain large amounts of mucin.

[0006] Colon mucinous cancer and other mucinous diseases also exhibit pathological characteristics associated with the excessive expression of mucus. Therefore, when treating mucin-related diseases, it is necessary to remove mucin to improve patient outcomes. For example, when treating pseudomyxoma peritonei, laparotomy can remove mucus, but it generally cannot be completely removed. Furthermore, multiple incision surgeries over a long period of time are a burden for patients. Therefore, mucolytic agents have been developed and used to treat mucin-related diseases. These agents decompose and destroy the mucin structure using physical and chemical methods, eliminating the gel-like structure. This allows mucin to be easily aspirated or removed from the body, and also facilitates the delivery of cancer cell-targeted drugs to target cells and exert their anti-cancer effects. In clinical practice, patients undergoing surgery often have extensive tumors in the abdominal cavity and hard mucus, leading to severe organ adhesions, increasing the difficulty of surgery and the incidence of adverse events. Therefore, softening or dissolving mucus before surgery can alleviate organ adhesions to some extent and reduce the risks of surgery.

[0007] This indicates that mucolytic agents play an important role in the treatment of mucin-related diseases, and there is a need to provide novel proteases and preparations thereof that can better degrade disease-related mucins. Summary of the Invention [Problem to be solved by the invention]

[0008] In order to solve the shortcomings of the prior art, the present application provides a serine protease EatA that selectively degrades mucin through the degrading activity of the discovered serine protease EatA. The serine protease EatA can effectively degrade mucin in mucus-related diseases, and when administered to a patient's body, it can exert the mucus-degrading function of the serine protease EatA, making it suitable for use in the treatment of mucus-related diseases. [Means for solving the problem]

[0009] Therefore, in a first aspect of the present application, there is provided a serine protease that selectively degrades mucin, wherein the serine protease is serine protease EatA, the protein comprises a passenger domain having serine protease EatA activity, and the amino acid sequence of the passenger domain is an amino acid sequence having at least 90% identity with SEQ ID NO:2.

[0010] In some specific embodiments, the amino acid sequence of the passenger domain is an amino acid sequence that has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% identity to SEQ ID NO:2.

[0011] In some preferred embodiments, the amino acid sequence of the passenger domain is the amino acid sequence set forth in SEQ ID NO: 2. The sequence set forth in SEQ ID NO: 2 is specifically as follows:

[0012] In the present application, when the passenger domain in the serine protease EatA protein is correctly folded, the serine protease EatA has the activity of the serine protease EatA (i.e., the activity of selectively degrading mucin), and the activity is obtained by the histidine-aspartate-serine catalytic triad. Thus, in some specific embodiments, the serine protease EatA is a serine protease EatA-EatAp having only the passenger domain.

[0013] In some embodiments, the serine protease EatA protein further comprises at least one of an N-terminal signal peptide and a C-terminal β-barrel structure.

[0014] In some embodiments, the amino acid sequence of the serine protease EatA protein having a passenger domain and a C-terminal β-barrel structure has at least 90% identity with SEQ ID NO:3.

[0015] In some specific embodiments, the amino acid sequence of the serine protease EatA protein having a passenger domain and a C-terminal β-barrel structure is an amino acid sequence that has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% identity to SEQ ID NO:3.

[0016] In some preferred embodiments, the amino acid sequence of the serine protease EatA protein having a passenger domain and a C-terminal β-barrel structure is the amino acid sequence set forth in SEQ ID NO: 3. The sequence set forth in SEQ ID NO: 3 is specifically as shown below.

[0017] In some embodiments, the full-length amino acid sequence of the serine protease EatA protein having a passenger domain, an N-terminal signal peptide, and a C-terminal β-barrel structure is an amino acid sequence that has at least 80% identity with SEQ ID NO:1.

[0018] In some specific embodiments, the full-length amino acid sequence of the serine protease EatA protein having the passenger domain, an N-terminal signal peptide, and a C-terminal β-barrel structure is an amino acid sequence that has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% identity to SEQ ID NO:1.

[0019] In some preferred embodiments, the full-length amino acid sequence of the serine protease EatA protein having the passenger domain, an N-terminal signal peptide, and a C-terminal β-barrel structure is an amino acid sequence that has at least 90% identity with SEQ ID NO:1.

[0020] In some more preferred embodiments, the full-length amino acid sequence of the serine protease EatA protein having the passenger domain, an N-terminal signal peptide, and a C-terminal β-barrel structure is the amino acid sequence set forth in SEQ ID NO: 1. The sequence set forth in SEQ ID NO: 1 is specifically as shown below.

[0021] In the present application, the serine protease EatA is an autotransporter derived from enterotoxigenic Escherichia coli (E. coli) of the Enterobacteriaceae family. The full-length protein contains an N-terminal signal peptide, a C-terminal β-barrel structure, and a passenger domain in the middle of the protein chain. The passenger domain has serine protease activity, which is obtained by a histidine-aspartic acid-serine catalytic triad. The Enterobacteriaceae serotype is O78:H11, and the enterotoxigenic E. coli strain is H10407. In the present application, the serine protease EatA is defined as including the passenger domain fragment EatA-EatAp having serine protease activity, which is the protein set forth in SEQ ID NO:2; the full-length serine protease EatA, which is the protein set forth in SEQ ID NO:1; and the signal peptide-less protease fragment, which is the protein set forth in SEQ ID NO:3.

