WO2022011717A1 - 针对新型冠状病毒的纳米抗体及其应用 - Google Patents
针对新型冠状病毒的纳米抗体及其应用 Download PDFInfo
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Images
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
- C07K16/1002—Coronaviridae
- C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/46—Hybrid immunoglobulins
Definitions
- the present invention relates to the technical field of biomedicine or biopharmaceuticals, and more particularly to a nanobody against novel coronavirus and its application.
- Coronaviruses have single-stranded, non-segmented, positive-polarity RNA genomes, and their viruses contain 4 major structural proteins: nucleocapsid (N) protein, transmembrane (M) protein, envelope (E) protein, and spine Spike (S) protein.
- N nucleocapsid
- M transmembrane
- E envelope
- S spine Spike
- the spike protein S plays a key role in virus attachment, fusion, entry, and spread, including the S1 subunit at the N-terminal responsible for viral receptor binding and the C-terminal S2 subunit responsible for virus-cell membrane fusion.
- S1 can be subdivided into N- Terminal domain (NTD) and receptor binding domain (RBD).
- Novel coronavirus is the causative agent of novel coronavirus pneumonia (COVID-19).
- Neutralizing antibodies are considered candidate therapies against COVID-19.
- the new coronavirus recognizes and binds to host cell surface receptors through the spike glycoprotein (S protein) on its surface, and neutralizing antibodies target this protein. After the neutralizing antibody drug is injected into the human body, it can preemptively bind to the spike protein (S protein) of the new coronavirus, so that the virus cannot infect human cells and is cleared by the immune system.
- S protein spike glycoprotein
- the purpose of the present invention is to provide a nanobody against novel coronavirus and its application.
- a first aspect of the present invention provides a nanobody against a novel coronavirus.
- the nanobody against the novel coronavirus can specifically bind to SARS-CoV2.
- the nanobody against the novel coronavirus can specifically bind to the SARS-Cov2 S protein.
- the complementarity determining region CDR of the Nanobody against the novel coronavirus is one or more selected from the following group:
- CDR1 shown in SEQ ID NO:127 CDR2 shown in SEQ ID NO:128, and CDR3 shown in SEQ ID NO:129;
- any one of the above amino acid sequences also includes at least one (such as 1-3, preferably 1-2, more preferably through addition, deletion, modification and/or substitution) 1) amino acid and a derivative sequence that retains the ability to specifically bind to the S protein of the new coronavirus.
- the nanobody against the novel coronavirus can specifically bind to the S protein of the novel coronavirus.
- the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.
- the nanobody against the novel coronavirus further includes a framework region FR.
- the framework region FR is one or more selected from the following group:
- FR1 shown in SEQ ID NO: 130 FR2 shown in SEQ ID NO: 131, FR3 shown in SEQ ID NO: 132, and FR4 shown in SEQ ID NO: 133;
- FR1 shown in SEQ ID NO:292 FR2 shown in SEQ ID NO:293, FR3 shown in SEQ ID NO:294, and FR4 shown in SEQ ID NO:295;
- FR1 shown in SEQ ID NO:310 FR2 shown in SEQ ID NO:311, FR3 shown in SEQ ID NO:312, and FR4 shown in SEQ ID NO:313.
- the amino acid sequence of the VHH chain of the Nanobody against the novel coronavirus is selected from the following group: SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:62, SEQ ID NO:71, SEQ ID NO:80, SEQ ID NO:89, SEQ ID NO:98, SEQ ID NO:107, SEQ ID NO:116, SEQ ID NO:125, SEQ ID NO:134, SEQ ID NO:143, SEQ ID NO:152, SEQ ID NO:161, SEQ ID NO:170, SEQ ID NO:179, SEQ ID NO:188, SEQ ID NO:197, SEQ ID NO:206, SEQ ID NO:215, SEQ ID NO:224, SEQ ID NO:233, SEQ ID NO:242, SEQ ID NO:251, SEQ ID NO: 260, SEQ ID NO:269, SEQ ID NO:278, SEQ ID NO:
- the nanobodies against the novel coronavirus include humanized antibodies, camel-derived antibodies, and chimeric antibodies.
- the second aspect of the present invention provides an antibody against the novel coronavirus, the antibody comprising one or more VHH chains of the nanobody against the novel coronavirus according to the first aspect of the present invention.
- the amino acid sequence of the VHH chain of the Nanobody against the novel coronavirus is selected from the following group: SEQ ID NO: 8, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:62, SEQ ID NO:71, SEQ ID NO:80, SEQ ID NO:89, SEQ ID NO:98, SEQ ID NO:107, SEQ ID NO:116, SEQ ID NO:125, SEQ ID NO:134, SEQ ID NO:143, SEQ ID NO:152, SEQ ID NO:161, SEQ ID NO:170, SEQ ID NO:179, SEQ ID NO:188, SEQ ID NO:197, SEQ ID NO:206, SEQ ID NO:215, SEQ ID NO:224, SEQ ID NO:233, SEQ ID NO:242, SEQ ID NO:251, SEQ ID NO: 260, SEQ ID NO:269, SEQ ID NO:278, SEQ ID NO:
- the antibody against the novel coronavirus can be a monomer, a bivalent antibody, and/or a multivalent antibody.
- the third aspect of the present invention provides a polynucleotide encoding a protein selected from the group consisting of the nanobody against the novel coronavirus according to the first aspect of the present invention, or the second aspect of the present invention the antibody.
- sequence of the polynucleotide is selected from the group consisting of: SEQ ID NO:9, SEQ ID NO:18, SEQ ID NO:27, SEQ ID NO:36, SEQ ID NO:45, SEQ ID NO:45, ID NO:54, SEQ ID NO:63, SEQ ID NO:72, SEQ ID NO:81, SEQ ID NO:90, SEQ ID NO:99, SEQ ID NO:108, SEQ ID NO:117, SEQ ID NO:126, SEQ ID NO:135, SEQ ID NO:144, SEQ ID NO:153, SEQ ID NO:162, SEQ ID NO:171, SEQ ID NO:180, SEQ ID NO:189, SEQ ID NO:198 , SEQ ID NO:207, SEQ ID NO:216, SEQ ID NO:225, SEQ ID NO:234, SEQ ID NO:243, SEQ ID NO:252, SEQ ID NO:261, SEQ ID NO:270, SEQ ID NO:270 ID NO: 279, SEQ ID NO:9,
- the present invention relates to a nucleic acid molecule encoding the nanobody against the novel coronavirus of the present invention.
- the nucleic acid of the present invention may be RNA, DNA or cDNA.
- the fourth aspect of the present invention provides an expression vector containing the polynucleotide of the third aspect of the present invention.
- the expression vector is selected from the group consisting of DNA, RNA, viral vector, plasmid, transposon, other gene transfer systems, or a combination thereof.
- the expression vector comprises a viral vector, such as lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.
- the fifth aspect of the present invention provides a host cell, the host cell contains the expression vector of the fourth aspect of the present invention, or the polynucleotide of the third aspect of the present invention is integrated into its genome.
- the host cells include prokaryotic cells or eukaryotic cells.
- the host cell is selected from the group consisting of: Escherichia coli, yeast cells, and mammalian cells.
- the sixth aspect of the present invention provides a method for producing a nanobody against a novel coronavirus, comprising the steps of:
- step (c) Optionally, purify and/or modify the nanobody against the novel coronavirus obtained in step (b).
- conjugation moiety selected from the group consisting of detectable labels, drugs, cytokines, radionuclides, enzymes, gold nanoparticles/nanorods, nanomagnetic particles, viral coat proteins or VLPs, or combinations thereof.
- the radionuclide includes:
- a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and/or
- a therapeutic isotope selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd- 103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb- 177, or a combination thereof.
- the coupling moiety is a detectable label.
- the immunoconjugate contains: a multivalent (eg, bivalent) VHH chain of the nanobody against the novel coronavirus according to the first aspect of the present invention.
- the multivalent means that the amino acid sequence of the immunoconjugate comprises multiple repeats of the nanobody VHH chain directed against the novel coronavirus as described in the first aspect of the present invention.
- the eighth aspect of the present invention provides the nanobody against the novel coronavirus described in the first aspect of the present invention, or the use of the antibody against the novel coronavirus as described in the second aspect of the present invention, for preparing: (1) Drugs for the prevention and/or treatment of diseases caused by novel coronavirus SARS-CoV2 infection; (2) Reagents for detection of novel coronavirus SARS-CoV2.
- the reagent shown is a diagnostic reagent, preferably, the diagnostic reagent is a detection sheet or a detection plate.
- the diagnostic reagent is used for: detecting the novel coronavirus SARS-CoV2 S protein or a fragment thereof in the sample.
- the ninth aspect of the present invention provides a pharmaceutical composition, the pharmaceutical composition contains:
- Nanobody against novel coronavirus according to the first aspect of the present invention is a Nanobody against novel coronavirus according to the second aspect of the present invention, or immunization according to the seventh aspect of the present invention conjugate;
- the tenth aspect of the present invention provides a recombinant protein, the recombinant protein has:
- the tag sequence includes Fc tag, HA tag and 6His tag.
- the recombinant protein specifically binds to SARS-CoV2.
- the eleventh aspect of the present invention provides a kit containing the nanobody against the novel coronavirus as described in the first aspect of the present invention, or the nanobody directed against the novel coronavirus as described in the second aspect of the present invention
- the antibody of the novel coronavirus, or the immunoconjugate according to the seventh aspect of the present invention is provided.
- a method for preventing and/or treating diseases caused by novel coronavirus SARS-CoV2 infection comprising administering to a subject in need the method described in the first aspect of the present invention Nanobodies against novel coronavirus, antibodies against novel coronavirus according to the second aspect of the present invention, or immunoconjugates according to the seventh aspect of the present invention.
- the subject includes mammals, such as humans.
- the disease caused by the novel coronavirus SARS-CoV2 infection is pneumonia.
- a method for in vitro detection of novel coronavirus SARS-CoV2, novel coronavirus SARS-CoV2 S protein, or fragments thereof in a sample comprising the steps of:
- the detection includes diagnostic or non-diagnostic.
- the sample is a blood sample or a throat swab sample, or a sample from other tissues and organs.
