WO2021253807A1 - Anti-novel coronavirus complex and preparation method therefor - Google Patents

Anti-novel coronavirus complex and preparation method therefor Download PDF

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WO2021253807A1
WO2021253807A1 PCT/CN2020/142599 CN2020142599W WO2021253807A1 WO 2021253807 A1 WO2021253807 A1 WO 2021253807A1 CN 2020142599 W CN2020142599 W CN 2020142599W WO 2021253807 A1 WO2021253807 A1 WO 2021253807A1
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coronavirus
nanobody
new
complex
protein
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谭相石
裴建武
张跃茹
向道凤
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杭州星鳌生物科技有限公司
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    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the technical field of biomedicine, and specifically relates to the composition and preparation method of a new type of anti-coronavirus complex and its application in drugs for preventing and treating coronavirus diseases.
  • antibody drugs As a biological preparation, antibody drugs have been successfully applied to the treatment of viruses such as HIV, Ebola virus, and MERS-CoV coronavirus. However, it often takes months or even years to develop neutralizing antibodies suitable for clinical use. Although some drugs have shown initial effectiveness, the final clinical results are still awaited. The development of innovative anti-coronavirus drugs and vaccines is imminent!
  • the body's first line of defense against pathogens is the innate immune system.
  • This immune system is composed of cells and other mechanisms that can resist non-specific virus infections, that is, recognize and respond to invading viruses in a universal way.
  • the human body's second line of defense against viruses is cellular immunity, including immune cells called T cells.
  • T cells include immune cells called T cells.
  • T cells Cells in the human body continuously display fragments of their internal proteins on the cell surface (antigen presentation) for T cells to check. Once the T cells identify possible viral fragments, the corresponding cells will be specific for T killer cells and viruses Sexual T cell expansion is eliminated.
  • Some cells, such as macrophages, are exclusively responsible for antigen presentation.
  • Interferon (IFN) is a hormone produced by the body after a virus infection.
  • Interferon is a cell signal transmission protein, a host-specific glycoprotein with antiviral function.
  • the interferon secreted by cells infected with the virus can interact with the relevant receptors on the surrounding uninfected cells, prompting these cells to synthesize antiviral proteins to prevent further infection, thereby playing an antiviral effect.
  • Viral DNA in infected mammalian cells can induce a strong endogenous immune response by stimulating the secretion of interferon.
  • Endoplasmic reticulum (ER) receptor protein (STING) is an essential factor for immune response to cytoplasmic DNA.
  • ER Endoplasmic reticulum
  • STING Endoplasmic reticulum
  • cGAMP cyclized cGMP-AMP dinucleotide synthase
  • cGAMP As a second messenger, cGAMP stimulates the induction of interferon INF-I through STING, mediates the activation of TBK1 and IRF-3, and then initiates the transcription of type I interferon (INF- ⁇ ) gene.
  • STING is a transmembrane protein of the endoplasmic reticulum, which has an ENPP1 hydrolase.
  • ENPP1 hydrolase can degrade STING's agonist 2'3'-cGAMP. Therefore, preventing the hydrolysis of the STING agonist cGAMP by ENPP1 is an inevitable choice to maintain the effective life and efficacy of the immune agonist.
  • nanoliposome particles As a drug carrier, nanoliposome particles have broad application prospects in terms of prolonging the half-life of drugs, enhancing drug efficacy, and targeting targeted drug delivery. However, how to prepare targeted nanoliposomes with good stability, high encapsulation efficiency and easy to escape the cell protection barrier is still a challenge.
  • Immune targeting liposomes such as monoclonal antibodies are a good development direction.
  • monoclonal antibodies have problems such as large molecular weight, expensive preparation, difficult to mass produce, and immune response, which also pose severe challenges. How to choose antibodies with small molecular weight, good tissue penetration ability, strong specificity, high affinity, weak human immunogenicity, and avoid the complement reaction caused by the Fc segment as a component of targeted liposomes is an ideal goal for scientists. In addition, it is easy to prepare, can be efficiently expressed in a prokaryotic system fermentor, saves preparation costs, and can be mass-produced on a large scale, which is also an important factor that we prefer to consider.
  • a new bifunctional integrated complex that contains a natural immune activator and a multivalent nanobody capable of neutralizing the spike protein of the new coronavirus.
  • the integrated complex has a two-pronged anti-coronavirus function.
  • This type of dual-functional anti-neo-coronavirus complex has remarkable features that gather multiple advantages. Exploratory studies have found that the integrated complex has significantly improved antiviral activity than a single immune activator or a new coronavirus neutralizing nanobody, and has potential application value in the preparation of drugs for the prevention and treatment of new coronavirus pneumonia.
  • the present invention provides a novel anti-new coronavirus bifunctional integrated compound and a preparation method thereof.
  • the new anti-coronavirus complex can significantly induce the specific immune function against the neocoronavirus, significantly and effectively neutralize the neocoronavirus and effectively inhibit the replication of the coronavirus and viral inflammation.
  • the novel anti-neo-coronavirus bifunctional integrated complex is composed of a recombinant anti-neo-coronavirus nanobody coupled liposome and an immune agonist, and the immune agonist is encapsulated in the liposome coupled with the recombinant nanobody.
  • the immune agonist is an agonist of STING in the natural immune pathway (cGAS-STING-cGAMP-IRF3 pathway), which is the cyclic dinucleotide 2'3'-cGAMP or its derivatives.
  • the anti-coronavirus nanobody refers to the variable region domain of the alpaca nanobody or monoclonal antibody against the neocoronavirus that can tightly bind to the spike S protein of the neocoronavirus and target and neutralize the neocoronavirus.
  • the nanobody targeting the S protein of the new coronavirus spike is SARS2-VHH, and its amino acid sequence is: SEQ ID NO:1.
  • the preparation method of the above-mentioned novel anti-new coronavirus bifunctional integrated complex mainly includes the following steps:
  • coronavirus infection diseases include, but are not limited to, viral pneumonia/respiratory inflammation, viral nephritis, viral encephalitis, viral enteritis, or viral hepatitis caused by human or animal infection with coronavirus.
  • Anti-coronavirus complex drugs can be separately prepared into unit preparations of different specifications or prepared into pharmaceutical preparations through pharmaceutically acceptable carriers, including but not limited to intravenous injection preparations, nasal drip preparations, intravenous drip preparations, intramuscular injection preparations, Subcutaneous injection preparations or oral preparations; oral preparations include but are not limited to capsules, tablets or granules.
  • the present invention comprehensively studies and optimizes the functions and advantages of natural immune agonists, liposomes, and nano-antibodies that are affinity and can neutralize the spike protein of the new coronavirus, and optimize the composition of a new type of bifunctional integrated complex against the new coronavirus.
  • the research of the present invention shows that the novel anti-coronavirus complex has potential application prospects in the prevention and treatment of coronavirus drugs.
  • This type of novel complex can pass novel natural immune agonists and nano-antibodies targeted to neutralize the novel coronavirus through liposomes.
  • the chemical bond coupling is integrated, and it exerts its significantly improved performance and pharmacodynamic function than a single immunoagonist/or nanobody.
  • the new anti-coronavirus complex can effectively activate/induce type I interferon; it can effectively neutralize the neocoronavirus and effectively inhibit the replication of the coronavirus and viral inflammation. It can be used to prevent and treat coronavirus infections, including new coronavirus pneumonia and other viral inflammations.
  • cyclic dinucleotide cGAMP (ie 2'3'-cGAMP) mentioned in the present invention refers to C 20 H 22 N 10 O 13 P 2 .2NH 4 unless otherwise specified.
  • the present invention refers to STING, which is the name of a specific protein. If not specified, it is consistent with most public documents, NCBI database and European gene database. Its GENE name is: TMEM173; GENE ID is: 340061; Other names disclosed by STING include: Transmembrane Protein 173, ERIS, MITA, MPYS, NET23, SAVI, STING, hMITA, hSTING.
  • the STING agonists mentioned herein include but are not limited to cGAMP (i.e. 2'3'-cGAMP, or c-AMP-GMP) and derivatives and mixtures thereof.
  • Example 1 Preparation of a novel anti-coronavirus complex
  • Cyclic dinucleotide cGAMP is synthesized by cyclic cGMP-AMP dinucleotide synthetase (cGAS) catalyzed by the activation conditions after binding to DNA according to literature methods. The purity is above 98% (Pingwei Li, et al., Immunity, 2013, 39(6), 1019-1031.).
  • the anti-coronavirus nanobody refers to the variable region domain of a nanobody that can tightly bind to the spike S protein of the novel coronavirus and target to neutralize the novel coronavirus or a monoclonal antibody against the novel coronavirus.
  • the nanobody targeting the spike S protein of the new coronavirus is that the anti-new coronavirus alpaca nanobody gene (SARS2-VHH) is synthesized by Biotech Co., Ltd.
  • the plasmid uses pET-22b(+) as the vector, and the SMT3 tag is added. , Carrying Amp+ resistance.
  • SMT3-SARS2-VHH protein amino acids are listed as follows: SEQ ID NO: 2
  • Anti-coronavirus nano antibodies are highly expressed in Escherichia coli, and the antibody protein purification method is purified by NiNTA affinity column, with a purity of 98%.
  • the SMT3-SARS2-VHH protein was digested with SUMO protease overnight, the SMT3 tag protein was removed by the second NiNTA affinity column, and the target protein that flowed through SARS2-VHH was collected, concentrated, lyophilized in a freeze dryer, and the powdered protein was lyophilized. (Purity above 98%) is stored in an ultra-low temperature refrigerator for later use.
