Anti-fibrotic peptide, vaccine, preparation method for peptide vaccine, and use thereof

Abstract

In order to explore more peptides for use as peptide vaccines against fibrotic diseases, provided are an anti-fibrotic peptide, a vaccine, a preparation method for a peptide vaccine, and a use thereof. Major histocompatibility complex-restricted peptides derived from six fibrosis-specific proteins MAF, FNDC3A, TOP2A, TNS3, APBB2 and OLFML2b are used as targets for the treatment and prevention of fibrotic diseases, and these six peptides are used as vaccines, so that multiple fibrotic diseases, such as pulmonary, hepatic, and pancreatic fibrosis, are significantly prevented and treated by inhibiting fibrosis-induced pathological changes and collagen fiber deposition.

Description

Antifibrotic peptides, vaccines, and preparation methods and applications of peptide vaccines. Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to antifibrotic peptides, vaccines, and methods for preparing and applying peptide vaccines. Background Technology

[0002] Fibrosis can occur in organs such as the lungs, liver, and pancreas, characterized by fibroblast proliferation, massive extracellular matrix accumulation, inflammatory damage, and tissue structural destruction. In other words, normal tissue is damaged and then undergoes abnormal repair, leading to structural abnormalities (scarring). The cause of most pulmonary fibrosis patients is unknown (idiopathic). This group of diseases is called idiopathic interstitial pneumonia (IIP), a large category of interstitial lung diseases. The most common type of IIP, with pulmonary fibrosis as the main manifestation, is idiopathic pulmonary fibrosis (IPF), a severe interstitial lung disease that leads to progressive loss of lung function. Furthermore, liver fibrosis is a pathophysiological process, referring to the abnormal proliferation of connective tissue in the liver caused by various pathogenic factors. Any liver injury involves a process of liver fibrosis during the liver's repair and healing process. If the damaging factors are not removed for a long time, the fibrotic process will continue and develop into cirrhosis. Simultaneously, pancreatic fibrosis is a persistent and permanent damage to pancreatic tissue and function caused by chronic pancreatitis. The pancreas exhibits varying degrees of acinar atrophy, pancreatic duct deformation, and fibrosis, clinically manifesting primarily as abdominal pain, diarrhea or steatorrhea, weight loss, and malnutrition—symptoms of pancreatic insufficiency. Additionally, renal fibrosis, myocardial fibrosis, and splenic fibrosis are also common fibrotic diseases.

[0003] Peptide vaccines are a novel type of vaccine that utilizes artificially synthesized short peptides to stimulate the immune system to produce an immune response against specific pathogens. These peptides are typically immunogenic fragments of the pathogen's proteins. Compared to traditional vaccines, peptide vaccines offer several advantages: First, because they do not contain the entire pathogen or its derivatives, there is no potential risk of infection; second, the synthesis process of peptide vaccines is relatively simple, allowing for rapid and flexible customization to address different variants; furthermore, because they involve only specific peptides, the risk of potential side effects and allergic reactions is lower. The research and development of peptide vaccines is in a rapid development phase, and they are being applied to the treatment or prevention of various infectious diseases, or major human diseases, including cancer and fibrosis. Currently, while there is considerable research on using peptides as vaccines for the treatment or prevention of fibrosis, exploring more peptides for use as anti-fibrotic vaccines has positive implications for clinical medical research. Summary of the Invention

[0004] In order to explore the use of peptide segments as peptide vaccines for fibrotic diseases, the purpose of this application is to provide new anti-fibrotic peptides, vaccines, and methods for preparing and applying peptide vaccines.

[0005] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows:

[0006] In a first aspect, the present invention provides an anti-fibrotic peptide derived from fibrosis-specific proteins such as MAF, FNDC3A, TOP2A, TNS3, APBB2, or OLFML2b, wherein the peptide is MAF. 116-124 FNDC3A 1101-1108 TOP2A 1118-1125 TNS3 119-127 APBB2 70-78 or OLFML2b 263-271 Its amino acid sequence is selected from at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.

[0007] peptide MAF 116-124 The amino acid sequence is shown in SEQ ID NO: 1: ALISNSHQL.

