WO2021227687A1 - 一种构建冠状病毒抗体的平台 - Google Patents

一种构建冠状病毒抗体的平台 Download PDF

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WO2021227687A1
WO2021227687A1 PCT/CN2021/084187 CN2021084187W WO2021227687A1 WO 2021227687 A1 WO2021227687 A1 WO 2021227687A1 CN 2021084187 W CN2021084187 W CN 2021084187W WO 2021227687 A1 WO2021227687 A1 WO 2021227687A1
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fusion protein
amino acid
chain
acid sequence
seq
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PCT/CN2021/084187
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French (fr)
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缪小牛
许英达
罗羿
袁志军
陈连娣
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普米斯生物技术(珠海)有限公司
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    • 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
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • 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
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • A—HUMAN NECESSITIES
    • 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
    • A61K47/50—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
    • A61K47/51—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 non-active ingredient being a modifying agent
    • A61K47/68—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 non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • C07K16/1002—Coronaviridae
    • 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
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/485—Exopeptidases (3.4.11-3.4.19)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/17—Metallocarboxypeptidases (3.4.17)
    • C12Y304/17023—Angiotensin-converting enzyme 2 (3.4.17.23)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • 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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to a platform for constructing coronavirus antibodies.
  • the 2019-nCoV coronavirus belongs to the genus Coronavirus of the Coronavirus family, and is a single-stranded positive-sense RNA virus with an envelope. Similar to other known coronaviruses, the 2019-nCoV coronavirus also completes the proliferation of progeny viruses through several processes such as adsorption, penetration, uncoating, biosynthesis, and assembly and release of progeny viruses.
  • the infection of host cells by the 2019-nCoV coronavirus starts when the spike glycoprotein on the surface of the virus envelope binds to the human angiotensin-converting enzyme 2 (ACE2) on the surface of the host cell, and then membrane fusion occurs. The virus enters the host cell.
  • ACE2 human angiotensin-converting enzyme 2
  • the single-stranded sense RNA of the virus's genetic material is released.
  • the translation produces polyproteins.
  • the two essential cysteine proteases of the 2019-nCoV coronavirus papain-like protease (PL pro ) and 3C-like protease (3C-like protease, 3CL pro ) cut and process polymers at specific sites Protein precursors produce multiple non-structural proteins that are very important to the life cycle of the virus.
  • the viral RNA replicates the nucleic acid material of the progeny virus, and a large number of required structural proteins are translated to complete the assembly and release of the progeny virus.
  • Any link or key enzyme in the life cycle of cells infected by the 2019-nCoV coronavirus can be used as research targets for antiviral drugs, such as the cysteine proteases PL pro and 3CL pro that hydrolyze and cleave polyprotein precursors, which are responsible for the completion of the product.
  • the transmission route of the 2019-nCoV virus has not been fully grasped. It is known to be spread through droplets and contact, and there is human-to-human transmission, medical staff infection, a certain risk of community transmission, and the virus may mutate. At present, there is no specific prevention and treatment method for the disease caused by the new coronavirus.
  • the purpose of the present invention is to provide an effective method for the diagnosis and treatment of SARS-CoV-2 coronavirus.
  • Another object of the present invention is to provide a technical means that can improve the neutralization ability of anti-SARS-Cov2 or other virus antibodies.
  • Another object of the present invention is to provide a technical means of antibody fusion protein capable of resisting SARS-Cov2 or other viruses.
  • a method for improving the neutralization ability of anti-coronavirus antibodies includes the steps of: combining the anti-coronavirus antibody to be improved with human angiotensin-converting enzyme 2 (ACE2) or a fragment thereof Perform fusion to obtain a fusion protein;
  • ACE2 human angiotensin-converting enzyme 2
  • the fusion protein Compared with the anti-coronavirus antibody alone, the fusion protein has an antigen neutralization ability increased by ⁇ 10 times (preferably, by ⁇ 20 times).
  • the coronavirus is SAR-CoV-2 virus.
  • the antibody specifically binds to the SARS-CoV-2 Spike 1 protein.
  • the amino acid sequence of the human ACE2 or its fragment is shown in SEQ ID NO: 1, 2, 3 or 4; or in the sequence shown in SEQ ID NO: 1, 2, 3 or 4. Based on the addition, deletion, modification and/or substitution of at least one amino acid, the derived sequence can retain the binding affinity of the SARS-CoV-2 Spike 1 protein.
  • the derivative sequence that has been added, deleted, modified and/or substituted at least one amino acid and can retain the binding affinity of SARS-CoV-2 Spike 1 is homology or sequence identity is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the amino acid sequence.
  • the human ACE2 or a fragment thereof is the full-length protein of the extracellular region of human ACE2.
  • amino acid sequence of the human ACE2 or a fragment thereof is shown in SEQ ID NO: 4.
  • the human ACE2 or its fragment is fused to the N-terminus of the heavy chain, the C-terminus of the heavy chain, the N-terminus of the light chain, and/or The C-terminus of the light chain.
  • the fusion protein has two pairs of peptide chains stacked with each other, each pair of peptide chains contains a heavy chain H chain and a light chain L chain, and all peptide chains are connected by disulfide bonds, any of which A pair of peptide chains has the structure of H chain and L chain as shown in formula I to V from C-terminal to C-terminal,
  • ACE2 stands for human ACE2 or its fragments
  • VH stands for the heavy chain variable region of the anti-SARS-Cov2 Spike 1 protein antibody
  • VL stands for the light chain variable region of the anti-SARS-Cov2 Spike 1 protein antibody
  • CH1, CH2, and CH3 represent the heavy chain constant regions CH1, CH2, and CH3 of the anti-SARS-Cov2 Spike 1 protein antibody, respectively;
  • CL stands for the light chain constant region of the anti-SARS-Cov2 Spike 1 protein antibody
  • the variant of (G 4 S) n includes: a variant of the (G 4 S) n linker sequence obtained by substituting amino acids with similar or similar properties in the sequence, such as One or more S are respectively mutated to T; or 1-3 amino acids are inserted into the sequence.
  • amino acid sequence of the connecting peptide sequence is shown in SEQ ID NO: 10.
  • the method is non-therapeutic and non-diagnostic.
  • the heavy chain constant regions CH1, CH2, and CH3, and the light chain constant region CL of the antibody are all derived from human IgG1 or IgG4, preferably human IgG1.
  • amino acid sequence of the VH-CH1-CH2-CH3 segment (that is, the heavy chain of the 89C8 antibody to be improved) in the formula I to the formula V is shown in SEQ ID NO: 11, or is The sequence of SEQ ID NO: 11 has a sequence identity of> 85% (preferably 90%, more preferably 95%).
  • amino acid sequence of the VL-CL segment in Formula I to Formula V (that is, the light chain of the 89C8 antibody to be improved) is shown in SEQ ID NO: 16, or is the same as SEQ ID NO:
  • the sequence of 16 has a sequence identity of> 85% (preferably 90%, more preferably 95%).
  • the H chain in the fusion protein has an amino acid sequence as shown in SEQ ID NO: 15, and the L chain in the fusion protein has an amino acid sequence as shown in SEQ ID NO: 16;
  • the H chain in the fusion protein has an amino acid sequence as shown in SEQ ID NO: 15, and the L chain in the fusion protein has an amino acid sequence as shown in SEQ ID NO: 20;
  • the H chain in the fusion protein has an amino acid sequence as shown in SEQ ID NO: 11
  • the L chain in the fusion protein has an amino acid sequence as shown in SEQ ID NO: 20.
  • amino acid sequence of the VH-CH1-CH2-CH3 segment (that is, the heavy chain of the CR3022 antibody to be improved) in the formula I to the formula V is shown in SEQ ID NO: 21, or is The sequence of SEQ ID NO: 21 has a sequence identity of> 85% (preferably 90%, more preferably 95%).
  • the amino acid sequence of the VL-CL segment (that is, the light chain of the CR3022 antibody to be improved) in Formula I to Formula V is shown in SEQ ID NO: 22, or is the same as SEQ ID NO:
  • the sequence of 22 has a sequence identity of> 85% (preferably 90%, more preferably 95%).
  • the H chain in the fusion protein has the amino acid sequence shown in SEQ ID NO: 26, and the L chain in the fusion protein has the amino acid sequence shown in SEQ ID NO: 22;
  • the H chain in the fusion protein has an amino acid sequence as shown in SEQ ID NO: 26, and the L chain in the fusion protein has an amino acid sequence as shown in SEQ ID NO: 30;
  • the H chain in the fusion protein has an amino acid sequence as shown in SEQ ID NO: 21, and the L chain in the fusion protein has an amino acid sequence as shown in SEQ ID NO: 30.
  • a fusion protein in the second aspect of the present invention, includes:
  • the human ACE2 or its fragments are located at the N-terminus of the heavy chain, the C-terminus of the heavy chain, and the N-terminus of the light chain of the SARS-Cov2 Spike 1 protein antibody. , And/or the C-terminus of the light chain.
  • an isolated polynucleotide which encodes the fusion protein according to the second aspect of the present invention.
  • the ratio of the polynucleotide encoding the H chain to the polynucleotide encoding the L chain is 1:2 to 3:1, preferably 2:1.
  • a vector which contains the polynucleotide according to the third aspect of the present invention.
  • the vector contains all the polynucleotides in the polynucleotide of the third aspect of the present invention at the same time.