  • Dissolve liposome materials including lecithin, cholesterol, 1,2-distearoyl-SN-glycerol-3-phosphoethanolamine-N-maleimide-polyethylene glycol 2000
  • chloroform Vacuum rotary evaporation in a water bath to dry the film, and then add (NH 4 ) 2 SO 4 hydration to make a unilamellar liposome, add an immunoagonist to the blank liposome, and then add the terminal sulfhydryl nanobody, Incubate overnight in the dark at room temperature, and then use a molecular sieve column to remove unencapsulated drugs and unlinked Nanobody protein.
  • the obtained complex has double-layer circular vesicles with good morphology.
  • the liposome diameter is about ⁇ 195nm, and the Zeta potential is ⁇ -25mV.
  • the immune agonist encapsulation rate is 78%, and it is stable under 4 degrees cold storage.
  • the freeze-dried powder of 3% sea bath sugar solution is stored in cold storage.
  • the gene of the recombinant new coronavirus S protein (Spike protein) RBD-SD1 domain was synthesized by a biotechnology service company.
  • SEQ ID NO: 3 ITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCAFGGVSVITPGTNASGGSGGS HHHHHHHH, the RBD-SD1 was cloned into the eukaryotic expression vector pTT5, RBD-SD1
  • the expression vector was used to transiently transfect FreeStyle293F cells (Thermo Fisher) with polyethyleneimine.
  • the protein was purified from the filtered cell supernatant using NiNTA affinity column resin, and then further purified by size exclusion chromatography using Superdex 200 (GE Healthcare).
  • SDS-Page shows that the purity of the protein is more than 95%.
  • Nanobody SARS2-VHH was prepared according to the method of Example 1(B).
  • SARS2-VHH with histidine tag was fixed in a single flow cell of the NTA sensor chip, and the level of each cycle was about each cycle 400 response units (RUs).
  • Example 3 Nanobody SARS2-VHH neutralizes the titer of pseudotyped new coronavirus
  • Example 1 Prepare a novel anti-coronavirus complex according to the method in Example 1.
  • the complex contains multivalent nano antibody SARS2-VHH, which is located on the surface of nano liposome particles.
  • the pseudotyped virus particles are effectively released in the cell supernatant, the supernatant containing the SARS-CoV-2 pseudotyped virus is collected, and the new anti-coronavirus containing the SARS2-VHH nanoantibody is serially diluted with DMEM containing 10% fetal bovine serum virus complex solution, which was incubated for 1 hour at 37 [deg.] C and pseudovirions, the mixture was then added to the monolayer Huh-7 cells (per well of a 96 10 4). After 12 hours of infection, change the medium and incubate for another 48 hours. Then, the cells were lysed, and the Bright-Glo Luciferase Assay System (Promega) was used to detect relative light units to calculate the luciferase activity.
  • the neutralization percentage is calculated as follows: the neutralization rate of non-infected cells is 100%; the neutralization rate of cells with only pseudovirus is 0%; use Prism (GraphPad) to perform nonlinear regression analysis on the obtained curve to calculate the half maximum inhibitory concentration (IC50) value.
  • the experimental results show that the multivalent nanobody on the surface of the new composite particles neutralizes the pseudotyped SARS-2-S virus with a half-maximal inhibitory concentration (IC50) value of 15ng/ml.
  • Example 4 The new anti-coronavirus complex inhibits the replication of coronavirus
  • PBMC cells namely human peripheral blood mononuclear cells, were purchased from Biotech Co., Ltd.
  • PBMC cells mainly include lymphocytes (T cell/B cell), monocytes, macrophages, dendritic cells and a small number of other cell types. Among them, lymphocytes account for a large part.
  • PBMC are normal primary cells, and the cells are a mixed system. At 37°C and 5% CO 2 , 100 U/ml penicillin (Sigma-Aldrich) and 100 mg/ml streptomycin (Sigma-Aldrich). Freezing conditions: 90% complete medium + 10% DMSO, liquid nitrogen storage. Sales company QC test: does not contain HIV-1, HBV, HCV, mycoplasma, bacteria, yeast and fungi.
  • Virus strain An attenuated virus strain suitable for laboratory use was purchased from ATCC in the United States: Coronavirus (ATCC VR-841), which is bronchopneumonia coronavirus. The virus experiment operation in this study was commissioned by American Animals Inc. Virus Laboratory in the United States.
  • Group A negative control group, PBMC;
  • Group B PBMC+cGAMP;
  • Group C PBMC+anti-coronavirus complex;
  • Group D PBMC+anti-coronavirus nanoantibody.
  • 100 micrograms/ml of the corresponding immune agonist cGAMP, anti-coronavirus complex and anti-coronavirus nanoantibodies were added to the cell fluids of the other groups.
  • the inhibition rate of the virus is selected as the evaluation index.
  • the activity titer of the virus was detected by fluorescence quantitative RT-PCR.
  • Table 1 The inhibitory effects of immune agonists, nanobodies and their bifunctional integrated complexes on viruses are listed in Table 1.
  • the results in Table 1 show that the new anti-coronavirus complex has a good inhibitory effect on the replication of coronavirus in PBMC cells.
  • the novel complex is significantly better than the single immune agonist cGAMP or the nano antibody SARS2-VHH in suppressing the virus.
  • the new anti-coronavirus complex shows a significantly improved anti-virus effect.
  • mice C57BL/6 mice, male, weighing 20-22g, 7-8 weeks old, SPF grade, all mice are free to forage and drink, and are raised at room temperature (23 ⁇ 2) degrees. Feed and water are autoclaved, and all experimental feeding processes are SPF grade.
  • Intranasal drip Put the mouse in a sufficiently deep anesthesia state, fix the mouse in a dorsal position, and slowly drip the VR-841 virus suspension through the inner wall of the mouse nostril. In order to ensure the maximum lung infection efficiency, drip The inlet volume is 60 ⁇ L (30 ⁇ L per nostril). The mouse was gently taken off the workbench, and the head and chest were raised slightly with folded paper towels to ensure smooth breathing of the mouse. After the mice wake up, they are put back into the squirrel cage.
  • Mouse immunity C57BL/6 male mice, 6-8 weeks, weighing 20-22 grams;
  • mice each group of 10 mice, a total of 4 groups, respectively.
  • A OVA + cGAMP; B: OVA + novel complex; C: OVA + Nanobody; D: OVA.
  • Each mouse was injected subcutaneously with 10 micrograms of OVA and 100 micrograms of different types of cGAMP, Nanobodies or novel complexes. They were immunized on the 1, 7, and 14 days respectively, and the lung lavage fluid and blood samples were obtained on the 21st day.
  • the ELISA method was used to determine the titer of the immune agonist and its complex with the nanobody as an adjuvant to induce antibody production.
  • the experimental results are shown in Table 3. The measurement results show that as an immune adjuvant, the effect of the novel immunoagonist nanobody complex is significantly higher than that of the immunoagonist cGAMP and nanobody.
  • Example 7 The new anti-coronavirus complex induces cellular immune titers
  • Isotype control flow cytometry antibody was purchased from eBiosciences, antibody magnetic strain was purchased from Militeny Biotech, and flow cytometer was purchased from BD company. After 14 days of immunization, the mouse spleen and lung tissues were taken, ground and crushed, and the cells were removed through 40 micron holes. Centrifuge at 1000 rpm for 10 minutes to separate unlysed immune cells. Separate DC (CD40 ⁇ CD80 ⁇ CD86 ⁇ MHCII) and T (CD8+) cells with antibody magnets, add corresponding FAC antibody (diluted with FACS buffer), isotype control Antibody was used as a negative control.
  • the flow cytometry results are shown in Table 4.
  • the results of flow cytometry showed that the immune agonist cGAMP and its novel complex with nanobody targeting liposomes can significantly activate dendritic cells, DC and T cells, and the novel immune agonist-nanobody complex The effect is significantly higher than that of using the immune agonist cGAMP alone.
  • mice were intraperitoneally injected with 1g/kg of the new immunostimulant-nanobody agent complex (prepared in normal saline solution) according to their body weight, and the toxicity and death of the mice within 14 days after administration were observed. It was found that after intraperitoneal injection of the mice, the mice moved normally. Within 14 days after the administration, the mice did not die. On the 15th day, all the mice were sacrificed, dissected, and visually inspected the various organs, and no obvious lesions were seen.
  • the new immunostimulant-nanobody agent complex prepared in normal saline solution

Abstract

Provided is an anti-novel coronavirus complex and a preparation method therefor. The complex is composed of an immunostimulant wrapped by a recombinant anti-novel coronavirus alpaca nanobody-coupled liposome. Said immunostimulant is encapsulated in the recombinant nanobody-coupled liposome.

Description

[根据细则37.2由ISA制定的发明名称]　一种抗新冠病毒复合物及其制备方法[Name of invention formulated by ISA according to Rule 37.2] 　An anti-coronavirus complex and its preparation method 技术领域Technical field
本发明属于生物医药技术领域，具体涉及一类新型抗新冠病毒复合物的组成、制备方法及其在防治冠状病毒疾病药物中的应用。The invention belongs to the technical field of biomedicine, and specifically relates to the composition and preparation method of a new type of anti-coronavirus complex and its application in drugs for preventing and treating coronavirus diseases.