[0008] Peptide FNDC3A 1101-1108 The amino acid sequence is shown in SEQ ID NO: 2: TSFRYSSL.

[0009] Peptide TOP2A 1118-1125 The amino acid sequence is shown in SEQ ID NO: 3: AGPTFNYL.

[0010] peptide TNS3 119-127 Its amino acid sequence is shown in SEQ ID NO: 4: SSPKSTLTL.

[0011] Peptide APBB2 70-78 Its amino acid sequence is shown in SEQ ID NO: 5: YALTNIQAA.

[0012] peptide OLFML2b 263-271 Its amino acid sequence is shown in SEQ ID NO: 6: SQQINSIEL.

[0013] Secondly, the present invention provides an antifibrotic peptide vaccine containing the aforementioned peptides and pharmaceutically acceptable adjuvant components.

[0014] Furthermore, the auxiliary component is an immune adjuvant.

[0015] Furthermore, the immune adjuvant is a 1V209-Cho-Lip liposome, which is prepared from cholesterol-modified 1V209 molecules 1V209-Cho, lipid components and cholesterol, wherein the structural formula of 1V209-Cho is shown in Formula II;

[0016] Furthermore, 1V209 is a synthetic small-molecule TLR7 agonist with the structural formula shown in Formula I.

[0017] The lipid component is at least one of lecithin, hydrogenated lecithin, or synthetic phospholipid.

[0018] Preferably, the lipid component is soybean lecithin or hydrogenated soybean lecithin.

[0019] Most preferably, the lipid component is hydrogenated soybean lecithin (HSPC).

[0020] The ratio of lipid components, cholesterol, and 1V209-Cho in the liposomes is 60-70:27-37:1-8.

[0021] The preferred ratio of lipid components, cholesterol, and 1V209-Cho in the liposomes is 63-68:30-35:2-4.

[0022] The optimal ratio of lipid components, cholesterol, and 1V209-Cho in the liposomes is 65:32:3.

[0023] Furthermore, the mass ratio of the peptide to the lipid delivery carrier is 5:2.

[0024] Thirdly, the present invention provides the use of the above-mentioned peptide or peptide vaccine in the preparation of medicaments for the prevention and / or treatment of fibrotic diseases.

[0025] Furthermore, in the aforementioned applications, the drug is a pharmaceutical preparation administered via injection, oral administration, nasal mucosa, lungs, rectum, oral mucosa, or skin.

[0026] Furthermore, the drug is an injectable preparation, and the injection route is at least one of intramuscular injection, intravenous injection, subcutaneous injection, intradermal injection, intramyocardial injection, or intraperitoneal injection.

[0027] Furthermore, the fibrotic diseases include at least one of pulmonary fibrosis, liver fibrosis, pancreatic fibrosis, kidney fibrosis, cardiac fibrosis, endometrial fibrosis, ocular fibrosis, splenic fibrosis, myelofibrosis, or skin fibrosis.

[0028] Furthermore, the pulmonary fibrosis includes at least one of idiopathic pulmonary fibrosis, secondary pulmonary fibrosis, hereditary pulmonary fibrosis, or other types of pulmonary fibrosis. Preferably, the pulmonary fibrosis is drug-induced, and the drug is at least one of bleomycin, amiodarone, or methotrexate.

[0029] Furthermore, the liver fibrosis is caused by viral hepatitis, alcoholic hepatitis, autoimmune diseases, fatty liver, malnutrition, chronic congestive heart failure, or drugs, as well as liver fibrosis of unknown etiology, or diseases induced by liver fibrosis. Preferably, the liver fibrosis is drug-induced, and the drug is at least one of carbon tetrachloride or phosphorus.

[0030] Furthermore, the pancreatic fibrosis is a disease induced by acute pancreatitis, chronic pancreatitis, pancreatic duct obstruction, chronic alcoholism, pancreatic ischemia, or pancreatic fibrosis. Preferably, the pancreatic fibrosis is drug-induced, and the drug is spirodiclofen.

[0031] Furthermore, in the application described, the drug inhibits the pathological changes caused by fibrosis, the massive proliferation of fibroblasts, and the deposition of collagen.

[0032] Fourthly, the present invention provides a combination anti-fibrotic drug containing the aforementioned peptide or peptide vaccine administered separately or simultaneously, along with other drugs for treating fibrotic diseases.