  • the vector contains the polynucleotide encoding the H chain and the polynucleotide encoding the L chain as described in the third aspect of the present invention, respectively.
  • the vector is an expression vector.
  • the vector includes a plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus, or other vectors.
  • a genetically engineered host cell contains the vector as described in the fourth aspect of the present invention or the genome integrates the polynucleotide as described in the third aspect of the present invention .
  • an antibody conjugate the antibody conjugate containing:
  • a coupling part coupled to the targeting part being selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, gold nanoparticles/nanorods , Nano magnetic particles, viral coat protein or VLP, or a combination thereof.
  • the targeting moiety and the coupling moiety are coupled through a chemical bond or a linker.
  • the radionuclide includes:
  • Diagnostic isotopes said diagnostic isotopes are selected from the following group: Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and/or
  • Therapeutic isotope said therapeutic isotope is selected from the following group: Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd- 103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133Yb-169, Yb-177, Or a combination.
  • the coupling moiety is a drug or a toxin.
  • the drug is a cytotoxic drug.
  • the cytotoxic drug is selected from the group consisting of anti-tubulin drugs, DNA minor groove binding reagents, DNA replication inhibitors, alkylating reagents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy Sensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.
  • cytotoxic drugs include, for example, DNA minor groove binding reagents, DNA alkylating reagents, and tubulin inhibitors.
  • Typical cytotoxic drugs include, for example, auristatins, camptothecin (camptothecins), dokamycin/duocarmycins, etoposides, maytansines and maytansinoids (e.g.
  • DM1 and DM4 taxanes (etoposides) taxanes), benzodiazepines or benzodiazepine containing drugs (such as pyrrolo[1,4] benzodiazepines (PBDs), indoline benzodiazepines (Indolinobenzodiazepines) and oxazolidinobenzodiazepines (oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.
  • PBDs pyrrolo[1,4] benzodiazepines
  • Indolinobenzodiazepines Indolinobenzodiazepines
  • oxazolidinobenzodiazepines oxazolidinobenzodiazepines
  • the toxin is selected from the following group:
  • Otostatin for example, Otostatin E, Otostatin F, MMAE, and MMAF
  • chlortetracycline mettancilol
  • octoxin for example, Otostatin E, Otostatin F, MMAE, and MMAF
  • Lastatin doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, autumn Narcissus, dihydroxy anthracisin diketone, actinomycin, diphtheria toxin, pseudomonas exotoxin (PE) A, PE40, acacia toxin, acacia toxin A chain, capsule lotus root toxin A chain, ⁇ -Sarcina, white tree toxin, mitogellin, retstrictocin, phenomycin, enoxomycin
  • the coupling portion is a detectable label.
  • the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computed tomography technology) contrast agents, or can produce detectable Product enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, magnetic nanoparticles, prodrugs Activating enzymes (such as DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutics (such as cisplatin).
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the immunoconjugate contains: multivalent (such as bivalent) (a).
  • the multivalent means that the amino acid sequence of the immunoconjugate includes multiple repeats (a).
  • an immune cell that expresses or is exposed outside the cell membrane with the fusion protein as described in the second aspect of the present invention.
  • the immune cells include NK cells and T cells.
  • the immune cells are derived from human or non-human mammals (such as mice).
  • a pharmaceutical composition which contains:
  • Active ingredient the active ingredient is selected from the group consisting of the fusion protein according to the second aspect of the present invention, the antibody conjugate according to the sixth aspect of the present invention, and the antibody conjugate according to the seventh aspect of the present invention Immune cells, or a combination thereof;
  • the pharmaceutical composition is a liquid preparation.
  • the pharmaceutical composition is an injection.
  • the pharmaceutical composition includes 0.01-99.99% of the fusion protein according to the second aspect of the present invention, the antibody conjugate according to the sixth aspect of the present invention, and the seventh aspect of the present invention.
  • the immune cell, or a combination thereof, and 0.01-99.99% of the pharmaceutical carrier according to the aspect, the percentage is the mass percentage of the pharmaceutical composition.
  • an active ingredient is provided for (a) preparation of diagnostic reagents or kits for SARS-CoV2 virus infection; and/or (b) preparation of prevention and/or treatment of SARS- CoV2 virus infection drugs;
  • the active ingredient is selected from the following group: the fusion protein according to the second aspect of the present invention, the antibody conjugate according to the sixth aspect of the present invention, the immune cell according to the seventh aspect of the present invention, or Its combination.
  • the diagnostic reagent is a test piece or a test plate.
  • the diagnostic reagent or kit is used to detect SARS-CoV-2 Spike1 protein in a sample.
  • a method for detecting SARS-CoV-2 virus or SARS-CoV-2 Spike 1 protein in a sample in vitro comprising the steps:
  • the detection includes diagnostic or non-diagnostic.
  • a kit which includes:
  • a first container containing the fusion protein according to the second aspect of the present invention containing the fusion protein according to the second aspect of the present invention.
  • a second container which contains a secondary antibody against the fusion protein according to the second aspect of the present invention
  • the kit contains a test plate, the test plate includes: a substrate (support plate) and a test strip, and the test strip contains the fusion protein as described in the fifth aspect of the present invention, as described in the sixth aspect of the present invention.
  • a method for treating viral infections of a subject in need including the steps:
  • (a) Prepare a fusion protein, the amino acid sequence of the fusion protein includes: an antibody against the surface protein of the virus, and a protein or a fragment thereof that mediates the infection of the virus in the body of the subject.
  • the virus includes: SARS, MERS, or other viruses.
  • the protein that mediates virus infection is, for example, ACE2 corresponding to SARS, or DPP2 corresponding to MERS.
  • a method for preparing an anti-coronavirus fusion protein which includes the steps of fusing an anti-coronavirus antibody with human angiotensin-converting enzyme 2 (ACE2) or a fragment thereof, thereby The fusion protein is obtained.
  • ACE2 human angiotensin-converting enzyme 2
  • Figure 1 shows the five basic structures of the fusion protein of the present invention.
  • Figure 2 shows the affinity of 89C8-ACE2-F1 fusion protein and 89C8 antibody to S1 protein, respectively.
  • Figure 3 shows the binding activity of 89C8-ACE2-F1 fusion protein to 293-Spike cells.
  • Figure 4 shows the blocking activity of 89C8-ACE2-F1 fusion protein on the binding of ACE2 to Spike 1 protein.
  • Figure 5 shows the neutralizing ability of 89C8-ACE2-F1 fusion protein, 89C8 and ACE2-Fc against the infective activity of pseudoviruses.
  • the inventors developed a method for improving the neutralization ability of anti-SARS-CoV2 S1 antibodies for the first time. Studies have shown that after the extracellular region of ACE2 is fused with the antibody, the affinity of the resulting fusion protein to the SARS-CoV2 S1 protein is significantly improved, and the neutralizing activity of the fusion protein to the virus is significantly enhanced.
  • a method for improving the neutralization ability of anti-coronavirus antibodies includes the steps of fusing the anti-coronavirus antibody to be improved with human angiotensin-converting enzyme 2 (ACE2) or a fragment thereof,
  • ACE2 human angiotensin-converting enzyme 2
  • a fusion protein is obtained; compared with the anti-coronavirus antibody alone, the antigen neutralization ability of the fusion protein is increased by ⁇ 10 times (preferably, by ⁇ 20 times).
  • the coronavirus described in the present invention is the SAR-CoV-2 virus. More preferably, the antibody specifically binds to the SARS-CoV-2 Spike 1 protein.
  • the amino acid sequence of the human ACE2 or a fragment thereof is shown in SEQ ID NO: 1, 2, 3 or 4; or based on the sequence shown in SEQ ID NO: 1, 2, 3 or 4
  • the human ACE2 or a fragment thereof refers to the full-length protein of the extracellular region of ACE2, and its amino acid sequence is shown in SEQ ID NO: 4.
  • the human ACE2 or a fragment thereof is fused to the N-terminus of the heavy chain, the C-terminus of the heavy chain, the N-terminus of the light chain, and/or the C-terminus of the light chain of the antibody. end.
  • the antibody to be improved can be any antibody that has high affinity with the SARS-CoV2 S1 protein.
  • the antibody is 89C8, the amino acid sequence of its heavy chain is shown in SEQ ID NO: 11, and the amino acid sequence of its light chain is shown in SEQ ID NO: 16.
  • the fusion protein has the H chain of the amino acid sequence shown in SEQ ID NO: 15 and the L chain of the amino acid sequence shown in SEQ ID NO: 16 (89C8-ACE -F1);
  • the fusion protein has an H chain with an amino acid sequence as shown in SEQ ID NO: 15 and an L chain (89C8-ACE-F2) with an amino acid sequence as shown in SEQ ID NO: 20;
  • the fusion protein has an H chain with the amino acid sequence shown in SEQ ID NO: 11 and an L chain (89C8-ACE-F3) with the amino acid sequence shown in SEQ ID NO: 20.
  • the antibody is CR3022, the amino acid sequence of its heavy chain is shown in SEQ ID NO: 21, and the amino acid sequence of its light chain is shown in SEQ ID NO: 22.
  • the fusion protein has the H chain of the amino acid sequence shown in SEQ ID NO: 26 and the L chain of the amino acid sequence shown in SEQ ID NO: 22 (CR3022-ACE -F1);
  • the fusion protein has an H chain with an amino acid sequence as shown in SEQ ID NO: 26, and an L chain (CR3022-ACE-F2) with an amino acid sequence as shown in SEQ ID NO: 30;
  • the fusion protein has an H chain with the amino acid sequence shown in SEQ ID NO: 21 and an L chain (CR3022-ACE-F3) with the amino acid sequence shown in SEQ ID NO: 30.