背景技术Background technique
目前对于新型冠状病毒所致疾病并没有特效治疗方法。新冠肺炎疫情发生以来，最受关注的焦点之一，就是可能对新冠肺炎有效的治疗方法与特效药物。但是，到目前为止，尚没有发现治疗新型冠状肺炎(COVID-19)的特效药物，也没有预防性药物和疫苗上市。人们迫切需要高效的药物来治愈COVID-19。根据目前的临床结果，可重新利用的小分子药物缺乏特异性，疗效受到影响。血浆治疗虽然表现出了一定的疗效，但恢复期血浆的供应受到限制。血浆治疗的有效成分是靶向性中和抗体。抗体药物作为一种生物制剂，已成功应用于HIV、埃博拉病毒、MERS-CoV冠状病毒等病毒的治疗。然而，要开发出适合临床使用的中和抗体，往往需要花费数月甚至数年的时间。尽管部分药物显示初步有效，但最终还有待临床结果。创新特效抗冠状病毒药物和疫苗的研发迫在眉睫！There is currently no specific treatment for the disease caused by the new coronavirus. Since the outbreak of the new crown pneumonia, one of the focuses of the most attention has been the possible effective treatment methods and specific drugs for the new crown pneumonia. However, so far, no specific drugs for the treatment of new coronary pneumonia (COVID-19) have been found, and no preventive drugs and vaccines have been on the market. People urgently need highly effective drugs to cure COVID-19. According to current clinical results, reusable small molecule drugs lack specificity and their efficacy is affected. Although plasma therapy has shown some curative effects, the supply of plasma during the recovery period is limited. The active ingredient of plasma therapy is targeted neutralizing antibodies. As a biological preparation, antibody drugs have been successfully applied to the treatment of viruses such as HIV, Ebola virus, and MERS-CoV coronavirus. However, it often takes months or even years to develop neutralizing antibodies suitable for clinical use. Although some drugs have shown initial effectiveness, the final clinical results are still awaited. The development of innovative anti-coronavirus drugs and vaccines is imminent!
最近的研究报道，科学家成功地从恢复期血浆中鉴定出多种针对新型冠状病毒SARS-CoV-2(导致呼吸道疾病COVID-19)的高强效中和抗体。由人体免疫***产生的中和抗体可以有效防止病毒感染细胞。来自动物研究的新结果显示， 这些中和抗体为COVID-19提供了一种潜在的治疗方法，以及短期预防手段。这标志着抵抗COVID-19疫情的一个重要里程碑。另外，从羊驼血液中也分离出多种抗冠状病毒的抗体，筛选出的纳米抗体可以交叉中和多种冠状病毒刺突蛋白，包括新冠病毒刺突蛋白。但是，这些中和抗体能否作为抗新冠病毒肺炎药物还要有待临床验证研究。Recent studies have reported that scientists have successfully identified a variety of highly potent neutralizing antibodies against the new coronavirus SARS-CoV-2 (causing respiratory disease COVID-19) from the plasma during the recovery period. The neutralizing antibodies produced by the human immune system can effectively prevent viruses from infecting cells. New results from animal studies show that these neutralizing antibodies provide a potential treatment and short-term prevention for COVID-19. This marks an important milestone in the fight against the COVID-19 epidemic. In addition, a variety of anti-coronavirus antibodies have also been isolated from the blood of alpaca, and the screened nano-antibodies can cross-neutralize a variety of coronavirus spike proteins, including the new coronavirus spike protein. However, whether these neutralizing antibodies can be used as anti-coronavirus pneumonia drugs has yet to be clinically validated.
人体抵御病原体的第一道防线是先天性免疫***，这一免疫***由能够抵御非特异性病毒感染的细胞和其他机制组成，即以一种通用方式来对入侵的病毒做出识别和反应。人体抗病毒的第二道防线是细胞免疫，包括了称为T细胞的免疫细胞。人体中的细胞不断地将其内部蛋白质的片断展示在细胞表面(抗原呈递)供T细胞来进行检查，一旦T细胞识别出可能的病毒片断，那么对应的细胞就会由T杀手细胞和病毒特异性T细胞扩增所消灭。诸如巨噬细胞在内的一些细胞专门负责抗原呈递。干扰素(IFN)是病毒感染之后由肌体所产生的一种激素，它能够通过杀死受感染细胞及其邻近细胞来逐步阻止病毒的复制。干扰素为细胞信号传送蛋白，是具有抗病毒功能的宿主特异性糖蛋白。细胞感染病毒后分泌的干扰素能够与周围未感染的细胞上的相关受体作用，促使这些细胞合成抗病毒蛋白防止进一步的感染，从而起到抗病毒的作用。The body's first line of defense against pathogens is the innate immune system. This immune system is composed of cells and other mechanisms that can resist non-specific virus infections, that is, recognize and respond to invading viruses in a universal way. The human body's second line of defense against viruses is cellular immunity, including immune cells called T cells. Cells in the human body continuously display fragments of their internal proteins on the cell surface (antigen presentation) for T cells to check. Once the T cells identify possible viral fragments, the corresponding cells will be specific for T killer cells and viruses Sexual T cell expansion is eliminated. Some cells, such as macrophages, are exclusively responsible for antigen presentation. Interferon (IFN) is a hormone produced by the body after a virus infection. It can gradually prevent the virus from replicating by killing the infected cell and its neighboring cells. Interferon is a cell signal transmission protein, a host-specific glycoprotein with antiviral function. The interferon secreted by cells infected with the virus can interact with the relevant receptors on the surrounding uninfected cells, prompting these cells to synthesize antiviral proteins to prevent further infection, thereby playing an antiviral effect.
在感染的哺乳动物细胞中病毒DNA能通过刺激干扰素分泌诱导内源强有力的免疫应答。内质网(ER)受体蛋白(STING)对胞质DNA的免疫应答是必需的因素。研究表明，环化cGMP-AMP二核苷酸合成酶(cGAS)在结合DNA后的活化条件下，内源性地催化cGAMP的合成。cGAMP作为第二信使通过STING刺激干扰素INF-I的感应，介导TBK1和IRF-3的活化，进而启动I型干 扰素(INF-β)基因的转录。STING是内质网的跨膜蛋白，内质网上具有一种ENPP1的水解酶。ENPP1水解酶可以降解STING的激动剂2’3’-cGAMP。因此，阻止STING激动剂cGAMP被ENPP1水解是保持免疫激动剂有效寿命和药效的必然选择。作为一种药物载体，纳米脂质体颗粒在延长药物半衰期，增强药效，靶向定点给药等方面具有广阔的应用前景。但是，如何制备稳定性好、包封率高、易于逃过细胞保护屏障的靶向纳米脂质体仍然是一个挑战。单克隆抗体等免疫靶向脂质体是一个很好的发展方向，但是，单克隆抗体有分子量大、制备成本昂贵且难于量产、免疫反应等问题，也形成了严峻挑战。如何选择分子量小，组织穿透能力好，特异性强，亲和力高，对人的免疫原性弱，避免Fc段引起的补体反应的抗体作为靶向脂质体的组成部分是科学家的理想目标。另外，容易制备，可以用原核***发酵罐高效表达，节省制备成本，可以大规模量产，也是我们优选考虑的重要因素。Viral DNA in infected mammalian cells can induce a strong endogenous immune response by stimulating the secretion of interferon. Endoplasmic reticulum (ER) receptor protein (STING) is an essential factor for immune response to cytoplasmic DNA. Studies have shown that cyclized cGMP-AMP dinucleotide synthase (cGAS) endogenously catalyzes the synthesis of cGAMP under the activation conditions after binding to DNA. As a second messenger, cGAMP stimulates the induction of interferon INF-I through STING, mediates the activation of TBK1 and IRF-3, and then initiates the transcription of type I interferon (INF-β) gene. STING is a transmembrane protein of the endoplasmic reticulum, which has an ENPP1 hydrolase. ENPP1 hydrolase can degrade STING's agonist 2'3'-cGAMP. Therefore, preventing the hydrolysis of the STING agonist cGAMP by ENPP1 is an inevitable choice to maintain the effective life and efficacy of the immune agonist. As a drug carrier, nanoliposome particles have broad application prospects in terms of prolonging the half-life of drugs, enhancing drug efficacy, and targeting targeted drug delivery. However, how to prepare targeted nanoliposomes with good stability, high encapsulation efficiency and easy to escape the cell protection barrier is still a challenge. Immune targeting liposomes such as monoclonal antibodies are a good development direction. However, monoclonal antibodies have problems such as large molecular weight, expensive preparation, difficult to mass produce, and immune response, which also pose severe challenges. How to choose antibodies with small molecular weight, good tissue penetration ability, strong specificity, high affinity, weak human immunogenicity, and avoid the complement reaction caused by the Fc segment as a component of targeted liposomes is an ideal goal for scientists. In addition, it is easy to prepare, can be efficiently expressed in a prokaryotic system fermentor, saves preparation costs, and can be mass-produced on a large scale, which is also an important factor that we prefer to consider.
经过多个方面因素的考虑和研究筛选，我们优选制备了一种新型双功能一体化复合物，该一体化复合物包含天然免疫激活剂和能中和新冠病毒刺突蛋白的多价纳米抗体，该一体化的复合物具有双管齐下地抗新冠病毒功能。该类双功能抗新冠病毒复合物具有集多种优势的显著特征。探索研究发现该一体化复合物具有比单一免疫激活剂或新冠病毒中和纳米抗体显著提高的抗病毒活性，在制备防治新冠病毒肺炎药物方面有潜在应用价值。After consideration of many factors and research and screening, we have preferably prepared a new bifunctional integrated complex that contains a natural immune activator and a multivalent nanobody capable of neutralizing the spike protein of the new coronavirus. The integrated complex has a two-pronged anti-coronavirus function. This type of dual-functional anti-neo-coronavirus complex has remarkable features that gather multiple advantages. Exploratory studies have found that the integrated complex has significantly improved antiviral activity than a single immune activator or a new coronavirus neutralizing nanobody, and has potential application value in the preparation of drugs for the prevention and treatment of new coronavirus pneumonia.