[0033] Fifthly, the present invention provides a method for preparing the above-mentioned peptide vaccine, which is prepared by mixing the above-mentioned peptide with a pharmaceutically acceptable auxiliary component.

[0034] Furthermore, the above-mentioned peptide vaccine is prepared by uniformly encapsulating the above-mentioned peptides in the lipid delivery carrier 1V209-Cho-Lip liposome using a microfluidic method.

[0035] Furthermore, the mass ratio of the peptide to the lipid delivery carrier is 5:2.

[0036] More preferably, the method for preparing the peptide vaccine includes the following steps:

[0037] The lipid components, cholesterol, and 1V209-Cho were mixed in an organic solvent in a certain proportion and used as the organic phase; the peptide aqueous solution was used as the aqueous phase. The organic phase and the aqueous phase were mixed by a microfluidic system to encapsulate the peptides, and then ultrafiltration was performed to obtain the purified peptide vaccine.

[0038] The ratio of lipid components, cholesterol, and 1V209-Cho is 60-70:27-37:1-8.

[0039] The preferred ratio of lipid components, cholesterol, and 1V209-Cho in liposomes is 63-68:30-35:2-4.

[0040] The optimal ratio of lipid components, cholesterol, and 1V209-Cho in liposomes is 65:32:3.

[0041] The volume ratio of the organic phase to the aqueous phase is 1:1 to 4; preferably, the volume ratio of the organic phase to the aqueous phase is 1:3.

[0042] The microfluidic operating temperature is 35–37°C.

[0043] The ultrafiltration time is 20-25 minutes, and the rotation speed is 3500-4000 rpm.

[0044] Beneficial effects: This invention utilizes fibrosis neoantigens as targets, specifically targeting the major histocompatibility complex restriction peptide MAF derived from six fibrosis-specific proteins: MAF, FNDC3A, TOP2A, TNS3, APBB2, and OLFML2b. 116-124 FNDC3A 1101-1108 TOP2A 1118-1125 TNS3 119-127 APBB2 70-78 or OLFML2b 263-271 Targeting the therapeutic and preventative agents of fibrosis, this peptide vaccine is developed for the prevention and treatment of various fibrotic diseases. Animal experiments have shown that the peptide vaccine of this invention, utilizing a lipid delivery system, can significantly treat drug-induced pulmonary, hepatic, and pancreatic fibrosis. Furthermore, it achieves its therapeutic effect by inhibiting the pathological changes and collagen fiber deposition caused by fibrosis. Therefore, the peptide vaccine of this invention provides a promising candidate vaccine for future research into preventative and therapeutic vaccines for fibrosis. Attached Figure Description

[0045] Figure 1 shows the results of treating bleomycin-induced pulmonary fibrosis in mice with PEP-VAC vaccines from six different protein sources in Example 1; a) Masson staining of lung tissue sections from mice vaccinated with PEP-VAC vaccines from six different protein sources and mice not vaccinated with PEP-VAC in the pulmonary fibrosis treatment model; b) Lung coefficients from mice vaccinated with PEP-VAC vaccines from six different protein sources and mice not vaccinated with PEP-VAC in the pulmonary fibrosis treatment model; c) Hydroxyproline content in lung tissue from mice vaccinated with PEP-VAC vaccines from six different protein sources and mice not vaccinated with PEP-VAC in the pulmonary fibrosis treatment model. Data are expressed as mean ± SEM. Scale bar: 50 μm.

[0046] Figure 2 shows the results of treating carbon tetrachloride-induced liver fibrosis in mice using the PEP-VAC vaccine in Example 2; a) Sirius red staining of liver tissue sections from mice vaccinated with MAF-VAC, FNDC3A-VAC, and unvaccinated with PEP-VAC in the liver fibrosis treatment model; b) Percentage of Sirius red-positive areas in mice vaccinated with MAF-VAC, FNDC3A-VAC, and unvaccinated with PEP-VAC in the liver fibrosis treatment model; c) Hydroxyproline content in liver tissue from mice vaccinated with MAF-VAC, FNDC3A-VAC, and unvaccinated with PEP-VAC in the liver fibrosis treatment model. Data are expressed as mean ± SEM. Scale bar: 50 μm.

