WO2023040835A1 - 一株羊驼源纳米抗体及其应用 - Google Patents

一株羊驼源纳米抗体及其应用 Download PDF

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WO2023040835A1
WO2023040835A1 PCT/CN2022/118494 CN2022118494W WO2023040835A1 WO 2023040835 A1 WO2023040835 A1 WO 2023040835A1 CN 2022118494 W CN2022118494 W CN 2022118494W WO 2023040835 A1 WO2023040835 A1 WO 2023040835A1
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cov
sars
nanobody
rbd
alpaca
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French (fr)
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王奇慧
高福
刘红辉
韩鹏程
仵丽丽
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中国科学院微生物研究所
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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • 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
    • C07K2317/567—Framework region [FR]
    • 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

Definitions

  • This application relates to the field of biomedicine, specifically to a strain of alpaca-derived nanobody and its application, more specifically, to an alpaca-derived nanobody or its antigen-binding fragment that binds to the SARS-CoV-2 RBD, and its encoding Polynucleotides, nucleic acid constructs comprising the polynucleotides, expression vectors comprising the nucleic acid constructs, preparation methods thereof, transformed cells, and pharmaceutical compositions comprising the above, and their use in the preparation of prevention, treatment and/or detection of new crowns Drug application for viral infections.
  • SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus
  • MERS-CoV Middle East Respiratory Syndrome Coronavirus
  • SARS-CoV Severe Acute Respiratory Syndrome Coronavirus
  • MERS-CoV Middle East Respiratory Syndrome Coronavirus
  • Neutralizing antibodies the drug mainly binds to antigens on the surface of pathogenic microorganisms, preventing specific molecules expressed by pathogenic microorganisms from binding to cell surface receptors to achieve a "neutralizing” effect.
  • SARS-CoV and SARS-CoV-2 viruses have a glycosylated spike protein (S) on the surface, the S protein can interact with the host cell receptor protein ACE2 and trigger membrane fusion, thus blocking the S protein
  • S glycosylated spike protein
  • RNA viruses such as the new coronavirus are prone to mutation and immune escape, and it is difficult for a single specific antibody to meet the long-term treatment needs.
  • conventional monoclonal antibodies also have certain defects in practical applications due to their excessive molecular weight.
  • the purpose of this application is to provide an alpaca-derived Nanobody or its antigen-binding fragment that binds to the SARS-CoV-2 RBD, a polynucleotide encoding it, a nucleic acid construct comprising the polynucleotide, a nucleic acid construct comprising the The expression vector, its preparation method, transformed cells, and the pharmaceutical composition containing the above, and their application in the preparation of drugs for preventing or treating the new coronavirus.
  • the alpaca-derived nanobody or its antigen-binding fragment of the application is a nanobody with high neutralization activity, which has a strong binding ability to the SARS-CoV-2 RBD protein and can effectively inhibit SARS-CoV-2 infection.
  • the nanobody has a small molecular weight ( ⁇ 15kDa), small immunogenicity, better solubility and stability, and longer CDR3 region, can be administered by aerosolization, can reach the lungs, and have faster onset of action, providing new crown or other coronavirus infections potential therapeutic strategies.
  • the application provides an alpaca-derived Nanobody or an antigen-binding fragment thereof that binds to the SARS-CoV-2 RBD, comprising a heavy chain variable region, the heavy chain variable region comprising the following CDRs:
  • the amino acid sequence is CDR1 as shown in SEQ ID NO: 1 (i.e., GFTLDYYAIG),
  • the amino acid sequence is CDR2 as shown in SEQ ID NO: 2 (i.e., CISSSDGSTSYADSVKG), and
  • the amino acid sequence is CDR3 as shown in SEQ ID NO: 3 (i.e., TPATYYSGRYYYQCPAGGMDY).
  • the heavy chain variable region further includes four framework regions FR1-4, and the FR1-4 and the CDR1, CDR2 and CDR3 are arranged alternately in sequence.
  • amino acid sequences of the FR1-4 are as SEQ ID NO:4 (i.e., QVQLQESGGGLVQPGGSLRLSCAVS), SEQ ID NO:5 (i.e., WFRQAPGKEREGVS), SEQ ID NO:6 (i.e., RFTISRDNAKNTVYLQMNSLKPEDTALYYCAA) and SEQ ID NO: 7 (i.e., WGQGTQVTVSS).
  • SEQ ID NO:4 i.e., QVQLQESGGGLVQPGGSLRLSCAVS
  • SEQ ID NO:5 i.e., WFRQAPGKEREGVS
  • SEQ ID NO:6 i.e., RFTISRDNAKNTVYLQMNSLKPEDTALYYCAA
  • SEQ ID NO: 7 i.e., WGQGTQVTVSS
  • amino acid sequence of the heavy chain variable region is shown in the following SEQ ID NO: 8:
  • underlined parts are the framework regions FR1-4 respectively, and the black marked parts are the CDR1, CDR2 and CDR3 of the heavy chain variable region respectively.
  • the present application provides a polynucleotide encoding the alpaca-derived Nanobody or an antigen-binding fragment thereof that binds to the SARS-CoV-2 RBD.
  • polynucleotide is DNA or mRNA.
  • polynucleotide has a nucleotide sequence as shown in SEQ ID NO:9:
  • the present application provides a nucleic acid construct comprising the polynucleotide described above.
  • the polynucleotide further comprises at least one expression regulatory element operably linked to the polynucleotide. Examples include histidine tags, stop codons, etc.
  • the present application provides an expression vector comprising the nucleic acid construct described above.
  • the present application provides a transformed cell comprising the polynucleotide as described in the second aspect above, the nucleic acid construct as described in the third aspect above or the expression vector as described in the fourth aspect above .
  • the present application provides a pharmaceutical composition, which comprises the alpaca-derived Nanobody or its antigen-binding fragment that binds to the SARS-CoV-2 RBD as described in the first aspect above, as described in the second aspect above.
  • a pharmaceutical composition which comprises the alpaca-derived Nanobody or its antigen-binding fragment that binds to the SARS-CoV-2 RBD as described in the first aspect above, as described in the second aspect above.
  • the pharmaceutical composition is in the form of nasal spray, oral formulation, suppository or parenteral formulation.
  • the nasal spray is selected from aerosol, spray and powder spray.
  • the oral preparation is selected from tablet, powder, pill, powder, granule, fine granule, soft/hard capsule, film-coated agent, pellet, sublingual tablet and ointment.
  • the parenteral preparation is a transdermal preparation, an ointment, a plaster, a liquid for external use, an injectable or a pushable preparation.
  • the present application provides an alpaca-derived nanobody or an antigen-binding fragment thereof that binds to the SARS-CoV-2 RBD as described in the first aspect above, the polynucleotide as described in the second aspect above,
  • the nucleic acid construct as described in the third aspect above, the expression vector as described in the fourth aspect above, or the transformed cell as described in the fifth aspect above, or the pharmaceutical composition as described in the sixth aspect above is used in the preparation of prophylaxis, Application in drugs for the treatment and/or detection of novel coronavirus infection.
  • the novel coronavirus is an original strain of SARS-CoV-2 and/or a mutant strain of SARS-CoV-2.
  • the mutant strain of SARS-CoV-2 is Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1) and/or Delta (B.1.617.2) strain.
  • the present application provides a method for preventing or treating a new coronavirus infection, which includes: administering a preventive or therapeutically effective amount of the SARS-CoV-2 as described in the first aspect above to a subject in need
  • a preventive or therapeutically effective amount of the SARS-CoV-2 as described in the first aspect above to a subject in need
  • the novel coronavirus is an original strain of SARS-CoV-2 and/or a mutant strain of SARS-CoV-2.
  • the mutant strain of SARS-CoV-2 is Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1) and/or Delta (B.1.617.2) strain.
  • the dosage of the active ingredients of the pharmaceutical composition of the present application varies according to the administration object, object organs, symptoms, administration methods, etc., and the type of dosage form, administration method, age and body weight of the patient, etc. may be considered.
