WO2022061594A1 - Sars-cov-2 spike protein binding molecule and use thereof - Google Patents

Sars-cov-2 spike protein binding molecule and use thereof Download PDF

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WO2022061594A1
WO2022061594A1 PCT/CN2020/117164 CN2020117164W WO2022061594A1 WO 2022061594 A1 WO2022061594 A1 WO 2022061594A1 CN 2020117164 W CN2020117164 W CN 2020117164W WO 2022061594 A1 WO2022061594 A1 WO 2022061594A1
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seq
cov
sars
spike protein
binding molecule
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PCT/CN2020/117164
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French (fr)
Chinese (zh)
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张军方
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深圳市因诺赛生物科技有限公司
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Priority to CN202111556186.5A priority Critical patent/CN114276443B/en
Priority to CN202210089563.7A priority patent/CN114292326B/en
Priority to PCT/CN2020/117164 priority patent/WO2022061594A1/en
Priority to CN202111554248.9A priority patent/CN114456260B/en
Priority to CN202111554250.6A priority patent/CN114437206B/en
Priority to CN202111556178.0A priority patent/CN114276442B/en
Priority to CN202080002271.4A priority patent/CN112513076B/en
Publication of WO2022061594A1 publication Critical patent/WO2022061594A1/en

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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
    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • 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
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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®
    • 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
    • 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
    • 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
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • 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

  • This solution relates to the field of biomedical technology, in particular to a novel coronavirus (SARS-COV-2) spike protein binding molecule and its application.
  • SARS-COV-2 novel coronavirus
  • SARS-COV-2 coronavirus
  • This scheme provides a novel coronavirus (SARS-COV-2) spike protein binding molecule and its application.
  • SARS-COV-2 coronavirus
  • the SARS-COV-2 spike protein binding molecule can specifically bind to the SARS-COV-2 spike protein and comprises at least one immunoglobulin single variable domain, CDR1 in the immunoglobulin single variable domain , CDR2 and CDR3 are selected from any one of the following combinations:
  • the immunoglobulin single variable domain is a single domain antibody.
  • the single domain antibody comprises an amino acid sequence that is at least 80% identical to any of SEQ ID NOs: 64-84.
  • the single domain antibody comprises an amino acid sequence that has at least 90% sequence identity to any of SEQ ID Nos: 64-84.
  • the single domain antibody comprises an amino acid sequence that is at least 99% identical to any of SEQ ID NOs: 64-84.
  • the single domain antibody comprises any one of the amino acid sequences of SEQ ID NOs: 64-84.
  • the SARS-COV-2 spike protein binding molecule further comprises an immunoglobulin Fc region.
  • an immunoglobulin Fc region in a SARS-COV-2 spike protein binding molecule allows the binding molecule to form a dimer while further extending the in vivo half-life of the molecule.
  • the Fc region used in this protocol can be derived from immunoglobulins of different subtypes, eg, IgG (IgGl, IgG2, IgG3 or IgG4 subtype), IgAl, IgA2, IgD, IgE or IgM.
  • the immunoglobulin Fc region is the Fc region of human IgGl.
  • the amino acid sequence of the immunoglobulin Fc region is SEQ ID NO:85.
  • the stability and biological activity of the binding molecule fused with the above-mentioned Fc region are further improved, and the KD value of its binding to the SARS-COV-2 spike protein is further reduced.
  • the SARS-COV-2 spike protein binding molecule comprises at least one amino acid sequence in SEQ ID NOs: 86-106.
  • This solution also provides a nucleic acid molecule encoding the SARS-COV-2 spike protein binding molecule, the nucleic acid molecule is RNA, DNA or cDNA, which can be obtained by artificial synthesis, or can be obtained from suitable natural sources. obtained separately.
  • This solution also provides an expression vector comprising the nucleic acid molecule and its expression control element.
  • the expression vector typically comprises at least one nucleic acid molecule provided in this protocol operably linked to one or more suitable expression control elements (promoter, enhancer, terminator, integration factor, selectable marker, leader sequence) , reporter genes, etc.).
  • suitable expression control elements promoter, enhancer, terminator, integration factor, selectable marker, leader sequence
  • reporter genes etc.
  • the present protocol also provides host cells comprising and expressing the nucleic acid molecule.
  • Said host cells are cells used for expressing heterologous proteins, including bacterial cells, fungal cells or mammalian cells.
  • This protocol also provides a method for obtaining the SARS-COV-2 spike protein binding molecule, including:
  • the SARS-COV-2 spike protein binding molecule expressed by the host cell is collected from the culture in step a.
  • SARS-COV-2 spike protein binding molecules of this protocol can also be obtained by other methods known in the art to generate proteins of known sequences, such as chemical synthesis.
  • This protocol also provides an immunoconjugate comprising the SARS-COV-2 spike protein binding molecule of any one of the above conjugated to a therapeutic moiety.
  • This solution also provides a pharmaceutical composition, comprising the SARS-COV-2 spike protein binding molecule and/or the immunoconjugate described in any one of the above, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition described in this scheme may also include other adjuvants and excipients as needed.
  • the "pharmaceutically acceptable carrier” includes any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion).
  • the active compound i.e. the binding molecule, the immunoconjugate
  • This scheme also provides the application of the pharmaceutical composition in the preparation of a medicine for treating or preventing novel coronavirus disease pneumonia.
  • This solution also provides a kit for detecting SARS-COV-2, comprising the SARS-COV-2 spike protein binding molecule described in any one of the above.
  • This scheme also provides a method for using the kit for detecting SARS-COV-2, in the case of the combination between the SARS-COV-2 spike protein binding molecule described in any of the above and the SARS-COV-2 spike protein Under the condition that the complex can be formed, the detection sample and the control sample are contacted with the SARS-COV-2 spike protein binding molecule described in any one of the above, and the formation of the complex is detected; Differences in complex formation judge the presence of SARS-COV-2 in the samples.
  • the SARS-COV-2 spike protein (SARS-COV-2-Spike protein) binding molecule provided in this solution can specifically bind to the SARS-COV-2-Spike protein and effectively block the SARS-COV-2-Spike protein It binds to the ACE2 receptor of human cells, thereby blocking the infection process of SARS-COV-2 on cells and inhibiting the infection and expansion of SARS-COV-2.
  • the SARS-COV-2-Spike protein binding molecule provided in this scheme also has the characteristics of good binding specificity with the SARS-COV-2-Spike protein, high biological activity and stability, and no toxic and side effects.
  • the SARS-COV-2-Spike protein binding molecule provided by this program can play a long-term inhibitory effect on SARS-COV-2 in vivo, and effectively avoid the recurrence or re-yang of SARS-COV-2 in vivo.
  • Figure 1 is an agarose gel electrophoresis image of the total RNA extracted in Example 1 of this scheme, wherein M: DNA marker 2000, and lane 1: total RNA;
  • Figure 2 is an agarose gel electrophoresis image of the PCR amplification product in Step 1 of the nested PCR amplification of the single-domain antibody gene in Example 1 of the present scheme, wherein M: DNA marker 2000, and lane 1: amplification product;
  • Figure 3 is an agarose gel electrophoresis image of the PCR amplification product in Step 2 of the nested PCR amplification of the single-domain antibody gene in Example 1 of the present scheme, wherein, DNA marker 2000, lane 1: amplification product;
  • Figure 4 is an agarose gel electrophoresis image of the colony PCR amplification product for measuring the library insertion rate in Example 1 of the present scheme, wherein M: DNA marker 2000; swimming lanes 1-8: 8 colonies picked;
  • Fig. 5 is a graph showing the change of viral load in the respiratory tract of rhesus monkeys in the treatment group and the control group with the change of days in Example 2 of the present scheme.
  • immunoglobulin single variable domain refers to substantially consisting of what are known in the art as “framework region 1" or “FR1”, “framework region 2” or “FR2”, “framework region 3” or An immunoglobulin domain consisting of "FR3”, and four “framework regions” of "framework region 4" or “FR4", wherein the framework region is composed of “complementarity determining region 1" or “CDR1”, which is called in the art, The three “complementarity determining regions” or “CDRs” of “complementarity determining region 2" or “CDR2", and “complementarity determining region 3" or “CDR3” are spaced apart.
  • an immunoglobulin single variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Immunoglobulin single variable domains confer antigen specificity to antibodies by having an antigen-binding site.
  • Single domain antibody refers to a type of antibody that lacks the light chain of the antibody and only has the variable region of the heavy chain. Because of its small molecular weight, it is also called Nanobody (Nanobody). Single-domain antibodies specifically bind epitopes without the need for additional antigen-binding domains. Single-domain antibodies are small, stable and efficient antigen-recognition units formed from a single variable domain of immunoglobulins.
  • the total number of amino acid residues in a single domain antibody will typically range from 110 to 120, often between 112 and 115. It should be noted, however, that smaller and longer sequences may also be suitable for the purposes described in this protocol.
  • the term "specificity” refers to the number of different types of antigens or epitopes that a particular antigen-binding molecule or antigen-binding protein (eg, immunoglobulin single variable domain of this scheme) molecule can bind.
  • the specificity of an antigen-binding molecule can be determined based on its affinity and/or avidity.
  • the affinity represented by the dissociation equilibrium constant (KD) of an antigen and an antigen-binding protein, is a measure of the binding strength between an epitope and an antigen-binding site on an antigen-binding protein: the smaller the KD value, the greater the affinity between the epitope and the antigen-binding molecule.
  • affinity can also be expressed as an association constant (KA), which is 1/KD).
  • affinity can be determined in a known manner depending on the particular antigen of interest.
  • Affinity is a measure of the strength of binding between an antigen-binding molecule (eg, an immunoglobulin, antibody, immunoglobulin single variable domain, or polypeptide containing the same) and a relevant antigen.
  • Avidity is related to both: the affinity between its antigen-binding site on the antigen-binding molecule, and the number of associated binding sites present on the antigen-binding molecule.
  • SARS-COV-2 spike protein binding molecule SARS-COV-2-Spike protein binding molecule
  • SARS-COV-2 spike protein binding molecule SARS-COV-2-Spike protein binding molecule
  • SARS-COV-2 spike protein binding molecule may comprise a single domain antibody as defined in this protocol, or a conjugate thereof, directed against the SARS-COV-2 spike protein.
  • SARS-COV-2 spike protein binding molecules also encompass so-called "SMIPs" ("small modular immunopharmaceuticals”), or immunoglobulin superfamily antibodies (IgSF) or CDR grafting molecules.
  • SMIPs small modular immunopharmaceuticals
  • IgSF immunoglobulin superfamily antibodies
  • the "SARS-COV-2 spike protein binding molecule" of the present scheme may comprise at least one immunoglobulin single variable domain such as a single domain antibody that binds to the SARS-COV-2 spike protein.
  • the "SARS-COV-2 spike protein binding molecule” of the present scheme may comprise two immunoglobulin single variable domains such as single domain antibodies that bind to the SARS-COV-2 spike protein.
  • SARS-COV-2 spike binding molecules containing more than one immunoglobulin single variable domain are also referred to as "formatted" SARS-COV-2 spike binding molecules.
  • Formatted SARS-COV-2 spike protein binding molecules may also contain linkers and/or moieties with effector functions, such as half-life extending moieties, in addition to binding to the immunoglobulin single variable domain of the SARS-COV-2 spike protein (eg, an immunoglobulin single variable domain that binds serum albumin), and/or a fusion partner (eg, serum albumin) and/or a conjugated polymer (eg, PEG) and/or an Fc region.
  • the "SARS-COV-2 spike protein binding molecules" of this protocol also encompass bispecific antibodies, which contain immunoglobulin single variable domains that bind different antigens.
  • the SARS-COV-2 spike protein binding molecule of the present protocol will be present at preferably 10-8 to 10-12 moles per liter (M), more preferably 10-9 to 10-11 as measured in a Biacore or KinExA assay moles/liter, even more preferably 10-10 to 10-12, even more preferably 10-11 to 10-12 or lower dissociation constant (KD). Any KD value greater than 10-4 M is generally considered to be indicative of non-specific binding.
  • Specific binding of an antigen-binding protein to an antigen or epitope can be assayed in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays described herein, and/or competitive binding assays (eg, enzyme immunization). Assay (EIA) and Sandwich Competitive Assay.
  • SPR surface plasmon resonance
  • EIA enzyme immunization
  • Amino acid residues will be represented according to standard three-letter or one-letter amino acid codes as well known and agreed in the art.
  • Said conservative amino acid substitutions are well known in the art, for example conservative amino acid substitutions are preferably one amino acid residue within the following groups (1)-(5) replaced by another amino acid residue within the same group: (1) smaller Aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (2) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (3) Polar positively charged residues: His, Arg and Lys; (4) larger aliphatic non-polar residues: Met, Leu, Ile, Val and Cys; and (5) aromatic residues: Phe, Tyr and Trp.
  • Particularly preferred conservative amino acid substitutions are as follows: Ala by Gly or Ser; Arg by Lys; Asn by Gln or His; Asp by Glu; Cys by Ser; Gln by Asn; Glu by Asp; Gly by Ala or Pro; His by Asn or Gln; Ile by Leu or Val; Leu by Ile or Val; Lys by Arg, Gln or Glu; Met by Leu, Tyr or Ile; Phe by Met, Leu or Tyr Substitution; Ser by Thr; Thr by Ser; Trp by Tyr; Tyr by Trp or Phe; Val by Ile or Leu.
  • Sequence identity between two polypeptide sequences indicates the percentage of identical amino acids between the sequences. Methods for assessing the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, identity can be determined using the BLAST program of the NCBI database. For the determination of sequence identity, see for example: Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.
  • a polypeptide or nucleic acid molecule is considered “substantially isolated” when another protein/polypeptide, another nucleic acid, another biological component or macromolecule or at least one contaminant, impurity or minor component) is isolated.