[0047] Figure 3 shows the effect of PEP-VAC vaccine on the treatment of pancreatic fibrosis induced by hygroscopic fibrosis in mice in Example 3; a) Masson staining of pancreatic tissue pathological sections from mice vaccinated with MAF-VAC, FNDC3A-VAC, and unvaccinated with PEP-VAC in the pancreatic fibrosis treatment model; b) Hydroxyproline content in pancreatic tissue from mice vaccinated with MAF-VAC, FNDC3A-VAC, and unvaccinated with PEP-VAC in the pancreatic fibrosis treatment model. Data are expressed as mean ± SEM. Scale bar: 50 μm.

[0048] Figure 4 shows the peptide MAF. 116-124 FNDC3A 1101-1108 TOP2A 1118-1125 TNS3 119-127 APBB2 70-78 OLFML2b 263-271 Structural diagram and structural formula; a) is the peptide MAF 116-124 FNDC3A 1101-1108 TOP2A 1118-1125 TNS3 119-127 APBB2 70-78 OLFML2b 263-271 Structural diagram; b) shows the peptide MAF. 116-124 FNDC3A 1101-1108 TOP2A 1118-1125 TNS3 119-127 APBB2 70-78 OLFML2b 263-271 The structural formula. Detailed Implementation

[0049] To make the technical problems, solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with the embodiments. Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art.

[0050] This invention uses MAF 116-124 FNDC3A 1101-1108 TOP2A 1118-1125 TNS3 119-127 APBB2 70-78 OLFML2b 263-271 Six peptides were identified as targets for the treatment and prevention of fibrotic diseases, and peptide vaccines were prepared using these six peptides for the treatment and prevention of fibrotic diseases.

[0051] In one specific embodiment of the invention, a major histocompatibility complex-restricted peptide derived from fibrosis-specific proteins, including MAF, is used. 116-124 FNDC3A 1101-1108 TOP2A 1118-1125 TNS3 119-127 APBB2 70-78 OLFML2b 263-271 A peptide vaccine, PEP-VAC, was prepared for the treatment and prevention of various fibrotic diseases. Its therapeutic efficacy in fibrotic diseases was further investigated using a lipid delivery system.

[0052] The peptide vaccine described in this invention is a novel vaccine that utilizes artificially synthesized short peptides to stimulate the immune system to produce an immune response against a specific pathogen. These peptides are typically immunogenic fragments within the pathogen's proteins. Compared to traditional vaccines, it offers several advantages: First, since it does not contain the entire pathogen or its derivatives, there is no potential risk of infection; second, the synthesis process is relatively simple and can be quickly and flexibly customized to address different variants; furthermore, because it involves only specific peptides, the risk of potential side effects and allergic reactions is lower.

[0053] The peptide vaccine described in this invention is prepared by adding pharmaceutically acceptable adjuvants, such as immune adjuvants, to the aforementioned six peptides. Any drug containing the above peptides and used as an anti-fibrotic agent, such as a peptide vaccine and its preparation method, falls within the scope of protection of this invention.

[0054] In one specific embodiment of the present invention, the peptides are used for anti-fibrosis via a lipid delivery carrier. More specifically, the above six peptides are uniformly encapsulated in a lipid delivery carrier 1V209-Cho-Lip liposome using a microfluidic method to prepare a peptide vaccine for the prevention and treatment of fibrosis.

[0055] The peptide vaccine is prepared by first using an organic mixture of 1V209-Cho, lipid components, and cholesterol as the organic phase, and a peptide solution as the aqueous phase. The peptides are uniformly encapsulated in 1V209-Cho-Lip liposomes using a microfluidic system, followed by ultrafiltration purification.

[0056] The lipid component is lecithin, hydrogenated lecithin, or synthetic phospholipid, preferably soybean lecithin or hydrogenated soybean lecithin, with hydrogenated soybean lecithin (HSPC) being the most preferred.

[0057] The molar ratio of lipid components, cholesterol, and 1V209-Cho in the liposomes is 60–70:27–37:1–8. A preferred ratio is 63–68:30–35:2–4. The most preferred ratio is 65:32:3.