  • the present invention also provides uses of the fusion protein, antibody conjugate ADC, recombinant protein, and/or immune cell of the present invention, for example, for preparing diagnostic preparations or preparing medicines.
  • the medicine is a medicine for preventing and/or treating SARS-CoV-2 virus infection.
  • the fusion protein or ADC of the present invention can be used for detection applications, for example, for detecting samples, thereby providing diagnostic information.
  • the samples (samples) used include cells, tissue samples and biopsy specimens.
  • the sample is a blood sample from the test subject.
  • biopsy used in the present invention shall include all kinds of biopsy known to those skilled in the art. Therefore, the biopsy used in the present invention may include, for example, excision samples of tumors, tissue samples prepared by endoscopic methods or organ puncture or needle biopsy.
  • the samples used in the present invention include fixed or preserved cell or tissue samples.
  • the present invention also provides a kit containing the fusion protein (or a fragment thereof) of the present invention.
  • the kit further includes a container, instructions for use, buffers, and the like.
  • the fusion protein of the present invention can be immobilized on a detection plate.
  • the invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned fusion protein or its active fragment or its fusion protein or its ADC or corresponding immune cells, and a pharmaceutically acceptable carrier.
  • these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH can be The nature of the formulated substance and the condition to be treated vary.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration.
  • the route of administration of the pharmaceutical composition of the present invention is preferably injection or oral administration.
  • the injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection.
  • the pharmaceutical composition is a variety of conventional dosage forms in the art, preferably in the form of solid, semi-solid or liquid, and can be an aqueous solution, non-aqueous solution or suspension, more preferably a tablet, capsule, or granule. , Injection or infusion, etc.
  • the fusion protein of the present invention can also be expressed in a cell by a nucleotide sequence for cell therapy, for example, the fusion protein is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.
  • CAR-T chimeric antigen receptor T cell immunotherapy
  • the pharmaceutical composition of the present invention is a pharmaceutical composition for preventing and/or treating SARS-CoV-2 virus infection.
  • the pharmaceutical composition of the present invention can be directly used to bind the SARS-CoV-2 Spike 1 protein molecule, and therefore can be used to prevent and treat diseases caused by viral infections.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned fusion protein (or conjugate thereof) of the present invention and a pharmaceutically Acceptable carrier or excipient.
  • a pharmaceutically Acceptable carrier or excipient include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under aseptic conditions.
  • the dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram/kg body weight to about 5 mg/kg body weight per day.
  • the fusion protein of the present invention can also be
  • the pharmaceutical composition of the present invention further includes one or more pharmaceutical carriers.
  • the pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and the pharmaceutical carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient.
  • the pharmaceutical excipients are conventional pharmaceutical excipients in the field, and preferably include pharmaceutically acceptable excipients, fillers or diluents. More preferably, the pharmaceutical composition includes 0.01-99.99% of the above-mentioned protein and 0.01-99.99% of a pharmaceutical carrier, and the percentage is a mass percentage of the pharmaceutical composition.
  • the administration amount of the pharmaceutical composition is an effective amount
  • the effective amount is an amount that can alleviate or delay the progression of the disease, degenerative or traumatic condition.
  • the effective amount can be determined on an individual basis and will be partly based on consideration of the symptoms to be treated and the results sought. Those skilled in the art can determine the effective amount by using the aforementioned factors such as individual basis and using no more than conventional experiments.
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases, does not exceed about 50 mg/kg body weight, Preferably the dosage is about 10 micrograms/kg body weight to about 20 mg/kg body weight.
  • the specific dosage should also consider factors such as the route of administration and the patient's health status, which are all within the skill range of a skilled physician.
  • the present invention provides the application of the above-mentioned pharmaceutical composition in the preparation of medicines for preventing and/or treating SARS-CoV-2 virus infection.
  • the SARS-CoV-2 virus-infected disease is pneumonia.
  • new coronavirus As used herein, the terms “new coronavirus”, “2019-nCov” or “SARS-CoV-2” are used interchangeably.
  • the 2019 new coronavirus is the seventh type of coronavirus that is known to infect humans and causes new coronary pneumonia. (COVID-19) is one of the serious infectious diseases that threaten human health around the world.
  • Coronavirus belongs to the Coronavirus family (Coronaviridae) of the Nidovirales (Nidovirales). It is an enveloped positive-stranded RNA virus, and its subfamily includes four genera of ⁇ , ⁇ , ⁇ and ⁇ .
  • HCoV-229E and HCoV-NL63 belong to the ⁇ genus coronavirus
  • HCoV-OC43, SARS-CoV, HCoV-HKU1, MERS-CoV and SARS-CoV-2 are all ⁇ genus coronavirus.
  • Virus. SARS-CoV-2 is also known as 2019-nCov.
  • the genome of this type of virus is a single-stranded positive-stranded RNA, which is one of the largest RNA viruses in the genome.
  • the codes include replicase, spike protein, envelope protein, envelope protein, and nucleocapsid protein.
  • the genome is translated into two peptide chains of several thousand amino acids, the precursor polyprotein (Polyprotein), and then the precursor protein is cleaved by proteases to produce non-structural proteins (such as RNA polymerase and unwinding). Enzymes) and structural proteins (such as spike proteins) and accessory proteins.
  • the fusion protein of the present invention can bind to the S1 protein with high affinity.
  • the present invention can block the interaction between S1 protein and ACE2, and the blocking activity is significantly higher than that of ACE2 protein.
  • the present invention can neutralize the cell infection activity of Spike1 pseudovirus, and the neutralization activity is significantly higher than that of monoclonal antibodies.
  • the method of the present invention can be used for the treatment of SARS-CoV2 and other viral infections (such as SARS, MERS, etc.) diseases.
  • Protein name Heavy chain name SEQ ID NO: Light chain name SEQ ID NO: 89C8-23-F1 89C8-23-F1 HC 12 89C8 LC 16 89C8-45-F1 89C8-45-F1 HC 13 89C8 LC 16 89C8-98-F1 89C8-98-F1 HC 14 89C8 LC 16 89C8-ACE2-F1 89C8-ACE2-F1 HC 15 89C8 LC 16 89C8-23-F2 89C8-23-F1 HC 12 89C8-23-F1 LC 17 89C8-45-F2 89C8-45-F1 HC 13 89C8-45-F1 LC 18 89C8-98-F2 89C8-98-F1 HC 14 89C8-98-F1 LC 19 89C8-ACE2-F2 89C8-ACE2-F1 HC 15 89C8
  • the ACE2 truncated protein containing 23 amino acids, 45 amino acids, and 98 amino acids and the full-length ACE2 extracellular domain protein were used as the targeting and neutralizing part of the fusion protein, and the S1 antibody was used as The targeting part of the fusion protein forms the S1 antibody/ACE2 extracellular domain fusion protein.
  • the structure of the fusion protein is shown in Figure 2.
  • the amino acid sequence of the ACE2 truncated and full-length protein is as follows:
  • the truncated protein or full-length protein of ACE2 of the present invention is connected to the anti-S1 antibody 89C8 or the reported anti-SARS S1 protein antibody CR3022 through (G 4 S) 4 G (patent number: US 2008/0014204 A1)
  • the C-terminus of the heavy chain (structure 1, F1), the N-terminus of the light chain (structure 3, F3), the C-terminus of the heavy chain and the N-terminus of the light chain (structure 2, F2), the N-terminus of the heavy chain (structure 4, F4) and the C-terminus of the light chain (structure 5, F5).
  • each fusion protein obtained was digested into the linearized pCDNA3.1 vector with homologous recombinase (purchased from Vazyme) and EcoR I/Not I, and the process was in accordance with the product specification.
  • the homologous recombination products were transformed into Top10 competent cells, coated with ampicillin resistant plates, incubated overnight at 37°C, and single clones were picked for sequencing.
  • the magnetic beads were resuspended (1-4 times the volume of magnetic beads) in an appropriate volume of binding buffer (PBS+0.1% Tween 20, pH 7.4) and added to the sample to be purified, incubated at room temperature for 1 hour, and gently shaken during the period.
  • the sample was placed on a magnetic stand (purchased from Beaver), the supernatant was discarded, and the magnetic beads were washed 3 times with binding buffer.
  • Add elution buffer (0.1M sodium citrate, pH 3.2) according to 3-5 times the volume of the magnetic beads and shake at room temperature for 5-10 minutes, put it back on the magnetic stand, collect the elution buffer, and transfer to neutralization. Mix well in the collection tube of buffer (1M Tris, pH 8.54).
  • Example 2 The binding activity of S1 antibody/ACE2 extracellular domain fusion protein and S1 protein
  • the AHC sensor was equilibrated in the analysis buffer for 30 minutes offline, and then tested online for 60 seconds to establish a baseline, and the purified S1 antibody/ACE2 extracellular domain (truncated) fusion protein obtained as described above was loaded online to the AHC sensor. Then put the sensor in 100nM S1-His antigen for 240s, then transfer the sensor to PBS to dissociate for 1000s. Use 1:1 combination model to analyze the kinetics.
