WO2022061594A1 - Molécule de liaison à la protéine de spicule du sars-cov-2 et son utilisation - Google Patents

Molécule de liaison à la protéine de spicule du sars-cov-2 et son utilisation Download PDF

Info

Publication number
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
Authority
WO
WIPO (PCT)
Prior art keywords
seq
cov
sars
spike protein
binding molecule
Prior art date
Application number
PCT/CN2020/117164
Other languages
English (en)
Chinese (zh)
Inventor
张军方
Original Assignee
深圳市因诺赛生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳市因诺赛生物科技有限公司 filed Critical 深圳市因诺赛生物科技有限公司
Priority to CN202080002271.4A priority Critical patent/CN112513076B/zh
Priority to CN202210089563.7A priority patent/CN114292326B/zh
Priority to CN202111554250.6A priority patent/CN114437206B/zh
Priority to CN202111556186.5A priority patent/CN114276443B/zh
Priority to PCT/CN2020/117164 priority patent/WO2022061594A1/fr
Priority to CN202111556178.0A priority patent/CN114276442B/zh
Priority to CN202111554248.9A priority patent/CN114456260B/zh
Publication of WO2022061594A1 publication Critical patent/WO2022061594A1/fr

Links

Classifications

    • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Communicable Diseases (AREA)
  • Microbiology (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention se rapporte au domaine technique de la biomédecine. En particulier, la présente invention concerne une molécule de liaison à la protéine de spicule du SARS-COV-2 et son utilisation. La molécule de liaison peut se lier de manière spécifique à une protéine de spicule du SARS-COV-2 et contient au moins un domaine variable unique d'immunoglobuline. La molécule de liaison à la protéine de spicule du SARS-COV-2 selon la présente invention peut bloquer de manière efficace la liaison de la protéine de spicule du SARS-COV-2 à un récepteur de l'ACE 2 de cellule humaine, ce qui permet de bloquer le processus d'infection par le SARS-COV-2 sur des cellules et d'inhiber l'infection par le SARS-COV-2 et l'amplification de ce dernier. La molécule de liaison exerce un effet à long terme d'inhibition du SARS-COV-2 in vivo et empêche de manière efficace le SARS-COV-2 d'une récurrence in vivo.
PCT/CN2020/117164 2020-09-23 2020-09-23 Molécule de liaison à la protéine de spicule du sars-cov-2 et son utilisation WO2022061594A1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
CN202080002271.4A CN112513076B (zh) 2020-09-23 2020-09-23 新型冠状病毒(sars-cov-2)刺突蛋白结合分子及其应用
CN202210089563.7A CN114292326B (zh) 2020-09-23 2020-09-23 新型冠状病毒(sars-cov-2)刺突蛋白结合分子及其应用
CN202111554250.6A CN114437206B (zh) 2020-09-23 2020-09-23 新型冠状病毒（sars-cov-2）刺突蛋白结合分子及其应用
CN202111556186.5A CN114276443B (zh) 2020-09-23 2020-09-23 新型冠状病毒（sars-cov-2）刺突蛋白结合分子及其应用
PCT/CN2020/117164 WO2022061594A1 (fr) 2020-09-23 2020-09-23 Molécule de liaison à la protéine de spicule du sars-cov-2 et son utilisation
CN202111556178.0A CN114276442B (zh) 2020-09-23 2020-09-23 新型冠状病毒（sars-cov-2）刺突蛋白结合分子及其应用
CN202111554248.9A CN114456260B (zh) 2020-09-23 2020-09-23 新型冠状病毒（sars-cov-2）刺突蛋白结合分子及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2020/117164 WO2022061594A1 (fr) 2020-09-23 2020-09-23 Molécule de liaison à la protéine de spicule du sars-cov-2 et son utilisation

Publications (1)

Publication Number Publication Date
WO2022061594A1 true WO2022061594A1 (fr) 2022-03-31

Family

ID=74952828

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/117164 WO2022061594A1 (fr) 2020-09-23 2020-09-23 Molécule de liaison à la protéine de spicule du sars-cov-2 et son utilisation

Country Status (2)

Country Link
CN (6) CN114456260B (fr)
WO (1) WO2022061594A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115057938A (zh) * 2022-06-24 2022-09-16 广东菲鹏制药股份有限公司 抗新型冠状病毒人源化多价结合蛋白及其应用

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MX2021004130A (es) 2020-04-02 2021-06-15 Regeneron Pharma Anticuerpos contra glicoproteína de espícula anti-sars-cov-2 y fragmentos de unión al antígeno.
CR20220660A (es) 2020-06-03 2023-02-17 Regeneron Pharma Métodos para tratar o prevenir infecciones por sars-cov2 y covid-19 con anticuerpos de glicoproteína espícular anti-sars-cov-2
CN113388031B (zh) * 2021-08-17 2021-11-09 上海浙江大学高等研究院 单克隆抗体35b5及其制备方法和用途
CN115160434B (zh) * 2022-05-26 2023-05-02 广东菲鹏制药股份有限公司 人源化单域抗体及其应用和药物

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111303279A (zh) * 2020-03-17 2020-06-19 中国医学科学院病原生物学研究所 一种针对新型冠状病毒的单域抗体及其应用
CN111303280A (zh) * 2020-03-22 2020-06-19 中国人民解放军军事科学院军事医学研究院 高中和活性抗SARS-CoV-2全人源单克隆抗体及应用
CN111423508A (zh) * 2020-03-31 2020-07-17 江苏省疾病预防控制中心(江苏省公共卫生研究院) 一种分离的抗病毒感染的SARS-CoV-2蛋白结合分子