  • a polypeptide or nucleic acid molecule is considered “substantially isolated” when it has been purified at least 2-fold, in particular at least 10-fold, more particularly at least 100-fold and up to 1000-fold or more.
  • a "substantially isolated” polypeptide or nucleic acid molecule is preferably substantially homogeneous as determined by a suitable technique, eg, a suitable chromatographic technique, such as polyacrylamide gel electrophoresis.
  • Alpacas were immunized with the new coronavirus Spike-RBD protein, and immunized at the 1st, 2nd, 4th, and 6th weeks, a total of 4 times, each immunization dose was 300ug.
  • RNA reverse transcription system is as follows:
  • reverse transcription was performed to obtain cDNA.
  • the reverse transcription conditions were: 42 °C, 30 min; 50 °C, 15 min; 70 °C, 15 min.
  • VHH Single domain antibody
  • Nested PCR was used to amplify the VHH gene as follows:
  • the PCR product was purified and concentrated by the DNA purification kit, and then subjected to agarose gel electrophoresis.
  • the obtained agarose gel electrophoresis diagram is shown in Figure 2.
  • the DNA product gel recovery kit was used to recover the 750bp band, which was quantified by UV spectrophotometer. , as the DNA template of Step 2;
  • the sequence of the amplification primer is: Primer For-2: 5 ⁇ -CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT-3 ⁇ (SEQ ID NO: 110); Primer Rev-2: 5'-GATGTGCAGCTGCAGGAGTCTGRGGAGG-3' (SEQ ID NO: 111).
  • the obtained PCR product was subjected to agarose gel electrophoresis, and the agarose gel electrophoresis diagram was shown in Figure 3, which was recovered by a DNA product gel recovery kit and quantified by an ultraviolet spectrophotometer. end up with about 500
  • the target gene (VHH) of bp was 200 ⁇ L at a concentration of 458 ng/ ⁇ L.
  • the obtained target gene and vector pHEN1 were double-enzyme digested with SfiI and Not1, and the digested target gene and pHEN1 fragment were ligated with T4 DNA ligase, then transformed into Escherichia coli electroporation competent cell TG1, and constructed for SARS.
  • S2-Lib Single domain antibody gene library of COV-2-Spike protein, named S2-Lib. A total of 15 transformations were performed, and after mixing, they were evenly spread on 6 ⁇ 150 mm petri dishes (LB solid medium containing ampicillin).
  • the calculated library capacity is 1.425 ⁇ 10 9 cfu.
  • the colony PCR system is as follows:
  • the colony PCR reaction conditions were as follows: 98°C for 10s, 50°C for 30s, and 72°C for 1 min, a total of 31 cycles.
  • S2-Lib gene library Take 10-100 times the library capacity of live cells from the above S2-Lib gene library for inoculation and culture. After culture to log phase, M13K07 phage is used for rescue. After rescue culture, the phage is collected by centrifugation, and the phage is purified by PEG-NaCl, namely A phage display library was obtained, named S2-PDL, with a titer of 3.5 ⁇ 10 13 cfu/mL. It can be directly used for subsequent affinity screening of specific phages.
  • the positive clones are enriched, and the purpose of screening Spike-RBD protein-specific antibodies in the antibody library by using the phage display library is achieved.
  • the obtained positive phages were sequenced to obtain antibody gene sequences.
  • the obtained antibody gene sequences were constructed on the pcDNA3.4 vector respectively, the antibody was expressed in HEK-293 cells, and the antibody in the supernatant of the medium was collected by purification with proteinA medium. Purified antibodies were incubated with Spike-RBD-coated plates for ELISA assays. Obtain antibodies that can specifically bind to Spike-RBD protein.
  • the obtained antibody sequences were analyzed according to the sequence alignment software Vector NTI.
  • the clones with the same CDR1, CDR2 and CDR3 sequences were regarded as the same antibody strain, and the clones with different CDR sequences were regarded as different antibody strains.
  • a total of 21 different single-domain antibody strains that can specifically bind to the Spike-RBD protein were obtained.
  • the single-domain antibody sequences are such as SEQ ID NOs: 64-84, which respectively carry 21 groups of CDR1-3 sequences in SEQ ID NOs: 1-63 , as shown in Table 2.
  • the blank is the OD450 value in the duplicate wells without antibody.
  • Spike-RBD protein and ACE2 protein were obtained by expressing in HEK293 cells (pCDNA4, Invitrogen, Cat V86220). Then use the Biotinlytion kit of Thermo Company to obtain the biotinylated ACE2 protein.
  • the purified 21 single-domain antibodies were added to the culture system. , 24 hours later, the cells were washed twice with PBS, and the 21 single-domain antibodies were mixed with the virus and added to the 96-well plate. Incubate at °C for 2 hours, and detect whether the VERO cells are infected with the virus on the 5th day (if the cells do not change, it means that the single-domain antibody can neutralize the virus and block the process of the virus infecting the VERO cells).
  • the test results are shown in Table 5.
  • the single-domain antibodies of 21 strains can effectively block the process of virus infection of cells at a concentration of more than 5 ⁇ g/ml, and some antibodies can still effectively block at a concentration of less than 0.02 ⁇ g/ml.
  • the process by which viruses infect cells According to the IC50 ( ⁇ g/ml) data obtained in Table 5, the obtained 21 single-domain antibodies can block the process of virus infection of cells, and are effective neutralizing antibodies.
  • the amino acid sequence of the human immunoglobulin (IgG1) on the protein database Uniprot the amino acid sequence of the human IgG1-Fc region (SEQ ID NO: 85) was obtained.
  • the nucleic acid fragment encoding human IgG1-Fc (nucleic acid sequence such as SEQ ID NO: 107) was obtained from the total RNA of human PBMC, and then the SARS-COV-2-Spike protein single domain antibody and Fc were obtained by overlapping PCR.
  • the nucleic acid fragment encoding the fusion protein was recombined into the vector pCDNA4 (Invitrogen, Cat V86220).
  • the successfully constructed pCDNA4 plasmid containing the nucleic acid fragment of the fusion protein of SARS-COV-2-Spike protein single domain antibody and Fc was transfected into HEK293 cells for expression.
  • the recombinant expression plasmid was diluted with Freestyle293 medium, and the PEI (Polyethylenimine) solution required for transformation was added.
  • the plasmid/PEI mixture was added to the HEK293 cell suspension, and placed at 37°C, 10% CO 2 , and a shaker at 100 rpm. cultured in medium; supplemented with 50 ⁇ g/L IGF-1.
  • SARS-COV-2-Spike protein single domain antibody and Fc fusion protein to SARS-COV-2-Spike protein was identified by SPR method.
  • the specific operation is as follows: the binding kinetics of the obtained 21 SARS-COV-2-Spike protein single-domain antibodies and Fc fusion proteins to spike-RBD are measured by surface plasmon resonance (SRP) method using a BIAcoreX100 instrument.
  • SRP surface plasmon resonance
  • the spike-RBD protein was directly coated on the CM5 biosensor chip to obtain approximately 1000 response units (RU).
  • the fusion protein of SARS-COV-2-Spike protein single domain antibody and Fc was three-fold serially diluted (1.37 nm to 1000 nm), injected at 25°C for 120 s, dissociated for 30 min, and regenerated by adding 10 mM glycine-HCl (pH 2.0) for 120 s.
  • a simple one-to-one Languir binding model (BIAcore Evaluation Software version 3.2) Calculate the on-rate (kon), dissociation rate (koff) and equilibrium dissociation constant (kD) of the fusion protein with the SARS-COV-2-Spike protein (in the ratio koff/kon calculate). The calculation results are shown in Table 6.
  • ACE2 protein was obtained by HEK293 cell expression.
  • the biotinylated protein ACE2-Biotin was obtained by using the Biotinlytion kit of Thermo Company.
  • the plate was coated with 0.5 ⁇ g/well of Spike-RBD protein at 4°C overnight, and then 21 strains of SARS-COV-2-Spike protein single domain antibody and Fc fusion protein 200ng and ACE2-Biotin 5ug were added to each well.
  • Control group 1 No fusion protein was added, and ACE2-Biotin was not added to control group 2, and the reaction was carried out at room temperature for 2 h. After washing, SA-HRP (purchased from Sigma) was added, and the reaction was performed at room temperature for 1 hour. After washing, a color developing solution was added, and the absorption value was read at a wavelength of 450 nm.
  • Table 7 The results are shown in Table 7.
  • Transient transfection of human HEK293 cells was used to obtain seven currently known coronaviruses (SARS-COV-2, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV ) plasmid of the full-length gene of Spike protein (pCDNA4, Invitrogen, Cat V86220) and transiently express Spike protein on membrane.
  • This plasmid makes the C-terminal of Spike protein fused with EGFP protein, so that the expression level of Spike protein on the membrane can be examined by the green fluorescence intensity.
  • the constructed cells were resuspended in 0.5% PBS-BSA Buffer, SARS-COV-2-Spike protein single domain antibody and Fc fusion protein were added, and a negative control was set at the same time, and incubated on ice for 20 min. After washing, eBioscience secondary antibody anti-hIg-PE was added and kept on ice for 20 min. After washing, the cells were resuspended in 500 ⁇ l of 0.5% PBS-BSABuffer and detected by flow cytometry. The results showed that the 21 SARS-COV-2-Spike protein single-domain antibody-Fc fusion proteins only specifically bound to the SARS-COV-2-Spike protein, but not to the Spike proteins of other coronaviruses.
  • the detection process of the new coronavirus load is as follows: take the throat swabs of the rhesus monkeys treated with drug treatment (treatment group) and the rhesus monkeys without drug treatment (control group), and extract the virus in the throat swabs.
  • RNA extraction kit (Qiagen) is used to extract SARS-COV-2 RNA according to the instructions, and the obtained RNA is dissolved in 50 ⁇ L elution buffer and used as a template for RT-PCR amplification.
  • the viral S-region gene was amplified with primers RBD-qF1 (5 ⁇ -CAATGGTTTAACAGGCACAGG-3 ⁇ , SEQ ID NO: 112) and RBD-qR1 (5 ⁇ -CTCAAGTGTCTGTGGATCACG-3 ⁇ , SEQ ID NO: 113).
  • Adopt HiScriptR II One Step qRT-PCR SYBRR Green Kit (Vazyme Biotech Co., Ltd) kit, operate according to the kit instructions, set the PCR amplification conditions as: 50°C 3min, 95°C 10s, 60°C 30s, 40 cycles, the PCR amplification used
  • the instrument is an ABI quantitative PCR instrument.

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Abstract

The present invention relates to the technical field of biomedicine. In particular, disclosed are a SARS-COV-2 spike protein binding molecule and the use thereof. The binding molecule can specifically bind to a SARS-COV-2 spike protein and contains at least one immunoglobulin single variable domain. The SARS-COV-2 spike protein binding molecule provided in the present invention can effectively block the binding of the SARS-COV-2 spike protein to a human cell ACE 2 receptor, thereby blocking the infection process of SARS-COV-2 on cells and inhibiting infection by and amplification of SARS-COV-2. The binding molecule exerts a long-term effect of inhibiting SARS-COV-2 in vivo and effectively prevents SARS-COV-2 from recurrence in vivo.

Description

新型冠状病毒（SARS-COV-2）刺突蛋白结合分子及其应用Novel coronavirus (SARS-COV-2) spike protein binding molecule and its application 技术领域technical field
本方案涉及生物医药技术领域，尤其涉及一种新型冠状病毒（SARS-COV-2）刺突蛋白结合分子及其应用。This solution relates to the field of biomedical technology, in particular to a novel coronavirus (SARS-COV-2) spike protein binding molecule and its application.
背景技术Background technique
目前对COVID-19临床上缺乏特异有效的治疗手段。此外，越来越多的研究显示，新型冠状病毒（SARS-COV-2）感染可能存在慢性携带状态；部分出院复阳病人也提示病毒可能会长期存在人体。目前尚不清楚长期携带存在的机制、时间等关键因素，防止SARS-COV-2卷土重来至关重要。Currently, there is no specific and effective treatment for COVID-19. In addition, more and more studies have shown that the new coronavirus (SARS-COV-2) infection may have a chronic carrier state; some discharged patients with positive symptoms also suggest that the virus may exist in the human body for a long time. At present, it is not clear the mechanism, time and other key factors of long-term carry-on existence, and it is crucial to prevent the resurgence of SARS-COV-2.
目前针对COVID-19尚无特效药物，亟需快速研制有效的药物。国内外众多研发机构都在针对COVID-19的治疗策略研究上分秒必争。虽然已发掘的瑞德西韦、法匹拉韦等广谱小分子抗病毒药物对COVID-19具有一定疗效，但由于针对SARS-COV-2并无特异性，治疗效果受限，难以成为COVID-19的特效药。At present, there is no specific drug for COVID-19, and there is an urgent need to develop effective drugs quickly. Many R&D institutions at home and abroad are racing against the clock on the research on treatment strategies for COVID-19. Although the broad-spectrum small-molecule antiviral drugs such as remdesivir and favipiravir that have been discovered have a certain effect on COVID-19, due to the lack of specificity for SARS-COV-2, the therapeutic effect is limited, and it is difficult to become a COVID-19 disease. -19 special medicine.
技术问题technical problem
本方案提供一种新型冠状病毒（SARS-COV-2）刺突蛋白结合分子及其应用。This scheme provides a novel coronavirus (SARS-COV-2) spike protein binding molecule and its application.