  • the AHC sensor was equilibrated in the analysis buffer for 30 minutes offline, and then tested online for 60 seconds to establish a baseline, and the purified S1 antibody/ACE2 extracellular region (full-length) fusion protein obtained as described above was loaded online to the AHC sensor. Then put the sensor in 100nM, 50nM, 25nM, 12.5nM S1-His antigen for 3600s, and then transfer the sensor to PBS to dissociate for 6400s. Use 1:1 combination model to analyze the kinetics.
  • the affinity of the antibody fusion protein (preferably structure 1, F1) constructed with the full-length ACE2 extracellular region and S1 is 10 times higher than that of 89C8.
  • Example 3 Binding of antibody/ACE2 extracellular domain fusion protein to Spike expressed on the cell surface
  • the binding activity of the purified antibody/ACE2 extracellular region fusion protein sample 89C8-ACE2-F1 with 293-Spike cells was detected.
  • the PLVX-puro vector cloned into MCS SARS-CoV-2 Spike protein full-length gene was transfected to produce 293T cells (293-Spike cells) overexpressing Spike protein. Treat the expanded cultured 293-Spike cells with trypsin, adjust the cell suspension density to 2 ⁇ 10 6 cells/ml, add 100 ⁇ l/well to a 96-well flow plate, block with 2% BSA and centrifuge for use to prepare the purified antibody
  • the fusion protein of /ACE2 extracellular domain was diluted with PBS, starting at 200nM, 3 times dilution, a total of 12 points.
  • Example 4 Antibody/ACE2 extracellular domain fusion protein blocks the binding activity of S1 protein and ACE2
  • CHO cells (CHO-ACE2 cells) overexpressing human ACE2 protein were produced by transfecting the PLVX-puro vector cloned into the full-length human ACE2 protein gene of MCS. Adjust the cell suspension density of the expanded cultured CHO-ACE2 cells to 2 ⁇ 10 6 cells/ml, add 100 ⁇ l/well to a 96-well flow plate, seal with 2% BSA and centrifuge for use. Dilute the purified sample with PBS, start at 200nM and dilute 3 times at 12 points. Add 60 ⁇ l/well of the above-mentioned diluted sample to a 96-well sample dilution plate, and at the same time add S1-mFc protein (purchased from sinobiologics) at 60 ⁇ l/well.
  • S1-mFc protein purchased from sinobiologics
  • the concentration is 0.1nM and incubated with the sample at 37°C.
  • Anti-mouse Fc-APC purchased from eBioscience
  • diluted 100 times with PBS was added to 100 ⁇ l/well, incubated at 4° C. for 30 min, and washed twice with PBS.
  • 100 ⁇ l/well was added to PBS to resuspend the cells, detected on a CytoFlex (Bechman) flow cytometer, and calculated the corresponding MFI.
  • Example 5 Antibody/ACE2 extracellular domain fusion protein blocks SARS-Cov2 Spike pseudovirus from infecting ACE2 overexpressing cells
  • the PLVX-puro vector cloned into the full-length human ACE2 protein gene of MCS was transfected to produce 293T cells (293-ACE2 cells) overexpressing human ACE2 protein.
  • the 293-ACE2 cells were seeded into a 96-well cell culture plate at 8000 cells/well and cultured overnight.
  • the mixture of serially diluted 89C8-ACE2-F1, 89C8 and ACE2-Fc samples and a final concentration of 1 ⁇ 10 5 TU/ml luciferase fluorescent reporter SARS-Cov2 Spike pseudovirus (purchased from Beijing Tiantan Pharmaceutical Biotechnology Development Company) Incubate at 37°C for 1h. Remove the 293-ACE2 cell culture medium, add the mixture of the above sample and pseudovirus, and incubate at 37°C for 48h. Detect cell fluorescence and calculate the neutralizing activity of the sample.
  • 89C8-ACE2-F1 can completely neutralize the infective activity of pseudoviruses, and 89C8 and ACE2-Fc have similar neutralizing activities.
  • the neutralizing activity of the antibody/ACE2 extracellular domain fusion protein 89C8-ACE2-F1 is about 60 times stronger than that of 89C8.

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Abstract

提供了一种抗SARS-CoV2 Spike 1的抗体与细胞受体ACE2胞外区的融合蛋白，用于治疗SARS-CoV2感染。还提供了该融合蛋白的表达载体和能够表达该融合蛋白的宿主细胞，以及该抗体的生产方法。

Description

一种构建冠状病毒抗体的平台 技术领域
本发明属于生物技术领域，具体涉及一种构建冠状病毒抗体的平台。
背景技术
在急性传染病中，绝大部分都是病毒性传染病，病毒性传染病的发病率高，死亡率也很高。由于检测和诊断手段有限，导致新病毒引发的新疫情爆发往往具有突发性、随机性和不可预测性等特点，一旦爆发，如无有效的防治手段，极易造成大规模流行，严重威胁人民健康生命安全。
2019-nCoV冠状病毒属于冠状病毒科冠状病毒属，为具有包膜的单链正义RNA病毒。和其他已知冠状病毒类似，2019-nCoV冠状病毒也经过吸附、穿入、脱壳、生物合成、子代病毒的组装与释放等几个过程完成子代病毒的增殖。2019-nCoV冠状病毒感染宿主细胞起始于病毒包膜表面的刺突糖蛋白与宿主细胞表面的人血管紧张素转化酶2(ACE2)结合，随后发生膜融合，病毒进入宿主细胞，在细胞溶酶体等细胞器作用下，释放出病毒的遗传物质单链正义RNA，在宿主细胞的线粒体、核糖体等蛋白质合成元件以及必须的原料等作用下，翻译产生多聚蛋白。之后，2019-nCoV冠状病毒的两大必需半胱氨酸蛋白酶：木瓜样蛋白酶(papain-like protease,PL  pro)和3C样蛋白酶(3C-like protease,3CL pro)在特定位点切割加工多聚蛋白前体，产生多个对病毒生命周期非常重要的非结构蛋白。在这些非结构蛋白的作用下，病毒RNA复制出子代病毒核酸物质，并大量翻译出所需的结构蛋白，完成子代病毒的组装和释放。2019-nCoV冠状病毒感染细胞的生命周期的任何环节或关键酶均可以作为抗病毒药物的研究靶点，如水解切割多聚蛋白前体的半胱氨酸蛋白酶PL pro和3CL pro，负责完成子代病毒遗传物质复制的RNA聚合酶等。
