WO2023225802A1 - 三叶因子2/干扰素α2融合蛋白及其防治病毒感染性疾病的应用 - Google Patents

三叶因子2/干扰素α2融合蛋白及其防治病毒感染性疾病的应用 Download PDF

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WO2023225802A1
WO2023225802A1 PCT/CN2022/094509 CN2022094509W WO2023225802A1 WO 2023225802 A1 WO2023225802 A1 WO 2023225802A1 CN 2022094509 W CN2022094509 W CN 2022094509W WO 2023225802 A1 WO2023225802 A1 WO 2023225802A1
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fusion protein
tff2
ifnα2
seq
peptide
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PCT/CN2022/094509
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French (fr)
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徐建青
张晓燕
翟贯星
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复旦大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins

Definitions

  • This application belongs to the field of biomedical technology, and specifically relates to the fusion protein of trefoil factor 2 (TFF2) and interferon ⁇ 2 (IFN ⁇ 2), its preparation and its application in the treatment and prevention of viral infection diseases.
  • TNF2 trefoil factor 2
  • IFN ⁇ 2 interferon ⁇ 2
  • Viral infections cause great harm to the life and health of humans and mammals, among which respiratory virus infections and enterovirus infections are the most common acute viral infections.
  • Enteroviruses are a class of positive-sense single-stranded RNA viruses associated with human and mammalian diseases that are transmitted through the intestines. Enteroviruses affect millions of people around the world each year and are often found in the respiratory secretions (such as saliva, sputum or nasal mucus) and feces of infected people.
  • Infections can cause a variety of symptoms, including: mild respiratory illness (common cold), hand, foot and mouth disease, acute hemorrhagic conjunctivitis, aseptic meningitis, myocarditis, severe neonatal sepsis-like illness, acute flaccid paralysis, and related of acute flaccid myelitis.
  • Both respiratory and enteroviruses are mucosal-infecting viruses. Although the characteristics and infection modes of different viruses are not completely consistent, their common pathogenic mechanisms are still traceable: on the one hand, direct virus infection causes target cell apoptosis or necrosis, damaging the structure and function of normal mucosa; on the other hand, the virus passes through Regulates the host immune response, inhibits the production of type I interferon and its signal transduction pathway, induces the secretion of a large number of inflammatory cytokines and chemokines, generates an inflammatory storm, recruits a large number of immune cells to infiltrate into mucosal tissue, severely damages the mucosal structure, and causes mucosal damage.
  • glucocorticoid is a type of steroid hormone.
  • Glucocorticoids are part of the feedback mechanism of the immune system and can reduce certain aspects of immune function, thereby effectively suppressing inflammation.
  • side effects are also significant, and currently used glucocorticoid drugs have non-selective effects that impair many healthy anabolic processes.
  • the side effects of long-term use of this drug include: iatrogenic hyperadrenocorticism, inducing or exacerbating infection or transferring potential infectious lesions in the body, causing peptic ulcers, inducing pancreatitis and fatty liver, iatrogenic adrenocortical insufficiency, Induces schizophrenia and epilepsy, femoral head necrosis, etc.
  • interferon As an antiviral drug, interferon has been widely used in various viral infections when used alone or in combination with other therapies. It has inhibitory effects on many currently known viruses. As a classic antiviral drug, IFN ⁇ 2 has been widely used clinically. Its ⁇ -2b spray has significant efficacy in treating acute upper respiratory tract infections in children and can effectively improve the clinical symptoms and signs of children (Chen Qing et al., Recombinant Analysis of the efficacy of human interferon ⁇ -2b spray in the treatment of acute upper respiratory tract infections in children, Biomedical Engineering and Clinical.
  • ⁇ -2b spray uses a low dose of 20 ⁇ g combined with oseltamivir to treat influenza A, It can downregulate the levels of cellular inflammatory factors, increase the virus negative conversion rate, promote symptom improvement, and is highly safe (Xu Guangfeng, evaluation of the efficacy of recombinant human interferon ⁇ -2b spray combined with oseltamivir in the treatment of influenza A, Shanxi Health Care Journal of the Academy of Sciences. 2020, 30(04)). Animal experiments and clinical studies have shown that the combination of IFN ⁇ 2 and ribavirin can effectively inhibit the replication of MERS-CoV and improve MERS symptoms and clinical outcomes.
  • a single subcutaneous dose of 1 ⁇ g/kg PEG IFN-alfa-2b + standard treatment can reduce the duration of viral disappearance and significantly improve clinical outcomes compared with standard treatment (Anuja Pandit, Efficacy and safety of pegylated interferon alfa-2b in moderate COVID- 19: A phase II, randomized, controlled, open-label study, Int J Infect Dis. 202104; 105: 516-521).
  • TFF2 As a small molecule peptide secreted by the gastrointestinal tract, TFF2 can participate in mucosal repair. Although it is overexpressed during inflammation, adding TFF2 will produce an anti-inflammatory effect, help create a microenvironment required for tissue repair, and promote tissue repair ( Abdelaziz Ghanemi et al., Trefoil factor family member 2(TFF2) as an inflammatory-induced and anti-inflammatory tissue repair factor, Animals (Basel). 2020 Sep 14; 10(9): 1646.). In preliminary studies, we found that TFF2, a host-secreted peptide, can reduce pathological damage, promote lung tissue damage repair, exert a protective effect, and improve the prognosis of influenza virus infection by inhibiting inflammatory responses (CN105582526B).
  • the present disclosure provides an effective active substance that can be used more safely and effectively for the prevention and/or treatment of viral infections, its application in pharmaceuticals, and methods for preventing and treating diseases.
  • a fusion protein comprising one or more fusion units, each fusion unit comprising:
  • TFF2 trefoil factor 2
  • interferon alpha 2 (IFN alpha 2) element comprising an IFN alpha 2 peptide or an active fragment thereof
  • the TFF2 element and the IFN ⁇ 2 element are fused at a molecular ratio of 1:1, and the TFF2 element is located at the N-terminus of the IFN ⁇ 2 element in each fusion unit.
  • the TFF2 peptide or active fragment thereof is derived from humans, primates, rodents (such as mice, rats, guinea pigs, hamsters), dogs, and cats.
  • the TFF2 peptide or active fragment thereof comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 8, SEQ ID NO: 12, or an active fragment thereof (e.g., with SEQ ID NO: 8 and SEQ ID NO: 12 Amino acid sequences with at least 80% sequence identity and TFF2 activity).
  • the TFF2 peptide or active fragment thereof is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 11, or an active fragment thereof (e.g., identical to SEQ ID NO :7 or SEQ ID NO:11 has at least 80% sequence identity and is capable of encoding an active TFF2 peptide nucleic acid molecule).
  • the IFNa2 peptide or active fragment thereof is derived from humans, primates, rodents (such as mice, rats, guinea pigs, hamsters), dogs, and cats.
  • the IFNa2 peptide or active fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 14, or an active fragment thereof (e.g., with SEQ ID NO: 10 and SEQ ID NO: 14 Amino acid sequences with at least 80% sequence identity and IFN ⁇ 2 activity).
  • the IFN ⁇ 2 peptide or active fragment thereof is encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: SEQ ID NO: 9, SEQ ID NO: 13, or an active fragment thereof (e.g., identical to SEQ ID NO :9 or SEQ ID NO:13 a nucleic acid molecule with at least 80% sequence identity and capable of encoding active IFN ⁇ 2 peptide).
  • the fusion protein further includes a linker connecting the TFF2 element to the IFN ⁇ 2 element and/or the constituent peptides in the element.
  • the linker is a flexible linker comprising n amino acid residues, where n is an integer from 2 to 300.
  • the linker is a glycine or glycine/serine linker. In some embodiments, the linker is selected from the amino acid sequence of Gn , (GS) n , (GGS) n , (GGGS) n , (GGGGS) n , or (GGGGGS) n , where n is 1, 2, 3, 4 , 5, 6, 7, 8, 9 or 10 integers.
  • the fusion protein contains one or more TFF2 peptides arranged contiguously or spaced apart and/or one or more IFNa2 peptides arranged contiguously or spaced apart.
  • the fusion protein further comprises an Fc region that does not contain mutations. In some embodiments, the fusion protein contains one or more mutations that reduce antibody-mediated ADCC and CDC activity. In some embodiments, the fusion protein contains the amino acid mutations D265A and N297G according to EU numbering.
  • the fusion protein also contains a signal peptide.
  • the signal peptide in the fusion protein is selected from the group consisting of tPA2 signal peptide, TFF2 signal peptide, IL-2 signal peptide, bPRL signal peptide, and CD33 signal peptide.
  • the fusion protein also contains labels, such as labels used for purification, detection, and localization, such as selected from fluorescent labels, non-radioactive nuclide labels, biotin-based labels, phosphorylation modification labels, Peptide tags.
  • labels such as labels used for purification, detection, and localization, such as selected from fluorescent labels, non-radioactive nuclide labels, biotin-based labels, phosphorylation modification labels, Peptide tags.
  • the fusion protein has the amino acid sequence of SEQ ID NO: 2 or has at least 80% sequence identity with said sequence. In some embodiments, the fusion protein is encoded by a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 or a nucleic acid molecule that has at least 80% sequence identity with said sequence.
  • an isolated nucleic acid molecule or a construct or vector comprising the nucleic acid molecule wherein the nucleic acid molecule encodes a fusion protein of the disclosure.
  • the nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 1 or has at least 80% sequence identity with said sequence.
  • the nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO: 2 or having at least 80% sequence identity with the amino acid sequence.
  • the vector is selected from viral vectors, mRNA vectors, and DNA vectors.
  • a cell comprising the fusion protein of any one of claims 1 to 7, or the nucleic acid molecule, construct or vector of claims 8 or 9.
  • composition comprising a fusion protein, nucleic acid molecule, construct, vector, and/or cell of the present disclosure; and a carrier.
  • fusion proteins in the preparation of for the prevention and/or treatment of viral infectious diseases.
  • a method for preventing and/or treating viral infectious diseases comprising administering to a subject in need a prophylactically or therapeutically effective amount of the fusion protein, nucleic acid molecule, construct of the present disclosure , vectors, cells, compositions or drugs containing the aforementioned substances.
  • fusion proteins, nucleic acid molecules, constructs, vectors, cells, compositions and/or medicaments of the present disclosure are provided for use in preventing and/or treating viral infectious diseases.
  • the viral infectious disease is selected from acute viral infections, such as respiratory viral infections and enteroviral infections.
  • the viral infectious disease is caused by one or more viruses selected from the group consisting of coronavirus, influenza virus, rhinovirus, adenovirus, parainfluenza virus, respiratory syncytial virus, coxsackie virus, Echovirus, novel enterovirus.
  • the fusion proteins, nucleic acid molecules, constructs, vectors, cells, compositions and/or medicaments of the present disclosure are administered as prophylactic drugs before viral infection occurs to prevent the occurrence of viral infection or reduce subsequent viral infection.
  • the severity of the infection is administered as prophylactic drugs before viral infection occurs to prevent the occurrence of viral infection or reduce subsequent viral infection.
  • the fusion proteins, nucleic acid molecules, constructs, vectors, cells, compositions and/or medicaments of the present disclosure are administered as therapeutic drugs after a viral infection has occurred to reduce the severity of the viral infection and disease.
  • the fusion proteins, nucleic acid molecules, constructs, vectors, cells, compositions and/or medicaments of the present disclosure serve as both prophylactic and therapeutic agents, either continuously or at intervals before and after the occurrence of a viral infection. Medication.
  • the fusion proteins, nucleic acid molecules, constructs, vectors, cells, compositions and/or medicaments of the present disclosure are in a form suitable for administration selected from the group consisting of: respiratory aerosol inhalation, nasal instillation, spray , oral, intramuscular and/or intravenous administration.
  • the fusion proteins, nucleic acid molecules, constructs, vectors, cells, compositions, and/or medicaments of the present disclosure are suitable for use alone or in combination with other antiviral drugs, immunological drugs, or viral therapies.
  • Figure 1 Construction of fusion protein expression vector and in vitro expression:
  • Figure 1A Construction map of the eukaryotic expression vectors pSV1.0 IFN ⁇ 2-TFF2-Fc, pSV1.0 TFF2-IFN ⁇ 2-Fc and pSV1.0 2xTFF2-IFN ⁇ 2-Fc for TFF2 and IFN ⁇ 2 fusion proteins;
  • Figure 1B Expression of TFF2 and IFN ⁇ 2 fusion proteins with different signal peptides in 293F suspension cell line and CHO-K1 cells, where Blank is the control cell, Cell is the cell, Sup is the supernatant, and the signal peptides are TFF2 and IL respectively.
  • Blank is the control cell
  • Cell is the cell
  • Sup is the supernatant
  • the signal peptides are TFF2 and IL respectively.
  • Figure 2 Verification of the fusion protein of TFF2 and IFN ⁇ 2 and its impact on viral protein expression and replication.
