Ephrin-b2 recombinant protein and vaccine, preparation method and application thereof

Abstract

The application belongs to the field of medicine and biotechnology, and particularly relates to an Ephrin-B2 recombinant protein and a vaccine, a preparation method and application thereof. In order to treat and prevent fibrosis diseases, the application is based on an Ephrin-B2 extracellular segment, expresses and purifies an Ephrin-B2 recombinant protein, and adds an adjuvant to prepare an Ephrin-B2 recombinant protein vaccine, which is used for the treatment and prevention of fibrosis diseases. Animal experiment results show that the Ephrin-B2 recombinant protein vaccine prepared by the application can induce obvious cellular immunity and humoral immunity in mice, effectively treat and prevent idiopathic pulmonary fibrosis, radiation-induced pulmonary fibrosis, and has obvious inhibitory effect on bleomycin-induced systemic sclerosis skin fibrosis. It can be seen that the Ephrin-B2 recombinant protein and the vaccine thereof provide a candidate scheme for future preventive and therapeutic vaccine conversion research of fibrosis diseases.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical biotechnology, specifically relating to Ephrin-B2 recombinant protein and its vaccine, preparation method and application. 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). Pulmonary fibrosis is a disease characterized by progressive scarring of lung tissue, with a poor prognosis, especially idiopathic pulmonary fibrosis (IPF), which usually results in death within 3-5 years of diagnosis. Currently, antifibrotic therapies for IPF are scarce; clinical treatment options are limited to antifibrotic drugs like nintedanib and pirfenidone to slow disease progression. Besides pulmonary fibrosis, common fibrotic diseases include systemic sclerosis with skin fibrosis, liver fibrosis, pancreatic fibrosis, kidney fibrosis, myocardial fibrosis, and splenic fibrosis.

[0003] EFNB2 encodes the transmembrane protein Ephrin-B2, which belongs to the ephrin ligand family, proteins previously described as being associated with pro-migration activity. Studies have shown that the EFNB2 gene is upregulated in fibroblasts of patients with IPF and systemic sclerosis-associated interstitial lung disease compared to gene expression in healthy lung fibroblasts. Recent studies have reported that soluble Ephrin-B2 is a pro-fibrotic mediator in pulmonary fibrosis and skin fibrosis, demonstrating that mice lacking Ephrin-B2 in fibroblasts are protected from both. The pro-fibrotic role of Ephrin-B2 in myocardial fibrosis has also been reported to be mediated through the interaction of Stat3 with TGF-β / Smad3 signaling. Therefore, Ephrin-B2 is a potential therapeutic target for fibrotic diseases.

[0004] Recombinant protein vaccines have been widely developed for the prevention and treatment of various diseases and viral infections due to their superior safety, high purity, and ease of manufacture. However, there are currently few reports of recombinant protein vaccines prepared using Ephrin-B2 protein as an antigen for the prevention and treatment of fibrotic diseases. Summary of the Invention

[0005] To treat and prevent fibrotic diseases, this invention develops a recombinant Ephrin-B2 protein and vaccine based on Ephrin-B2 as a target, for the treatment and prevention of fibrotic diseases such as idiopathic pulmonary fibrosis, radiation-induced pulmonary fibrosis, systemic sclerosis skin fibrosis, liver fibrosis, and pancreatic fibrosis.

[0006] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows:

[0007] In a first aspect, the present invention provides a recombinant Ephrin-B2 protein for the prevention and / or treatment of fibrotic diseases, which contains an extracellular domain of the Ephrin-B2 protein.

[0008] Furthermore, the amino acid sequence of the extracellular segment of the Ephrin-B2 protein is as shown in SEQ ID No. 1 or SEQ ID No. 2, or has more than 80% homology with SEQ ID No. 1 or SEQ ID No. 2 and has the same or similar biological activities.

[0009] Furthermore, the Ephrin-B2 recombinant protein also contains an acidic domain of the SARS-CoV-2S2 subunit.

[0010] More preferably, the amino acid sequence of the acidic domain of the SARS-CoV-2S2 subunit is as shown in SEQ ID No. 3, or has more than 80% homology with SEQ ID No. 3 and has the same or similar biological activities.

[0011] More preferably, the amino acid sequence of the Ephrin-B2 recombinant protein is shown in SEQ ID No. 8 or SEQ ID No. 10.

[0012] In a second aspect, the present invention provides a protein precursor of recombinant Ephrin-B2 protein for the prevention and / or treatment of fibrotic diseases, wherein a signal peptide and / or protein tag are linked to the recombinant Ephrin-B2 protein.

[0013] Furthermore, the protein tag is selected from at least one of the following: histidine tag, Trx protein tag, glutathione transferase tag, ubiquitin-like modified protein tag, maltose-binding protein tag, c-Myc protein tag, Avi tag protein tag, and nitrogen-utilizing substance A protein tag.

[0014] Furthermore, a protease recognition region with a cleavage protein tag is also attached to the Ephrin-B2 recombinant protein for the prevention and / or treatment of fibrosis.

[0015] More preferably, the protease is selected from at least one of the following: enterokinase, TEV protease, thrombin, coagulation factor Xa, carboxypeptidase A, and rhinovirus 3c protease.

[0016] More preferably, the amino acid sequence of the protein precursor is selected from at least one of SEQ ID No. 7 or SEQ ID No. 9.

[0017] Furthermore, the Ephrin-B2 recombinant protein or protein precursor is obtained by codon optimization of the target gene encoding the Ephrin-B2 recombinant protein, ligating it to an expression vector to form a recombinant plasmid, and then introducing the recombinant plasmid into host cells for transfection, culture, expression, and purification.

[0018] Furthermore, the expression system is selected from at least one of the baculovirus-insect cell expression system, yeast cell expression system, or mammalian cell expression system.

[0019] More preferably, the baculovirus-insect cell expression vector is pFastBacI.

[0020] More preferably, the expression vector for the yeast cells is a Pichia pastoris expression vector.

[0021] More preferably, the Pichia pastoris expression vector is pPIC9k or pPICZα.

[0022] More preferably, the expression vector for mammalian cells is a CHO cell expression vector.

[0023] More preferably, the CHO cell expression vector is pTT5 or FTP-002.

[0024] Furthermore, the host cell containing the recombinant vector is an insect cell or a mammalian cell.

[0025] More preferably, the insect cells are selected from at least one of sf9 cells, sf21 cells, and Hi5 cells.

[0026] More preferably, the mammalian cell is a CHO cell.

[0027] Thirdly, the present invention provides an Ephrin-B2 recombinant protein vaccine for the prevention and / or treatment of fibrotic diseases, which contains the aforementioned Ephrin-B2 recombinant protein or protein precursor, as well as pharmaceutically acceptable adjuvant components.

[0028] Furthermore, the ratio of the protein antigen to the auxiliary component is 20:25 to 100 (g / L).

[0029] Furthermore, the auxiliary component is an immune adjuvant.

[0030] Preferably, the immune adjuvant is selected from at least one of the following: squalene oil-in-water emulsion, aluminum salt, calcium salt, plant saponins, plant polysaccharides, monophosphate lipid A, muramyl dipeptide, muramyl tripeptide, recombinant cholera toxin, GM-CSF cytokines, lipids, cationic liposome materials, and CpG ODN.

[0031] More preferably:

[0032] The squalene oil-in-water emulsion is an adjuvant similar to MF59.

[0033] The aluminum salt is selected from at least one of aluminum hydroxide and alum.