迄今为止，2019-nCoV病毒的传播途径未完全掌握，已知能通过飞沫和接触传播，且存在人传人、医务人员感染，一定社区传播风险，且病毒存在变异的可能。目前对于新型冠状病毒所致疾病没有特异的预防和治疗方法。
目前，针对SARS-CoV-2(同2019-nCoV)冠状病毒导致的严重肺炎疾病尚无特效的疫苗和抗病毒药物。这些感染性疾病严重影响了人类的生命健康，研发效果好的抗病毒药物迫在眉睫。针对SARS-CoV-2冠状病毒开发出低毒高效 的抗病毒药物，以满足国内外SARS-CoV-2冠状病毒感染患者的临床需求，具有重大的社会意义。
综上所述，本领域迫切需要开发针对SARS-CoV-2冠状病毒有效诊断和治疗方法，以用于诊断和治疗新型冠状病毒感染引起的肺炎。
发明内容
本发明的目的就是提供一种高效的针对SARS-CoV-2冠状病毒有效诊断和治疗方法。
本发明的另一目的是提供一种能够改进抗SARS-Cov2或其他病毒抗体中和能力的技术手段。
本发明的另一目的是提供一种能够抗SARS-Cov2或其他病毒的抗体融合蛋白技术手段。
在本发明的第一方面，提供了一种改进抗冠状病毒抗体中和能力的方法，包括步骤：将所述待改进的抗冠状病毒抗体与人血管紧张素转化酶2(ACE2)或其片段进行融合，从而得到一融合蛋白；
所述的融合蛋白与单独的所述抗冠状病毒抗体相比，抗原中和能力提高≥10倍(较佳地，提高≥20倍)。
在另一优选例中，所述的冠状病毒是SAR-CoV-2病毒。
在另一优选例中，所述的抗体特异性地结合SARS-CoV-2 Spike 1蛋白。
在另一优选例中，所述的人ACE2或其片段的氨基酸序列如SEQ ID NO:1、2、3或4所示；或在SEQ ID NO:1、2、3或4所示序列的基础上，经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留SARS-CoV-2 Spike 1蛋白结合亲和力的衍生序列。
在另一优选例中，所述的经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留SARS-CoV-2 Spike 1结合亲和力的衍生序列为同源性或序列相同性为至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的氨基酸序列。
在另一优选例中，所述的人ACE2或其片段是人ACE2的胞外区全长蛋白。
在另一优选例中，所述的人ACE2或其片段的氨基酸序列如SEQ ID NO:4所示。
在另一优选例中，所述的融合蛋白中，所述的人ACE2或其片段被融合至所述抗体的重链的N端、重链的C端、轻链的N端，和/或轻链的C端。
在另一优选例中，所述的融合蛋白具有相互堆成的两对肽链，每对肽链含有重链H链和轻链L链，所有的肽链均由二硫键相连，其中任意一对肽链从C端到C端具有如式I至V所示的H链和L链的结构，
Figure PCTCN2021084187-appb-000001
Figure PCTCN2021084187-appb-000002
Figure PCTCN2021084187-appb-000003
Figure PCTCN2021084187-appb-000004
Figure PCTCN2021084187-appb-000005
其中，
L1、L2、L3、L4、L5和L6均为连接肽序列，其序列各自独立地为(G 4S) n或其变体，其中，n为正整数(例如1、2、3、4、5或6)，优选地，n＝4；
ACE2代表人ACE2或其片段；
VH代表抗SARS-Cov2 Spike 1蛋白抗体的重链可变区；
VL代表抗SARS-Cov2 Spike 1蛋白抗体的轻链可变区；
CH1、CH2和CH3分别代表抗SARS-Cov2 Spike 1蛋白抗体的重链恒定区CH1、CH2和CH3；
CL代表抗SARS-Cov2 Spike 1蛋白抗体的轻链恒定区；
“～”代表二硫键；
“-”代表肽键。
在另一优选例中，所述(G 4S) n的变体包括：将所述序列中性能相近或相似的氨基酸进行取代所获得的(G 4S) n接头序列的变体，如将一个或多个S分别突变为T；或者在所述序列中插入1-3个氨基酸。
在另一优选例中，所述连接肽序列的氨基酸序列如SEQ ID NO:10所示。
在另一优选例中，所述的方法是非治疗性，并且是非诊断性的。
在另一优选例中，所述抗体的重链恒定区CH1、CH2和CH3、以及轻链恒定区CL均来源于人IgG1或IgG4，优选为人IgG1。
在另一优选例中，所述式I至式V中的VH-CH1-CH2-CH3区段(即待改进的89C8抗体的重链)的氨基酸序列如SEQ ID NO:11所示，或与SEQ ID NO:11的序列具有≥85％(优选地90％，更优选地95％)的序列同一性。
在另一优选例中，所述式I至式V中的VL-CL区段(即待改进的89C8抗体的轻链)的氨基酸序列如SEQ ID NO:16所示，或与SEQ ID NO:16的序列具有≥85％(优选地90％，更优选地95％)的序列同一性。
在另一优选例中，所述融合蛋白中的H链具有如SEQ ID NO:15所示的氨基酸序列，并且融合蛋白中的L链具有如SEQ ID NO:16所示的氨基酸序列；
或者，所述融合蛋白中的H链具有如SEQ ID NO:15所示的氨基酸序列，并且融合蛋白中的L链具有如SEQ ID NO:20所示的氨基酸序列；
或者，所述融合蛋白中的H链具有如SEQ ID NO:11所示的氨基酸序列，并且融合蛋白中的L链具有如SEQ ID NO:20所示的氨基酸序列。
在另一优选例中，所述式I至式V中的VH-CH1-CH2-CH3区段(即待改进的CR3022抗体的重链)的氨基酸序列如SEQ ID NO:21所示，或与SEQ ID NO:21的序列具有≥85％(优选地90％，更优选地95％)的序列同一性。
在另一优选例中，所述式I至式V中的VL-CL区段(即待改进的CR3022抗体的轻链)的氨基酸序列如SEQ ID NO:22所示，或与SEQ ID NO:22的序列具有≥85％(优选地90％，更优选地95％)的序列同一性。
在另一优选例中，所述融合蛋白中的H链具有如SEQ ID NO:26所示的氨基酸序列，并且融合蛋白中的L链具有如SEQ ID NO:22所示的氨基酸序列；
或者，所述融合蛋白中的H链具有如SEQ ID NO:26所示的氨基酸序列，并且融合蛋白中的L链具有如SEQ ID NO:30所示的氨基酸序列；
或者，所述融合蛋白中的H链具有如SEQ ID NO:21所示的氨基酸序列，并且融合蛋白中的L链具有如SEQ ID NO:30所示的氨基酸序列。
在本发明的第二方面，提供了一种融合蛋白，所述的融合蛋白的序列中包含：
(a)抗SARS-Cov2 Spike 1蛋白抗体的序列；和
(b)人ACE2或其片段的序列。
在另一优选例中，所述的融合蛋白中，所述的人ACE2或其片段位于所述SARS-Cov2 Spike 1蛋白抗体的重链的N端、重链的C端、轻链的N端，和/或轻链的C端。
在本发明的第三方面，提供了一种分离的多核苷酸，所述多核苷酸编码如本发明第二方面所述的融合蛋白。
在另一优选例中，所述的多核苷酸中，编码H链的多核苷酸和编码L链的多核苷酸的比例为1:2至3:1，优选地为2:1。
在本发明的第四方面，提供了一种载体，所述载体含有如本发明第三方面所述的多核苷酸。
在另一优选例中，所述载体同时含有如本发明第三方面所述多核苷酸中的所有多核苷酸。
在另一优选例中，所述载体分别含有如本发明第三方面所述的编码H链的多核苷酸和编码L链的多核苷酸。
在另一优选例中，所述载体为表达载体。
在另一优选例中，所述载体包括质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、或其他载体。
在本发明的第五方面，提供了一种遗传工程化的宿主细胞，所述宿主细胞含有如本发明第四方面所述的载体或基因组中整合有如本发明第三方面所述的多核苷酸。
在本发明的第六方面，提供了一种抗体偶联物，所述抗体偶联物含有：
(a)靶向部分，所述靶向部分包括如本发明第二方面所述的融合蛋白；和
(b)与所述靶向部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、金纳米颗粒/纳米棒、纳米磁粒、病毒外壳蛋白或VLP，或其组合。
在另一优选例中，所述的靶向部分与所述的偶联部分通过化学键或接头进行偶联。
在另一优选例中，所述的放射性核素包括：
(i)诊断用同位素，所述的诊断用同位素选自下组：Tc-99m、Ga-68、F-18、I-123、I-125、I-131、In-111、Ga-67、Cu-64、Zr-89、C-11、Lu-177、Re-188、或其组合；和/或
(ii)治疗用同位素，所述的治疗用同位素选自下组：Lu-177、Y-90、Ac-225、As-211、Bi-212、Bi-213、Cs-137、Cr-51、Co-60、Dy-165、Er-169、Fm-255、Au-198、Ho-166、I-125、I-131、Ir-192、Fe-59、Pb-212、Mo-99、Pd-103、P-32、K-42、Re-186、Re-188、Sm-153、Ra223、Ru-106、Na24、Sr89、Tb-149、Th-227、Xe-133Yb-169、Yb-177、或其组合。
在另一优选例中，所述偶联部分为药物或毒素。
在另一优选例中，所述的药物为细胞毒性药物。
在另一优选例中，所述的细胞毒性药物选自下组：抗微管蛋白药物、DNA小沟结合试剂、DNA复制抑制剂、烷化试剂、抗生素、叶酸拮抗物、抗代谢药物、化疗增敏剂、拓扑异构酶抑制剂、长春花生物碱、或其组合。
特别有用的细胞毒性药物类的例子包括，例如，DNA小沟结合试剂、DNA烷基化试剂、和微管蛋白抑制剂、典型的细胞毒性药物包括、例如奥瑞他汀(auristatins)、喜树碱(camptothecins)、多卡霉素/倍癌霉素(duocarmycins)、依托泊甙(etoposides)、美登木素(maytansines)和美登素类化合物(maytansinoids)(例如DM1和DM4)、紫杉烷(taxanes)、苯二氮卓类(benzodiazepines)或者含有苯二氮卓的药物(benzodiazepine containing drugs)(例如吡咯并[1,4]苯二氮卓类(PBDs)，吲哚啉苯并二氮卓类(indolinobenzodiazepines)和噁唑烷并苯并二氮卓类(oxazolidinobenzodiazepines))、长春花生物碱(vinca alkaloids)、或其组合。