[0022] In the present application, when Escherichia coli is used as an expression vector, the full-length EatA protein is a membrane protein, and the middle part (passenger domain) is transported outside the membrane by self-transport, and the passenger domain is released by self-cleavage. The protein released into the medium is a target protein having serine protease activity EatA (serine protease EatA-EatAp having only the passenger domain).

[0023] Mucolytic agents are protease-containing drugs that can affect and act on mucin, causing the hydrolysis of glycoproteins and mucin proteins, thereby breaking down gel-like mucus. The serine protease EatA is a member of the Enterobacteriaceae serine autotransporter family and is used as an antigen for preventing severe diarrhea caused by Enterobacteriaceae diarrheal bacteria, inducing an immune response and producing antibodies upon injection. The inventors have found through research that the serine protease EatA has a very long stability period in vitro and destroys the gel-like physical state of mucin, turning it into a liquid. Therefore, the serine protease EatA of the present application can be used to produce mucolytic agents.

[0024] Homologs of the serine protease EatA can be found in other species and strains using standard search methods in the art, and the sequence similarity of homologs can be determined using standard algorithms. For example, when comparing the similarity of two proteins, calculations can be performed using the widely used programs blastN and blastX, whose algorithms were proposed by Karlin and Altschul and allow the introduction of small gaps to achieve maximum matching in percent sequence identity between two nucleic acid or protein molecules. Thus, the serine protease EatA described herein may be replaced with a homolog that shares a high amino acid sequence identity (at least 80% identity).

[0025] In some embodiments, the optimal reaction conditions for the serine protease EatA to decompose mucin are a reaction temperature of 34 to 37°C, a pH of 7 to 8, and a reaction coefficient of 10,000 to 1,000,000. For example, in some specific embodiments, the optimal reaction conditions for the serine protease EatA to decompose mucin are a reaction temperature of 37°C, a pH of 7.2 to 7.4 (a pH of 7.2 to 7.4 is closer to the pH of the human body environment, particularly the tissues and peritoneal cavity), and a reaction coefficient of 100 to 10,000.

[0026] In the present application, when the reaction conditions were limited to the above range, the mucin-decomposing effect of the serine protease EatA was optimal in in vitro experiments.

[0027] The mucin to be degraded in this application is a gel-like semi-solid that is initially insoluble in water. However, as it slowly hydrates, the relative average density of the mucin used in the examples of this application approaches 1.0. Therefore, the following definition is used when measuring concentration: The reaction coefficient is defined as follows: When 1000 μL (1, y) of mucin is present in a fixed reaction system, the mucin density is 1 (1 g / mL, ρ), and the system contains serine protease EatA at a total mass concentration of 1 μg / mL (1 μg, z). After incubating the reaction system at 37°C for 10 hours, a mucin dissolution value w of 100% indicates complete decomposition (where w represents the mass percentage of dissolved mucus), and the reaction coefficient at this time is 1. That is, the reaction coefficient is calculated as follows: Equation 1: Reaction coefficient δ = (y / (x × z)) × w × ρ y>x, y~δ: In other words, when the mass ratio of the enzyme to the substrate mucin is known, the volume of the reaction system must not exceed the corresponding reaction coefficient, and the value representing the volume of the reaction system should always be greater than the value representing the volume of the mucin.

[0028] Therefore, the range of the reaction coefficient used is expected to be, for example, 1 to 100, 100 to 1,000, 1,000 to 10,000, 10,000 to 100,000, 100,000 to 1,000,000, and so on, and is 1 to 1,000,000.

[0029] The above reaction coefficient structure is ethically reasonable. Mucin spontaneously hydrates and dissolves slowly in large volumes of aqueous solution. To mitigate the effects of hydration during enzymatic digestion, the reaction coefficient refers to the strength of the nonlinear interaction of the reaction system with the substrate mucin when all mucus is degraded into a soluble liquid 10 hours after the addition of serine protease EatA. It does not directly represent enzyme activity. Its value is related to the volume of the reaction system, the amount of substrate mucin, the mass concentration and activity of the enzyme itself, and the reaction time. Although mucin density varies depending on pathological conditions, its volume is used as a consistent parameter for calculations.

[0030] It should be noted that the reaction coefficients given in the examples are preferred embodiments and indicate the measurements and concentrations at which EatA degrades mucus / mucin, and are not intended to be limiting.

[0031] In some embodiments, the serine protease EatA has specific serine protease activity and is used to selectively degrade mucins.

[0032] In the present application, the serine protease EatA can specifically degrade mucin but does not degrade non-target proteins (e.g., foreign proteins in mucus). Previous studies have shown that EatA does not degrade common glycoproteins in the human body, such as gelatin, lactoferrin, and CD43, or non-human proteins, such as BSA and BSM. Experimental results by the present inventors further demonstrated that EatA does not degrade foreign proteins contained in clinically derived mucin, but selectively degrades only gel-like mucin, converting the semi-solid gel into a solution that is easily respirable or absorbed by the human body. Therefore, the serine protease EatA of the present application can be suitably used to degrade mucin in mucus-related diseases without damaging other proteins in the human body.