[0034] The calcium salt mentioned is tricalcium phosphate.

[0035] The plant saponins mentioned are QS-21 or ISCOM.

[0036] The plant polysaccharide mentioned is Astragalus polysaccharide.

[0037] The lipid is selected from at least one of the following: phosphatidylethanolamine, phosphatidylcholine, cholesterol, and dioleoylphosphatidylethanolamine. The cationic liposome material is selected from at least one of the following: (2,3-dioleoyloxypropyl)trimethylammonium chloride, N-[1-(2,3-dioleoylchloro)propyl]-N,N,N-trimethylamine chloride, cationic cholesterol, dimethyl-2,3-dioleenoyloxypropyl-2-(2-spermineformylamino)ethylammonium trifluoroacetate, trimethyldodecylammonium bromide, trimethyltetradecylammonium bromide, trimethylhexadecylammonium bromide, or dimethylbisoctadecylammonium bromide.

[0038] Fourthly, the present invention provides a pharmaceutical composition for the prevention and / or treatment of fibrotic diseases, comprising the above-mentioned Ephrin-B2 recombinant protein, protein precursor, or protein vaccine.

[0039] Furthermore, the dosage form of the Ephrin-B2 recombinant protein vaccine or pharmaceutical composition is an injection, nasal drop, spray, or inhaler.

[0040] Preferably, the injection route of the vaccine or drug composition is at least one of intramuscular injection, intravenous injection, subcutaneous injection, intradermal injection, intramyocardial injection, or intraperitoneal injection.

[0041] Fifthly, the present invention provides a method for preparing the above-mentioned Ephrin-B2 recombinant protein or protein precursor, comprising the following steps: codon optimization of the target gene encoding the Ephrin-B2 recombinant protein, ligation with an expression vector to form a recombinant plasmid, and introduction of the recombinant plasmid into a host cell for transfection, culture, expression and purification.

[0042] Furthermore, in the method for preparing the Ephrin-B2 recombinant protein or protein precursor, the expression system is selected from at least one of the baculovirus-insect cell expression system, yeast cell expression system, or mammalian cell expression system.

[0043] More preferably, the baculovirus-insect cell expression vector is pFastBacI.

[0044] More preferably, the expression vector for the yeast cells is a Pichia pastoris expression vector.

[0045] More preferably, the Pichia pastoris expression vector is pPIC9k or pPICZα.

[0046] More preferably, the expression vector for mammalian cells is a CHO cell expression vector.

[0047] More preferably, the CHO cell expression vector is pTT5 or FTP-002.

[0048] Furthermore, the host cell containing the recombinant vector is an insect cell or a mammalian cell.

[0049] More preferably, the insect cells are selected from at least one of sf9 cells, sf21 cells, and Hi5 cells.

[0050] More preferably, the mammalian cell is a CHO cell.

[0051] In a sixth aspect, the present invention provides the use of the above-described Ephrin-B2 recombinant protein, protein precursor, protein vaccine, or pharmaceutical composition in the preparation of medicaments for the prevention and / or treatment of fibrotic diseases.

[0052] Furthermore, the fibrotic disease is a disease caused by fibrosis, including at least one of pulmonary fibrosis, skin fibrosis, liver fibrosis, pancreatic fibrosis, kidney fibrosis, cardiac fibrosis, endometrial fibrosis, ocular fibrosis, splenic fibrosis, and myelofibrosis.

[0053] More preferably, the pulmonary fibrosis includes at least one of idiopathic pulmonary fibrosis, radiation-induced pulmonary fibrosis, secondary pulmonary fibrosis, hereditary pulmonary fibrosis, or other pulmonary fibrosis diseases.

[0054] More preferably, the pulmonary fibrosis is caused by a drug, said drug being at least one of bleomycin, amiodarone, or methotrexate.

[0055] More preferably, the skin fibrosis is systemic sclerosis skin fibrosis or keloid scars.

[0056] More preferably, the liver fibrosis disease is caused by viral hepatitis, alcoholic hepatitis, non-alcoholic hepatitis, cirrhosis, autoimmune diseases, fatty liver, malnutrition, chronic congestive heart failure, or drug-induced liver fibrosis, other liver fibrosis of unknown etiology, or diseases induced by liver fibrosis.

[0057] More preferably, the liver fibrosis is caused by a drug, wherein the drug is at least one of carbon tetrachloride or phosphorus.

[0058] More preferably, the pancreatic fibrosis is a disease induced by acute pancreatitis, chronic pancreatitis, pancreatic duct obstruction, chronic alcoholism, pancreatic ischemia, or pancreatic fibrosis.

[0059] More preferably, the pancreatic fibrosis is caused by a drug, namely, hyoscyamine.

[0060] The amino acid sequence involved in this invention is shown below:

[0061] SEQ ID NO.1: Human Ephrin-B2 (25-187aa)

[0062] SKSIVLEPIYWNSSNSKFLPGQGLVLYPQIGDKLDIICPKVDSKTVGQYEYYKVYMVDKDQADRCTIKKENTPLLNCAKPDQDIKFTIKFQEFSPNLWGLEFQKNKDYYIISTSNGSLEGLDNQEGGVCQTRAMKILMKVGQDASSAGSTRNKDPTRRPELEA

[0063] SEQ ID NO.2: Human Ephrin-B2 (25-164aa)

[0064] SKSIVLEPIYWNSSNSKFLPGQGLVLYPQIGDKLDIICPKVDSKTVGQYEYYKVYMVDKDQADRCTIKKENTPLLNCAKPDQDIKFTIKFQEFSPNLWGLEFQKNKDYYIISTSNGSLEGLDNQEGGVCQTRAMKILMKV

[0065] SEQ ID NO.3: SARS-CoV-2 acidic domain

[0066] DPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLI DLQE

[0067] SEQ ID NO.4: GP67 secretion signal peptide

[0068] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAAD

[0069] SEQ ID NO.5: Trx protein tag

[0070] SIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKV DVDECEDIATEYNINSMPTFVFVKNSKKIEEFSGANVDKLRNTIIKLKLAGSGSGHM

[0071] SEQ ID NO.6: TEV protease

[0072] ENLYFQ

[0073] SEQ ID NO.7: pFastBacI-GP67-Trx-6X His-TEV-Ephrin-B2(25-187aa)-AD protein expression design amino acid full sequence:

[0074] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSIHIKDSDDLKNRLAEAGDKLVVIDFMATWCGPCKMIGPKLDEMANEMSDSIVVLKVDVDECEDIATEYNINSMPTFVFVKNSKKIEEFSGANVDKLRNTIIKLKLAGSGSGHMHHHHHHSSGENLYFQSKSIVLEPIYWNSSNSKFLPGQGLVLYPQ IGDKLDIICPKVDSKTVGQYEYYKVYMVDKDQADRCTIKKENTPLLNCAKPDQDIKFTIKFQEFSPNLWGLEFQKNKDYYIISTSNGSLEGLDNQEGGV CQTRAMKILMKVGQDASSAGSTRNKDPTRRPELEADPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQE

[0075] SEQ ID NO.8: Ephrin-B2 (25-187aa)-AD amino acid sequence (i.e., the Ephrin-B2 protein vaccine antigen sequence of this invention)

[0076] SKSIVLEPIYWNSSNSKFLPGQGLVLYPQIGDKLDIICPKVDSKTVGQYEYYKVYMVDKDQADRCTIKKENTPLLNCAKPDQDIKFTIKFQEFSPNLWGLEFQKNKDYYIISTSNGSLEGLDNQEGGVCQTRAMKILMKVGQDASSAGSTRNKDPTRRPELEADPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQE

[0077] SEQ ID NO. 9: Full-length amino acid sequence designed for protein expression of pFastBacI-GP67-Ephrin-B2(25-164aa)-6×His:

[0078] MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADSKSIVLEPIYWNSSNSKFLPGQGLVLYPQIGDKLDIICPKVDSKTVGQYEYYKVYMVDKDQADRCTIKKENTPLLNCAKPDQDIKFTIKFQEFSPNLWGLEFQKNKDYYIISTSNGSLEGLDNQEGGVCQTRAMKILMKVHHHHHH

[0079] SEQ ID NO. 10: Ephrin-B2(25-164aa)-6×His (i.e., the Ephrin-B2 monomer protein sequence in the present invention)

[0080] SKSIVLEPIYWNSSNSKFLPGQGLVLYPQIGDKLDIICPKVDSKTVGQYEYYKVYMVDKDQADRCTIKKENTPLLNCAKPDQDIKFTIKFQEFSPNLWGLEFQKNKDYYIISTSNGSLEGLDNQEGGVCQTRAMKILMKVHHHHHH

[0081]

[0082] SEQ ID NO.12: Optimized GP67-Ephrin-B2(25-164aa)-6×His nucleotide sequence

[0083] ATGCTGCTGGTCAACCAGTCCCACCAGGGTTTCAACAAGGAACACACTAGCAAGATGGTGAGCGCCATCGTCCTGTACGTGCTGCTGGCTGCTGCCGCTCACTCCGCTTTCGCTGCCGACTCCAAGAGCATCGTCCTGGAACCCATCTACTGGAACTCCAGCAACTCCAAGTTCCTGCCCGGTCAGGGTCTGGTGCTGTACCCCCAGATCGGTGACAAGCTGGACATCATCTGCCCCAAGGTGGACAGCAAGACCGTGGGTCAGTACGAGTACTACAAGGTGTACATGGTCGATAAGGACCAGGCCGACCGTTGCACTATCAAGAAGGAAAACACCCCTCTGCTGAACTGCGCTAAGCCCGACCAGGACATCAAGTTCACTATCAAGTTCCAGGAATTTTCCCCTAACCTGTGGGGTCTGGAATTTCAGAAGAACAAGGACTACTACATCATCTCCACTAGCAACGGCTCCCTGGAGGGCCTGGACAACCAGGAGGGCGGTGTGTGCCAGACTCGTGCCATGAAGATCCTGATGAAGGTCCACCACCACCACCATCACTAA

[0084] Beneficial Effects: This invention uses the extracellular domain of Ephrin-B2 protein, which binds to a slightly acidic protein domain (AD) of the SARS-CoV-2 antigen, as the protein antigen. The amino acid sequence is shown in SEQ ID NO.8. A recombinant Ephrin-B2 protein capable of preventing and treating various fibrotic diseases was prepared. A highly safe adjuvant, preferably MF59-like adjuvant, was selected to prepare an Ephrin-B2 recombinant protein vaccine capable of preventing and treating various fibrotic diseases. Animal experiments show that the Ephrin-B2 recombinant protein vaccine prepared in this invention has a significant inhibitory effect on idiopathic pulmonary fibrosis, radiation-induced pulmonary fibrosis, and systemic sclerosis skin fibrosis in mouse models of lung and skin fibrosis treatment and prevention. It can significantly improve the survival rate of mice with lung and skin fibrosis and induce significant humoral and cellular immunity in mice, thus playing a preventive and therapeutic role in fibrotic diseases. Furthermore, this vaccine has excellent biocompatibility, providing a candidate for future clinical translation research of preventive and therapeutic vaccines for fibrotic diseases. Attached Figure Description

[0085] Figure 1 The diagram shows the design for the preparation of the Ephrin-B2 recombinant protein in Example 1: a) Schematic diagram of the construction of the Ephrin-B2 recombinant protein; b) Representative elution chromatogram of the Ephrin-B2-AD recombinant protein using a Superdex 200 Increase 10 / 300GL column (GE Healthcare); c) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) image of the Ephrin-B2-AD recombinant protein; d) Representative elution chromatogram of the Ephrin-B2 monomeric recombinant protein using a Superdex 200 Increase 10 / 300GL column (GE Healthcare); e) SDS-PAGE image of the Ephrin-B2 monomeric recombinant protein.

[0086] Figure 2 Figure 1 shows the results of treating BLM-induced pulmonary fibrosis in mice with the Ephrin-B2 recombinant protein vaccine in Example 2; a) BLM modeling and immunization timeline in the treated mice; b) Survival curve of mice after BLM modeling; c) Visual image of mouse lungs after sacrifice on day 27 after BLM modeling; d) Lung weight and lung coefficient of the treated pulmonary fibrosis model mice; e) Masson staining and Ashcroft score of lung tissue pathological sections from the treated pulmonary fibrosis model mice. Data are expressed as mean ± SD. Statistical differences: *P≤0.05, **P≤0.01, ***P≤0.001 and ****P≤0.0001.

[0087] Figure 3Figures showing the results of using the Ephrin-B2 recombinant protein vaccine in Example 3 to prevent BLM-induced pulmonary fibrosis in mice: a) Immunization and BLM modeling timeline in the prevention model mice; b) Percentage change in mouse body weight after BLM modeling; c) Visual image of mouse lungs after sacrifice on day 27 after BLM modeling; d) Lung weight and lung coefficient in the pulmonary fibrosis prevention model mice; e) Masson staining and Ashcroft score of lung tissue sections from the pulmonary fibrosis prevention model mice. Data are expressed as mean ± SD. Statistical differences: *P≤0.05, **P≤0.01, ***P≤0.001, and ****P≤0.0001.

[0088] Figure 4 Figure 4 shows the results of humoral and cellular immunity analysis in mice after immunization with the Ephrin-B2 recombinant protein vaccine: a) Ephrin-B2 antibody titers in mouse serum on days 21 and 27 post-vaccination; b) CD4+ levels in the spleen after vaccination. + T and CD8 + c) Changes in the proportion of T cells; c) Central memory (CD44) in the spleen after vaccination. + CD62L + CD4 + T and CD8 + Changes in T cells; d) Follicular helper T cells (CD3+) after vaccination + CD19 - CD4 + CXCR5 + PD-1 + Changes in ) ; e) Germinal center B cells ((CD3) after vaccination - CD19 + GL7 + CD95 + Changes in INF-γ were observed in: f) T cells secreting INF-γ 48 h after stimulation with 10 μg / mL Ephrin-B2 protein monomer (25-164 aa); g) T cells secreting Granzyme B 48 h after stimulation with 10 μg / mL Ephrin-B2 protein monomer (25-164 aa); h) INF-γ ElisaSpot cells stimulated with 10 μg / mL Ephrin-B2 protein monomer for 24 h. Data are expressed as mean ± SD. Statistical differences were defined as: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.

[0089] Figure 5Figures showing the results of adoptive immunotherapy based on Ephrin-B2 recombinant protein vaccine in Example 5 to alleviate pulmonary fibrosis in mice: a) Schematic diagram of T-cell adoptive therapy; b) Survival curve of mice treated with T-cell adoptive therapy; c) Percentage change in body weight of mice treated with T-cell adoptive therapy; d) Visual image of the lungs of mice treated with T-cell adoptive therapy; e) Lung weight and lung coefficient of mice treated with T-cell adoptive therapy; f) Masson staining of lung tissue pathological sections of mice treated with T-cell adoptive therapy; g) Schematic diagram of serum adoptive therapy; h) Survival curve of mice treated with serum adoptive therapy; i) Percentage change in body weight of mice treated with serum adoptive therapy; j) Visual image of mice treated with serum adoptive therapy; k) Lung weight and lung coefficient of mice treated with serum adoptive therapy; l) Masson staining of lung tissue pathological sections of mice treated with serum adoptive therapy.