- a method for preparing a recombinant polypeptide wherein the recombinant polypeptide is a nanobody against a novel coronavirus as described in the first aspect of the present invention, a nanobody as described in the second aspect of the present invention
- the antibody against the new coronavirus includes:
- Figures 1A, 1B, 1C, and 1D show the detection results of FACS detection of candidate antibodies for ACE2/SARS-CoV2 S-RBD blocking activity, respectively.
- Figure 2 shows the results of ELISA detection of candidate nanobodies binding to SARS-CoV1 S-RBD.
- Figure 3 shows the results of ELISA detection of candidate nanobodies binding to SARS-WIV1 S-RBD.
- Figure 4 shows the results of ELISA detection of candidate Nanobodies binding to MERS S-RBD.
- Figures 5A, 5B, 5C, 5D, 5E, 5F, 5G, 5H, 5I, 5J, 5K, 5L, 5M and 5N show ELISA detection of the binding of candidate Nanobodies to different S protein mutants, respectively.
- Figure 6 shows the results of FACS detection of the blocking activity of candidate nanobodies against SARS-WIV1 S-RBD/ACE2.
- Figures 7A, 7B, 7C, 7D, 7E, 7F, 7G and 7H respectively show the results of FACS detection of the blocking activity of candidate Nanobodies on the interaction of different S protein mutants with ACE2.
- Figure 8 shows the detection results of the neutralizing activity of candidate Nanobodies against euviruses.
- Figure 9 shows the results of FACS detection of the blocking activity of candidate nanobody combinations against ACE2/SARS-CoV2 S-RBD.
- the present invention utilizes human SARS-CoV2 S protein to immunize camels to obtain a high-quality immune nanobody gene library. Then, the SARS-CoV2 S protein molecule was coupled to the ELISA plate to display the correct spatial structure of the SARS-CoV2 S protein. This form of antigen was used to screen the immune nanobody gene library (camel heavy chain antibody phage display gene) using phage display technology. library), thereby obtaining the SARS-Cov2 S protein-specific nanobody gene.
- the relevant experimental results show that the nanobody against the novel coronavirus obtained by the present invention can effectively bind to the SARS-CoV2 S protein while blocking the interaction with its ligand. The present invention has been completed on this basis.
- the terms “antibody of the present invention”, “antibody of the present invention”, “nanobody against novel coronavirus of the present invention”, “nanobody against novel coronavirus of the present invention”, “nanobody against novel coronavirus” “Antibody” and “nanobody against novel coronavirus” have the same meaning and can be used interchangeably, and both refer to antibodies that specifically recognize and bind to SARS-CoV2 S protein (including human SARS-CoV2 S protein).
- Each numerical value in the table represents the sequence number, that is, "1" represents “SEQ ID NO: 1", and the sequence numbers of CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4 shown in the table are the numbers of their amino acid sequences.
- single domain antibody As used herein, the terms “single domain antibody”, “VHH”, “nanobody”, “single domain antibody” (sdAb, or nanobody) have the same meaning and are used interchangeably, It refers to cloning the variable region of the antibody heavy chain to construct a single-domain antibody (VHH) composed of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete functions. Usually, an antibody that naturally lacks light chain and heavy chain constant region 1 (CH1) is obtained first, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting of only one heavy chain variable region.
- VHH single-domain antibody
- CH1 light chain and heavy chain constant region 1
- variable means that certain portions of the variable regions of an antibody differ in sequence that contribute to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved parts of the variable regions are called the framework regions (FRs).
- CDRs complementarity determining regions
- FRs framework regions
- the variable domains of native heavy and light chains each contain four FR regions, which are generally in a b-sheet configuration, connected by three CDRs that form linking loops, and in some cases may form part of the b-sheet structure.
- immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules combined with the antibodies of the present invention or fragments thereof to form the conjugate.
- the present invention also includes cell surface markers or antigens that bind to the nanobodies against the novel coronavirus or fragments thereof.
- the heavy chain variable region of the antibody includes three complementarity determining regions CDR1, CDR2, and CDR3.
- the present invention also provides other protein or fusion expression products with the antibodies of the present invention.
- the present invention includes any protein or protein conjugate and fusion expression product (ie, immunoconjugate and fusion expression product) having a variable region-containing heavy chain, as long as the variable region is associated with the heavy chain of an antibody of the invention
- the variable regions are identical or at least 90% homologous, preferably at least 95% homologous.
- variable regions which are separated into four framework regions (FRs), four FR amino acids
- FRs framework regions
- FRs framework regions
- the sequence is relatively conservative and does not directly participate in the binding reaction.
- CDRs form a circular structure, and the ⁇ -sheets formed by the FRs in between are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody.
- Which amino acids make up the FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type.
- variable regions of the heavy chains of the antibodies of the invention are of particular interest because at least some of them are involved in binding antigen. Accordingly, the present invention includes those molecules having CDR-bearing antibody heavy chain variable regions, as long as their CDRs have greater than 90% (preferably greater than 95%, optimally greater than 98%) homology to the CDRs identified herein sex.
- fragment refers to polypeptides that retain substantially the same biological function or activity of an antibody of the invention.
- a polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide with another compound (such as a compound that prolongs the half-life of a polypeptide, e.g.
- polyethylene glycol polyethylene glycol
- an additional amino acid sequence fused to the polypeptide sequence such as a leader sequence or a secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or with 6His-tagged fusion protein.
- the antibody of the present invention refers to a polypeptide comprising the above-mentioned CDR region with SARS-CoV2 S protein binding activity.
- the term also includes variant forms of the polypeptides comprising the above-mentioned CDR regions having the same function as the antibodies of the present invention. These variants include (but are not limited to): deletion of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitution with amino acids of similar or similar properties generally does not alter the function of the protein. As another example, the addition of one or more amino acids to the C-terminus and/or N-terminus generally does not alter the function of the protein.
- the term also includes active fragments and active derivatives of the antibodies of the invention.
- Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNAs capable of hybridizing with the DNA encoding the antibody of the present invention under conditions of high or low stringency
- the encoded protein, and the polypeptide or protein obtained using the antiserum against the antibody of the present invention are included in the polypeptide.
- the invention also provides other polypeptides, such as fusion proteins comprising antibodies or fragments thereof.
- the present invention also includes fragments of the antibodies of the present invention.
- the fragment has at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of an antibody of the invention.
- “conservative variants of the antibody of the present invention” means that compared with the amino acid sequence of the antibody of the present invention, there are at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3
- the amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
- These conservatively variant polypeptides are best produced by amino acid substitutions according to Table 2.
- the present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof.
- the polynucleotides of the present invention may be in the form of DNA or RNA.
- DNA forms include cDNA, genomic DNA or synthetic DNA.
- DNA can be single-stranded or double-stranded.
- DNA can be the coding or non-coding strand.
- Polynucleotides encoding mature polypeptides of the invention include: coding sequences encoding only the mature polypeptide; coding sequences and various additional coding sequences for the mature polypeptide; coding sequences (and optional additional coding sequences) for the mature polypeptide and non-coding sequences .
- polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide or a polynucleotide that also includes additional coding and/or non-coding sequences.
- the present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
- the present invention relates to polynucleotides that are hybridizable under stringent conditions to the polynucleotides of the present invention.
- stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, more Hybridization occurs when it is more than 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
- the full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
- a feasible method is to use artificial synthesis to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments of very long sequences are obtained by synthesizing multiple small fragments followed by ligation.
- the coding sequence of the heavy chain and the expression tag (such as 6His) can also be fused together to form a fusion protein.
- Biomolecules nucleic acids, proteins, etc.
- Biomolecules include biomolecules in isolated form.
- DNA sequences encoding the proteins of the present invention can be obtained entirely by chemical synthesis.
- This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.
- mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
- the present invention also relates to vectors comprising suitable DNA sequences as described above together with suitable promoter or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.
- the obtained transformants can be cultured by conventional methods to express the polypeptides encoded by the genes of the present invention.
- the medium used in the culture can be selected from various conventional media depending on the host cells used. Cultivation is carried out under conditions suitable for growth of the host cells. After the host cells have grown to an appropriate cell density, the promoter of choice is induced by a suitable method (eg, temperature switching or chemical induction), and the cells are cultured for an additional period of time.
- recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
- recombinant proteins can be isolated and purified by various isolation methods utilizing their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic disruption, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
- Antibodies of the invention may be used alone, or may be conjugated or conjugated to a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.
- Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or those capable of producing detectable products. enzymes.
- Coronaviruses have single-stranded, non-segmented, positive-polarity RNA genomes, and their viruses contain 4 major structural proteins: nucleocapsid (N) protein, transmembrane (M) protein, envelope (E) protein, and spine Spike (S) protein.
- N nucleocapsid
- M transmembrane
- E envelope
- S spine Spike
- the spike protein S plays a key role in virus attachment, fusion, entry, and spread, including the S1 subunit at the N-terminal responsible for viral receptor binding and the C-terminal S2 subunit responsible for virus-cell membrane fusion.
- S1 can be subdivided into N- Terminal domain (NTD) and receptor binding domain (RBD).
- the coronavirus first binds to the cell membrane receptor (ACE2/DPP4) through the viral RBD, triggering the conformational change of the S2 subunit, and the virus fuses to invade the cell.
- ACE2/DPP4 cell membrane receptor
- a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms/kg body weight, and in most cases no more than about 50 mg/kg body weight, Preferably the dose is about 10 micrograms/kg body weight to about 10 mg/kg body weight.
- the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.
- Colloidal gold labeling can be performed using methods known to those skilled in the art.
- the antibody against the SARS-CoV2 S protein is labeled with colloidal gold to obtain an antibody labeled with colloidal gold.
- the nanobody against the novel coronavirus of the present invention can effectively bind the SARS-CoV2 S protein.
- the present invention also relates to a method for detecting the SARS-CoV2 S protein.
- the method steps are roughly as follows: obtaining a cell and/or tissue sample; dissolving the sample in a medium; detecting the level of SARS-CoV2 S protein in the solubilized sample.
- the present invention also provides a kit containing the antibody (or fragment thereof) or detection plate of the present invention.
- the kit further includes a container, an instruction manual, a buffer, and the like.
- the present invention also provides a detection kit for detecting the level of SARS-CoV2 S protein
- the kit includes an antibody that recognizes the SARS-CoV2 S protein, a lysis medium for dissolving the sample, general reagents and buffers required for detection, Such as various buffers, detection labels, detection substrates, etc.
- the detection kit may be an in vitro diagnostic device.