技术解决方案technical solutions
该SARS-COV-2刺突蛋白结合分子，能特异性结合SARS-COV-2刺突蛋白且包含至少一个免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域中的CDR1、CDR2和CDR3选自如下组合中的任意一组：The SARS-COV-2 spike protein binding molecule can specifically bind to the SARS-COV-2 spike protein and comprises at least one immunoglobulin single variable domain, CDR1 in the immunoglobulin single variable domain , CDR2 and CDR3 are selected from any one of the following combinations:
1）SEQ ID NO:1所示的CDR1，SEQ ID NO:2所示的CDR2和SEQ ID NO:3所示的CDR3；1) CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 2 and CDR3 shown in SEQ ID NO: 3;
2）SEQ ID NO:4所示的CDR1，SEQ ID NO:5所示的CDR2和SEQ ID NO:6所示的CDR3；2) CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5 and CDR3 shown in SEQ ID NO:6;
3）SEQ ID NO:7所示的CDR1，SEQ ID NO:8所示的CDR2和SEQ ID NO:9所示的CDR3；3) CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8 and CDR3 shown in SEQ ID NO:9;
4）SEQ ID NO:10所示的CDR1，SEQ ID NO:11所示的CDR2和SEQ ID NO:12所示的CDR3；4) CDR1 shown in SEQ ID NO: 10, CDR2 shown in SEQ ID NO: 11 and CDR3 shown in SEQ ID NO: 12;
5）SEQ ID NO:13所示的CDR1，SEQ ID NO:14所示的CDR2和SEQ ID NO:15所示的CDR3；5) CDR1 shown in SEQ ID NO: 13, CDR2 shown in SEQ ID NO: 14 and CDR3 shown in SEQ ID NO: 15;
6）SEQ ID NO:16所示的CDR1，SEQ ID NO:17所示的CDR2和SEQ ID NO:18所示的CDR3；6) CDR1 shown in SEQ ID NO: 16, CDR2 shown in SEQ ID NO: 17 and CDR3 shown in SEQ ID NO: 18;
7）SEQ ID NO:19所示的CDR1，SEQ ID NO:20所示的CDR2和SEQ ID NO:21所示的CDR3；7) CDR1 shown in SEQ ID NO: 19, CDR2 shown in SEQ ID NO: 20 and CDR3 shown in SEQ ID NO: 21;
8）SEQ ID NO:22所示的CDR1，SEQ ID NO:23所示的CDR2和SEQ ID NO:24所示的CDR3；8) CDR1 shown in SEQ ID NO:22, CDR2 shown in SEQ ID NO:23 and CDR3 shown in SEQ ID NO:24;
9）SEQ ID NO:25所示的CDR1，SEQ ID NO:26所示的CDR2和SEQ ID NO:27所示的CDR3；9) CDR1 shown in SEQ ID NO:25, CDR2 shown in SEQ ID NO:26 and CDR3 shown in SEQ ID NO:27;
10）SEQ ID NO:28所示的CDR1，SEQ ID NO:29所示的CDR2和SEQ ID NO:30所示的CDR3；10) CDR1 shown in SEQ ID NO:28, CDR2 shown in SEQ ID NO:29 and CDR3 shown in SEQ ID NO:30;
11）SEQ ID NO:31所示的CDR1，SEQ ID NO:32所示的CDR2和SEQ ID NO:33所示的CDR3；11) CDR1 shown in SEQ ID NO:31, CDR2 shown in SEQ ID NO:32 and CDR3 shown in SEQ ID NO:33;
12）SEQ ID NO:34所示的CDR1，SEQ ID NO:35所示的CDR2和SEQ ID NO:36所示的CDR3；12) CDR1 shown in SEQ ID NO:34, CDR2 shown in SEQ ID NO:35 and CDR3 shown in SEQ ID NO:36;
13）SEQ ID NO:37所示的CDR1，SEQ ID NO:38所示的CDR2和SEQ ID NO:39所示的CDR3；13) CDR1 shown in SEQ ID NO:37, CDR2 shown in SEQ ID NO:38 and CDR3 shown in SEQ ID NO:39;
14）SEQ ID NO:40所示的CDR1，SEQ ID NO:41所示的CDR2和SEQ ID NO:42所示的CDR3；14) CDR1 shown in SEQ ID NO:40, CDR2 shown in SEQ ID NO:41 and CDR3 shown in SEQ ID NO:42;
15）SEQ ID NO:43所示的CDR1，SEQ ID NO:44所示的CDR2和SEQ ID NO:45所示的CDR3；15) CDR1 shown in SEQ ID NO:43, CDR2 shown in SEQ ID NO:44 and CDR3 shown in SEQ ID NO:45;
16）SEQ ID NO:46所示的CDR1，SEQ ID NO:47所示的CDR2和SEQ ID NO:48所示的CDR3；16) CDR1 shown in SEQ ID NO:46, CDR2 shown in SEQ ID NO:47 and CDR3 shown in SEQ ID NO:48;
17）SEQ ID NO:49所示的CDR1，SEQ ID NO:50所示的CDR2和SEQ ID NO:51所示的CDR3；17) CDR1 shown in SEQ ID NO:49, CDR2 shown in SEQ ID NO:50 and CDR3 shown in SEQ ID NO:51;
18）SEQ ID NO:52所示的CDR1，SEQ ID NO:53所示的CDR2和SEQ ID NO:54所示的CDR3；18) CDR1 shown in SEQ ID NO:52, CDR2 shown in SEQ ID NO:53 and CDR3 shown in SEQ ID NO:54;
19）SEQ ID NO:55所示的CDR1，SEQ ID NO:56所示的CDR2和SEQ ID NO:57所示的CDR3；19) CDR1 shown in SEQ ID NO:55, CDR2 shown in SEQ ID NO:56 and CDR3 shown in SEQ ID NO:57;
20）SEQ ID NO:58所示的CDR1，SEQ ID NO:59所示的CDR2和SEQ ID NO:60所示的CDR3；20) CDR1 shown in SEQ ID NO:58, CDR2 shown in SEQ ID NO:59 and CDR3 shown in SEQ ID NO:60;
21）SEQ ID NO:61所示的CDR1，SEQ ID NO:62所示的CDR2和SEQ ID NO:63所示的CDR3。21) CDR1 shown in SEQ ID NO:61, CDR2 shown in SEQ ID NO:62 and CDR3 shown in SEQ ID NO:63.
作为一种实施例，所述免疫球蛋白单一可变域为单域抗体。As an example, the immunoglobulin single variable domain is a single domain antibody.
作为一种实施例，所述单域抗体包含与SEQ ID NO:64-84中任一序列具有至少80％的序列相同性的氨基酸序列。As an example, the single domain antibody comprises an amino acid sequence that is at least 80% identical to any of SEQ ID NOs: 64-84.
作为一种实施例，所述单域抗体包含与SEQ ID NO:64-84中任一序列具有至少90％的序列相同性的氨基酸序列。As one example, the single domain antibody comprises an amino acid sequence that has at least 90% sequence identity to any of SEQ ID NOs: 64-84.
作为一种实施例，所述单域抗体包含与SEQ ID NO:64-84中任一序列具有至少99％的序列相同性的氨基酸序列。As one example, the single domain antibody comprises an amino acid sequence that is at least 99% identical to any of SEQ ID NOs: 64-84.
作为一种实施例，所述单域抗体包含 SEQ ID NO:64-84中的任意一种氨基酸序列。As an example, the single domain antibody comprises any one of the amino acid sequences of SEQ ID NOs: 64-84.
作为一种实施例，所述SARS-COV-2刺突蛋白结合分子还包含免疫球蛋白Fc区。SARS-COV-2刺突蛋白结合分子中包含免疫球蛋白Fc区可以使所述结合分子形成二聚体，同时进一步延长所述分子的体内半衰期。用于本方案的Fc区可以来自不同亚型的免疫球蛋白，例如，IgG(IgG1、IgG2、IgG3或IgG4亚型)、IgA1、IgA2、IgD、IgE或IgM。As an example, the SARS-COV-2 spike protein binding molecule further comprises an immunoglobulin Fc region. The inclusion of an immunoglobulin Fc region in a SARS-COV-2 spike protein binding molecule allows the binding molecule to form a dimer while further extending the in vivo half-life of the molecule. The Fc region used in this protocol can be derived from immunoglobulins of different subtypes, eg, IgG (IgGl, IgG2, IgG3 or IgG4 subtype), IgAl, IgA2, IgD, IgE or IgM.
作为一种实施例，所述免疫球蛋白Fc区是人IgG1的Fc区。As an example, the immunoglobulin Fc region is the Fc region of human IgGl.
作为一种实施例，所述免疫球蛋白Fc区的氨基酸序列为SEQ ID NO:85。与上述Fc区融合后的结合分子，稳定性和生物活性进一步提高，并进一步降低了其与SARS-COV-2刺突蛋白结合的KD值。As an example, the amino acid sequence of the immunoglobulin Fc region is SEQ ID NO:85. The stability and biological activity of the binding molecule fused with the above-mentioned Fc region are further improved, and the KD value of its binding to the SARS-COV-2 spike protein is further reduced.
作为一种实施例，所述SARS-COV-2刺突蛋白结合分子包含SEQ ID NO:86-106中的至少一种氨基酸序列。As an example, the SARS-COV-2 spike protein binding molecule comprises at least one amino acid sequence in SEQ ID NOs: 86-106.
本方案还提供编码所述的SARS-COV-2刺突蛋白结合分子的核酸分子，所述核酸分子为RNA、DNA或cDNA，其可以通过人工合成的方式获得，也可从适合的天然来源加以分离获得。This solution also provides a nucleic acid molecule encoding the SARS-COV-2 spike protein binding molecule, the nucleic acid molecule is RNA, DNA or cDNA, which can be obtained by artificial synthesis, or can be obtained from suitable natural sources. obtained separately.
本方案还提供包含所述核酸分子及其表达调控原件的表达载体。所述表达载体通常包含至少一种本方案提供的核酸分子，其可操作地连接至一个或多个适合的表达调控元件（启动子、增强子、终止子、整合因子、选择标记物、前导序列、报告基因等）。针对在特定宿主细胞中的表达对所述元件及其序列进行选择为本领域技术人员的常识。This solution also provides an expression vector comprising the nucleic acid molecule and its expression control element. The expression vector typically comprises at least one nucleic acid molecule provided in this protocol operably linked to one or more suitable expression control elements (promoter, enhancer, terminator, integration factor, selectable marker, leader sequence) , reporter genes, etc.). The selection of such elements and their sequences for expression in a particular host cell is within the general knowledge of those skilled in the art.
本方案还提供包含所述核酸分子并进行表达的宿主细胞。所述的宿主细胞为用于表达异源蛋白的细胞，包括细菌细胞、真菌细胞或哺乳动物细胞。The present protocol also provides host cells comprising and expressing the nucleic acid molecule. Said host cells are cells used for expressing heterologous proteins, including bacterial cells, fungal cells or mammalian cells.
本方案还提供获得所述SARS-COV-2刺突蛋白结合分子的方法，包括：This protocol also provides a method for obtaining the SARS-COV-2 spike protein binding molecule, including:
a、在允许所述SARS-COV-2刺突蛋白结合分子表达的条件下培养权利要求16所述的宿主细胞；a, culturing the host cell of claim 16 under conditions that allow the expression of the SARS-COV-2 spike protein binding molecule;
b、从步骤a的培养物中收集由所述宿主细胞表达的SARS-COV-2刺突蛋白结合分子。b. The SARS-COV-2 spike protein binding molecule expressed by the host cell is collected from the culture in step a.
将特定的核酸分子重组到表达载体上并通过转化或转染方法进入宿主细胞中表达、选择标记物、诱导蛋白表达的方法、培养条件等在本领域中是已知的。同时蛋白结合分子的分离及纯化技术为本领域技术人员所公知。此外，本方案的SARS-COV-2刺突蛋白结合分子也可以通过本领域已知的其它产生已知序列的蛋白质的方法获得，例如化学合成。Recombination of specific nucleic acid molecules into expression vectors and expression in host cells by transformation or transfection methods, selectable markers, methods of inducing protein expression, culture conditions, and the like are known in the art. At the same time, the separation and purification techniques of protein-binding molecules are well known to those skilled in the art. In addition, the SARS-COV-2 spike protein binding molecules of this protocol can also be obtained by other methods known in the art to generate proteins of known sequences, such as chemical synthesis.
本方案还提供了一种免疫缀合物，包含与治疗性部分缀合的上述任一项所述的SARS-COV-2刺突蛋白结合分子。This protocol also provides an immunoconjugate comprising the SARS-COV-2 spike protein binding molecule of any one of the above conjugated to a therapeutic moiety.
本方案还提供了一种药物组合物，包含上述任一项所述的SARS-COV-2刺突蛋白结合分子和/或所述的免疫缀合物，以及药学上可接受的载体。本方案所述的药物组合物根据需要还可以包括其它佐剂和辅料等。所述的“药学上可接受的载体”包括生理学相容的任何溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和吸收延迟剂等。该载体适合于静脉内、肌内、皮下、肠胃外、脊柱或表皮施用(如通过注射或输注)。根据施用途径，可将活性化合物即结合分子、免疫缀合物包裹于一种材料中，以保护该化合物免受可使该化合物失活的酸和其他天然条件的作用，为本领域技术人员公知。This solution also provides a pharmaceutical composition, comprising the SARS-COV-2 spike protein binding molecule and/or the immunoconjugate described in any one of the above, and a pharmaceutically acceptable carrier. The pharmaceutical composition described in this scheme may also include other adjuvants and excipients as needed. The "pharmaceutically acceptable carrier" includes any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion). Depending on the route of administration, the active compound, i.e. the binding molecule, the immunoconjugate, can be encapsulated in a material to protect the compound from the action of acids and other natural conditions that can inactivate the compound, as known to those skilled in the art .
本方案还提供了所述药物组合物在制备治疗或预防新型冠状病毒病肺炎药物中的应用。This scheme also provides the application of the pharmaceutical composition in the preparation of a medicine for treating or preventing novel coronavirus disease pneumonia.