[0058] The mass ratio of peptide to lipid delivery carrier is 5:2.

[0059] The fibrotic disease described in this invention is induced by a variety of factors. After these factors cause tissue damage, during the repair process, collagen, extracellular matrix, and fibronectin produced by myofibroblasts are deposited in the tissue, leading to tissue remodeling and severely affecting organ function.

[0060] The fibrotic diseases described in this invention include at least one of pulmonary fibrosis, liver fibrosis, pancreatic fibrosis, kidney fibrosis, cardiac fibrosis, endometrial fibrosis, ocular fibrosis, splenic fibrosis, myelofibrosis, or skin fibrosis.

[0061] Pulmonary fibrosis includes at least one of idiopathic pulmonary fibrosis, secondary pulmonary fibrosis, hereditary pulmonary fibrosis, or other types of pulmonary fibrosis. In a preferred embodiment of the present invention, the pulmonary fibrosis is caused by a drug, said drug being at least one of bleomycin, amiodarone, or methotrexate.

[0062] In one specific embodiment of the present invention, a mouse pulmonary fibrosis model was constructed by sublingual bleomycin infusion. The results showed that intramuscular injection of PEP-VAC vaccine could effectively treat collagen deposition, increased hydroxyproline content, and pathological changes in lung tissue caused by bleomycin in mice.

[0063] Liver fibrosis refers to the abnormal proliferation of connective tissue in the liver caused by various pathogenic factors. Any liver injury involves a process of liver fibrosis during the liver's repair and healing process. If the damaging factors are not removed for a long time, the fibrosis process will continue and eventually develop into cirrhosis. Viral hepatitis, alcoholic hepatitis, autoimmune diseases, fatty liver, malnutrition, chronic congestive heart failure, or drugs can all cause liver fibrosis. Furthermore, liver fibrosis also includes other types of liver fibrosis with unknown causes, or diseases induced by liver fibrosis. In a preferred embodiment of the present invention, the liver fibrosis is caused by a drug, wherein the drug is at least one of carbon tetrachloride or phosphorus.

[0064] In one specific embodiment of the present invention, a mouse liver fibrosis model was constructed by intraperitoneal injection of carbon tetrachloride. The results showed that intramuscular injection of PEP-VAC vaccine could effectively treat the pathological changes and collagen deposition in mouse liver tissue caused by carbon tetrachloride.

[0065] Pancreatic fibrosis is a persistent and permanent damage to pancreatic tissue and function caused by various factors. The pancreas exhibits varying degrees of acinar atrophy, pancreatic duct deformation, and fibrosis. Clinically, it mainly manifests as abdominal pain, diarrhea or steatorrhea, weight loss, and malnutrition—symptoms of pancreatic insufficiency. The pancreatic fibrosis described in this invention is a disease induced by acute pancreatitis, chronic pancreatitis, pancreatic duct obstruction, chronic alcoholism, pancreatic ischemia, or pancreatic fibrosis. In a preferred embodiment of this invention, the pancreatic fibrosis is drug-induced, and the drug is spirochetin.

[0066] In one specific embodiment of the present invention, a mouse liver fibrosis model was constructed by intraperitoneal injection of hymenoplasmin. The results showed that intramuscular injection of PEP-VAC vaccine could effectively treat the pathological changes and collagen deposition in mouse pancreatic tissue caused by hymenoplasmin.

[0067] Renal fibrosis is caused by factors such as hypertension, glomerulonephritis, systemic lupus erythematosus, scleroderma, kidney transplant rejection, pyelonephritis, kidney stones, hyperlipidemia, diabetes, hyperuricemia, and hypercalciuria, as well as other types of renal fibrosis with unknown causes, and diseases induced by renal fibrosis.

[0068] Cardiac fibrosis is a condition characterized by cardiac fibrosis, cardiac remodeling, and myocardial hypertrophy caused by ischemic heart disease, hypertension, viral myocarditis, metabolic cardiomyopathy, Keshan disease, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, and arrhythmias, as well as cardiac fibrosis of unknown etiology and diseases induced by cardiac fibrosis.

[0069] Endometrial fibrosis is a fibrotic lesion of the endometrium caused by various reasons, such as endometriosis, as well as diseases induced by endometrial fibrosis.

