WO2023174122A1 - 一种长效抑制人冠状病毒膜融合的高分子及其应用 - Google Patents

一种长效抑制人冠状病毒膜融合的高分子及其应用 Download PDF

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WO2023174122A1
WO2023174122A1 PCT/CN2023/080301 CN2023080301W WO2023174122A1 WO 2023174122 A1 WO2023174122 A1 WO 2023174122A1 CN 2023080301 W CN2023080301 W CN 2023080301W WO 2023174122 A1 WO2023174122 A1 WO 2023174122A1
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human coronavirus
membrane fusion
long
polymer
inhibits human
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PCT/CN2023/080301
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English (en)
French (fr)
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王瑞
徐巍
姜世勃
陆路
段倩玉
夏帅
王茜
黄金
里卡多罗塞约
卢水秀
滕灵燕
姚雪莲
史孟君
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北京华金瑞清生物医药技术有限公司
复旦大学
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Publication of WO2023174122A1 publication Critical patent/WO2023174122A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • 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
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag

Definitions

  • This application relates to the field of medicine, specifically, to a polymer that can long-actingly inhibit human coronavirus membrane fusion and its application.
  • Coronaviruses can be divided into four genera, namely ⁇ -, ⁇ -, ⁇ -, and ⁇ -, and ⁇ -coronaviruses are further divided into four lineages: A, B, C, and D.
  • coronaviruses that can infect humans, including HCoV-229E and HCoV-NL63 of the alpha-coronavirus family, HCoV-OC43 and HCoV-HKU1 in the beta-coronavirus lineage A, and beta-coronavirus lineage B.
  • SARS-CoV and SARS-CoV-2, and MERS-CoV in the beta-coronavirus lineage C are 7 types of coronaviruses that can infect humans, including HCoV-229E and HCoV-NL63 of the alpha-coronavirus family, HCoV-OC43 and HCoV-HKU1 in the beta-coronavirus lineage A, and beta-coronavirus lineage B.
  • anti-human coronavirus membrane fusion polymers are peptides or proteins, and peptide or protein drugs face the problem of degradation after being taken.
  • Some traditional long-lasting technologies can partially solve the above degradation problems to a certain extent.
  • the application of traditional long-lasting peptide technology (such as PEG chemical modification and serum albumin/Fc fusion technology, etc.) requires the introduction of irrelevant groups or proteins that are more than 10 times larger than the volume of anti-human coronavirus membrane fusion polymers. molecules will cause the loss of activity of anti-human coronavirus membrane fusion polymers, and the long-term effect is still limited.
  • the purpose of this application is to provide a long-lasting human coronavirus membrane fusion polymer that can effectively inhibit the human coronavirus membrane fusion process, thereby achieving the effect of inhibiting human coronavirus infection, and has long-term activity in the body.
  • Another purpose of this application is to provide the application of a long-acting human coronavirus membrane fusion polymer, which can be used to prepare drugs for treating human coronavirus infection.
  • embodiments of the present application provide a long-lasting polymer that inhibits human coronavirus membrane fusion, including the following two parts: a polypeptide that inhibits human coronavirus membrane fusion and a mimetic antibody that targets serum albumin.
  • the polypeptide and mimetic antibody pass through Connecting molecules connect.
  • embodiments of the present application provide the use of a polymer that long-actingly inhibits human coronavirus membrane fusion in the preparation of drugs for treating human coronavirus infection.
  • This application provides a long-term polymer that inhibits human coronavirus membrane fusion, which can effectively interfere with or block the membrane fusion process of human coronavirus entering host cells, thereby achieving the effect of inhibiting human coronavirus infection, and in vivo Long-lasting activity.
  • the long-lasting human coronavirus membrane fusion polymer provided by this application can be transferred into E. coli using genetic engineering technology for mass production, which is more economical and economical.
  • the long-acting human coronavirus membrane fusion polymer provided in this application still has a virus inhibitory effect even if it contains a non-functional tag sequence that facilitates production and purification.
  • the long-acting human coronavirus membrane fusion polymer provided in this application has good pharmaceutical properties and can be further developed into an economical and applicable anti-new coronavirus drug.
  • Figure 1 shows the SDS-PAGE electrophoresis results of purified FL-EK1 and mimetic antibody AB in Experimental Example 1 of this application;
  • Figure 2 is the detection result of the effect of FL-EK1 on the formation of six-helix (6-HB) by HR1P and HR2P in Experimental Example 1 of the present application;
  • Figure 3 shows the inhibitory effect of FL-EK1 mediated on cell-cell fusion mediated by SARS-CoV-2 S and HCoV-OC43 S proteins respectively in Experimental Example 2 of this application;
  • Figure 4 shows the FL-EK1-mediated inhibitory effect on SARS-CoV-2 and HCoV-OC43 S pseudovirus respectively in Experimental Example 2 of this application;
  • Figure 5 shows the FL-EK1-mediated inhibitory effect on SARS-CoV-2 and HCoV-OC43 S live virus infection respectively in Experimental Example 2 of this application;
  • Figure 6 shows the FL-EK1-mediated inhibitory effect on SARS-CoV-2 Delta mutant strain live virus infection in Experimental Example 2 of the present application;
  • Figure 7 shows the anti-SARS-CoV-2 pseudovirus activity detection results of serum samples collected at different time points in Experimental Example 3 of the present application;
  • Figure 8 shows the concentrations of FL-EK1 and EK1 in mouse serum samples estimated in Experimental Example 3 of the present application and their respective half-lives.
  • the present application relates to a long-acting polymer that inhibits human coronavirus.
  • the polymer includes a part that can inhibit human coronavirus and a part that makes the inhibitory effect of the polymer long-lasting.
  • the part that inhibits human coronavirus is generally one or more polypeptides or proteins that can interfere with or block a certain step in the entry of human coronavirus into host cells, such as the membrane fusion step.
  • the envelope glycoprotein completes a membrane fusion process.
  • the S1 subunit realizes recognition and adsorption to the receptor, and the S2 subunit mainly mediates the fusion of the virus envelope and the cell membrane.
  • the two heptad repeat sequences (HR1 and HR2) on the S2 subunit play an important role in this process.
  • HR1 and HR2 The two heptad repeat sequences (HR1 and HR2) on the S2 subunit play an important role in this process.
  • the conformation of the S2 subunit undergoes a series of changes, eventually forming a hairpin trimer (or six-helix bundle) structure, in which three HR2 helices are attached in an antiparallel manner. into the groove of the central trimer formed by the polymerization of three HR1 helices.