- the antibody of the present invention has higher neutralizing activity to SARS-CoV2 true virus
- the quality of the antibody of the present invention is stable before and after atomization.
- control antibody VHH72 is derived from the published article Daniel Wrapp et al. Structural basis for the effective neutralization of betaconavirus by single-domain Camelid antibodies [J]. Cell, 2020, 181(6).
- the library capacity of the constructed library was 4.7 ⁇ 10 9 CFU, 3.3 ⁇ 10 9 CFU, 1.3 ⁇ 10 9 CFU and 1.3 ⁇ 10 8 CFU, and the insertion rates were 100%, 91.7%, 95.8% and 95.8%, respectively.
- library screening was performed, and phage enrichment with antibody genes was obtained from 4 libraries after 3 rounds of screening. 400 clones were selected from each library for PE-ELISA identification, the obtained positive clones were sequenced, and 206 nanobodies with different sequences were finally obtained. The supernatant was selected and the Nanobodies (35 strains) with blocking activity were initially screened for expression in E. coli.
- the expression method see the method description in Example 4 of Patent 2018101521076.
- Example 3 Identification of the binding activity of candidate Nanobodies to S protein of different types of viruses
- Example 5 Activity detection of candidate nanobodies blocking SARS WIV1 S-RBD and ACE2
- Example 6 Activity detection of candidate nanobodies blocking different S protein mutants and ACE2
- the Vero E6 cells at 1.5E5 cells / per well were seeded into 24-well plates, placed 37 °C, incubated overnight in 5% CO 2 incubator.
- Antibodies were serially diluted in DMEM medium containing 2% FBS (2-DMEM). Dilute the SARS-CoV2 virus solution with 2% FBS+DMEM medium.
- the medium of Vero cells in the 24-well plate was removed, and 200 ⁇ L of antibody-virus mixture was added.
- the plates were placed in 37 °C 5% CO 2 incubator for one hour.
Abstract
本发明公开了一种针对新型冠状病毒的纳米抗体及其应用。具体地，本发明提供了一种抗针对新型冠状病毒的纳米抗体及其序列。本发明还提供了编码上述纳米抗体、相应的表达载体和能够表达该纳米抗体的宿主细胞，以及本发明纳米抗体的生产方法。本发明纳米抗体能够特异性结合人SARS-CoV2；本发明纳米抗体具有良好的ACE2/SARS-CoV2 S-RBD阻断活性；本发明纳米抗体能够结合新冠病毒的多种突变体；本发明纳米抗体对SARS-CoV2真病毒中和活性较高；本发明抗体经雾化前后质量稳定。
Description
本发明涉及生物医学或生物制药技术领域，更具体地涉及一种针对新型冠状病毒的纳米抗体及其应用。
冠状病毒(CoV)具有单链、非节段的正极性RNA基因组，其病毒含有4种主要结构蛋白：核衣壳(N)蛋白、跨膜(M)蛋白、包膜(E)蛋白和刺突(S)蛋白。其中刺突蛋白S对病毒附着、融合、进入、传播发挥关键作用，包含N端负责病毒受体结合的S1亚基和C端负责病毒-细胞膜融合的S2亚基，S1可细分为N-末端结构域(NTD)和受体结合结构域(RBD)。
新冠病毒(SARS-CoV2)是新型冠状病毒肺炎(COVID-19)的病原体。中和抗体被认为是对抗COVID-19候选疗法。就其原理来说，新冠病毒通过其表面的刺突糖蛋白(S蛋白)识别并结合宿主细胞表面受体，而中和抗体正是靶向该蛋白。中和抗体药物注射进人体后，能抢先与新冠病毒的刺突蛋白(S蛋白)结合，使病毒无法感染人体细胞，从而被免疫***清除。
众多抗体药物研发企业和高校院所都竞相开展了中和抗体药物的研发，靶点基本都是新冠病毒的S蛋白，且绝大比例研究集中在S蛋白的RBD结合区域。从3月中旬第一个新冠病毒中和抗体问世，至今已经有多个具备真病毒中和活性的中和抗体发表。复旦大学应天雷组以及中国医学科学院病原生物学研究所均公布了针对新型冠状病毒的纳米抗体。纳米抗体作为新一代抗体诊断及治疗中的新兴力量，具有稳定性高、人源化简单、生产快、成本低、易于纯化等特点，在免疫实验、诊断与治疗中，发挥着超乎想象的巨大功能。同时，纳米抗体由于体积小而理化性质优异，可开发为雾化吸入制剂，理论上尤其适用于新冠肺炎等呼吸***疾病的治疗。
针对目前尚无治疗新冠肺炎的特效药的现状，研发一款对ACE2/S-RBD具备较高阻断活性，且能够与多种病毒突变体结合的纳米抗体，将具备极高的市场价值及临床应用价值。
发明内容
本发明的目的在于提供一种针对新型冠状病毒的纳米抗体及其应用。
本发明的第一方面，提供了一种针对新型冠状病毒的纳米抗体。
在另一优选例中，所述针对新型冠状病毒的纳米抗体能够特异性结合SARS-CoV2。
在另一优选例中，所述针对新型冠状病毒的纳米抗体能够特异性结合SARS-Cov2 S蛋白。
在另一优选例中，所述针对新型冠状病毒的纳米抗体的互补决定区CDR为选自下组的一种或多种：
(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；
(2)SEQ ID NO:10所示的CDR1、SEQ ID NO:11所示的CDR2、和SEQ ID NO:12所示的 CDR12；
(3)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；
(4)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；
(5)SEQ ID NO:37所示的CDR1、SEQ ID NO:38所示的CDR2、和SEQ ID NO:39所示的CDR12；
(6)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；
(7)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；
(8)SEQ ID NO:64所示的CDR1、SEQ ID NO:65所示的CDR2、和SEQ ID NO:66所示的CDR12；
(9)SEQ ID NO:73所示的CDR1、SEQ ID NO:74所示的CDR2、和SEQ ID NO:75所示的CDR3；
(10)SEQ ID NO:82所示的CDR1、SEQ ID NO:83所示的CDR2、和SEQ ID NO:84所示的CDR3；
(11)SEQ ID NO:91所示的CDR1、SEQ ID NO:92所示的CDR2、和SEQ ID NO:93所示的CDR12；
(12)SEQ ID NO:100所示的CDR1、SEQ ID NO:101所示的CDR2、和SEQ ID NO:102所示的CDR3；
(13)SEQ ID NO:109所示的CDR1、SEQ ID NO:110所示的CDR2、和SEQ ID NO:111所示的CDR3；
(14)SEQ ID NO:118所示的CDR1、SEQ ID NO:119所示的CDR2、和SEQ ID NO:120所示的CDR12；
(15)SEQ ID NO:127所示的CDR1、SEQ ID NO:128所示的CDR2、和SEQ ID NO:129所示的CDR3；
(16)SEQ ID NO:136所示的CDR1、SEQ ID NO:137所示的CDR2、和SEQ ID NO:138所示的CDR3；
(17)SEQ ID NO:145所示的CDR1、SEQ ID NO:146所示的CDR2、和SEQ ID NO:47所示的CDR12；
(18)SEQ ID NO:154所示的CDR1、SEQ ID NO:155所示的CDR2、和SEQ ID NO:156所示的CDR3；
(19)SEQ ID NO:163所示的CDR1、SEQ ID NO:164所示的CDR2、和SEQ ID NO:165所示的CDR3；
(20)SEQ ID NO:172所示的CDR1、SEQ ID NO:173所示的CDR2、和SEQ ID NO:174所 示的CDR12；
(21)SEQ ID NO:181所示的CDR1、SEQ ID NO:182所示的CDR2、和SEQ ID NO:183所示的CDR3；
(22)SEQ ID NO:190所示的CDR1、SEQ ID NO:191所示的CDR2、和SEQ ID NO:192所示的CDR3；
(23)SEQ ID NO:199所示的CDR1、SEQ ID NO:200所示的CDR2、和SEQ ID NO:201所示的CDR12；
(24)SEQ ID NO:208所示的CDR1、SEQ ID NO:209所示的CDR2、和SEQ ID NO:210所示的CDR3；
(25)SEQ ID NO:217所示的CDR1、SEQ ID NO:218所示的CDR2、和SEQ ID NO:219所示的CDR3；
(26)SEQ ID NO:226所示的CDR1、SEQ ID NO:227所示的CDR2、和SEQ ID NO:228所示的CDR12；
(27)SEQ ID NO:235所示的CDR1、SEQ ID NO:236所示的CDR2、和SEQ ID NO:237所示的CDR3；
(28)SEQ ID NO:244所示的CDR1、SEQ ID NO:245所示的CDR2、和SEQ ID NO:246所示的CDR3；
(29)SEQ ID NO:253所示的CDR1、SEQ ID NO:254所示的CDR2、和SEQ ID NO:255所示的CDR12；