[0070] Ocular fibrosis is a fibrotic disease of the retina caused by eye trauma, eye surgery, and diabetes, as well as diseases induced by ocular fibrosis.

[0071] Myelofibrosis includes idiopathic and drug-induced myelofibrosis, polycythemia vera, chronic myeloid leukemia, Hodgkin's disease, and diseases induced by myelofibrosis.

[0072] The following specific embodiments will be provided to explain the solution of the present invention. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0073] peptide MAF 116-124 (Amino acid sequence: ALISNSHQL), FNDC3A 1101-1108 (Amino acid sequence: TSFRYSSL), TOP2A 1118-1125 (Amino acid sequence: AGPTFNYL), TNS3 119-127 (Amino acid sequence: SSPKSTLTL), APBB2 70-78 (Amino acid sequence: YALTNIQAA), OLFML2b 263-271 (Amino acid sequence: SQQINSIEL) The dry powder was purchased from Jier Biochemical Co., Ltd. 1V209-Cho was prepared from commercially available 1V209 (Selleck) according to the article Nano Lett. 2021, 21, 7960-7969 or patent CN118217247A; the kit for detecting tissue hydroxyproline content was purchased from Nanjing Jiancheng Bioengineering Institute. Male C57BL / 6 mice (8-10 weeks old) were purchased from Beijing Vital River Laboratory Animal Science Co., Ltd. All animal experiments were conducted in accordance with the guidelines evaluated and approved by the Ethics Committee of Sichuan University.

[0074] 1V209-Cho was prepared by the following synthetic method:

[0075] Step a: Cholesterol, EDCI, and DMAP were dissolved in 10 mL of DCM at a ratio of 1 eq: 2 eq: 0.1 eq. Then, 1.2 eq of BOC-aminobutyric acid was added. After reacting at room temperature for 24 hours, 25 mL of DCM was added to extract the organic layer. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain the crude product. The crude product was purified by silica gel chromatography using petroleum ether / ethyl acetate to obtain product 1.

[0076] Step b: Product 1 was dissolved in 5 mL of DCM and 10% TFA was added. The mixture was stirred at room temperature for 1 h, then 25 mL of DCM was added to extract the organic layer. The organic layer was washed with brine, dried with anhydrous sodium sulfate, and concentrated under vacuum to obtain product 2.

[0077] In step c, 1V209, HATU, and TEA were dissolved in 5 mL of DMF at a ratio of 1 eq: 1.2 eq: 2 eq. Then, product 2 dissolved in DMF was added at a volume of 1.2 eq. After reacting at room temperature for 48 hours, the solvent DMF was removed by rotary evaporation to obtain the crude product. The crude product was then purified by silica gel column chromatography using dichloromethane / methanol to obtain 1V209-Cho.

[0078] Preparation of PEP-VAC vaccine

[0079] The PEP-VAC vaccines, including MAF-VAC, FNDC3A-VAC, TOP2A-VAC, TNS3-VAC, APBB2-VAC, and OLFML2b-VAC vaccines, were prepared using a liposome delivery system and a microfluidic approach. The liposome delivery carrier, consisting of lipid components HSPC, cholesterol, and 1V209-Cho at a molar ratio of 65:32:3, was dissolved in anhydrous ethanol and dimethyl sulfoxide (DMSO) as the organic phase (anhydrous ethanol to DMSO volume ratio of 10:1). Peptides dissolved in physiological saline were used as the aqueous phase, with a peptide-to-liposome delivery carrier mass ratio of 5:2. The organic and aqueous phases were then rapidly passed through a microfluidic chip at a volume ratio of 1:3 at 37°C. Finally, the microfluidically mixed liquid was ultrafiltered at 4000 rpm for 20 minutes to remove water, organic solvents, and unencapsulated peptides, resulting in a purified MAF-VAC vaccine solution. The volume of the liposome-coated peptides was then adjusted to 1.2 mL using physiological saline, completing the preparation of the peptide vaccine. This process is also applicable to FNDC3A-VAC, TOP2A-VAC, TNS3-VAC, APBB2-VAC, and OLFML2b-VAC, simply by using the corresponding peptide solution. Ultrafiltration ensures that the vaccine solution contains only the active ingredient, without unencapsulated peptides or other impurities, thereby improving the purity and stability of the vaccine.