  • the patient's symptoms and the like are determined according to the doctor's judgment.
  • the present application provides a method for detecting the new coronavirus, which includes using the alpaca-derived Nanobody or its antigen-binding fragment that binds to the SARS-CoV-2 RBD as described in the first aspect above.
  • the novel coronavirus is an original strain of SARS-CoV-2 and/or a mutant strain of SARS-CoV-2.
  • the mutant strain of SARS-CoV-2 is Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617.1) and/or Delta (B.1.617.2) strain.
  • This application is aimed at the development of nano-antibody drugs for the new coronavirus, through simultaneous immunization of alpacas with SARS-CoV-2 RBD protein and SARS-CoV-2 NTD protein, construction of antibody libraries, and screening of specific nano-antibodies using phage display technology.
  • -CoV-2 RBD protein was used as "bait”, and a nanobody that specifically binds SARS-CoV-2 RBD with high affinity was screened, which was named R14 in this paper.
  • the R14 of the present application can be combined with SARS-CoV-2 RBD with high affinity, and its binding constant is less than 1E-10M, and in pseudovirus neutralization experiment, can neutralize SARS-CoV-2 pseudovirus with high activity, these are It shows that R14 is a novel coronavirus (SARS-CoV-2) alpaca-derived nanobody that can bind to SARS-CoV-2 RBD with high affinity and has high neutralizing activity.
  • SARS-CoV-2 novel coronavirus
  • This application provides potential new nanobody drugs for the clinical prevention, treatment and detection of novel coronaviruses (including original strains and a series of mutant strains).
  • Figure 1 is a schematic diagram of the SARS-CoV-2 RBD-his protein molecular sieve chromatography and SDS-PAGE identification results described in Example 1 of the present application;
  • Figure 2 is a schematic diagram of the SARS-CoV-2 NTD-his protein molecular sieve chromatography and SDS-PAGE identification results described in Example 1 of the present application;
  • Fig. 3 is a schematic diagram of molecular sieve chromatography and SDS-PAGE identification results of Nanobody R14 described in Example 4 of the present application;
  • Fig. 4 is the kinetic curve of the Nanobody R14 determined in Example 5 of the present application in conjunction with the SARS-CoV-2 prototype strain and its mutant strains Alpha, Beta, Gamma, Kappa, Delta RBD; wherein, the dotted line refers to the original Data, the solid line refers to the kinetic curve after fitting;
  • Figure 5 is the kinetic curve of Nanobody R14 binding to the RBD of the related coronavirus GD/1/2019 determined in Example 5 of the present application; wherein, the dotted line refers to the original data, and the solid line refers to the fitted kinetic curve;
  • Figure 6 is a schematic diagram of the effect of Nanobody R14 neutralizing VSV-SARS-CoV-2 pseudovirus infection determined in Example 7 of the present application, wherein A is Nanobody R14 neutralizing the pseudovirus of the prototype strain SARS-CoV-2WT Effect diagram of infection; B is the effect diagram of nanobody R14 neutralizing pseudovirus infection of SARS-CoV-2 mutant strain Alpha (B.1.1.7); C is nanobody R14 neutralizing SARS-CoV-2 mutant virus The effect diagram of pseudovirus infection of strain Beta (B.1.351); D is the effect diagram of nanobody R14 and the pseudovirus infection of SARS-CoV-2 mutant strain Gamma (P.1); E is the effect diagram of Nanobody R14 and SARS-CoV-2 mutant strain Kappa (B.1.617.1) pseudovirus infection effect diagram; F is Nanobody R14 and SARS-CoV-2 mutant strain Delta (B.1.617.2) pseudovirus infection Rendering of viral infection.
  • A is Nanobody R14 neutralizing the pseudovirus of the prototype strain SARS-CoV-2
  • Figure 7 shows the neutralizing activity of the Nanobody R14 detected in Example 9 of the present application to the pseudovirus of the original strain of the new coronavirus before and after nebulization.
  • Figure 8 shows the efficacy of the Nanobody R14 detected in Example 10 of the present application in preventing SARS-CoV-2 infection in mice.
  • Nemobody that is, “heavy chain single domain antibody”
  • VHH variable domain of heavy chain of heavy-chain antibody
  • nanobodies Due to the biophysical advantages of nanobodies themselves, they can be easily nebulized and delivered directly to the lungs through an inhaler, thereby treating infections caused by respiratory viruses, and are considered to be very promising antibody drugs.
  • Specific binding when referring to a ligand/receptor, antibody/antigen or other binding pair refers to the determination of the presence or absence of said protein in a heterogeneous population of proteins and/or other biological agents, such as the nano Antibody binding reaction to SARS-CoV-2 RBD protein.
  • a specific ligand/antigen binds to a specific receptor/antibody and does not bind in significant amounts to other proteins present in the sample.
  • Chemical materials such as reagents, enzymes, medium, antibiotics and milk used in the following examples of the application are all commercially available products, for example, TRIzol is purchased from Invitrogen, and the Superscript II First-Strand Synthesis System for RT-PCR kit is purchased from Invitrogen.
  • pCAGGS vectors are purchased from MiaoLingPlasmid
  • 293F cells, HEK293T cells, etc. are purchased from ATCC
  • electrocompetent E. coli TG1 cells were purchased from Lucigen
  • VCSM13 helper phages were purchased from StrataGene
  • plasmid pMES4 was purchased from Addgene
  • protein A chips were purchased from GE Healthcare
  • Vero cells were purchased from ATCC CCL81.
  • primers Some synthetic biological materials, such as primers, sequences and other materials that require artificial synthesis, are entrusted to a synthetic company to complete.
  • the primers (SED ID NO: 13-18) in this application were synthesized by Beijing Qingke Biotechnology Co., Ltd.
  • SARS-CoV-2 RBD protein and the SARS-CoV-2 NTD protein of the present application were obtained from the inventor's laboratory (see Example 1).
  • the 3' end of the SARS-CoV-2 NTD protein coding sequence (as shown in SEQ ID NO: 11) is connected with the coding sequence of 6 histidine tags (hexa-His-tag) and the translation stop codon TGA, through the EcoRI and XhoI restriction endonuclease sites, it was constructed into the pCAGGS vector, transfected into 293F cells, and expressed the SARS-CoV-2 NTD-his protein.
  • the SDS-PAGE identification size of the SARS-CoV-2 RBD-his protein is about 30KD, and the result is shown in Figure 1; the SDS-PAGE identification size of the SARS-CoV-2 NTD-his protein is about 60KD, and the result is shown in the figure 2.
  • the RBD domain of the Alpha mutant strain contains the N501Y mutation
  • the RBD domain of the Beta mutant strain contains K417N, E484K and N501Y mutations
  • the RBD domain of the Gamma mutant strain contains K417T, E484K and N501Y mutations Mutations
  • the RBD domain of the Kappa mutant strain contains L452R and E484Q mutations
  • the RBD domain of the Delta mutant strain contains L452R and T478K mutations.
  • the SARS-CoV-2 RBD protein and SARS-CoV-2NTD protein with 6 histidine tags prepared in Example 1 were each 200 ⁇ g, respectively diluted with PBS to a final volume of 1 mL, and emulsified with 1 mL of complete Freund’s adjuvant For 5 minutes, multi-point subcutaneous injections were performed at the same time for immunization, and then immunization was performed every two weeks. On the 12th day after the fourth immunization, 50-60 mL of blood was collected to separate PBMCs (peripheral blood mononuclear cells). The isolated PBMCs were added to 1 mL TRIzol (purchased from Invitrogen), and total RNA was extracted according to the instructions.
  • PBMCs peripheral blood mononuclear cells
  • the Superscript II First-Strand Synthesis System for RT-PCR kit (purchased from Invitrogen) was used to synthesize cDNA with a random primer oligo-dT 12-18 primer.