[0090] Figure 6 Figure 6 shows the results of treating radiation-induced pulmonary fibrosis in mice with the Ephrin-B2 recombinant protein vaccine: a) Schematic diagram of immunization time and radiation-induced model; b) Mouse survival curve; c) Immunoblotting of fibrosis markers and immunoglobulins; d) Mouse lung weight and lung coefficient; e) Representative H&E, Masson, and histochemical staining of mouse lung tissue pathological sections; f) Hydroxy-CoA content.

[0091] Figure 7 Figure 7 shows the results of treating BLM-induced skin fibrosis in mice with the Ephrin-B2 recombinant protein vaccine: a) Schematic diagram of BLM-induced skin fibrosis and drug administration time; b) Statistical diagram of dermal thickness; c) Representative diagram of Masson staining of dermal layer.

[0092] Figure 8 Figure 8 shows the in vivo safety evaluation results of the Ephrin-B2 recombinant protein vaccine in Example 8: a) Schematic diagram of mouse immunization program; b) Complete blood cell count of mice, including white blood cells (WBC), neutrophils (NEUT), lymphocytes (LYMPH), monocytes (MONO), eosinophils (EOS), basophils (BASO), large unstained cells (LUC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin content (MCH), platelet count (PLT), and mean platelet volume (MPV).

[0093] Figure 9The image shows the serum biochemical parameters of mice used in the in vivo safety evaluation of the Ephrin-B2 recombinant protein vaccine in Example 8, including albumin-to-globulin ratio (AG), albumin (ALB), alkaline phosphatase (ALP), alanine aminotransferase (ALT), amylase (AMY), aspartate aminotransferase (AST), direct bilirubin (BILD), total bilirubin (BILT), total cholesterol (CHOL), creatine kinase-MB isoenzyme (CK-MB), creatinine (CREA), globulin (GLB), blood glucose (GLU), high-density lipoprotein cholesterol (HDL-C), lactate dehydrogenase (LDH), low-density lipoprotein cholesterol (LDL-C), total protein (TP), triglycerides (TRIGL), uric acid (UA), and urea (UREA).

[0094] Figure 10 The images show pathological sections of important organs from mice used in the in vivo safety evaluation of the Ephrin-B2 recombinant protein vaccine in Example 8, including the heart, liver, spleen, lungs, kidneys, skin, and muscle at the vaccine injection site, stained with H&E. The scale bar represents 400 μm. Detailed Implementation

[0095] 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.

[0096] Definitions:

[0097] Lipid monophosphate A (MPL), recombinant cholera toxin (rCTB), astragalus polysaccharide (APS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), cholesterol (Chol), dioleoylphosphatidylethanolamine (DOPE), (2,3-dioleoyloxypropyl)trimethylammonium chloride (DOTAP), N-[1-(2,3-dioleoylchloro)propyl]-N,N,N-trimethylamine chloride (DOTMA), cationic cholesterol (DC-Chol), trifluoroacetic acid dimethyl-2,3-dioleenoyloxypropyl-2-(2-spermineformylamino)ethylammonium (DOSPA), trimethyldodecylammonium bromide (DTAB), trimethyltetradecylammonium bromide (TTAB), trimethylhexadecylammonium bromide (CTAB), dimethylbisoctadecylammonium bromide (DDAB), CpG ODN (a nucleotide sequence containing unmethylated cytosine and guanine dinucleotides as the core sequence, artificially synthesized CpG).

[0098] The auxiliary components described in this invention refer to substances included in the dosage form other than the active ingredient. These auxiliary components possess certain physiological activities, but their addition does not alter the dominant role of the recombinant protein or pharmaceutical composition in the disease treatment process; rather, it merely exerts an auxiliary effect. These auxiliary effects are simply the utilization of the known activity of the component, a common adjunctive therapy method in the pharmaceutical field. If the aforementioned auxiliary components are used in combination with the protein or pharmaceutical composition of this invention, they should still fall within the scope of protection of this invention.

[0099] In some embodiments of the present invention, the extracellular domain of Ephrin-B2 is used as a vaccine antigen for the preparation of Ephrin-B2 recombinant protein vaccines.

[0100] In some specific embodiments of the present invention, the amino acid sequence of the extracellular segment of the Ephrin-B2 protein is the 25th-187th or 25th-164th amino acid of the extracellular segment of Ephrin-B2, and its amino acid sequence is as shown in SEQ ID NO.1 or SEQ ID NO.2, or has more than 80% homology with SEQ ID NO.1 or SEQ ID NO.2 and has the same or similar biological activities.

[0101] Preferably, the 80% or more homology and having the same or similar biological activity can be at least 85%, 90%, 95%, 98%, and 99% similarity to SEQ ID NO.1 or SEQ ID NO.2.

[0102] In some specific embodiments of the present invention, the extracellular domain (positions 25-187) of Ephrin-B2 is directly linked to an acidic domain AD (amino acids 1139-1202) of the SARS-CoV-2S2 subunit as the antigen of the Ephrin-B2 recombinant protein vaccine, and its specific amino acid sequence is shown in SEQ ID NO.8.

[0103] Due to its numerous advantages, including high safety, the insect cell-baculovirus expression vector system is widely used in research on recombinant protein expression, the production of human and veterinary vaccines, and pharmaceutical applications. Therefore, in some specific embodiments of this invention, the baculovirus insect expression system is used for protein purification and preparation.

[0104] MF59-like adjuvant is an oil-in-water emulsion used in vaccines. In 1997, the trivalent influenza vaccine Fluad, using MF59 as an adjuvant, was approved in Europe, becoming the first adjuvant to be included in novel human vaccines after aluminum adjuvants. MF59 adjuvant can induce a stronger humoral immune response and has superior immunogenicity than aluminum adjuvant. Although some studies have indicated that local reactions (such as redness and pain at the injection site) may occur after vaccination with MF59 adjuvant vaccines, these reactions are usually mild and transient. Large-scale use experience shows that the application of MF59 in influenza vaccines is safe and does not significantly increase the risk of serious adverse reactions. Therefore, in some specific embodiments of the present invention, MF59-like adjuvant is combined with Ephrin-B2 recombinant protein to prepare an Ephrin-B2 recombinant protein vaccine to enhance immunogenicity for the prevention and treatment of fibrotic diseases.

[0105] Fibrosis can be 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 lung tissue, leading to tissue remodeling and seriously affecting organ function.

[0106] The fibrotic diseases described in this invention include at least one of pulmonary fibrosis, skin fibrosis, liver fibrosis, pancreatic fibrosis, kidney fibrosis, cardiac fibrosis, endometrial fibrosis, ocular fibrosis, splenic fibrosis, or myelofibrosis.

[0107] Pulmonary fibrosis includes at least one of idiopathic pulmonary fibrosis, radiation-induced 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 drug-induced, and the drug is at least one of bleomycin, amiodarone, or methotrexate.