本方案还提供了一种用于检测SARS-COV-2的试剂盒，包含上述任一项所述的SARS-COV-2刺突蛋白结合分子。This solution also provides a kit for detecting SARS-COV-2, comprising the SARS-COV-2 spike protein binding molecule described in any one of the above.
本方案还提供了所述用于检测SARS-COV-2的试剂盒的使用方法，在上述任一项所述的SARS-COV-2刺突蛋白结合分子与SARS-COV-2刺突蛋白之间能够形成复合物的条件下，使检测样品和对照样品接触上述任一项所述的SARS-COV-2刺突蛋白结合分子，检测复合物的形成；通过所述检测样品与对照样品之间复合物形成的差异判断样品中SARS-COV-2的存在。This scheme also provides a method for using the kit for detecting SARS-COV-2, in the case of the combination between the SARS-COV-2 spike protein binding molecule described in any of the above and the SARS-COV-2 spike protein Under the condition that the complex can be formed, the detection sample and the control sample are contacted with the SARS-COV-2 spike protein binding molecule described in any one of the above, and the formation of the complex is detected; Differences in complex formation judge the presence of SARS-COV-2 in the samples.
有益效果beneficial effect
本方案提供的SARS-COV-2刺突蛋白（SARS-COV-2-Spike蛋白）结合分子能够特异性的结合SARS-COV-2-Spike蛋白，并有效阻断SARS-COV-2-Spike蛋白与人体细胞ACE2受体的结合，进而阻断SARS-COV-2对细胞的感染过程，抑制SARS-COV-2的传染和扩增。且本方案提供的SARS-COV-2-Spike蛋白结合分子还具有与SARS-COV-2-Spike蛋白结合的特异性好，生物活性和稳定性高以及无毒副作用的特点。同时本方案提供的SARS-COV-2-Spike蛋白结合分子可以在体内发挥长效抑制SARS-COV-2的作用，有效避免SARS-COV-2在活体内复发或复阳。The SARS-COV-2 spike protein (SARS-COV-2-Spike protein) binding molecule provided in this solution can specifically bind to the SARS-COV-2-Spike protein and effectively block the SARS-COV-2-Spike protein It binds to the ACE2 receptor of human cells, thereby blocking the infection process of SARS-COV-2 on cells and inhibiting the infection and expansion of SARS-COV-2. In addition, the SARS-COV-2-Spike protein binding molecule provided in this scheme also has the characteristics of good binding specificity with the SARS-COV-2-Spike protein, high biological activity and stability, and no toxic and side effects. At the same time, the SARS-COV-2-Spike protein binding molecule provided by this program can play a long-term inhibitory effect on SARS-COV-2 in vivo, and effectively avoid the recurrence or re-yang of SARS-COV-2 in vivo.
附图说明Description of drawings
图1是本方案实施例1中提取的总RNA的琼脂糖凝胶电泳图，其中，M：DNA marker 2000，泳道1：总RNA；Figure 1 is an agarose gel electrophoresis image of the total RNA extracted in Example 1 of this scheme, wherein M: DNA marker 2000, and lane 1: total RNA;
图2是本方案实施例1中巢式PCR扩增单域抗体基因的Step1中的PCR扩增产物的琼脂糖凝胶电泳图，其中，M：DNA marker 2000，泳道1：扩增产物；Figure 2 is an agarose gel electrophoresis image of the PCR amplification product in Step 1 of the nested PCR amplification of the single-domain antibody gene in Example 1 of the present scheme, wherein M: DNA marker 2000, and lane 1: amplification product;
图3是本方案实施例1中巢式PCR扩增单域抗体基因的Step2中的PCR扩增产物的琼脂糖凝胶电泳图，其中，DNA marker 2000，泳道1：扩增产物；Figure 3 is an agarose gel electrophoresis image of the PCR amplification product in Step 2 of the nested PCR amplification of the single-domain antibody gene in Example 1 of the present scheme, wherein, DNA marker 2000, lane 1: amplification product;
图4是本方案实施例1中测算文库***率的菌落PCR扩增产物的琼脂糖凝胶电泳图，其中，M：DNA marker 2000；泳道1-8：挑取的8个菌落；Figure 4 is an agarose gel electrophoresis image of the colony PCR amplification product for measuring the library insertion rate in Example 1 of the present scheme, wherein M: DNA marker 2000; Swimming lanes 1-8: 8 colonies picked;
图5是本方案实施例2中随天数变化治疗组与对照组恒河猴呼吸道中的病毒载量变化图。Fig. 5 is a graph showing the change of viral load in the respiratory tract of rhesus monkeys in the treatment group and the control group with the change of days in Example 2 of the present scheme.
本发明的实施方式Embodiments of the present invention
定义definition
除非另有指示或定义，否则所有所用术语均具有本领域中的通常含义，该含义将为本领域技术人员所了解。此外，除非另有说明，否则未具体详述的所有方法、步骤、技术及操作均可以且已经以本身已知的方式进行，该方式将为本领域技术人员所了解。Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art, as would be understood by those skilled in the art. Furthermore, unless otherwise indicated, all methods, steps, techniques, and operations not specifically recited can and have been performed in manners known per se, which would be understood by those skilled in the art.
如本文所用的术语“免疫球蛋白单一可变结构域”是指基本上由本领域所称的“框架区1”或“FR1”、“框架区2”或“FR2”、“框架区3”或“FR3”、及“框架区4”或“FR4”的四个“框架区”组成的免疫球蛋白结构域，其中所述框架区由本领域所称的“互补决定区1”或“CDR1”、“互补决定区2”或“CDR2”、及“互补决定区3”或“CDR3”的三个“互补决定区”或“CDR”间隔开。因此，免疫球蛋白单一可变结构域的一般结构或序列可如下表示为：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。免疫球蛋白单一可变结构域因具有抗原结合位点而赋予抗体对抗原的特异性。The term "immunoglobulin single variable domain" as used herein refers to substantially consisting of what are known in the art as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or An immunoglobulin domain consisting of "FR3", and four "framework regions" of "framework region 4" or "FR4", wherein the framework region is composed of "complementarity determining region 1" or "CDR1", which is called in the art, The three "complementarity determining regions" or "CDRs" of "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3" are spaced apart. Thus, the general structure or sequence of an immunoglobulin single variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Immunoglobulin single variable domains confer antigen specificity to antibodies by having an antigen-binding site.
传统IgG抗体分子一般由轻链和重链组成，轻链包含1个可变区(VL)和1个恒定区(CL)，重链包含1个可变区(VH)和3个恒定区(CH1，CH2，CH3)。单域抗体(Single domain antibody，sdAb)，是指缺失抗体轻链而只有重链可变区的一类抗体，因其分子量小，也被称为纳米抗体(Nanobody)。单域抗体特异性结合表位而无需其他抗原结合结构域。单域抗体为由免疫球蛋白单一可变结构域形成的小型稳定及高效的抗原识别单元。Traditional IgG antibody molecules are generally composed of light chain and heavy chain, the light chain contains 1 variable region (VL) and 1 constant region (CL), and the heavy chain contains 1 variable region (VH) and 3 constant regions ( CH1, CH2, CH3). Single domain antibody (Single domain antibody, sdAb) refers to a type of antibody that lacks the light chain of the antibody and only has the variable region of the heavy chain. Because of its small molecular weight, it is also called Nanobody (Nanobody). Single-domain antibodies specifically bind epitopes without the need for additional antigen-binding domains. Single-domain antibodies are small, stable and efficient antigen-recognition units formed from a single variable domain of immunoglobulins.
本领域公知对于单域抗体中的各CDR中的氨基酸残基的总数可能不同。It is well known in the art that the total number of amino acid residues in each CDR in a single domain antibody may vary.
单域抗体中的氨基酸残基的总数将通常在110至120范围内，常常介于112与115之间。然而应注意较小及较长序列也可适于本方案所述的目的。The total number of amino acid residues in a single domain antibody will typically range from 110 to 120, often between 112 and 115. It should be noted, however, that smaller and longer sequences may also be suitable for the purposes described in this protocol.
获得结合特定抗原或表位的单域抗体的方法，先前已公开于以下文献中：R.van derLinden et al.,Journal of Immunological Methods,240(2000)185–195；Li et al.,JBiol Chem .,287(2012)13713–13721；Deffar et al .,African Journal of Biotechnology Vol.8(12),pp.2645-2652,17June,2009和WO94/04678。Methods for obtaining single-domain antibodies that bind specific antigens or epitopes have been previously disclosed in: R. van der Linden et al., Journal of Immunological Methods, 240 (2000) 185-195; Li et al., JBiol Chem ., 287 (2012) 13713–13721; Deffar et al., African Journal of Biotechnology Vol. 8(12), pp. 2645-2652, 17 June, 2009 and WO 94/04678.
此外，本领域技术人员还将了解，有可能将一个或多个上述CDR“移植”于其他“支架”(包括但不限于人支架或非免疫球蛋白支架)上。适于所述CDR移植的支架及技术在本领域中是已知的。In addition, those skilled in the art will also appreciate that it is possible to "graft" one or more of the above-described CDRs onto other "scaffolds" including, but not limited to, human scaffolds or non-immunoglobulin scaffolds. Scaffolds and techniques suitable for such CDR transplantation are known in the art.
一般而言，术语“特异性”是指特定抗原结合分子或抗原结合蛋白(例如本方案的免疫球蛋白单一可变结构域)分子可结合的不同类型抗原或表位的数目。可基于抗原结合分子的亲和力和/或亲合力确定其特异性。由抗原与抗原结合蛋白的解离平衡常数(KD)所表示的亲和力，是表位与抗原结合蛋白上抗原结合位点之间结合强度的量度：KD值越小，表位与抗原结合分子之间的结合强度越强(或者，亲和力也可表示为缔合常数(KA)，其为1/KD)。如本领域技术人员将了解，取决于具体感兴趣的抗原，可以以已知方式测定亲和力。亲合力为抗原结合分子(例如免疫球蛋白、抗体、免疫球蛋白单一可变结构域或含有其的多肽)与相关抗原之间结合强度的量度。亲合力与以下两者有关：与其抗原结合分子上的抗原结合位点之间的亲和力，以及存在于抗原结合分子上的相关结合位点的数目。In general, the term "specificity" refers to the number of different types of antigens or epitopes that a particular antigen-binding molecule or antigen-binding protein (eg, immunoglobulin single variable domain of this scheme) molecule can bind. The specificity of an antigen-binding molecule can be determined based on its affinity and/or avidity. The affinity, represented by the dissociation equilibrium constant (KD) of an antigen and an antigen-binding protein, is a measure of the binding strength between an epitope and an antigen-binding site on an antigen-binding protein: the smaller the KD value, the greater the affinity between the epitope and the antigen-binding molecule. (Alternatively, affinity can also be expressed as an association constant (KA), which is 1/KD). As will be appreciated by those skilled in the art, affinity can be determined in a known manner depending on the particular antigen of interest. Affinity is a measure of the strength of binding between an antigen-binding molecule (eg, an immunoglobulin, antibody, immunoglobulin single variable domain, or polypeptide containing the same) and a relevant antigen. Avidity is related to both: the affinity between its antigen-binding site on the antigen-binding molecule, and the number of associated binding sites present on the antigen-binding molecule.
本方案所用术语“SARS-COV-2刺突蛋白结合分子（SARS-COV-2-Spike蛋白结合分子）”意指任何能够特异性结合SARS-COV-2刺突蛋白的分子。SARS-COV-2刺突蛋白结合分子可以包括针对SARS-COV-2刺突蛋白的如本方案定义的单域抗体或其缀合物。SARS-COV-2刺突蛋白结合分子还涵盖所谓的“SMIP”(“小模块免疫药物”)，或者免疫球蛋白超家族抗体(IgSF)或CDR移植分子。The term "SARS-COV-2 spike protein binding molecule (SARS-COV-2-Spike protein binding molecule)" as used in this protocol means any molecule that can specifically bind to the SARS-COV-2 spike protein. The SARS-COV-2 spike protein binding molecule may comprise a single domain antibody as defined in this protocol, or a conjugate thereof, directed against the SARS-COV-2 spike protein. SARS-COV-2 spike protein binding molecules also encompass so-called "SMIPs" ("small modular immunopharmaceuticals"), or immunoglobulin superfamily antibodies (IgSF) or CDR grafting molecules.
本方案的“SARS-COV-2刺突蛋白结合分子”可以包含至少一个结合SARS-COV-2刺突蛋白的免疫球蛋白单一可变结构域如单域抗体。在一些实施方案中，本方案的“SARS-COV-2刺突蛋白结合分子”可以包含两个结合SARS-COV-2刺突蛋白的免疫球蛋白单一可变结构域如单域抗体。含有一个以上的免疫球蛋白单一可变结构域的SARS-COV-2刺突蛋白结合分子亦称为“格式化的”SARS-COV-2刺突蛋白结合分子。格式化的SARS-COV-2刺突蛋白结合分子除结合SARS-COV-2刺突蛋白的免疫球蛋白单一可变结构域外也可包含接头和/或具有效应器功能的部分，例如半衰期延长部分(如结合血清白蛋白的免疫球蛋白单一可变结构域)、和/或融合配偶体(如血清白蛋白)和/或缀合的聚合物(如PEG)和/或Fc区。本方案的“SARS-COV-2刺突蛋白结合分子”还涵盖双特异性抗体，其含有结合不同抗原的免疫球蛋白单一可变结构域。The "SARS-COV-2 spike protein binding molecule" of the present scheme may comprise at least one immunoglobulin single variable domain such as a single domain antibody that binds to the SARS-COV-2 spike protein. In some embodiments, the "SARS-COV-2 spike protein binding molecule" of the present scheme may comprise two immunoglobulin single variable domains such as single domain antibodies that bind to the SARS-COV-2 spike protein. SARS-COV-2 spike binding molecules containing more than one immunoglobulin single variable domain are also referred to as "formatted" SARS-COV-2 spike binding molecules. Formatted SARS-COV-2 spike protein binding molecules may also contain linkers and/or moieties with effector functions, such as half-life extending moieties, in addition to binding to the immunoglobulin single variable domain of the SARS-COV-2 spike protein (eg, an immunoglobulin single variable domain that binds serum albumin), and/or a fusion partner (eg, serum albumin) and/or a conjugated polymer (eg, PEG) and/or an Fc region. The "SARS-COV-2 spike protein binding molecules" of this protocol also encompass bispecific antibodies, which contain immunoglobulin single variable domains that bind different antigens.