  • cDNA as a template, PCR experiments were performed using specific primers CALL001 and CALL002 (primers are listed in Table 1), and the bands with a size of 700 bp were cut and recovered.
  • the purified DNA was used as a template to perform nested PCR using nested primers VHH-BACK and PMCF to amplify the nanobody (VHHs) sequence, and recover and purify the VHHs sequence with a size of about 400 bp.
  • the VHHs fragment was ligated into plasmid pMES4 by the double enzyme digestion method through the restriction sites PstI and BstEII.
  • the purified cloning vector was mixed with electrocompetent E.coli TG1 cells, and the cloning vector was transformed into electrocompetent E.coli TG1 cells using an electroporator (BIO-RAD Electrotransformer MicroPulser).
  • electroporator BIO-RAD Electrotransformer MicroPulser
  • Example 3 Screening of specific Nanobodies by phage display technology
  • the 96-well plate coated with the SARS-CoV-2 RBD-His antigen was prepared again, and the second round of panning was performed to enrich the phage expressing specific nanobodies, and a total of 3 rounds of panning were performed.
  • After each round of panning randomly select different single colonies from the agar plate with colonies, culture them in a shaker at 37°C, then add VCSM13 helper phages to expand overnight, centrifuge the culture medium the next day, and take the phage supernatant for ELISA
  • OD 450nM >0.2 it was judged as a positive reaction, and the corresponding clone was taken, and the plasmid was sequenced using specific primers MP57 and GIII (primers are shown in Table 2), and the sequences encoding VHHs in the plasmid were obtained. Through sequence determination, the core coding sequence of R14 was obtained.
  • the 5' end of the core coding sequence of R14 obtained in Example 3 was connected to the coding sequence of QVQLQ (CAGGTGCAGCTGCAG), and the 3' end was connected to the coding sequence of QVTVSS (CAGGTGACCGTGAGCTCT), Obtain the nucleotide sequence such as SEQ ID NO: 9, that is, the coding sequence of the Nanobody R14 of the present application; then connect the signal peptide (ATGCACAGCAGCGCCCTGCTGTGCTGCCTGGTTCTGCTGACCGGAGTGAGGGCC, SEQ ID NO: 27) at its 5' end, and connect 6 at the 3' end.
  • the coding sequence of a histidine tag (hexa-His-tag) and the translation stop codon TGA were constructed into the pCAGGS vector through the restriction endonuclease sites EcoRI and XhoI, transfected into 293F cells, and cultured for 5 days , collected the supernatant, centrifuged at 5000rpm for 30min, filtered through a 0.22 ⁇ m filter membrane, and was subjected to nickel ion affinity chromatography (HisTrap TM excel ((GE Healthcare)) and gel filtration chromatography (Superdex TM 75Increase 10/300GL column (GE Healthcare)) after purification, obtain relatively pure target protein.Purpose peak is determined by SDS-PAGE, and the result is shown in Figure 3, obtains the Nanobody R14 of purification.
  • Embodiment 5 Surface plasmon resonance technology detects the binding ability of antibody and viral antigen RBD
  • the protein A chip (purchased from GE Healthcare) was selected, and the SARS-CoV-2 prototype strain with hFc tag obtained in Example 1 and its variant strains (Alpha, Beta, Gamma, Kappa, Delta), and the RBD protein of related coronavirus GD/1/2019 were immobilized on the chip, and the immobilization amount was about 100RU. 2 PO 4 , 0.05% Tween) doubly dilute the R14 protein, and load the samples one by one from low concentration to high concentration.
  • the kinetic curves of antibody R14 binding to the SARS CoV-2 prototype strain and its mutant strains Alpha, Beta, Gamma, Kappa, Delta RBD proteins are shown in Figure 4, and the kinetics of R14 binding to the RBD of the related coronavirus GD/1/2019
  • the learning curve is shown in Figure 5.
  • the equilibrium dissociation constant (K D ) between antibody R14 and each viral antigen RBD is shown in Table 3 below, and the results in Table 3 show that the K D between antibody R14 and each viral antigen RBD is less than 0.1 nM.
  • the calculation of binding kinetic constants was performed using BIAevaluation software 8K (Biacore, Inc.) software.
  • Nanobody R14 has a broad spectrum and is capable of interacting with the prototype strain of SARS-CoV-2 and its variant strains (Alpha, Beta, Gamma, Kappa, Delta), as well as the related coronavirus GD/1/2019. RBD binds with higher affinity.
  • Embodiment 6 the packaging of SARS-CoV-2 original strain and mutant strain pseudovirus
  • SARS-CoV-2 prototype strain (WT) and mutant strains (Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.617. 1) and Delta (B.1.617.2)), the gene at the last 18 amino acids of the S protein was removed, and the remaining sequence of the S protein was synthesized (synthesis service provided by Suzhou Jinweizhi), and SARS-CoV-2-WT-S- Nuclei of del18, B.1.1.7-S-del18, B.1.351-S-del18, P.1-S-del18, B.1.617.1-S-del18, B.1.617.2-S-del18 genes Nucleotide sequence, the sequence is shown in SEQ ID NO:19 ⁇ 24 respectively.
  • SARS-CoV-2 original strain and mutant strain pseudovirus are as follows:
  • HEK293T cells ATCC CRL-3216 cells
  • the culture medium is DMEM medium containing 10% FBS.
  • Toxification Add the pseudovirus packaging skeleton virus G*VSV-delG (purchased from Wuhan Privy Brain Science and Technology Co., Ltd.) to the above transfected HEK293T cells, incubate at 37°C for 2h, and change the culture medium (containing 10% FBS) DMEM medium), and VSV-G antibody was added (the hybridoma cells expressing the antibody were purchased from ATCC cell bank), and the culture was continued for 30 h in the incubator.
  • G*VSV-delG purchased from Wuhan Privy Brain Science and Technology Co., Ltd.
  • Toxin collection the supernatant was collected and centrifuged at 3000rpm for 10min, filtered through a 0.45 ⁇ m sterile filter in an ultra-clean workbench to remove cell debris, aliquoted, and stored in a -80°C refrigerator.
  • SARS-CoV-2 prototype strain SARS-CoV-2WT
  • mutant strains Alpha(B.1.1.7), Beta(B.1.351), Gamma(P.1), Kappa(B.1.617) were obtained respectively. .1) and Delta (B.1.617.2)) pseudoviruses.
  • Embodiment 7 Nanobody R14 neutralizes the detection of SARS-CoV-2 pseudovirus infection
  • the purified Nanobody R14 obtained in Example 4 was diluted 5-fold from 5 ⁇ g/mL to the ninth gradient (2.56 pg/mL) and 1.6 ⁇ 10 4 TCID 50.
  • a series of SARS-CoV- 2 Pseudoviruses of the original strain and the mutant strain were mixed separately, mixed and incubated at 37° C. for 1 hour, and then added to a 96-well plate pre-inoculated with Vero cells (purchased from ATCC CCL81). After incubation for 18-20 hours, it was detected by CQ1 Confocal Quantitative Image Cytometer (Yokogawa). According to the number of cells with GFP fluorescence, the neutralization ability of the antibody against the pseudoviruses of the above-mentioned series of original and mutant strains of SARS-CoV-2 was calculated. .
  • IC 50 ( ⁇ g/mL) a is the half-inhibitory concentration of R14 Nanobody.
  • the nanobody R14 can neutralize the pseudoviruses of the above-mentioned series of SARS-CoV-2 original strains and mutant strains with high neutralization activity.
  • nanobody R14 can be used as a high-level and active new coronavirus (SARS-CoV-2) alpaca-derived nanobody.
  • SARS-CoV-2 active new coronavirus
  • Example 8 Detection of Nanobody R14 Neutralizing SARS-CoV-2 Live Virus Infection
  • the neutralization effect of the nanobody R14 on the live virus of the new coronavirus was determined through a live virus neutralization test based on the cytopathic effect (CPE); the specific procedure is as follows:
  • Example 9 Detection of the stability of Nanobody R14 before and after nebulization
  • the nanobody R14 was nebulized, and then the antibody after the nebulization was collected using an all-glass SKC (Eighty Four, PA, USA) containing 20mL PBS, and pressed Carry out pseudovirus neutralization test described in embodiment 7.