  • hairpin trimer or six-helix bundle
  • the HR1 and HR2 regions can bind to exogenously added HR1, HR2 or similar polypeptides, which prevents the relationship between HR1 and HR2 of the S2 subunit itself in the intermediate state of the hairpin precursor.
  • the interaction results in the S2 subunit ultimately being unable to transform into a hairpin trimer structure, which inhibits membrane fusion.
  • EK1 is a pan-coronavirus membrane fusion inhibitor.
  • EK1 contains a 36-amino acid sequence.
  • EK1 is a long polypeptide, the production process is complex and costly, and it does not have a thermally stable protein tertiary structure, so its half-life in the body may be short, which may limit the clinical application of EK1. Therefore, the development of long-acting human coronavirus membrane fusion inhibitors is an urgent problem we face that needs to be solved.
  • the polymers involved in this application also include a small protein with serum albumin targeting function, that is, the aforementioned mimetic antibody AB.
  • a small protein with serum albumin targeting function that is, the aforementioned mimetic antibody AB.
  • This technology uses artificially modified small protein molecules (generally only about 100 residues) with serum albumin targeting and genetic recombination with polypeptides that need to extend the half-life. On this basis, further rational design has greater potential. The probability will not significantly reduce the original activity of the polypeptide. After the obtained fusion protein drug enters the blood circulation system, most of it is adsorbed to serum albumin, and a small part remains in a free state.
  • the adsorbed fusion protein drug avoids degradation or excretion by means of reversible binding to human serum albumin (half-life: 19-20 days). As the free fusion protein drug is consumed or cleared, the adsorbed fusion protein drug is released from It gradually dissociates from serum albumin, thus maintaining the drug concentration in the blood and maintaining the drug effect for a long time.
  • Prototype proteins that can be used to produce such artificial targeting proteins include: Staphylococcus aureus A domain protein (US5831012, EP0739353), human fibronectin (US6818418 EP1266025), etc.
  • a long-lasting polymer that inhibits human coronavirus membrane fusion characterized by comprising the following two parts: a polypeptide that inhibits human coronavirus membrane fusion and a mimetic antibody targeting serum albumin.
  • the above polypeptide and the above mimetic antibody are connected through a connecting molecule connect.
  • the above-mentioned mimetic antibody, linker molecule and polypeptide are connected together in the form of a fusion protein, and the amino acid sequence of the above-mentioned polymer is SEQ ID NO: 4.
  • the above-mentioned polymer also carries additional non-functional labels. After the polymer is added to the label, it is beneficial to improve production efficiency and reduce production costs.
  • the above-mentioned non-functional tag includes a plurality of histidines, and the amino acid sequence of the above-mentioned polymer with a non-functional tag is SEQ ID NO: 5.
  • the above-mentioned mimetic antibody is a polypeptide or protein composed of natural amino acids or unnatural amino acids and its derivatives with the ability to target serum albumin.
  • the above-mentioned mimetic antibody is a mutant derived from human fibronectin domain FN3.
  • the above-mentioned polypeptide is derived from EK1 or a derivative thereof.
  • the above-mentioned linking molecule has a molecular weight between 300Da and 5500Da and is composed of natural amino acids or unnatural amino acids. It is not easy to connect if the molecular weight is too large or too small. The molecular weight of this linking molecule is within this range and it is easier to connect to the polypeptide EK1 and the mimetic antibody AB.
  • a recombinant vector containing the above nucleic acid molecule A recombinant vector containing the above nucleic acid molecule.
  • a genetically engineered bacterium containing the above nucleic acid molecule A genetically engineered bacterium containing the above nucleic acid molecule.
  • SEQ ID NO: 1 is the amino acid sequence of the polypeptide EK1;
  • SEQ ID NO: 2 is the amino acid sequence of the linker;
  • SEQ ID NO: 3 is the amino acid sequence of the mimetic antibody AB;
  • SEQ ID NO: 4 is the amino acid sequence of the polymer AB-EK1;
  • SEQ ID NO: 5 is the amino acid sequence of the polymer FL-EK1 with a non-functional tag.
  • a method for preparing long-lasting polymers that inhibit human coronavirus membrane fusion including the following steps:
  • the mimetic antibody AB was screened by the special internal technology of Beijing Huajin Ruiqing Company, and the peptide EK1 and the linker molecule were screened by Fudan University.
  • the target gene i.e., the gene connection fragment consisting of the mimetic antibody AB, the linker molecule with a non-functional tag, and the polypeptide EK1 and the plasmid vector (pET-28a) were provided by Beijing Huajin Ruiqing Company; Escherichia coli BL21 (DE3) competent Purchased from Beijing Tiangen Company.
  • Sterilized water high pressure deionized water
  • Plasmid pET-28a-FL-EK1 plasmid was provided by Beijing Huajin Ruiqing Company
  • Escherichia coli BL21(DE3) competent form was purchased from Beijing Tiangen Company
  • Ni column protein purification process is as follows:
  • the ultrasonic crushing conditions are as follows: ultrasonic power 300W, working time 3s, interval 5s, total ultrasonic 30min. Centrifuge at 10000rpm/min for 30min.
  • a tag is added in front of the connecting molecule sequence of the polymer.
  • the sequence list of the polymer after adding the tag is SEQ ID NO: 5.
  • the final linker molecule is named FL, and the untagged polymer sequence list is SEQ ID NO: 4.
  • the labeled polymer was used for experiments, namely pET-28a-FL-EK1.
  • Embodiment 1 is basically the same as Embodiment 1, except for the following points: 1.
  • the ultrasonic power is 250W, the working time is 2s, the interval is 4s, and the total ultrasonic time is 25min. Centrifuge at 8000rpm/min for 35min; 2.
  • the washing buffer is composed of 40mM NaH 2 PO 4 , 250mM NaCl and 55mM imidazole;
  • the striping buffer is composed of 40M NaH 2 PO 4 , 250mM NaCl and 250mM imidazole; 3.
  • the pH of the washing buffer and striping buffer is 7.5.
  • Embodiment 1 is basically the same as Embodiment 1, except for the following points: 1.
  • the ultrasonic power is 250W, the working time is 4s, the interval is 6s, and the total ultrasonic time is 35min. Centrifuge at 12000rpm/min for 25min; 2.
  • the washing buffer is composed of 60mM NaH 2 PO 4 , 350mM NaCl and 65mM imidazole;
  • the striping buffer is composed of 60M NaH 2 PO 4 , 350mM NaCl and 350mM imidazole; 3.
  • the pH of the washing buffer and striping buffer is 8.5.