[0033] In some embodiments, the degraded mucin is present in at least one of pseudomyxoma peritonei mucin, chronic obstructive pulmonary disease mucin, ovarian mucinous cancer mucin, colon mucinous cancer mucin, and appendix cancer mucin, and is preferably present in pseudomyxoma peritonei mucin.

[0034] In this study, the mucins degraded by the serine protease EatA were not denatured and purified mucins (e.g., MUC2), but rather mucins present in disease-associated mucus. Unlike denatured and purified mucins (e.g., MUC2), mucins present in disease-associated mucus are involved in mucus formation with tissues, particularly in the formation of gel-like mucus in PMPs. MUC2 forms large polymers with itself and other mucin family molecules, and the mucin family exhibits different modification patterns in disease. Therefore, compared with denatured and purified mucins, mucins present in disease-associated mucus are structurally more complex and less susceptible to degradation. For example, while elastase and trypsin can both be used to purify and obtain mucin molecules suitable for SDS-PAGE, their efficiency in degrading mucins decreases when mucus is formed. In contrast, the serine protease EatA described in the present application can efficiently degrade mucin in mucus-related diseases, and can be used in mucus-related diseases that express the mucin, particularly MUC2-type mucin, such as pseudomyxoma peritonei, to degrade mucin, eliminate its gel-like properties, and achieve specific therapeutic effects.

[0035] In this application, specific therapeutic effects include, but are not limited to, facilitating the removal of mucin, exposing cancer cells or lesions, facilitating the realization of the effects of other chemotherapeutic agents, and reducing the impact on the patient.

[0036] When the serine protease EatA described herein is used to treat mucinous diseases such as pseudomyxoma peritonei, the serine protease EatA described herein has stable activity over a long period of time. Therefore, after being introduced into the peritoneal cavity, it maintains effective mucolytic activity for a long period of time, digesting or reducing the mass and volume of gel-like solids formed by large amounts of mucin under minimally or non-invasive conditions. This is easier and less invasive than open surgery, and avoids the infection and trauma that can result from open surgery. The mucolytic activity of serine protease EatA can be achieved by introducing it into a specific lesion area in the patient's body. The introduction method can be tailored to the type of disease and the target area. For example, in the case of pseudomyxoma peritonei, it can be achieved by injection. A specific concentration of EatA protease can be directly injected into the peritoneal cavity or into a tumor or cancerous cyst to degrade mucin in the peritoneal cavity or target area, ultimately achieving a certain therapeutic effect. Such a therapeutic effect may be a reduction in the mass of mucin within its environment and concomitant reduction in its effects on the patient.

[0037] Furthermore, when the serine protease EatA described in the present application degrades mucus in mucus-related diseases, the resulting mucus is a watery liquid, eliminating its gel-like properties. Furthermore, the mucus in the affected area of ​​the patient can be easily removed and aspirated from the body using a needle or a specific aspirator, facilitating the direct delivery of other therapeutic agents, such as anticancer drugs, to the lesion and cancer cells, thereby improving the efficacy of treatment, reducing the impact and burden on patients, and improving their quality of life. Compared with conventional mucin-degrading preparations in the field, such as preparations containing bromelain, the serine protease EatA described in the present application has superior properties, specifically, it exhibits better selectivity, does not degrade non-target proteins, has higher degradation efficiency, and is more easily aspirated using a pipette, which is more consistent with the expected degradation products.

[0038] In a second aspect of the present application, there is provided a pharmaceutical composition comprising the serine protease according to the first aspect of the present application.

[0039] In some embodiments, the pharmaceutical composition further comprises at least one other agent selected from a chemotherapy, a radiotherapy agent, an enzyme, and a chemical salt.

[0040] In the present application, serine protease EatA can be used in combination with one or more other therapeutically effective drugs, such as common cytotoxic chemotherapy drugs like cisplatin and gemcitabine, allowing the drugs to target cancer cells more directly and effectively, improving the efficacy of chemotherapy. Furthermore, because the protease activity of serine protease EatA, inhibited by PMSF, allows it to exert its mucolytic function, in specific embodiments, it can be used in combination with other enzymes and chemical agents with mucin glycolytic activity.

[0041] In some specific embodiments, the chemotherapeutic and radiotherapeutic agents are at least one selected from gemcitabine, cisplatin, adriamycin, fluorouracil, paclitaxone, paclitaxel, and oxaliplatin; the enzymes are at least one selected from N-acetylgalactosidase, galactosidase, glucosidase, neuraminidase, and specific endomucin; and the chemical bases are at least one selected from sodium bicarbonate, carbocysteine, N-acetylcysteine, and ambroxol.

[0042] In a third aspect of the present application, there is provided use of the serine protease according to the first aspect of the present application or the pharmaceutical composition according to the second aspect in the manufacture of a medicament for treating a mucus-related disease and / or a medicament for improving the therapeutic effect of a mucus-related disease and the quality of life of a patient.