  • Example 10 Detection of the effect of R14 nanobody on the prevention of new coronavirus infection in vivo
  • administration is performed in the following groups:
  • Intranasal administration group After 5 mice were anesthetized, 50 ⁇ L of 2 mg/ml Nanobody R14 was instilled into the nostrils of the mice with a dose of 5 mg/kg;
  • Nebulization administration group 8 mice were placed in the nebulization chamber, and the nanobody R14 of 20 mg/mL was continuously nebulized. After 10 minutes, the mice were immediately taken out, 5 of which were put into the mouse cage, and the remaining 3 Lung perfusion test was performed only after anesthesia to determine the amount of inhaled antibody, and the inhaled dose was 0.25mg/kg;
  • Control group 5 mice were given 200 ⁇ L of PBS by nasal drop;
  • mice 6 hours after the administration, the mice were anesthetized again, and the live virus (5 ⁇ 10 5 TCID50) of the original strain of the new coronavirus was infected intranasally;
  • mice were dissected, lung tissue was taken, RNA was extracted, and viral load was measured.
  • the results are shown in Figure 8.
  • the results in Figure 8 show that the Nanobody R14 of the present application can effectively reduce the viral load in the lungs and effectively prevent the infection of the new coronavirus in mice, whether it is administered through nasal drops or aerosolized.
  • This application provides an alpaca-derived nanobody or its antigen-binding fragment that binds to the SARS-CoV-2 RBD, its preparation method, related products, and its application in the preparation of drugs for the prevention or treatment of new coronaviruses.
  • the alpaca-derived nanobody of the present application is a nanobody with high neutralization activity, has a strong binding ability with the SARS-CoV-2 RBD protein, and can effectively inhibit SARS-CoV-2 infection.
  • the nanobody also has a small molecular weight ( ⁇ 15kDa), less immunogenicity, better solubility and stability, longer CDR3 region, etc., can be administered by aerosolization, can reach the lungs, and have faster onset, providing a potential for new crown or other coronavirus infections treatment strategy.

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Abstract

提供了一种与SARS-CoV-2 RBD结合的羊驼源纳米抗体或其抗原结合片段及其应用，该纳米抗体能有效抑制SARS-CoV-2假病毒感染，可用于预防、治疗和/或检测SARS-CoV-2感染。

Description

一株羊驼源纳米抗体及其应用
交叉引用
本申请要求于2021年9月16日提交的、申请号为202111086956.4、发明名称为“一株羊驼源纳米抗体及其应用”的发明专利申请的优先权益，其全部内容通过引用并入本文。
技术领域
本申请涉及生物医药领域，具体涉及一株羊驼源纳米抗体及其应用，更具体地，涉及一种与SARS-CoV-2 RBD结合的羊驼源纳米抗体或其抗原结合片段、编码其的多核苷酸、包含该多核苷酸的核酸构建体、包含该核酸构建体的表达载体、其制备方法、转化的细胞以及包含上述的药物组合物，以及它们在制备预防、治疗和/或检测新冠病毒感染的药物中的应用。
背景技术
2019年12月以来，由冠状病毒科(family Coronaviridae)的新型冠状病毒(SARS-CoV-2)引起的疫情在全球范围内仍不断蔓延。除此之外，同属冠状病毒科的严重急性呼吸综合征冠状病毒(SARS-CoV)、中东呼吸综合征冠状病毒(MERS-CoV)等也是针对人类呼吸系统的主要病原体，主要通过飞沫、气溶胶和接触等方式传播，其传染性强，易引起群众恐慌，因此这类引起呼吸系统疾病的病毒严重危害公共卫生安全，尤其近年来呼吸性传染病的频发、病毒的不断变异，对人民群众的身体健康、生命安全和国民经济发展、社会稳定造成极大威胁。
中和抗体，该药物主要是通过与病原微生物表面的抗原结合，阻止病原微生物表达的特定分子与细胞表面受体结合，达到“中和”的效果。SARS-CoV和SARS-CoV-2病毒表面都具有糖基化的刺突蛋白(spike protein,S)，该S蛋白能与宿主细胞受体蛋白ACE2相互作用并触发膜融合，因此阻断S蛋白与ACE2的结合是治疗新冠病毒感染的有效途径。
然而旨在阻断病毒与宿主细胞受体的抗体策略，仍需要进一步优化和升级。一方面，新冠病毒这样的RNA病毒具有易突变、易发生免疫逃逸等特点，单一特异性抗体很难满足长久的治疗需求。另外，常规的单克隆抗体因其分子量过大，在实际应用中也存在一定的缺陷。
发明内容
发明目的
本申请的目的在于提供一种与SARS-CoV-2 RBD结合的羊驼源纳米抗体或其抗原结合片段、编码其的多核苷酸、包含该多核苷酸的核酸构建体、包含该核酸构建体的表达载体、其制备方法、转化的细胞以及包含上述的药物组合物，以及它们在制备预防或治疗新冠病毒的药物中的应用。本申请的羊驼源纳米抗体或其抗原结合片段为高中和活性的纳米抗体，与SARS-CoV-2 RBD蛋白的结合能力强，能有效抑制SARS-CoV-2感染，该纳米抗体具有分子量小(～15kDa)、免疫原性小、更好的溶解度和稳定性、较长的CDR3区的优点，可以雾化给药，能直达肺部，起效更快，为新冠或其他冠状病毒感染提供了潜在的治疗策略。