通常，本方案的SARS-COV-2刺突蛋白结合分子将以如于Biacore或KinExA测定中测量的优选10-8至10-12摩尔/升(M)、更优选10-9至10-11摩尔/升、甚至更优选10-10至10-12、甚至更优选10-11 至10-12 或更低的解离常数(KD)。任何大于10-4 M的KD值一般都视为指示非特异性结合。抗原结合蛋白对抗原或表位的特异性结合可以以已知的任何适合方式来测定，包括例如本文所述的表面等离子体共振术(SPR)测定、和/或竞争性结合测定(例如酶免疫测定(EIA)及夹心式竞争性测定。Typically, the SARS-COV-2 spike protein binding molecule of the present protocol will be present at preferably 10-8 to 10-12 moles per liter (M), more preferably 10-9 to 10-11 as measured in a Biacore or KinExA assay moles/liter, even more preferably 10-10 to 10-12, even more preferably 10-11 to 10-12 or lower dissociation constant (KD). Any KD value greater than 10-4 M is generally considered to be indicative of non-specific binding. Specific binding of an antigen-binding protein to an antigen or epitope can be assayed in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays described herein, and/or competitive binding assays (eg, enzyme immunization). Assay (EIA) and Sandwich Competitive Assay.
氨基酸残基将根据如本领域中公知且达成一致的标准三字母或一字母氨基酸编码加以表示。所述保守氨基酸取代在本领域中是公知的，例如保守氨基酸取代优选是以下组(1)-(5)内的一个氨基酸被同一组内的另一氨基酸残基所取代：(1)较小脂族非极性或弱极性残基：Ala、Ser、Thr、Pro及Gly；(2)极性带负电残基及其(不带电)酰胺：Asp、Asn、Glu及Gln；(3)极性带正电残基：His、Arg及Lys；(4)较大脂族非极性残基：Met、Leu、Ile、Val及Cys；及(5)芳族残基：Phe、Tyr及Trp。特别优选的保守氨基酸取代如下：Ala被Gly或Ser取代；Arg被Lys取代；Asn被Gln或His取代；Asp被Glu取代；Cys被Ser取代；Gln被Asn取代；Glu被Asp取代；Gly被Ala或Pro取代；His被Asn或Gln取代；Ile被Leu或Val取代；Leu被Ile或Val取代；Lys被Arg、Gln或Glu取代；Met被Leu、Tyr或Ile取代；Phe被Met、Leu或Tyr取代；Ser被Thr取代；Thr被Ser取代；Trp被Tyr取代；Tyr被Trp或Phe取代；Val被Ile或Leu取代。Amino acid residues will be represented according to standard three-letter or one-letter amino acid codes as well known and agreed in the art. Said conservative amino acid substitutions are well known in the art, for example conservative amino acid substitutions are preferably one amino acid residue within the following groups (1)-(5) replaced by another amino acid residue within the same group: (1) smaller Aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (2) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (3) Polar positively charged residues: His, Arg and Lys; (4) larger aliphatic non-polar residues: Met, Leu, Ile, Val and Cys; and (5) aromatic residues: Phe, Tyr and Trp. Particularly preferred conservative amino acid substitutions are as follows: Ala by Gly or Ser; Arg by Lys; Asn by Gln or His; Asp by Glu; Cys by Ser; Gln by Asn; Glu by Asp; Gly by Ala or Pro; His by Asn or Gln; Ile by Leu or Val; Leu by Ile or Val; Lys by Arg, Gln or Glu; Met by Leu, Tyr or Ile; Phe by Met, Leu or Tyr Substitution; Ser by Thr; Thr by Ser; Trp by Tyr; Tyr by Trp or Phe; Val by Ile or Leu.
两个多肽序列之间的“序列相同性”指示序列之间相同氨基酸的百分比。用于评价氨基酸或核苷酸之间的序列相同性程度的方法是本领域技术人员已知的。例如，氨基酸序列相同性通常使用序列分析软件来测量。例如，可使用NCBI数据库的BLAST程序来确定相同性。对于序列相同性的确定，可以参见例如：Sequence Analysis in Molecular Biology,von Heinje,G.,Academic Press,1987和Sequence Analysis Primer,Gribskov,M.and Devereux,J.,eds.,M Stockton Press,NewYork,1991。"Sequence identity" between two polypeptide sequences indicates the percentage of identical amino acids between the sequences. Methods for assessing the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, identity can be determined using the BLAST program of the NCBI database. For the determination of sequence identity, see for example: Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.
相比于其天然生物来源和/或获得该多肽或核酸分子的反应介质或培养基，当其已与至少一种在该来源或介质(培养基)中通常与之相关的其他组分(例如另一蛋白/多肽、另一核酸、另一生物组分或大分子或至少一种污染物、杂质或微量组分)分离时，多肽或核酸分子视为“基本上分离的”。特别地，多肽或核酸分子在其已纯化至少2倍、特别是至少10倍、更特别是至少100倍且多达1000倍或1000倍以上时被视为“基本上分离的”。经适合的技术(例如适合色谱技术，如聚丙烯酰胺凝胶电泳)确定，“基本上分离的”多肽或核酸分子优选基本上为均质的。Compared to its natural biological source and/or the reaction medium or medium from which the polypeptide or nucleic acid molecule is obtained, when it has been combined with at least one other component (e.g., A polypeptide or nucleic acid molecule is considered "substantially isolated" when another protein/polypeptide, another nucleic acid, another biological component or macromolecule or at least one contaminant, impurity or minor component) is isolated. In particular, a polypeptide or nucleic acid molecule is considered "substantially isolated" when it has been purified at least 2-fold, in particular at least 10-fold, more particularly at least 100-fold and up to 1000-fold or more. A "substantially isolated" polypeptide or nucleic acid molecule is preferably substantially homogeneous as determined by a suitable technique, eg, a suitable chromatographic technique, such as polyacrylamide gel electrophoresis.
实施例1Example 1
筛选针对SARS-COV-2-Spike蛋白的单域抗体Screening of single domain antibodies against SARS-COV-2-Spike protein
1.1文库的构建1.1 Construction of the library
1.1.1免疫1.1.1 Immunization
用新型冠状病毒的Spike-RBD蛋白免疫羊驼，并分别在第1、2、4、6周免疫，共免疫4次，每次免疫剂量为300ug。Alpacas were immunized with the new coronavirus Spike-RBD protein, and immunized at the 1st, 2nd, 4th, and 6th weeks, a total of 4 times, each immunization dose was 300ug.
1.1.2提取总RNA1.1.2 Extraction of total RNA
取第6周免疫后的羊驼的外周血50ml，分离淋巴细胞，用Trizol提取淋巴细胞的总RNA，采用紫外分光光度计检测提取的RNA的结果为：OD260/280＝1.97，OD260/230＝2.14，说明提取的RNA没有明显降解，纯度较好；总RNA浓度为937.5ng/μL。用提取的总RNA进行琼脂糖凝胶电泳，结果如图1所示，可以看到28S和18S两条条带。Take 50 ml of peripheral blood of the 6th week immunized alpaca, separate lymphocytes, extract the total RNA of lymphocytes with Trizol, and use UV spectrophotometer to detect the extracted RNA. The results are: OD260/280=1.97, OD260/230= 2.14, indicating that the extracted RNA is not significantly degraded and the purity is good; the total RNA concentration is 937.5ng/μL. The extracted total RNA was subjected to agarose gel electrophoresis. The results are shown in Figure 1. Two bands of 28S and 18S can be seen.
1.1.3 RNA反转录1.1.3 RNA reverse transcription
RNA反转录体系如下：The RNA reverse transcription system is as follows:
Step 1：Step 1:
Figure dest_path_image001
Figure dest_path_image001
混匀后，65℃保温5 min，迅速冰浴；After mixing, incubate at 65°C for 5 min, and quickly take an ice bath;
Step 2 Step 2
Figure dest_path_image002
Figure dest_path_image002
混匀后，进行反转录得到cDNA，反转录条件为：42℃，30 min；50℃，15 min ；70℃，15 min。After mixing, reverse transcription was performed to obtain cDNA. The reverse transcription conditions were: 42 °C, 30 min; 50 °C, 15 min; 70 °C, 15 min.
1.1.4 单域抗体（VHH）基因扩增1.1.4 Single domain antibody (VHH) gene amplification
采用巢式PCR扩增VHH基因，方法如下：Nested PCR was used to amplify the VHH gene as follows:
Step1 Step1
Figure dest_path_image003
Figure dest_path_image003
混匀后，进行PCR反应，反应条件：98℃ 10s，50℃ 30s，72℃ 1min，共20个循环。扩增引物的序列为：Primer For-1: 5´-GTCCTGGCTGCTCTTCTACAAGG-3´（SEQ ID NO:108）；Primer Rev-1: 5´-GGTACGTGCTGTTGAACTGTTCC-3´（SEQ ID NO:109）。After mixing, carry out PCR reaction, reaction conditions: 98°C for 10s, 50°C for 30s, 72°C for 1 min, a total of 20 cycles. The sequences of the amplification primers are: Primer For-1: 5'-GTCCTGGCTGCTCTTCTACAAGG-3' (SEQ ID NO: 108); Primer Rev-1: 5'-GGTACGTGCTGTTGAACTGTTCC-3' (SEQ ID NO: 109).
PCR产物经DNA纯化试剂盒纯化浓缩后，进行琼脂糖凝胶电泳，得到的琼脂糖凝胶电泳图如图2所示，采用DNA产物凝胶回收试剂盒回收750bp条带，紫外分光光度计定量，作为Step 2的DNA模板；The PCR product was purified and concentrated by the DNA purification kit, and then subjected to agarose gel electrophoresis. The obtained agarose gel electrophoresis diagram is shown in Figure 2. The DNA product gel recovery kit was used to recover the 750bp band, which was quantified by UV spectrophotometer. , as the DNA template of Step 2;
Step 2Step 2
Figure dest_path_image004
Figure dest_path_image004
混匀后，进行PCR反应，反应条件：98℃ 10s，55℃ 30s，72℃ 30s，共20个循环。扩增引物的序列为：Primer For-2:5´-CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT-3´（SEQ ID NO:110）；Primer Rev-2:5´-GATGTGCAGCTGCAGGAGTCTGGRGGAGG-3´（SEQ ID NO:111）。After mixing, carry out PCR reaction, reaction conditions: 98°C for 10s, 55°C for 30s, 72°C for 30s, a total of 20 cycles. The sequence of the amplification primer is: Primer For-2: 5´-CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT-3´ (SEQ ID NO: 110); Primer Rev-2: 5'-GATGTGCAGCTGCAGGAGTCTGRGGAGG-3' (SEQ ID NO: 111).
得到的PCR产物进行琼脂糖凝胶电泳，琼脂糖凝胶电泳图如图3所示，采用DNA产物凝胶回收试剂盒回收，紫外分光光度计定量。最终得到约500 bp的目的基因（VHH）200μL，浓度为458ng/μL。The obtained PCR product was subjected to agarose gel electrophoresis, and the agarose gel electrophoresis diagram was shown in Figure 3, which was recovered by a DNA product gel recovery kit and quantified by an ultraviolet spectrophotometer. end up with about 500 The target gene (VHH) of bp was 200 μL at a concentration of 458 ng/μL.
1.1.5文库转化1.1.5 Library transformation
将得到的目的基因和载体pHEN1采用SfiI和Not1进行双酶切，将酶切后的目的基因和pHEN1片段采用T4 DNA连接酶连接后，转化至大肠杆菌电穿孔感受态细胞TG1中，构建针对SARS-COV-2-Spike蛋白的单域抗体基因文库，命名为S2-Lib。共转化15次，混合后均匀涂布于6块Ф150 mm的培养皿（含氨苄青霉素的LB固体培养基）中。The obtained target gene and vector pHEN1 were double-enzyme digested with SfiI and Not1, and the digested target gene and pHEN1 fragment were ligated with T4 DNA ligase, then transformed into Escherichia coli electroporation competent cell TG1, and constructed for SARS. - Single domain antibody gene library of COV-2-Spike protein, named S2-Lib. A total of 15 transformations were performed, and after mixing, they were evenly spread on 6 Ф150 mm petri dishes (LB solid medium containing ampicillin).
分别取0.1μL，0.01μL，0.001μL和0.0001μL混合后的转化液均匀涂布于Ф90mm的培养皿（含氨苄青霉素的LB固体培养基），用于文库库容量的计算（以菌落数为30-300的平板为准计数），如表1所示，计算库容量为1.425×10 9cfu。 Take 0.1μL, 0.01μL, 0.001μL and 0.0001μL of the mixed transformation solution and spread them evenly on a Ф90mm petri dish (LB solid medium containing ampicillin) for the calculation of the library capacity (take the number of colonies as 30 -300 plates quasi count), as shown in Table 1, the calculated library capacity is 1.425 × 10 9 cfu.