  • This embodiment is basically the same as Example 1, except that the washing buffer is composed of 45mM NaH 2 PO 4 , 300mM NaCl and 60mM imidazole; the striping buffer is composed of 50M NaH 2 PO 4 , 280mM NaCl and 280mM imidazole.
  • Nondenaturing polyacrylamide gel electrophoresis buffer (Native buffer) was purchased from Beijing Tianenze Company; HR1P and HR2P peptides were synthesized by Nanjing Jietai Biotechnology Co., Ltd.
  • FN-PAGE non-denaturing polyacrylamide gel electrophoresis
  • the protein materials used in this experimental example and all subsequent experimental examples are the purified proteins in Example 1. Purified The SDS-PAGE electrophoresis results of FL-EK1 and mimetic antibody AB are shown in Figure 1. It was identified that they were indeed FL-EK1 and mimetic antibody AB in the experiment of this example.
  • the FN-PAGE experimental process is as follows:
  • the first lane is HR1P
  • the second lane is HR2P
  • the third lane is +HR2P
  • the fourth lane is FL-EK1
  • the fifth, sixth and seventh lanes are all FL-EK1+HR1+ HR2, the difference is that the concentration of FL-EK1 is different, and the concentration of FL-EK1 in lanes five, six and seven gradually increases.
  • the strip that as the concentration of FL-EK1 increases, it interferes with the formation of the six-helix bundle (6-HB) formed by HR1P and HR2P.
  • the experimental results show that FL-EK1 can bind to HR1P and compete with HR2P to affect the formation of 6-HB, and as the concentration of FL-EK1 increases, the amount of 6-HB formed gradually decreases.
  • Cells 293T cells, Hul-7 (SARS-CoV-2 cell fusion target cells), Calu-3 (SARS-CoV-2 target cells), RD cells (HCoV-OC43 target cells).
  • 96-well flat culture plate (corning), 96-well round-bottom culture plate (corning), 5 ⁇ cell lysis buffer (Promega), Luciferase Assay System (Promega), Vigofect transfection reagent, EasyPure Viral DNA/RNA Kit, One Step PrimeScript RT-PCR Kit (Perfect Real Time, TaKaRa).
  • Viruses SARS-CoV-2 pseudovirus, live virus, SARS-CoV-2 mutant Delta live virus.
  • the cell-cell fusion experimental steps are as follows:
  • the experimental steps for live virus inhibition are as follows:
  • Both cell-cell fusion mediated by CoV-2 S protein have inhibitory effects, and the inhibitory effect of FL-EK1 on cell-cell fusion mediated by SARS-CoV-2 S protein is stronger than that of EK1 on SARS-CoV-2 S Inhibition of protein-mediated cell-cell fusion; the IC 50 of FL-EK1 and EK1 on cell-cell fusion mediated by HCoV-OC43 S protein are 398.2nM and 246.4nM, respectively, indicating that FL-EK1 and EK1 have an inhibitory effect on HCoV-OC43 S protein.
  • Both cell-cell fusion mediated by OC43 S protein have inhibitory effects, and the inhibitory effect of EK1 on cell-cell fusion mediated by HCoV-OC43 S protein is stronger than that of FL-EK1 on cell-cell fusion mediated by HCoV-OC43 S protein. Inhibition of fusion.
  • IC 50 are 1592.6nM and 3261.2nM respectively, indicating that both FL-EK1 and EK1 have inhibitory effects on HCoV-OC43 S pseudovirus, and the inhibitory effect of FL-EK1 on HCoV-OC43 S pseudovirus is stronger than that of EK1 on HCoV-OC43 S Inhibitory effects of pseudoviruses.
  • the IC 50 of FL-EK1 and EK1 against HCoV-OC43 S live virus are 29.4nM and 66.8nM respectively, indicating that FL-EK1 and EK1 are active against HCoV-OC43 S All viruses have inhibitory effects, and the inhibitory effect of FL-EK1 on HCoV-OC43 S live virus is stronger than the inhibitory effect of EK1 on HCoV-OC43 S live virus.
  • mice BALB/C mice, FL-EK1 recombinant protein, EK1 peptide, Caco-2 cells and SARS-CoV-2 pseudovirus.
  • mice were injected intraperitoneally with FL-EK1 and EK1, and their drug metabolism processes in the mice were detected respectively.
  • binding to serum albumin can substantially increase the half-life of a polypeptide or protein.
  • This experimental example tests the pharmacokinetics of FL-EK1 and EK1 in mice. After intraperitoneal injection of FL-EK1 and EK1 in mice, the anti-SARS-CoV-2 pseudovirus in serum samples collected at different time points The serum inhibitory activity of is shown in Figure 7. The estimated concentrations of FL-EK1 and EK1 in mouse serum samples and their respective half-lives are shown in Figure 8. It can be seen that the half-life of FL-EK1 is 30h and the half-life of EK1 is 1.8h, showing the half-life requirements of FL-EK1. About 15.7 times longer than EK1.
  • the embodiments of the present application provide a long-term polymer that inhibits human coronavirus membrane fusion and its preparation method and application.
  • the polymer includes part of EK1 that can inhibit human coronavirus and makes the polymer.
  • the partial mimetic antibody AB and a linker molecule are used to extend the inhibitory effect of the molecule.
  • the polypeptide EK1 and the mimetic antibody AB are connected through the linker molecule.
  • This polymer can effectively interfere with or block the membrane fusion process of human coronavirus entering host cells, thereby achieving the effect of inhibiting human coronavirus infection, and the inhibition time is long-term, because this polymer can be used to prepare treatments for human coronavirus infection. Long-acting membrane fusion peptide drugs.
  • FL-EK1 and EK1 both have inhibitory effects on cell-cell fusion mediated by SARS-CoV-2 S protein and HCoV-OC43 S protein.
  • FL-EK1 inhibits cell-cell fusion mediated by SARS-CoV-2 S protein.
  • the inhibitory effect is stronger than that of EK1 on cell-cell fusion mediated by SARS-CoV-2 S protein; the inhibitory effect of EK1 on cell-cell fusion mediated by HCoV-OC43 S protein is stronger than that of FL-EK1 on HCoV-OC43 Inhibition of S protein-mediated cell-cell fusion.
  • FL-EK1 and EK1 have inhibitory effects on both SARS-CoV-2 S pseudovirus and HCoV-OC43 S pseudovirus, and FL-EK1 has both inhibitory effects on SARS-CoV-2 S pseudovirus and HCoV-OC43 S pseudovirus. Stronger than EK1's inhibitory effect on SARS-CoV-2 S pseudovirus HCoV-OC43 S pseudovirus.