[0043] Because serine protease EatA has protease activity, it can be used as a drug or medicinal ingredient for treating mucus-related diseases. Therefore, the present application provides a novel use of serine protease EatA in treating mucus-related diseases, such as pseudomyxoma peritonei and chronic obstructive pulmonary disease. Administration of an effective dose of serine protease EatA to the lesion area of ​​the mucus-related disease, such as the peritoneal cavity and respiratory tract, can degrade mucin in the lesion area and reduce or eliminate the mass of gel-like proteins, facilitating the removal of large amounts of mucin and achieving the goal of treating mucus-related diseases. Furthermore, administration of an effective dose of serine protease EatA to degrade mucin eliminates the mucus covering and sequestration of cancer cells and lesions, enhancing the effects of cytotoxic drugs and facilitating the access of other chemotherapeutic drugs, such as cisplatin and gemcitabine, to lesions and cancer cells, thereby improving the therapeutic efficacy of mucus-related diseases and the quality of life of patients. Therefore, the serine protease according to the first aspect or the pharmaceutical composition according to the second aspect of the present application can be used in the manufacture of a drug for treating a mucus-related disease and / or a drug for improving the therapeutic effect of a mucus-related disease and the quality of life of patients. A drug comprising the serine protease according to the first aspect or the pharmaceutical composition according to the second aspect degrades mucin and destroys the gel-like protein formed by mucin, making it easier to remove mucin and facilitating the direct delivery of other therapeutic agents, such as anticancer drugs, to the lesion and cancer cells, thereby improving the efficacy of treatment, reducing the impact and burden on patients, and improving their quality of life.

[0044] In this application, the above-mentioned mucin-related diseases include not only mucinous carcinoma, defined by the World Health Organization as a malignant tumor composed of gastrointestinal cells containing intracytoplasmic mucin, but also diseases in which healthy cells, tissues, and organs under normal conditions lose their normal control related to mucus expression due to changes in their function or environment, resulting in the production of large amounts of mucus and the formation of pathological tissue, or in which mucin is secreted and cells, tissues, and organs are enveloped from the outside, preventing the realization of normal physiological functions such as immune recognition or the infiltration and delivery of therapeutic drugs.

[0045] In some embodiments, the mucus-related disease is at least one selected from chronic obstructive pulmonary disease, colon mucinous cancer, lung cancer, liver cancer, gastric cancer, appendix cancer, peritoneal cancer, prostate cancer, colon cancer, small intestine cancer, lymphoma, ovarian cancer, adenocarcinoma, and asthma, and the ovarian cancer includes ovarian mucinous cancer and the peritoneal cancer includes pseudomyxoma peritonei. The appendix cancer may be, for example, appendix mucinous adenocarcinoma.

[0046] Research has shown that the present serine protease EatA has efficient and highly specific degrading activity against mucus derived from pseudomyxoma peritonei. Furthermore, several other mucinous diseases also exhibit large or small amounts of pseudomyxoma peritonei-associated mucin, depending on the type of disease. Treatable pseudomyxoma peritonei is most often caused by cancer cells originating from the colon, appendix, or ovary metastasizing to the peritoneal cavity, secreting large amounts of mucus from tumor cells. These cancer cells originate from various sites, including the colon, rectum, pancreas, breast, lung, gallbladder, and ovaries, but most clinical reports have shown that they originate from the appendix. The large amounts of mucus secreted by these tumor cells into the peritoneal cavity are difficult to degrade and ultimately accumulate, affecting other organs and increasing the severity and mortality of the disease. Conventional methods of mucin removal using open surgery often result in incomplete treatment, requiring multiple surgeries as the disease progresses, leading to increased incidence and mortality.

[0047] According to the above definition of mucus in mucus-related diseases, the mucus in pseudomyxoma peritonei refers to a colorless, transparent to amber-colored, jelly-like, semi-solid gel-like substance with three phenotypes (soft, medium-hard, and hard) formed after tumor or cancer forms on the surface of the peritoneum, primarily composed of MUC2, MUC5AC, and MUC5B. It is characterized by the proliferation of tumor-secreting mucin-secreting cells in the peritoneal cavity on the surface of the peritoneum, producing mucin-like ascites. In ovarian mucinous carcinoma, mucus refers to the white to amber substance secreted by tumors or cancers in the ovaries into the pathological area. Some mucus is chocolate-brown, translucent or transparent, and has a very high viscosity, ranging from several thousand to tens of thousands of mPa·s, and is a syrup-like, semi-solid or fluid. It is characterized by a viscous mixture mainly composed of large amounts of coating mucin adhering to the surface of ovarian mucinous cancer cells or adenoid cysts. In chronic obstructive pulmonary disease, mucus, also known as sputum, is a mucous substance secreted by epithelial / glandular cells in the lungs or respiratory tract. Its main component is mucin, which adheres to the surfaces of the respiratory tract and trachea to form mucus. Unlike normal physiological conditions, sputum in chronic obstructive pulmonary disease has a higher content and viscosity, which often leads to lung and tracheal obstruction.

[0048] In general, the route of administration of the drug should be determined by taking into consideration the physical characteristics of the subject, such as the type of disease and health condition, and this is obvious to clinicians and engineers. In some embodiments, the route of administration of the drug is selected from injection, oral administration, and spray administration.

