CN116041498A - Single-domain antibody specifically binding SARS-CoV-2 spike protein and application thereof - Google Patents
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- 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
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- 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]
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- 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®
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a single domain antibody specifically binding SARS-CoV-2 spike protein and its application. In particular, single domain antibodies that are effective against a variety of novel coronavirus SARS-CoV-2 mutants are disclosed. The invention successfully obtains broad-spectrum neutralization single-domain antibodies B3A3 and I3A10 which specifically bind SARS-CoV-2 spike protein RBD by utilizing phage antibody library technology. The single domain antibody of the present invention has high antigen affinity, obvious inhibition to SARS-CoV-2 main epidemic strain, broad spectrum neutralizing antibody and high neutralizing activity. The single domain antibody of the present invention may be prepared into specific antibody medicine for preventing and treating coronavirus pneumonia (COVID-19), SARS-CoV-2 diagnosis reagent or reagent kit, etc. and has wide application foreground and important significance in medicine application, clinical diagnosis, etc.
Description
The application is a divisional application with the application number of 202111195168.9 and the application date of 2021, 10 and 13, and the invention and the creation name of the novel coronavirus SARS-CoV-2 broad-spectrum neutralizing antibody and the application thereof.
Technical Field
The invention belongs to the field of biological medicine, and relates to a single domain antibody specifically binding SARS-CoV-2 spike protein and application thereof. In particular to a single domain antibody for effectively inhibiting various novel coronavirus SARS-CoV-2 mutant strains and application thereof.
Background
Antibodies (abs) are effector immune molecules produced by proliferation and differentiation of B cells into plasma cells after specific stimulation by B cell epitopes, and mediate liquid immunity, and are a class of immunoglobulins (Ig) that specifically bind to antigens. Ig is specifically identified and combined with corresponding antigen epitope through an antigen binding groove formed by the CDR of the V region of Ig and a lock-key (lock-key) complementary relation. When the antibody binds to the surface of the pathogen, or to a critical epitope of a bacterial toxin, the virulence structure of the pathogen or toxin is blocked, disabling the infectious ability of the virus and disabling the toxin, known as neutralization. Most antibodies are generated by sending signals for locking antigens to T-lymphocytes to excite cellular immune response and kill viruses, while neutralizing antibodies are antibodies which are generated by B lymphocytes and can be combined with antigens on the surface of pathogenic microorganisms, are antibodies which are generated by specific aiming at virus neutralizing epitopes, can be directly targeted to the virus neutralizing epitopes, so that the viruses lose the capacity of binding to receptors, can effectively remove extracellular pathogens (most bacteria) and free viruses in a lytic replication period, and are therefore key to the display of immune protection mechanisms by most prophylactic vaccines. An ideal vaccine should induce antibodies capable of neutralizing most, if not all, of the transmitted families of such viruses, such antibodies being termed broad-spectrum neutralizing antibodies (broadly neutralizing antibody, bnAb). bnAb generally functions by targeting epitopes that are highly conserved and exposed on variable viral surface proteins. The specific binding capacity of antibodies to antigens makes them of great importance in disease diagnosis and immune control. Antibody drugs composed of antibody substances (including whole antibody molecules and antibody fragments with therapeutic functions) are one of the important means for targeted therapy, and have become the most promising and valuable hot spot field in the biomedical industry at present.
Heavy chain antibodies (heavy chain antibody, hcAb) are a naturally occurring type of antibody that was first reported by Hamers doctor at the university of Brussels free Belgium and its team in 1993 (Hamers-Casterman C, atarouch T, muydermans Set al. Naturaly occurring antibodies devoid of light char. 1993; 363:446-8.). Heavy chain antibodies are unique antibody types that camelids or cartilaginous fish possess, whose antibody domains naturally lack the light chain, consisting of only two heavy chains. The antigen recognition function of heavy chain antibodies is largely determined by the variable region (VHH) of heavy chain antibodies. VHH alone recognizes antigens and is therefore also known as Single-domain antibodies (sdabs). The molecular weight of single domain antibodies is only about 13-15kDa, the diameter is about 2.5nm, the length is 4nm, and the antibody fragments with antigen binding function, which are the smallest so far, have the ability to bind antigen and the stability, are basically consistent with complete antibodies or have higher specific antigen affinity. Compared with the traditional antibody, the single-domain antibody has a plurality of unique properties, such as good stability, can reach special antigen epitopes, random combination of block modes, low production cost and the like. The conventional method for obtaining single domain antibodies is composed of numerous steps such as multiple immunization of camelids, B lymphocyte isolation, VHH region amplification, display library construction and screening. With the development of synthetic biology, it becomes possible to construct high-quality randomized large-capacity single domain antibody libraries based on total synthesis. At present, VHH single domain antibodies have been widely used in the research of miniaturized genetic engineering antibodies, the development of new drugs and the diagnosis and treatment of diseases due to the advantages of stable structure, small molecules, good solubility, tolerance to various adverse environments, good preparation stability, easy humanization and the like. The research and development of the single domain antibody have very broad prospect and important significance in the fields of medicine application, clinical diagnosis and the like.