  • FL-EK1 and EK1 both have inhibitory effects on SARS-CoV-2 S live virus and HCoV-OC43 S live virus, and FL-EK1 has both inhibitory effects on SARS-CoV-2 S live virus and HCoV-OC43 S live virus.
  • This application provides a method for preparing a long-acting human coronavirus membrane fusion polymer. After the polymer is subjected to a series of rational designs through genetic engineering technology, its biological activity can be retained and the polymer can be maximized for human use. Inhibition of the coronavirus membrane fusion process; in addition, this preparation The method is simple in process and easy to operate, and can maximize the inhibitory effect of the long-lasting human coronavirus membrane fusion polymer.

Abstract

本申请提出了一种长效抑制人冠状病毒膜融合的高分子及其应用，涉及医药领域。一种长效抑制人冠状病毒膜融合的高分子，包括以下两部分：抑制人冠状病毒膜融合的多肽和靶向血清白蛋白的拟抗体，多肽和拟抗体通过连接分子连接。该高分子能够有效地干扰或阻断人冠状病毒进入宿主细胞的膜融合过程，从而达到抑制人冠状病毒感染的作用，且在体内活性持久。该高分子即使在带有便于生产纯化的非功能性标签序列情况下，该高分子也仍然具备病毒抑制效果。该高分子的以上特征，使其具备了良好的成药性，可进一步开发成为经济适用的抗新冠病毒药品。

Description

一种长效抑制人冠状病毒膜融合的高分子及其应用 技术领域
本申请涉及医药领域，具体而言，涉及一种长效抑制人冠状病毒膜融合的高分子及其应用。
背景技术
冠状病毒可分为四个属，即α-、β-、γ-和δ-，β-冠状病毒进一步被分为四个谱系：A、B、C和D。目前，可以感染人类的冠状病毒有7种，包括α-冠状病毒家族的HCoV-229E和HCoV-NL63，β-冠状病毒A系中的HCoV-OC43和HCoV-HKU1，β-冠状病毒B系中的SARS-CoV和SARS-CoV-2，以及β-冠状病毒C系中MERS-CoV。自2019年12月以来，由SARS-CoV-2感染引起的新型冠状病毒病(COVID-19)已在全球范围内蔓延。尤其是最近一系列备受关注的SARS-CoV-2突变体(VOC)，包括Alpha(B.1.1.7)、Beta(B.1.351)、Gamma(P.1)、Delta(B.1.617.2)和Omicron(B.1.1.529)的出现，导致其传播性增强并且对当前COVID-19疫苗和抗体疗法的敏感性降低。
很多抗人冠状病毒的膜融合的高分子是多肽或蛋白，而多肽或蛋白药物在服用后都面临被降解的问题。一些传统的长效化技术可以从一定程度上部分解决上述降解问题。但是，应用传统的多肽长效化技术(如PEG化学修饰和血清白蛋白/Fc融合技术等)，需要引入比抗人冠状病毒膜融合的高分子的体积大10倍以上的无关基团或蛋白分子，会导致抗人冠状病毒膜融合的高分子的活性丧失，且长效化效果依然有限。
申请内容
本申请的目的在于提供一种长效人冠状病毒膜融合的高分子，该高分子能够有效地抑制人冠状病毒膜融合过程，从而达到抑制人冠状病毒感染的作用，且在体内活性长久。
本申请的另一目的在于提供一种长效人冠状病毒膜融合的高分子的应用，该高分子可以用于制备治疗人冠状病毒感染的药物。
本申请解决其技术问题是采用以下技术方案来实现的。
一方面，本申请实施例提供一种长效抑制人冠状病毒膜融合的高分子，包括以下两部分：抑制人冠状病毒膜融合的多肽和靶向血清白蛋白的拟抗体，多肽和拟抗体通过连接分子连接。
另一方面，本申请实施例提供一种长效抑制人冠状病毒膜融合的高分子在制备治疗人冠状病毒感染的药物中的应用。
相对于现有技术，本申请的实施例至少具有如下优点或有益效果：
1、本申请提供的一种长效抑制人冠状病毒膜融合的高分子，能够有效地干扰或阻断人冠状病毒进入宿主细胞的膜融合过程，从而达到抑制人冠状病毒感染的作用，且体内活性持久。
2、本申请提供的一种长效人冠状病毒膜融合的高分子，可以利用基因工程技术将该高分子转入大肠杆菌中进行批量化生产，更加的经济节约。
3、本申请提供的一种长效人冠状病毒膜融合的高分子，即使在带有便于生产纯化的非功能性标签序列情况下，也仍然具备病毒抑制效果。
4、本申请提供的一种长效人冠状病毒膜融合的高分子，具备良好的成药性，可进一步开发成为经济适用的抗新冠病毒药品。
附图说明
为了更清楚地说明本申请实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本申请的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为本申请实验例1中纯化后FL-EK1和拟抗体AB的SDS‐PAGE电泳结果；
图2为本申请实验例1中FL-EK1对HR1P和HR2P形成六螺旋(6-HB)影响的检测结果；
图3为本申请实验例2中FL-EK1介导的分别对SARS-CoV-2 S和HCoV-OC43 S蛋白介导的细胞-细胞融合的抑制效果；
图4为本申请实验例2中FL-EK1介导的分别对SARS-CoV-2和HCoV-OC43 S假病毒的抑制效果；
图5为本申请实验例2中FL-EK1介导的分别对SARS-CoV-2和HCoV-OC43 S活病毒感染的抑制效果；
图6为本申请实验例2中FL-EK1介导的对SARS-CoV-2 Delta突变株活病毒感染的抑制效果；