[0049] In the present application, among the above-mentioned administration routes, injection administration may be, for example, intraperitoneal injection administration, oral administration may be, for example, fast-dissolving tablets, and spray administration may be, for example, nasal or oral inhalation by aerosol. In particular, for injection administration, the drug-carrying medium may be a pharmaceutically acceptable carrier or excipient, such as physiological saline, phosphate buffer, Tris-HCl buffer, Ringer's solution, glucose injection buffer, and water-propylene glycol solution. Those skilled in the art can easily modify the methods for preparing compositions and drugs for non-gastrointestinal and intraperitoneal administration.

[0050] In the present application, the effective dose of the above-mentioned drug should be determined based on various factors, such as the type of mucin-related disease and the amount of mucus, the prescribed treatment period, etc. For example, the appropriate dose may be determined based on various factors, including, but not limited to, the individual's physical characteristics, such as age, weight, and sex, whether the drug is used as a single agent or as an adjunctive therapy, the progression of the disease or symptom to be treated, i.e., the pathological state, and other factors that will be obvious to those skilled in the art.

[0051] The medicaments of the present application can be used in any patient or individual. In some embodiments, the patient is a mammal, most typically a human patient. However, it will be appreciated that other mammalian individuals may also benefit from the present invention, where appropriate. Thus, these patients and individuals may include mice, rats, cats, dogs, bears, cows, horses, and other mammals deserving of treatment. [Effects of the Invention]

[0052] The present application provides the following beneficial effects: The serine protease described in the present application is serine protease EatA, which can selectively degrade disease-related mucins and is stable for a long period of time. Therefore, the serine protease and a pharmaceutical composition containing the serine protease can be used as a drug or active ingredient in the treatment of mucin-secreting cancers, such as pseudomyxoma peritonei (PMP) and other mucin-related diseases. [Brief explanation of the drawings]

[0053] [Figure 1] FIG. 1 shows the results of mucin degradation in the gel-like mucin of the control group in Example 1 without the addition of EatA. [Figure 2] FIG. 1 shows the results of mucin degradation when EatA was added to gel-like mucin in the experimental group of Example 1. [Figure 3] FIG. 1 shows the results of mucin degradation by bromelain (BroM) and serine protease EatA in Example 2. [Figure 4] FIG. 1 is a temperature curve diagram of mucin decomposition by serine protease EatA in Example 3. [Figure 5] FIG. 1 is a pH curve diagram of mucin degradation by serine protease EatA in Example 3. [Figure 6] FIG. 1 is a time curve diagram of mucin degradation by serine protease EatA in Example 3. [Figure 7] FIG. 1 shows the results of decomposition of foreign proteins mixed in mucin by bromelain (BroM) and serine protease EatA in Example 4. [Figure 8] FIG. 10 shows the results of the final mucin residue amounts in the experimental group (mucus + EatA), the control group without enzyme addition (mucus + PBS), and the bromelain experimental group (mucus + bromelain) in Example 5. [Figure 9] FIG. 1 shows the results of abdominal examination of mice in the experimental group (mucus + EatA), the control group without enzyme addition (mucus + PBS), and the bromelain experimental group (mucus + bromelain) in Example 5. [Figure 10] FIG. 10 is a box plot of the degradation of mucus from ovarian mucinous carcinoma and mucus from appendix mucinous adenocarcinoma by the serine protease EatA of Example 6. [Figure 11] FIG. 10 shows the results of degradation of mucus from appendix mucinous adenocarcinoma (MAA) by serine protease EatA in Example 6. DETAILED DESCRIPTION OF THE INVENTION

[0054] In order to facilitate understanding of the present invention, the present invention will be described in more detail below using examples. These examples are for illustrative purposes only and do not limit the scope of use of the present invention. Unless otherwise specified, all raw materials or components used in the present invention can be purchased or prepared by conventional methods.

[0055] Because mucins of different origins and mucins associated with mucinous diseases have different expression patterns and post-translational modifications, this study used mucins of different clinical pathological origins and their mixtures. Preferably, mucin derived from clinical pseudomyxoma peritonei was used. In this study, gel-like mucin was first collected from the peritoneal cavity of clinical patients with pseudomyxoma peritonei and precipitated with 30-100% ammonium sulfate. After protein precipitation, cells, tissue debris, and adipose tissue that may have been contaminated during the mucin collection process were removed. The sample was then dialyzed and filtered using a 100 kDa dialysis bag. Using sulfate test paper, sulfate ion concentrations in the dialysate and inside the dialysis bag were confirmed to be below 200 mg / L. Upon completion of dialysis, gel-like mucin was observed. This sample was collected as a substrate for in vitro experiments to examine the mucin degradation ability of the serine protease EatA. In some cases, experiments were performed using mucin directly collected from the peritoneal cavity of clinical patients without any purification treatment.

[0056] Example 1 In a 500μL reaction system, 0.30g of gel-like mucin was accurately weighed, and approximately 80μg of serine protease EatA (reaction coefficient 133) was added to the experimental group, while no serine protease EatA was added to the control group. Both the experimental and control groups were then incubated at 37℃ for 3 hours, and the mucin degradation status was observed after incubation. The mucin degradation results for the control and experimental groups are shown in Figure 1 and Figure 2, respectively.