发明内容Summary of the invention
本发明提供了一种新型抗新冠病毒的双功能一体化复合物及其制备方法。该新型抗新冠病毒复合物可显著诱发抗新冠病毒的特异免疫功能，显著有效 地中和新冠病毒及有效抑制冠状病毒复制和病毒性炎症。The present invention provides a novel anti-new coronavirus bifunctional integrated compound and a preparation method thereof. The new anti-coronavirus complex can significantly induce the specific immune function against the neocoronavirus, significantly and effectively neutralize the neocoronavirus and effectively inhibit the replication of the coronavirus and viral inflammation.
该新型抗新冠病毒双功能一体化复合物由重组的抗新冠病毒纳米抗体偶联脂质体与免疫激动剂构成，所述免疫激动剂包封于重组纳米抗体偶联的脂质体中。The novel anti-neo-coronavirus bifunctional integrated complex is composed of a recombinant anti-neo-coronavirus nanobody coupled liposome and an immune agonist, and the immune agonist is encapsulated in the liposome coupled with the recombinant nanobody.
免疫激动剂为天然免疫通路(cGAS-STING-cGAMP-IRF3通路)STING的激动剂，既环二核苷酸2’3’-cGAMP或其衍生物。The immune agonist is an agonist of STING in the natural immune pathway (cGAS-STING-cGAMP-IRF3 pathway), which is the cyclic dinucleotide 2'3'-cGAMP or its derivatives.
抗新冠病毒纳米抗体是指，能与新冠病毒刺突S蛋白紧密结合，且靶向中和新冠病毒的羊驼纳米抗体或抗新冠病毒的单克隆抗体的可变区结构域。The anti-coronavirus nanobody refers to the variable region domain of the alpaca nanobody or monoclonal antibody against the neocoronavirus that can tightly bind to the spike S protein of the neocoronavirus and target and neutralize the neocoronavirus.
优选地，靶向新冠病毒刺突S蛋白的纳米抗体为SARS2-VHH,其氨基酸序列为：SEQ ID NO:1。Preferably, the nanobody targeting the S protein of the new coronavirus spike is SARS2-VHH, and its amino acid sequence is: SEQ ID NO:1.
Figure PCTCN2020142599-appb-000001
Figure PCTCN2020142599-appb-000001
上述新型抗新冠病毒双功能一体化复合物的制备方法，主要包括如下步骤：The preparation method of the above-mentioned novel anti-new coronavirus bifunctional integrated complex mainly includes the following steps:
(1)运用原核表达载体，在大肠杆菌中重组表达、纯化抗新冠病毒S蛋白的纳米抗体SARS2-VHH；(1) Using a prokaryotic expression vector, recombinantly express and purify the SARS2-VHH nanoantibody against the new coronavirus S protein in E. coli;
(2)对重组抗新冠病毒S蛋白的纳米抗体SARS2-VHH进行巯基化；(2) Sulfhydrylation of the recombinant anti-new coronavirus S protein nanobody SARS2-VHH;
(3)巯基化的重组纳米抗体SARS2-VHH与脂质体化学键偶联融合， 进而包封免疫激动剂。(3) The sulfhydrylized recombinant nanobody SARS2-VHH is coupled and fused with the chemical bond of the liposome, thereby encapsulating the immune agonist.
上述新型抗新冠病毒复合物在制备预防和/或治疗冠状病毒感染疾病药物中的应用。Application of the aforementioned novel anti-coronavirus complex in the preparation of medicines for the prevention and/or treatment of coronavirus infections.
优选地，冠状病毒感染疾病包括但不限于人或动物感染冠状病毒引起的病毒性肺炎/呼吸道炎症、病毒性肾炎、病毒性脑炎、病毒性肠炎或病毒性肝炎。Preferably, coronavirus infection diseases include, but are not limited to, viral pneumonia/respiratory inflammation, viral nephritis, viral encephalitis, viral enteritis, or viral hepatitis caused by human or animal infection with coronavirus.
抗新冠病毒复合物药物可单独制备成不同规格的单位制剂或通过药学上可接受的载体制备成药物制剂，包括但不限于静脉注射制剂、鼻腔滴注制剂、静脉滴注制剂、肌肉注射制剂、皮下注射制剂或口服制剂；口服制剂包括但不限于胶囊、片剂或颗粒剂。Anti-coronavirus complex drugs can be separately prepared into unit preparations of different specifications or prepared into pharmaceutical preparations through pharmaceutically acceptable carriers, including but not limited to intravenous injection preparations, nasal drip preparations, intravenous drip preparations, intramuscular injection preparations, Subcutaneous injection preparations or oral preparations; oral preparations include but are not limited to capsules, tablets or granules.
本发明综合研究优化天然免疫激动剂、脂质体的作用和优点，亲和且能中和新冠病毒刺突蛋白的纳米抗体，优化组成一种新型抗新冠病毒的双功能一体化复合物。The present invention comprehensively studies and optimizes the functions and advantages of natural immune agonists, liposomes, and nano-antibodies that are affinity and can neutralize the spike protein of the new coronavirus, and optimize the composition of a new type of bifunctional integrated complex against the new coronavirus.
本发明研究表明，新型抗新冠病毒复合物在预防和治疗冠状病毒药物中有潜在应用前景，该类新型复合物可将新型天然免疫激动剂及靶向中和新冠病毒的纳米抗体通过脂质体化学键偶联融为一体，发挥其比单一免疫激动剂/或纳米抗体显著提高的性能和药效功能。该新型抗新冠病毒复合物可以有效激活/诱导I型干扰素；能高效中和新冠病毒、有效抑制冠状病毒复制及病毒性炎症。可用于预防和治疗冠状病毒感染疾病包括新冠病毒性肺炎等病毒性炎症。The research of the present invention shows that the novel anti-coronavirus complex has potential application prospects in the prevention and treatment of coronavirus drugs. This type of novel complex can pass novel natural immune agonists and nano-antibodies targeted to neutralize the novel coronavirus through liposomes. The chemical bond coupling is integrated, and it exerts its significantly improved performance and pharmacodynamic function than a single immunoagonist/or nanobody. The new anti-coronavirus complex can effectively activate/induce type I interferon; it can effectively neutralize the neocoronavirus and effectively inhibit the replication of the coronavirus and viral inflammation. It can be used to prevent and treat coronavirus infections, including new coronavirus pneumonia and other viral inflammations.
本发明提及的环二核苷酸cGAMP(即2’3’-cGAMP)，如不加特殊说明，均指 C 20H 22N 10O 13P 2.2NH 4。 The cyclic dinucleotide cGAMP (ie 2'3'-cGAMP) mentioned in the present invention refers to C 20 H 22 N 10 O 13 P 2 .2NH 4 unless otherwise specified.
本发明提及STING，为特定蛋白质名称，如不加说明，均与多数公开文献及NCBI数据库、欧洲基因数据库一致。其GENE名为：TMEM173；GENE ID为：340061；STING公开的其它命名包括：Transmembrane Protein 173,ERIS,MITA,MPYS,NET23,SAVI,STING,hMITA,hSTING。The present invention refers to STING, which is the name of a specific protein. If not specified, it is consistent with most public documents, NCBI database and European gene database. Its GENE name is: TMEM173; GENE ID is: 340061; Other names disclosed by STING include: Transmembrane Protein 173, ERIS, MITA, MPYS, NET23, SAVI, STING, hMITA, hSTING.
本文提及的STING激动剂，包括但不限制于cGAMP(即2’3’-cGAMP，或c-AMP-GMP)以及其衍生物及混合物。The STING agonists mentioned herein include but are not limited to cGAMP (i.e. 2'3'-cGAMP, or c-AMP-GMP) and derivatives and mixtures thereof.
具体实施方式detailed description
下面通过实施例具体说明本发明的内容。在本发明中，以下所述的实施例是为了更好地阐述本发明，并不是用来限制本发明的范围。The content of the present invention will be described in detail through the following examples. In the present invention, the following embodiments are used to better illustrate the present invention, and are not used to limit the scope of the present invention.
实施例1：新型抗新冠病毒复合物的制备Example 1: Preparation of a novel anti-coronavirus complex
(A)天然免疫激动剂的制备。环二核苷酸cGAMP按文献方法在结合DNA后的活化条件下，由环化cGMP-AMP二核苷酸合成酶(cGAS)催化合成。纯度在98％以上(Pingwei Li，et al.,Immunity,2013,39(6),1019-1031.)。(A) Preparation of innate immune agonist. Cyclic dinucleotide cGAMP is synthesized by cyclic cGMP-AMP dinucleotide synthetase (cGAS) catalyzed by the activation conditions after binding to DNA according to literature methods. The purity is above 98% (Pingwei Li, et al., Immunity, 2013, 39(6), 1019-1031.).
(B)纳米抗体的制备。抗新冠病毒纳米抗体是指，能与新冠病毒刺突S蛋白紧密结合，且靶向中和新冠病毒的纳米抗体或抗新冠病毒的单克隆抗体的可变区结构域。(B) Preparation of Nanobodies. The anti-coronavirus nanobody refers to the variable region domain of a nanobody that can tightly bind to the spike S protein of the novel coronavirus and target to neutralize the novel coronavirus or a monoclonal antibody against the novel coronavirus.