Coronaviridae (coronaviridae) is a family of single-stranded positive-strand RNA viruses that primarily infect vertebrates, whose genomes are relatively large among the known RNA viruses. Coronaviruses are commonly infected via the respiratory or fecal route and can cause a variety of diseases such as: common cold, bronchitis, pneumonia, gastroenteritis, heart disease, etc. Certain coronaviruses have a high mortality rate and a high transmission rate, and can cause serious social and public health problems. Coronavirus particles are typically characterized by an electron microscope that exhibits a "imperial crown" like morphology formed by numerous Spike proteins (S proteins) distributed on the viral envelope. The coronaviridae contains 4 viral genera, respectively: alpha coronavirus genus, beta coronavirus genus, gamma coronavirus genus and delta coronavirus genus. Coronaviruses associated with human infection are mainly alpha and beta coronavirus members, specific virus members including: hCoV-229E, hCoV-NL63, hCoV-OC43, hCoV-HKU1, SARS-CoV, MERS-CoV and SARS-CoV-2.
The novel coronavirus (SARS-CoV-2, severe acute respiratory syndrome coronavirus 2) is a member of the genus beta coronavirus, a new pathogen, with about 80% homology to the SARS-CoV genome, mainly causing human novel coronavirus pneumonia (COVID-19). SARS-CoV-2 virus can infect a variety of cells in vitro, including Vero-E6, huh7, calu-3, and differentiated human airway epithelial cells, among others. SARS-CoV-2 virus is a enveloped positive-strand RNA virus with a viral particle diameter of about 80-140 nm comprising four major structural proteins: surface spike protein (S protein), envelope protein (E protein), membrane protein (M protein), nucleocapsid protein (N protein), among these four proteins, spike protein (S protein) is the most important. The S protein is one of the major proteins forming the viral "corona" morphology, mediating SARS-CoV-2 entry into the cell. The S protein of SARS-CoV-2 consists of 1273 amino acids and structurally belongs to type I membrane fusion protein, and is divided into two regions of S1 and S2. The S1 region mainly comprises the receptor binding region (receptor binding domain, RBD) and the N-terminal domain (NTD), while the S2 region is essential for membrane fusion. A potential receptor for SARS-CoV-2 is human angiotensin converting enzyme 2 (human angiotensin converting enzyme, ACE 2). SARS-CoV-2 binds to human cell surface receptor ACE2 through S protein RBD, just like a key is matched with a lock, and opens the channel into host. Therefore, the RBD structure of SARS-CoV-2 virus determines its binding efficiency to the potential receptor ACE2 and the species specificity of infection, and is an important neutralizing antibody recognition and development target.
SARS-CoV-2 is an RNA virus that is prone to error during replication, and large numbers of replications can cause multiple variations. Once a well-adapted variant appears, it may cause widespread spread. The subunit S1 of SARS-CoV-2 spike protein is considered a mutation hotspot and may have high clinical relevance in terms of toxicity, transmissibility and host immune escape. With the increasing spread of SARS-CoV-2 virus, more and more mutants are produced, and mutation sites have been present in hundreds of thousands. In order to normalize the naming problem of mutant strains, the World Health Organization (WHO) has introduced a new naming system, which is classified into new Coronamutant Strains (VOCs) and mutant strains to be observed (VOIs) of interest according to differences in transmissibility and pathogenicity. WHO recommends the use of greek letters alpha, beta, gamma, delta, lambda, etc. to identify these important SARS-CoV-2 mutant strains. Some countries also define which mutant strains their current VOCs and VOIs include based on their popularity in the country. More and more mutant strains produce various results, including accelerated transmission and altered pathogenicity, and another direct consequence is that some monoclonal antibodies developed earlier have significantly reduced drug failure or neutralization ability, leading to reduced protection of the vaccine.
In view of this, research and development are more effective, and the broad-spectrum neutralizing antibodies against various mutant strains have important scientific significance and wide application prospects for the development of clinical treatment and diagnostic reagents for diseases.
Disclosure of Invention
The invention aims to provide a broad-spectrum neutralization single-domain antibody capable of effectively inhibiting the activities of various novel coronavirus SARS-CoV-2 mutant strains and application thereof. The technical problems to be solved are not limited to the technical subject matter as described, and other technical subject matter not mentioned herein will be clearly understood by those skilled in the art from the following description.
To solve the above technical problem, the present invention provides, first of all, a single domain antibody that specifically binds to SARS-CoV-2 spike protein, which consists of a heavy chain variable region (VHH) comprising a complementarity determining region selected from A1) or A2):
a1 Amino acid sequences are complementarity determining region CDR1 at positions 26-34 of SEQ ID No.1, complementarity determining region CDR2 at positions 51-57 of SEQ ID No.1, and complementarity determining region CDR3 at positions 96-113 of SEQ ID No.1, respectively;
a2 Amino acid sequences are complementarity determining region CDR1 at positions 26-34 of SEQ ID No.2, complementarity determining region CDR2 at positions 51-57 of SEQ ID No.2, and complementarity determining region CDR3 at positions 96-115 of SEQ ID No.2, respectively.