[0057] As can be seen from Figures 1 and 2, in the control group to which serine protease EatA was not added, clumps of gel-like protein molecules remained after 3 hours and could not be sucked out using a pipette.Compared to the control group, the experimental group to which serine protease EatA was added showed clear decomposition of the gel-like mucin.The gel-like mucin in the experimental group was decomposed into a watery solution that could be sucked out using a pipette, and the clumps of gel disappeared when viewed under a stereomicroscope.

[0058] Example 2 Bromelain, a broad-spectrum protease with a wide range of degrading activities, can degrade MUC2 mucin and is used for anti-inflammatory and mucus degradation. In this study, we compared the degradative activities of bromelain (BroM) and the serine protease EatA against gel-like mucin at the same substrate / enzyme ratio.

[0059] 0.10 g of mucin was accurately weighed and added to 500 μL of phosphate buffer solution (PBS) in a reaction system. Two enzymes (bromelain (BroM) and serine protease EatA) were added, and the reaction was completed after 6 hours at 37°C. The entire product was placed upside down on a 40 μm cell filter, and the liquid was absorbed from the other side of the filter using absorbent paper. The gel-like product was carefully inverted 3-4 times to remove as much of the flowing liquid as possible, and the mass of the remaining gel-like mucin was measured. This experiment was repeated with three samples at each sampling point, and the results are shown in Figure 3. As can be seen from Figure 3, as the mass concentration of EatA enzyme increased over 6 hours, the mass loss of the gel-like mucin increased, and the residual amount rapidly decreased, indicating that the gel-like mucin was degraded. When 40 μg of EatA was added, the mass loss of the gel-like mucin averaged 90.33% (calculated based on the initial mass of 0.1 g), indicating almost complete decomposition. In addition, the mass of the gel-like mucin increased in the early stage due to its water absorption. As for bromelain, at the substrate / enzyme ratio, the mass of the gel-like mucin increased after it absorbed water, and as the BroM enzyme concentration increased, the mass loss was negative, indicating that the degradation efficiency was low and the gel-like mucin was only slightly degraded.

[0060] Example 3 In this example, the operating environment for mucin degradation by serine protease EatA was investigated by obtaining decomposition curves under different pH, temperature, and reaction time conditions when serine protease EatA decomposes mucin.

[0061] In the experiment, 0.10 g of gel-like mucin was accurately weighed out for each reaction, and 20 μg of serine protease EatA was added. All reactions were carried out in 500 μL of solution. The remaining gel-like mucin was weighed out using the same method as in Example 2. A control group was run under the same operating conditions, except that serine protease EatA was not added. Each group had three reactions as statistical replicates. Due to the hydration properties of gel-like mucin, it absorbs water and swells when in solution, and when finally weighed, even the control group showed a certain weight gain.

[0062] The temperature gradient experiment was conducted in saline solution with a pH of 7.1, and the results are shown in Figure 4. When conducting the pH experiment, the reaction system used was a solution system with different pH values ​​prepared based on 0.9% saline, and the reaction temperature was 37°C. The results are shown in Figure 5. The reaction time for both experiments was 6 hours.

[0063] In the experiment on the decomposition time, it was determined to be carried out in a PBS buffer solution of pH 7.2 to 7.4 at 37°C according to the temperature and pH curves, and the results are shown in FIG.

[0064] As can be seen from Figure 4, in the control group to which no enzyme was added, there was no significant change in the mass of the gel-like mucin after incubation was completed, even when the temperature was changed. However, in all experimental groups to which enzyme was added, the gel-like mucin was decomposed, and the enzyme activity was greatest at 34-37°C. As the control group showed a tendency to decompose to some extent (mass loss) even at temperatures above 41°C, although the effect of temperature on proteins was confirmed, it cannot be said that the increase in decomposition efficiency at that stage was solely related to the enzyme.

[0065] As can be seen from Figure 5, the degradation of gel-like mucin by serine protease EatA under different pH conditions revealed that the degradation efficiency of gel-like mucin by serine protease EatA was maximum around pH = 8. In the control group, the mass loss was almost unchanged regardless of the pH.

[0066] As can be seen from Figure 6, after the addition of serine protease EatA, the mass loss of the gel-like mucin initially decreased to a negative value, indicating that its mass increased due to water absorption. As time passed, the mass loss increased, reaching 80% at 10.5 hours, indicating that the degradation of the substrate gel-like mucin by serine protease EatA continued with increasing incubation time.

[0067] As can be seen from the above, the reaction temperature is preferably 34 to 37°C and the pH is preferably 7 to 8 as the operating environment for decomposing gel-like mucin with serine protease EatA.

[0068] (Example 4: Specificity of degradation by serine protease EatA) Unprocessed mucin derived from clinical patients was decellularized using a simple procedure. 0.20 g of the above mucin was accurately weighed and added to 1 mL of PBS solution. Three to four 1 mm diameter magnetic beads were added, frozen at -30°C, and homogenized by shaking to disrupt the gel-like form of the mucin and release the patient-derived foreign proteins contained within the gel. 100 μL of the homogenized mucin solution was added to 2 to 10 μg of protease (bromelain or serine protease EatA) and incubated in a 200 μL reaction system. After 6 hours of incubation, samples were taken and analyzed by SDS-PAGE. The total protein content after disruption was determined to be the patient-derived foreign proteins in the patient's peritoneal / intraperitoneal mucin. The results are shown in Figure 7.