在另一优选例中，所述的毒素选自下组：
耳他汀类(例如，耳他汀E、耳他汀F、MMAE和MMAF)、金霉素、类美坦西醇、篦麻毒素、篦麻毒素A-链、考布他汀、多卡米星、多拉司他汀、阿霉素、柔红霉素、紫杉醇、顺铂、cc1065、溴化乙锭、丝裂霉素、依托泊甙、替诺泊甙(tenoposide)、长春新碱、长春碱、秋水仙素、二羟基炭疽菌素二酮、放线菌素、白喉毒素、假单胞菌外毒素(PE)A、PE40、相思豆毒素、相思豆毒素A链、蒴莲根毒素A链、α-八叠球菌、白树毒素、迈托毒素(mitogellin)、局限曲菌素(retstrictocin)、酚霉素、依诺霉素、麻疯树毒蛋白(curicin)、巴豆毒素、卡奇霉素、肥皂草(Sapaonaria  officinalis)抑制剂、糖皮质激素、或其组合。
在另一优选例中，所述偶联部分为可检测标记物。
在另一优选例中，所述偶联物选自：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶、放射性核素、生物毒素、细胞因子(如IL-2)、抗体、抗体Fc片段、抗体scFv片段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、前药激活酶(如DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))、化疗剂(如顺铂)。
在另一优选例中，所述免疫偶联物含有：多价(如二价)的(a)。
在另一优选例中，所述多价是指在所述免疫偶联物的氨基酸序列中包含多个重复的(a)。
在本发明的第七方面，提供了一种免疫细胞，所述免疫细胞表达或在细胞膜外暴露有如本发明第二方面所述的融合蛋白。
在另一优选例中，所述的免疫细胞包括NK细胞、T细胞。
在另一优选例中，所述的免疫细胞来自人或非人哺乳动物(如鼠)。
在本发明的第八方面，提供了一种药物组合物，所述药物组合物含有：
(i)活性成分，所述活性成分选自下组：如本发明第二方面所述的融合蛋白、如本发明第六方面所述的抗体偶联物、如本发明第七方面所述的免疫细胞，或其组合；以及
(ii)药学上可接受的载体。
在另一优选例中，所述的药物组合物为液态制剂。
在另一优选例中，所述的药物组合物为注射剂。
在另一优选例中，所述的药物组合物包括0.01～99.99％的如本发明第二方面所述的融合蛋白、如本发明第六方面所述的抗体偶联物、如本发明第七方面所述的免疫细胞，或其组合，以及0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。
在本发明的第九方面，提供了一种活性成分的用途，被用于(a)制备SARS-CoV2病毒感染的诊断试剂或试剂盒；和/或(b)制备预防和/或治疗SARS-CoV2 病毒感染的药物；
其中，所述活性成分选自下组：如本发明第二方面所述的融合蛋白、如本发明第六方面所述的抗体偶联物、如本发明第七方面所述的免疫细胞，或其组合。
在另一优选例中，所述的诊断试剂为检测片或检测板。
在另一优选例中，所述诊断试剂或试剂盒用于：检测样品中SARS-CoV-2 Spike1蛋白。
在本发明的第十方面，提供了一种制备如本发明第二方面所述的融合蛋白的方法，包括步骤：
(i)在适合表达的条件下，培养如本发明第五方面所述的宿主细胞；和
(ii)从培养物中分离出所述的重组多肽。
在本发明的第十一方面，提供了一种体外检测样品中SARS-CoV-2病毒或SARS-CoV-2 Spike 1蛋白的方法，所述方法包括步骤：
(1)在体外，将所述样品与如本发明第二方面所述的融合蛋白接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在SARS-CoV-2病毒或SARS-CoV-2 Spike 1蛋白。
在另一优选例中，所述的检测包括诊断性的或非诊断性的。
在本发明的第十二方面，提供了一种试剂盒，所述试剂盒中包括：
(1)第一容器，所述第一容器中含有如本发明第二方面所述的融合蛋白；和/或
(2)第二容器，所述第二容器中含有抗如本发明第二方面所述融合蛋白的二抗；
或者，
所述试剂盒含有一检测板，所述检测板包括：基片(支撑板)和测试条，所述的测试条含有如本发明第五方面所述的融合蛋白、如本发明第六方面所述的抗体偶联物、如本发明第七方面所述的免疫细胞，或其组合。
在本发明的第十三方面，提供了一种治疗有所需要的对象的病毒感染类疾病 的方法，包括步骤：
(a)制备一融合蛋白，所述融合蛋白的氨基酸序列中包含：抗病毒表面蛋白的抗体，以及所述对象体内介导所述病毒侵染的蛋白或其片段。
在另一优选例中，所述的病毒包括：SARS、MERS，或其他病毒。
在另一优选例中，所述的介导病毒侵染的蛋白例如SARS所对应的ACE2，或MERS所对应的DPP2等。
在本发明的第十四方面，提供了一种抗冠状病毒的融合蛋白的制备方法，包括步骤：将一抗冠状病毒抗体与人血管紧张素转化酶2(ACE2)或其片段进行融合，从而得到所述融合蛋白。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了本发明融合蛋白的5种基本结构。
图2显示了89C8-ACE2-F1融合蛋白和89C8抗体分别与S1蛋白的亲和力。
图3显示了89C8-ACE2-F1融合蛋白与293-Spike细胞的结合活性。
图4显示了89C8-ACE2-F1融合蛋白对ACE2与Spike 1蛋白结合的阻断活性。
图5显示了89C8-ACE2-F1融合蛋白、89C8和ACE2-Fc对假病毒的侵染活性的中和能力。
具体实施方式
本发明人经过广泛而深入的研究，经过大量的筛选，首次开发了一种改进抗SARS-CoV2 S1抗体的中和能力的方法。研究表明，将ACE2胞外区与所述抗体进行融合后，所得的融合蛋白与SARS-CoV2 S1蛋白的亲和力显著提高，并且所述融合蛋白对病毒的中和活性显著增强。
在此基础上，完成了本发明。
本发明的改进方法(或平台)
在本发明中，提供了一种改进抗冠状病毒抗体中和能力的方法，包括步骤：将所述待改进的抗冠状病毒抗体与人血管紧张素转化酶2(ACE2)或其片段进行融合，从而得到一融合蛋白；所述的融合蛋白与单独的所述抗冠状病毒抗体相比，抗原中和能力提高≥10倍(较佳地，提高≥20倍)。
优选地，本发明所述的冠状病毒是SAR-CoV-2病毒。更加优选地，所述的抗体特异性地结合SARS-CoV-2 Spike 1蛋白。
在一个实施方式中，所述的人ACE2或其片段的氨基酸序列如SEQ ID NO:1、2、3或4所示；或在SEQ ID NO:1、2、3或4所示序列的基础上，经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留SARS-CoV-2 Spike 1蛋白结合亲和力的衍生序列。特别优选地，所述的人ACE2或其片段是指ACE2胞外区的全长蛋白，其氨基酸序列如SEQ ID NO:4所示。
优选地，所述的融合蛋白中，所述的人ACE2或其片段被融合至所述抗体的重链的N端、重链的C端、轻链的N端，和/或轻链的C端。
在所述的融合蛋白中，待改进的抗体可以是与SARS-CoV2 S1蛋白具有高亲和力的任何抗体。
在一个实施方式中，所述的抗体是89C8，其重链的氨基酸序列如SEQ ID NO:11所示，并且其轻链的氨基酸序列如SEQ ID NO:16所示。
当所述的抗体为89C8时，所述的融合蛋白中具有如SEQ ID NO:15所示的氨基酸序列的H链，以及如SEQ ID NO:16所示的氨基酸序列的L链(89C8-ACE-F1)；
或者，所述的融合蛋白具有如SEQ ID NO:15所示的氨基酸序列的H链，以及如SEQ ID NO:20所示的氨基酸序列的L链(89C8-ACE-F2)；
或者，所述的融合蛋白具有如SEQ ID NO:11所示的氨基酸序列的H链，以及如SEQ ID NO:20所示的氨基酸序列的L链(89C8-ACE-F3)。
在一个实施方式中，所述的抗体是CR3022，其重链的氨基酸序列如SEQ ID NO:21所示，并且其轻链的氨基酸序列如SEQ ID NO:22所示。
当所述的抗体为CR3022时，所述的融合蛋白中具有如SEQ ID NO:26所示的氨基酸序列的H链，以及如SEQ ID NO:22所示的氨基酸序列的L链(CR3022-ACE-F1)；
或者，所述的融合蛋白具有如SEQ ID NO:26所示的氨基酸序列的H链，以及如SEQ ID NO:30所示的氨基酸序列的L链(CR3022-ACE-F2)；
或者，所述的融合蛋白具有如SEQ ID NO:21所示的氨基酸序列的H链，以 及如SEQ ID NO:30所示的氨基酸序列的L链(CR3022-ACE-F3)。
应用
本发明还提供了本发明融合蛋白、抗体偶联物ADC、重组蛋白、和/或免疫细胞的用途，例如用于制备诊断制剂或制备药物。
较佳地，所述的药物是用于预防和/或治疗SARS-CoV-2病毒感染的疾病的药物。
检测用途和试剂盒
本发明的融合蛋白或其ADC可用于检测应用，例如用于检测样本，从而提供诊断信息。
本发明中，所采用的样本(样品)包括细胞、组织样本和活检标本。
较佳地，所述的样本是来自于检测对象的血液样本。
本发明使用的术语“活检”应包括本领域技术人员已知的所有种类的活检。因此本发明中使用的活检可以包括例如肿瘤的切除样本、通过内窥镜方法或器官的穿刺或针刺活检制备的组织样本。
本发明中使用的样本包括固定的或保存的细胞或组织样本。
本发明还提供了一种指含有本发明的融合蛋白(或其片段)的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。在优选例中，本发明的融合蛋白可以固定于检测板。
药物组合物
本发明还提供了一种组合物。在优选例中，所述的组合物是药物组合物，它含有上述的融合蛋白或其活性片段或其融合蛋白或其ADC或相应的免疫细胞，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。
配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。典型地，本发明所述的药物组合物的给药途径较佳地为注射给药或口服给药。所述注射给药较佳地包括静脉注 射、肌肉注射、腹腔注射、皮内注射或皮下注射等途径。所述的药物组合物为本领域常规的各种剂型，较佳地为固体、半固体或液体的形式，可以为水溶液、非水溶液或混悬液，更佳地为片剂、胶囊、颗粒剂、注射剂或输注剂等。