  • Figure 2A Verification of the fusion protein of TFF2 and IFN ⁇ 2 on polyacrylamide gel electrophoresis (PAGE) after purification;
  • FIG. 2B&2C Effect of TFF2 and IFN ⁇ 2 fusion protein on the expression of interferon-induced transmembrane protein 3 (IFITM3) protein in the lung epithelial cell line A549;
  • IFITM3 interferon-induced transmembrane protein 3
  • Figure 2D&2E Effect of TFF2 and IFN ⁇ 2 fusion protein on the replication of influenza virus PR8 in the lung epithelial cell line A549 in vitro.
  • FIG. 3 Effect of TFF2 and IFN ⁇ 2 fusion protein on the expression of virus-induced inflammation-related factors.
  • FIG. 3A Effect of TFF2 and IFN ⁇ 2 fusion protein on PR8-induced COX-2 expression
  • FIG. 3B Effect of TFF2 and IFN ⁇ 2 fusion protein on IL-6 expression caused by PR8;
  • Figure 3C Effect of TFF2 and IFN ⁇ 2 fusion protein on LPS-induced iNOS expression in the lung epithelial cell line A549 in vitro.
  • FIG. 4 Protective effects of TFF2-IFN ⁇ 2-Fc and IFN ⁇ 2-TFF2-Fc on animals challenged with influenza virus.
  • FIG. 4A Challenge and administration models of TFF2-IFN ⁇ 2-Fc and IFN ⁇ 2-TFF2-Fc influenza viruses
  • FIG. 4B Survival rate curve and body weight change curve of mice after PR8 infection (TFF2-IFN ⁇ 2-Fc);
  • Figure 4C Survival rate curve and body weight change curve of mice after PR8 infection (IFN ⁇ 2-TFF2-Fc);
  • FIG. 4D Challenge and administration models of TFF2-IFN ⁇ 2-Fc and 2xTFF2-IFN ⁇ 2-Fc influenza viruses
  • FIG. 4E Survival rate curve of mice after PR8 infection (TFF2-IFN ⁇ 2-Fc, 2xTFF2-IFN ⁇ 2-Fc);
  • Figure 4F Body weight change curve of mice after PR8 infection (TFF2-IFN ⁇ 2-Fc, 2xTFF2-IFN ⁇ 2-Fc).
  • the inventors have constructed various forms of TFF2 and IFN ⁇ 2 fusion proteins, and tested and compared the antiviral, inflammation-reducing, animal protection effects and other functions of individual TFF2 or IFN ⁇ 2 polypeptides and various fusion proteins. and screening, overcoming various technical difficulties and obtaining a specific fusion protein form with correct structure and excellent effects.
  • the present disclosure provides a TFF2 and IFN ⁇ 2 fusion protein with a specific structure and ratio, and verifies its excellent preventive and therapeutic effects on viral infectious diseases.
  • the fusion protein of the present application has both excellent antiviral effect and the effect of inhibiting excessive inflammation. Its effect is significantly better than that of TFF2 or IFN ⁇ 2 alone, has a synergistic effect, and is also significantly better than other structures and ratios of TFF2 and IFN ⁇ 2. fusion protein.
  • TFF2 polypeptide and IFN ⁇ 2 polypeptide were fused and expressed in various forms, the successful expression of the fusion protein was verified through in vitro experiments, and the inhibitory effect of TFF2 and IFN ⁇ 2 fusion protein on influenza virus replication and its effect on inflammation were tested. Decreasing effect of sex factor secretion. Further studies in the mouse influenza infection model found that aerosol inhalation of TFF2 and IFN ⁇ 2 fusion protein can improve the survival rate of influenza-infected mice and reduce the weight loss of mice.
  • the fusion protein containing TFF2 polypeptide and IFN ⁇ 2 polypeptide fused at a molecular ratio of 1:1, with the TFF2 polypeptide located at the N-terminus of IFN ⁇ 2, has the most excellent effect.
  • the test results show that the anti-viral and excessive inflammation-inhibiting effects of this specific type of fusion protein in vivo and in vitro are significantly better than those of TFF2 or IFN ⁇ 2 alone. It has a synergistic effect and is also significantly better than other structures and ratios of TFF2 and IFN ⁇ 2 fusion proteins.
  • the inventors constructed the eukaryotic expression vectors pSV1.0 IFN ⁇ 2-TFF2-Fc, pSV1.0 TFF2-IFN ⁇ 2-Fc and pSV1 .0 2xTFF2-IFN ⁇ 2-Fc, express it in 293T, use Western Blot to verify the expression of the fusion protein, and express it in 293F cells, collect the supernatant, and then use Pure was used for purification, Coomassie Brilliant Blue was used to identify the purity of the purified protein, and the protein concentration was determined by BCA.
  • Western blot detected the effect of TFF2 and IFN ⁇ 2 fusion protein on the replication of influenza virus PR8 in the lung epithelial cell line A549 in vitro, proving that the fusion protein can reduce virus replication.
  • mice were infected with influenza virus H1N1 strain PR8 strain, and the weight changes and survival rate of mice after PR8 infection were observed. It was found that the TFF2 and IFN ⁇ 2 fusion protein treatment group could significantly improve the survival rate of mice, reduce weight loss, and improve the symptoms of highly pathogenic influenza infection. On this basis, we conducted a preventive experiment with TFF2 and IFN ⁇ 2 fusion protein to further verify its preventive and protective effects against influenza virus H1N1 strain PR8 strain challenge. These results fully prove that TFF2 and IFN ⁇ 2 fusion protein plays an important protective role in acute viral infection injury models induced by respiratory viruses or other factors.
  • TFF2 and IFN ⁇ 2 fusion protein Since the mechanism of tissue damage caused by other acute viral infections is similar to that caused by influenza virus infection, the protective effect of TFF2 and IFN ⁇ 2 fusion protein is not limited to respiratory tissue damage caused by influenza virus, including respiratory tissue damage caused by other acute viral infections, and Intestinal diseases such as enteroviruses that cause acute symptoms.
  • preventive administration of disease control personnel and high-risk groups involved in the management of acute viral infection epidemics can effectively reduce the risks and damage suffered by the above-mentioned personnel.
  • this application is further improved and optimized on the basis of previous research.
  • TFF2 and IFN ⁇ 2 in a specific form, it can exert multi-molecule and multi-functional effects at the same time, and the effect is significantly better than administering a single drug separately.
  • the formation of a single molecule through fusion can not only play a multifunctional role, but also reduce the complexity of multi-protein combinations, improve efficacy, and play a dual role of antiviral and reducing inflammation.
  • the fusion protein of TFF2 and IFN ⁇ 2 of a single molecule the antiviral, anti-inflammatory and protective functions of the fusion protein in viral infectious diseases, especially acute viral infectious diseases, were analyzed.
  • the specific fusion protein of the present disclosure targets the common pathogenic mechanism of acute viral infection.
  • the two fusion components complement each other and unexpectedly exert an effect that is significantly better than what can be obtained by giving the two components alone.
  • TFF2 is a host small molecule polypeptide that is highly conserved in different species.
  • the mature human TFF2 molecule consists of 106 amino acids, has a molecular weight of approximately 12kD, and contains two symmetrical polypeptides of approximately 40 amino acid residues. It consists of a special conserved sequence, including 6 cysteine residues forming three intra-chain disulfide bonds (cys1-cys5, cys2-cys4 and cys3-cys6), resulting in a specific and stable clover-shaped structure that is acid-resistant and resistant to It is heat-resistant and resistant to protease hydrolysis, which has great advantages in transportation.
  • IFN ⁇ 2 is also a polypeptide secreted by the host and has been widely used clinically. Therefore, the fusion expression of TFF2 and IFN ⁇ 2 not only has clear activity and good safety, but also has a high drug yield, and has high application potential in the prevention and treatment of acute respiratory virus infections and enterovirus infections.
  • This disclosure proposes for the first time an intervention strategy for TFF2 and IFN ⁇ 2 fusion protein, which adopts a three-in-one strategy of antiviral + anti-inflammatory + repair promotion, which can be used for broad-spectrum prevention and treatment of acute infectious diseases caused by viral infections.
  • the fusion protein can not only be administered systemically, but can also be administered locally by atomization and inhalation. It is highly targeted, has quick effects, has few side effects, and can exert better effects.
  • the disclosed product has low cost and is easy to promote and apply in economically weak countries or regions. It can also be used as a technical reserve for the country to prevent and control infectious diseases caused by new emergent viruses, and has high economic value. social value and political significance.
  • the present disclosure provides a fusion protein of trefoil factor 2 and interferon ⁇ 2.
  • the fusion protein can inhibit the replication of the virus, reduce tissue inflammation, reduce tissue damage, and promote the functional repair of lung tissue. It can be used to prepare treatments and/or Or drugs to prevent acute viral infections, which can significantly improve the prognosis caused by acute viral infections.
  • the present disclosure provides a fusion protein based on TFF2 and IFN ⁇ 2.
  • the inventor has proved through experiments that the TFF2 and IFN ⁇ 2 fusion protein molecule can play a protective role in influenza virus PR8 infection, reduce the morbidity and mortality, and alleviate the inflammatory symptoms of respiratory tract infection, thereby being able to respond to the epidemic of new respiratory viral infections. It plays an important role in the prevention and treatment of viral infections and severe infections for which there are no effective treatments.
  • the TFF2 and IFN ⁇ 2 fusion protein targets the common pathogenic mechanism of acute viral infection and exerts an antiviral effect by inhibiting the inflammatory response, promoting mucosal tissue repair, reducing tissue damage, and inhibiting the replication function of the virus.
  • the protective effect of TFF2 and IFN ⁇ 2 fusion protein is not limited to respiratory tissue damage caused by influenza virus, including damage caused by other viral infections.
  • Preventive treatment should be provided to disease control personnel and high-risk groups involved in the management of acute viral infection epidemics. Medicine can effectively reduce the risk and damage suffered by the above-mentioned people.
  • mammal may include humans, primates, rodents (eg, rats, mice, guinea pigs, hamsters), domesticated animals, or livestock mammals.
  • the term "element” refers to an amino acid sequence that forms part of a fusion protein.
  • the term “unit” refers to the basic fragment of an element constituting a function.
  • the TFF2 element of the fusion protein may contain one or more contiguous or spaced TFF2 units, each of which may produce the desired TFF2-related functional activity.
  • TFF2 element and “TFF2 protein (polypeptide)" are used interchangeably and refer to a natural (eg, mammalian source), recombinant or synthetic TFF2 polypeptide sequence that forms part of a fusion protein.
  • Natural TFF2 is highly conserved in mammals, has a specific and stable clover-shaped structure, and has certain mucosal repair and inflammation inhibitory effects.
  • TFF2 polypeptides also include naturally occurring variants and fragments of TFF2 (eg, splice variants or allelic variants), as well as non-naturally occurring variants with native TFF2 activity.
  • TFF2 polypeptides are known. See, for example, GenBank accession numbers (human Gene ID: 7032; mouse Gene ID: 21785).
  • the TFF2 element herein may comprise the amino acid sequence of SEQ ID NO: 8 (human TFF2) or be encoded by a nucleic acid molecule comprising SEQ ID NO: 7, comprise the amino acid sequence of SEQ ID NO: 12 (mouse TFF2) or comprise
  • the nucleic acid molecule encoding described in SEQ ID NO: 11 may be a homologous sequence with the same or similar activity as these proteins (for example, homologous sequences can be obtained through databases or comparison software known in the art), variants, or modified form.
  • the TFF2 polypeptide can be selected from: (a) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 8 or 12 (for example, a polypeptide with a sequence shown in SEQ ID NO: 8 or 12); or (b) in ( a) A protein or polypeptide derived from (a) that has the activity of mucosal repair and inflammation inhibition through substitution, deletion or addition of one or several amino acids in the defined amino acid sequence.
  • IFN ⁇ 2 polypeptide
  • IFN ⁇ 2 protein polypeptide
  • IFN ⁇ 2 polypeptides known in the art can be used in this application, as well as natural variants and fragments thereof (eg, splice variants or allelic variants), as well as non-natural variants with IFN ⁇ 2 activity.
  • nucleotide and amino acid sequences of native IFN[alpha]2 polypeptides are known. See, for example, GenBank accession numbers (human Gene ID: 3440; mouse Gene ID: 15965).
  • the IFN ⁇ 2 protein used herein may comprise the amino acid sequence of SEQ ID NO: 10 (human IFN ⁇ 2) or be encoded by a nucleic acid molecule comprising SEQ ID NO: 9, may comprise the amino acid sequence of SEQ ID NO: 14 (mouse IFN ⁇ 2) or Encoded by the nucleic acid molecule comprising SEQ ID NO: 13, or may be a homologous sequence with the same or similar activity as these proteins (for example, homologous sequences can be obtained through databases or comparison software known in the art), variations body or modified form.
  • the IFN ⁇ 2 polypeptide can be selected from: (a) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 10 or 14 (for example, a polypeptide with a sequence shown in SEQ ID NO: 10 or 14); or (b) in ( a) A protein or polypeptide derived from (a) that has the activity of mucosal repair and inflammation inhibition through substitution, deletion or addition of one or several amino acids in the defined amino acid sequence.