[0108] In one specific embodiment of the present invention, a mouse pulmonary fibrosis model was constructed by intratracheal instillation of bleomycin. Animal experiments showed that intramuscular injection of Ephrin-B2 recombinant protein vaccine can not only effectively prevent and treat BLM-induced pulmonary fibrosis, but also effectively stimulate humoral and cellular immune responses in mice, and can also alleviate pulmonary fibrosis in mice through adoptive immunotherapy.

[0109] Skin fibrosis is mainly systemic sclerosis dermatofibrosis (scleroderma) or keloids. In one specific embodiment of the present invention, intramuscular injection of the Ephrin-B2 recombinant protein vaccine can effectively treat BLM-induced systemic sclerosis dermatofibrosis.

[0110] 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 fibrotic process will continue and eventually develop into cirrhosis. Viral hepatitis, alcoholic hepatitis, non-alcoholic hepatitis, autoimmune diseases, fatty liver, malnutrition, chronic congestive heart failure, or drugs can all cause liver fibrosis. Furthermore, liver fibrosis also includes liver fibrosis of unknown etiology 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.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] Endometrial fibrosis is a fibrotic lesion of the endometrium caused by various reasons, such as endometriosis, as well as diseases induced by endometrial fibrosis.

[0115] 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.

[0116] Myelofibrosis includes idiopathic and drug-induced myelofibrosis, polycythemia vera, chronic myeloid leukemia, Hodgkin's disease, and diseases induced by myelofibrosis.

[0117] 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.

[0118] The main materials used in the following embodiments are as follows:

[0119] Bleomycin (HY-108345) used for establishing a mouse pulmonary fibrosis model was purchased from MedChemExpress. SIM-SF serum-free medium for culturing sf9 insect cells was purchased from Yiqiao Shenzhou, and RPMI 1640 medium used for cell culture was purchased from Gibco. Antibodies used for flow cytometry analysis were purchased from Biolegend. Lymphocyte separation medium was purchased from Dakota. CD3-positive T cell isolation kit was purchased from STEMCELL, Catalog #19851A. Male C57BL / 6 mice (6-10 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.

[0120] Example 1: Preparation of Ephrin-B2 recombinant protein

[0121] 1. Expression of recombinant Ephrin-B2 protein

[0122] The recombinant protein was prepared using the Bac-to-Bac baculovirus expression system (Invitrogen) through secretory expression.

[0123] 1) Ephrin-B2-AD protein expression sequence design: A slightly acidic protein domain (AD) (amino acid sequence shown in SEQ ID NO.1) from the SARS-CoV-2 antigen was linked to the C-terminus of human Ephrin-B2 (25-187 aa, amino acid sequence as shown in SEQ ID NO.1). This domain was then cloned into a modified pFastBacI vector. This vector contained a GP67 secretion signal peptide (amino acid sequence shown in SEQ ID NO.4) at the N-terminus. Following the GP67 signal peptide was a Trix protein tag (amino acid sequence shown in SEQ ID NO.5) tandemly to aid in the correct folding of the target protein. Following the Trix tag was a 6x His tag (HHHHHH) tandemly to aid in protein purification. Following the 6x His tag was a Tobacco Etch Virus (TEV) protease (amino acid sequence shown in SEQ ID NO.6) tandemly for tag excision. The construction diagram is shown below. Figure 1 As shown in a.

[0124] The pFastBacI containing the target gene was named pFastBacI-GP67-Trx-6x His-TEV-Ephrin-B2(25-187aa)-AD, with the amino acid sequence shown in SEQ ID No. 7. The target gene underwent codon optimization for an insect cell expression system (the optimized nucleotide sequence is shown in SEQ ID No. 11). After synthesis at Anhui General Biotechnology Co., Ltd., the gene was constructed between the BamHI and HindIII restriction sites of the pFastBacI vector.

[0125] 2) Design of Ephrin-B2 monomeric protein expression sequence: Human Ephrin-B2 (25-164 aa, amino acid sequence as shown in SEQ ID NO.2) was cloned into a modified pFastBacI vector containing a GP67 secretion signal peptide (amino acid sequence as shown in SEQ ID NO.4) at the N-terminus. The target gene Ephrin-B2 (25-164 aa) was tandemly linked after the GP67 signal peptide, followed by a 6x His protein tag for purification. The pFastBacI containing the target gene was named pFastBacI-GP67-Ephrin-B2(25-164 aa)-6x His, amino acid sequence as shown in SEQ ID NO.9. The target gene underwent codon optimization for an insect cell expression system (optimized nucleotide sequence as shown in SEQ ID NO.12). The gene was synthesized by Anhui General Biotechnology Co., Ltd. and constructed between the BamHI and HindIII restriction sites of the pFastBacI vector.

[0126] 3) The target genes from steps 1) and 2) were synthesized at Anhui General Biotechnology Co., Ltd. and constructed into the pFastBacI vector between the BamHI and HindIII restriction sites. After sequencing verification, the synthesized plasmid was transformed into competent DH10bac E. coli cells to produce bacmids. Correctly identified bacmids were extracted and transfected into Sf9 cells according to the instructions of the transfection reagent (Beyotime, catalog number: C0551). After transfection, Sf9 cells were cultured at 27℃ for 96 hours. The supernatant containing recombinant baculovirus was collected and labeled as P1 generation virus. The P1 generation virus was used to amplify Sf9 cells 2-3 times at a volume ratio of 1:20. The P4 generation virus was then added to a solution at a volume ratio of 1:30 to a density of 1.5 × 10⁻⁶ cells. 6 Sf9 cells in logarithmic growth phase were used for large-scale protein expression. The supernatant was collected 72 hours after the virus was added for protein purification.

[0127] 4) Protein purification: The collected culture supernatant was first purified using a 5 mL HisTrap exchange column (GE Healthcare). The pre-purified protein was then tagged with TEV protease at a ratio of 1 U: 1 mg protein at 4°C for 18 h. The tagged protein was removed again using a HisTrap exchange column. The flow-through liquid containing Ephrin-B2 protein was concentrated by ultrafiltration and further purified using a Superdex 200 Inrease 10 / 300GL chromatography column. The purified recombinant protein was subjected to SDS-PAGE electrophoresis and its purity was verified by Coomassie Brilliant Blue staining. Figure 1 b and c are representative elution chromatograms and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) images of Ephrin-B2-AD recombinant protein using a Superdex 200 Increase 10 / 300GL column (GE Healthcare), respectively. Figure 1 d and e are representative elution chromatograms and SDS-PAGE images of Ephrin-B2 monomeric recombinant protein using a Superdex 200 Increase 10 / 300GL column (GE Healthcare), respectively.

[0128] Through the above experimental procedures, high-purity Ephrin-B2-AD protein (amino acid sequence as shown in SEQ ID NO. 8) and Ephrin-B2 monomeric protein (amino acid sequence as shown in SEQ ID NO. 10) were finally obtained. Ephrin-B2-AD protein was used as an antigen with MF59 adjuvant to prepare an Ephrin-B2 recombinant protein vaccine, which was used for subsequent studies such as animal immunization described below. All vaccines used below were prepared from Ephrin-B2-AD antigen (SEQ ID NO. 8). Ephrin-B2 monomeric protein was only used for antibody coating in specific antibody detection ELISA experiments and as an antigen stimulus in Ephrin-B2 specific T-cell immunoassay.