图7为本申请实验例3中不同时间点采集的血清样品的抗SARS-CoV-2假病毒的活性检测结果；
图8为本申请实验例3中估算的小鼠血清样品中FL-EK1和EK1的浓度及各自的半衰期。
具体实施方式
为使本申请实施例的目的、技术方案和优点更加清楚，下面将对本申请实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。
本申请涉及一种长效抑制人冠状病毒的高分子，该高分子包括能抑制人冠状病毒的部分和使该高分子的抑制作用长效化的部分。抑制人冠状病毒的部分一般是一个或多个多肽或蛋白，该多肽或蛋白可以干扰或阻断人冠状病毒进入宿主细胞的某一步骤，例如膜融合步骤。人冠状病毒侵入靶细胞时由囊膜糖蛋白介导完成一个膜融合过程。在这个过程中，S1亚基实现识别及与受体吸附，S2亚基主要介导病毒囊膜和细胞膜的融合。在S2亚基上的2个七肽重复序列(HR1和HR2)在这一过程中发挥着重要的作用。当人冠状病毒侵染靶细胞时，S2亚基的构象发生一系列变化，最终形成一种发卡三聚体(或称六螺旋束)的结构，即3个HR2螺旋以反向平行的方式附着到3个HR1螺旋聚合形成的中心三聚体的沟槽中。推测在膜融合发生前存在一个HR1和HR2区暴露但未形成最终的发卡结构的发卡前体中间态。在中间态时，HR1和HR2区能结合外源加入的HR1、HR2或类似多肽，这就使得处在发卡前体中间态的S2亚基本身的HR1和HR2之间不能发生 相互作用，从而导致S2亚基最终不能转变形成发卡三聚体的结构，即抑制了膜融合。
EK1是一种泛冠状病毒膜融合抑制剂，EK1含有36个氨基酸序列，作为膜融合抑制剂，具有很高的抗病毒能力，可以广泛的抗击各种人冠状病毒。但是由于EK1属于长多肽，生产工艺复杂且成本高昂，并且不具备具有热稳定性的蛋白质三级结构，在体内的半衰期可能较短，从而可能限制EK1的临床应用。因此，研发长效人冠状病毒膜融合抑制剂是我们面临的一个亟待解决的问题。除抑制人冠状病毒的部分外，本申请所涉及的高分子还包括一个具有血清白蛋白靶向功能的小蛋白，即前述的拟抗体AB。近年来，基于靶向血清白蛋白的多肽长效化技术路线受到重视。这一技术采用人工改造后的，具有血清白蛋白靶向性的小蛋白分子(一般仅有约100残基)与需要延长半衰期的多肽基因重组，在此基础上进一步理性设计后具有较大的几率不会大幅度降低多肽原有活性。所得到的融合蛋白药物进入血液循环系统后，绝大部分被吸附到血清白蛋白上，少部分保持游离状态。被吸附的融合蛋白药物借助与人血清白蛋白的可逆性结合作用(半衰期：19‐20天(避免被降解或排泄。随着游离的融合蛋白药物被消耗或清除，吸附状态的融合蛋白药物从血清白蛋白上逐渐解离下来，从而维持了血液中的药物浓度，长时间地维持药效。可用于产生这类人工靶向蛋白的原型蛋白包括：金黄色葡萄球菌A结构域蛋白(US5831012、EP0739353)、人纤连蛋白(US6818418 EP1266025)等。
下面将参考具体实施例来详细说明本申请。
一种长效抑制人冠状病毒膜融合的高分子，其特征在于，包括以下两部分：抑制人冠状病毒膜融合的多肽和靶向血清白蛋白的拟抗体，上述多肽和上述拟抗体通过连接分子连接。
在本申请的实施例中，上述拟抗体、连接分子和多肽以融合蛋白形式连在一起，上述高分子的氨基酸序列为SEQ ID NO：4。
在本申请的实施例中，上述高分子还带有额外的非功能性标签。该高分子加入该标签后，有利于提高生产效率和降低生产成本。
在本申请的实施例中，上述非功能性标签包含有多个组氨酸，上述带有非功能性标签的高分子的氨基酸序列为SEQ ID NO：5。
在本申请的实施例中，上述拟抗体是具有靶向血清白蛋白能力的、由天然氨基酸或非天然氨基酸组成的多肽或蛋白及其衍生物。
在本申请的实施例中，上述拟抗体是来自于人纤连蛋白结构域FN3的突变体。
在本申请的实施例中，上述多肽来自于EK1或其衍生物。
在本申请的实施例中，上述连接分子为分子量在300Da-5500Da之间的、由天然氨基酸或非天然氨基酸组成。分子量太大或太小都不易于连接，本连接分子的分子量在此范围内更易于与多肽EK1和拟抗体AB连接。
一种分离的核酸分子，其编码任一上述的一种长效抑制人冠状病毒膜融合的高分子中的多肽或蛋白。
一种包含上述核酸分子的重组载体。
一种包含上述核酸分子的基因工程菌。
一种包含任一上述的一种长效抑制人冠状病毒膜融合的高分子的药物组合。
一种长效抑制人冠状病毒膜融合的高分子在制备治疗人冠状病毒感染的药物中的应用。
序列对照表如表1所示。
表1序列对照表
如表1所示，其中，SEQ ID NO：1为多肽EK1的氨基酸序列；SEQ ID NO：2为连接分子(Linker)的氨基酸序列；SEQ ID NO：3为拟抗体AB的氨基酸序列；SEQ ID NO：4为高分子AB-EK1的氨基酸序列；SEQ ID NO：5为带有非功能性标签的高分子FL-EK1的氨基酸序列。
以下结合实施例对本申请的特征和性能作进一步的详细描述。
实施例1
一种长效抑制人冠状病毒膜融合的高分子的制备方法，包括以下步骤：
1、FL-EK1基因工程菌的构建与表达
1.1、实验材料和试剂
拟抗体AB由北京华金瑞清公司内部特殊技术筛选得到，多肽EK1和连接分子由复旦大学筛选得到。目的基因(即由拟抗体AB、带有非功能性标签的连接分子和多肽EK1的基因连接片段)和质粒载体(pET-28a)均由北京华金瑞清公司提供；大肠杆菌BL21(DE3)感受态购自北京Tiangen公司。
1.2、实验步骤
将带有目的基因的质粒载体，即质粒pET-28a-FL-EK1转化到BL21(DE3)感受态中，培养12‐16h，挑取4‐5个克隆菌株，加入到含卡那霉素抗性的LB培养基中过夜培养。次日，将5ml过夜培养菌液加入500mlLB培养基中，然后在30℃，220rpm/min的条件培养4h左右，当OD值达到0.6时，加入0.2mM的IPTG诱导蛋白表达，在16℃温度下继续诱导12h左右，将培养好后的菌液在6000rpm/min的条件下进行离心10min，收集菌体。对收集好的菌体，用30ml的PBS缓冲液重悬菌体，再次离心后，弃上清，菌体冻于‐80℃冰箱。
2、蛋白纯化
2.1、实验材料和试剂
灭菌水：高压去离子水
质粒：pET-28a-FL-EK1质粒由北京华金瑞清公司提供