  • the TFF2 polypeptide element and IFN ⁇ 2 polypeptide element in the fusion protein of the present disclosure are preferably encoded by human genes or their homologous genes or family genes.
  • Variant forms of proteins or polypeptides in the present disclosure include (but are not limited to): one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, optimally 1 to 20) Deletion, insertion and/or substitution of 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acids, as well as the addition of one or several ( Usually within 20, preferably within 10, more preferably within 5) amino acids.
  • substitutions with amino acids with similar or similar properties generally do not alter the function of the protein or polypeptide.
  • adding one or several amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein or polypeptide.
  • the fusion protein may or may not include the initial methionine residue while still having its desired properties. required viral infection prevention and treatment activity.
  • Variant forms of polypeptides include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and proteins encoded by sequences that can hybridize to their protein coding sequences under high or low stringency conditions.
  • the protein or polypeptide of the invention may be glycosylated or may be non-glycosylated.
  • fusion protein refers to an amino acid molecule comprising at least one TFF2 polypeptide and at least one IFNa2 polypeptide fused.
  • the fusion protein of the present disclosure can be produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher animals, insect and mammalian cells; preferably eukaryotic hosts) using recombinant technology, or can also be produced artificially, for example, using full-sequence synthesis. Or fragments are synthesized and spliced together.
  • the TFF2 peptide in the fusion protein herein can be connected to the N-terminal or C-terminal of the IFN ⁇ 2 peptide.
  • the TFF2 peptide can be connected to the N-terminal (ie, upstream) of the IFN ⁇ 2 peptide.
  • the fusion protein herein may include one or more TFF2 peptides and/or IFN ⁇ 2 peptides, for example, TFF2 peptide and IFN ⁇ 2 peptide are fused at a molecular ratio of 5:1 to 1:5, such as 1:1 or 2:1. Fusion is preferred in a 1:1 molecular ratio.
  • the TFF2 peptide in the fusion protein herein is connected to the N-terminus of the IFN ⁇ 2 peptide, and the TFF2 peptide and the IFN ⁇ 2 peptide are fused in a molecular ratio of 1:1.
  • the fusion protein of the present application may also include an Fc region.
  • Fc region or “Fc fragment” refers to the Fc segment of an immunoglobulin used in a fusion protein.
  • the Fc region has substantially the same amino acid sequence as a native or variant immunoglobulin Fc fragment and has substantially the same biological activity as the native Fc fragment.
  • the Fc region may also contain a hinge region.
  • the Fc region can be derived from, for example, IgG or IgA.
  • the Fc segment of the fusion protein can extend the half-life of the functional protein in plasma, improve the stability of the molecule, specifically bind to Fc receptors in the body, and exert corresponding biological functions. effect.
  • the Fc segment can specifically bind to protein A, simplifying the purification steps of Fc fusion proteins, and is of great significance in the development and preparation of related biological products.
  • the Fc region used in the fusion proteins herein may be free of mutations, or may contain one or more mutations, such as mutations that reduce antibody-mediated ADCC and CDC activity, such as the amino acid mutations D265A and N297G/N297Q according to EU numbering.
  • the fusion protein herein may also include a signal peptide, such as an amino acid sequence that has the function of guiding the secretion, localization and/or delivery of the fusion protein, and is generally 5-50 amino acids in length.
  • the signal peptide can be selected from, for example: tPA2 signal peptide, TFF2 signal peptide, IL-2 signal peptide, bPRL signal peptide CD33 protein signal peptide, etc.
  • the fusion protein herein may also include labels, such as labels used for purification, detection, and localization, such as selected from fluorescent labels, non-radioactive nuclide labels, biotin-based labels, phosphorylation modification labels, and peptide tags.
  • labels such as labels used for purification, detection, and localization, such as selected from fluorescent labels, non-radioactive nuclide labels, biotin-based labels, phosphorylation modification labels, and peptide tags.
  • Each polypeptide element or peptide unit within an element in the fusion protein herein can be connected through a linker.
  • Flexible linkers are preferably used in this application to allow interactions between polypeptide elements or peptide units.
  • the linker that can be used in the fusion protein of the present application can contain 2 to 300 amino acid residues, such as 5 to 100, 10 to 50, or 15 to 3 amino acid residues.
  • Exemplary linkers may be glycine linkers, such as (G) n or glycine/serine linkers, such as the amino acid sequence of (GS) n , (GGS) n , (GGGS) n , (GGGGS) n or (GGGGGS) n , where n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the fusion protein of the present invention can easily prepare using various known methods. These methods include but are not limited to: recombinant DNA methods, artificial synthesis, etc. [See Murray KM, Dahl SLAnn; Pharmacother 1997 Nov; 31(11): 1335-8].
  • the fusion proteins herein can be produced by direct synthesis of peptides using solid-phase techniques, or the fragments of the fusion protein can be chemically synthesized separately and then chemically linked to produce the full-length molecule.
  • vectors for producing fusion proteins are also contemplated herein.
  • vector and “recombinant expression vector” are used interchangeably and refer to bacterial plasmids, phages, yeast plasmids, animal cell viruses, mammalian cell viruses or other vectors well known in the art, which can be expressed in host cells. Copy and express the target protein.
  • expression vectors containing fusion protein coding sequences and appropriate transcription/translation control signals can be used to construct expression vectors containing fusion protein coding sequences and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc.
  • the DNA sequence can be effectively linked to an appropriate promoter in an expression vector to direct mRNA synthesis.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • pSV1.0 vector, pcDNA3.1 vector, pIRES2-EGFP vector, and AdMaxTM expression system can be used herein.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green color for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green color for eukaryotic cell culture.
  • GFP Fluorescent protein
  • tetracycline or ampicillin resistance in E. coli tetracycline or ampicillin resistance in E. coli.
  • Vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins or polypeptides.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as an animal cell.
  • Representative examples include: animal cells, such as 293F cells, CHO cells, etc.; Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast.
  • Enhancers are DNA cis-acting factors, usually about 10 to 300 base pairs in length, that act on promoters to enhance gene transcription. Those of ordinary skill in the art will know how to select appropriate vectors, promoters, enhancers and host cells.
  • the fusion protein can be expressed within the cell or on the cell membrane or secreted out of the cell.
  • the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitating agents (salting out method), centrifugation, pure method, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.
  • This article also provides a product, which contains an effective amount of the fusion protein herein, a vector containing a molecule encoding the fusion protein, a host cell, and a pharmaceutically or physiologically acceptable carrier.
  • active substance refers to the fusion protein herein, its encoding nucleic acid molecule, a construct or vector containing the nucleic acid molecule, a host cell, or a composition of the foregoing.
  • the products herein are useful for preventing or treating diseases associated with viral infections and/or symptoms thereof.
  • the term “contains” or “includes” includes “comprising,” “consisting essentially of,” and “consisting of.”
  • the term “pharmaceutically acceptable” ingredients are substances suitable for use in humans and/or animals without undue adverse side effects (e.g., toxicity, irritation, and allergic reactions), i.e., with a reasonable benefit/risk ratio.
  • the term “effective amount” refers to an amount that produces a function or activity in humans and/or animals and is acceptable to humans and/or animals.
  • the term "pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent, including various excipients and diluents. This term refers to pharmaceutical carriers that do not themselves require the active ingredient and are not unduly toxic upon administration. Suitable carriers are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington’s Pharmaceutical Sciences, Mack Pub. Co., N.J. 1991.
  • compositions may contain liquids such as water, saline, glycerin and ethanol.
  • these carriers may also contain auxiliary substances, such as fillers, disintegrants, lubricants, glidants, effervescent agents, wetting agents or emulsifiers, flavoring agents, pH buffering substances, etc.
  • these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous medium, wherein the pH is usually about 5-8, preferably about 6-8.
  • unit dosage form refers to the preparation of the composition of the present invention into a dosage form required for a single administration for convenience of administration, including but not limited to various solid dosage forms (such as tablets, lyophilized powders), liquids dosage forms (such as solutions), capsules, and sustained-release formulations.
  • the composition is in unit dosage form or multiple dosage form, and the content of the active substance is 0.01 to 2000 mg/dose, preferably 0.1 to 1500 mg/dose, and more preferably 1 to 1000 mg/dose.
  • 1 to 6 doses of the composition of the present invention are administered every day, preferably 1 to 3 doses; most preferably, the dose taken per day is 1 dose.
  • the pharmaceutical composition of the present invention can be made into various dosage forms according to needs, and the doctor can determine the dose that is beneficial to the patient based on factors such as the patient's type, age, weight, general disease status, administration method, etc., through respiratory atomization inhalation, instillation, etc. Administer by nasal, spray, oral, intramuscular and/or intravenous administration.
  • the active substances or products of the present invention can be used in combination with each other, and can also be combined with other drugs and treatment methods for the prevention and treatment of infectious diseases (especially acute viral infections).
  • infectious diseases especially acute viral infections
  • other drugs or methods clinically used for the treatment of acute viral infections can be used simultaneously or successively.
  • the other drugs or methods include but are not limited to: Prevent further damage, regulate local area function, anti-inflammation, administer glucocorticoids, non-steroidal anti-inflammatory drugs NSAID, etc.
  • Example 1 Construction and design of fusion protein plasmids and eukaryotic expression
  • the gene sequence is first cloned based on the amino acid sequence of human TFF2 (as shown in SEQ ID NO: 8) and the amino acid sequence of human IFN ⁇ 2 protein (as shown in SEQ ID NO: 10), and the different forms of recombinant plasmids are After transfection into 293T cells, the eukaryotic expression of the TFF2 and IFN ⁇ 2 fusion protein was detected by WB, and then expressed in large quantities in 293F cells. The expression supernatant was harvested and purified through HiTrap MabSelect SuRe columns. The purity of the target protein was identified after collection. , ultrafiltrate and replace with PBS to obtain highly pure TFF2 and IFN ⁇ 2 fusion protein. The specific steps are as follows:
  • TFF2 has different repeat clones, and a human Fc fragment is added at the end ( Figure 1A).
  • the recombinant plasmid with correct sequencing was transfected into 293T cells.
  • the transfection reagent was TurboFect, and the culture medium was DMEM complete culture medium (10% FBS and 1% P/S). After culturing in the 37°C incubator for 24 hours, the cells were taken out and collected.
  • SDS loading buffer collect the cells into EP tubes, wash the cells with PBS buffer, add loading buffer, heat in a boiling water bath for 10 minutes to denature the protein, centrifuge briefly and then coagulate through SDS-polyacrylic acid amine Proteins were separated by gel electrophoresis (SDS-PAGE), and the concentration of the separation gel was 10%.
  • the electrophoresis voltage is 70V and the time is 30 to 40 minutes (marked by the separation of markers). After the bromophenol blue migrates to the position of the separation gel, adjust the voltage to 110V until the bromophenol blue migrates to the bottom of the gel. Then proceed Rapid transfer, constant current 400mA, time 50 minutes.
  • Example 2 Purification of fusion protein and in vitro antiviral and anti-inflammatory functions
  • the correctly sequenced TFF2 and IFN ⁇ 2 recombinant plasmids were transfected into 293F suspension cells. Specifically, use SMM 293-TI serum-free medium to suspend culture 293F cells, add 1% penicillin/streptomycin antibiotics to the medium; inoculate into fresh medium at a density of 5x10 cells/mL each time, and culture When the density reaches 3x10 6 cells/mL or above, experiments or passages are carried out. The cell shaker is filled with 5% carbon dioxide and the rotation speed is 125 rpm/min. The cell density when transiently transfected with fusion protein particles is 1x10 6 cells/mL. Use PEI for transfection.
  • the ratio of DNA:PEI is 1:3.5.
  • plasmid in a 1.5mL EP tube, dilute it with 150mM sodium chloride to 40ng/ ⁇ L, mix well, add PEI, and vortex. After thorough mixing, incubate at room temperature for 15 to 30 minutes, and add DNA/PEI mix to the cells; 24 hours later, add anti-cell clumping agent (Anti-Clumping Agent, Thermo Fisher, 0010057AE) at a ratio of 1:1000; culture on a cell shaker. Collect the supernatant and cells after 5 to 7 days. After WB detection, the fusion protein was mainly secreted in the supernatant, so the supernatant was directly used for purification.
  • anti-cell clumping agent Anti-Clumping Agent, Thermo Fisher, 0010057AE
  • a protein purifier Pure purifies the fusion protein, using HiTrap MabSelect SuRe 1mL column, operating by running UNICORN software, and filtering all buffers with 0.22 ⁇ m filter membrane. First, clean the pump and pipeline with ultrapure water, then connect the column and clean it, and rinse the ethanol with at least 5 times the volume of distilled water. Binding buffer (0.02M sodium phosphate, 0.15M NaCl, pH 7.2) was used for column equilibration through the A1 line, and the B1 pump was filled with elution buffer (0.1M sodium citrate, pH 3.0). After equilibrium, load the sample through pipeline A1.