解决方案
为实现上述目的，本申请提供了如下技术方案：
第一方面，本申请提供了一种与SARS-CoV-2 RBD结合的羊驼源纳米抗体或其抗原结合片段，包含重链可变区，所述重链可变区包含以下的CDR：
氨基酸序列如SEQ ID NO:1(即，GFTLDYYAIG)所示的CDR1，
氨基酸序列如SEQ ID NO:2(即，CISSSDGSTSYADSVKG)所示的CDR2，以及
氨基酸序列如SEQ ID NO:3(即，TPATYYSGRYYYQCPAGGMDY)所示的CDR3。
优选地，所述重链可变区还包括4个框架区FR1-4，所述FR1-4与所述CDR1、CDR2和CDR3按顺序交错排列。
在一个优选的实施方案中，所述FR1-4的氨基酸序列分别如SEQ ID NO:4(即，QVQLQESGGGLVQPGGSLRLSCAVS)、SEQ ID NO:5(即，WFRQAPGKEREGVS)、SEQ ID NO:6(即，RFTISRDNAKNTVYLQMNSLKPEDTALYYCAA)和SEQ ID NO:7(即，WGQGTQVTVSS)所示。
进一步优选地，所述重链可变区的氨基酸序列如以下SEQ ID NO:8所示：
Figure PCTCN2022118494-appb-000001
Figure PCTCN2022118494-appb-000002
其中，下划线部分分别为框架区FR1-4，标黑部分分别为重链可变区的CDR1、CDR2和CDR3。
第二方面，本申请提供一种多核苷酸，其编码所述的与SARS-CoV-2 RBD结合的羊驼源纳米抗体或其抗原结合片段。
进一步地，所述多核苷酸为DNA或mRNA。
进一步地，所述多核苷酸具有如SEQ ID NO:9所示的核苷酸序列：
Figure PCTCN2022118494-appb-000003
Figure PCTCN2022118494-appb-000004
第三方面，本申请提供一种核酸构建体，其包含所述的多核苷酸。
进一步优选地，所述多核苷酸还包含与所述多核苷酸可操作地连接的至少一个表达调控元件。例如组氨酸标签、终止密码子等。
第四方面，本申请提供一种表达载体，其包含所述的核酸构建体。
第五方面，本申请提供了一种转化的细胞，其包括如上述第二方面所述的多核苷酸、如上述第三方面所述的核酸构建体或如上述第四方面所述的表达载体。
第六方面，本申请提供了一种药物组合物，其包含如上述第一方面所述的与SARS-CoV-2 RBD结合的羊驼源纳米抗体或其抗原结合片段、如上述第二方面所述的多核苷酸、如上述第三方面所述的核酸构建体、如上述第四方面所述的表达载体或如上述第五方面所述的转化的细胞，以及药学上可接受的载体和/或赋形剂。
进一步优选地，所述药物组合物为鼻喷剂、口服制剂、栓剂或胃肠外制剂的形式。
进一步优选地，所述鼻喷剂选自气雾剂、喷雾剂和粉雾剂。
进一步优选地，所述口服制剂选自片剂、粉末剂、丸剂、散剂、颗粒剂、细粒剂、软/硬胶囊剂、薄膜包衣剂、小丸剂、舌下片和膏剂。
进一步优选地，所述胃肠外制剂为经皮剂、软膏剂、硬膏剂、外用液剂、可注射或可推注制剂。
第七方面，本申请提供了一种如上述第一方面所述的与SARS-CoV-2 RBD结合的羊驼源纳米抗体或其抗原结合片段、如上述第二方面所述的多核苷酸、如上述第三方面所述的核酸构建体、如上述第四方面所述的表达载体或如上述第五方面所述的转化的细胞或如上述第六方面所述的药物组合物在制备预防、治疗和/或检测新冠病毒感染的药物中的应用。
进一步优选地，所述新冠病毒为SARS-CoV-2原始毒株和/或SARS-CoV-2变异毒株。
进一步优选地，所述SARS-CoV-2变异毒株为Alpha(B.1.1.7)、Beta(B.1.351)、Gamma(P.1)、Kappa(B.1.617.1)和/或Delta(B.1.617.2)毒株。
第八方面，本申请提供了一种预防或治疗新冠病毒感染的方法，其包括：向有需要 的受试者施用预防或治疗有效量的如上述第一方面所述的与SARS-CoV-2 RBD结合的羊驼源纳米抗体或其抗原结合片段、如上述第二方面所述的多核苷酸、如上述第三方面所述的核酸构建体、如上述第四方面所述的表达载体或如上述第五方面所述的转化的细胞或如上述第六方面所述的药物组合物。
优选地，所述新冠病毒为SARS-CoV-2原始毒株和/或SARS-CoV-2变异毒株。
进一步优选地，所述SARS-CoV-2变异毒株为Alpha(B.1.1.7)、Beta(B.1.351)、Gamma(P.1)、Kappa(B.1.617.1)和/或Delta(B.1.617.2)毒株。
本申请的药物组合物的有效成分的给药量，根据给药对象、对象脏器、症状、给药方法等不同而存在差异，可以考虑剂型的种类、给药方法、患者的年龄和体重、患者的症状等，根据医生的判断来确定。
第九方面，本申请提供了一种检测新冠病毒的方法，其包括使用如上述第一方面所述的与SARS-CoV-2 RBD结合的羊驼源纳米抗体或其抗原结合片段。
优选地，所述新冠病毒为SARS-CoV-2原始毒株和/或SARS-CoV-2变异毒株。
进一步优选地，所述SARS-CoV-2变异毒株为Alpha(B.1.1.7)、Beta(B.1.351)、Gamma(P.1)、Kappa(B.1.617.1)和/或Delta(B.1.617.2)毒株。
有益效果
本申请针对新冠病毒进行纳米抗体药物开发，通过用SARS-CoV-2 RBD蛋白和SARS-CoV-2 NTD蛋白同时免疫羊驼、构建抗体文库、利用噬菌体展示技术筛选特异性纳米抗体等，以SARS-CoV-2 RBD蛋白作为“钓饵”，筛选到以高亲和力特异性结合SARS-CoV-2 RBD的纳米抗体，本文命名为R14。本申请的R14能以高亲和力与SARS-CoV-2RBD结合，其结合常数小于1E-10M，并且在假病毒中和实验中，能以高中和活性中和SARS-CoV-2假病毒，这些均表明：R14是能够以高亲和力与SARS-CoV-2 RBD结合的、并具有高中和活性的新型冠状病毒(SARS-CoV-2)羊驼源纳米抗体。
本申请为新型冠状病毒(包括原始毒株以及一系列变异毒株)的临床预防、治疗和检测提供了潜在的纳米抗体新药。
附图说明
一个或多个实施例通过与之对应的附图中的图片进行示例性说明，这些示例性说明并不构成对实施例的限定。在这里专用的词“示例性”意为“用作例子、实施例或说明性”。这里作为“示例性”所说明的任何实施例不必解释为优于或好于其它实施例。
图1是本申请实施例1中记载的SARS-CoV-2 RBD-his蛋白分子筛层析和SDS-PAGE鉴定结果示意图；
图2是本申请实施例1中记载的SARS-CoV-2 NTD-his蛋白分子筛层析和 SDS-PAGE鉴定结果示意图；
图3是本申请实施例4中记载的纳米抗体R14的分子筛层析和SDS-PAGE鉴定结果示意图；
图4是本申请实施例5中所测定的纳米抗体R14结合SARS-CoV-2原型毒株及其变异毒株Alpha、Beta、Gamma、Kappa、Delta RBD的动力学曲线；其中，虚线是指原始数据，实线是指拟合后的动力学曲线；
图5是本申请实施例5中测定的纳米抗体R14结合相关冠状病毒GD/1/2019的RBD的动力学曲线；其中，虚线是指原始数据，实线是指拟合后的动力学曲线；
图6是本申请实施例7所测定的纳米抗体R14中和VSV-SARS-CoV-2假病毒感染的效果示意图，其中，A为纳米抗体R14中和原型毒株SARS-CoV-2WT的假病毒感染的效果图；B为纳米抗体R14中和SARS-CoV-2变异毒株Alpha(B.1.1.7)的假病毒感染的效果图；C为纳米抗体R14中和SARS-CoV-2变异毒株Beta(B.1.351)的假病毒感染的效果图；D为纳米抗体R14中和SARS-CoV-2变异毒株Gamma(P.1)的假病毒感染的效果图；E为纳米抗体R14中和SARS-CoV-2变异毒株Kappa(B.1.617.1)的假病毒感染的效果图；F为纳米抗体R14中和SARS-CoV-2变异毒株Delta(B.1.617.2)的假病毒感染的效果图。
图7显示了本申请实施例9中所检测的纳米抗体R14在雾化前、后对新冠病毒原始毒株假病毒的中和活性。