(30)SEQ ID NO:262所示的CDR1、SEQ ID NO:263所示的CDR2、和SEQ ID NO:264所示的CDR3；
(31)SEQ ID NO:271所示的CDR1、SEQ ID NO:272所示的CDR2、和SEQ ID NO:273所示的CDR3；
(32)SEQ ID NO:280所示的CDR1、SEQ ID NO:281所示的CDR2、和SEQ ID NO:282所示的CDR12；
(33)SEQ ID NO:289所示的CDR1、SEQ ID NO:290所示的CDR2、和SEQ ID NO:291所示的CDR3；
(34)SEQ ID NO:298所示的CDR1、SEQ ID NO:299所示的CDR2、和SEQ ID NO:300所示的CDR12；和
(35)SEQ ID NO:307所示的CDR1、SEQ ID NO:308所示的CDR2、和SEQ ID NO:309所示的CDR3。
在另一优选例中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个(如1-3个，较佳地1-2个，更佳地1个)氨基酸并能保留与新型冠状病毒的S蛋白特异性结合能力的衍生序列。
在另一优选例中，所述针对新型冠状病毒的纳米抗体能够与新型冠状病毒的S蛋白特异性结合。
在另一优选例中，所述的CDR1、CDR2和CDR3由VHH链的框架区FR1、FR2、FR3和FR4所隔开。
在另一优选例中，所述针对新型冠状病毒的纳米抗体还包括框架区FR。
在另一优选例中，所述的框架区FR为选自下组的一种或多种：
(1)SEQ ID NO:4所示的FR1、SEQ ID NO:5所示的FR2、SEQ ID NO:6所示的FR3、和SEQ ID NO:7所示的FR4；
(2)SEQ ID NO:13所示的FR1、SEQ ID NO:14所示的FR2、SEQ ID NO:15所示的FR3、和SEQ ID NO:16所示的FR4；
(3)SEQ ID NO:22所示的FR1、SEQ ID NO:23所示的FR2、SEQ ID NO:24所示的FR3、和SEQ ID NO:25所示的FR4；
(4)SEQ ID NO:31所示的FR1、SEQ ID NO:32所示的FR2、SEQ ID NO:33所示的FR3、和SEQ ID NO:34所示的FR4；
(5)SEQ ID NO:40所示的FR1、SEQ ID NO:41所示的FR2、SEQ ID NO:42所示的FR3、和SEQ ID NO:43所示的FR4；
(6)SEQ ID NO:49所示的FR1、SEQ ID NO:50所示的FR2、SEQ ID NO:51所示的FR3、和SEQ ID NO:52所示的FR4；
(7)SEQ ID NO:58所示的FR1、SEQ ID NO:59所示的FR2、SEQ ID NO:60所示的FR3、和SEQ ID NO:61所示的FR4；
(8)SEQ ID NO:67所示的FR1、SEQ ID NO:68所示的FR2、SEQ ID NO:69所示的FR3、和SEQ ID NO:70所示的FR4；
(9)SEQ ID NO:76所示的FR1、SEQ ID NO:77所示的FR2、SEQ ID NO:78所示的FR3、和SEQ ID NO:79所示的FR4；
(10)SEQ ID NO:85所示的FR1、SEQ ID NO:86所示的FR2、SEQ ID NO:87所示的FR3、和SEQ ID NO:88所示的FR4；
(11)SEQ ID NO:94所示的FR1、SEQ ID NO:95所示的FR2、SEQ ID NO:96所示的FR3、和SEQ ID NO:97所示的FR4；
(12)SEQ ID NO:103所示的FR1、SEQ ID NO:104所示的FR2、SEQ ID NO:105所示的FR3、和SEQ ID NO:106所示的FR4；
(13)SEQ ID NO:112所示的FR1、SEQ ID NO:113所示的FR2、SEQ ID NO:114所示的FR3、和SEQ ID NO:115所示的FR4；
(14)SEQ ID NO:121所示的FR1、SEQ ID NO:122所示的FR2、SEQ ID NO:123所示的FR3、和SEQ ID NO:124所示的FR4；
(15)SEQ ID NO:130所示的FR1、SEQ ID NO:131所示的FR2、SEQ ID NO:132所示的FR3、和SEQ ID NO:133所示的FR4；
(16)SEQ ID NO:139所示的FR1、SEQ ID NO:140所示的FR2、SEQ ID NO:141所示的FR3、和SEQ ID NO:142所示的FR4；
(17)SEQ ID NO:148所示的FR1、SEQ ID NO:149所示的FR2、SEQ ID NO:150所示的FR3、和SEQ ID NO:151所示的FR4；
(18)SEQ ID NO:157所示的FR1、SEQ ID NO:158所示的FR2、SEQ ID NO:159所示的FR3、和SEQ ID NO:160所示的FR4；
(19)SEQ ID NO:166所示的FR1、SEQ ID NO:167所示的FR2、SEQ ID NO:168所示的FR3、和SEQ ID NO:169所示的FR4；
(20)SEQ ID NO:175所示的FR1、SEQ ID NO:176所示的FR2、SEQ ID NO:177所示的FR3、和SEQ ID NO:178所示的FR4；
(21)SEQ ID NO:184所示的FR1、SEQ ID NO:185所示的FR2、SEQ ID NO:186所示的FR3、和SEQ ID NO:187所示的FR4；
(22)SEQ ID NO:193所示的FR1、SEQ ID NO:194所示的FR2、SEQ ID NO:195所示的FR3、和SEQ ID NO:196所示的FR4；
(23)SEQ ID NO:202所示的FR1、SEQ ID NO:203所示的FR2、SEQ ID NO:204所示的FR3、和SEQ ID NO:205所示的FR4；
(24)SEQ ID NO:211所示的FR1、SEQ ID NO:212所示的FR2、SEQ ID NO:123所示的FR3、和SEQ ID NO:214所示的FR4；
(25)SEQ ID NO:220所示的FR1、SEQ ID NO:221所示的FR2、SEQ ID NO:222所示的FR3、和SEQ ID NO:223所示的FR4；
(26)SEQ ID NO:229所示的FR1、SEQ ID NO:230所示的FR2、SEQ ID NO:231所示的FR3、和SEQ ID NO:232所示的FR4；
(27)SEQ ID NO:238所示的FR1、SEQ ID NO:239所示的FR2、SEQ ID NO:240所示的FR3、和SEQ ID NO:241所示的FR4；
(28)SEQ ID NO:247所示的FR1、SEQ ID NO:248所示的FR2、SEQ ID NO:249所示的FR3、和SEQ ID NO:250所示的FR4；
(29)SEQ ID NO:256所示的FR1、SEQ ID NO:257所示的FR2、SEQ ID NO:258所示的FR3、和SEQ ID NO:259所示的FR4；
(30)SEQ ID NO:265所示的FR1、SEQ ID NO:266所示的FR2、SEQ ID NO:267所示的FR3、和SEQ ID NO:268所示的FR4；
(31)SEQ ID NO:274所示的FR1、SEQ ID NO:275所示的FR2、SEQ ID NO:276所示的FR3、和SEQ ID NO:277所示的FR4；
(32)SEQ ID NO:283所示的FR1、SEQ ID NO:284所示的FR2、SEQ ID NO:285所示的FR3、和SEQ ID NO:286所示的FR4；
(33)SEQ ID NO:292所示的FR1、SEQ ID NO:293所示的FR2、SEQ ID NO:294所示的FR3、和SEQ ID NO:295所示的FR4；
(34)SEQ ID NO:301所示的FR1、SEQ ID NO:302所示的FR2、SEQ ID NO:303所示的FR3、和SEQ ID NO:304所示的FR4；和
(35)SEQ ID NO:310所示的FR1、SEQ ID NO:311所示的FR2、SEQ ID NO:312所示的FR3、和SEQ ID NO:313所示的FR4。
在另一优选例中，所述的针对新型冠状病毒的纳米抗体的VHH链的氨基酸序列选自下组：SEQ ID NO:8、SEQ ID NO:17、SEQ ID NO:26、SEQ ID NO:35、SEQ ID NO:44、SEQ ID NO:53、SEQ ID NO:62、SEQ ID NO:71、SEQ ID NO:80、SEQ ID NO:89、SEQ ID NO:98、SEQ ID NO:107、SEQ ID NO:116、SEQ ID NO:125、SEQ ID NO:134、SEQ ID NO:143、SEQ ID NO:152、SEQ ID NO:161、SEQ ID NO:170、SEQ ID NO:179、SEQ ID NO:188、SEQ ID NO:197、SEQ ID NO:206、SEQ ID NO:215、SEQ ID NO:224、SEQ ID NO:233、SEQ ID NO:242、SEQ ID NO:251、SEQ ID NO:260、SEQ ID NO:269、SEQ ID NO:278、SEQ ID NO:287、SEQ ID NO:296、SEQ ID NO:305、SEQ ID NO:314、或其组合。
在另一优选例中，所述的针对新型冠状病毒的纳米抗体包括人源化抗体、骆驼源抗体、嵌合抗体。
本发明的第二方面，提供一种针对新型冠状病毒的抗体，所述抗体包括一个或多个如本发明第一方面所述的针对新型冠状病毒的纳米抗体的VHH链。
在另一优选例中，所述的针对新型冠状病毒的纳米抗体的VHH链的氨基酸序列选自下组：SEQ ID NO:8、SEQ ID NO:17、SEQ ID NO:26、SEQ ID NO:35、SEQ ID NO:44、SEQ ID NO:53、SEQ ID NO:62、SEQ ID NO:71、SEQ ID NO:80、SEQ ID NO:89、SEQ ID NO:98、SEQ ID NO:107、SEQ ID NO:116、SEQ ID NO:125、SEQ ID NO:134、SEQ ID NO:143、SEQ ID NO:152、SEQ ID NO:161、SEQ ID NO:170、SEQ ID NO:179、SEQ ID NO:188、SEQ ID NO:197、SEQ ID NO:206、SEQ ID NO:215、SEQ ID NO:224、SEQ ID NO:233、SEQ ID NO:242、SEQ ID NO:251、SEQ ID NO:260、SEQ ID NO:269、SEQ ID NO:278、SEQ ID NO:287、SEQ ID NO:296、SEQ ID NO:305、SEQ ID NO:314、或其组合。
在另一优选例中，所述的针对新型冠状病毒的抗体，可以为单体、二价抗体、和/或多价抗体。
本发明的第三方面，提供一种多核苷酸，所述多核苷酸编码选自下组的蛋白质：如本发明第一方面所述的针对新型冠状病毒的纳米抗体，或本发明第二方面所述的抗体。