  • the sample is also centrifuged at 12000xg and filtered with a 0.22 ⁇ m filter before loading. Load the sample at a rate of 0.5 mL/min and collect the flow-through liquid. After loading the sample, wash with binding buffer until the baseline is flat, then switch to B1 for elution with 100% eluent, collect the eluent until the baseline is basically flat, add 100 to 200 ⁇ L of medium to each 1.5 mL EP tube and solution (Tris-HCl, pH 9.0).
  • a BSA standard curve was made using the BCA quantitative kit method. Based on the optical density (OD) value of the standard, the concentration of TFF2 and IFN ⁇ 2 fusion protein was quantified and aliquoted. Then freeze at -80 degrees.
  • IFITM3 interferon-induced transmembrane protein 3
  • WB Figure 2B
  • IFITM3 belongs to the interferon-inducible gene ISG, which can be induced by interferon and viruses.
  • the fusion protein can stimulate the expression of IFITM3, indicating that the fusion protein can induce interferon activity.
  • IFN ⁇ 2-TFF2-Fc can induce strong IFITM3 expression
  • TFF2-IFN ⁇ 2-Fc and 2xTFF2-IFN ⁇ 2-Fc have slightly weaker IFITM3 expression levels. Further combined with subsequent in vivo and in vitro experiments to determine whether the interferon-inducing activity of the fusion protein is moderate.
  • COX-2 protein (Fig. 3A).
  • the activity of COX-2 in normal tissue cells is extremely low. When cells are stimulated by inflammation, its expression level in inflammatory cells can increase to 10 to 80 times the normal level.
  • the expression level of COX-2 in A549 cells increased significantly under the influence of PR8 virus, proving that TFF2-IFN ⁇ 2-Fc and IFN ⁇ 2-TFF2-Fc fusion proteins can also reduce epoxidation while inhibiting the replication of PR8.
  • the expression of enzyme COX-2, IFN ⁇ 2 can also reduce the level of cyclooxygenase COX-2, TFF2 alone cannot inhibit virus replication, and therefore cannot reduce the level of virus-induced COX-2.
  • Inflammatory factor standard protein is freeze-dried powder. First dissolve the lyophilized powder in sterile distilled water according to the label instructions, and then dilute it with standard diluent. After full dissolution, the concentration of the mother solution is 1000pg/mL. Shake the standard gently for 5 minutes before diluting. The next day, the plate was washed three times with 300 ⁇ L/well washing buffer (0.05% Tween-20 in PBS), and blocked with ELISA blocking solution (10% FBS in PBS) for 1 hour. Add sample: Add 100 ⁇ L/well of diluted cytokine standard.
  • the standard concentration gradient used is: 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.25pg/mL, 15.6pg/mL, 7.8 pg/mL, complete the double dilution of the standard in the imported EP tube. Then 100 ⁇ L/well of sample was added to the wells. After the samples and standards were incubated for 2 hours, the samples were washed 5 times with washing buffer. Add detection antibody: 100 ⁇ L/well add diluted 100 ⁇ L detection antibody + SAv-HRP reagent to each well. Incubate at room temperature for 1 hour. Wash away the liquid in the wells. Leave the washing buffer for 1 minute and then discard the liquid in the wells.
  • TFF2-IFN ⁇ 2-Fc fusion protein can significantly reduce the level of IL-6 inflammation induced by PR8 at doses of 0.2 and 1 ⁇ g/mL, while at a dose of 5 ⁇ g/mL, It was also comparable to the inflammatory levels of IL-6 secreted by IFN ⁇ 2 treatment.
  • IFN ⁇ 2-TFF2-Fc can induce higher IL-6 inflammation levels in a dose-dependent manner.
  • IFN ⁇ 2 itself has an antiviral effect, the level of IL-6 inflammation also appears under its action, indicating that different sequences of TFF2 and IFN ⁇ 2 can affect the level of IL-6 inflammation induced by IFN ⁇ 2.
  • RAW264.7 cells plate them on a 12-well plate the day before, 2.5x10 5 cells/well, and add fusion protein 4 to 6 hours in advance. Incubate for 4 hours with fusion protein concentrations of 0.2, 1, and 5 ⁇ g/mL, then add LPS 1ng/mL, wash the cells with PBS for 24 hours, digest with trypsin for 2 to 3 minutes, terminate the medium, pipet down, wash again with PBS, and add 100 ⁇ L 1x loading buffer, boiling water bath for 10 minutes, detect the expression of iNOS by WB, under LPS stimulation of RAW264.7 cells, TFF2-IFN ⁇ 2-Fc produced lower iNOS than IFN ⁇ 2 ( Figure 3C).
  • the level of iNOS may be an indicator of the degree of inflammation in the body.
  • TFF2-IFN ⁇ 2-Fc can significantly reduce the expression of iNOS.
  • IFN ⁇ 2-TFF2-Fc can promote the expression level of inflammation, and as the dose increases, it gradually induces an increase in the expression level of iNOS.
  • the structure of IFN ⁇ 2 in the center of TFF2-IFN ⁇ 2-Fc can affect the function of IFN ⁇ 2, and IFN ⁇ 2 can induce higher levels of iNOS.
  • influenza virus H1N1 strain PR8 (in the P2 laboratory) was used to infect C57 mice through intranasal instillation. Administration was administered 6 hours before infection ( Figure 4A). 0.5g Tribromoethanol was used for anesthesia before infection.
  • mice were weighed for 14 consecutive days, and the survival and survival status of the mice were observed.
  • influenza virus H1N1 strain PR8 (in the P2 laboratory) was used to infect C57 mice through intranasal instillation. Administration was administered 12 hours before infection (Figure 4D). 0.5g Trisamine was used for anesthesia before infection. Tribromoethanol + 1 mL 2-Methyl-2-Butanol + 39 mL water are prepared into a 40 mL solution. Anesthetize the mouse with 300 ⁇ L/mouse and challenge the virus at a dose of 500 TCID50/mouse. , the challenge time is day 0, and aerosol administration is performed 6 hours, 2, 4, and 6 days after the challenge, and the dosage is 1 ⁇ g/g and 5 ⁇ g/g.
  • mice were weighed for 14 consecutive days, and the survival and survival status (weight changes) of the mice were observed. Survival rate analysis (Figure 4E) showed that PR8-infected mice began to die on the 7th day after infection, and the final survival rate of mice in the PBS group was 30%.