30％Bis‐Acr聚丙烯酰胺凝胶：购自Bio‐rad公司
Ni纯化柱：购自Qiagen公司
大肠杆菌BL21(DE3)感受态购自北京Tiangen公司
2.2、实验步骤
Ni柱纯化蛋白过程如下：
(1)、取冻存后的菌液，室温放置至解冻，用纯化试剂binding buffer(50mM NaH2PO4，pH8.0，300mM NaCl，10mM咪唑)30ml重悬菌体，漩涡振荡器震10min，加入150μl的10％Titron100‐PBS后，颠倒震荡，放置于冰水混合物中。
(2)、超声破碎，超声破碎条件如下：超声功率300W，工作时间3s，间隔5s，共计超声30min。10000rpm/min离心30min。
(3)、取离心后的破碎后的菌液上清，使用0.45μm的滤膜过滤。同时取出平衡后的Ni纯化柱，将1mlNi柱材混入上清中，冰上水平震荡45min。
(4)、加入蛋白纯化柱，Ni柱琼脂糖颗粒在溶液中逐渐沉降，上清至少过柱2遍，使得蛋白与亲和纯化柱充分结合。
(5)、使用washing buffer(50mM NaH2P04，pH8.0，300mM NaCI，60mM咪唑)溶液洗脱杂蛋白，洗脱体积约为50ml。
(6)、使用striping buffer(50mM NaH2P04，pH8.0，300mM NaCI，300mM咪唑)洗脱目的蛋白。收集洗脱下来的各个组分，置于4℃冰箱用PBS透析过夜，冻存于‐80℃冰箱。
(7)、使用完毕的Ni纯化柱使用5ml6M盐酸胍处理剥脱柱上蛋白，直至binding buffer平衡后，加入20％乙醇溶液保存于4℃冰箱。
这里需要说明的是为了将该高分子更好的进行蛋白纯化，在该高分子的连接分子序列前面加了一段标签，加好标签后的高分子序列表为SEQ ID NO：5，加好标签后的连接分子将之命名为FL，不加标签的高分子序列表为SEQ ID NO：4。在本申请后面的实验例中，均采用加好标签的高分子进行实验，即pET-28a-FL-EK1。
实施例2
本实施例与实施例1基本相同，区别在于以下几点：1、超声功率250W，工作时间2s，间隔4s，共计超声25min。8000rpm/min离心35min；2、washing buffer由40mM NaH2P04、250mM NaCl和55mM咪唑组成；striping buffer由40M NaH2PO4、250mM NaCl和250mM咪唑组成；3、washing buffer和striping buffer的pH为7.5。
实施例3
本实施例与实施例1基本相同，区别在于以下几点：1、超声功率250W，工作时间4s，间隔6s，共计超声35min。12000rpm/min离心25min；2、washing buffer由60mM NaH2P04、350mM NaCl和65mM咪唑组成；striping buffer由60M NaH2PO4、350mM NaCl和350mM咪唑组成；3、washing buffer和striping buffer的pH为8.5。
实施例4
本实施例与实施例1基本相同，区别在于：washing buffer由45mM NaH2P04、300mM NaCl和60mM咪唑组成；striping buffer由50M NaH2PO4、280mM NaCl和280mM咪唑组成。
实验例1
非变性聚丙烯酰胺凝胶电泳缓冲液(Native buffer)购自北京天恩泽公司；HR1P、HR2P多肽由南京杰泰生物科技有限公司合成。
使用FN-PAGE(非变性聚丙烯酰胺凝胶电泳)检测FL-EK1对六螺旋形成的影响，本实验例以及后面所有的实验例所用的蛋白材料均为实施例1中纯化后的蛋白，纯化后FL-EK1、拟抗体AB的SDS‐PAGE电泳结果如图1所示，经鉴定其确为本实施例实验的FL-EK1和拟抗体AB。
FN-PAGE实验过程如下：
(1)、配制18％的分离胶，5％的浓缩胶
(2)、配制HR1P、FL-EK1(4.2μM)、HR1P/FL-EK1混合物(FL-EK1的终浓度分别为：4.2μM、16.7μM和66.7μM)，使HR1P终浓度为120μM。置于37℃放置1h。
(3)、配制HR2P(40μM)、向HR1P/FL-EK1混合物中加入HR2P，继续37℃放置30min。
(4)、加入N-PAGE专用loading buffer,在电压125V的条件下，于冰水浴中电泳3h。
(5)、考马斯亮蓝染色N-PAGE胶30min后，脱色液脱色。
(6)、使用ImageJ软件进行灰度值分析，其结果如图2所示。
从图2中可以看出，第一泳道为HR1P，第二泳道为HR2P，第三泳道为+HR2P，第四泳道为FL-EK1，第五、六和七泳道均为FL-EK1+HR1+HR2，区别是FL-EK1浓度不同，第五、六和七泳道FL-EK1浓度逐渐增加。从条带上可见，随着FL-EK1浓度的增加，干扰了HR1P和HR2P形成的六螺旋束(6-HB)的形成。该实验结果表明：FL-EK1可与HR1P结合，与HR2P竞争来影响6-HB的形成，且随着FL-EK1浓度的增加，6-HB的形成量逐渐减少。
实验例2
SARS-CoV-2、HCoV-OC43的细胞-细胞融合实验及假病毒/活病毒抑制实验。
实验材料如下：
细胞：293T细胞、Hul-7(SARS-CoV-2细胞融合靶细胞)、Calu-3(SARS-CoV-2的靶细胞)、RD细胞(HCoV-OC43的靶细胞)。
培养基：含10％FBS的DMEM。
96孔平地培养板(corning)、96孔圆底培养板(corning)、5×细胞裂解液(Promega)，Luciferase Assay System(Promega)、Vigofect转染试剂、EasyPure Viral DNA/RNA Kit试剂盒、One Step PrimeScript RT-PCR Kit(Perfect Real Time，TaKaRa)。
病毒：SARS-CoV-2假病毒、活病毒、SARS-CoV-2突变株Delta活病毒。
细胞-细胞融合实验步骤如下：
(1)、将292T细胞铺于六孔板内至24h后长至70％-80％，将编码EGFP的质粒pAAV-IRES-EGFP与Vigofect转染试剂共转染293T细胞，12h后更换新鲜培养基，再培养24h后，收集表达绿色荧光的293T细胞作为效应细胞。