图8显示了本申请实施例10中所检测的纳米抗体R14在小鼠体内预防新冠病毒感染的功效。
具体实施方式
为使本申请实施例的目的、技术方案和优点更加清楚，下面将对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。除非另有其它明确表示，否则在整个说明书和权利要求书中，术语“包括”或其变换如“包含”或“包括有”等等将被理解为包括所陈述的元件或组成部分，而并未排除其它元件或其它组成部分。
另外，为了更好的说明本申请，在下文的具体实施方式中给出了众多的具体细节。本领域技术人员应当理解，没有某些具体细节，本申请同样可以实施。在一些实施例中，对于本领域技术人员熟知的原料、元件、方法、手段等未作详细描述，以便于凸显本申请的主旨。
以下，对本申请进行详述。
定义
“纳米抗体”，即“重链单域抗体”，该类抗体只包含一个重链可变区(VHH，variable domain of heavy chain of heavy-chain antibody)，相比于其他抗体，轻链天然缺失。
由于纳米抗体自身的生物物理优势，可以很容易地对其进行雾化并通过吸入器直接递送到肺部，从而治疗呼吸系统病毒引起的感染，被认为是非常有潜力的抗体类药物。
当提及配体/受体、抗体/抗原或其它结合对时，“特异性”结合是指在蛋白和/或其它生物试剂的异质群体中确定是否存在所述蛋白，例如本申请的纳米抗体与SARS-CoV-2 RBD蛋白的结合反应。因此，在所指定的条件下，特定的配体/抗原与特定的受体/抗体结合，并且并不以显著量与样品中存在的其它蛋白结合。
本申请以下实施例中所使用的试剂、酶、培养基、抗生素和牛奶等化学材料均为市售产品，例如，TRIzol购自Invitrogen，Superscript II First-Strand Synthesis System for RT-PCR试剂盒购自Invitrogen。
一些常用的生物材料，如感受态细胞、载体、辅助噬菌体、待转化的细胞等也为市售产品，例如，pCAGGS载体购自MiaoLingPlasmid；293F细胞、HEK293T细胞等购自ATCC；电感受态E.coli TG1细胞购自Lucigen；VCSM13辅助噬菌体购自StrataGene；质粒pMES4购自Addgene；protein A芯片购自GE Healthcare；Vero细胞购自ATCC CCL81。
一些合成类生物材料，例如引物、序列等需要人工合成的材料，均委托合成公司完成，例如，本申请中的引物(SED ID NO:13～18)由北京擎科生物科技有限公司合成。
本申请的SARS-CoV-2 RBD蛋白、SARS-CoV-2 NTD蛋白为发明者实验室获得(参见实施例1)。
实施例1：病毒抗原蛋白的表达与纯化
在SARS-CoV-2原型毒株S蛋白(GenBank登录号：MN908947.3)RBD结构域(R319-F541区段)的编码序列(如SEQ ID NO:10所示)的5’端连上信号肽(ATGTTTGTGTTTCTTGTGCTTCTTCCTCTTGTGTCATCACAATGC，SEQ ID NO:25)，3’端连上6个组氨酸标签(hexa-His-tag)的编码序列及翻译终止密码子TGA，通过限制性内切酶位点EcoRI和XhoI，将其构建入pCAGGS载体中，转染至293F细胞中，进行SARS-CoV-2 RBD-his蛋白的表达。同样地，在SARS-CoV-2 NTD蛋白编码序列(如SEQ ID NO:11所示)的3’端连上6个组氨酸标签(hexa-His-tag)的编码序列及翻译终止密码子TGA，通过EcoRI和XhoI限制性内切酶位点，将其构建入pCAGGS载体中，转染至293F细胞中，进行SARS-CoV-2 NTD-his蛋白的表达。
含有目的蛋白的细胞培养液经镍离子亲和层析(HisTrap  TM excel((GE Healthcare))和凝胶过滤层析(Superdex TM 200Increase 10/300GL column(GE Healthcare))纯化后，可以获得较纯的目的蛋白。SARS-CoV-2 RBD-his蛋白的SDS-PAGE鉴定大小为30KD左右， 结果如图1；SARS-CoV-2 NTD-his蛋白的SDS-PAGE鉴定大小为60KD左右，结果如图2。
分别地，在SARS-CoV-2原型毒株S蛋白(GenBank登录号是MN908947.3)上的RBD结构域(R319-F541区段)的编码序列(如SEQ ID NO:10所示)以及在该序列基础上通过点突变分别构建的、变异毒株(Alpha、Beta、Gamma、Kappa、Delta)的RBD结构域的编码序列的5’端连上信号肽(ATGTTTGTGTTTCTTGTGCTTCTTCCTCTTGTGTCATCACAATGC，SEQ ID NO:25)，3’端连上人源Fc标签(hFc)的编码序列(如SEQ ID NO:12所示)及翻译终止密码子TGA，通过连接EcoRI和XhoI构建入pCAGGS载体中，转染至293F细胞中，进行SARS-CoV-2RBD-hFc蛋白的表达，用于表面等离子共振分析。其中，相对于原型毒株，Alpha变异毒株的RBD结构域包含N501Y突变，Beta变异毒株的RBD结构域包含K417N、E484K和N501Y突变，Gamma变异毒株的RBD结构域包含K417T、E484K和N501Y突变，Kappa变异毒株的RBD结构域包含L452R和E484Q突变，Delta变异毒株的RBD结构域包含L452R和T478K突变。
在相关冠状病毒(Sarbecovirus)GD/1/2019的S蛋白(GISAID数据库登录号：EPI_ISL_410721)上的RBD结构域(R315-F537区段)的编码序列的5’端连上信号肽(ATGTTTGTGTTCCTGGTGCTGCTGCCCCCGGTGAGCTCTCAGTGC，SEQ ID NO:26)，3’端连上人源Fc标签(hFc)的编码序列(如SEQ ID NO:12所示)及翻译终止密码子TGA，通过连接EcoRI和XhoI构建入pCAGGS载体中，转染至293F细胞中，进行SARS-CoV-2RBD-hFc蛋白的表达，用于表面等离子共振分析。
实施例2：羊驼免疫和抗体文库构建
实施例1中制备的带有6个组氨酸标签的SARS-CoV-2 RBD蛋白和SARS-CoV-2NTD蛋白各200μg，分别用PBS稀释至终体积1mL，分别加1ml完全弗氏佐剂乳化5min，同时进行皮下多点注射进行免疫，之后每两周进行一次免疫，第四次免疫后的第12天，采集50-60mL的血液，分离PBMCs(外周血单个核细胞)。将分离的PBMCs加入1mL TRIzol(购自Invitrogen)，按照说明书的步骤提取总RNA。以提取的总RNA为模板，使用Superscript II First-Strand Synthesis System for RT-PCR试剂盒(购自Invitrogen)，用随机引物oligo-dT 12-18引物合成cDNA。以cDNA为模板，使用特异性引物CALL001和CALL002(引物如表1)进行PCR试验，对700bp大小的条带进行切胶并回收。纯化后的DNA作为模板，使用巢式引物VHH-BACK和PMCF进行巢式PCR，来扩增纳米抗体(VHHs)序列，回收纯化大小在400bp左右VHHs序列。
使用双酶切法，通过限制性酶切位点PstⅠ和BstEⅡ，将VHHs片段连接入质粒pMES4。将纯化的克隆载体和电感受态E.coli TG1细胞混合，使用电转仪(BIO-RAD电转化仪 MicroPulser)将克隆载体转化入电感受态E.coli TG1细胞，全部涂布在含有氨苄青霉素的选择性培养基上，37℃过夜培养后，收集所有的菌落于LB培养基中，离心并弃上清，用LB重悬细胞，此为抗体文库。
表1、反应引物
Figure PCTCN2022118494-appb-000005
实施例3：噬菌体展示技术筛选特异性的纳米抗体
取实施例2已转染重组质粒的E.coli TG1，以感染复数(multiplicity of infection,MOI)约为20的比例，加入VCSM13辅助噬菌体，过夜培养后，4000rpm离心，取上清，0.22μm的膜过滤后，按体积比1:4加入PEG6000/NaCl，混合后，4℃下放置至少1小时，8000×g离心30min，弃上清，沉淀用PBS重悬，即为收集的噬菌体颗粒，测定噬菌体效价。