  • the TFF2-IFN ⁇ 2-Fc fusion protein protected all mice at a dose of 1 ⁇ g/g. Death from influenza; as shown in Figure 4F, PR8-infected mice continued to lose weight. On the 7th day, most mice lost more than 10 to 30% of their body weight.
  • 2xTFF2-IFN ⁇ 2-Fc had no protective effect at either the 1 ⁇ g/g dose or the 5 ⁇ g/g dose, indicating that the structure of 2xTFF2 in 2xTFF2-IFN ⁇ 2-Fc will affect the function of IFN ⁇ 2 and thus the protective effect.
  • TFF2-IFN ⁇ 2-Fc fusion protein can produce significantly better effects than TFF2 alone, IFN ⁇ 2 alone and other forms of fusion proteins in preventing and protecting mice from weight loss and death caused by influenza virus.

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Abstract

本公开提供了三叶因子2/干扰素α2融合蛋白及其防治病毒感染性疾病的应用。具体而言，本公开提供了一种融合蛋白，其包含：含三叶因子2(TFF2)肽的TFF2元件，以及与TFF2元件融合的、含干扰素α2(IFNα2)肽的IFNα2元件。本公开还提供了此类融合蛋白在预防和/或治疗病毒感染性疾病(例如急性呼吸道/肠道病毒感染)中的应用。

Description

三叶因子2/干扰素α2融合蛋白及其防治病毒感染性疾病的应用 技术领域
本申请属于生物医学技术领域，具体涉及三叶因子2(TFF2)与干扰素α2(IFNα2)的融合蛋白、及其制备和在治疗及预防病毒感染疾病方面的应用。
背景技术
病毒感染对于人类和哺乳动物生命和健康造成了极大危害，其中呼吸道病毒感染与肠道病毒感染是最常见的急性病毒感染。
急性呼吸道病毒感染易引发疫情，这些疫情严重威胁人类的生命健康，也给人民的生活和社会经济发展造成了不利影响，迫切需要开发针对类似疫情的防控措施。而肠道病毒是一类与人类和哺乳动物疾病相关的正义单链RNA病毒，其通过肠道进行传播。肠道病毒每年影响全世界数百万人，通常存在于感染者的呼吸道分泌物(例如唾液、痰或鼻粘液)和粪便中。感染可导致多种症状，包括：轻度呼吸道疾病(普通感冒)、手足口病、急性出血性结膜炎、无菌性脑膜炎、心肌炎、严重新生儿败血症样疾病、急性弛缓性麻痹、和相关的急性弛缓性脊髓炎。
呼吸道与肠道病毒均为粘膜感染病毒。尽管不同病毒的特性和感染方式不完全一致，然而其共性致病机制依然有迹可循：一方面病毒直接感染造成靶细胞凋亡或坏死、损害正常粘膜的结构和功能；另一方面病毒通过调控宿主免疫反应，抑制I型干扰素产生及其信号转导通路，大量诱导炎症细胞因子与趋化因子分泌，产生炎症风暴，招募大量免疫细胞浸润到粘膜组织，严重破坏粘膜结构，导致粘膜损伤、炎症渗出等，最终导致宿主致病或致死。因此，针对呼吸道与肠道病毒感染的共性致病机制，需要在限制病毒复制的同时，抑制炎症风暴，促进粘膜损伤修复，以达到改善患者预后的目的。
目前，针对病毒感染所诱导的炎症反应，常见的抑炎药物是糖皮质激素，其是一类类固醇激素。糖皮质激素是免疫系统反馈机制的一部分，可降低免疫功能的某些方面，由此可有效抑炎。然而，其副作用也很明显，目前使用的糖 皮质激素药物具有非选择性作用，会损害许多健康的合成代谢过程。长期服用该药物的副作用包括：医源性肾上腺皮质机能亢进症，诱发或加重感染或使体内潜在的感染病灶转移，造成消化性溃疡，诱发胰腺炎和脂肪肝，医源性肾上腺皮质功能不全，诱发精神分裂症和癫痫，股骨头坏死等。
干扰素作为抗病毒药物，单独或与其他疗法联合使用已在各种病毒感染中被广泛应用，其对目前已知的许多病毒均有抑制作用。IFNα2作为一种经典抗病毒药物，在临床上已经被广泛应用，其α-2b喷雾剂在治疗儿童急性上呼吸道感染中，疗效显著，能有效改善患儿临床症状和体征(陈青等，重组人干扰素α-2b喷雾剂治疗儿童急性上呼吸道感染疗效分析，生物医学工程与临床.2019，23(04))；α-2b喷雾剂以低剂量20μg联合奥司他韦治疗甲型流感，可下调细胞炎症因子水平，提高病毒转阴率，促进症状改善，并且安全性高(许光锋，重组人干扰素α-2b喷雾剂联合奥司他韦治疗甲型流感的疗效评价，山西卫生健康职业学院学报.2020，30(04))。动物实验和临床研究都表明IFNα2与利巴韦林联用可有效抑制MERS-CoV的复制、改善MERS症状和临床结果。在2003的SARS治疗方案和最新版《新型冠状病毒肺炎诊疗方案(试行第八版)》中，均推荐使用IFNα2治疗(成人每次500万U或相当剂量，加入灭菌注射用水2mL，每日2次，雾化吸入，疗程不超过10天)。Anuja Pandit在2020年进行了聚乙二醇干扰素α-2b(PEG IFN-α2b)在中度COVID-19中的II期研究评估其疗效与安全性。单剂量1μg/kg的PEG IFN-α2b皮下给药+标准治疗相比标准治疗能减少病毒消失的持续时间，显着改善临床结果(Anuja Pandit，Efficacy and safety of pegylated interferon alfa-2b in moderate COVID-19：A phase II，randomized，controlled，open-label study，Int J Infect Dis.202104；105：516-521)。
TFF2作为胃肠道分泌的小分子多肽，能参与黏膜修复，虽然它在炎症过程中过度表达，但添加TFF2会产生降炎作用，有助于创造组织修复所需的微环境，促进组织修复(Abdelaziz Ghanemi等，Trefoil factor family member 2(TFF2)as an inflammatory-induced and anti-inflammatory tissue repair factor，Animals(Basel).2020 Sep 14；10(9)：1646.)。在前期研究中，我们发现一种宿主分泌肽TFF2能通过抑制炎症反应，减轻病理损伤，促进肺组织损伤修复，发 挥保护效果，改善流感病毒感染的预后(CN105582526B)。在2019年紧急启动的新冠肺炎临床研究中，我们将TFF2与I型干扰素κ(IFN-k)联用，采用雾化吸入的方式治疗中度新冠肺炎患者，结果表明这种组合治疗能够显著缩短COVID-19患者的核酸阴转时间，提升核酸转阴患者的比例，加快患者的CT好转，减少患者的住院时间。同时，接受治疗的患者血浆中的炎症细胞因子水平快速下降(EClinicalMedicine 2020：100478/100547；中国专利申请号：202010239633.3)。
在人类与病毒感染的长期斗争中，仍然迫切需要开发出更安全有效防治病毒感染的药物和方法。
发明内容
本公开中正是提供了一种能够更安全有效用于病毒感染的预防和/或治疗的有效活性物质、其在制药中的应用和防治疾病的方法。
在本公开的一个方面中，提供了一种融合蛋白，其包含一个或多个融合单元，每个融合单元包括：
(a)三叶因子2(TFF2)元件，所述TFF2元件包含TFF2肽或其活性片段；
(b)干扰素α2(IFNα2)元件，所述IFNα2元件包含IFNα2肽或其活性片段，
其中，所述TFF2元件和IFNα2元件以1∶1的分子比融合，且在各融合单元中所述TFF2元件位于所述IFNα2元件的N端。
在一些实施方式中，TFF2肽或其活性片段来源于人、灵长类动物、啮齿类动物(如小鼠、大鼠、豚鼠、仓鼠)、犬、猫。
在一些实施方式中，TFF2肽或其活性片段包含选自下组的氨基酸序列：SEQ ID NO：8、SEQ ID NO：12、或其活性片段(例如与SEQ ID NO：8和SEQ ID NO：12具有至少80％序列同一性且具有TFF2活性的氨基酸序列)。
在一些实施方式中，TFF2肽或其活性片段由包含选自下组的核苷酸序列的核酸分子编码：SEQ ID NO：7、SEQ ID NO：11、或其活性片段(例如与SEQ ID NO：7或SEQ ID NO：11具有至少80％序列同一性且能够编码活性TFF2肽的 核酸分子)。
在一些实施方式中，IFNα2肽或其活性片段来源于人、灵长类动物、啮齿类动物(如小鼠、大鼠、豚鼠、仓鼠)、犬、猫。
在一些实施方式中，IFNα2肽或其活性片段包含选自下组的氨基酸序列：SEQ ID NO：10、SEQ ID NO：14、或其活性片段(例如与SEQ ID NO：10和SEQ ID NO：14具有至少80％序列同一性且具有IFNα2活性的氨基酸序列)。
在一些实施方式中，IFNα2肽或其活性片段由包含选自下组的核苷酸序列的核酸分子编码：SEQ ID NO：9、SEQ ID NO：13、或其活性片段(例如与SEQ ID NO：9或SEQ ID NO：13具有至少80％序列同一性且能够编码活性IFNα2肽的核酸分子)。
在一些实施方式中，融合蛋白还包含连接TFF2元件和IFNα2元件和/或元件中组成肽段的接头。
在一些实施方式中，接头为包含n个氨基酸残基的柔性接头，n为2～300的整数。
在一些实施方式中，接头是甘氨酸或甘氨酸/丝氨酸接头。在一些实施方式中，接头选自G n、(GS) n、(GGS) n、(GGGS) n、(GGGGS) n或(GGGGGS) n的氨基酸序列，其中n是1、2、3、4、5、6、7、8、9或10的整数。
在一些实施方式中，融合蛋白包含一个或多个连续或间隔排列的TFF2肽和/或一个或多个连续或间隔排列的IFNα2肽。
在一些实施方式中，融合蛋白还包含Fc区，所述Fc区不含突变。在一些实施方式中，融合蛋白包含一个或多个降低抗体介导的ADCC和CDC活性的突变。在一些实施方式中，融合蛋白包含根据EU编号方式的氨基酸突变D265A和N297G。
在一些实施方式中，融合蛋白还包含信号肽。在一些实施方式中，融合蛋白中的信号肽选自tPA2信号肽、TFF2信号肽、IL-2信号肽、bPRL信号肽、CD33信号肽。
在一些实施方式中，融合蛋白还包含标记物，例如用于纯化、检测、定位的标记物，如选自荧光标记物、非放射性核素标记物、生物素类标记物、磷酸 化修饰标记、肽标签。
在一些实施方式中，融合蛋白具有SEQ ID NO：2的氨基酸序列或与所述序列具有至少80％的序列同一性。在一些实施方式中，融合蛋白由具有SEQ ID NO：1的核苷酸序列的核酸分子或与所述序列具有至少80％的序列同一性的核酸分子编码。
在本公开的一些方面中，提供了一种分离的核酸分子或包含所述核酸分子的构建体或载体，其中，所述核酸分子编码本公开的融合蛋白。在一些实施方式中，所述核酸分子具有SEQ ID NO：1的核苷酸序列或与所述序列具有至少80％的序列同一性。在一些实施方式中，所述核酸分子编码具有SEQ ID NO：2的氨基酸序列或与所述氨基酸序列具有至少80％的序列同一性的多肽。
在一些实施方式中，载体选自病毒载体、mRNA载体、DNA载体。
在本公开的一些方面中，提供了一种细胞，其包含如权利要求1～7中任一项所述的融合蛋白、或如权利要求8或9所述的核酸分子、构建体或载体。
在本公开的一些方面中，提供了一种组合物，其包含本公开的融合蛋白、核酸分子、构建体、载体和/或细胞；以及载剂。
在本公开的一些方面中，提供了本公开的融合蛋白、核酸分子、构建体、载体、细胞和/或组合物在制备用于预防和/或治疗病毒感染性疾病中的应用。
在本公开的一些方面中，提供了一种预防和/或治疗病毒感染性疾病的方法，所述方法包括给予有需要的对象预防或治疗有效量的本公开的融合蛋白、核酸分子、构建体、载体、细胞、组合物或包含前述物质的药物。
在本公开的一些方面中，提供了本公开的融合蛋白、核酸分子、构建体、载体、细胞、组合物和/或药物，其用于预防和/或治疗病毒感染性疾病。
在一些实施方式中，病毒感染性疾病选自急性病毒感染，例如呼吸道病毒感染和肠道病毒感染。
在一些实施方式中，病毒感染性疾病由选自下组的一种或多种病毒引起：冠状病毒、流感病毒、鼻病毒、腺病毒、副流感病毒、呼吸道合胞病毒、柯萨奇病毒、埃可病毒、新型肠道病毒。
在一些实施方式中，本公开的融合蛋白、核酸分子、构建体、载体、细胞、 组合物和/或药物作为预防性药物在病毒感染发生前预防性给药以预防病毒感染发生或降低后续病毒感染的严重程度。
在一些实施方式中，本公开的融合蛋白、核酸分子、构建体、载体、细胞、组合物和/或药物作为治疗性药物在病毒感染发生后给药以降低病毒感染与疾病的严重程度。
在一些实施方式中，本公开的融合蛋白、核酸分子、构建体、载体、细胞、组合物和/或药物既作为预防性药物，也作为治疗性药物，在病毒感染发生之前和之后连续或间隔给药。
在一些实施方式中，本公开的融合蛋白、核酸分子、构建体、载体、细胞、组合物和/或药物的形式适于选自下组的给药方式：呼吸道雾化吸入、滴鼻、喷雾、口服、肌注和/或静脉给药。
在一些实施方式中，本公开的融合蛋白、核酸分子、构建体、载体、细胞、组合物和/或药物适于单独使用或与其他抗病毒药物、免疫药物或病毒疗法联合使用。
本领域的技术人员可对前述的技术方案和技术特征进行任意组合而不脱离本发明的发明构思和保护范围。本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
下面结合附图对本发明作进一步说明，其中这些显示仅为了图示说明本发明的实施方案，而不是为了局限本发明的范围。
图1：融合蛋白表达载体的构建以及体外表达：
图1A：TFF2与IFNα2融合蛋白真核表达载体pSV1.0 IFNα2-TFF2-Fc、pSV1.0 TFF2-IFNα2-Fc和pSV1.0 2xTFF2-IFNα2-Fc的构建图谱；