优选地，靶向新冠病毒刺突S蛋白的纳米抗体为，抗新冠病毒羊驼纳米抗体基因(SARS2-VHH)由生物科技有限公司合成，质粒采用pET-22b(+)为载体， 添加SMT3标签，携带Amp+抗性。SMT3-SARS2－VHH蛋白氨基酸列如下：SEQ ID NO:2Preferably, the nanobody targeting the spike S protein of the new coronavirus is that the anti-new coronavirus alpaca nanobody gene (SARS2-VHH) is synthesized by Biotech Co., Ltd. The plasmid uses pET-22b(+) as the vector, and the SMT3 tag is added. , Carrying Amp+ resistance. SMT3-SARS2-VHH protein amino acids are listed as follows: SEQ ID NO: 2
Figure PCTCN2020142599-appb-000002
Figure PCTCN2020142599-appb-000002
抗新冠病毒纳米抗体用大肠杆菌高效表达，抗体蛋白纯化方法用NiNTA亲和柱纯化，纯度达98％。SMT3-SARS2－VHH蛋白再用SUMO蛋白酶酶切过夜后，经第二次NiNTA亲和柱去除SMT3标签蛋白，收集流穿SARS2－VHH目标蛋白，浓缩，用冷冻干燥机冻干，冻干粉蛋白(纯度达98％以上)于超低温冰箱保存备用。Anti-coronavirus nano antibodies are highly expressed in Escherichia coli, and the antibody protein purification method is purified by NiNTA affinity column, with a purity of 98%. After the SMT3-SARS2-VHH protein was digested with SUMO protease overnight, the SMT3 tag protein was removed by the second NiNTA affinity column, and the target protein that flowed through SARS2-VHH was collected, concentrated, lyophilized in a freeze dryer, and the powdered protein was lyophilized. (Purity above 98%) is stored in an ultra-low temperature refrigerator for later use.
(C)[纳米抗体－脂质体－免疫激动剂]复合物的制备。首先将SARS2-VHH纳米抗体末端巯基化，在纳米抗体蛋白溶液中搅动下滴加巯基化试剂(Traut’s reagent)，水浴中搅动下避光孵育1小时。用脱盐柱除去过量巯基化试剂。用Ellman方法测定纳米抗体蛋白上的巯基，验证纳米抗体巯基化成功。将脂质体材料(包括卵磷脂、胆固醇、1,2-二硬脂酰-SN-甘油-3-磷酰乙醇胺-N-马来酰亚胺-聚乙二醇2000)，溶解于氯仿中，在水浴中真空旋转蒸发干成膜，然后加入(NH 4) 2SO 4水化制成单室脂质体，向空白脂质体中加入免疫激动剂，然后加入末端巯基化的纳米抗体，室温避光孵育过夜，再用分子筛柱除去未包封的药物和未连接的纳米抗体蛋白。用TEM电镜检测所得到的复合物，双层圆形囊泡，形态良好，脂质体直径约为～195nm，Zeta电位～－25mV。免疫激动剂包封率为 78％，4度冷藏条件下稳定，3％海澡糖溶液的冻干粉冷藏保存。 (C) Preparation of [Nanobody-Liposome-Immune Agonist] Complex. Firstly, the SARS2-VHH Nanobody is sulfhylated at the end, and Traut's reagent is added dropwise to the Nanobody protein solution under agitation, and incubated in a water bath for 1 hour under agitation and protected from light. Use a desalting column to remove excess sulfhydryl reagent. The Ellman method was used to determine the sulfhydryl group on the Nanobody protein to verify the successful sulfhydrylation of the Nanobody. Dissolve liposome materials (including lecithin, cholesterol, 1,2-distearoyl-SN-glycerol-3-phosphoethanolamine-N-maleimide-polyethylene glycol 2000) in chloroform , Vacuum rotary evaporation in a water bath to dry the film, and then add (NH 4 ) 2 SO 4 hydration to make a unilamellar liposome, add an immunoagonist to the blank liposome, and then add the terminal sulfhydryl nanobody, Incubate overnight in the dark at room temperature, and then use a molecular sieve column to remove unencapsulated drugs and unlinked Nanobody protein. Detected by TEM electron microscope, the obtained complex has double-layer circular vesicles with good morphology. The liposome diameter is about ~195nm, and the Zeta potential is ~-25mV. The immune agonist encapsulation rate is 78%, and it is stable under 4 degrees cold storage. The freeze-dried powder of 3% sea bath sugar solution is stored in cold storage.
实施例2：纳米抗体SARS2-VHH与新冠病毒S蛋白亲和作用研究(1)重组新冠病毒S蛋白RBD－SD1结构域的制备Example 2: Study on the Affinity of Nanobody SARS2-VHH with S protein of New Coronavirus (1) Preparation of RBD-SD1 Domain of S protein of Recombinant New Coronavirus
重组新冠病毒S蛋白(Spike protein，刺突蛋白)RBD－SD1结构域(S蛋白第319-591氨基酸序列如下)基因有生物技术服务公司合成。SEQ ID NO：3 ITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCAFGGVSVITPGTNASGGSGGS HHHHHHHH，该RBD-SD1基因克隆到pTT5真核表达载体上，RBD－SD1结构域基因序列N端前加信号肽(MGVLLTQRTLLSLVLALLFPSMASM)，在信号肽前加入Kozak序列，末端加8XHis组氨酸标签，基因合成由生物技术公司服务完成。表达载体用于使用聚乙烯亚胺瞬时转染FreeStyle293F细胞(Thermo Fisher)。使用NiNTA亲和柱树脂从过滤的细胞上清液中纯化蛋白质，然后使用Superdex 200通过尺寸排阻色谱进行进一步纯化(GE Healthcare)。纯化蛋白质的缓冲液使用50mM Tris pH 8.0,200mM NaCl,洗杂蛋白和洗脱目的蛋白时，分别使用含有20mM咪唑/200mM咪唑的上述缓冲液。SDS－Page显示蛋白纯度达95％以上。 The gene of the recombinant new coronavirus S protein (Spike protein) RBD-SD1 domain (the 319-591 amino acid sequence of the S protein is as follows) was synthesized by a biotechnology service company. SEQ ID NO: 3 ITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNFNFNGLTGTGVLTPSSKRFQPFQQFGRDVSDFTDSVRDPKTSEILDISPCAFGGVSVITPGTNASGGSGGS HHHHHHHH, the RBD-SD1 was cloned into the eukaryotic expression vector pTT5, RBD-SD1 gene sequence prior to N-terminal domain plus signal peptide (MGVLLTQRTLLSLVLALLFPSMASM), a Kozak sequence before the signal peptide plus terminal 8XHis Histidine tag, gene synthesis is completed by the service of biotechnology company. The expression vector was used to transiently transfect FreeStyle293F cells (Thermo Fisher) with polyethyleneimine. The protein was purified from the filtered cell supernatant using NiNTA affinity column resin, and then further purified by size exclusion chromatography using Superdex 200 (GE Healthcare). Use 50mM Tris pH 8.0, 200mM NaCl as the buffer for protein purification, and use the above-mentioned buffer containing 20mM imidazole/200mM imidazole when washing the protein and eluting the target protein. SDS-Page shows that the purity of the protein is more than 95%.
(2)纳米抗体SARS2-VHH按实施例1(B)方法制备。(2) Nanobody SARS2-VHH was prepared according to the method of Example 1(B).
(3)纳米抗体SARS2-VHH与新冠病毒S蛋白RBD-SD1的亲和作用(3) The affinity of the nanobody SARS2-VHH and the new coronavirus S protein RBD-SD1
运用表面等离子体共振(SPR)方法，使用Biacore X100(GE Healthcare)，将带有组氨酸标签的SARS2-VHH固定在NTA传感器芯片的单个流通池中，每个循环的水平约为每个循环400个响应单位(RUs)。使用0.35M EDTA和0.1M NaOH，然后使用0.5mM NiCl 2双重再生芯片。将三个仅包含运行缓冲液的样品(分别由10mM HEPES pH 8.0、150mM NaCl和0.005％Tween 20组成)注入配体和参比流通池，然后依次从50-2nM稀释SARS-CoV-2RBD-SD1，重复浓度为3nM。使用Biacore X100评估软件，将所得数据扣除两次参考值并拟合为1：1结合模型。拟合结果显示，纳米抗体SARS2-VHH与新冠病毒S蛋白RBD-SD1具有很好的亲和作用，亲和常数为：K D＝19nM,ka＝9.12X 10 7M -1S -1,kd＝1.73S -1。 Using the surface plasmon resonance (SPR) method, using Biacore X100 (GE Healthcare), the SARS2-VHH with histidine tag was fixed in a single flow cell of the NTA sensor chip, and the level of each cycle was about each cycle 400 response units (RUs). Use 0.35M EDTA and 0.1M NaOH, and then use 0.5mM NiCl 2 double regeneration chip. Inject three samples containing only running buffer (composed of 10mM HEPES pH 8.0, 150mM NaCl, and 0.005% Tween 20) into the ligand and reference flow cell, and then dilute SARS-CoV-2RBD-SD1 from 50-2nM in sequence , The repeat concentration is 3nM. Using Biacore X100 evaluation software, the obtained data was subtracted twice from the reference value and fitted into a 1:1 combination model. The fitting results show that the Nanobody SARS2-VHH has a good affinity with the new coronavirus S protein RBD-SD1, and the affinity constant is: K D =19nM, ka = 9.12X 10 7 M -1 S -1 , kd = 1.73S -1 .