[0069] As can be seen from Figure 7, in the BroM (bromelain)-added group, when several μg of BroM enzyme was added, the foreign proteins derived from the patient's peritoneal cavity tended to degrade as the amount of enzyme added increased. In other words, the degradation of foreign proteins was observed in the BroM-added group. The control group (the three middle bands marked "0") matched the protein bands obtained with the addition of serine protease EatA, indicating that these proteins derived from the peritoneal cavity were not degraded during the process of mucin degradation by adding serine protease EatA, demonstrating the specificity of the mucin degradation.

[0070] Example 5: In vivo degradative function of serine protease EatA on mucin Since good effects were obtained in in vitro experiments, in this example, in vivo experiments using animals were carried out to confirm that serine protease EatA has the in vivo decomposition function for mucin.

[0071] Twenty 6- to 8-week-old C57 male mice were used in the experiment. Five groups were established: the experimental group (mucus + EatA, 5 mice), the no-enzyme control group (mucus + PBS, 5 mice), the bromelain experimental group (mucus + bromelain, 5 mice), the negative sham-surgery group (open surgery only, no mucin or enzymes, 3 mice), and the reference group (open surgery, EatA protease, 2 mice). Based on previous rat studies and the reference average mass of intraperitoneal mucin in patients with pseudomyxoma peritonei and the response coefficients from in vitro experiments, the maximum allowable intraperitoneal injection volume for mouse experiments was set at 2 mL. In the mouse experiments, 0.30 g of gel-like mucin derived from human patients, briefly washed with PBS buffer, was implanted into the mice, and the general behavior of the mice was observed and confirmed. After 24 hours, the mice were injected with 80 μg each of bromelain and serine protease EatA dissolved in PBS. After 48 hours, the mice were sacrificed and laparotomized (see Figure 9 for abdominal cavity examination results). All gel-like mucin was removed from the mice's bodies and weighed using the method described in Example 2. The final residual amounts of gel-like mucin were statistically compared among the three groups to which gel-like mucin was added as a substrate: the experimental group (mucus + EatA), the control group (mucus + PBS) without enzyme addition, and the bromelain experimental group (mucus + bromelain). The results are shown in Figure 8.

[0072] As can be seen from Figure 8, the group of mice treated with serine protease EatA had the smallest mass of remaining gel-like mucin, indicating that the gel-like mucin was most degraded, with a degradation efficiency of 78% (calculated based on an initial inoculation amount of 0.30 g. The mass of gel-like mucin increased in the abdominal cavity due to reabsorption of water). In contrast, the mass of remaining gel-like mucin in the BroM and control groups still exceeded 0.30 g, making it impossible to calculate its degradation efficiency. This demonstrates that serine protease EatA is far superior to BroM in degrading gel-like mucin in mice.

[0073] The red frame in Figure 9 shows that after the gel-like mucin was transplanted into the abdominal cavity of a mouse, it migrated to different locations in the abdominal cavity as the mouse moved, and its final volume after decomposition also corresponds to that shown in Figure 8.

[0074] During the experiment, changes in body weight were recorded for all groups before and after the experiment, and no differences were found in the results. Abdominal examinations performed after sacrificing the mice revealed no abnormalities. The mice injected with serine protease EatA survived for more than 100 days from the start of the experiment without any adverse events, and showed no abnormalities in either behavior or body weight compared to the uninjected group.

[0075] Example 6: Degradation of serine protease EatA in ovarian mucinous carcinoma (MOC) mucus and appendix mucinous adenocarcinoma (MAA) mucus In this example, the serine protease EatA was used to degrade mucus from ovarian mucinous carcinoma and mucus from appendix mucinous adenocarcinoma. 0.50 mL of clinically collected mucus from the two types of mucinous carcinoma was directly measured in a test tube, and 50 μg of EatA serine protease dissolved in 0.25 mL of PBS buffer was added. The samples were then incubated in a 750 μL reaction system at 37°C and 600 rpm for 20 hours. Each mucinous carcinoma mucus sample had a control group in which an equal volume of PBS alone was added. A total of four groups were tested, with each group having three replicates. After incubation, the viscosity of the experimental samples was measured using an Anton Paar rheometer with a PP25 probe and a 0.45 mm sample spacing. Each sample was run for 20 seconds, and after pre-shearing at a shear frequency of 5 rad / s, a total of 12 data points were obtained at a shear frequency of 10 rad / s, with readings taken every 5 seconds within 60 seconds, resulting in a total of 36 sample points for each group of samples. The statistical results for all data points for each group of samples are shown in box plots in Figure 10.

[0076] As can be seen from Figure 10, the viscosity of the ovarian mucinous cancer mucus collected clinically during the experiment was approximately 2000 mPa·s. After incubation with serine protease EatA, the viscosity decreased to approximately 670 mPa·s. In the case of appendix mucinous adenocarcinoma MAA, the viscosity of the clinically collected mucus was approximately 1200 mPa·s. After the addition of mucin, the viscosity decreased to approximately 7 mPa·s. This indicates that the viscosity of the mucus derived from these two different cancers was significantly reduced after the reaction, and that serine protease EatA has a very clear viscosity-reducing effect.