图1B：不同信号肽的TFF2与IFNα2融合蛋白在293F悬浮细胞系中及CHO-K1细胞中的表达，其中Blank为对照细胞，Cell为细胞，Sup为上清液，信号肽分别为TFF2、IL-2信号肽、tPA2信号肽。
图2：TFF2与IFNα2的融合蛋白的验证及其对病毒蛋白表达和复制的影 响。
图2A：TFF2与IFNα2的融合蛋白在纯化后在聚丙烯酰胺凝胶电泳(PAGE)上的验证；
图2B&2C：TFF2与IFNα2融合蛋白在肺上皮细胞系A549中对干扰素诱导的跨膜蛋白3(IFITM3)蛋白的表达的影响；
图2D&2E：TFF2与IFNα2融合蛋白对流感病毒PR8在体外肺上皮细胞系A549中复制的影响。
图3：TFF2与IFNα2融合蛋白对病毒诱导的炎症相关因子表达的影响。
图3A：TFF2与IFNα2融合蛋白对PR8诱导的COX-2的表达的影响；
图3B：TFF2与IFNα2融合蛋白对PR8引起的IL-6的表达的影响；
图3C：TFF2与IFNα2融合蛋白对LPS诱导体外肺上皮细胞系A549中iNOS表达的影响。
***表示p＜0.001。
图4：TFF2-IFNα2-Fc、IFNα2-TFF2-Fc对流感病毒攻毒动物的保护作用。
图4A：TFF2-IFNα2-Fc、IFNα2-TFF2-Fc流感病毒的攻毒及给药模型；
图4B：PR8感染后小鼠的生存率曲线与体重变化曲线(TFF2-IFNα2-Fc)；
图4C：PR8感染后小鼠的生存率曲线与体重变化曲线(IFNα2-TFF2-Fc)；
图4D：TFF2-IFNα2-Fc、2xTFF2-IFNα2-Fc流感病毒的攻毒及给药模型；
图4E：PR8感染后小鼠的生存率曲线(TFF2-IFNα2-Fc、2xTFF2-IFNα2-Fc)；
图4F：PR8感染后小鼠的体重变化曲线(TFF2-IFNα2-Fc、2xTFF2-IFNα2-Fc)。
具体实施方式
本发明人通过长期而深入的研究，构建了各种形式的TFF2与IFNα2融合蛋白，通过对单独TFF2或IFNα2多肽和各种融合蛋白的抗病毒、降低炎症、动物保护效果等功能的测试、比较和筛选，克服各种技术难度，获得了具有正确结构、优异效果的特定融合蛋白形式。由此，本公开中提供了具有特定结构 和配比的TFF2与IFNα2融合蛋白，并验证了其对病毒感染性疾病的优异预防和治疗效果。本申请的融合蛋白既具有优异的抗病毒效果，又具有抑制过度炎症的效果，其效果显著优于单独使用TFF2或IFNα2，具有协同效果，且也显著优于其他结构和配比的TFF2和IFNα2融合蛋白。
具体而言，本公开中将TFF2多肽与IFNα2多肽以多种形式融合表达，通过体外实验验证了融合蛋白的成功表达，并测试了TFF2与IFNα2融合蛋白对流感病毒复制的抑制作用，以及对炎性因子分泌的降低作用。小鼠流感感染模型中进一步研究发现雾化吸入TFF2与IFNα2融合蛋白能提高流感感染小鼠的生存率，减少小鼠体重丢失。而在所构建的多种融合蛋白中，所含TFF2多肽和IFNα2多肽以1：1的分子比融合且TFF2多肽位于IFNα2的N端的融合蛋白具有最为优异的效果。测试结果表明，该特定类型融合蛋白的抗病毒、抑制过度炎症的体内外效果显著优于单独使用TFF2或IFNα2，具有协同效果，且也显著优于其他结构和配比的TFF2和IFNα2融合蛋白。
更具体而言，为了开发新型抗病毒药物并验证其在抗病毒感染防治中的效果，发明人构建了真核表达载体pSV1.0 IFNα2-TFF2-Fc、pSV1.0 TFF2-IFNα2-Fc和pSV1.0 2xTFF2-IFNα2-Fc，在293T中对其进行表达，采用Western Blot验证融合蛋白的表达，并在293F细胞中表达，收集上清，然后采用
Figure PCTCN2022094509-appb-000001
pure进行纯化，考马斯亮蓝对纯化的蛋白纯度进行鉴定，并通过BCA测定蛋白浓度。在体外细胞实验中Western blot检测了TFF2与IFNα2融合蛋白对流感病毒PR8在体外肺上皮细胞系A549中复制的影响，证明了融合蛋白能够降低病毒的复制。
对流感病毒H1N1毒株PR8毒株感染小鼠，观察PR8感染后小鼠体重变化和生存率。发现TFF2与IFNα2融合蛋白处理组能显著提高小鼠的生存率，减少体重丢失，改善高致病性流感感染的症状。在此基础上，我们进行了TFF2与IFNα2融合蛋白的预防实验，进一步验证其对流感病毒H1N1毒株PR8毒株攻毒的预防及保护效果。这些结果充分证明TFF2与IFNα2融合蛋白在呼吸道病毒或其他因素诱导的急性病毒感染损伤模型中发挥了重要的保护作用。
由于其他的急性病毒感染引起的组织损伤的机理与流感病毒感染类似，因 此TFF2与IFNα2融合蛋白的保护作用并不局限于流感病毒造成的呼吸道组织损伤，包括其他急性病毒感染引起的呼吸道损伤，以及肠道病毒等引发急性症状的肠道疾病。并且，对参与急性病毒感染疫情处理的疾控人员、高风险人群的进行预防性给药，可有效降低上述人员的风险以及受到的损害。
综上，本申请是在前期研究基础上进一步改进和优化，通过将TFF2与IFNα2以特定形式融合，能够同时发挥多分子、多功能的作用，效果显著优于分别给予单一药物。通过融合形成单一分子既能够发挥多功能的作用，也能够降低多蛋白组合的复杂性，提升疗效，能够发挥抗病毒与降低炎症的双重作用。通过单一分子的TFF2与IFNα2的融合蛋白，解析该融合蛋白在病毒感染性疾病，尤其是急性病毒感染性疾病中的抗病毒、抑炎与保护功能。本公开的特定融合蛋白针对急性病毒感染的共性致病机制，一方面可以发挥IFNα2广谱抑制病毒复制的功能，另一方面通过TFF2抑制炎症细胞因子风暴，促进粘膜损伤修复，改善预后，发挥抗病毒与促修复的作用。同时，两种融合组分间相辅相成，出乎意料地发挥了显著优于两者单独给予所能获得的效果。通过调整该融合蛋白中TFF2与IFNα2的表达比例，有助于消除IFNα2诱导炎症细胞因子风暴的副反应，达到安全有效的干预目的。
另外，TFF2是一种宿主小分子多肽，在不同物种中具有高度的保守性，例如成熟的人TFF2分子由106个氨基酸组成，分子量约为12kD，含有两个对称的由约40个氨基酸残基组成的特殊保守序列，包含6个半胱氨酸残基形成三个链内二硫键(cys1-cys5，cys2-cys4及cys3-cys6)，从而产生特异而稳定的三叶草型结构，耐酸、耐热且抗蛋白酶水解，在运输上极具优势。IFNα2也是宿主分泌的多肽，在临床上已被广泛应用。因此，将TFF2与IFNα2进行融合表达，不仅活性明确，安全性良好，且具有较高的成药率，在急性呼吸道病毒感染及肠道病毒感染的预防与治疗中具有较高的应用潜力。
本公开中首次提出TFF2与IFNα2融合蛋白的干预策略，采用抗病毒+抑炎+促修复三效合一策略，可用于广谱预防和治疗病毒感染引起的急性传染病。融合蛋白不仅可以系统给药，还可以进行局部雾化吸入给药，针对性强、见效快、副作用小，能发挥更好的效果。另外，本公开的产品成本低，易在经济较 薄弱的国家或地区推广和应用，也可作为国家防控新发突发病毒引起的传染病的一种技术储备，具有较高的经济价值、社会价值和政治意义。
本公开中提供了三叶因子2与干扰素α2的融合蛋白，利用此类融合蛋白能抑制病毒的复制、降低组织炎症减少组织损伤和促进肺组织功能修复等优点，其可用于制备治疗和/或预防急性病毒感染疾病的药物，该药物对急性病毒感染引起的预后有明显的改善效果。
与现有技术相比，本公开提供了基于TFF2与IFNα2的融合蛋白。发明人通过试验证明了所述TFF2与IFNα2融合蛋白分子能在流感病毒PR8感染中起保护作用，减少发病率和死亡率，减轻呼吸道感染炎症症状，由此可在应对呼吸道新发病毒性感染的疫情中起重要作用，尤其对预防与治疗尚无有效治疗药物的病毒性感染以及重症感染尤为有益。TFF2与IFNα2融合蛋白针对急性病毒感染的共性致病机制，通过抑制炎症反应，促进粘膜组织修复，减少组织损伤，同时抑制病毒的复制功能，发挥抗病毒作用。因此，TFF2与IFNα2融合蛋白的保护作用并不局限于流感病毒造成的呼吸道组织损伤，包括其他病毒感染引起的损伤，对参与急性病毒感染疫情处理的疾控人员、高风险人群的进行预防性给药，可有效降低上述人员的风险以及受到的损害。
本文中提供的所有数值范围旨在清楚地包括落在范围端点之间的所有数值及它们之间的数值范围。可对本发明提到的特征或实施例提到的特征进行组合。本说明书所揭示的所有特征可与任何组合物形式并用，说明书中所揭示的各个特征，可以任何可提供相同、均等或相似目的的替代性特征取代。因此除有特别说明，所揭示的特征仅为均等或相似特征的一般性例子。
如本文所用，“含有”、“具有”或“包括”包括了“包含”、“主要由......构成”、“基本上由......构成”、和“由......构成”；“主要由......构成”、“基本上由......构成”和“由......构成”属于“含有”、“具有”或“包括”的下位概念。
如本文所用，“哺乳动物”可包括人、灵长类动物、啮齿动物(例如大鼠、小鼠、豚鼠、仓鼠)、驯养动物或畜牧哺乳动物。
TFF2元件和IFNα2元件
如本文所用，术语“元件”是指构成融合蛋白中一部分的氨基酸序列。术语“单元”是指元件构成功能的基本片段。例如，融合蛋白的TFF2元件中可包含一个或多个连续或间隔的TFF2单元，它们各自可产生所需的TFF2相关功能活性。
如本文所用，术语“TFF2元件”和“TFF2蛋白(多肽)”可互换使用，是指构成融合蛋白一部分的天然(例如哺乳动物来源)、重组或合成的TFF2多肽序列。天然TFF2在哺乳动物中具有高度保守性，具有特异而稳定的三叶草型结构，具有一定的黏膜修复和炎症抑制作用。TFF2多肽也包含TFF2的天然变体和片段(例如剪接变体或等位基因变体)，以及具有天然TFF2活性的非天然存在变体。
天然TFF2多肽的核苷酸和氨基酸序列是已知的。参见，例如GenBank登录号(人Gene ID：7032；鼠Gene ID：21785)。
本文的TFF2元件可包含SEQ ID NO：8的氨基酸序列(人TFF2)或由包含SEQ ID NO：7所述的核酸分子编码，包含SEQ ID NO：12的氨基酸序列(小鼠TFF2)或由包含SEQ ID NO：11所述的核酸分子编码，或可为与这些蛋白质具有相同或类似活性的同源序列(例如可通过本领域已知的数据库或比对软件获得同源序列)、变异体或修饰形式。例如，所述TFF2多肽可选自：(a)包含SEQ ID NO：8或12所示氨基酸序列的多肽(例如序列如SEQ ID NO：8或12所示的多肽)；或(b)在(a)限定的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有黏膜修复和炎症抑制作用的活性的由(a)衍生的蛋白质或多肽。
如本文所用，术语“IFNα2(多肽)”和“IFNα2蛋白(多肽)”可互换使用，是指是指天然(例如哺乳动物来源)、重组或合成的IFNα2多肽。如背景部分中所述，IFNα2的结构和功能在本领域中已有一定的研究和了解。本申请中可采用本领域中已知的IFNα2多肽，也可采用其天然变体和片段(例如剪接变体或等位基因变体)，以及具有IFNα2活性的非天然变体。
天然IFNα2多肽的核苷酸和氨基酸序列是已知的。参见，例如GenBank登录号(人Gene ID：3440；鼠Gene ID：15965)。
本文所用的IFNα2蛋白可包含SEQ ID NO：10的氨基酸序列(人IFNα2) 或由包含SEQ ID NO：9所述的核酸分子编码，可包含SEQ ID NO：14的氨基酸序列(小鼠IFNα2)或由包含SEQ ID NO：13所述的核酸分子编码，或可为与这些蛋白质具有相同或类似活性的同源序列(例如可通过本领域已知的数据库或比对软件获得同源序列)、变异体或修饰形式。例如，所述IFNα2多肽可选自：(a)包含SEQ ID NO：10或14所示氨基酸序列的多肽(例如序列如SEQ ID NO：10或14所示的多肽)；或(b)在(a)限定的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有黏膜修复和炎症抑制作用的活性的由(a)衍生的蛋白质或多肽。
本公开融合蛋白中的TFF2多肽元件和IFNα2多肽元件优选由人基因或其同源基因或家族基因编码。本公开中蛋白质或多肽的变异形式包括(但并不限于)：一个或多个(通常为1～50个，较佳地1～30个，更佳地1～20个，最佳地1～10个，例如1、2、3、4、5、6、7、8、9或10个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质或多肽的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质或多肽的功能，例如融合蛋白可包括或不包括起始的甲硫氨酸残基而仍然具有其所需的病毒感染防治活性。
多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与其蛋白编码序列杂交的序列所编码的蛋白。根据重组生产方案所用的宿主，本发明的蛋白质或多肽可以是糖基化的，或可以是非糖基化的。
融合蛋白
如本文所用，术语“融合蛋白”，是指包含相融合的至少一个TFF2多肽和至少一个IFNα2多肽的氨基酸分子。本公开融合蛋白可以使用重组技术从原核或真核宿主(例如，细菌、酵母、高等动物、昆虫和哺乳动物细胞；优选真核宿主)中产生，也可通过人工合成产生，例如采用全序列合成或片段合成后拼 接而成。
本文的融合蛋白中的TFF2肽可连接于IFNα2肽的N端或C端，优选TFF2肽可连接于IFNα2肽的N端(即上游)。本文的融合蛋白可包含一个或多个TFF2肽和/或IFNα2肽，例如TFF2肽和IFNα2肽以5∶1～1∶5的分子比例融合，如1∶1或2∶1的分子比例融合，优选以1∶1的分子比例融合。
在本申请的优选实施方式中，本文的融合蛋白中的TFF2肽连接于IFNα2肽的N端，且TFF2肽和IFNα2肽以1∶1的分子比例融合。
本申请的融合蛋白还可包含Fc区。如本文所用，术语“Fc区”或“Fc片段”是指用于融合蛋白中的免疫球蛋白Fc段。在一些实施方式中，Fc区具有与天然的或变异的免疫球蛋白Fc片段基本上相同的氨基酸序列，并且具有与天然Fc片段基本上相同的生物活性。除了免疫球蛋白的CH2和CH3区以外，Fc区还可包含铰链区。Fc区可来源于例如IgG或IgA。