[0129] Example 2: Efficacy study of Ephrin-B2 recombinant protein vaccine in treating bleomycin (BLM)-induced pulmonary fibrosis

[0130] To evaluate the efficacy of the Ephrin-B2 recombinant protein vaccine in treating pulmonary fibrosis in mice, male C57 mice aged 9-10 weeks (weighing 24-26g) and SPF-grade were purchased from Vital River Pharmaceuticals. After acclimatizing to an SPF environment for one week, the mice were divided into different dosage groups according to their weight, such as a 25g dosage group (24.5g-25.5g) and a 26g dosage group (25.5g-26.5g). BLM was prepared at a modeling dose of 3mg / kg, with a modeling volume of 50μL per mouse. The BLM concentration corresponding to the 25g dosage group was 1.5mg / mL, the 26g dosage group was 1.56mg / mL, the 27g dosage group was 1.62mg / mL, and the 28g dosage group was 1.68mg / mL. The model was established by intratracheal infusion of BLM after isoflurane anesthesia. The modeling time was recorded as day 0, and immunizations were administered on days 1, 8, and 15. The dosage, grouping, and immunization methods of the Ephrin-B2 recombinant protein vaccine are as follows:

[0131] Normal control group: No bleomycin model was established, and no medication was administered;

[0132] Model group: Bleomycin was used to establish the model; 100 μL PBS was injected intramuscularly for immunization;

[0133] Adjuvant group: Bleomycin model was established; 50 μL MF59 + 50 μL PBS was administered intramuscularly for immunization;

[0134] Vaccine group: Bleomycin model was established; 50μL MF59 + 20μg Ephrin-B2-AD recombinant protein was administered via intramuscular injection for immunization.

[0135] After modeling, mouse weight and survival were monitored. Mice were sacrificed on day 27, and lung tissue was isolated for pathological assessment. Figure 2 As shown in b, the survival rate of mice in the vaccine group was significantly improved compared to the model group and the adjuvant group. Figure 2 The lung weight and lung coefficient (lung coefficient = lung wet weight / body weight * 100%) of the vaccine group shown in d were significantly lower than those of the model group. Figure 2 e represents the Masson staining results for lung pathology. In the BLM-induced pulmonary fibrosis treatment model, the collagen in the pulmonary fibrosis tissue of mice in the vaccine group was significantly lower than that in the model group and the adjuvant group.

[0136] Example 3: Efficacy experiment of Ephrin-B2 recombinant protein vaccine in preventing BLM-induced pulmonary fibrosis

[0137] To evaluate the efficacy of the Ephrin-B2 recombinant protein vaccine in preventing pulmonary fibrosis in mice, SPF-grade 6-week-old mice were purchased from Vital River Pharmaceuticals. After acclimatization in an SPF environment, mice were immunized on days 0, 14, and 21. On day 28, mice were divided into different dosage groups based on their body weight, for example, a 25g dosage group (24.5g-25.5g) and a 26g dosage group (25.5g-26.5g). The drug was prepared at a dosage of 3mg / kg and a modeling volume of 50uL per mouse. The corresponding BLM concentrations were 1.5mg / mL for the 25g dosage group, 1.56mg / mL for the 26g dosage group, 1.62mg / mL for the 27g dosage group, and 1.68mg / mL for the 28g dosage group. The model was established by intratracheal instillation of BLM after isoflurane anesthesia. The dosage, grouping, and immunization methods of the Ephrin-B2 recombinant protein vaccine are as follows:

[0138] Normal control group: No bleomycin model was established, and no medication was administered;

[0139] Model group: Bleomycin was used to establish the model; 100 μL PBS was injected intramuscularly for immunization;

[0140] Adjuvant group: Bleomycin model was established; 50 μL MF59 + 50 μL PBS was administered intramuscularly for immunization;

[0141] Vaccine group: Bleomycin model was established; 50μL MF59 + 20μg Ephrin-B2-AD recombinant protein was administered via intramuscular injection for immunization.

[0142] After modeling, mouse weight and survival were monitored. Mice were sacrificed on day 27, and lung tissue was isolated for pathological assessment. Figure 3 As shown in b, regardless of whether it is the model group, adjuvant group, or vaccine group, the mice's weight first decreased and then recovered after BLM modeling, indicating that intratracheal infusion of BLM caused acute damage to mice in the short term and the modeling was stable and uniform. Figure 3 The lung weight and lung coefficient (lung coefficient = lung wet weight / body weight * 100%) of the vaccine group shown in d were significantly lower than those of the model group. Figure 3 e represents the Masson staining results for lung pathology. In the BLM-induced pulmonary fibrosis prevention model, the collagen in the pulmonary fibrosis tissue of mice in the vaccine group was significantly lower than that in the model group and the adjuvant group.

[0143] Example 4: Analysis of humoral and cellular immunity in mice after immunization with Ephrin-B2 recombinant protein vaccine

[0144] To evaluate the immune response activated in mice following immunization with the Ephrin-B2 recombinant protein vaccine, SPF-grade 6-week-old mice were purchased from Vital River and acclimatized to an SPF environment. They were then immunized via intramuscular injection on days 0, 14, and 21. The groups and immunization doses were as follows:

[0145] Control group: 100 μL PBS, administered via intramuscular injection;

[0146] Adjuvant group: 50 μL MF59 + 50 μL PBS, administered via intramuscular injection;

[0147] Vaccine group: 50μL MF59 + 20μg Ephrin-B2-AD recombinant protein, administered via intramuscular injection.

[0148] On day 21 (after two doses of vaccine) and day 27 (after three doses of vaccine), orbital blood was collected for antibody titer testing. After incubating at room temperature for 2 hours, the collected orbital blood was centrifuged at 6000 rpm for 10 minutes at 4°C. The supernatant serum was used for ELISA antibody detection. Ephrin-B2 monomeric protein (25-164 aa) was diluted at a concentration of 5 μg / mL in carbonate coating buffer, and 100 μL / well was added to uncoated ELISA plates. The plates were coated overnight at 4°C. The next day, the serum was serially diluted and used for ELISA. A positive result was defined as a mean OD value of the current dilution of the sample being tested being greater than or equal to the cutoff value. The cutoff value was calculated as 2.1 × the mean OD value of the unimmunized serum at the current dilution (if less than 0.05, it was calculated as 0.05). The results are as follows: Figure 4 As shown in a, the antibody titer of Ephrin-B2 in mouse serum increased significantly on days 21 and 27 after vaccination.

[0149] Mice were sacrificed on day 28, and inguinal lymph nodes and spleens were isolated for humoral and cellular immune analysis. Lymphocytes were isolated from the spleen using lymphocyte separation medium, and CD4 counts in the spleen after vaccination were further detected using flow cytometry. + and CD8 + Changes in the proportion of T cells Figure 4 b) Central memory T cells ( Figure 4 c) Activation of follicular helper T cells (Tfh) (4d) and germinal center B cells (GC B) (4e). To further investigate the activation of cellular immunity by the vaccine, lymphocytes isolated from the spleen were uniformly treated with Ephrin-B2 monomeric protein (25-164aa) at a concentration of 10 μg / mL / 10 6Cells were incubated at various concentrations for 48 hours and then collected for flow cytometry analysis. Golgi blocking reagent was added 8 hours before cell collection. Collected cells underwent cell viability and extracellular marker staining, followed by fixation and perforation for intracellular cytokine staining. Results showed that the vaccine group secreted INF-γ (…). Figure 4 f) and Granzyme B ( Figure 4 The proportion of T cells in all groups was significantly increased. INF-γ ELISA was performed according to the MABTECH kit instructions (catalog number: 3321-4HST-2), with an Ephrin-B2 monomeric protein stimulation concentration of 10 μg / mL and a stimulation time of 24 h. Figure 4 h).