表1Table 1
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在上述用于计算库容量的培养皿中随机挑选8个菌落，进行菌落PCR，并将PCR产物进行琼脂糖凝胶电泳，测算文库的目的基因***率，琼脂糖凝胶电泳如图4所示，说明文库***率为100%，文库实际库容量为1.425×10 9cfu。 Eight colonies were randomly selected in the above-mentioned petri dish for calculating the library capacity, colony PCR was performed, and the PCR products were subjected to agarose gel electrophoresis to calculate the target gene insertion rate of the library. The agarose gel electrophoresis is shown in Figure 4. , indicating that the insertion rate of the library is 100%, and the actual library capacity of the library is 1.425×10 9 cfu.
菌落PCR体系如下：The colony PCR system is as follows:
Figure dest_path_image006
Figure dest_path_image006
菌落PCR反应条件为：98℃ 10s，50℃ 30s，72℃ 1min，共31个循环。The colony PCR reaction conditions were as follows: 98°C for 10s, 50°C for 30s, and 72°C for 1 min, a total of 31 cycles.
1.1.6文库救援1.1.6 Library Rescue
从上述S2-Lib基因文库中取10-100倍库容量的活细胞进行接种培养，培养至对数期后采用M13K07噬菌体进行救援，救援培养后，离心收集噬菌体，采用PEG-NaCl纯化噬菌体，即得噬菌体展示文库，命名为S2-PDL，滴度为3.5×10 13cfu/mL。可直接用于后续特异性噬菌体的亲和筛选。 Take 10-100 times the library capacity of live cells from the above S2-Lib gene library for inoculation and culture. After culture to log phase, M13K07 phage is used for rescue. After rescue culture, the phage is collected by centrifugation, and the phage is purified by PEG-NaCl, namely A phage display library was obtained, named S2-PDL, with a titer of 3.5×10 13 cfu/mL. It can be directly used for subsequent affinity screening of specific phages.
1.2针对SARS-COV-2-Spike蛋白单域抗体的筛选1.2 Screening of single domain antibodies against SARS-COV-2-Spike protein
用Spike-RBD蛋白（刺突蛋白受体结合区蛋白）3μg/孔包被平板，4℃放置过夜；用1wt%脱脂奶粉室温封闭2h，加入100μl噬菌体(8×10 11tfu，来自1.1.6所构建的噬菌体展示文库S2-PDL)，在室温下作用1h。之后用PBST(PBS中含有0.05vt%吐温20)洗脱5遍，以洗掉不结合的噬菌体；用三乙基胺(100mM)将与Spike-RBD蛋白特异性结合的噬菌体解离下，并感染处于对数期生长的大肠杆菌TG1，产生并纯化噬菌体用于下一轮的筛选。相同筛选过程重复3轮。由此，阳性的克隆被富集，达到了利用噬菌体展示文库筛取抗体库中Spike-RBD蛋白特异抗体的目的。并将获得的阳性的噬菌体进行测序，获得抗体基因序列。 Coat the plate with 3 μg/well of Spike-RBD protein (spike protein receptor binding domain protein), and place it at 4°C overnight; block with 1 wt% nonfat dry milk at room temperature for 2 h, and add 100 μl of bacteriophage (8×10 11 tfu, from 1.1.6 The constructed phage display library S2-PDL) was acted on for 1 h at room temperature. After 5 times of elution with PBST (PBS containing 0.05vt% Tween 20), the unbound phage was washed away; the phage specifically bound to the Spike-RBD protein was dissociated with triethylamine (100 mM), E. coli TG1 in log phase growth was infected, and phages were produced and purified for the next round of screening. The same screening process was repeated for 3 rounds. Thus, the positive clones are enriched, and the purpose of screening Spike-RBD protein-specific antibodies in the antibody library by using the phage display library is achieved. The obtained positive phages were sequenced to obtain antibody gene sequences.
将获得的抗体基因序列分别构建在pcDNA3.4载体上，用HEK-293细胞表达抗体，用proteinA介质纯化收集培养基上清中的抗体。纯化后的抗体与包被Spike-RBD的板孵育进行ELISA测定。获得可特异性结合Spike-RBD蛋白的抗体。The obtained antibody gene sequences were constructed on the pcDNA3.4 vector respectively, the antibody was expressed in HEK-293 cells, and the antibody in the supernatant of the medium was collected by purification with proteinA medium. Purified antibodies were incubated with Spike-RBD-coated plates for ELISA assays. Obtain antibodies that can specifically bind to Spike-RBD protein.
根据序列比对软件Vector NTI分析获得的抗体序列。把CDR1、CDR2、CDR3序列均相同的克隆视为同一抗体株，而CDR序列不同的克隆视为不同抗体株。共获得21个不同的可特异性结合Spike-RBD蛋白的单域抗体株，单域抗体序列如SEQ ID NO:64-84，分别携带SEQ ID NO:1-63中的21组CDR1-3序列，具体如表2所示。The obtained antibody sequences were analyzed according to the sequence alignment software Vector NTI. The clones with the same CDR1, CDR2 and CDR3 sequences were regarded as the same antibody strain, and the clones with different CDR sequences were regarded as different antibody strains. A total of 21 different single-domain antibody strains that can specifically bind to the Spike-RBD protein were obtained. The single-domain antibody sequences are such as SEQ ID NOs: 64-84, which respectively carry 21 groups of CDR1-3 sequences in SEQ ID NOs: 1-63 , as shown in Table 2.
表2Table 2
Figure dest_path_image007
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21个不同的单域抗体株与包被Spike-RBD的板孵育进行ELISA测定，测定的单域抗体与Spike-RBD反应后的复孔中的OD450的值如表3所示。Twenty-one different single-domain antibody strains were incubated with Spike-RBD-coated plates for ELISA assay. The measured OD450 values in duplicate wells after the single-domain antibody reacted with Spike-RBD are shown in Table 3.
表3table 3
Figure dest_path_image008
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其中，空白为不加抗体的复孔中的OD450值。Wherein, the blank is the OD450 value in the duplicate wells without antibody.
由表3中的数据可知单域抗体与Spike-RBD蛋白进行了结合反应。From the data in Table 3, it can be seen that the single domain antibody has a binding reaction with Spike-RBD protein.
1.3针对SARS-COV-2-Spike蛋白的单域抗体的评价鉴定1.3 Evaluation and identification of single domain antibodies against SARS-COV-2-Spike protein
1.3.1单域抗体在宿主菌大肠杆菌中表达、纯化1.3.1 Single domain antibody expression and purification in host bacteria E. coli
将获得的21个不同CDR1-3的单域抗体的基因编码序列分别重组至表达载体PET32b(Novagen，产品号：69016-3)中，并将测序鉴定正确的重组质粒分别转化到表达型宿主菌BL1(DE3)(天根生化科技，产品号：CB105-02)中，将其涂布在含有100μg/mL的氨苄青霉素的LB平板上，37℃过夜。挑选单菌落接种、培养过夜，第二天将过夜菌种转接扩增，37℃摇床培养至OD值达到0.5-1时，加入0.5mM IPTG诱导，28℃摇床培养过夜。第二天，离心收集菌体，并将收集的菌体破碎获得抗体粗提液。然后纯化21株单域抗体蛋白，使其纯度达到90%以上。The obtained gene coding sequences of 21 single-domain antibodies of different CDR1-3 were recombined into the expression vector PET32b (Novagen, product number: 69016-3), and the correct recombinant plasmids identified by sequencing were respectively transformed into the expression host bacteria. BL1(DE3) (Tiangen Biochemical Technology, product number: CB105-02), spread it on LB plates containing 100 μg/mL ampicillin, overnight at 37°C. A single colony was selected for inoculation and cultured overnight. On the second day, the overnight strain was transferred and amplified. When the OD value reached 0.5-1 on a shaker at 37°C, 0.5mM IPTG was added for induction, and the culture was shaken at 28°C overnight. The next day, the cells were collected by centrifugation, and the collected cells were crushed to obtain crude antibody extracts. Then 21 strains of single-domain antibody proteins were purified to a purity of more than 90%.
1.3.2竞争ELISA考察21株SARS-COV-2-Spike蛋白单域抗体对Spike-RBD蛋白与受体ACE2结合的阻断效果1.3.2 Competitive ELISA to investigate the blocking effect of 21 SARS-COV-2-Spike protein single-domain antibodies on the binding of Spike-RBD protein to receptor ACE2
先通过HEK293细胞(pCDNA4，Invitrogen，Cat V86220)表达获得 Spike-RBD蛋白与ACE2蛋白。再利用Thermo公司的Biotinlytion试剂盒，得到生物素化的ACE2蛋白。Spike-RBD protein and ACE2 protein were obtained by expressing in HEK293 cells (pCDNA4, Invitrogen, Cat V86220). Then use the Biotinlytion kit of Thermo Company to obtain the biotinylated ACE2 protein.
用Spike-RBD蛋白0.5μg/孔，4℃过夜包被平板，之后每孔加入100ng的1.3.1纯化所得的单域抗体以及5μg生物素化的ACE2蛋白，并设置对照组，对照组1的孔中不加入单域抗体，对照组2的孔中不加入生物素化的ACE2蛋白，室温下反应2h。之后加入SA-HRP(购自Sigma公司)，室温反应1小时后加入显色液，450nm波长读取吸收值。当样品OD值比对照OD值<0.8时，则认为单域抗体有阻断效果。Coat the plate with Spike-RBD protein 0.5μg/well overnight at 4°C, then add 100ng of the single-domain antibody purified in 1.3.1 and 5μg of biotinylated ACE2 protein to each well, and set up a control group, control group 1 No single domain antibody was added to the wells, and no biotinylated ACE2 protein was added to the wells of control group 2, and the reaction was performed at room temperature for 2 h. Then, SA-HRP (purchased from Sigma) was added, and the color developing solution was added after the reaction at room temperature for 1 hour, and the absorption value was read at a wavelength of 450 nm. When the OD value of the sample is less than the control OD value of <0.8, the single domain antibody is considered to have a blocking effect.
结果如表4所示，21株不同的单域抗体株均表现出对Spike蛋白/ACE2蛋白相互作用的阻断效应。The results are shown in Table 4, 21 different single-domain antibody strains all showed a blocking effect on the interaction of Spike protein/ACE2 protein.
表4Table 4
Figure dest_path_image009
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在生物安全级别为P3的实验室，通过用病毒感染VERO细胞模型，将纯化得到的21株单域抗体分别加入到培养体系中，具体操作为：将10 4/孔VERO细胞加到96孔板，24小时后，用PBS洗细胞2次，将21株单域抗体分别与病毒混合加入96孔板，抗体初始浓度为10μg/mL，再分别2倍稀释10个梯度，5个复孔，37℃孵育2小时，第5天检测VERO细胞是否感染病毒（若细胞不发生病变说明单域抗体具有中和病毒并阻断病毒感染VERO细胞的过程）。 In a laboratory with a biosafety level of P3, by infecting the VERO cell model with the virus, the purified 21 single-domain antibodies were added to the culture system. , 24 hours later, the cells were washed twice with PBS, and the 21 single-domain antibodies were mixed with the virus and added to the 96-well plate. Incubate at ℃ for 2 hours, and detect whether the VERO cells are infected with the virus on the 5th day (if the cells do not change, it means that the single-domain antibody can neutralize the virus and block the process of the virus infecting the VERO cells).
检测结果如表5所示，21株单域抗体在5μg/ml以上的浓度下均可有效阻断病毒感染细胞的过程，且部分抗体在低于0.02μg/ml的浓度下依然可以有效阻断病毒感染细胞的过程。根据表5中得到的IC50（μg/ml）数据说明所获得的21株单域抗体能够阻断病毒感染细胞的过程，为有效的中和抗体。The test results are shown in Table 5. The single-domain antibodies of 21 strains can effectively block the process of virus infection of cells at a concentration of more than 5 μg/ml, and some antibodies can still effectively block at a concentration of less than 0.02 μg/ml. The process by which viruses infect cells. According to the IC50 (μg/ml) data obtained in Table 5, the obtained 21 single-domain antibodies can block the process of virus infection of cells, and are effective neutralizing antibodies.
表5table 5
Figure dest_path_image010
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注：“+”表示可以阻断病毒感染细胞，“-”表示不能阻断病毒感染细胞。Note: "+" means that it can block virus infection of cells, "-" means that it cannot block virus infection of cells.
实施例2Example 2
1.1制备SARS-COV-2-Spike蛋白单域抗体的Fc融合蛋白1.1 Preparation of Fc fusion protein of SARS-COV-2-Spike protein single domain antibody
根据蛋白数据库Uniprot上人免疫球蛋白(IgG1)的恒定区氨基酸序列，得到人IgG1-Fc区氨基酸序列(SEQ ID NO:85)。通过逆转录PCR，从人PBMC总RNA中获得编码人IgG1-Fc的核酸片段（核酸序列如SEQ ID NO:107），再通过overlapping PCR得到SARS-COV-2-Spike蛋白单域抗体与Fc的融合蛋白的编码核酸片段，并重组至载体pCDNA4(Invitrogen，Cat V86220)。According to the amino acid sequence of the constant region of human immunoglobulin (IgG1) on the protein database Uniprot, the amino acid sequence of the human IgG1-Fc region (SEQ ID NO: 85) was obtained. Through reverse transcription PCR, the nucleic acid fragment encoding human IgG1-Fc (nucleic acid sequence such as SEQ ID NO: 107) was obtained from the total RNA of human PBMC, and then the SARS-COV-2-Spike protein single domain antibody and Fc were obtained by overlapping PCR. The nucleic acid fragment encoding the fusion protein was recombined into the vector pCDNA4 (Invitrogen, Cat V86220).