将2×10 11个上述收集的噬菌体与等体积的5％(w/v)脱脂牛奶混合，加到包被有SARS-CoV-2 RBD-His抗原的96孔板中，室温孵育1h后，用0.2M甘氨酸洗脱特异性的噬菌体，并用Tris-HCl(pH 9.1)中和洗脱的噬菌体。然后用该噬菌体感染E.coli TG1细胞，并对噬菌体进行扩增。再次准备包被有SARS-CoV-2 RBD-His抗原的96孔板，进行第2轮淘选，来富集表达有特异性纳米抗体的噬菌体，共进行3轮淘选。每轮淘选后，从长有菌落的琼脂平板上随机挑选不同单一菌落，在37℃摇床中培养，随后加入VCSM13辅助噬菌体过夜扩培，第二天离心培养液，取噬菌体上清进行ELISA实验，当OD 450nM>0.2时，判定为阳性反应，取对应的克隆，使用特异性引物MP57和GⅢ对质粒进行测序(引物如表2)，获得其质粒中编码VHHs的序列。通过序列测定，获得R14的核心编码序列。
表2、反应引物
Figure PCTCN2022118494-appb-000006
实施例4：纳米抗体R14的表达
为了使R14的重链可变区更加完整，在实施例3获得的R14的核心编码序列的5’端连上QVQLQ的编码序列(CAGGTGCAGCTGCAG)，3’端连上QVTVSS的编码序列(CAGGTGACCGTGAGCTCT)，得到如SEQ ID NO:9的核苷酸序列，即本申请的纳米抗体R14的编码序列；然后在其5’端连上信号肽(ATGCACAGCAGCGCCCTGCTGTGCTGCCTGGTTCTGCTGACCGGAGTGAGGGCC， SEQ ID NO:27)，3’端连上6个组氨酸标签(hexa-His-tag)的编码序列及翻译终止密码子TGA，通过限制性内切酶位点EcoRI和XhoI，将其构建入pCAGGS载体中，转染293F细胞，培养5天后，收集上清，经过5000rpm离心30min后，经过0.22μm滤膜过滤后，经镍离子亲和层析(HisTrap  TM excel((GE Healthcare))和凝胶过滤层析(Superdex TM 75Increase 10/300GL column(GE Healthcare))纯化后，获得较纯的目的蛋白。目的峰通过SDS-PAGE确定，结果如图3，得到纯化的纳米抗体R14。
实施例5：表面等离子共振技术检测抗体与病毒抗原RBD的结合能力
表面等离子共振分析利用Biacore 8K(Biacore Inc.)进行。具体步骤如下：
选用protein A芯片(购自GE Healthcare)，通过protein A芯片与hFc的亲和力将实施例1得到的带有hFc标签的SARS-CoV-2原型毒株及其变异毒株(Alpha、Beta、Gamma、Kappa、Delta)、以及相关冠状病毒GD/1/2019的RBD蛋白固定在芯片上，固定量约为100RU，用PBST缓冲液(2.7mM KCl，137mM NaCl，4.3mM Na 2HPO 4，1.4mM KH 2PO 4，0.05％吐温)倍比稀释R14蛋白，从低浓度到高浓度逐一上样。抗体R14结合SARS CoV-2原型毒株及其变异毒株Alpha、Beta、Gamma、Kappa、Delta RBD蛋白的动力学曲线如图4所示，R14结合相关冠状病毒GD/1/2019的RBD的动力学曲线如图5所示。抗体R14与各病毒抗原RBD之间的平衡解离常数(K D)如以下表3，表3结果显示：抗体R14与各病毒抗原RBD之间的K D均小于0.1nM。结合动力学常数的计算是利用BIAevaluation software 8K(Biacore，Inc.)软件进行。这些结果说明，纳米抗体R14具有广谱性，且能够和SARS-CoV-2原型毒株及其变异毒株(Alpha、Beta、Gamma、Kappa、Delta)、以及相关冠状病毒GD/1/2019的RBD以较高的亲和力结合。
表3、纳米抗体R14与SARS-CoV-2及其变异毒株RBD的结合动力学常数
Figure PCTCN2022118494-appb-000007
实施例6：SARS-CoV-2原始毒株及变异毒株假病毒的包装
1)分别将编码SARS-CoV-2原型毒株(WT)及变异毒株(Alpha(B.1.1.7)、Beta(B.1.351)、Gamma(P.1)、Kappa(B.1.617.1)和Delta(B.1.617.2))的S蛋白后18位氨基酸的基因去掉，S蛋白其余序列进行合成(由苏州金唯智提供合成服务)，得到SARS-CoV-2-WT-S-del18、B.1.1.7-S-del18、B.1.351-S-del18、P.1-S-del18、B.1.617.1-S-del18、B.1.617.2-S-del18基因的核苷酸序列，序列分别如SEQ ID NO:19～24所示。
2)分别将1)中获得的该蛋白基因克隆到pCAGGS载体上，得到表达质粒pCAGGS-SARS-CoV-2-WT-S-del18、pCAGGS-B.1.1.7-S-del18、pCAGGS-B.1.351-S-del18、pCAGGS-P.1-S-del18、pCAGGS-B.1.617.1-S-del18、pCAGGS-B.1.617.2-S-del18。
SARS-CoV-2原始毒株及变异毒株假病毒的包装步骤如下：
a.细胞准备：在10cm细胞培养皿中铺HEK293T细胞(ATCC CRL-3216细胞)，使第二天细胞汇合密度至80％左右。培养液为含10％FBS的DMEM培养基。
b.转染：取上述步骤2)中的各S蛋白的表达质粒，用PEI转染30μg质粒/10cm细胞培养皿，目的质粒与PEI按1:3比例混匀后转染，4-6h换培养液(含10％FBS的DMEM培养基)，37℃培养24h。
c.加毒：将假病毒包装骨架病毒G*VSV-delG(购自武汉枢密脑科学技术有限公司)加入上述转染后的HEK293T细胞，37℃孵育2h，换培养液(含10％FBS的DMEM培养基)，并加入VSV-G抗体(表达该抗体的杂交瘤细胞购自ATCC细胞库)，在培养箱中继续培养30h。
d.收毒：收上清3000rpm离心10min,在超净工作台中经0.45μm无菌滤器过滤，去除细胞碎片，分装，-80℃冰箱冻存。
分别得到SARS-CoV-2原型毒株(SARS-CoV-2WT)及变异毒株(Alpha(B.1.1.7)、Beta(B.1.351)、Gamma(P.1)、Kappa(B.1.617.1)及Delta(B.1.617.2))的假病毒。
实施例7：纳米抗体R14中和SARS-CoV-2假病毒感染的检测
将实施例4得到的纯化的纳米抗体R14从5μg/mL开始5倍倍比稀释至第9个梯度(2.56pg/mL)与1.6×10 4TCID 50实施例6获得的一系列SARS-CoV-2原始毒株及变异毒株的假病毒分别混合，在37℃混合孵育1h，然后加入到预先接种Vero细胞(购自ATCC CCL81)的96孔板中。孵育18～20小时后，通过CQ1Confocal Quantitative Image Cytometer(Yokogawa)检测。根据带GFP荧光的细胞数，计算抗体对上述一系列SARS-CoV-2原始毒株及变异毒株的假病毒的中和能力，其结果分别如图6A～6F所示，结果统计如表4。
表4、纳米抗体R14对新冠病毒的假病毒中和效果
Figure PCTCN2022118494-appb-000008
Figure PCTCN2022118494-appb-000009
*其中，IC 50(μg/mL) a为R14纳米抗体的半抑制浓度。
由表4可知，纳米抗体R14能以高中和活性中和上述一系列SARS-CoV-2原始毒株及变异毒株的假病毒。
综上，纳米抗体R14能够作为高中和活性的新型冠状病毒(SARS-CoV-2)羊驼源纳米抗体。
实施例8：纳米抗体R14中和SARS-CoV-2活病毒感染的检测
本实施例中，通过基于细胞病变效应(CPE)的活病毒中和试验，测定纳米抗体R14对新冠病毒活病毒的中和效果；具体程序如下：