(2)、提前一晚将Hul-7细胞及RD细胞调整至2×104个/孔，96孔平底板中每孔加入100μl细胞，置于37℃含5％CO2的培养箱培养。
(3)、FL-EK1、多肽EK1、拟抗体AB在96孔圆底板内进行倍比稀释，同时设定细胞对照孔和病毒(效应细胞)对照孔。
(4)、收集表达绿色荧光的效应细胞等体积加入药物稀释版中，置于37℃含5％CO2的培养箱孵育30min。
(5)、吸取100μl/孔的混合液到96孔靶细胞板中，培养2-6h后在荧光显微镜下观察细胞融合情况，并进行计数。
假病毒抑制实验步骤如下：
(1)、将Calu-3细胞调整到1×104个/孔，将RD细胞调整到8000个/孔，96孔平底板中每孔加入100μl细胞，置于37℃含5％CO2的培养箱过夜。
(2)、FL-EK1、EK1和拟抗体AB在96孔圆底板内进行倍比稀释，同时设定细胞对照孔和病毒对照孔。
(3)、将‐80℃解冻的病毒株充分混匀，1：1等量加入药物稀释板中，置于37℃，含5％CO2的培养箱孵育30min。
(4)、吸取100μl/孔的培养基到96孔靶细胞板中，培养12h后弃去原培养基，加入新的含10％FBS的DMEM培养基，继续37℃培养2天。
(5)、弃去上清，加入50μl稀释好的细胞裂解液1h后，吸取35μl上清至96孔白板中，加入35μl的Luciferase，检测OD450的吸光度值。
活病毒抑制实验步骤如下：
(1)、细胞准备和药物准备同假病毒抑制实验(1)、(2)。
(2)、将‐80℃解冻的病毒株充分混匀，将活病毒按照100倍TCID50值(即50％组织感染剂量)1：1等量加入药物稀释板中置于含有5％CO2，37℃培养箱中2h。弃去96孔细胞板内上清，加入含2％FBS的DMEM，置于含有5％CO2,37℃培养箱中继续培养4h。
(3)、对于SARS-CoV-2活病毒细胞板，按照EasyPure Viral DNA/RNA Kit试剂盒提取样品RNA。采用探针法和One Step PrimeScript RT-PCR Kit(Perfect Real Time)(RT-PCR试剂盒)检测SARS-CoV-2活病毒及SARS-CoV-2突变株Delta(B.1.617.2)活病毒N基因的拷贝数。
(4)、对于HCoV-OC43活病毒细胞板，弃去原有上清，加入CCK8(100μL/孔)，置于37℃培养2h左右，检测每孔OD450吸光度。
FL-EK1介导的对SARS-CoV-2 S蛋白和HCoV-OC43 S蛋白介导的细胞-细胞融合的抑制效果分别如图3A和3B所示；
FL-EK1介导的对SARS-CoV-2 S假病毒和HCoV-OC43 S假病毒的抑制效果分别如图4A和4B所示；
FL-EK1介导的对SARS-CoV-2 S活病毒和HCoV-OC43 S活病毒的抑制效果分别如图5A和5B所示；
FL-EK1介导的对SARS-CoV-2 Delta突变株活病毒的抑制效果如图6所示；
从图3A和图3B中可以看出FL-EK1和EK1对SARS-CoV-2 S蛋白的介导的细胞-细胞融合的IC50分别为68.5nM和393.5nM，表明FL-EK1和EK1对SARS-CoV-2 S蛋白介导的细胞-细胞融合均具有抑制作用，且FL-EK1对SARS-CoV-2 S蛋白介导的细胞-细胞融合的抑制作用强于EK1对SARS-CoV-2 S蛋白介导的细胞-细胞融合的抑制作用；FL-EK1和EK1对HCoV-OC43 S蛋白介导的细胞-细胞融合的IC50分别为398.2nM和246.4nM，表明FL-EK1和EK1对HCoV-OC43 S蛋白介导的细胞-细胞融合均具有抑制作用，且EK1对HCoV-OC43 S蛋白介导的细胞-细胞融合的抑制作用强于FL-EK1对HCoV-OC43 S蛋白介导的细胞-细胞融合的抑制作用。
从图4A和图4B中可以看出FL-EK1和EK1对SARS-CoV-2 S假病毒的IC50分别为90.6nM和491.9nM，表明FL-EK1和EK1对SARS-CoV-2 S假病毒均具有抑制作用，且FL-EK1对SARS-CoV-2 S假病毒的抑制作用强于EK1对SARS-CoV-2 S假病毒的抑制作用；FL-EK1和EK1对HCoV-OC43 S假病毒的IC50分别为1592.6nM和3261.2nM，表明FL-EK1和EK1对HCoV-OC43 S假病毒均具有抑制作用，且FL-EK1对HCoV-OC43 S假病毒的抑制作用强于EK1对HCoV-OC43 S假病毒的抑制作用。
从图5A和图5B中可以看出FL-EK1和EK1对SARS-CoV-2 S活病毒的IC50分别为25.3nM和96.0nM，表明FL-EK1和EK1对SARS-CoV-2 S活病毒均具有抑制作用，且FL-EK1对SARS-CoV-2 S活病毒的抑制作用强 于EK1对SARS-CoV-2 S活病毒的抑制作用；FL-EK1和EK1对HCoV-OC43 S活病毒的IC50分别为29.4nM和66.8nM，表明FL-EK1和EK1对HCoV-OC43 S活病毒均具有抑制作用，且FL-EK1对HCoV-OC43 S活病毒的抑制作用强于EK1对HCoV-OC43 S活病毒的抑制作用。
从图6可以看出FL-EK1和EK1对SARS-CoV-2 Delta突变株活病毒的IC50分别为281.2nM和38.9nM，表明FL-EK1和EK1对SARS-CoV-2 Delta突变株活病毒均具有抑制作用，且EK1对SARS-CoV-2 Delta突变株活病毒的抑制作用强于FL-EK1对SARS-CoV-2 Delta突变株活病毒的抑制作用。
实验例3
小鼠半衰期实验
实验材料：BALB/C小鼠、FL-EK1重组蛋白、EK1多肽、Caco-2细胞和SARS-CoV-2假病毒。
实验过程如下：
采取小鼠腹腔注射FL-EK1与EK1，分别检测其在小鼠体内的药物代谢过程。
(1)、腹腔注射前，采阴性血清作为阴性对照。
(2)、腹腔注射药物：FL-EK1及EK1的药物注射剂量分别为40mg/kg和8.25mg/kg。
(3)、腹腔注射EK1后的8min、0.5h、1h、3h、7h、12h及注射FL-EK1后的8min、0.5h、1h、3h、7h、24h、72h、96h眼眶采集小鼠血液。