实施例3：纳米抗体SARS2-VHH中和假型新冠病毒效价研究Example 3: Nanobody SARS2-VHH neutralizes the titer of pseudotyped new coronavirus
(1)按照实施例1方法制备新型抗新冠病毒复合物。该复合物含有多价纳米抗体SARS2-VHH，位于纳米脂质体颗粒表面。(1) Prepare a novel anti-coronavirus complex according to the method in Example 1. The complex contains multivalent nano antibody SARS2-VHH, which is located on the surface of nano liposome particles.
(2)假型新冠病毒的制备及中和活性研究(2) Study on the preparation and neutralization activity of pseudotyped new coronavirus
为了确定新型抗新冠病毒复合物颗粒表面纳米抗体SARS2-VHH的中和活性，进行了假型病毒中和试验。按照文献方法，(Wu et al.,Identification of Human Single-Domain Antibodies against SARS-CoV-2,Cell Host&Microbe(2020),https://doi.org/10.1016/j.chom.2020.04)，将pcDNA3.1-SARS-CoV-2-S(编码SARS-CoV-2S蛋白)和pNL4-3.luc.RE(载有表达HIV-1骨架的荧光素酶报告基因)表达载体共转染到293T细胞。假型病毒颗粒有效地释放在细胞上清液中，收集含有SARS-CoV-2假型病毒的上清液，用含有10％胎牛血清的DMEM系列稀释含有纳米抗体SARS2-VHH的新型抗新冠病毒复合物溶液，将其与假病毒在37℃下孵育1小时，然后将混合物添加至单层Huh-7细胞(96孔板中每孔10 4个)。感染12小时后，换培养基，然后再孵育48小时。然后，裂解细胞，使 用Bright-Glo荧光素酶测定***(Promega)以检测相对光单位计算荧光素酶活性。中和百分率按如下方法计算：无感染细胞中和率按100％；只有假病毒的细胞其中和率为0％；使用Prism(GraphPad)对所得曲线进行非线性回归分析，以计算半数最大抑制浓度(IC50)值。 In order to determine the neutralizing activity of the SARS2-VHH nanobody on the surface of the novel anti-coronavirus complex particles, a pseudotype virus neutralization test was carried out. According to the literature method, (Wu et al., Identification of Human Single-Domain Antibodies against SARS-CoV-2, Cell Host&Microbe (2020), https://doi.org/10.1016/j.chom.2020.04), pcDNA3. 1-SARS-CoV-2-S (encoding SARS-CoV-2S protein) and pNL4-3.luc.RE (carrying a luciferase reporter gene expressing HIV-1 backbone) expression vectors were co-transfected into 293T cells. The pseudotyped virus particles are effectively released in the cell supernatant, the supernatant containing the SARS-CoV-2 pseudotyped virus is collected, and the new anti-coronavirus containing the SARS2-VHH nanoantibody is serially diluted with DMEM containing 10% fetal bovine serum virus complex solution, which was incubated for 1 hour at 37 [deg.] C and pseudovirions, the mixture was then added to the monolayer Huh-7 cells (per well of a 96 10 4). After 12 hours of infection, change the medium and incubate for another 48 hours. Then, the cells were lysed, and the Bright-Glo Luciferase Assay System (Promega) was used to detect relative light units to calculate the luciferase activity. The neutralization percentage is calculated as follows: the neutralization rate of non-infected cells is 100%; the neutralization rate of cells with only pseudovirus is 0%; use Prism (GraphPad) to perform nonlinear regression analysis on the obtained curve to calculate the half maximum inhibitory concentration (IC50) value.
实验结果显示该新型复合物颗粒表面多价纳米抗体中和假型SARS－2－S病毒的半数最大抑制浓度(IC50)值为15ng/ml。The experimental results show that the multivalent nanobody on the surface of the new composite particles neutralizes the pseudotyped SARS-2-S virus with a half-maximal inhibitory concentration (IC50) value of 15ng/ml.
实施例4：新型抗新冠病毒复合物抑制冠状病毒的复制Example 4: The new anti-coronavirus complex inhibits the replication of coronavirus
PBMC细胞：PBMC细胞，即人外周血单个核细胞，购买于生物科技有限公司。PBMC cells: PBMC cells, namely human peripheral blood mononuclear cells, were purchased from Biotech Co., Ltd.
PBMC细胞主要包括淋巴细胞(T cell/B cell)，单核细胞，巨噬细胞，树突状细胞和其他少量细胞类型。其中淋巴细胞占很大一部分。PBMC属于正常原代细胞，细胞是混合体系。在37℃和5％CO 2下，100U/ml青霉素(Sigma-Aldrich)和100mg/ml链霉素(Sigma-Aldrich)。冻存条件:90％完全培养基+10％DMSO，液氮储存。销售公司QC检测:不含有HIV-1、HBV、HCV、支原体、细菌、酵母和真菌。 PBMC cells mainly include lymphocytes (T cell/B cell), monocytes, macrophages, dendritic cells and a small number of other cell types. Among them, lymphocytes account for a large part. PBMC are normal primary cells, and the cells are a mixed system. At 37°C and 5% CO 2 , 100 U/ml penicillin (Sigma-Aldrich) and 100 mg/ml streptomycin (Sigma-Aldrich). Freezing conditions: 90% complete medium + 10% DMSO, liquid nitrogen storage. Sales company QC test: does not contain HIV-1, HBV, HCV, mycoplasma, bacteria, yeast and fungi.
病毒株：适合在实验室使用的减毒病毒株购于美国ATCC公司：冠状病毒(ATCC VR－841)，该病毒为支气管肺炎冠状病毒。该研究中病毒实验操作委托美国American Animals Inc.病毒实验室完成。Virus strain: An attenuated virus strain suitable for laboratory use was purchased from ATCC in the United States: Coronavirus (ATCC VR-841), which is bronchopneumonia coronavirus. The virus experiment operation in this study was commissioned by American Animals Inc. Virus Laboratory in the United States.
PBMC细胞培养操作：PBMC cell culture operation:
1)复苏细胞：将含1mL细胞悬液的冻存管在37℃水浴中迅速摇晃解冻，加入4mL培养基混合均匀。在1000RPM条件下离心4分钟，弃去上清液，补加1-2mL培养基后吹匀。然后将所有细胞悬液加入培养瓶中培养过夜。第二天换液并检查细胞密度。1) Resuscitate the cells: Shake the cryotube containing 1mL of cell suspension in a 37°C water bath quickly to thaw, add 4mL of medium and mix well. Centrifuge at 1000 RPM for 4 minutes, discard the supernatant, add 1-2 mL of medium and blow well. Then all the cell suspension was added to the culture flask and cultured overnight. Change the medium the next day and check the cell density.
2)细胞传代：细胞密度达80％-90％，进行传代培养。弃去培养上清，用不含钙、镁离子的PBS润洗细胞1-2次。加1ml消化液(0.25％Trypsin-0.53mM EDTA)于培养瓶中，置于37℃培养箱中消化1-2分钟，然后在显微镜下观察细胞消化情况，若细胞大部分变圆并脱落，迅速拿回操作台，轻敲几下培养瓶后加少量培养基终止消化。按8ml/瓶补加培养基，轻轻打匀后吸出，在1000RPM条件下离心4分钟，弃去上清液，补加1-2mL培养液后吹匀。将细胞悬液按1：2比例分到新的含8ml培养基的新瓶中。细胞活度测定：Fixable Red Dead Cell染色试剂盒(生命技术公司)测定细胞活力，使用0.5μl染料在1ml PBS中在黑暗中将细胞染色5分钟。然后将它们用PBS洗涤两次，并在FACS Calibur上测量。作为死细胞的阳性对照，PBMC在95℃煮沸20分钟，并用相同的程序染色以确定死细胞的峰。2) Cell passage: the cell density reaches 80%-90%, and the cells are subcultured. The culture supernatant was discarded, and the cells were rinsed 1-2 times with PBS without calcium and magnesium ions. Add 1ml of digestion solution (0.25% Trypsin-0.53mM EDTA) to the culture flask and place it in a 37℃ incubator for digestion for 1-2 minutes, and then observe the cell digestion under the microscope. If most of the cells become round and fall off, quickly Take back to the operating table, tap the culture flask a few times and add a small amount of medium to stop the digestion. Add culture medium at 8ml/bottle, mix gently and aspirate it, centrifuge at 1000RPM for 4 minutes, discard the supernatant, add 1-2mL culture solution and blow well. Divide the cell suspension into a new bottle containing 8ml of culture medium in a ratio of 1:2. Cell viability measurement: Fixable Red Dead Cell Staining Kit (Life Technology Company) to determine cell viability, stain the cells for 5 minutes in the dark with 0.5μl dye in 1ml PBS. Then they were washed twice with PBS and measured on FACS Calibur. As a positive control for dead cells, PBMC was boiled at 95°C for 20 minutes and stained with the same procedure to determine the peak of dead cells.