[0080] Example 1: Efficacy of PEP-VAC in treating pulmonary fibrosis in mice

[0081] To investigate the therapeutic effect of PEP-VAC on pulmonary fibrosis in mice, mice were intramuscularly injected with PEP-VAC (50 μg MAF) dissolved in physiological saline on days 0, 7, and 14. 116-124 Or FNDC3A 1101-1108 Or TOP2A 1118-1125 TNS3 119-127 or APBB2 70-78 or OLFML2b 263-271A mouse model of pulmonary fibrosis was established by sublingual administration of 3 mg / kg bleomycin to each mouse on day 1 (equivalent to 1.25 μg / kg mice). Twenty-eight days after bleomycin administration, mice were sacrificed, and lung tissue was dissected to assess case characteristics. Masson staining results, as shown in Figure 1a, indicated a significant reduction in lung collagen fibers in mice immunized with the six PEP-VAC treatments in the treatment model. Furthermore, as shown in Figure 1b, the six PEP-VAC immunizations significantly inhibited the increase in lung coefficient (lung weight / body weight) induced by pulmonary fibrosis in the treatment model. Since hydroxyproline is a major component of collagen tissue and an important indicator for assessing the degree of pulmonary fibrosis, we further analyzed the hydroxyproline content in the lungs of mice treated with the six PEP-VACs using a hydroxyproline kit, as shown in Figure 1c. The results showed that in the treatment model, all six PEP-VAC vaccines reduced the hydroxyproline content in the lungs of bleomycin-treated mice. Based on the above results, in the treatment model, PEP-VAC, derived from multiple fibrosis-specific proteins, can effectively alleviate bleomycin-induced pulmonary fibrosis in mice, and is a potential candidate vaccine for the treatment of idiopathic pulmonary fibrosis.

[0082] Example 2: Efficacy of PEP-VAC in treating liver fibrosis in mice

[0083] MAF 116-124 With FNDC3A 1101-1108 The prepared PEP-VAC showed good therapeutic effects in pulmonary fibrosis in mice. To further verify the ability of these two peptide vaccines to treat liver fibrosis in mice, mice were intramuscularly injected with PEP-VAC (50 μg MAF) on days 0, 14, and 21. 116-124 Or FNDC3A 1101-1108 A mouse liver fibrosis model was established by intraperitoneal injection of 100 μL of 20% volume concentration carbon tetrachloride (dissolved in corn oil) every 3 days, starting from day 1. 72 hours after the 12th injection, the mice were sacrificed, and liver tissue was dissected to assess case characteristics. Sirius red staining results, as shown in Figure 2a, indicate that in the treatment model, mice receiving MAF... 116-124 Or FNDC3A 1101-1108 Mice treated with the prepared PEP-VAC vaccine showed a significant reduction in liver tissue collagen after carbon tetrachloride treatment. Furthermore, the Sirius red staining results were analyzed using ImageJ image analysis software, as shown in Figure 2b. In the treatment model, the magnification of collagen by MAF... 116-124 Or FNDC3A 1101-1108 The prepared PEP-VAC vaccine also significantly inhibited the increase of collagen in liver fibrosis tissue induced by carbon tetrachloride. Furthermore, analysis of hydroxyproline content in mouse liver tissue using a hydroxyproline kit showed, as shown in Figure 2c, that in the treatment model, collagen levels increased significantly due to MAF. 116-124Or FNDC3A 1101-1108 The prepared PEP-VAC vaccine significantly reduced the hydroxyproline content in mouse liver tissue treated with carbon tetrachloride.

[0084] Example 3: Efficacy of PEP-VAC in treating pancreatic fibrosis in mice

[0085] MAF 116-124 With FNDC3A 1101-1108 The prepared PEP-VAC showed good therapeutic effects in mouse models of pulmonary and liver fibrosis. To further verify the ability of these two peptide vaccines to treat pancreatic fibrosis in mice, mice were intramuscularly injected with PEP-VAC (50 μg MAF) on days 0, 14, and 21. 116-124 Or FFNDC3A 1101-1108 (Mice) were injected intraperitoneally with 50 μg / kg of lecithin starting on day 1, for four consecutive weeks, on Mondays, Wednesdays, and Fridays, six times a day, with one-hour intervals between injections, to establish a mouse model of pancreatic fibrosis. 72 hours after the last injection, the mice were sacrificed, and pancreatic tissue was dissected to assess case characteristics. Masson staining results, as shown in Figure 3a, indicate that in the treatment model, mice receiving MAF... 116-124 Or FNDC3A 1101-1108 Mice treated with the prepared PEP-VAC vaccine showed a significant reduction in liver collagen after treatment with hygroscopic lecithin. Furthermore, analysis of hydroxyproline content in mouse pancreatic tissue using a hydroxyproline kit, as shown in Figure 3b, revealed that in the treatment model, MAF... 116-124 Or FNDC3A 1101-1108 The prepared PEP-VAC vaccine significantly reduced the hydroxyproline content in the pancreatic tissue of mice treated with taurine.

Claims

1. Anti-fibrotic peptide stretch, characterized in that: peptide derived from the fibrosis-specific MAF, FNDC3A, TOP2A, TNS3, APBB2 or OLFML2b protein, said peptide being a MAF 116-124 , FNDC3A 1101-1108 , TOP2A 1118-1125 , TNS3 119-127 , APBB2 70-78 or OLFML2b 263-271 , the amino acid sequence of which is selected from at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO:

6.

2. A peptide segment vaccine against fibrosis, characterized in that: The peptide segment of claim 1 and a pharmaceutically acceptable auxiliary ingredient.

3. The anti-fibrotic peptide segment vaccine of claim 2, characterized by: The auxiliary component is an immunoadjuvant; further, the immunoadjuvant is 1V209-Cho-Lip liposome which is prepared from a cholesterol-modified 1V209 molecule 1V209-Cho, a lipid component and cholesterol, wherein the structural formula of 1V209-Cho is shown as formula II; 4. The anti-fibrotic peptide segment vaccine of claim 3, characterized by: 1V209 is a synthetic small molecule TLR7 agonist with the structure shown in Formula I, 5. The anti-fibrotic peptide segment vaccine according to claim 3 or 4, characterized in that: at least one of the following is satisfied: The lipid component is at least one of lecithin, hydrogenated lecithin or synthetic phospholipid; preferably, the lipid component is soy lecithin or hydrogenated soy lecithin; most preferably, the lipid component is HSPC; The ratio between the lipid component, cholesterol and IV209-Cho in the liposome is 60-70: 27-37: 1-8 in molar ratio; preferably, the ratio is 63-68: 30-35: 2-4 in molar ratio; most preferably, the ratio is 65: 32: 3; The mass ratio of the peptide segment and the lipid delivery carrier is 5:

2.

6. Use of the peptide segment of claim 1 or the peptide segment vaccine of any one of claims 2-5 in the preparation of a drug for preventing and / or treating fibrotic diseases.

7. Use according to claim 6, characterized in that: The drug is a pharmaceutical preparation for injection, oral, nasal mucosa, lung, rectum, oral mucosa or skin administration; further, the drug is an injection preparation, and the injection route is at least one of intramuscular injection, intravenous injection, subcutaneous injection, intradermal injection, intramyocardial injection or intraperitoneal injection.

8. Use according to claim 6 or 7, characterized in that: The fibrotic disease includes at least one of pulmonary fibrosis, liver fibrosis, pancreatic fibrosis, kidney fibrosis, cardiac fibrosis, endometrial fibrosis, ocular fibrosis, splenic fibroproliferative disease, bone marrow fibrosis or skin fibrosis.

9. A combination of drugs against fibrosis, characterized in that: The drug contains the peptide segment of claim 1 or the peptide segment vaccine of any one of claims 2-5 administered separately or simultaneously, and other anti-fibrotic disease drugs.

10. A method for preparing the peptide segment vaccine of any one of claims 2-5, which comprises mixing the peptide segment of claim 1 with a pharmaceutically acceptable auxiliary ingredient; further, the peptide segment is uniformly encapsulated in the lipid delivery carrier IV209-Cho-Lip liposome by microfluidic method.

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