将构建成功的含有SARS-COV-2-Spike蛋白单域抗体与Fc的融合蛋白的核酸片段的pCDNA4质粒转染HEK293细胞进行表达。具体是将重组表达质粒用Freestyle293培养基稀释并加入转化所需PEI(Polyethylenimine)溶液，将质粒/PEI混合物分别加入HEK293细胞悬液中，放置在37℃，10%的CO 2，100rpm的摇床中培养；补加50μg/L IGF-1。4h后补加EX293培养基、2mM谷氨酰胺和50μg/L IGF-1，120rpm摇培。24h后加3.8mM VPA。培养5天后，收集表达培养上清液，通过Protein A亲和层析法，纯化得到SARS-COV-2-Spike蛋白单域抗体与Fc的融合蛋白。获得的21个SARS-COV-2-Spike蛋白单域抗体与Fc的融合蛋白的序列如SEQ ID NO:86-106。 The successfully constructed pCDNA4 plasmid containing the nucleic acid fragment of the fusion protein of SARS-COV-2-Spike protein single domain antibody and Fc was transfected into HEK293 cells for expression. Specifically, the recombinant expression plasmid was diluted with Freestyle293 medium, and the PEI (Polyethylenimine) solution required for transformation was added. The plasmid/PEI mixture was added to the HEK293 cell suspension, and placed at 37°C, 10% CO 2 , and a shaker at 100 rpm. cultured in medium; supplemented with 50 μg/L IGF-1. After 4 h, supplemented with EX293 medium, 2 mM glutamine and 50 μg/L IGF-1, and cultured with shaking at 120 rpm. 3.8mM VPA was added after 24h. After culturing for 5 days, the expression culture supernatant was collected and purified by Protein A affinity chromatography to obtain a fusion protein of SARS-COV-2-Spike protein single domain antibody and Fc. The sequences of the obtained fusion proteins of 21 SARS-COV-2-Spike protein single-domain antibodies and Fc are shown in SEQ ID NOs: 86-106.
1.2鉴定SARS-COV-2-Spike蛋白单域抗体与Fc的融合蛋白（SEQ ID NO:86-106）的功能1.2 Identify the function of the fusion protein of SARS-COV-2-Spike protein single domain antibody and Fc (SEQ ID NO: 86-106)
通过SPR法鉴定SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白对SARS-COV-2-Spike蛋白的结合能力。具体操作是：将获得的21株SARS-COV-2-Spike蛋白单域抗体与Fc的融合蛋白针对spike-RBD的结合动力学通过表面等离振子共振(SRP)方法，使用BIAcoreX100仪器测量，将spike-RBD蛋白直接包被于CM5生物传感器芯片上以获得大约1000应答单位(response units，RU)。对于动力学测量，将SARS-COV-2-Spike蛋白单域抗体与Fc的融合蛋白用HBS-EP+1×缓冲液(GE，cat#BR-1006-69)三倍连续稀释(1.37nm至1000nm)，在25℃进样120s，解离时间为30min，加入10mM甘氨酸-HCl(pH2.0)再生120s。使用简单一对一Languir结合模型(BIAcore Evaluation Software version 3.2)计算出融合蛋白与SARS-COV-2-Spike蛋白的结合速率(kon)、解离速率(koff)和平衡解离常数(kD)（以比率koff/kon 计算）。计算结果如表6所示。The binding ability of SARS-COV-2-Spike protein single domain antibody and Fc fusion protein to SARS-COV-2-Spike protein was identified by SPR method. The specific operation is as follows: the binding kinetics of the obtained 21 SARS-COV-2-Spike protein single-domain antibodies and Fc fusion proteins to spike-RBD are measured by surface plasmon resonance (SRP) method using a BIAcoreX100 instrument. The spike-RBD protein was directly coated on the CM5 biosensor chip to obtain approximately 1000 response units (RU). For kinetic measurements, the fusion protein of SARS-COV-2-Spike protein single domain antibody and Fc was three-fold serially diluted (1.37 nm to 1000 nm), injected at 25°C for 120 s, dissociated for 30 min, and regenerated by adding 10 mM glycine-HCl (pH 2.0) for 120 s. Using a simple one-to-one Languir binding model (BIAcore Evaluation Software version 3.2) Calculate the on-rate (kon), dissociation rate (koff) and equilibrium dissociation constant (kD) of the fusion protein with the SARS-COV-2-Spike protein (in the ratio koff/kon calculate). The calculation results are shown in Table 6.
表6Table 6
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由表6可知，SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白对SARS-COV-2-Spike蛋白的结合速率较高的，解离速率较低，平衡解离常数KD小于1.58E-10，说明融合蛋白能更快速的结合SARS-COV-2-Spike蛋白并很难解离下来，进一步说明SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白作为一个阻断型抗体，具有极佳的阻断效果。It can be seen from Table 6 that the binding rate of SARS-COV-2-Spike protein single domain antibody and Fc fusion protein to SARS-COV-2-Spike protein is higher, the dissociation rate is lower, and the equilibrium dissociation constant KD is less than 1.58E -10, indicating that the fusion protein can bind the SARS-COV-2-Spike protein more quickly and is difficult to dissociate, further indicating that the SARS-COV-2-Spike protein single domain antibody and the Fc fusion protein are a blocking antibody, Has an excellent blocking effect.
1.3通过竞争ELISA法鉴定SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白对Spike蛋白/ACE2的相互作用的阻断能力1.3 Identification of the ability of SARS-COV-2-Spike protein single domain antibody and Fc fusion protein to block the interaction of Spike protein/ACE2 by competitive ELISA
利用HEK293细胞表达获得ACE2蛋白。利用Thermo公司的Biotinlytion试剂盒，得到生物素化的蛋白ACE2-Biotin。ACE2 protein was obtained by HEK293 cell expression. The biotinylated protein ACE2-Biotin was obtained by using the Biotinlytion kit of Thermo Company.
用Spike-RBD蛋白0.5μg/孔4℃过夜包被平板，之后每孔加入获得的21株SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白200ng和ACE2-Biotin 5ug，对照组1中不加入融合蛋白，对照组2中不加入ACE2-Biotin，室温下反应2h。洗涤之后加入SA-HRP(购自Sigma公司)，室温反应1小时，洗涤之后加入显色液，450nm波长读取吸收值。结果如表7所示。The plate was coated with 0.5 μg/well of Spike-RBD protein at 4°C overnight, and then 21 strains of SARS-COV-2-Spike protein single domain antibody and Fc fusion protein 200ng and ACE2-Biotin 5ug were added to each well. Control group 1 No fusion protein was added, and ACE2-Biotin was not added to control group 2, and the reaction was carried out at room temperature for 2 h. After washing, SA-HRP (purchased from Sigma) was added, and the reaction was performed at room temperature for 1 hour. After washing, a color developing solution was added, and the absorption value was read at a wavelength of 450 nm. The results are shown in Table 7.
表7Table 7
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表7中的结果显示，SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白能有效阻断Spike蛋白与ACE2之间的相互作用。The results in Table 7 show that SARS-COV-2-Spike protein single domain antibody and Fc fusion protein can effectively block the interaction between Spike protein and ACE2.
1.4分析SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白对Spike蛋白结合的特异性1.4 Analysis of the specificity of SARS-COV-2-Spike protein single domain antibody and Fc fusion protein binding to Spike protein
利用人HEK293细胞通过瞬时转染，获得带有目前已知的7种冠状病毒（SARS-COV-2、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、MERS-CoV）Spike蛋白全长基因的质粒(pCDNA4,Invitrogen，Cat V86220)，并于膜上瞬时表达Spike蛋白。该质粒使得Spike蛋白C端融合EGFP蛋白，从而可以通过绿色荧光强度来考察膜上Spike蛋白的表达水平。将构建好的细胞重悬于0.5%的PBS-BSA Buffer中，加入SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白，同时设置阴性对照，冰上孵育20min。洗涤后加入eBioscience二抗anti-hIg-PE，冰上20min。洗涤后将细胞重悬于500μl的0.5％PBS-BSABuffer中，流式细胞仪进行检测。结果显示，21株SARS-COV-2-Spike蛋白单域抗体-Fc融合蛋白只特异性结合SARS-COV-2-Spike蛋白，而不与其他冠状病毒的Spike蛋白结合。Transient transfection of human HEK293 cells was used to obtain seven currently known coronaviruses (SARS-COV-2, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV ) plasmid of the full-length gene of Spike protein (pCDNA4, Invitrogen, Cat V86220) and transiently express Spike protein on membrane. This plasmid makes the C-terminal of Spike protein fused with EGFP protein, so that the expression level of Spike protein on the membrane can be examined by the green fluorescence intensity. The constructed cells were resuspended in 0.5% PBS-BSA Buffer, SARS-COV-2-Spike protein single domain antibody and Fc fusion protein were added, and a negative control was set at the same time, and incubated on ice for 20 min. After washing, eBioscience secondary antibody anti-hIg-PE was added and kept on ice for 20 min. After washing, the cells were resuspended in 500 μl of 0.5% PBS-BSABuffer and detected by flow cytometry. The results showed that the 21 SARS-COV-2-Spike protein single-domain antibody-Fc fusion proteins only specifically bound to the SARS-COV-2-Spike protein, but not to the Spike proteins of other coronaviruses.
1.5SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白阻断SARS-COV-2感染恒河猴1.5 SARS-COV-2-Spike protein single domain antibody and Fc fusion protein block SARS-COV-2 infection in rhesus monkeys
将感染SARS-COV-2病毒并出现症状的12只恒河猴中的6只（治疗组）利用本方案提供的SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白（SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白100μg/恒河猴）进行给药治疗，另外6只（对照组）不进行给药治疗。在治疗后的6天内，每天进行一次呼吸道病毒载量的检测。经检测，治疗组的6只恒河猴呼吸道中新冠病毒的平均载量相比对照组显著降低，如图5所示。继续对治疗组的6只恒河猴进行观察，每隔一周对其症状和呼吸道病毒载量进行检测。持续观察2周后，检测不出其呼吸道中的病毒载量，也未出现相应的发病症状。持续观察3个月，治疗组的6只恒河猴体内的病毒均无复发的情况，说明上述SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白可在体内发挥长效作用，且可以避免感染新型冠状病毒的活体治愈后出现复阳。其中新冠病毒的载量的检测过程为：分别采取给药治疗的恒河猴（治疗组）和未进行给药治疗的恒河猴（对照组）的咽拭子，提取咽拭子中的病毒的核酸进行检测，检测过程为：采用RNA提取试剂盒（Qiagen）按照说明书操作提取SARS-COV-2的RNA，将获得的RNA溶解在50μL洗脱buffer并作为模板进行RT-PCR扩增。用引物RBD-qF1（5´-CAATGGTTTAACAGGCACAGG-3´，SEQ ID NO:112）和RBD-qR1（5´-CTCAAGTGTCTGTGGATCACG-3´，SEQ ID NO:113）扩增病毒S区基因。采用HiScriptR II One Step qRT-PCR SYBRRGreen Kit (Vazyme Biotech Co.,Ltd)试剂盒，根据试剂盒说明书进行操作，设置PCR扩增条件为；50℃ 3min，95℃ 10s，60℃ 30s，40个循环，所用PCR扩增仪为ABI定量PCR仪。Six of the 12 rhesus monkeys (the treatment group) infected with SARS-COV-2 virus and showing symptoms were treated with the SARS-COV-2-Spike protein single domain antibody and Fc fusion protein (SARS-COV- 2-Spike protein single domain antibody and Fc fusion protein 100μg/rhesus monkey) were administered, and the other 6 (control group) were not administered. Respiratory viral load testing was performed once a day for 6 days after treatment. After testing, the average load of 2019-nCoV in the respiratory tract of 6 rhesus monkeys in the treatment group was significantly lower than that in the control group, as shown in Figure 5. The 6 rhesus monkeys in the treatment group continued to be observed and tested for symptoms and respiratory viral load every other week. After 2 weeks of continuous observation, the viral load in the respiratory tract could not be detected, and the corresponding symptoms did not appear. The 6 rhesus monkeys in the treatment group were observed for 3 months without recurrence of the virus, indicating that the above-mentioned SARS-COV-2-Spike protein single domain antibody and Fc fusion protein can play a long-term effect in vivo, and can Avoid re-positive after the recovery of the living body infected with the new coronavirus. The detection process of the new coronavirus load is as follows: take the throat swabs of the rhesus monkeys treated with drug treatment (treatment group) and the rhesus monkeys without drug treatment (control group), and extract the virus in the throat swabs. The detection process is as follows: RNA extraction kit (Qiagen) is used to extract SARS-COV-2 RNA according to the instructions, and the obtained RNA is dissolved in 50 μL elution buffer and used as a template for RT-PCR amplification. The viral S-region gene was amplified with primers RBD-qF1 (5´-CAATGGTTTAACAGGCACAGG-3´, SEQ ID NO: 112) and RBD-qR1 (5´-CTCAAGTGTCTGTGGATCACG-3´, SEQ ID NO: 113). Adopt HiScriptR II One Step qRT-PCR SYBRR Green Kit (Vazyme Biotech Co., Ltd) kit, operate according to the kit instructions, set the PCR amplification conditions as: 50°C 3min, 95°C 10s, 60°C 30s, 40 cycles, the PCR amplification used The instrument is an ABI quantitative PCR instrument.
上述体内实验结果表明，本方案SARS-COV-2-Spike蛋白单域抗体与Fc融合蛋白在对感染SARS-COV-2的恒河猴体内表现出显著的长效抑制SARS-COV-2感染细胞并扩增的效果，且经过治疗后的恒河猴的复阳率为0。The above in vivo experimental results show that the SARS-COV-2-Spike protein single-domain antibody and Fc fusion protein of this scheme showed significant long-term inhibition of SARS-COV-2 infected cells in rhesus monkeys infected with SARS-COV-2. And the effect of amplification, and the re-positive rate of rhesus monkeys after treatment was 0.
以上所述仅为本方案的较佳实施例而已，并不用以限制本方案，凡在本方案的精神和原则之内所作的任何修改、等同替换或改进等，均应包含在本方案的保护范围之内。The above descriptions are only the preferred embodiments of this scheme, and are not intended to limit this scheme. Any modification, equivalent replacement or improvement made within the spirit and principles of this scheme shall be included in the protection of this scheme. within the range.