将培养好的PBMC细胞分成4组(每组n＝4个样)。A组，阴性对照组，PBMC；B组，PBMC+cGAMP；C组，PBMC+抗新冠病毒复合物；D组，PBMC+抗新冠病毒纳米抗体。除了A组作为对照不加药外，其它组细胞液中分别加入100微克/毫升的相应的免疫激动剂cGAMP、抗新冠病毒复合物和抗新冠病毒纳米抗体。每一个细胞样品中加入10微升的病毒液，三周后检测复合物抗病毒复制情况。选择对病毒的抑制率作为评价指标。病毒的活性滴度用荧光定量RT－PCR检测。免疫激动剂、纳米抗体及其双功能一体化复合物对病毒的抑制效果列于表1.The cultured PBMC cells were divided into 4 groups (n=4 samples in each group). Group A, negative control group, PBMC; Group B, PBMC+cGAMP; Group C, PBMC+anti-coronavirus complex; Group D, PBMC+anti-coronavirus nanoantibody. Except for group A as a control without drug addition, 100 micrograms/ml of the corresponding immune agonist cGAMP, anti-coronavirus complex and anti-coronavirus nanoantibodies were added to the cell fluids of the other groups. Add 10 microliters of virus solution to each cell sample, and detect the antiviral replication of the complex after three weeks. The inhibition rate of the virus is selected as the evaluation index. The activity titer of the virus was detected by fluorescence quantitative RT-PCR. The inhibitory effects of immune agonists, nanobodies and their bifunctional integrated complexes on viruses are listed in Table 1.
表1.新型抗新冠病毒复合物对冠状病毒的抑制作用Table 1. The inhibitory effect of the new anti-coronavirus complex on the coronavirus
Figure PCTCN2020142599-appb-000003
Figure PCTCN2020142599-appb-000003
表1结果显示，新型抗新冠病毒复合物对冠状病毒在PBMC细胞中的复制有很好的抑制作用。该新型复合物比单一的免疫激动剂cGAMP或纳米抗体SARS2-VHH抑制病毒效果明显都好。新型抗新冠病毒复合物显示出显著提高的抑制病毒效果。The results in Table 1 show that the new anti-coronavirus complex has a good inhibitory effect on the replication of coronavirus in PBMC cells. The novel complex is significantly better than the single immune agonist cGAMP or the nano antibody SARS2-VHH in suppressing the virus. The new anti-coronavirus complex shows a significantly improved anti-virus effect.
实施例5.新型抗新冠病毒复合物对小鼠肺炎的抑制作用Example 5. Inhibitory effect of new anti-coronavirus complex on pneumonia in mice
实验动物：C57BL/6小鼠，雄性，体重20－22g，7-8周龄，SPF级，所有小鼠均自由觅食和饮水，在室温(23±2)度下饲养。饲料及水均经高压灭菌处理，全部实验饲养过程为SPF级。Experimental animals: C57BL/6 mice, male, weighing 20-22g, 7-8 weeks old, SPF grade, all mice are free to forage and drink, and are raised at room temperature (23±2) degrees. Feed and water are autoclaved, and all experimental feeding processes are SPF grade.
动物分组：Animal grouping:
将30只小鼠随机分为5组(n＝6)，具体分组为：A组，正常对照组；B组，肺炎模型组，PBS；C组，给药组，新型抗新冠病毒复合物(10mg/kg)；D组，给药组，纳米抗体SARS2-VHH(10mg/kg)；E组，给药组，免疫激动剂cGAMP(10mg/kg)。30 mice were randomly divided into 5 groups (n=6), specifically grouped into: group A, normal control group; group B, pneumonia model group, PBS; group C, administration group, new anti-coronavirus complex ( 10mg/kg); D group, administration group, Nanobody SARS2-VHH (10mg/kg); E group, administration group, immunoagonist cGAMP (10mg/kg).
肺炎病毒模型小鼠建立：Establishment of pneumonia virus model mice:
鼻内滴注。使小鼠处于足够深的麻醉状态，将小鼠以背卧姿势固定，并慢慢地将VR－841病毒悬液通过小鼠鼻孔内壁逐滴滴入，为保证最大的肺部感染效率，滴入体积为60μL(每个鼻孔30μL)。将小鼠轻轻从工作台拿下，并将头部和胸部用折叠的纸巾小幅度垫高，以保证小鼠顺畅的呼吸。待小鼠苏醒后，放回鼠笼。肺组织病理学观察：取左肺大叶组织，切两半，用苦味酸溶液固定24小时后，脱水、透明、石蜡包埋，行5μm厚切片，然后进行苏木精－伊红(HE)染色，光镜下观察小鼠肺组织的病理形态学变化。Intranasal drip. Put the mouse in a sufficiently deep anesthesia state, fix the mouse in a dorsal position, and slowly drip the VR-841 virus suspension through the inner wall of the mouse nostril. In order to ensure the maximum lung infection efficiency, drip The inlet volume is 60μL (30μL per nostril). The mouse was gently taken off the workbench, and the head and chest were raised slightly with folded paper towels to ensure smooth breathing of the mouse. After the mice wake up, they are put back into the squirrel cage. Pathological observation of lung histopathology: Take the left lung large leaf tissue, cut in half, fix with picric acid solution for 24 hours, dehydrated, transparent, paraffin embedded, 5μm thick section, and then hematoxylin-eosin (HE) Dyeing, observe the pathomorphological changes of mouse lung tissue under light microscope.
小鼠肺泡灌洗液获得方法：Method of obtaining mouse alveolar lavage fluid:
取等体积PBS沿小鼠气管注射后吸出，反复几次，获得肺泡灌洗液。空白组和肺炎模型组腹腔注射等体积的DMEM。收集的血清，于－80度保存。采用ELISA法，检测TNF-alpha、IL-1beta浓度。终止反应后，将酶标板放入酶标仪槽内，选择450nm波长检测，确定标准品和空白对照区域，检测相应的光密度值，然后绘制标准曲线并计算相应的浓度。在小鼠肺炎模型中，促炎性细胞因子IL-1beta和TNF-alpha在血清和肺泡灌洗液中的含量都显著上升，三组给药后均不同程度地降低了促炎因子的含量。不同药物分别显示抑制小鼠肺炎作用的结果见表2.Take an equal volume of PBS and inject it along the mouse trachea and aspirate it, repeat it several times to obtain alveolar lavage fluid. The blank group and the pneumonia model group were intraperitoneally injected with equal volume of DMEM. The collected serum is stored at -80°C. ELISA method was used to detect the concentration of TNF-alpha and IL-1beta. After terminating the reaction, put the microtiter plate into the microplate reader slot, select 450nm wavelength for detection, determine the standard and blank control area, detect the corresponding optical density value, and then draw the standard curve and calculate the corresponding concentration. In the mouse model of pneumonia, the levels of pro-inflammatory cytokines IL-1beta and TNF-alpha in serum and alveolar lavage fluid both increased significantly. After the three groups were administered, the levels of pro-inflammatory factors were reduced to varying degrees. The results of different drugs showing the effect of inhibiting pneumonia in mice are shown in Table 2.
实验结果表明，新型免疫激动剂－纳米抗体新型复合物抗小鼠肺炎促炎细胞因子的作用显著优于单独使用免疫激动剂或纳米抗体。该类新型免疫激动剂纳米抗体复合物药物具有抗小鼠肺炎损伤的作用。The experimental results show that the novel immune agonist-nanobody complex has a significantly better effect on the anti-inflammatory cytokine of mouse pneumonia than the immune agonist or nanobody alone. This new type of immune agonist nano-antibody complex drug has the effect of anti-pneumonia injury in mice.
小鼠肺组织病理学观察可见(附图1，H&E染色肺组织石蜡切片)：与正常组小鼠相比，肺炎模型组小鼠肺部炎细胞浸润增加，肺泡间隔明显增厚。免疫激动剂及其与纳米抗体新型复合物给药后小鼠肺部炎症症状明显有所缓解，新型 复合物显示显著进步的药效。The pathological observation of mouse lung tissue showed that (Figure 1, H&E stained lung tissue paraffin section): Compared with the normal group of mice, the lungs of the pneumonia model group increased infiltration of inflammatory cells, and the alveolar septum was significantly thickened. After the administration of the immune agonist and its new complex with nano-antibody, the pulmonary inflammation in mice was significantly relieved, and the new complex showed significantly improved efficacy.
表2.新型免疫激动剂纳米抗体复合物对小鼠肺炎的抑制作用Table 2. Inhibitory effects of novel immunoagonist nanobody complexes on pneumonia in mice
Figure PCTCN2020142599-appb-000004
Figure PCTCN2020142599-appb-000004
实施例6.新型抗新冠病毒复合物诱发免疫功能Example 6. A novel anti-coronavirus complex induces immune function
小鼠免疫：C57BL/6雄性小鼠，6-8周，体重20-22克；Mouse immunity: C57BL/6 male mice, 6-8 weeks, weighing 20-22 grams;
小鼠分组：每10只一组，共4组，分别为，Grouping of mice: each group of 10 mice, a total of 4 groups, respectively,
A：OVA+cGAMP；B:OVA+新型复合物；C：OVA+纳米抗体；D：OVA。A: OVA + cGAMP; B: OVA + novel complex; C: OVA + Nanobody; D: OVA.
每只小鼠皮下注射10微克OVA和100微克的不同种类的cGAMP、纳米抗体或新型复合物。分别在第1，7，14天各免疫一次，在第21天获得肺灌洗液和取血样。用ELISA法测定免疫激动剂及其与纳米抗体复合物作为佐剂诱导产生抗体的效价。实验结果见表3.测定结果显示，作为免疫佐剂，新型免疫激动剂纳米抗体复合物的效果显著高于免疫激动剂cGAMP和纳米抗体。Each mouse was injected subcutaneously with 10 micrograms of OVA and 100 micrograms of different types of cGAMP, Nanobodies or novel complexes. They were immunized on the 1, 7, and 14 days respectively, and the lung lavage fluid and blood samples were obtained on the 21st day. The ELISA method was used to determine the titer of the immune agonist and its complex with the nanobody as an adjuvant to induce antibody production. The experimental results are shown in Table 3. The measurement results show that as an immune adjuvant, the effect of the novel immunoagonist nanobody complex is significantly higher than that of the immunoagonist cGAMP and nanobody.