[0077] Furthermore, Figure 11 shows the results of decomposition of mucus from mucinous adenocarcinoma of the appendix (MAA). Because the appendix cancer mucus in this sample was transparent, its viscosity reduction could be determined by detecting the uniformity of the solution. As can be seen from the results after incubation, after incubation with serine protease, the mucus sample solution became uniform, and impurities in the sample that were not decomposed by EatA settled to the bottom after standing. In contrast, in the control group, the gel-like mucus was largely retained, and the impurities in it were unable to settle to the bottom.

[0078] Furthermore, the results of related research by the applicant have demonstrated that the serine protease EatA of the present application has some degree of decomposition effect on mucus (phlegm) from patients with chronic obstructive pulmonary disease, mucus from colon mucinous cancer, etc. From the above examples, it can be seen that the serine protease of the present application is expected to be widely used in the medical field and has significant advantages over other drugs on the market and preparations under development as pharmaceuticals.

[0079] It should be noted that the above-described examples are merely illustrative of the present application and are not intended to limit the present application in any way. While the present application is described with reference to exemplary embodiments, it should be understood that the terms used therein are for descriptive and interpretative purposes only and are not intended to limit the present application. The present application may be modified within the scope of the appended claims, and the present invention may be modified without departing from the scope and spirit of the present application. While specific methods, materials, and examples are described herein, the present application is not limited to the disclosed specific examples, but rather extends to all methods and uses having the same functionality.

Claims

1. A serine protease that selectively degrades mucin, the serine protease is serine protease EatA, the protein comprising a passenger domain having serine protease EatA activity, and the amino acid sequence of the passenger domain is an amino acid sequence having at least 90% identity with SEQ ID NO:2; A serine protease characterized by:

2. The serine protease according to claim 1, wherein the amino acid sequence of the passenger domain is the amino acid sequence shown in SEQ ID NO:

2.

3. The serine protease according to claim 1, wherein the serine protease EatA protein further comprises at least one of an N-terminal signal peptide and a C-terminal β-barrel structure.

4. The serine protease according to claim 3, characterized in that the amino acid sequence of the serine protease EatA protein having the passenger domain and a C-terminal β-barrel structure is an amino acid sequence having at least 90% identity with SEQ ID NO:

3.

5. The serine protease according to claim 3, wherein the amino acid sequence of the serine protease EatA protein having the passenger domain and a C-terminal β-barrel structure is the amino acid sequence shown in SEQ ID NO:

3.

6. The serine protease according to claim 3, wherein the full-length amino acid sequence of the serine protease EatA protein having the passenger domain, the N-terminal signal peptide, and the C-terminal β-barrel structure is an amino acid sequence having at least 80% identity with SEQ ID NO:

1.

7. The serine protease according to claim 3, wherein the full-length amino acid sequence of the serine protease EatA protein having the passenger domain, the N-terminal signal peptide, and the C-terminal β-barrel structure is an amino acid sequence having at least 90% identity with SEQ ID NO:

1.

8. The serine protease according to claim 3, wherein the full-length amino acid sequence of the serine protease EatA protein having the passenger domain, the N-terminal signal peptide, and the C-terminal β-barrel structure is the amino acid sequence shown in SEQ ID NO:

1.

9. The serine protease described in claim 1, characterized in that the serine protease EatA has specific serine protease activity and is used to selectively degrade mucin, and the degraded mucin is present in at least one of peritoneal pseudomyxoma mucus, chronic obstructive pulmonary disease mucus, ovarian mucinous cancer mucus, colon mucinous cancer mucus, and appendix cancer mucus.

10. A pharmaceutical composition comprising the serine protease of claim 1 and another drug, The other drug is at least one selected from a chemotherapeutic agent, a radiotherapeutic agent, an enzyme, and a chemical salt agent, the chemotherapeutic agent and the radiotherapeutic agent are at least one selected from gemcitabine, cisplatin, adriamycin, fluorouracil, paclitaxone, paclitaxel, and oxaliplatin, the enzyme is at least one selected from N-acetylgalactosidase, galactosidase, glucosidase, neuraminidase, and specific endomucin, and the chemical salt agent is at least one selected from sodium bicarbonate, carbocysteine, N-acetylcysteine, and ambroxol; Pharmaceutical compositions.

11. 12. Use of the serine protease of claim 1 or the pharmaceutical composition of claim 10 in the manufacture of a drug for treating a mucus-related disease and / or a drug for improving the therapeutic effect of a mucus-related disease and the quality of life of a patient, The mucus-related disease is at least one selected from chronic obstructive pulmonary disease, colon mucinous cancer, lung cancer, liver cancer, gastric cancer, appendix cancer, peritoneal cancer, prostate cancer, colon cancer, small intestine cancer, lymphoma, ovarian cancer, adenocarcinoma, and asthma, the ovarian cancer includes ovarian mucinous cancer, and the peritoneal cancer includes pseudomyxoma peritonei. use.

12. The use according to claim 11, wherein the administration route of the drug is selected from injection, oral administration and spray administration, and preferably the injection administration is intraperitoneal injection administration.

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