Claims (20)

  1. 一种SARS-COV-2刺突蛋白结合分子，其特征在于：能特异性结合SARS-COV-2刺突蛋白且包含至少一个免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域中的CDR1、CDR2和CDR3选自如下组合中的任意一组： A SARS-COV-2 spike protein binding molecule, characterized in that: it can specifically bind to the SARS-COV-2 spike protein and comprises at least one immunoglobulin single variable domain, the immunoglobulin single variable The CDR1, CDR2 and CDR3 in the domain are selected from any one of the following combinations:
    1）SEQ ID NO:1所示的CDR1，SEQ ID NO:2所示的CDR2和SEQ ID NO:3所示的CDR3；1) CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 2 and SEQ ID NO: 2 CDR3 shown in ID NO: 3;
    2）SEQ ID NO:4所示的CDR1，SEQ ID NO:5所示的CDR2和SEQ ID NO:6所示的CDR3；2) CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5 and SEQ ID NO:5 CDR3 shown in ID NO: 6;
    3）SEQ ID NO:7所示的CDR1，SEQ ID NO:8所示的CDR2和SEQ ID NO:9所示的CDR3；3) CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8 and SEQ ID NO:8 CDR3 shown in ID NO: 9;
    4）SEQ ID NO:10所示的CDR1，SEQ ID NO:11所示的CDR2和SEQ ID NO:12所示的CDR3；4) CDR1 shown in SEQ ID NO: 10, CDR2 shown in SEQ ID NO: 11 and SEQ ID NO: 11 CDR3 shown in ID NO: 12;
    5）SEQ ID NO:13所示的CDR1，SEQ ID NO:14所示的CDR2和SEQ ID NO:15所示的CDR3；5) CDR1 shown in SEQ ID NO: 13, CDR2 shown in SEQ ID NO: 14 and SEQ ID NO: 14 CDR3 shown in ID NO: 15;
    6）SEQ ID NO:16所示的CDR1，SEQ ID NO:17所示的CDR2和SEQ ID NO:18所示的CDR3；6) CDR1 shown in SEQ ID NO: 16, CDR2 shown in SEQ ID NO: 17 and SEQ ID NO: 17 CDR3 shown in ID NO: 18;
    7）SEQ ID NO:19所示的CDR1，SEQ ID NO:20所示的CDR2和SEQ ID NO:21所示的CDR3；7) CDR1 shown in SEQ ID NO: 19, CDR2 shown in SEQ ID NO: 20 and SEQ ID NO: 20 CDR3 shown in ID NO: 21;
    8）SEQ ID NO:22所示的CDR1，SEQ ID NO:23所示的CDR2和SEQ ID NO:24所示的CDR3；8) CDR1 shown in SEQ ID NO:22, CDR2 shown in SEQ ID NO:23 and SEQ ID NO:23 CDR3 shown in ID NO: 24;
    9）SEQ ID NO:25所示的CDR1，SEQ ID NO:26所示的CDR2和SEQ ID NO:27所示的CDR3；9) CDR1 shown in SEQ ID NO: 25, CDR2 shown in SEQ ID NO: 26 and SEQ ID NO: 26 CDR3 shown in ID NO: 27;
    10）SEQ ID NO:28所示的CDR1，SEQ ID NO:29所示的CDR2和SEQ ID NO:30所示的CDR3；10) CDR1 shown in SEQ ID NO: 28, CDR2 shown in SEQ ID NO: 29 and SEQ ID NO: 29 CDR3 shown in ID NO: 30;
    11）SEQ ID NO:31所示的CDR1，SEQ ID NO:32所示的CDR2和SEQ ID NO:33所示的CDR3；11) CDR1 shown in SEQ ID NO:31, CDR2 shown in SEQ ID NO:32 and SEQ ID NO:32 CDR3 shown in ID NO: 33;
    12）SEQ ID NO:34所示的CDR1，SEQ ID NO:35所示的CDR2和SEQ ID NO:36所示的CDR3；12) CDR1 shown in SEQ ID NO:34, CDR2 shown in SEQ ID NO:35 and SEQ ID NO:35 CDR3 shown in ID NO: 36;
    13）SEQ ID NO:37所示的CDR1，SEQ ID NO:38所示的CDR2和SEQ ID NO:39所示的CDR3；13) CDR1 shown in SEQ ID NO:37, CDR2 shown in SEQ ID NO:38 and SEQ ID NO:38 CDR3 shown in ID NO: 39;
    14）SEQ ID NO:40所示的CDR1，SEQ ID NO:41所示的CDR2和SEQ ID NO:42所示的CDR3；14) CDR1 shown in SEQ ID NO:40, CDR2 shown in SEQ ID NO:41 and SEQ ID NO:41 CDR3 shown in ID NO: 42;
    15）SEQ ID NO:43所示的CDR1，SEQ ID NO:44所示的CDR2和SEQ ID NO:45所示的CDR3；15) CDR1 shown in SEQ ID NO:43, CDR2 shown in SEQ ID NO:44 and SEQ ID NO:44 CDR3 shown in ID NO: 45;
    16）SEQ ID NO:46所示的CDR1，SEQ ID NO:47所示的CDR2和SEQ ID NO:48所示的CDR3；16) CDR1 shown in SEQ ID NO:46, CDR2 shown in SEQ ID NO:47 and SEQ ID NO:47 CDR3 shown in ID NO: 48;
    17）SEQ ID NO:49所示的CDR1，SEQ ID NO:50所示的CDR2和SEQ ID NO:51所示的CDR3；17) CDR1 shown in SEQ ID NO:49, CDR2 shown in SEQ ID NO:50 and SEQ ID NO:50 CDR3 shown in ID NO: 51;
    18）SEQ ID NO:52所示的CDR1，SEQ ID NO:53所示的CDR2和SEQ ID NO:54所示的CDR3；18) CDR1 shown in SEQ ID NO:52, CDR2 shown in SEQ ID NO:53 and SEQ ID NO:53 CDR3 shown in ID NO: 54;
    19）SEQ ID NO:55所示的CDR1，SEQ ID NO:56所示的CDR2和SEQ ID NO:57所示的CDR3；19) CDR1 shown in SEQ ID NO:55, CDR2 shown in SEQ ID NO:56 and SEQ ID NO:56 CDR3 shown in ID NO: 57;
    20）SEQ ID NO:58所示的CDR1，SEQ ID NO:59所示的CDR2和SEQ ID NO:60所示的CDR3；20) CDR1 shown in SEQ ID NO:58, CDR2 shown in SEQ ID NO:59 and SEQ ID NO:59 CDR3 shown in ID NO: 60;
    21）SEQ ID NO:61所示的CDR1，SEQ ID NO:62所示的CDR2和SEQ ID NO:63所示的CDR3。21) CDR1 shown in SEQ ID NO:61, CDR2 shown in SEQ ID NO:62 and SEQ ID NO:62 CDR3 shown in ID NO:63.
  2. 如权利要求1所述的SARS-COV-2刺突蛋白结合分子，其特征在于：所述免疫球蛋白单一可变域为单域抗体。 The SARS-COV-2 spike protein binding molecule of claim 1, wherein the immunoglobulin single variable domain is a single domain antibody.
  3. 如权利要求2所述的SARS-COV-2刺突蛋白结合分子，其特征在于：所述单域抗体包含与SEQ ID NO:64-84中任一序列具有至少80％的序列相同性的氨基酸序列。 The SARS-COV-2 spike protein binding molecule of claim 2, wherein the single domain antibody comprises an amino acid having at least 80% sequence identity with any sequence in SEQ ID NOs: 64-84 sequence.
  4. 如权利要求2所述的SARS-COV-2刺突蛋白结合分子，其特征在于：所述单域抗体包含与SEQ ID NO:64-84中任一序列具有至少90％的序列相同性的氨基酸序列。 The SARS-COV-2 spike protein binding molecule of claim 2, wherein the single-domain antibody comprises an amino acid having at least 90% sequence identity with any sequence in SEQ ID NOs: 64-84 sequence.
  5. 如权利要求2所述的SARS-COV-2刺突蛋白结合分子，其特征在于：所述单域抗体包含与SEQ ID NO:64-84中任一序列具有至少99％的序列相同性的氨基酸序列。 The SARS-COV-2 spike protein binding molecule of claim 2, wherein the single domain antibody comprises an amino acid having at least 99% sequence identity with any sequence in SEQ ID NOs: 64-84 sequence.
  6. 如权利要求2所述的SARS-COV-2刺突蛋白结合分子，其特征在于：所述单域抗体包含SEQ ID NO:64-84中的任意一种氨基酸序列。 The SARS-COV-2 spike protein binding molecule of claim 2, wherein the single domain antibody comprises any one of the amino acid sequences of SEQ ID NOs: 64-84.
  7. 如权利要求1-6任一项所述的SARS-COV-2刺突蛋白结合分子，其特征在于：还包含免疫球蛋白Fc区。 The SARS-COV-2 spike protein binding molecule according to any one of claims 1-6, further comprising an immunoglobulin Fc region.
  8. 如权利要求7所述的SARS-COV-2刺突蛋白结合分子，其特征在于：所述免疫球蛋白Fc区是人免疫球蛋白Fc区。 The SARS-COV-2 spike protein binding molecule of claim 7, wherein the immunoglobulin Fc region is a human immunoglobulin Fc region.
  9. 如权利要求8所述的SARS-COV-2刺突蛋白结合分子，其特征在于：所述免疫球蛋白Fc区是人IgG1的Fc区。 The SARS-COV-2 spike protein binding molecule of claim 8, wherein the immunoglobulin Fc region is the Fc region of human IgG1.
  10. 如权利要求9所述的SARS-COV-2刺突蛋白结合分子，其特征在于：所述免疫球蛋白Fc区的氨基酸序列为SEQ ID NO:85。 The SARS-COV-2 spike protein binding molecule of claim 9, wherein the amino acid sequence of the immunoglobulin Fc region is SEQ ID NO:85.
  11. 如权利要求10所述的SARS-COV-2刺突蛋白结合分子，其特征在于：包含SEQ ID NO:86-106中的至少一种氨基酸序列。The SARS-COV-2 spike protein binding molecule of claim 10, characterized in that it comprises at least one amino acid sequence in SEQ ID NOs: 86-106.
  12. 编码权利要求1-11任一项所述的SARS-COV-2刺突蛋白结合分子的核酸分子。A nucleic acid molecule encoding the SARS-COV-2 spike protein binding molecule of any one of claims 1-11.
  13. 包含权利要求12所述的核酸分子及其表达调控原件的表达载体。An expression vector comprising the nucleic acid molecule of claim 12 and an expression control element thereof.
  14. 包含权利要求12所述的核酸分子并进行表达的宿主细胞。A host cell comprising and expressing the nucleic acid molecule of claim 12.
  15. 获得权利要求1-11中任一项所述的SARS-COV-2刺突蛋白结合分子的方法，其特征在于，包括：The method for obtaining the SARS-COV-2 spike protein binding molecule described in any one of claims 1-11, characterized in that, comprising:
    a、在允许所述SARS-COV-2刺突蛋白结合分子表达的条件下培养权利要求14所述的宿主细胞；a, culturing the host cell of claim 14 under conditions that allow the expression of the SARS-COV-2 spike protein binding molecule;
    b、从步骤a的培养物中收集由所述宿主细胞表达的SARS-COV-2刺突蛋白结合分子。b. The SARS-COV-2 spike protein binding molecule expressed by the host cell is collected from the culture in step a.
  16. 一种免疫缀合物，其特征在于，包含与治疗性部分缀合的权利要求1-11任一项所述的SARS-COV-2刺突蛋白结合分子。An immunoconjugate, characterized by comprising the SARS-COV-2 spike protein binding molecule of any one of claims 1-11 conjugated to a therapeutic moiety.
  17. 一种药物组合物，其特征在于，包含权利要求1-11任一项所述的SARS-COV-2刺突蛋白结合分子和/或权利要求16所述的免疫缀合物，以及药学上可接受的载体。A pharmaceutical composition, characterized in that, comprising the SARS-COV-2 spike protein binding molecule described in any one of claims 1-11 and/or the immunoconjugate described in claim 16, and a pharmaceutically acceptable accepted vector.
  18. 权利要求17所述的药物组合物在制备治疗或预防新型冠状病毒病肺炎药物中的应用。The application of the pharmaceutical composition of claim 17 in the preparation of a medicine for treating or preventing novel coronavirus disease pneumonia.
  19. 一种用于检测SARS-COV-2的试剂盒，其特征在于，包含权利要求1-11任一项所述的SARS-COV-2刺突蛋白结合分子。A test kit for detecting SARS-COV-2, characterized in that it comprises the SARS-COV-2 spike protein binding molecule described in any one of claims 1-11.
  20. 权利要求19所述的试剂盒的非诊断目的的使用方法，其特征在于，在权利要求1-11任一项所述的SARS-COV-2刺突蛋白结合分子与SARS-COV-2刺突蛋白之间能够形成复合物的条件下，使检测样品和对照样品接触权利要求1-11任一项所述的SARS-COV-2刺突蛋白结合分子，检测复合物的形成；通过所述检测样品与对照样品之间复合物形成的差异判断样品中SARS-COV-2的存在。 The non-diagnostic use method of the test kit of claim 19, wherein the SARS-COV-2 spike protein binding molecule described in any one of claims 1-11 and the SARS-COV-2 spike Under the condition that complexes can be formed between the proteins, the detection sample and the control sample are contacted with the SARS-COV-2 spike protein binding molecule described in any one of claims 1-11, and the formation of the complex is detected; Differences in complex formation between samples and control samples judge the presence of SARS-COV-2 in the samples.
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