本发明所述融合蛋白也可以是由核苷酸序列在细胞内表达用于的细胞治疗，比如，所述融合蛋白用于嵌合抗原受体T细胞免疫疗法(CAR-T)等。
本发明所述的药物组合物是用于预防和/或治疗SARS-CoV-2病毒感染的疾病的药物组合物。
本发明的药物组合物可直接用于结合SARS-CoV-2 Spike 1蛋白分子，因而可用于预防和治疗病毒感染所引发的疾病。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的融合蛋白(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约5毫克/千克体重。此外，本发明的融合蛋白还可与其他治疗剂一起使用。
本发明中，较佳地，本发明所述的药物组合物还包括一种或多种药用载体。所述的药用载体为本领域常规药用载体，所述的药用载体可以为任意合适的生理学或药学上可接受的药物辅料。所述的药物辅料为本领域常规的药物辅料，较佳的包括药学上可接受的赋形剂、填充剂或稀释剂等。更佳地，所述的药物组合物包括0.01～99.99％的上述蛋白质和0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。
本发明中，较佳地，所述的药物组合物的施用量为有效量，所述有效量为能够缓解或延迟疾病、退化性或损伤性病症进展的量。所述有效量可以以个体基础来测定，并将部分基于待治疗症状和所寻求结果的考虑。本领域技术人员可以通过使用个体基础等上述因素和使用不超过常规的实验来确定有效量。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50 毫克/千克体重，较佳地该剂量是约10微克/千克体重-约20毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明提供上述药物组合物在制备预防和/或治疗SARS-CoV-2病毒感染的疾病的药物中的应用。较佳地，所述SARS-CoV-2病毒感染的疾病为肺炎。
冠状病毒
如本文所用，术语“新型冠状病毒”、“2019-nCov”或“SARS-CoV-2”可互换使用，该2019新型冠状病毒是已知感染人的第7种冠状病毒，并且造成新冠肺炎(COVID-19)，是威胁全球人类健康的严重传染性疾病之一。
冠状病毒(Coronavirus，CoV)属于套式病毒目(Nidovirales)冠状病毒科(Coronaviridae)，是一种有包膜的正链RNA病毒，其亚科包含α、β、δ及γ四属。
目前已知的感染人的冠状病毒中，HCoV-229E和HCoV-NL63属于α属冠状病毒，HCoV-OC43、SARS-CoV、HCoV-HKU1、MERS-CoV和SARS-CoV-2均为β属冠状病毒。SARS-CoV-2也被称为2019-nCov。
2003年和2012年分别爆发的高致病性冠状病毒“非典”SARS-CoV和“中东呼吸综合征”MERS-CoV均属于β属冠状病毒。2019年年底爆发的新型冠状病毒(SARS-CoV-2)与SARS-CoV有约80％相似性、与MERS-CoV有40％的相似性，也属于β属冠状病毒。
该类病毒的基因组是一条单股正链RNA，是基因组最大的RNA病毒之一，编码包括复制酶、刺突蛋白、囊膜蛋白、包膜蛋白和核壳蛋白等。在病毒复制的初始阶段，基因组被翻译成两条长达几千个氨基酸的肽链即前体多聚蛋白(Polyprotein)，随后前体蛋白被蛋白酶切割生成非结构蛋白(如RNA聚合酶和解旋酶)和结构蛋白(如刺突蛋白)及辅助蛋白。
本发明的主要优点包括：
1)本发明融合蛋白可以和S1蛋白以高亲和力结合。
2)本发明可以阻断S1蛋白和ACE2的相互作用，阻断活性比ACE2蛋白明显提高。
3)本发明可以中和Spike1假病毒的细胞侵染活性，中和活性比单抗明显 提高。
4)本发明的方法可以用于SARS-CoV2及其他病毒感染(如SARS、MERS等)类疾病的治疗。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
本发明融合蛋白名称以及本发明的序列
在本发明中，涉及到多个融合蛋白,其名称以及对应的重链和轻链的名称总结于表1中。
表1融合蛋白列表
蛋白名称 重链名称 SEQ ID NO: 轻链名称 SEQ ID NO:
89C8-23-F1 89C8-23-F1 HC 12 89C8 LC 16
89C8-45-F1 89C8-45-F1 HC 13 89C8 LC 16
89C8-98-F1 89C8-98-F1 HC 14 89C8 LC 16
89C8-ACE2-F1 89C8-ACE2-F1 HC 15 89C8 LC 16
89C8-23-F2 89C8-23-F1 HC 12 89C8-23-F1 LC 17
89C8-45-F2 89C8-45-F1 HC 13 89C8-45-F1 LC 18
89C8-98-F2 89C8-98-F1 HC 14 89C8-98-F1 LC 19
89C8-ACE2-F2 89C8-ACE2-F1 HC 15 89C8-ACE2-F1 LC 20
89C8-23-F3 89C8 HC 11 89C8-23-F1 LC 17
89C8-45-F3 89C8 HC 11 89C8-45-F1 LC 18
89C8-98-F3 89C8 HC 11 89C8-98-F1 LC 19
89C8-ACE2-F3 89C8 HC 11 89C8-ACE2-F1 LC 20
CR3022-23-F1 CR3022-23-F1 HC 23 CR3022 LC 22
CR3022-45-F1 CR3022-45-F1 HC 24 CR3022 LC 22
CR3022-98-F1 CR3022-98-F1 HC 25 CR3022 LC 22
CR3022-ACE2-F1 CR3022-ACE2-F1 HC 26 CR3022 LC 22
CR3022-23-F2 CR3022-23-F1 HC 23 CR3022-23-F1 LC 27
CR3022-45-F2 CR3022-45-F1 HC 24 CR3022-45-F1 LC 28
CR3022-98-F2 CR3022-98-F1 HC 25 CR3022-98-F1 LC 29
CR3022-ACE2-F2 CR3022-ACE2-F1 HC 26 CR3022-ACE2-F1 LC 30
CR3022-23-F3 CR3022 HC 21 CR3022-23-F1 LC 27
CR3022-45-F3 CR3022 HC 21 CR3022-45-F1 LC 28
CR3022-98-F3 CR3022 HC 21 CR3022-98-F1 LC 29
CR3022-ACE2-F3 CR3022 HC 21 CR3022-ACE2-F1 LC 30
表2本发明涉及的序列列表
Figure PCTCN2021084187-appb-000006
Figure PCTCN2021084187-appb-000007
Figure PCTCN2021084187-appb-000008
Figure PCTCN2021084187-appb-000009
Figure PCTCN2021084187-appb-000010
实施例1：IgG-ACE2融合蛋白的克隆和表达
研究表明，ACE2和CAR-CoV-2 S1蛋白的结合位点集中在其N端的23个氨基酸中。
在本实施例中，使用了含有23个氨基酸、45个氨基酸、98个氨基酸的ACE2截短蛋白以及ACE2胞外区全长蛋白作为融合蛋白中靶向及中和作用的部分，将S1抗体作为融合蛋白的靶向部分，形成S1抗体/ACE2胞外区融合蛋白。融合蛋白的结构如图2所示。ACE2截短及全长蛋白的氨基酸序列如下：
23个氨基酸的ACE2截短蛋白序列(SEQ ID NO:1)：
Figure PCTCN2021084187-appb-000011
45个氨基酸的ACE2截短蛋白序列(SEQ ID NO:2)：
Figure PCTCN2021084187-appb-000012
98个氨基酸的ACE2截短蛋白序列(SEQ ID NO:3)：
Figure PCTCN2021084187-appb-000013
ACE2胞外区全长序列(SEQ ID NO:4)：
Figure PCTCN2021084187-appb-000014
利用分子克隆技术将本发明ACE2的截短蛋白或者全长蛋白通过(G 4S) 4G连接到抗S1抗体89C8或已报道的抗SARS S1蛋白抗体CR3022(专利号：US 2008/0014204 A1)的重链C端(结构1，F1)、轻链N端(结构3，F3)、同时连到重链C端和轻链N端(结构2，F2)、重链N端(结构4，F4)及轻链C端(结构5，F5)。
随后，将所得的各融合蛋白的基因序列利用同源重组酶(购自Vazyme)和EcoR I/Not I双酶切线性化的pCDNA3.1载体中，流程按照商品说明书。同源重组产物化转入Top10感受态细胞，涂布氨苄抗性平板，37℃培养过夜，挑取单克隆测序。
根据所需转染体积传代HEK293细胞，转染前一天将细胞密度调整至1.2×10 6个细胞/ml。取适量的MEM(购自Gibco)培养基作为转染缓冲液，加入重组基因质粒，质粒配对如表1所示，混匀，过滤除菌。按PEI(购自Polysciences)：质粒＝3:1比例加入PEI，混匀后室温孵育20min，倒入HEK293细胞中，36.5℃，8％CO 2培养，等细胞活力降低到60％以下离心收集上清利用 蛋白A磁珠(购自金斯瑞)分选法纯化目的蛋白。将磁珠用适当体积的结合缓冲液(PBS+0.1％吐温20，pH 7.4)重悬(1-4倍磁珠体积)后加入至待纯化样品中，室温孵育1h，期间温柔振荡。样品置于磁力架上(购自海狸)，弃去上清，磁珠用结合缓冲液清洗3遍。按照磁珠体积的3-5倍体积加入洗脱缓冲液(0.1M柠檬酸钠，pH3.2)室温振荡5-10min，置回磁力架上，收集洗脱缓冲液，转移至已加入中和缓冲液(1M Tris，pH 8.54)的收集管中混匀。
从而得到结构如图1所示的融合蛋白。
实施例2：S1抗体/ACE2胞外区融合蛋白和S1蛋白的结合活性
AHC传感器在分析缓冲液中线下平衡30min，然后线上检测60s建立基线，在线加载如上所述获得的经纯化的S1抗体/ACE2胞外区(截短)融合蛋白至AHC传感器上。再将传感器放入100nM的S1-His抗原中作用240s，之后将传感器转移至PBS中解离1000s。使用1:1结合模型进行动力学的分析。