在另一优选例中，所述多核苷酸的序列选自下组：SEQ ID NO:9、SEQ ID NO:18、SEQ ID NO:27、SEQ ID NO:36、SEQ ID NO:45、SEQ ID NO:54、SEQ ID NO:63、SEQ ID NO:72、SEQ ID NO:81、SEQ ID NO:90、SEQ ID NO:99、SEQ ID NO:108、SEQ ID NO:117、SEQ ID NO:126、SEQ ID NO:135、SEQ ID NO:144、SEQ ID NO:153、SEQ ID NO:162、SEQ ID NO:171、SEQ ID NO:180、SEQ ID NO:189、SEQ ID NO:198、SEQ ID NO:207、SEQ ID NO:216、SEQ ID NO:225、SEQ ID NO:234、SEQ ID NO:243、SEQ ID NO:252、SEQ ID NO:261、SEQ ID NO:270、SEQ ID NO:279、SEQ ID NO:288、SEQ ID NO:297、SEQ ID NO:306、SEQ ID NO:315、或其组合。
在另一优选例中，本发明涉及编码本发明的针对新型冠状病毒的纳米抗体的核酸分子。本发明的核酸可为RNA、DNA或cDNA。
本发明的第四方面，提供一种表达载体，所述表达载体含有本发明的第三方面所述的多核苷酸。
在另一优选例中，所述的表达载体选自下组：DNA、RNA、病毒载体、质粒、转座子、其他基因转移***、或其组合。优选地，所述表达载体包括病毒载体，如慢病毒、腺病毒、AAV病毒、逆转录病毒、或其组合。
本发明的第五方面，提供一种宿主细胞，所述宿主细胞含有本发明的第四方面所述的表达载体，或其基因组中整合有本发明的第三方面所述的多核苷酸。
在另一优选例中，所述的宿主细胞包括原核细胞或真核细胞。
在另一优选例中，所述的宿主细胞选自下组：大肠杆菌、酵母细胞、哺乳动物细胞。
本发明的第六方面，提供一种产生针对新型冠状病毒的纳米抗体的方法，包括步骤：
(a)在适合产生纳米抗体的条件下，培养如本发明的第五方面所述的宿主细胞，从而获得含针对新型冠状病毒的纳米抗体的培养物；
(b)从所述培养物中分离和/或回收所述的针对新型冠状病毒的纳米抗体；和
(c)任选地，对步骤(b)获得的针对新型冠状病毒的纳米抗体进行纯化和/或修饰。
本发明的第七方面，提供一种免疫偶联物，所述免疫偶联物含有：
(a)本发明第一方面所述的针对新型冠状病毒的纳米抗体，或如本发明第二发明所述的针对新型冠状病毒的抗体；和
(b)选自下组的偶联部分：可检测标记物、药物、细胞因子、放射性核素、酶、金纳米颗粒/纳米棒、纳米磁粒、病毒外壳蛋白或VLP，或其组合。
在另一优选例中，所述的放射性核素包括：
(i)诊断用同位素，所述的诊断用同位素选自下组：Tc-99m、Ga-68、F-18、I-123、I-125、I-131、In-111、Ga-67、Cu-64、Zr-89、C-11、Lu-177、Re-188，或其组合；和/或
(ii)治疗用同位素，所述的治疗用同位素选自下组：Lu-177、Y-90、Ac-225、As-211、Bi-212、Bi-213、Cs-137、Cr-51、Co-60、Dy-165、Er-169、Fm-255、Au-198、Ho-166、I-125、I-131、Ir-192、Fe-59、Pb-212、Mo-99、Pd-103、P-32、K-42、Re-186、Re-188、Sm-153、Ra223、Ru-106、Na24、Sr89、Tb-149、Th-227、Xe-133、Yb-169、Yb-177，或其组合。
在另一优选例中，所述偶联部分为可检测标记物。
在另一优选例中，所述偶联部分选自下组：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂，或能够产生可检测产物的酶、放射性核素、生物毒素、细胞因子(如IL-2等)、抗体、抗体Fc片段、抗体scFv片段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))或任何形式的纳米颗粒。
在另一优选例中，所述免疫偶联物含有：多价(如二价)的如本发明的第一方面所述的针对新型冠状病毒的纳米抗体的VHH链。
在另一优选例中，所述多价是指在所述免疫偶联物的氨基酸序列中包含多个重复的如本发明的第一方面所述针对新型冠状病毒的纳米抗体VHH链。
本发明的第八方面，提供了本发明的第一方面所述的针对新型冠状病毒的纳米抗体，或如本发明的第二方面所述的针对新型冠状病毒的抗体的用途，用于制备：(1)预防和/或治疗新型冠状病毒SARS-CoV2感染引起的疾病的药物；(2)检测新型冠状病毒SARS-CoV2的试剂。
在另一优选例中，所示试剂为诊断试剂，较佳地，所述的诊断试剂为检测片或检测板。
在另一优选例中，所述诊断试剂用于：检测样品中的新型冠状病毒SARS-CoV2 S蛋白或其片段。
本发明的第九方面，提供了一种药物组合物，所述药物组合物含有：
(i)如本发明的第一方面所述的针对新型冠状病毒的纳米抗体、如本发明的第二方面所述的针对新型冠状病毒的抗体，或如本发明的第七方面所述的免疫偶联物；和
(ii)药学上可接受的载体。
本发明的第十方面，提供了一种重组蛋白，所述的重组蛋白具有：
(i)如本发明的第一方面所述的针对新型冠状病毒的纳米抗体，或如本发明的第三方面所述的针对新型冠状病毒的抗体；和
(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括Fc标签、HA标签和6His标签。
在另一优选例中，所述的重组蛋白特异性结合于SARS-CoV2。
本发明的第十一方面，提供了一种试剂盒，所述试剂盒含有如本发明的第一方面所述的针对新型冠状病毒的纳米抗体，或如本发明的第二方面所述的针对新型冠状病毒的抗体，或如本发明的第七方面所述的免疫偶联物。
在本发明的第十二方面，提供了一种预防和/或治疗新型冠状病毒SARS-CoV2感染引起的疾病的方法，所述方法包括，给需要的对象施用如本发明第一方面所述的针对新型冠状病毒的纳米抗体、如本发明的第二方面所述的针对新型冠状病毒的抗体，或如本发明的第七方面所述的免疫偶联物。
在另一优选例中，所述的对象包括哺乳动物，如人。
在另一优选例中，所述的新型冠状病毒SARS-CoV2感染引起的疾病为肺炎。
在本发明的第十三方面，提供了一种体外检测样品中新型冠状病毒SARS-CoV2、新型冠状病毒SARS-CoV2 S蛋白、或其片段的方法，所述方法包括步骤：
(1)在体外，将所述样品与如本发明第一方面所述的针对新型冠状病毒的纳米抗体、如本发明的第二方面所述的针对新型冠状病毒的抗体，或如本发明的第七方面所述的免疫偶联物接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在新型冠状病毒SARS-CoV2、新型冠状病毒SARS-CoV2 S蛋白、或其片段。
在另一优选例中，所述的检测包括诊断性的或非诊断性的。
在本发明的第十四方面，提供了一种针对新型冠状病毒感染的诊断方法，包括步骤：
(i)从诊断对象获取一样品，将所述的样品与如本发明第一方面所述的针对新型冠状病毒的纳米抗体、如本发明的第二方面所述的针对新型冠状病毒的抗体，或如本发明的第七方面所述的免疫偶联物接触；和
(ii)检测是否形成抗原-抗体复合物，其中形成复合物就表示所述的对象为新型冠状病毒确诊患者。
在另一优选例中，所述的样品为血液样品或咽拭子样品，或其他组织器官中的样品。
在本发明的第十五方面，提供了一种重组多肽的制备方法，所述的重组多肽是如本发明第一方面所述的针对新型冠状病毒的纳米抗体、如本发明的第二方面所述的针对新型冠状病毒的抗体，所述的方法包括：
(a)在适合表达的条件下，培养如本发明第五方面所述的宿主细胞；和
(b)从培养物中分离出所述的重组多肽。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
图1A、1B、1C和1D分别显示了FACS检测候选抗体对ACE2/SARS-CoV2 S-RBD阻断活性的检测检测结果。
图2显示了ELISA检测候选纳米抗体与SARS-CoV1 S-RBD结合结果。
图3显示了ELISA检测候选纳米抗体与SARS-WIV1 S-RBD结合结果。
图4显示了ELISA检测候选纳米抗体与MERS S-RBD结合结果。
图5A、5B、5C、5D、5E、5F、5G、5H、5I、5J、5K、5L、5M和5N分别显示了ELISA检测候选纳米抗体与不同S蛋白突变体的结合。
图6显示了FACS检测候选纳米抗体对SARS-WIV1 S-RBD/ACE2的阻断活性结果。
图7A、7B、7C、7D、7E、7F、7G和7H分别显示了FACS检测候选纳米抗体对不同S蛋白突变体与ACE2相互作用的阻断活性结果。
图8显示了候选纳米抗体对真病毒的中和活性检测结果。
图9显示了FACS检测候选纳米抗体组合对ACE2/SARS-CoV2 S-RBD的阻断活性结果。
本发明人通过广泛而深入的研究，经过大量的筛选，成功获得多个针对新型冠状病毒的纳米抗体。具体地，本发明利用人源的SARS-CoV2 S蛋白免疫骆驼，获得高质量的免疫纳米抗体基因文库。然后将SARS-CoV2 S蛋白分子偶联在酶标板上，展示SARS-CoV2 S蛋白的正确空间结构，以此形式的抗原利用噬菌体展示技术筛选免疫纳米抗体基因库(骆驼重链抗体噬菌体展示基因库)，从而获得了SARS-Cov2 S蛋白特异性的纳米抗体基因。此外，相关实验结果表明，本发明获得的针对新型冠状病毒的纳米抗体能够有效地与SARS- CoV2 S蛋白结合，同时阻断与其配体的相互作用。在此基础上完成了本发明。
术语
如本文所用，术语“本发明抗体”、“本发明的抗体”、“本发明的针对新型冠状病毒的纳米抗体”、“本发明针对新型冠状病毒的纳米抗体”、“针对新型冠状病毒的纳米抗体”、“针对新型冠状病毒的纳米抗体”具有相同的含义，可互换使用，均指特异性识别和结合于SARS-CoV2 S蛋白(包括人SARS-CoV2 S蛋白)的抗体。
本发明的纳米抗体的抗体编号以及对应的序列编号如下表1所示。
表1
注：表中各个数值表示序列编号，即“1”表示“SEQ ID NO:1”，表中显示的CDR1、CDR2、CDR3、FR1、FR2、FR3、FR4的序列编号为其氨基酸序列的编号。
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用，术语“单域抗体”、“VHH”、“纳米抗体(nanobody)”、“单域抗体”(single domain antibody,sdAb，或纳米抗体nanobody)具有相同的含义并可互换使用，指克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体(VHH)，它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体(VHH)。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈b-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分b折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的 依赖于抗体的细胞毒性。
如本领域技术人员所知，免疫偶联物及融合表达产物包括：药物、毒素、细胞因子(cytokine)、放射性核素、酶和其他诊断或治疗分子与本发明的抗体或其片段结合而形成的偶联物。本发明还包括与所述的针对新型冠状病毒的纳米抗体或其片段结合的细胞表面标记物或抗原。
如本文所用，术语“重链可变区”与“VH”可互换使用。
如本文所用，术语“可变区”与“互补决定区(complementarity determining region,CDR)”可互换使用。