[0150] Example 5: Efficacy of adoptive immunotherapy based on Ephrin-B2 recombinant protein vaccine in alleviating pulmonary fibrosis in mice.

[0151] To evaluate the mechanism of action of the Ephrin-B2 recombinant protein vaccine in treating pulmonary fibrosis, serum and T cells from the Ephrin-B2-AD recombinant protein vaccine were adopted to treat pulmonary fibrosis. Donor mice were immunized with the recombinant protein vaccine on days 0, 14, and 21. Donor mice were divided into two groups, immunized with MF59 adjuvant and the Ephrin-B2-AD recombinant protein vaccine, respectively. Mice were sacrificed on day 27, and CD3-positive T cells from serum and spleen were aseptically isolated and intravenously transferred to fibrotic recipient mice. Adoption was performed three times, on days 3, 6, and 9 post-modeling.

[0152] The grouping and immunization methods for T-cell adoptive mice are as follows:

[0153] Normal control group: No bleomycin model was established, and no medication was administered;

[0154] Model group: Bleomycin was used to establish the model, and 100 μL of sterile PBS was transferred via the tail vein.

[0155] Adjuvant group - T cells: Bleomycin model was established, with 2 x 10^6 adoptive adjuvant donors via tail vein. 3 CD3 + T cells;

[0156] Vaccine group - T cells: Bleomycin model was established, with 2 x 10⁻⁶ vaccine donors via tail vein adoption. 3 CD3 + T cells.

[0157] The grouping and immunization methods for adoptive mice are as follows:

[0158] Normal control group: No bleomycin model was established, and no medication was administered;

[0159] Model group: Bleomycin was used to establish the model, and 200 μL of sterile PBS was transferred via the tail vein.

[0160] Adjuvant group-serum: Bleomycin model was established, and 200 μL of serum was transferred from the tail vein to the adjuvant donor group;

[0161] Vaccine group-serum: Bleomycin model was established, and 200 μL of serum from the adoptive vaccine donor group was transferred via tail vein.

[0162] After establishing the recipient mouse model, the mice's body weight and survival were monitored. On day 27, the mice were sacrificed, and lung tissue was isolated for pathological assessment. For example... Figure 5 As shown, T cells in mice immunized with adoptive Ephrin-B2 recombinant protein preparation ( Figure 5 af) and serum ( Figure 5 Both gI and gI can improve the survival rate of mice and reduce the occurrence and development of pulmonary fibrosis in mice.

[0163] Example 6: Efficacy experiment of Ephrin-B2 recombinant protein vaccine in treating radiation-induced pulmonary fibrosis

[0164] To evaluate the efficacy of the Ephrin-B2 recombinant protein vaccine in treating radiation-induced pulmonary fibrosis in mice, 10-week-old (uniformly weighted) male C57 mice of SPF grade were purchased from Vital River and fed to an average weight of 30 g / mouse in an SPF environment. After anesthesia, a 20 Gry local radiation exposure was performed on a 1 cm x 1 cm area of ​​the mouse's chest. The modeling time was recorded as day 0. Intramuscular immunizations were administered at week 1 (day 7), week 3 (day 21), week 5 (day 35), and week 9 (day 45). The dosage, grouping, and immunization methods of the Ephrin-B2 recombinant protein vaccine are as follows:

[0165] Normal control group: No radiation modeling was performed, and no drugs were administered;

[0166] Model group: Radiation-induced modeling; immunization via intramuscular injection of 100uL PBS;

[0167] Adjuvant group: Radiation modeling was performed; 50 μL MF59 + 50 μL PBS was administered intramuscularly for immunization;

[0168] Vaccine group: Radiation modeling was performed; 50 μL MF59 + 20 μg Ephrin-B2-AD recombinant protein was administered via intramuscular injection for immunization.

[0169] After modeling, survival was monitored. Mice were sacrificed at week 24 post-modeling, and lung tissue was isolated for pathological assessment. Figure 6 mouse survival curve and Figure 6As shown in the lung weight and lung coefficient of mice at week 24, the survival rate of mice in the vaccine group was significantly improved compared with the model group and the adjuvant group. Figure 6 c. Fibrosis markers, immunoproteins, immunoblotting Figure 6 Representative images of lung tissue pathology in e mice and Figure 6 As shown in the figure of hydroxyproline content in mouse lungs, compared with the irradiation model group, the collagen in the pulmonary fibrosis tissue of the vaccine group was significantly lower than that of the model group and the adjuvant group, indicating that the vaccine significantly improved radiation-induced pulmonary fibrosis.

[0170] Example 7: Efficacy study of Ephrin-B2 recombinant protein vaccine in treating BLM-induced skin fibrosis in systemic sclerosis.

[0171] To evaluate the efficacy of the Ephrin-B2 recombinant protein vaccine in treating BLM-induced systemic sclerosis cutaneous fibrosis in mice, 8-week-old (uniform weight) male BALB / c mice of SPF grade were purchased from Vital River and acclimatized to an SPF environment for one week. After shaving the back, a 1cm x 1cm square dosing zone was marked on the back of each mouse. BLM (100μg / 100μl / mouse / day) was injected subcutaneously from each of the four corners daily for 28 consecutive days to model systemic sclerosis. The first day of subcutaneous BLM injection was designated as day 0. Intramuscular immunizations were administered on days 2, 9, and 16. The dosage, grouping, and immunization methods for the Ephrin-B2 recombinant protein vaccine are as follows:

[0172] Normal control group: Subcutaneous injection of physiological saline (100 μl / animal / day), no other medication;

[0173] Model group: Model established by subcutaneous injection of BLM (100μg / 100μl / animal / day); immunized by intramuscular injection of 100μL PBS;

[0174] Adjuvant group: Model was established by subcutaneous injection of BLM (100μg / 100μl / animal / day); immunization was performed by intramuscular injection of 50μL MF59 + 50μL PBS;

[0175] Vaccine group: Model was established by subcutaneous injection of BLM (100μg / 100μl / animal / day); 50μL MF59 + 20μg Ephrin-B2-AD recombinant protein was administered via intramuscular injection.

[0176] Mice were subcutaneously injected with BLM for 28 consecutive days, and their condition was observed daily. On day 28, the mice were sacrificed, and skin from the modeling area was collected, fixed, and used for Masson staining to observe dermal thickness. Figure 7As shown in b and 7c, the dermal thickness in the model group was significantly higher than that in the control group, indicating that the skin hardening model was successfully constructed; the dermal thickness in the vaccine group was significantly lower than that in the model group, indicating that the vaccine has a certain therapeutic effect on BLM-induced skin fibrosis.

[0177] Example 8: Safety Evaluation of Ephrin-B2 Recombinant Protein Vaccine

[0178] To assess the safety of the Ephrin-B2 recombinant protein vaccine, SPF-grade 6-week-old mice were purchased from Vital Rivers. After acclimatization to an SPF environment, the mice were immunized intramuscularly three times according to the prophylactic vaccine immunization protocol: a normal control group (physiological saline), an adjuvant group (50 μL MF59 + 50 μL PBS), and a vaccine group (50 μL MF59 + 20 μg Ephrin-B2-AD recombinant protein). One week after the third immunization, fresh anticoagulated whole blood and serum were collected. Complete blood cell counts were performed. Figure 8 ) and serum biochemical tests ( Figure 9 There were no statistically significant differences among the normal control group, adjuvant group, and vaccine group. H&E staining of pathological sections showed no obvious pathological changes in the heart, liver, spleen, lungs, kidneys, skin, and muscles of the immunized mice. Figure 10 The above results indicate that the vaccine has good safety.