实验结果如下表所示，使用截短的ACE2构建的抗体融合蛋白和S1的亲和力与抗体89C8相似，没有产生加和作用。
表2 S1抗体/ACE2胞外区(截短)融合蛋白候选分子亲和力
编号 K D(M) kon(1/Ms) kdis(1/s)
89C8-23-F1 1.10E-08 3.93E+04 4.32E-04
89C8-45-F1 8.50E-09 4.65E+04 3.93E-04
89C8-98-F1 1.60E-08 3.19E+04 5.00E-04
89C8-23-F2 3.30E-08 2.41E+04 7.86E-04
89C8-45-F2 3.50E-08 1.95E+04 6.77E-04
89C8-98-F2 1.20E-07 7.70E+03 9.37E-04
89C8-23-F3 3.40E-08 2.53E+04 8.67E-04
89C8-45-F3 2.60E-08 2.01E+04 5.29E-04
89C8-98-F3 9.60E-08 8.25E+03 7.88E-04
89C8 7.60E-09 5.04E+04 3.85E-04
ACE2-Fc 2.40E-07 7.34E+04 1.78E-02
另外，AHC传感器在分析缓冲液中线下平衡30min，然后线上检测60s建立基线，在线加载如上所述获得的经纯化的S1抗体/ACE2胞外区(全长)融合蛋白至AHC传感器上。再将传感器放入100nM、50nM、25nM、12.5nM的S1-His 抗原中作用3600s，之后将传感器转移至PBS中解离6400s。使用1:1结合模型进行动力学的分析。
实验结果如图2所示，使用全长的ACE2胞外区构建的抗体融合蛋白(优选结构1，F1)和S1的亲和力比89C8高10倍。
实施例3：抗体/ACE2胞外区融合蛋白与细胞表面表达的Spike结合
在本实施例中，检测纯化获得的抗体/ACE2胞外区融合蛋白样品89C8-ACE2-F1与293-Spike细胞结合活性。
通过转染克隆到MCS的SARS-CoV-2 Spike蛋白全长基因的PLVX-puro载体产生过表达Spike蛋白的293T细胞(293-Spike细胞)。用胰蛋白酶处理扩大培养的293-Spike细胞，并调整细胞悬液密度至2×10 6个细胞/ml，100μl/孔加入96孔流式板，使用2％BSA封闭后离心备用将纯化的抗体/ACE2胞外区融合蛋白用PBS稀释，200nM开始3倍稀释共12个点。将上述稀释好的样品100μl/孔加入上述带有细胞的96孔流式板中，室温孵育60min，PBS清洗两次。100μl/孔加入用PBS稀释100倍的羊F(ab’)2抗人IgG-Fc(PE)(购自Abcam)，4℃孵育30min，PBS清洗两次。100μl/孔加入PBS重悬细胞，在CytoFlex(Bechman)流式细胞仪上进行检测并计算对应的MFI。
实验结果如图3所示。可见，89C8-ACE2-F1与293-Spike细胞有很好的结合活性。
实施例4：抗体/ACE2胞外区融合蛋白阻断S1蛋白和ACE2结合的活性
通过转染克隆到MCS的人ACE2蛋白全长基因的PLVX-puro载体产生过表达人ACE2蛋白的CHO细胞(CHO-ACE2细胞)。将扩大培养的CHO-ACE2细胞调整细胞悬液密度至2×10 6个细胞/ml，100μl/孔加入96孔流式板，使用2％BSA封闭后离心备用。将纯化的样品用PBS稀释，200nM开始3倍稀释共12个点，将上述稀释好的样品60μl/孔加入96孔样品稀释板，同时60μl/孔加入S1-mFc蛋白(购自sinobiologics)，终浓度为0.1nM，与样品37℃孵育。将共孵育样品100μl/孔加入上述带有细胞的96孔流式板中，室温孵育60min，PBS清洗3次。100μl/孔加入用PBS稀释100倍的Anti-mouse Fc-APC(购自eBioscience)，4℃孵育30min，PBS清洗两次。100μl/孔加入PBS重悬细胞，在CytoFlex(Bechman)流式细胞仪上进行检测并计算对应的MFI。
在如上方法的测定实验中，实验结果如图4所示。其结果表明，89C8-ACE2-F1可以阻断ACE2与Spike蛋白的结合，且阻断水平优于对照样品ACE2-Fc及89C8。
实施例5：抗体/ACE2胞外区融合蛋白阻断SARS-Cov2 Spike假病毒侵染ACE2过表达细胞实验
通过转染克隆到MCS的人ACE2蛋白全长基因的PLVX-puro载体产生过表达人ACE2蛋白的293T细胞(293-ACE2细胞)。将293-ACE2细胞按照8000个细胞/孔接种入96孔细胞培养板中，培养过夜。梯度稀释的89C8-ACE2-F1、89C8和ACE2-Fc样品和终浓度为1×10 5TU/ml的luciferase荧光报告基因的SARS-Cov2 Spike假病毒(购买自北京天坛药物生物技术开发公司)混合，37℃孵育1h。去除293-ACE2细胞培养基，并加入上述样品和假病毒的混合液，37℃孵育48h。检测细胞荧光并计算样品的中和活性。
实验结果如图5所示。
其中，89C8-ACE2-F1、89C8和ACE2-Fc均可以完全中和假病毒的侵染活性，其中89C8和ACE2-Fc的中和活性相似。抗体/ACE2胞外区融合蛋白89C8-ACE2-F1的中和活性比89C8强约60倍。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (11)

  1. 一种抗冠状病毒的融合蛋白的制备方法，其特征在于，包括步骤：将一抗冠状病毒抗体与人血管紧张素转化酶2(ACE2)或其片段进行融合，从而得到所述融合蛋白。
  2. 如权利要求1所述的方法，其特征在于，所述的人ACE2或其片段是人ACE2的胞外区全长蛋白或其片段蛋白。
  3. 如权利要求1所述的方法，其特征在于，所述的融合蛋白中，所述的人ACE2或其片段被融合至所述抗体的重链的N端、重链的C端、轻链的N端，和/或轻链的C端。
  4. 如权利要求1所述的方法，其特征在于，所述融合蛋白中的H链具有如SEQ ID NO:15所示的氨基酸序列，并且融合蛋白中的L链具有如SEQ ID NO:16所示的氨基酸序列；
    或者，所述融合蛋白中的H链具有如SEQ ID NO:15所示的氨基酸序列，并且融合蛋白中的L链具有如SEQ ID NO:20所示的氨基酸序列；
    或者，所述融合蛋白中的H链具有如SEQ ID NO:11所示的氨基酸序列，并且融合蛋白中的L链具有如SEQ ID NO:20所示的氨基酸序列。
  5. 如权利要求1所述的方法，其特征在于，所述融合蛋白中的H链具有如SEQ ID NO:26所示的氨基酸序列，并且融合蛋白中的L链具有如SEQ ID NO:22所示的氨基酸序列；
    或者，所述融合蛋白中的H链具有如SEQ ID NO:26所示的氨基酸序列，并且融合蛋白中的L链具有如SEQ ID NO:30所示的氨基酸序列；
    或者，所述融合蛋白中的H链具有如SEQ ID NO:21所示的氨基酸序列，并且融合蛋白中的L链具有如SEQ ID NO:30所示的氨基酸序列。
  6. 一种融合蛋白，其特征在于，所述的融合蛋白的序列中包含：
    (a)抗SARS-Cov2 Spike 1蛋白抗体的序列；和
    (b)人ACE2或其片段的序列。
  7. 一种抗体偶联物，其特征在于，所述抗体偶联物含有：
    (a)靶向部分，所述靶向部分包括如权利要求6所述的融合蛋白；和
    (b)与所述靶向部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、金纳米颗粒/纳米棒、纳米磁粒、 病毒外壳蛋白或VLP，或其组合。
  8. 一种免疫细胞，其特征在于，所述免疫细胞表达或在细胞膜外暴露有如权利要求6所述的融合蛋白。
  9. 一种药物组合物，其特征在于，所述药物组合物含有：
    (i)活性成分，所述活性成分选自下组：如权利要求6所述的融合蛋白、如权利要求7所述的抗体偶联物、如权利要求8所述的免疫细胞，或其组合；以及
    (ii)药学上可接受的载体。
  10. 一种活性成分的用途，其特征在于，被用于(a)制备SARS-CoV2病毒感染的诊断试剂或试剂盒；和/或(b)制备预防和/或治疗SARS-CoV2病毒感染的药物；
    其中，所述活性成分选自下组：如权利要求6所述的融合蛋白、如权利要求7所述的抗体偶联物、如权利要求8所述的免疫细胞，或其组合。
  11. 一种融合蛋白的制备方法，其特征在于，所述的融合蛋白的序列中包含：
    (a)抗病毒(SARS、MERS或其他病毒)表面蛋白抗体；和
    (b)机体内介导病毒侵染的蛋白(如SARS的ACE2、MERS的DPP4等)或其片段。
PCT/CN2021/084187 2020-05-15 2021-03-30 一种构建冠状病毒抗体的平台 WO2021227687A1 (zh)

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Citations (2)

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WO2006044410A2 (en) * 2004-10-14 2006-04-27 Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services A32 monoclonal antibody fusion proteins for use as hiv inhibitors and vaccines
CN104450654A (zh) * 2007-06-12 2015-03-25 阿派隆生物制剂股份公司 Ace2多肽

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WO2006044410A2 (en) * 2004-10-14 2006-04-27 Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services A32 monoclonal antibody fusion proteins for use as hiv inhibitors and vaccines
CN104450654A (zh) * 2007-06-12 2015-03-25 阿派隆生物制剂股份公司 Ace2多肽

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