在本发明的一个优选的实施方式中，所述抗体的重链可变区包括包括三个互补决定区CDR1、CDR2、和CDR3。
在本发明的一个优选的实施方式中，所述抗体的重链包括上述重链可变区和重链恒定区。
在本发明中，术语“本发明抗体”、“本发明蛋白”、或“本发明多肽”可互换使用，都指特异性结合SARS-CoV2 S蛋白的多肽，例如具有重链可变区的蛋白或多肽。它们可含有或不含起始甲硫氨酸。
本发明还提供了具有本发明抗体的其他蛋白质或融合表达产物。具体地，本发明包括具有含可变区的重链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联物及融合表达产物)，只要该可变区与本发明抗体的重链可变区相同或至少90％同源性，较佳地至少95％同源性。
一般，抗体的抗原结合特性可由位于重链可变区的3个特定的区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
本发明抗体的重链的可变区特别令人感兴趣，因为它们中至少部分涉及结合抗原。因此，本发明包括那些具有带CDR的抗体重链可变区的分子，只要其CDR与此处鉴定的CDR具有90％以上(较佳地95％以上，最佳地98％以上)的同源性。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标 签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明抗体指具有SARS-CoV2 S蛋白结合活性的、包括上述CDR区的多肽。该术语还包括具有与本发明抗体相同功能的、包含上述CDR区的多肽的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、***和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明抗体的活性片段和活性衍生物。
该多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。
本发明还提供了其他多肽，如包含抗体或其片段的融合蛋白。除了几乎全长的多肽外，本发明还包括了本发明抗体的片段。通常，该片段具有本发明抗体的至少约50个连续氨基酸，较佳地至少约50个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。
在本发明中，“本发明抗体的保守性变异体”指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表2进行氨基酸替换而产生。
表2
| 最初的残基 | 代表性的取代 | 优选的取代 |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。
编码本发明的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC,0.1％SDS,60℃；或(2)杂交时加有变性剂,如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上,更好是95％以上时才发生杂交。并且，可杂交的多核苷酸编码的多肽与成熟多肽有相同的生物学功能和活性。
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。此外，还可将重链的编码序列和表达标签(如6His)融合在一起，形成融合蛋白。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高 等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔,脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的抗体可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。
用于诊断目的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可与本发明抗体结合或偶联的治疗剂包括但不限于：1.放射性核素；2.生物毒；3.细胞因子如IL-2等；4.金纳米颗粒/纳米棒；5.病毒颗粒；6.脂质体；7.纳米磁粒；8.前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))等。
SARS-CoV2 S蛋白
冠状病毒(CoV)具有单链、非节段的正极性RNA基因组，其病毒含有4种主要结构蛋白：核衣壳(N)蛋白、跨膜(M)蛋白、包膜(E)蛋白和刺突(S)蛋白。其中刺突蛋白S对病毒附着、融合、进入、传播发挥关键作用，包含N端负责病毒受体结合的S1亚基和C端负责病毒-细胞膜融合的S2亚基，S1可细分为N-末端结构域(NTD)和受体结合结构域(RBD)。冠状病毒首先通过病毒RBD和细胞膜受体(ACE2/DPP4)结合，触发S2亚基构象变化，病毒融合侵入细胞。
药物组合物
本发明还提供了一种组合物。优选地，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白，以及药学上可接受的载体。通常，可将这些物质配制于 无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：腹膜内、静脉内、或局部给药。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约10微克/千克体重-约50毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约10毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
针对新型冠状病毒的纳米抗体
在本发明中，所述针对新型冠状病毒的纳米抗体包括单体、二价体(二价抗体)、四价体(四价抗体)、和/或多价体(多价抗体)。
在本发明的一个优选例中，所述针对新型冠状病毒的纳米抗体包括一条或多条具有如SEQ ID NO:8、SEQ ID NO:17、SEQ ID NO:26、SEQ ID NO:35、SEQ ID NO:44、SEQ ID NO:53、SEQ ID NO:62、SEQ ID NO:71、SEQ ID NO:80、SEQ ID NO:89、SEQ ID NO:98、SEQ ID NO:107、SEQ ID NO:116、SEQ ID NO:125、SEQ ID NO:134、SEQ ID NO:143、SEQ ID NO:152、SEQ ID NO:161、SEQ ID NO:170、SEQ ID NO:179、SEQ ID NO:188、SEQ ID NO:197、SEQ ID NO:206、SEQ ID NO:215、SEQ ID NO:224、SEQ ID NO:233、SEQ ID NO:242、SEQ ID NO:251、SEQ ID NO:260、SEQ ID NO:269、SEQ ID NO:278、SEQ ID NO:287、SEQ ID NO:296、SEQ ID NO:305、SEQ ID NO:314所示的氨基酸序列的VHH链。
标记的抗体
在本发明的一个优选例中，所述抗体带有可检测标记物。更佳地，所述的标记物选自下组：同位素、胶体金标记物、有色标记物或荧光标记物。
胶体金标记可采用本领域技术人员已知的方法进行。在本发明的一个优选的方案中，针对SARS-CoV2 S蛋白的抗体用胶体金标记，得到胶体金标记的抗体。
本发明的针对新型冠状病毒的纳米抗体能够有效结合SARS-CoV2 S蛋白。
检测方法
本发明还涉及检测SARS-CoV2 S蛋白蛋白的方法。该方法步骤大致如下：获得细胞和/或组织样本；将样本溶解在介质中；检测在所述溶解的样本中SARS-CoV2 S蛋白蛋白的水平。
在本发明的检测方法中，所使用的样本没有特别限制，代表性的例子是存在于细胞 保存液中的含细胞的样本。
试剂盒
本发明还提供了一种含有本发明的抗体(或其片段)或检测板的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。
本发明还提供了用于检测SARS-CoV2 S蛋白水平的检测试剂盒，该试剂盒包括识别SARS-CoV2 S蛋白的抗体，用于溶解样本的裂解介质，检测所需的通用试剂和缓冲液，如各种缓冲液、检测标记、检测底物等。该检测试剂盒可以是体外诊断装置。
应用
如上所述，本发明的抗体有广泛生物应用价值和临床应用价值，其应用涉及到与SARS-CoV2 S蛋白相关的疾病的诊断和治疗、基础医学研究、生物学研究等多个领域。一个优选的应用是用于针对SARS-CoV2 S蛋白的临床诊断、预防和治疗。
本发明的主要优点包括：
(a)本发明抗体能够特异性结合人SARS-CoV2；
(b)本发明抗体具有良好的ACE2/SARS-CoV2 S-RBD阻断活性；
(c)本发明抗体能够结合新冠病毒的多种突变体；
(d)本发明抗体对SARS-CoV2真病毒中和活性较高；
(e)本发明抗体经雾化前后质量稳定。
下面的具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如(Sambrook和Russell等人，分子克隆：实验室手册(Molecular Cloning-A Laboratory Manual)(第三版)(2001)CSHL出版社)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例中对照抗体VHH72来源于已发表文章Daniel Wrapp等.单域Camelid抗体有效中和betaconavirus的结构基础[J].细胞,2020,181(6)。
实施例1：SARS-CoV2 S蛋白特异性纳米抗体的筛选
为了获得特异性针对人SARS-CoV2 S蛋白的纳米抗体，首先利用哺乳动物细胞HEK293F瞬转表达人SARS-CoV2 S蛋白，经亲和纯化后用于骆驼免疫。简要地，将纯化的SARS-CoV2 S蛋白免疫两只新疆双峰驼，7次免疫结束后从骆驼外周血中分离total RNA，随后反转录和PCR扩增出VHH基因，再将VHH基因克隆至噬菌体载体pMECS上，转化至TG1中构建成噬菌体展示文库。构建的文库库容分别为4.7×10
9CFU、3.3×10
9CFU、1.3×10
9CFU、1.3×10
8CFU，***率分别为100％、91.7％、95.8％、95.8％。随后进行文库筛选，4个文库均经3轮筛选获得含抗体基因的噬菌体富集。从每个文库挑选400颗克隆进行PE-ELISA鉴定，将获得的阳性克隆进行测序，最终获得206种序列不同的纳米抗体。挑选上清液初筛具有阻断活性的的纳米抗体(35株)利用大肠杆菌表达，表达方法参见专利2018101521076实施例4中的方法描述。
实施例2：SARS-CoV2 S蛋白纳米抗体对ACE2/S-RBD的阻断活性检测
将培养好的293F/ACE2稳转细胞分装至96孔板中，每孔3E5个细胞，3000rpm离心3min去掉上清液，加入稀释好的各抗体及S-RBD-biotin蛋白孵育20min，抗体浓度从160ug/mL开始3倍梯度，共稀释12个梯度。离心弃上清，加入稀释的SA-PE抗体，4℃孵育20min。再次离心弃上清，每孔加入200uL PBS重悬细胞，流式细胞仪检测样本PE信号。
结果如图1A-1D所示，结果表明：32株候选抗体具有良好的ACE2/SARS-CoV2 S-RBD阻断活性。
实施例3：候选纳米抗体与不同类型病毒S蛋白的结合活性鉴定