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2019286406B2 (en) * 2018-06-12 2023-04-13 Kbio Holdings Limited Virus and antigen purification and conjugation
CN111647077B (zh) * 2020-06-02 2021-02-09 深圳市因诺赛生物科技有限公司 新型冠状病毒(sars-cov-2)刺突蛋白结合分子及其应用
CN117752779A (zh) * 2020-06-15 2024-03-26 睿丰康生物医药科技(浙江)有限公司 一种冠状病毒刺突蛋白包膜替换型载体疫苗及其构建方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111303279A (zh) * 2020-03-17 2020-06-19 中国医学科学院病原生物学研究所 一种针对新型冠状病毒的单域抗体及其应用
CN111303280A (zh) * 2020-03-22 2020-06-19 中国人民解放军军事科学院军事医学研究院 高中和活性抗SARS-CoV-2全人源单克隆抗体及应用
CN111423508A (zh) * 2020-03-31 2020-07-17 江苏省疾病预防控制中心(江苏省公共卫生研究院) 一种分离的抗病毒感染的SARS-CoV-2蛋白结合分子

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
RUI SHI ET AL.: "A Human Neutralizing Antibody Targets the Receptor-Binding Site of SARS-CoV-2", NATURE, vol. 584, no. 7819, 26 May 2020 (2020-05-26), XP037211681, DOI: 10.1038/s41586-020-2381-y *
SETH J. ZOST ET AL.: "Potently neutralizing and protective human antibodies against SARS-CoV-2", NATURE, vol. 584, no. 7821, 15 July 2020 (2020-07-15), XP037223576, DOI: 10.1038/s41586-020-2548-6 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115057938A (zh) * 2022-06-24 2022-09-16 广东菲鹏制药股份有限公司 抗新型冠状病毒人源化多价结合蛋白及其应用
CN115057938B (zh) * 2022-06-24 2023-01-06 广东菲鹏制药股份有限公司 抗新型冠状病毒人源化多价结合蛋白及其应用

Also Published As

Publication number Publication date
CN114276442B (zh) 2023-05-16
CN114276443B (zh) 2023-05-16
CN112513076A (zh) 2021-03-16
CN112513076B (zh) 2022-03-18
CN114276443A (zh) 2022-04-05
CN114437206A (zh) 2022-05-06
CN114292326A (zh) 2022-04-08
CN114456260B (zh) 2023-05-12
CN114276442A (zh) 2022-04-05
CN114292326B (zh) 2023-05-12
CN114437206B (zh) 2023-05-16
CN114456260A (zh) 2022-05-10

Similar Documents

Publication Publication Date Title
US20230331822A1 (en) SARS-COV-2 spike protein binding molecule and application thereof
CN113264998B (zh) 抗新冠病毒SARS-CoV-2表面S1蛋白的单链抗体及其应用
WO2022061594A1 (fr) Molécule de liaison à la protéine de spicule du sars-cov-2 et son utilisation
CN112094343B (zh) 与SARS-CoV-2 RBD结合的羊驼源纳米抗体
CN112390879B (zh) 靶向SARS-CoV-2的抗体及其制备方法和应用
CN113150129B (zh) 抗新冠病毒SARS-CoV-2表面S2蛋白的单链抗体及其应用
AU2019323389B2 (en) Anti-BCMA single domain antibodies and application thereof
WO2017201766A1 (fr) Anticorps monoclonal humanisé anti-pd-1 humain et son utilisation
CN113045647B (zh) 新冠病毒SARS-CoV-2的中和性抗体及其应用
JP6253986B2 (ja) コレクション及びその使用方法
WO2023125520A1 (fr) NANOCORPS DÉRIVÉ DU CHAMEAU À HAUTE AFFINITÉ POUR DES SOUCHES MUTANTES α, β, γ ET δ DE SARS-COV-2
CN111378048A (zh) 针对中东呼吸综合征冠状病毒的抗体-多肽双特异性免疫治疗剂
CN111303286B (zh) 一种anti-CD19的全人源抗体或抗体片段及其嵌合抗原受体和应用
WO2023216623A1 (fr) NANOANTICORPS À HAUTE AFFINITÉ DÉRIVÉ DU CHAMEAU DIRIGÉ CONTRE LES MUTANTS DU SARS-COV-2 α, γ, δ ET ο
US20240150460A1 (en) Anti-nkp30 antibody and application thereof
US20240117012A1 (en) Anti-sars-cov-2 antibody
WO2022067020A1 (fr) Plateforme d'ingénierie d'anticorps acellulaires et anticorps neutralisants contre le sars-cov-2
CN114539395A (zh) SARS-CoV-2野生型毒株和α突变株骆驼源高亲和力纳米抗体
CN114957469B (zh) 一种抗NKp30抗体及其应用
WO2023035226A1 (fr) Anticorps anti-ang2, son procédé de préparation et son utilisation
CN115043940A (zh) 抗人血清白蛋白抗体及其应用
CN116023482A (zh) 靶向冠状病毒的中和抗体、其抗原结合片段及其用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20954448

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20954448

Country of ref document: EP

Kind code of ref document: A1