表3.新型抗新冠病毒复合物诱发免疫效价Table 3. Immune titers induced by novel anti-coronavirus complexes
Figure PCTCN2020142599-appb-000005
Figure PCTCN2020142599-appb-000005
实施例7新型抗新冠病毒复合物诱发细胞免疫效价Example 7 The new anti-coronavirus complex induces cellular immune titers
小鼠饲养与病毒感染免疫、采血等见实施例5。同型对照流式抗体购自eBiosciences,抗体磁株购于Militeny Biotech,流式细胞仪购于BD公司，免疫14天后取小鼠脾脏、肺组织，分别研磨捣碎，过40微米孔脱过滤细胞，1000rpm离心10分钟，分离未被裂解的免疫细胞，用抗体磁株分离DC(CD40\CD80\CD86\MHCII)、T(CD8+)细胞，加入对应的FAC抗体(用FACS缓冲液稀释)，同型对照抗体作为阴性对照，抗体加入后孵育1小时后离心，用PBS清洗，用流式细胞仪分析样品，分选合适的细胞，测定选定细胞的荧光强度(MFI)，流式结果见表4.流式细胞测定结果显示，免疫激动剂cGAMP及其与纳米抗体靶向脂质体的免疫激动剂新型复合物均能显著活化树突状细胞DC和T细胞，新型免疫激动剂－纳米抗体复合物的效果显著高于单独使用免疫激动剂cGAMP。See Example 5 for the breeding of mice, virus infection immunity, and blood sampling. Isotype control flow cytometry antibody was purchased from eBiosciences, antibody magnetic strain was purchased from Militeny Biotech, and flow cytometer was purchased from BD company. After 14 days of immunization, the mouse spleen and lung tissues were taken, ground and crushed, and the cells were removed through 40 micron holes. Centrifuge at 1000 rpm for 10 minutes to separate unlysed immune cells. Separate DC (CD40\CD80\CD86\MHCII) and T (CD8+) cells with antibody magnets, add corresponding FAC antibody (diluted with FACS buffer), isotype control Antibody was used as a negative control. After the antibody was added, it was incubated for 1 hour and then centrifuged, washed with PBS, analyzed by flow cytometry, sorted suitable cells, and measured the fluorescence intensity (MFI) of the selected cells. The flow cytometry results are shown in Table 4. The results of flow cytometry showed that the immune agonist cGAMP and its novel complex with nanobody targeting liposomes can significantly activate dendritic cells, DC and T cells, and the novel immune agonist-nanobody complex The effect is significantly higher than that of using the immune agonist cGAMP alone.
表4.新型抗新冠病毒复合物诱发免疫细胞活化效价Table 4. The titer of immune cell activation induced by the novel anti-coronavirus complex
Figure PCTCN2020142599-appb-000006
Figure PCTCN2020142599-appb-000006
实施例8新型抗新冠病毒复合物的急性毒性研究Example 8 Study on the acute toxicity of the new anti-coronavirus complex
实验材料Experimental Materials
ICR小鼠10只(购于上海斯莱克实验动物有限责任公司[实验动物质量合格证号：SCXK(沪)2007-0005])，雌雄各半，体重20～22g，动物以颗粒饲料喂养，自由摄食和饮水。10 ICR mice (purchased from Shanghai Slack Laboratory Animal Co., Ltd. [Experimental Animal Quality Certificate No.: SCXK (Shanghai) 2007-0005]), half male and half male, weighing 20-22g, animals are fed with pellet feed, free Eat and drink.
实验方法experimental method
ICR小鼠按体重分别腹腔注射1g/kg的新型免疫激动-纳米抗体剂复合物(生理盐水液配制)，观察给药后小鼠14天内的毒性反应及死亡情况。结果发现，小鼠腹腔注射给药后，小鼠活动正常。给药后14天内，小鼠未出现死亡，第15天，全部小鼠处死，解剖，肉眼检查各脏器，均未见明显病变。ICR mice were intraperitoneally injected with 1g/kg of the new immunostimulant-nanobody agent complex (prepared in normal saline solution) according to their body weight, and the toxicity and death of the mice within 14 days after administration were observed. It was found that after intraperitoneal injection of the mice, the mice moved normally. Within 14 days after the administration, the mice did not die. On the 15th day, all the mice were sacrificed, dissected, and visually inspected the various organs, and no obvious lesions were seen.
实验结果Experimental result
上述急性毒性实验结果表明，腹腔注射给药最大耐受量MTD不低于1g/Kg，说明新型免疫激动剂复合物药物的急性毒性低。The above-mentioned acute toxicity experiment results show that the maximum tolerated dose MTD for intraperitoneal injection is not less than 1g/Kg, indicating that the acute toxicity of the new immune agonist complex drugs is low.
附图说明Description of the drawings
附图1.新型免疫激动剂--纳米抗体复合物抑制小鼠肺炎效果(肺组织石蜡切片H HE &E染色)Figure 1. Novel immune agonist-nanobody complex suppresses pneumonia in mice (paraffin section of lung tissue H HE &E staining)

Claims (4)

  1. 一种新型抗新冠病毒的双功能一体化复合物，其特征在于：由重组的抗新冠病毒的羊驼纳米抗体偶联脂质体包裹的免疫激动剂构成，所述免疫激动剂包封于重组纳米抗体偶联的脂质体中。所述免疫激动剂为STING的激动剂，既环二核苷酸2’3’-cGAMP或其衍生物；抗新冠病毒的重组羊驼纳米抗体，其特征为，能与新冠病毒刺突S蛋白紧密结合，且靶向中和新冠病毒的纳米抗体或抗新冠病毒的单克隆抗体的可变区结构域。A new type of bifunctional integrated complex against neocoronavirus, which is characterized in that it is composed of a recombinant anticoronavirus alpaca nanobody coupled with an immunoagonist encapsulated in liposomes, and the immunoagonist is encapsulated in a recombinant Nanobody conjugated in liposomes. The immune agonist is an agonist of STING, which is a cyclic dinucleotide 2'3'-cGAMP or its derivative; a recombinant alpaca nano antibody against the new coronavirus, which is characterized by being able to spike the S protein with the new coronavirus Tightly bind and target the variable region domains of Nanobodies that neutralize the new coronavirus or monoclonal antibodies against the new coronavirus.
    优选地，靶向新冠病毒刺突S蛋白的纳米抗体为SARS2-VHH,其氨基酸序列为：QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGLGTVVSEWDYDYYLDYWGQGTQVTVSS。Preferably, the nanobody targeting the spike S protein of the new coronavirus is SARS2-VHH, and its amino acid sequence is: QVQLQESGGGLVQAGGSLRLSCAASGRTFSEYAMGWFRQAPGKEREFVATISWSGGSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCAAAGLGTVVSEWDYDYYLDVTWGVTQVTQGT.
  2. 根据权利要求1所述的新型抗新冠病毒双功能一体化复合物的制备方法，其特征在于：The method for preparing a novel anti-new coronavirus bifunctional integrated complex according to claim 1, characterized in that:
    (1)运用原核表达载体，在大肠杆菌中重组表达、运用镍亲和层析柱纯化抗新冠病毒S蛋白的纳米抗体SARS2-VHH；(1) Using prokaryotic expression vector, recombinant expression in Escherichia coli, using nickel affinity chromatography column to purify the anti-new coronavirus S protein nanoantibody SARS2-VHH;
    (2)对重组抗新冠病毒S蛋白的纳米抗体SARS2-VHH进行巯基化；(2) Sulfhydrylation of the recombinant anti-new coronavirus S protein nanobody SARS2-VHH;
    (3)巯基化的重组纳米抗体SARS2-VHH与脂质体化学键偶联融合，进而包封免疫激动剂。(3) The thiolated recombinant nanobody SARS2-VHH is coupled to the liposome by chemical bond coupling and fusion, thereby encapsulating the immune agonist.
  3. 根据权利要求1所述的新型抗新冠病毒复合物在制备预防和/或治疗冠状病毒感染疾病药物中的应用，其特征在于：The use of the novel anti-coronavirus complex according to claim 1 in the preparation of drugs for preventing and/or treating coronavirus infections, characterized in that:
    冠状病毒感染疾病包括但不限于人或动物感染冠状病毒引起的病毒性肺炎/呼吸道炎症、病毒性肾炎、病毒性脑炎、病毒性肠炎或病毒性肝炎。Coronavirus infection diseases include, but are not limited to, viral pneumonia/respiratory inflammation, viral nephritis, viral encephalitis, viral enteritis, or viral hepatitis caused by human or animal infection with coronavirus.
  4. 根据权利要求1所述的新型抗新冠病毒复合物可单独制备成不同规格的单位制剂或通过药学上可接受的载体制备成药物制剂，包括但不限于静脉注射制剂、鼻腔滴注制剂、静脉滴注制剂、肌肉注射制剂、皮下注射制剂或口服制剂；口服制剂包括但不限于胶囊、片剂或颗粒剂。The novel anti-coronavirus complex according to claim 1 can be separately prepared into unit preparations of different specifications or prepared into pharmaceutical preparations through pharmaceutically acceptable carriers, including but not limited to intravenous injection preparations, nasal drip preparations, intravenous drips Injection preparations, intramuscular injection preparations, subcutaneous injection preparations or oral preparations; oral preparations include but are not limited to capsules, tablets or granules.
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