将1ug/mL SARS-CoV1 S-RBD蛋白、MERS S-RBD蛋白、SARS WIV1 S-RBD蛋白包被酶标版过夜。PBST洗5遍，每孔加入300uL 1％BSA室温封闭2小时。PBST洗5遍，然后将梯度稀释的SARS-CoV2 S-RBD纳米抗体加入室温孵育1小时。PBST洗5遍，加入100uL稀释的mouse anti-HA antibody室温孵育1小时。PBST洗5遍，加入100uL碱性磷酸酶标记的抗鼠抗体，37℃放置30分钟。加入显色液显色，酶标仪在405nm处测吸光值。
纳米抗体与SARS-CoV1 S-RBD结合的结果如图2所示，所有候选纳米抗体能识别SARS-CoV-1 S-RBD。
纳米抗体与SARS-WIV1 S-RBD结合的结果如图3所示，Nb16-68和Nb11-59能够识别SARS WIV1 S-RBD。
纳米抗体与MERS S-RBD结合的结果如图4所示，所有抗体不识别MERS S-RBD。
实施例4：纳米抗体与不同S蛋白突变体的结合鉴定
根据已报到的新冠病毒在传播过程中出现的SARS-CoV2 S-RBD突变位点(V483A、G476S、A435S、R408I、V367F、N354D、V341I、Q321L、S359N、Q409E、K458R、I472V、Y508H、H519P、K378R)，将制备好的各突变体蛋白以1ug/mL包被酶标版过夜。PBST洗5遍，每孔加入300uL 1％BSA室温封闭2小时。PBST洗5遍，然后将梯度稀释的SARS-CoV2 S-RBD纳米抗体加入室温孵育1小时。PBST洗5遍，加入100uL稀释的mouse anti-HA antibody室温孵育1小时。PBST洗5遍，加入100uL碱性磷酸酶标记的抗鼠抗体，37℃放置30分钟。加入显色液显色，酶标仪在405nm处测吸光值。
结果如图5A-5N所示，Nb16-75不能与突变体N354D结合，其余候选的纳米抗体均能与多种突变体结合。
实施例5：候选纳米抗体阻断SARS WIV1 S-RBD与ACE2的活性检测
挑选阻断型纳米抗体进行SARS WIV1 S-RBD/ACE2阻断活性鉴定。将培养好的293F/ACE2稳转细胞分装至96孔板中，每孔3E5个细胞，3000rpm离心3min去掉上清液，加入稀释好的各抗体及SARS WIV1 S-RBD-biotin蛋白孵育20min。离心弃上清，加入稀释的SA-PE抗体，4℃孵育20min。再次离心弃上清，每孔加入200uL PBS重悬细胞，流式细胞仪检测样本PE信号。
结果如图6所示，候选抗体Nb11-59和Nb16-68具有良好的阻断活性，且阻断活性优于对照抗体VHH72。
实施例6：候选纳米抗体阻断不同S蛋白突变体与ACE2的活性检测
挑选阻断型纳米抗体进行S-RBD突变体阻断活性鉴定。将不同突变体蛋白进行生物素标记，用于阻断活性检测。将培养好的293F/ACE2稳转细胞分装至96孔板中，每孔3E5个细胞，3000rpm离心3min去掉上清液，加入稀释好的各抗体及生物素标记的的突变体蛋白孵育20min。离心弃上清，加入稀释的SA-PE抗体，4℃孵育20min。再次离心弃上清，每孔加入200uL PBS重悬细胞，流式细胞仪检测样本PE信号。
结果如图7A-7H所示，7个SARS-CoV2-S-RBD纳米抗体均能阻断多种突变体与293F/ACE2细胞的结合，且都阻断效果均优于对照抗体VHH72。
实施例7：候选抗体与Protein A的结合测定
将1mg/mL候选纳米抗体与100uL Protein A亲和柱混合室温孵育30min，然后离心取上清测定蛋白含量。
结果如下表3所示：多个抗体能与Protein A亲和柱结合。
表3纳米抗体与Protein A结合效率
实施例8：候选纳米抗体的Tm值测定
取2μL 200×SYPRO Orange protein Gel stain染料与38μL 1mg/mL待检纳米抗体混合，冰上避光孵育15分钟。每个供试品3个复孔，每孔10μL加入PCR管中，置于Q-PCR仪上(25℃保持30s，Melt curve 25℃to 98℃，每5s增加0.5℃，扫描模式为FAM)检测。
结果统计如下表4所示。
表4纳米抗体的Tm值
| 抗体编号 | Tm(℃) | 抗体编号 | Tm(℃) |
| Nb3-85 | 57℃ | Nb15-16 | 57℃ |
| Nb4-14 | 57℃ | Nb15-25 | 57℃ |
| Nb4-43 | 57℃ | Nb15-41 | 57℃ |
| Nb7-17 | 57℃ | Nb15-44 | 58℃ |
| Nb8-65 | 57℃ | Nb15-48 | 57℃ |
| Nb8-87 | 58℃ | Nb15-51 | 58℃ |
| Nb9-22 | 52℃ | Nb15-59 | 58℃ |
| Nb9-56 | 57℃ | Nb15-61 | 58℃ |
| Nb10-46 | 57℃ | Nb15-67 | 58℃ |
| Nb10-51 | 57℃ | Nb15-78 | 58℃ |
| Nb11-25 | 57℃ | Nb15-84 | 58℃ |
| Nb11-59 | 58℃ | Nb15-90 | 58℃ |
| Nb12-21 | 57℃ | Nb16-52 | 58℃ |
| Nb13-10 | 57℃ | Nb16-68 | 58℃ |
| Nb13-58 | 57℃ | Nb16-6 | 58℃ |
| Nb13-8 | 57℃ | Nb16-75 | 57℃ |
| Nb14-24 | 57℃ | Nb16-95 | 58℃ |
| Nb14-33 | 57℃ |
实施例9：候选纳米抗体的真病毒中和实验
将Vero E6细胞以1.5E5个/每孔接种到24孔板中，置于37℃，5％CO
2培养箱中培养过夜。用含2％FBS的DMEM培养基(2-DMEM)梯度稀释抗体。用2％FBS+DMEM培养基将SARS-CoV2病毒液稀释。将稀释的病毒液每孔250μL加到等体积稀释的抗体中，充分混合后置于37℃培养箱中孵育1小时。去除24孔板中Vero细胞的培养基，加入200μL抗体-病毒混合液。将孔板置于37℃ 5％CO
2培养箱中孵育1小时。去除孵育后的抗体-病毒混合液，用0.5mL PBS洗一遍细胞后，加入0.5mL用2％FBS+DMEM稀释的0.9％羧甲基纤维素，置于37℃5％CO
2培养箱中培养3天。用20％甲醛-PBS加到培养好的细胞孔中孵育1小时，灭活病毒和固定细胞。用水冲洗细胞去除甲醛-PBS及羧甲基纤维素。每孔中加入结晶紫溶液孵育10分钟。用水冲洗孔板以去除结晶紫溶液。将24孔板倒置在纸巾上，使其完全干燥，拍照并统计噬斑数量。根据抗体稀释度计算抗体中和病毒的效价。
结果如图8所示，7株候选抗体均具有真病毒阻断活性，其中Nb11-59、Nb16-18的活性最优，Nb16-52、Nb15-61次之。
实施例10：抗体组合对ACE2/S-RBD的阻断活性检测
依据鸡尾酒疗法原理，将候选纳米抗体两两组合后，检测其对ACE2/S-RBD的阻断作用。将培养好的293F/ACE2稳转细胞分装至96孔板中，每孔3E5个细胞，3000rpm离心3min去掉上清液，加入稀释好的组合抗体(抗体两两组合，混合后的抗体终浓度为1ug/mL)及2.5ug/mL S-RBD-biotin蛋白孵育20min。离心弃上清，加入稀释的SA-PE抗体，4℃孵育20min。再次离心弃上清，每孔加入200uL PBS重悬细胞，流式细胞仪检测样本PE信号。
结果如图9所示，部分候选抗体组合后具有更好的阻断活性。
实施例11：抗体雾化前后纯度鉴定
选取抗体Nb11-59、Nb16-68配置为10mg/mL，使其置于pH7.5的10mM Tris缓冲液中，同时添加0.05％吐温80。吸取3mL抗体置于飞利浦inspire go雾化器中，开启雾化器雾化，同时用50mL离心管收集雾化后的液体。取雾化后液体样品进行HPLC-SEC检测。
检测结果统计如下表5所示。
表5纳米抗体雾化前后SEC结果
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
Claims (10)
- 一种针对新型冠状病毒的纳米抗体，其特征在于，所述针对新型冠状病毒的纳米抗体的VHH链的互补决定区CDR为选自下组的一种或多种：(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；(2)SEQ ID NO:10所示的CDR1、SEQ ID NO:11所示的CDR2、和SEQ ID NO:12所示的CDR12；(3)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；(4)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；(5)SEQ ID NO:37所示的CDR1、SEQ ID NO:38所示的CDR2、和SEQ ID NO:39所示的CDR12；(6)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；(7)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；(8)SEQ ID NO:64所示的CDR1、SEQ ID NO:65所示的CDR2、和SEQ ID NO:66所示的CDR12；(9)SEQ ID NO:73所示的CDR1、SEQ ID NO:74所示的CDR2、和SEQ ID NO:75所示的CDR3；(10)SEQ ID NO:82所示的CDR1、SEQ ID NO:83所示的CDR2、和SEQ ID NO:84所示的CDR3；(11)SEQ ID NO:91所示的CDR1、SEQ ID NO:92所示的CDR2、和SEQ ID NO:93所示的CDR12；(12)SEQ ID NO:100所示的CDR1、SEQ ID NO:101所示的CDR2、和SEQ ID NO:102所示的CDR3；(13)SEQ ID NO:109所示的CDR1、SEQ ID NO:110所示的CDR2、和SEQ ID NO:111所示的CDR3；(14)SEQ ID NO:118所示的CDR1、SEQ ID NO:119所示的CDR2、和SEQ ID NO:120所示的CDR12；(15)SEQ ID NO:127所示的CDR1、SEQ ID NO:128所示的CDR2、和SEQ ID NO:129所示的CDR3；(16)SEQ ID NO:136所示的CDR1、SEQ ID NO:137所示的CDR2、和SEQ ID NO:138所示的CDR3；(17)SEQ ID NO:145所示的CDR1、SEQ ID NO:146所示的CDR2、和SEQ ID NO:47所示的CDR12；(18)SEQ ID NO:154所示的CDR1、SEQ ID NO:155所示的CDR2、和SEQ ID NO:156所示的CDR3；(19)SEQ ID NO:163所示的CDR1、SEQ ID NO:164所示的CDR2、和SEQ ID NO:165所示的CDR3；(20)SEQ ID NO:172所示的CDR1、SEQ ID NO:173所示的CDR2、和SEQ ID NO:174所示的CDR12；(21)SEQ ID NO:181所示的CDR1、SEQ ID NO:182所示的CDR2、和SEQ ID NO:183所示的CDR3；(22)SEQ ID NO:190所示的CDR1、SEQ ID NO:191所示的CDR2、和SEQ ID NO:192所示的CDR3；(23)SEQ ID NO:199所示的CDR1、SEQ ID NO:200所示的CDR2、和SEQ ID NO:201所示的CDR12；(24)SEQ ID NO:208所示的CDR1、SEQ ID NO:209所示的CDR2、和SEQ ID NO:210所示的CDR3；(25)SEQ ID NO:217所示的CDR1、SEQ ID NO:218所示的CDR2、和SEQ ID NO:219所示的CDR3；(26)SEQ ID NO:226所示的CDR1、SEQ ID NO:227所示的CDR2、和SEQ ID NO:228所示的CDR12；(27)SEQ ID NO:235所示的CDR1、SEQ ID NO:236所示的CDR2、和SEQ ID NO:237所示的CDR3；(28)SEQ ID NO:244所示的CDR1、SEQ ID NO:245所示的CDR2、和SEQ ID NO:246所示的CDR3；(29)SEQ ID NO:253所示的CDR1、SEQ ID NO:254所示的CDR2、和SEQ ID NO:255所示的CDR12；(30)SEQ ID NO:262所示的CDR1、SEQ ID NO:263所示的CDR2、和SEQ ID NO:264所示的CDR3；(31)SEQ ID NO:271所示的CDR1、SEQ ID NO:272所示的CDR2、和SEQ ID NO:273所示的CDR3；(32)SEQ ID NO:280所示的CDR1、SEQ ID NO:281所示的CDR2、和SEQ ID NO:282所示的CDR12；(33)SEQ ID NO:289所示的CDR1、SEQ ID NO:290所示的CDR2、和SEQ ID NO:291所示的CDR3；(34)SEQ ID NO:298所示的CDR1、SEQ ID NO:299所示的CDR2、和SEQ ID NO:300所示的CDR12；和(35)SEQ ID NO:307所示的CDR1、SEQ ID NO:308所示的CDR2、和SEQ ID NO:309所示的CDR3。
- 如权利要求1所述的针对新型冠状病毒的纳米抗体，其特征在于，所述针对新型冠状病毒的纳米抗体的VHH链还包括框架区FR，所述的框架区FR为选自下组的一种或多种：(1)SEQ ID NO:4所示的FR1、SEQ ID NO:5所示的FR2、SEQ ID NO:6所示的FR3、和SEQ ID NO:7所示的FR4；(2)SEQ ID NO:13所示的FR1、SEQ ID NO:14所示的FR2、SEQ ID NO:15所示的FR3、和SEQ ID NO:16所示的FR4；(3)SEQ ID NO:22所示的FR1、SEQ ID NO:23所示的FR2、SEQ ID NO:24所示的FR3、和SEQ ID NO:25所示的FR4；(4)SEQ ID NO:31所示的FR1、SEQ ID NO:32所示的FR2、SEQ ID NO:33所示的FR3、和SEQ ID NO:34所示的FR4；(5)SEQ ID NO:40所示的FR1、SEQ ID NO:41所示的FR2、SEQ ID NO:42所示的FR3、和SEQ ID NO:43所示的FR4；(6)SEQ ID NO:49所示的FR1、SEQ ID NO:50所示的FR2、SEQ ID NO:51所示的FR3、和SEQ ID NO:52所示的FR4；(7)SEQ ID NO:58所示的FR1、SEQ ID NO:59所示的FR2、SEQ ID NO:60所示的FR3、和SEQ ID NO:61所示的FR4；(8)SEQ ID NO:67所示的FR1、SEQ ID NO:68所示的FR2、SEQ ID NO:69所示的FR3、和SEQ ID NO:70所示的FR4；(9)SEQ ID NO:76所示的FR1、SEQ ID NO:77所示的FR2、SEQ ID NO:78所示的FR3、和SEQ ID NO:79所示的FR4；(10)SEQ ID NO:85所示的FR1、SEQ ID NO:86所示的FR2、SEQ ID NO:87所示的FR3、和SEQ ID NO:88所示的FR4；(11)SEQ ID NO:94所示的FR1、SEQ ID NO:95所示的FR2、SEQ ID NO:96所示的FR3、和SEQ ID NO:97所示的FR4；(12)SEQ ID NO:103所示的FR1、SEQ ID NO:104所示的FR2、SEQ ID NO:105所示的FR3、和SEQ ID NO:106所示的FR4；(13)SEQ ID NO:112所示的FR1、SEQ ID NO:113所示的FR2、SEQ ID NO:114所示的FR3、和SEQ ID NO:115所示的FR4；(14)SEQ ID NO:121所示的FR1、SEQ ID NO:122所示的FR2、SEQ ID NO:123所示的FR3、和SEQ ID NO:124所示的FR4；(15)SEQ ID NO:130所示的FR1、SEQ ID NO:131所示的FR2、SEQ ID NO:132所示的FR3、和SEQ ID NO:133所示的FR4；(16)SEQ ID NO:139所示的FR1、SEQ ID NO:140所示的FR2、SEQ ID NO:141 所示的FR3、和SEQ ID NO:142所示的FR4；(17)SEQ ID NO:148所示的FR1、SEQ ID NO:149所示的FR2、SEQ ID NO:150所示的FR3、和SEQ ID NO:151所示的FR4；(18)SEQ ID NO:157所示的FR1、SEQ ID NO:158所示的FR2、SEQ ID NO:159所示的FR3、和SEQ ID NO:160所示的FR4；(19)SEQ ID NO:166所示的FR1、SEQ ID NO:167所示的FR2、SEQ ID NO:168所示的FR3、和SEQ ID NO:169所示的FR4；(20)SEQ ID NO:175所示的FR1、SEQ ID NO:176所示的FR2、SEQ ID NO:177所示的FR3、和SEQ ID NO:178所示的FR4；(21)SEQ ID NO:184所示的FR1、SEQ ID NO:185所示的FR2、SEQ ID NO:186所示的FR3、和SEQ ID NO:187所示的FR4；(22)SEQ ID NO:193所示的FR1、SEQ ID NO:194所示的FR2、SEQ ID NO:195所示的FR3、和SEQ ID NO:196所示的FR4；(23)SEQ ID NO:202所示的FR1、SEQ ID NO:203所示的FR2、SEQ ID NO:204所示的FR3、和SEQ ID NO:205所示的FR4；(24)SEQ ID NO:211所示的FR1、SEQ ID NO:212所示的FR2、SEQ ID NO:123所示的FR3、和SEQ ID NO:214所示的FR4；(25)SEQ ID NO:220所示的FR1、SEQ ID NO:221所示的FR2、SEQ ID NO:222所示的FR3、和SEQ ID NO:223所示的FR4；(26)SEQ ID NO:229所示的FR1、SEQ ID NO:230所示的FR2、SEQ ID NO:231所示的FR3、和SEQ ID NO:232所示的FR4；(27)SEQ ID NO:238所示的FR1、SEQ ID NO:239所示的FR2、SEQ ID NO:240所示的FR3、和SEQ ID NO:241所示的FR4；(28)SEQ ID NO:247所示的FR1、SEQ ID NO:248所示的FR2、SEQ ID NO:249所示的FR3、和SEQ ID NO:250所示的FR4；(29)SEQ ID NO:256所示的FR1、SEQ ID NO:257所示的FR2、SEQ ID NO:258所示的FR3、和SEQ ID NO:259所示的FR4；(30)SEQ ID NO:265所示的FR1、SEQ ID NO:266所示的FR2、SEQ ID NO:267所示的FR3、和SEQ ID NO:268所示的FR4；(31)SEQ ID NO:274所示的FR1、SEQ ID NO:275所示的FR2、SEQ ID NO:276所示的FR3、和SEQ ID NO:277所示的FR4；(32)SEQ ID NO:283所示的FR1、SEQ ID NO:284所示的FR2、SEQ ID NO:285所示的FR3、和SEQ ID NO:286所示的FR4；(33)SEQ ID NO:292所示的FR1、SEQ ID NO:293所示的FR2、SEQ ID NO:294所示的FR3、和SEQ ID NO:295所示的FR4；(34)SEQ ID NO:301所示的FR1、SEQ ID NO:302所示的FR2、SEQ ID NO:303 所示的FR3、和SEQ ID NO:304所示的FR4；和(35)SEQ ID NO:310所示的FR1、SEQ ID NO:311所示的FR2、SEQ ID NO:312所示的FR3、和SEQ ID NO:313所示的FR4。
- 如权利要求1所述的针对新型冠状病毒的纳米抗体，其特征在于，所述针对新型冠状病毒的纳米抗体的VHH链的氨基酸序列选自下组：SEQ ID NO:8、SEQ ID NO:17、SEQ ID NO:26、SEQ ID NO:35、SEQ ID NO:44、SEQ ID NO:53、SEQ ID NO:62、SEQ ID NO:71、SEQ ID NO:80、SEQ ID NO:89、SEQ ID NO:98、SEQ ID NO:107、SEQ ID NO:116、SEQ ID NO:125、SEQ ID NO:134、SEQ ID NO:143、SEQ ID NO:152、SEQ ID NO:161、SEQ ID NO:170、SEQ ID NO:179、SEQ ID NO:188、SEQ ID NO:197、SEQ ID NO:206、SEQ ID NO:215、SEQ ID NO:224、SEQ ID NO:233、SEQ ID NO:242、SEQ ID NO:251、SEQ ID NO:260、SEQ ID NO:269、SEQ ID NO:278、SEQ ID NO:287、SEQ ID NO:296、SEQ ID NO:305、SEQ ID NO:314、或其组合。
- 一种针对新型冠状病毒的抗体，其特征在于，所述抗体包括一个或多个如权利要求3所述的针对新型冠状病毒的纳米抗体。
- 如权利要求4所述的针对新型冠状病毒的抗体，其特征在于，所述抗体包括单体、二价抗体、和/或多价抗体。
- 一种多核苷酸，其特征在于，所述多核苷酸编码选自下组的蛋白质：权利要求1所述的针对新型冠状病毒的纳米抗体，或权利要求4所述的抗体。
- 一种表达载体，其特征在于，所述表达载体含有权利要求6所述的多核苷酸。
- 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求7所述的表达载体，或其基因组中整合有权利要求6所述的多核苷酸。
- 一种产生针对新型冠状病毒的纳米抗体的方法，其特征在于，包括步骤：(a)在适合产生纳米抗体的条件下，培养如权利要求8所述的宿主细胞，从而获得含针对新型冠状病毒的纳米抗体的培养物；(b)从所述培养物中分离和/或回收所述的针对新型冠状病毒的纳米抗体；和(c)任选地，对步骤(b)获得的针对新型冠状病毒的纳米抗体进行纯化和/或修饰。
- 一种免疫偶联物，其特征在于，所述免疫偶联物含有：(a)如权利要求1所述的针对新型冠状病毒的纳米抗体，或如权利要求4所述的抗体；和(b)选自下组的偶联部分：可检测标记物、药物、细胞因子、放射性核素、酶、金纳米颗粒/纳米棒、纳米磁粒、病毒外壳蛋白或VLP，或其组合。
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