Claims

1. A recombinant Ephrin-B2 protein for the prevention and / or treatment of fibrotic diseases, characterized in that, The SARS-CoV-2 protein contains an extracellular domain of Ephrin-B2 protein and an acidic domain of the S2 subunit of SARS-CoV-2. The amino acid sequence of the extracellular domain of Ephrin-B2 protein is shown in SEQ ID NO.1 or SEQ ID NO.

2. The amino acid sequence of the acidic domain of the S2 subunit of SARS-CoV-2 is shown in SEQ ID NO.

3.

2. The Ephrin-B2 recombinant protein according to claim 1, characterized in that, The amino acid sequence of the Ephrin-B2 recombinant protein is shown in SEQ ID NO.

8.

3. A protein precursor of recombinant Ephrin-B2 protein for the prevention and / or treatment of fibrotic diseases, characterized in that, A signal peptide and / or protein tag are attached to the Ephrin-B2 recombinant protein of claim 1.

4. The protein precursor according to claim 3, characterized in that, The protein tag is selected from at least one of the following: histidine tag, Trx protein tag, glutathione transferase tag, ubiquitin-like modified protein tag, maltose-binding protein tag, c-Myc protein tag, Avi tag protein tag, and nitrogen source utilization substance A protein tag.

5. The protein precursor according to claim 4, characterized in that, The Ephrin-B2 recombinant protein also has a protease recognition region with a protein tag removed; the protease is selected from at least one of the following: enterokinase, TEV protease, thrombin, coagulation factor Xa, carboxypeptidase A, and rhinovirus 3c protease.

6. The protein precursor according to claim 3, characterized in that, The amino acid sequence of the protein precursor is shown in SEQ ID NO.

7.

7. The Ephrin-B2 recombinant protein according to claim 1, characterized in that, The Ephrin-B2 recombinant protein is obtained by codon optimization of the target gene encoding the Ephrin-B2 recombinant protein or protein precursor, ligating it to an expression vector to form a recombinant plasmid, and then introducing the recombinant plasmid into host cells for transfection, culture, expression, and purification.

8. The Ephrin-B2 recombinant protein according to claim 7, characterized in that, The expression system is selected from at least one of the baculovirus-insect cell expression system, yeast cell expression system, or mammalian cell expression system.

9. The Ephrin-B2 recombinant protein according to claim 8, characterized in that, The baculovirus-insect cell expression vector is pFastBacI; the yeast cell expression vector is Pichia pastoris expression vector pPIC9k or pPICZα; and the mammalian cell expression vector is CHO cell expression vector pTT5 or FTP-002.

10. The Ephrin-B2 recombinant protein according to claim 7, characterized in that, The host cell is an insect cell or a mammalian cell.

11. The Ephrin-B2 recombinant protein according to claim 10, characterized in that, The insect cells are selected from at least one of sf9 cells, sf21 cells, and Hi5 cells; the mammalian cells are CHO cells.

12. An Ephrin-B2 recombinant protein vaccine for the prevention and / or treatment of fibrotic diseases, characterized in that, It contains the recombinant Ephrin-B2 protein as described in any one of claims 1-2 and 7-11, or the protein precursor as described in any one of claims 3-6, and pharmaceutically acceptable auxiliary components.

13. The Ephrin-B2 recombinant protein vaccine according to claim 12, characterized in that, The auxiliary component is an immune adjuvant; the immune adjuvant is selected from at least one of the following: squalene oil-in-water emulsion, aluminum salt, calcium salt, plant saponins, plant polysaccharides, monophosphate lipid A, muramyl dipeptide, muramyl tripeptide, recombinant cholera toxin, GM-CSF cytokines, lipids, cationic liposome materials, and CpG ODN.

14. The Ephrin-B2 recombinant protein vaccine according to claim 13, characterized in that, Satisfy at least one of the following: The squalene oil-in-water emulsion is an MF59-like adjuvant. The aluminum salt is selected from at least one of aluminum hydroxide and alum; The calcium salt mentioned is tricalcium phosphate; The plant saponins mentioned are QS-21 or ISCOM; The plant polysaccharide mentioned is Astragalus polysaccharide; The lipids are selected from at least one of the following: phosphatidylethanolamine, phosphatidylcholine, cholesterol, and dioleoylphosphatidylethanolamine; The cationic liposome material is selected from at least one of the following: (2,3-dioleoyloxypropyl)trimethylammonium chloride, N-[1-(2,3-dioleoylchloro)propyl]-N,N,N-trimethylamine chloride, cationic cholesterol, trifluoroacetic acid dimethyl-2,3-dioleenoyloxypropyl-2-(2-speramidino)ethylammonium, trimethyldodecylammonium bromide, trimethyltetradecylammonium bromide, trimethylhexadecylammonium bromide, or dimethylbisoctadecylammonium bromide.

15. A pharmaceutical composition for the prevention and / or treatment of fibrotic diseases, characterized in that, A vaccine containing the Ephrin-B2 recombinant protein as described in any one of claims 1-2, 7-11, the protein precursor as described in any one of claims 3-6, or the recombinant protein vaccine as described in any one of claims 12-14.

16. The Ephrin-B2 recombinant protein vaccine according to any one of claims 12 to 14, characterized in that, Protein vaccines are available in injection, nasal drops, spray, or inhalation formulations.

17. The Ephrin-B2 recombinant protein vaccine according to claim 16, characterized in that, The injection route is at least one of the following: intramuscular injection, intravenous injection, subcutaneous injection, intradermal injection, intramyocardial injection, or intraperitoneal injection.

18. The pharmaceutical composition according to claim 15, characterized in that, The dosage form of the drug composition is injection, nasal drops, spray, or inhaler.

19. The pharmaceutical composition according to claim 18, characterized in that, The injection route is at least one of the following: intramuscular injection, intravenous injection, subcutaneous injection, intradermal injection, intramyocardial injection, or intraperitoneal injection.

20. A method for preparing the Ephrin-B2 recombinant protein according to any one of claims 1-2 and 7-11, or the protein precursor according to any one of claims 3-6, characterized in that, The process includes the following steps: after codon optimization, the target gene encoding the recombinant Ephrin-B2 protein or protein precursor is ligated to an expression vector to form a recombinant plasmid. The recombinant plasmid is then introduced into host cells for transfection, culture, expression, and purification.

21. Use of the Ephrin-B2 recombinant protein according to any one of claims 1-2, 7-11, the protein precursor according to any one of claims 3-6, the recombinant protein vaccine according to any one of claims 12-14, 16-17, or the pharmaceutical composition according to any one of claims 15, 18-19 in the preparation of a medicament for the prevention and / or treatment of fibrotic diseases.

22. The use according to claim 21, characterized in that, The fibrotic diseases include at least one of pulmonary fibrosis, skin fibrosis, liver fibrosis, pancreatic fibrosis, kidney fibrosis, cardiac fibrosis, endometrial fibrosis, ocular fibrosis, splenic fibrosis, and myelofibrosis.

23. The use according to claim 22, characterized in that, The pulmonary fibrosis includes at least one of idiopathic pulmonary fibrosis, radiation-induced pulmonary fibrosis, secondary pulmonary fibrosis, hereditary pulmonary fibrosis, or other pulmonary fibrosis diseases; the skin fibrosis disease is systemic sclerosis skin fibrosis or keloids.

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