与单克隆抗体中Fc段功能类似，融合蛋白的Fc段可以起到延长功能蛋白在血浆内的半衰期、提高分子的稳定性、特异性结合体内的Fc受体，并发挥相应的生物学功能等作用。除此之外，Fc段可以特异性结合proteinA，简化了Fc融合蛋白的纯化步骤，在相关生物制品的研发制备具有重要意义。用于本文融合蛋白中的Fc区可不含突变，或者包含一个或多个突变，如降低抗体介导的ADCC和CDC活性的突变，例如根据EU编号方式的氨基酸突变D265A和N297G/N297Q。
本文的融合蛋白还可包含信号肽，诸如具有引导融合蛋白分泌、定位和/或输送功能的氨基酸序列，其长度通常为5-50个氨基酸。在一些实施方式中，信号肽可选自例如：tPA2信号肽、TFF2信号肽、IL-2信号肽、bPRL信号肽CD33蛋白信号肽等。
本文的融合蛋白还可包含标记物，例如用于纯化、检测、定位的标记物，如选自荧光标记物、非放射性核素标记物、生物素类标记物、磷酸化修饰标记、肽标签。
本文融合蛋白中各多肽元件或元件中的肽单元可通过接头连接。在本申请中优选采用柔性接头，以使得多肽元件或肽单元之间可存在相互作用。可用于 本申请融合蛋白中的接头可包含2～300个氨基酸残基，例如5～100个、10～50个、15～3个氨基酸残基。示例性的接头可为甘氨酸接头，如(G) n或甘氨酸/丝氨酸接头，如(GS) n、(GGS) n、(GGGS) n、(GGGGS) n或(GGGGGS) n的氨基酸序列，其中n是1、2、3、4、5、6、7、8、9或10的整数。
根据本申请中所提供的序列以及本领域中的技术，本技术领域人员可方便地用各种已知方法制得本发明的融合蛋白。这些方法例如但不限于：重组DNA法，人工合成等[参见Murray KM，Dahl SLAnn；Pharmacother 1997 Nov；31(11)：1335-8]。例如，本文的融合蛋白可用固相技术通过直接合成肽而加以生产，也可以分别化学合成融合蛋白的各片段，然后用化学方法加以连接以产生全长的分子。
载体和宿主
本文还涉及包含用于产生融合蛋白的载体，以及用该载体经基因工程产生的宿主细胞。
通过常规的重组DNA技术(Science，1984；224：1431)，可利用本发明的编码序列可用来表达或生产重组的融合蛋白。一般来说有以下步骤：
(1)用本文所述的编码融合蛋白的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2)在合适的培养基中培养宿主细胞；
(3)从培养基或细胞中分离、纯化目标融合蛋白。
本发明中，术语“载体”与“重组表达载体”可互换使用，指本领域熟知的细菌质粒、噬菌体、酵母质粒、动物细胞病毒、哺乳动物细胞病毒或其它载体，其能在宿主细胞内复制并表达目标蛋白。
本领域的技术人员熟知的方法能用于构建含融合蛋白编码序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。本文中可采用诸如使用pSV1.0载体、pcDNA3.1载体、 pIRES2-EGFP载体、AdMaxTM表达系统。
此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质或多肽。宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如动物细胞。代表性例子有：动物细胞，如293F细胞、CHO细胞等；大肠杆菌，链霉菌属、农杆菌；真菌细胞如酵母。
本发明的多核苷酸在高等真核细胞中表达时，如果在载体中插入增强子序列时将会使转录得到增强。增强子是DNA的顺式作用因子，通常大约有10到300个碱基对，作用于启动子以增强基因的转录。本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。
通过上述方法，融合蛋白可在细胞内或在细胞膜上表达或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、
Figure PCTCN2022094509-appb-000002
pure法、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
药物或试剂盒
本文还提供了一种产品，其中含有有效量的本文的融合蛋白、包含融合蛋白编码分子的载体、宿主细胞，以及药学上或生理学上可接受的载剂。如本文所用，术语“活性物质”是指本文的融合蛋白、其编码核酸分子、包含该核酸分子的构建体或载体、宿主细胞或前述的组合物。
在较佳的实施方案中，本文的产品可用于预防或治疗与病毒感染相关的疾病和/或其征状。如本文所用，术语“含有”或“包括”包括了“包含”、“基本上由......构成”、和“由......构成”。如本文所用，术语“药学上可接受的”成分是适 用于人和/或动物而无过度不良副反应(如毒性、刺激和变态反应)的，即有合理的效益/风险比的物质。如本文所用，术语“有效量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。
如本文所用，术语“药学上可接受的载剂”指用于治疗剂给药的载剂，包括各种赋形剂和稀释剂。该术语指这样一些药剂载剂：它们本身并不是必要的活性成分，且施用后没有过分的毒性。合适的载剂是本领域普通技术人员所熟知的。在《雷明顿药物科学》(Remington’s Pharmaceutical Sciences，Mack Pub.Co.，N.J.1991)中可找到关于药学上可接受的赋形剂的充分讨论。
在组合物中药学上可接受的载剂可含有液体，如水、盐水、甘油和乙醇。另外，这些载剂中还可能存在辅助性的物质，如填充剂、崩解剂、润滑剂、助流剂、泡腾剂、润湿剂或乳化剂、矫味剂、pH缓冲物质等。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性介质中，其中pH通常约为5～8，较佳地，pH约为6～8。
如本文所用，术语“单位剂型”是指为了施用方便，将本发明的组合物制备成单次施用所需的剂型，包括但不限于各种固体剂(如片剂、冻干粉末)、液体剂(如溶液)、胶囊剂、缓释剂。
在本发明的另一优选实施方式中，所述组合物为单位剂型或多剂型，且其中活性物质的含量为0.01～2000mg/剂，优选0.1～1500mg/剂，更优选1～1000mg/剂。在本发明的另一个优选例中，每天施用1～6剂本发明的组合物，优选施用1～3剂；最优选的，每天服用的剂量为1剂。
本发明的药物组合物可根据需要制成各种剂型，并可由医师根据患者种类、年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的剂量，通过是呼吸道雾化吸入、滴鼻、喷雾、口服、肌注和/或静脉给药等方式进行施用。
为了提高用药效果，本发明的活性物质或产品之间可相互间联合应用，还可以与其它药物和治疗手段联合，用于感染性疾病(尤其是急性病毒感染)的预防和治疗。例如，如果本发明的融合蛋白用于预防和/或治疗急性病毒感染，则可同时或先后采用临床上用于急性病毒感染治疗的其他药物或方法，所述其他药物或方法包括但不限于：预防进一步的伤害、调节局部区域功能、抗炎症、 施用糖皮质激素、非甾体抗炎药NSAID等。
实施例
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。本领域技术人员可对本发明做出适当的修改、变动，这些修改和变动都在本发明的范围之内。
下列实施例中未注明具体条件的实验方法，可采用本领域中的常规方法，例如参考《分子克隆实验指南》(第三版，纽约，冷泉港实验室出版社，New York：Cold Spring Harbor Laboratory Press，1989)或按照供应商所建议的条件。DNA的测序方法为本领域常规的方法，也可由商业公司提供测试。
除非另外说明，否则百分比和份数按重量计算。除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
实施例1：融合蛋白质粒的构建和设计以及真核表达
本实施例首先根据人源TFF2的氨基酸序列(如SEQ ID NO：8所示)与人源IFNα2蛋白的氨基酸序列(如SEQ ID NO：10所示)克隆基因序列，将重组的不同形式的质粒转染进入293T细胞后，再通过WB检测TFF2与IFNα2融合蛋白的真核表达，随后在293F细胞中进行大量表达，收获表达上清，并通过HiTrap MabSelect SuRe柱进行纯化，收集目的蛋白后鉴定纯度，超滤置换为PBS，得到高纯度的TFF2与IFNα2融合蛋白，其具体步骤如下：
通过将人源TFF2序列与干扰素α2通过3个G4S进行连接，并且TFF2具有不同的重复克隆，并在末端加入人源的Fc片段(图1A)。将测序正确的重组质粒转染进入293T细胞，转染试剂为TurboFect，培养基为DMEM完全培养基(10％FBS和1％P/S)，37℃孵箱培养24h后将细胞取出，收集细胞加入SDS上样缓冲液，收集细胞到EP管中，并以PBS缓冲液清洗细胞，加入上样缓冲液，并在沸水浴中加热10分钟使蛋白质变性，瞬时离心后通过SDS-聚 丙烯酸胺凝胶电泳(SDS-PAGE)分离蛋白，分离胶浓度为10％。电泳的电压为70V，时间为30～40分钟(以marker开始分离为标志)，待溴酚蓝迁移到分离胶位置后，将电压调至110V，直至溴酚蓝迁移到胶底部位置，之后进行快速转膜，恒流400mA，时间50分钟。转膜完毕后，将PVDF正面膜(与胶接触的面)作好记号，并置于5％的脱脂奶粉中室温封闭2小时。后加入合适稀释比例的一抗(TFF2，Proteintech：13681-1-AP，1∶1000；IFNα2，SantaCruz，sc-73305，1∶1000；β-actin，ABclonal，AC028，1∶5000)，采用5％脱脂奶粉稀释，置于摇床4℃孵育过夜。用0.05％PBST洗膜后加入二抗(羊抗兔(1∶5000)；羊抗鼠(1∶5000))，采用5％脱脂奶粉的PBST稀释，室温摇床孵育1小时后洗膜，对膜进行ECL显色，PVDF膜采用定量分析仪曝光2分钟，记录并分析显色结果。
结果表明(图1B左上)，细胞大量表达TFF2与IFNα2融合蛋白，采用TFF2信号肽时融合蛋白主要表达于细胞中，另一部分则分泌到上清中。
为了大量获得分泌的融合蛋白，我们对TFF2与IFNα2重组质粒的信号肽进行了替换，将其本身的TFF2信号肽替换为IL-2信号肽(图1B右上)及tPA2信号肽(图1B左下，右下)，通过对不同的信号肽的上清分泌进行比较，发现tPA2信号肽能够更有效地促进融合蛋白的分泌，因此在后续在融合蛋白表达中使用tPA2信号肽。
实施例2：融合蛋白的纯化及体外抗病毒和抑炎功能
为了大量获得重组蛋白，将测序正确的TFF2与IFNα2重组质粒转染进入293F悬浮细胞中。具体而言，使用SMM 293-TI无血清培养基来悬浮培养293F细胞，培养基中添加1％的青霉素/链霉素抗生素；每次以5x10 5个细胞/mL密度接种至新鲜培养基，培养至3x10 6个细胞/mL以上密度时进行实验或传代，细胞摇床通5％的二氧化碳，转速为125rpm/min。融合蛋白质粒瞬时转染时细胞密度为1x10 6个细胞/mL。采用PEI进行转染，转染过程中DNA∶PEI的比例为1∶3.5，取适量的质粒于1.5mL EP管，用150mM的氯化钠稀释至40ng/μL，混匀之后加入PEI，漩涡震荡充分混匀后，室温孵育15～30min，将DNA/PEI  mix加入细胞中；24小时之后按1∶1000加入抗细胞结团剂(Anti-Clumping Agent，Thermo Fisher，0010057AE)；在细胞摇床上培养5～7天收集上清与细胞。经WB检测，融合蛋白主要分泌在上清中，因此直接采用上清进行纯化。
采用蛋白质纯化仪
Figure PCTCN2022094509-appb-000003
Pure对融合蛋白进行纯化，柱子选用HiTrap MabSelect SuRe 1mL柱，通过运行UNICORN软件进行操作，所有缓冲液0.22μm滤膜过滤。首先，用超纯水清洗泵与管路，随后接入柱子并清洗，用至少5倍体积的蒸馏水冲洗乙醇。采用结合缓冲液(0.02M磷酸钠，0.15M NaCl，pH 7.2)进行通过A1管路进行柱子平衡，B1泵充满洗脱缓冲液(0.1M sodium citrate，pH 3.0)。平衡好之后通过管路A1进行上样，样品在上样前也通过12000xg离心，0.22μm滤膜过滤。以0.5mL/min速率进行上样，同时收集流穿液。上样完成后，用结合缓冲液洗至基线平，随后切换到B1进行100％洗脱液进行洗脱，收集洗脱液，直至基线基本平，每个1.5mL EP管加入100～200μL的中和液(Tris-HCl，pH 9.0)。再用5个CV的柱体积洗脱再生柱子，用3个CV的结合缓冲液，5个CV的0.1～0.5MNaOH清洗柱子，再用5～10个CV的结合缓冲液，重新平衡，20％乙醇洗净柱子，4℃保存。对于纯化后的融合蛋白，通过10kDa的超滤离心管进行置换为PBS缓冲液。为了定量TFF2与IFNα2融合蛋白的纯度，按照上述方法进行SDS-PAGE后，采用Quick Blue快速染胶液染色30～60min，用ddH 2O洗涤过夜(图2A)。为了定量TFF2与IFNα2融合蛋白的浓度，采用BCA定量试剂盒的方法，做出了BSA的标准曲线，根据标准品的光密度(OD)值，定量出了TFF2与IFNα2融合蛋白的浓度，分装后-80度冻存。
在A549细胞进行融合蛋白的干扰素活性实验，前一天铺板，24孔板，1x10 5个细胞/孔，加入融合蛋白，24h细胞PBS洗一遍，胰酶消化2～3min，培养基终止，吹打下来，PBS再洗一遍，加入100μL 1x上样缓冲液，沸水浴10min，WB检测干扰素诱导的跨膜蛋白3(IFITM3)蛋白的表达(图2B)。IFITM3属于干扰素诱导基因ISG，可以被干扰素和病毒诱导表达，融合蛋白能够刺激IFITM3的表达，表明融合蛋白具有诱导干扰素活性的作用。不同顺序的TFF2与IFNα2融合具有不同的体外活性，IFNα2-TFF2-Fc能够诱导较强的IFITM3 的表达，而TFF2-IFNα2-Fc与2xTFF2-IFNα2-Fc，其IFITM3表达水平稍弱。进一步结合后续体内外试验来确定融合蛋白的干扰素诱导活性是否适中。