(4)、样本室温放置1h后，分离血清，冻于‐80℃冰箱。
(5)、56℃放置30min，灭活血清中的补体及酶类，分别做1：40、1：80、1：160、1：320、1：640、1：1280倍稀释后，做假病毒抑制试验，同实验例2实验过程。
(6)、通过抑制率计算出导致50％抑制SARS-CoV-2假病毒的小鼠血清稀释倍数(IC50)。
(7)、基于导致50％抑制SARS-CoV-2假病毒的小鼠血清稀释倍数及实施例2计算出的FL-EK1及EK1体外抑制SARS-CoV-2假病毒的IC50值，估算出血清中FL-EK1及EK1的浓度。
(8)、使用MODFIT软件，根据不同时间点血清中FL-EK1及EK1的浓度，计算出FL-EK1及EK1的体内半衰期及其他药代动力学参数。
如前面申请内容中讨论过，与血清白蛋白的结合可以大幅度增加多肽或蛋白的半衰期。本实验例对FL-EK1和EK1在小鼠中的药代动力学进行检测，在小鼠腹腔内注射FL-EK1和EK1后，不同时间点采集的血清样品的抗SARS-CoV-2假病毒的血清抑制活性如图7所示。估算的小鼠血清样品中FL-EK1和EK1的浓度及各自的半衰期如图8所示，可以看出FL-EK1的半衰期是30h，EK1的半衰期是1.8h，显示了FL-EK1的半衰期要比EK1长约15.7倍。应该特别指出的是，因为人的血清白蛋白的半衰期(19至21天)要比小鼠血清白蛋白的半衰期长得多，融合蛋白(FL-EK1)在人体内的半衰期很可能要比FL-EK1在小鼠上测得的半衰期更长。
综上，本申请实施例的一种长效抑制人冠状病毒膜融合的高分子及其制备方法与应用，该高分子包括能抑制人冠状病毒的部分EK1和使该高分 子的抑制作用长效化的部分拟抗体AB以及一个连接分子，多肽EK1和拟抗体AB通过连接分子连接。该高分子能够有效地干扰或阻断人冠状病毒进入宿主细胞的膜融合过程，从而达到抑制人冠状病毒感染的作用，且抑制时间长久，因为该高分子可以用于制备治疗人冠状病毒感染的长效膜融合多肽类药物。
FL-EK1和EK1对SARS-CoV-2 S蛋白和HCoV-OC43 S蛋白介导的细胞-细胞融合均具有抑制作用，FL-EK1对SARS-CoV-2 S蛋白介导的细胞-细胞融合的抑制作用强于EK1对SARS-CoV-2 S蛋白介导的细胞-细胞融合的抑制作用；EK1对HCoV-OC43 S蛋白介导的细胞-细胞融合的抑制作用强于FL-EK1对HCoV-OC43 S蛋白介导的细胞-细胞融合的抑制作用。FL-EK1和EK1对SARS-CoV-2 S假病毒和HCoV-OC43 S假病毒均具有抑制作用，且FL-EK1对SARS-CoV-2 S假病毒和HCoV-OC43 S假病毒的抑制作用均强于EK1对SARS-CoV-2 S假病毒HCoV-OC43 S假病毒的抑制作用。FL-EK1和EK1对SARS-CoV-2 S活病毒和HCoV-OC43 S活病毒均具有抑制作用，且FL-EK1对SARS-CoV-2 S活病毒和HCoV-OC43 S活病毒的抑制作用均强于EK1对SARS-CoV-2 S活病毒和HCoV-OC43 S活病毒的抑制作用。此外，FL-EK1和EK1在小鼠中的药代动力学结果显示，FL-EK1在小鼠中的半衰期是30h，EK1的半衰期是1.8h，显示了FL-EK1的半衰期要比EK1长约15.7倍。人的血清白蛋白的半衰期(19至21天)要比小鼠血清白蛋白的半衰期长得多，因此，融合蛋白(FL-EK1)在人体内的半衰期很可能要比EK1的半衰期更长得多。
本申请提供的一种长效人冠状病毒膜融合的高分子的制备方法，将该高分子通过基因工程技术进行一系列的理性设计后，可以保留其生物活性，最大化发挥该高分子对人冠状病毒膜融合过程的抑制作用；此外，该制备 方法工艺简单、便于操作，且可以最大化发挥该长效人冠状病毒膜融合的高分子的抑制作用。
以上所描述的实施例是本申请一部分实施例，而不是全部的实施例。本申请的实施例的详细描述并非旨在限制要求保护的本申请的范围，而是仅仅表示本申请的选定实施例。基于本申请中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。

Claims (13)

  1. 一种长效抑制人冠状病毒膜融合的高分子，其特征在于，包括以下两部分：抑制人冠状病毒膜融合的多肽和靶向血清白蛋白的拟抗体，所述多肽和所述拟抗体通过连接分子连接。
  2. 根据权利要求1所述的一种长效抑制人冠状病毒膜融合的高分子，其特征在于，所述拟抗体、连接分子和多肽以融合蛋白形式连在一起，所述高分子的氨基酸序列为SEQ ID NO：4。
  3. 根据权利要求2所述的一种长效抑制人冠状病毒膜融合的高分子，其特征在于，所述高分子还带有额外的非功能性标签。
  4. 根据权利要求3所述的一种长效抑制人冠状病毒膜融合的高分子，其特征在于，所述非功能性标签包含有多个组氨酸，所述带有非功能性标签的高分子的氨基酸序列为SEQ ID NO：5。
  5. 根据权利要求2所述的一种长效抑制人冠状病毒膜融合的高分子，其特征在于，所述拟抗体是具有靶向血清白蛋白能力的、由天然氨基酸或非天然氨基酸组成的多肽或蛋白及其衍生物。
  6. 根据权利要求5所述的一种长效抑制人冠状病毒膜融合的高分子，其特征在于，所述拟抗体是来自于人纤连蛋白结构域FN3的突变体。
  7. 根据权利要求1所述的一种长效抑制人冠状病毒膜融合的高分子，其特征在于，所述多肽来自于EK1或其衍生物。
  8. 根据权利要求1所述的一种长效抑制人冠状病毒膜融合的高分子， 其特征在于，所述连接分子为分子量在300Da-5500Da之间的、由天然氨基酸或非天然氨基酸组成。
  9. 一种分离的核酸分子，其编码权利要求2-8任一所述的一种长效抑制人冠状病毒膜融合的高分子中的多肽或蛋白。
  10. 一种包含权利要求9所述核酸分子的重组载体。
  11. 一种包含权利要求9所述核酸分子的基因工程菌。
  12. 一种包含权利要求1-8任一所述的一种长效抑制人冠状病毒膜融合的高分子的药物组合。
  13. 一种如权利要求1-12任一项所述的一种长效抑制人冠状病毒膜融合的高分子在制备治疗人冠状病毒感染的药物中的应用。
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