将纳米抗体R14以2倍倍比稀释至第11个梯度，每个梯度4个重复孔，每孔50μL，将各稀释液与等体积的100TCID 50的SARS-CoV-2原始毒株或其变异毒株Alpha、Beta和Delta于37℃孵育；1小时后，将混合物加入到悬浮的Vero细胞中，并在37℃下继续孵育3天；观察和记录细胞病变情况；使用GraphPad Prism 7.0计算该纳米抗体抑制新冠病毒活病毒感染的IC 50。
上述实验在中国疾病预防控制中心的生物安全三级实验室(BSL3)中进行。
纳米抗体R14对新冠病毒原始毒株及其变异毒株的活病毒的中和效果见以下表5。
表5、纳米抗体R14对新冠病毒活病毒的中和效果
Figure PCTCN2022118494-appb-000010
表5结果显示：本申请纳米抗体R14对新冠病毒原始毒株及其变异毒株的活病毒均具有良好的抑制效果。
实施例9：纳米抗体R14雾化前后稳定性的检测
使用Aerogen Solo(Aerogen Inc.,Chicago,USA)雾化器，将纳米抗体R14进行雾化，然后使用含有20mL PBS的全玻璃SKC(Eighty Four,PA,USA)收集雾化后的抗体，并按实施例7所述进行假病毒中和试验。
结果如图7所示，图7结果表明：本申请的纳米抗体R14在雾化前、后对新冠病毒原始毒株的假病毒的中和活性保持稳定，提示该抗体适于通过雾化途径给药。
实施例10：R14纳米抗体在体内预防新冠病毒感染的效果检测
本实施例中，在7-8周龄的雌性BALB/c小鼠(购自北京维通利华实验动物技术有限公司)中，检测本申请纳米抗体R14预防新冠病毒感染的功效；具体程序如下：
1)对7-8周的雌性BALB/c小鼠进行麻醉，滴鼻感染重组腺病毒Ad5-hACE2(按照Jing Sun et al.,“Generation of a Broadly Useful Model for COVID-19Pathogenesis,Vaccination,and Treatment”Cell.2020Aug 6；182(3):734-743中描述的方法制得)；
2)第5天，对小鼠进行给药和攻毒；
具体地，按以下分组进行给药：
滴鼻给药组(IN)：将5只小鼠麻醉后，用移液枪往小鼠鼻孔滴注50μL 2mg/ml的纳米抗体R14，给药剂量为5mg/kg；
雾化给药组(IH)：8只小鼠置于雾化仓中，持续雾化20mg/mL的纳米抗体R14，10分钟后，立即取出小鼠，其中5只放入鼠笼，剩余3只麻醉后进行灌肺试验，来测定吸入抗体的量，吸入剂量为0.25mg/kg；
对照组(Control)：对5只小鼠通过滴鼻的方式给予200μL的PBS；
给药后6小时，再次将小鼠麻醉，滴鼻感染新冠病毒原始毒株的活病毒(5×10 5TCID50)；
3)第10天，解剖小鼠，取肺组织，提RNA，测病毒载量。
上述实验在中国疾病预防控制中心的生物安全三级实验室(BSL3)中进行。
结果如图8所示，图8结果显示，本申请的纳米抗体R14无论是通过滴鼻给药还是通过雾化给药，均能有效降低肺部病毒载量，有效预防小鼠感染新冠病毒。
最后应说明的是：以上实施例仅用以说明本申请的技术方案，而非对其限制；尽管参照前述实施例对本申请进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本申请各实施例技术方案的精神和范围。
工业实用性
本申请提供了一种与SARS-CoV-2 RBD结合的羊驼源纳米抗体或其抗原结合片段、其制备方法、相关产品及其在制备预防或新冠病毒的药物中的应用。本申请的羊驼源纳米抗体为高中和活性的纳米抗体，与SARS-CoV-2 RBD蛋白的结合能力强，能有效抑制SARS-CoV-2感染，此外，该纳米抗体还具有分子量小(～15kDa)、免疫原性小、更好的溶解度和稳定性、较长的CDR3区等优点，可以雾化给药，能直达肺部，起效更快，为新冠或其他冠状病毒感染提供了潜在的治疗策略。

Claims (16)

  1. 一种与SARS-CoV-2 RBD结合的羊驼源纳米抗体或其抗原结合片段，其包含重链可变区，
    所述重链可变区包含以下的CDR：氨基酸序列如SEQ ID NO:1所示的CDR1，氨基酸序列如SEQ ID NO:2所示的CDR2，以及氨基酸序列如SEQ ID NO:3所示的CDR3。
  2. 根据权利要求1所述的与SARS-CoV-2 RBD结合的羊驼源纳米抗体或其抗原结合片段，其特征在于，所述重链可变区还包括4个框架区FR1-4，所述FR1-4与所述CDR1、CDR2和CDR3按顺序交错排列；
    优选地，所述FR1-4分别如SEQ ID NO:4、5、6、7所示。
  3. 根据权利要求1所述的与SARS-CoV-2 RBD结合的羊驼源纳米抗体或其抗原结合片段，其特征在于，所述重链可变区的氨基酸序列如SEQ ID NO:8所示。
  4. 一种多核苷酸，其编码权利要求1至3任一项所述的与SARS-CoV-2 RBD结合的羊驼源纳米抗体或其抗原结合片段。
  5. 根据权利要求4所述的多核苷酸，其特征在于，所述多核苷酸为DNA或mRNA；
    优选地，所述多核苷酸具有如SEQ ID NO:9所示的核苷酸序列。
  6. 一种核酸构建体，其包含权利要求4或5所述的多核苷酸。
  7. 根据权利要求6所述的核酸构建体，其特征在于，还包含与所述多核苷酸可操作地连接的至少一个表达调控元件。
  8. 一种表达载体，其包含权利要求6或7所述的核酸构建体。
  9. 一种转化的细胞，其包括权利要求4或5所述的多核苷酸、权利要求6或7所述的核酸构建体或权利要求8所述的表达载体。
  10. 一种药物组合物，其包含权利要求1至3任一项所述的与SARS-CoV-2 RBD结合的羊驼源纳米抗体或其抗原结合片段、权利要求4或5所述的多核苷酸、权利要求6或7所述的核酸构建体、权利要求8所述的表达载体或权利要求9所述的转化的细胞，以及药学上可接受的载体和/或赋形剂。
  11. 根据权利要求10所述的药物组合物，其特征在于，所述药物组合物为鼻喷剂、口服制剂、栓剂或胃肠外制剂的形式；
    优选地，所述鼻喷剂选自气雾剂、喷雾剂和粉雾剂；
    优选地，所述口服制剂选自片剂、粉末剂、丸剂、散剂、颗粒剂、细粒剂、软/硬胶囊剂、薄膜包衣剂、小丸剂、舌下片和膏剂；
    优选地，所述胃肠外制剂为经皮剂、软膏剂、硬膏剂、外用液剂、可注射或可推注 制剂。
  12. 一种权利要求1至3任一项所述的与SARS-CoV-2 RBD结合的羊驼源纳米抗体或其抗原结合片段、权利要求4或5所述的核苷酸序列、权利要求6或7所述的核酸构建体、权利要求8所述的表达载体、权利要求9所述的转化的细胞或权利要求10或11所述的药物组合物在制备预防、治疗和/或检测新冠病毒感染的药物中的应用。
  13. 根据权利要求12所述的应用，其特征在于，所述新冠病毒为SARS-CoV-2原始毒株和/或SARS-CoV-2变异毒株；
    优选地，所述SARS-CoV-2变异毒株为Alpha(B.1.1.7)、Beta(B.1.351)、Gamma(P.1)、Kappa(B.1.617.1)和/或Delta(B.1.617.2)毒株。
  14. 一种预防或治疗新冠病毒感染的方法，其包括：向有需要的受试者施用预防或治疗有效量的权利要求1至3任一所述的与SARS-CoV-2 RBD结合的羊驼源纳米抗体或其抗原结合片段、权利要求4或5所述的核苷酸序列、权利要求6或7所述的核酸构建体、权利要求8所述的表达载体、权利要求9所述的转化的细胞或权利要求10或11所述的药物组合物。
  15. 一种检测新冠病毒的方法，其包括使用权利要求1至3任一项所述的与SARS-CoV-2 RBD结合的羊驼源纳米抗体或其抗原结合片段。
  16. 根据权利要求14或15所述的方法，其中，所述新冠病毒为SARS-CoV-2原始毒株和/或SARS-CoV-2变异毒株；
    优选地，所述SARS-CoV-2变异毒株为Alpha(B.1.1.7)、Beta(B.1.351)、Gamma(P.1)、Kappa(B.1.617.1)和/或Delta(B.1.617.2)毒株。
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