在A549细胞进行融合蛋白的抗病毒实验，前一天铺板，24孔板，1x10 5个细胞/孔，提前4～6h加入融合蛋白，第二天按照MOI＝5的比例感染细胞，感染2h，PBS洗一次，替换为10％FBS的DMEM培养基，24h细胞PBS洗一遍，胰酶消化2～3min，培养基终止，吹打下来，PBS再洗一遍，加入100μL 1x上样缓冲液，沸水浴10min，WB检测NP蛋白的表达(图2C)。结果表明融合蛋白能够降低病毒的复制作用，并且随着融合蛋白浓度的增加，抗病毒作用逐渐增强，而TFF2单独并不能影响病毒的复制作用。
WB检测COX-2蛋白的表达(图3A)。COX-2在正常组织细胞内的活性极低，当细胞受到炎症等刺激时，其在炎症细胞中的表达水平可升高至正常水平的10～80倍。实验中，A549细胞在PR8病毒作用下，COX-2的表达水平呈现显著提高，证明了TFF2-IFNα2-Fc与IFNα2-TFF2-Fc融合蛋白在抑制PR8的复制过程中，也能降低环氧化酶COX-2的表达，IFNα2也能够降低环氧化酶COX-2的水平，TFF2单独并不能抑制病毒的复制，因此无法降低病毒诱导的COX-2的水平。
在A549细胞通过ELISA检测PR8感染后上清炎症因子IL-6的表达(图3B)。细胞铺板，24孔板，1x10 5个细胞/孔，12孔，24h收集上清。检测前包被板子，用4℃预冷的ELISA包被液(ELISA包被液：10mM碳酸钠(Na 2CO 3)，30mM碳酸氢钠(NaHCO 3)，溶液pH＝9.6，用0.2μm的滤膜过滤除菌后，4℃保存)。在ELISA板的每孔加入1∶250稀释的100μL捕获抗体溶液，4℃过夜。炎症因子标准品蛋白为冻干粉。先按标签说明将冻干粉用无菌蒸馏水溶解，再用标准品稀释液进行倍比稀释。充分溶解后母液浓度为1000pg/mL。稀释前将标准品轻轻振荡5min。第二天，板子加入300μL/孔洗涤缓冲液(0.05％吐温-20的PBS)洗3次，ELISA封闭液(10％FBS的PBS)封闭1h。加样：100μL/孔加入稀释后的细胞因子标准品，使用标准品浓度梯度为：500pg/mL、250pg/mL、125pg/mL、62.5pg/mL、31.25pg/mL、15.6pg/mL、7.8pg/mL，在进口EP管中完成对标准品的倍比稀释。随后100μL/孔加入样品至孔。样品及标 准品孵育2h后，洗涤缓冲液洗5次。加检测抗体：100μL/well加入稀释后的在每孔中加入100μL检测抗体+SAv-HRP试剂。在室温下孵育1小时。洗掉孔内液体，洗涤缓冲液停留1min后弃去孔内液体，重复7次，最后一次在滤纸上扣干。显色：100μL/孔加入TMB，室温避光孵育30min。终止反应：迅速100μL/孔加入终止溶液终止反应。读板：加入终止溶液后10min内在450nm处读值。根据样品的吸光值在坐标上找出对应的浓度。
结果表明，与IFNα2μg/mL处理组相比，TFF2-IFNα2-Fc融合蛋白在0.2，1μg/mL剂量处理时，能够显著降低PR8诱导的IL-6炎症水平，而在5μg/mL的剂量时，也与IFNα2处理所分泌的IL-6炎症水平相持平。而与IFNα2相比，IFNα2-TFF2-Fc则能够诱导的较高的IL-6炎症水平，并且有剂量依赖性。IFNα2本身虽然具有抗病毒作用，但是在其作用下，IL-6的炎症水平也呈现，表明不同顺序的TFF2与IFNα2能够影响IFNα2所诱导的IL-6炎症水平。在RAW264.7细胞中，前一天铺板，12孔板，2.5x10 5个细胞/孔，提前4～6h加入融合蛋白。以融合蛋白0.2、1、5μg/mL的浓度进行孵育4h，然后加入LPS 1ng/mL，24h细胞PBS洗一遍，胰酶消化2～3min，培养基终止，吹打下来，PBS再洗一遍，加入100μL 1x上样缓冲液，沸水浴10min，通过WB检测iNOS的表达，在RAW264.7细胞进行LPS刺激下，与IFNα2相比TFF2-IFNα2-Fc产生较低的iNOS(图3C)。
iNOS其含量的高低可能最为一种机体内炎症程度的指标，结果显示：与IFNα2相比，TFF2-IFNα2-Fc能显著减少iNOS的表达。IFNα2-TFF2-Fc能够促进炎症的表达水平，并且随着剂量升高，逐渐诱导iNOS的表达水平的升高，TFF2-IFNα2-Fc中IFNα2居中的结构能够影响IFNα2的功能发挥，IFNα2能够诱导较高的iNOS，IFNα2居中的结构能够降低其诱导的iNOS水平，而IFNα2-TFF2-Fc中IFNα2在前的结构则对IFNα2功能发挥无影响，因此随着剂量升高，诱导的iNOS水平也随之升高。该结果表明TFF2-IFNα2-Fc融合蛋白能够诱导适宜的iNOS水平，降低了过度炎症反应的风险。
实施例3：融合蛋白的动物攻毒保护及预防实验
本实施例采用流感病毒H1N1病毒株PR8(在P2试验室中)通过滴鼻的方法感染C57小鼠，感染前提前6h进行给药(图4A)感染前麻醉采用0.5g三溴乙醇(Tribromoethanol)+1mL 2-甲基2-丁醇(2-Methyl-2-Butanol)+39mL水配置成40mL的溶液，300μL/只的剂量麻醉小鼠，以1000TCID50/只的剂量进行攻毒，攻毒时间为第0天，攻毒后6h、第2天、第4天、第6天进行雾化给药，每次给药剂量为0.2、1、5μg/g。连续14天称量小鼠体重，观察小鼠存活情况及生存状态。生存率分析(图4B与图4C)显示，PR8感染的小鼠在感染后第8天开始死亡，13天全部死亡，TFF2-IFNα2-Fc融合蛋白能够保护40％左右的小鼠免于流感死亡。如图4B与图4C所示，PR8感染的小鼠体重持续下降，在第10天大部分小鼠体重丢失20～30％以上，融合蛋白组在第11天开始体重逐渐增加，而IFNα2-TFF2-Fc 0.5μg/g组的体重则一直下降。与TFF2-IFNα2-Fc相比，IFNα2-TFF2-Fc整体的保护效果不佳，可能因其诱导较高的炎症反应而降低了保护效果。
融合蛋白的动物的预防实验采用流感病毒H1N1病毒株PR8(在P2试验室中)通过滴鼻的方法感染C57小鼠，感染前提前12h进行给药(图4D)，感染前麻醉采用0.5g三溴乙醇(Tribromoethanol)+1mL 2-甲基-2-丁醇(2-Methyl-2-Butanol)+39mL水配置成40mL的溶液，300μL/只麻醉小鼠，以500TCID50/只的剂量进行攻毒，攻毒时间为第0天，攻毒后6h、第2天、第4天、第6天进行雾化给药，给药剂量为1μg/g与5μg/g。连续14天称量小鼠体重，观察小鼠存活情况及生存状态(体重变化)。生存率分析(图4E)显示，PR8感染的小鼠在感染后第7天开始死亡，最后PBS组小鼠存活率30％，TFF2-IFNα2-Fc融合蛋白在1μg/g剂量具有保护全部小鼠的流感死亡；如图4F所示，PR8感染的小鼠体重持续下降，在第7天，大部分小鼠体重丢失10～30％以上，融合蛋白TFF2-IFNα2-Fc在5μg/g的剂量下体重最大下降10％左右，在1μg/g的剂量下体重基本不下降。2xTFF2-IFNα2-Fc在1μg/g剂量以及5μg/g剂量下，均无保护效果，表明2xTFF2-IFNα2-Fc中2xTFF2的结构会影响IFNα2的功能发挥进而影响保护效果。
上述结果证明了，融合蛋白TFF2-IFNα2-Fc融合蛋白在预防及保护小鼠因 流感病毒导致的体重流失和死亡中可产生显著优于TFF2单独、IFNα2单独以及其他形式融合蛋白的效果。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
附：序列信息
Figure PCTCN2022094509-appb-000004

Claims (15)

  1. 一种融合蛋白，其包含一个或多个融合单元，每个融合单元包括：
    (a)三叶因子2(TFF2)元件，所述TFF2元件包含TFF2肽或其活性片段；
    (b)干扰素α2(IFNα2)元件，所述IFNα2元件包含IFNα2肽或其活性片段，
    其中，所述TFF2元件和IFNα2元件以1∶1的分子比融合，且在各融合单元中所述TFF2元件位于所述IFNα2元件的N端。
  2. 如权利要求1所述的融合蛋白，其中，所述TFF2肽或其活性片段来源于人、灵长类动物、啮齿类动物(如小鼠、大鼠、豚鼠、仓鼠)、犬、猫：和/或
    所述TFF2肽或其活性片段包含选自下组的氨基酸序列：SEQ ID NO：8、SEQ ID NO：12、或其活性片段(例如与SEQ ID NO：8和SEQ ID NO：12具有至少80％序列同一性且具有TFF2活性的氨基酸序列)；和/或
    所述TFF2肽或其活性片段由包含选自下组的核苷酸序列的核酸分子编码：SEQ ID NO：7、SEQ ID NO：11、或其活性片段(例如与SEQ ID NO：7或SEQ ID NO：11具有至少80％序列同一性且能够编码活性TFF2肽的核酸分子)。
  3. 如权利要求1所述的融合蛋白，其中，所述IFNα2肽或其活性片段来源于人、灵长类动物、啮齿类动物(如小鼠、大鼠、豚鼠、仓鼠)、犬、猫：和/或
    所述IFNα2肽或其活性片段包含选自下组的氨基酸序列：SEQ ID NO：10、SEQ ID NO：14、或其活性片段(例如与SEQ ID NO：10和SEQ ID NO：14具有至少80％序列同一性且具有IFNα2活性的氨基酸序列)；和/或
    所述IFNα2肽或其活性片段由包含选自下组的核苷酸序列的核酸分子编码：SEQ ID NO：9、SEQ ID NO：13、或其活性片段(例如与SEQ ID NO：9或SEQ ID NO：13具有至少80％序列同一性且能够编码活性IFNα2肽的核酸分子)。
  4. 如权利要求1所述的融合蛋白，其中，所述融合蛋白还包含连接所述TFF2元件和所述IFNα2元件和/或元件中组成肽段的接头，
    例如，所述接头为包含n个氨基酸残基的柔性接头，n为2～300的整数；
    例如，所述接头是甘氨酸或甘氨酸/丝氨酸接头，如G n、(GS) n、(GGS) n、(GGGS) n、(GGGGS) n或(GGGGGS) n的氨基酸序列，其中n是1、2、3、4、5、6、7、8、9或10的整数。
  5. 如权利要求1所述的融合蛋白，其中，所述融合蛋白包含一个或多个连续或间隔排列的TFF2肽和/或一个或多个连续或间隔排列的IFNα2肽。
  6. 如权利要求1所述的融合蛋白，其中，所述融合蛋白还包含Fc区，所述Fc区不含突变，或者包含一个或多个降低抗体介导的ADCC和CDC活性的突变，例如根据EU编号方式的氨基酸突变D265A和N297G；和/或
    所述融合蛋白还包含信号肽，例如选自tPA2信号肽、TFF2信号肽、IL-2信号肽、bPRL信号肽、CD33信号肽；和/或
    所述融合蛋白还包含标记物，例如用于纯化、检测、定位的标记物，如选自荧光标记物、非放射性核素标记物、生物素类标记物、磷酸化修饰标记、肽标签。
  7. 如权利要求1所述的融合蛋白，其中，所述融合蛋白具有SEQ ID NO：2的氨基酸序列或与所述序列具有至少80％的序列同一性；和/或
    所述融合蛋白由具有SEQ ID NO：1的核苷酸序列的核酸分子或与所述序列具有至少80％的序列同一性的核酸分子编码。
  8. 分离的核酸分子或包含所述核酸分子的构建体或载体，其中，所述核酸分子编码权利要求1～7中任一项所述的融合蛋白。
  9. 如权利要求8所述的核酸分子、构建体或载体，所述核酸分子具有SEQ ID NO：1的核苷酸序列或与所述序列具有至少80％的序列同一性；和/或
    所述核酸分子编码具有SEQ ID NO：2的氨基酸序列或与所述氨基酸序列具有至少80％的序列同一性的多肽；和/或
    所述载体选自病毒载体、mRNA载体、DNA载体。
  10. 一种细胞，其包含如权利要求1～7中任一项所述的融合蛋白、或如权利要求8或9所述的核酸分子、构建体或载体。
  11. 一种组合物，其包含如权利要求1～7中任一项所述的融合蛋白、如权利要求8或9所述的核酸分子、构建体或载体或者如权利要求10所述的细胞；以及载剂。
  12. 如权利要求1～7中任一项所述的融合蛋白、如权利要求8或9所述的核酸分子、构建体或载体、如权利要求10所述的细胞或如权利要求11所述的组合物在制备用于预防和/或治疗病毒感染性疾病中的应用。
  13. 如权利要求12所述的应用，其中，所述病毒感染性疾病选自急性病毒感染，例如呼吸道病毒感染和肠道病毒感染；和/或
    所述病毒感染性疾病由选自下组的一种或多种病毒引起：冠状病毒、流感病毒、鼻病毒、腺病毒、副流感病毒、呼吸道合胞病毒、柯萨奇病毒、埃可病毒、新型肠道病毒。
  14. 如权利要求12所述的应用，其中，所述药物作为预防性药物在病毒感染发生前预防性给药以预防病毒感染发生或降低后续病毒感染的严重程度；
    所述药物作为治疗性药物在病毒感染发生后给药以降低病毒感染与疾病的严重程度；和/或
    所述药物既作为预防性药物，也作为治疗性药物，在病毒感染发生之前和 之后连续或间隔给药。
  15. 根据权利要求12所述的应用，其中，所述药物的剂型适于选自下组的给药方式：呼吸道雾化吸入、滴鼻、喷雾、口服、肌注和/或静脉给药；和/或
    所述药物适于单独使用或与其他抗病毒药物、免疫药物或病毒疗法联合使用。
PCT/CN2022/094509 2022-05-23 2022-05-23 三叶因子2/干扰素α2融合蛋白及其防治病毒感染性疾病的应用 WO2023225802A1 (zh)

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CN101578373A (zh) * 2006-09-06 2009-11-11 费斯生物制药公司 融合肽治疗组合物
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