The single domain antibody may be a broad spectrum neutralizing single domain antibody that specifically binds to the SARS-CoV-2 spike protein (S protein) receptor binding region (receptor binding domain, RBD). The single domain antibody consists of a heavy chain variable region, also known as a VHH antibody.
Further, the single domain antibodies of the invention also include a framework region.
The amino acid sequence of the framework region FR1 of the single domain antibody B3A3 is shown in the 1 st-25 th positions of SEQ ID No. 1; the amino acid sequence of FR2 is shown as SEQIDPositions 35-50 of No. 1; the amino acid sequence of FR3 is shown as SEQIDPositions 58-95 of No. 1; the amino acid sequence of FR4 is shown in positions 114-124 of SEQ ID No. 1.
The amino acid sequence of the framework region FR1 of the single domain antibody I3A10 is shown in positions 1-25 of SEQ ID No. 2; the amino acid sequence of FR2 is shown in the 35 th-50 th positions of SEQ ID No. 2; the amino acid sequence of FR3 is shown in 58-95 positions of SEQ ID No. 2; the amino acid sequence of FR4 is shown in positions 116-126 of SEQ ID No.2.
In the single domain antibody, the amino acid sequence of the heavy chain variable region may be SEQ ID No.1 or SEQ ID No.2.
The single domain antibody (heavy chain variable region) having the amino acid sequence of SEQ ID No.1 was designated B3A3 and the single domain antibody (heavy chain variable region) having the amino acid sequence of SEQ ID No.2 was designated I3A10.
The invention also provides a biomaterial associated with the single domain antibody, which biomaterial may be any one of the following C1) to C5):
c1 A nucleic acid molecule encoding a heavy chain variable region of said single domain antibody;
c2 An expression cassette comprising C1) said nucleic acid molecule;
c3 A recombinant vector comprising C1) said nucleic acid molecule, or a recombinant vector comprising C2) said expression cassette;
c4 A recombinant microorganism comprising C1) said nucleic acid molecule, or a recombinant microorganism comprising C2) said expression cassette, or a recombinant microorganism comprising C3) said recombinant vector;
c5 A cell line containing the nucleic acid molecule of C1), or a cell line containing the expression cassette of C2), or a cell line containing the recombinant vector of C3).
Wherein the recombinant microorganism of C4) and the cell line of C5) express the single domain antibody.
In the above biological material, the nucleic acid molecule may be any one of the following:
d1 A DNA molecule having a nucleotide sequence of SEQ ID No. 3;
d2 A DNA molecule having a nucleotide sequence of SEQ ID No. 4;
d3 A DNA molecule which has 75% or more identity to the nucleotide sequence defined in D1) or D2) and which encodes the heavy chain variable region of said single domain antibody.
The DNA molecule shown in SEQ ID No.3 encodes a single domain antibody B3A3 shown in SEQ ID No. 1;
the DNA molecule shown in SEQ ID No.4 encodes the single domain antibody I3A10 shown in SEQ ID No.2.
The 75% or more identity may be 80%, 85%, 90% or 95% or more identity.
Herein, identity refers to identity of nucleotide sequences. The 80% identity or more may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
Variants of the single domain antibodies of the invention having improved affinity and/or potency may be obtained by employing methods known in the art and are included within the scope of the invention. For example, amino acid substitutions may be used to obtain antibodies with further improved avidity. Alternatively, codon optimization of the nucleotide sequence may also be used to improve translational efficiency in expression systems used to produce antibodies. In addition, polynucleotides comprising sequences that optimize antibody specificity or neutralizing activity by applying directed evolution to any of the nucleic acid sequences of the present invention are also within the scope of the present invention.
Vectors described herein are well known to those of skill in the art and include, but are not limited to: plasmids, phages (e.g., lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), artificial chromosomes (e.g., yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs), P1 Artificial Chromosomes (PACs), or Ti plasmid artificial chromosomes (TACs), etc.), viral vectors (e.g., retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, or herpesviruses (e.g., herpes simplex viruses), etc.). In one embodiment of the invention, the vector may specifically be pET-28b.
The microorganism described herein may be a yeast, bacterium, algae or fungus. Wherein the bacteria may be derived from Escherichia, erwinia, agrobacterium (Agrobacterium), flavobacterium (Flavobacterium), alcaligenes (Alcaligenes), pseudomonas, bacillus (Bacillus), etc. In one embodiment of the invention, the microorganism may specifically be E.coli BL21 (DE 3).
The cells (host cells) refer to cells that can be used to introduce vectors, including but not limited to: eukaryotic cells (e.g., yeast cells, aspergillus), animal cells (e.g., mammalian cells, insect cells), plant cells, or prokaryotic cells.
In one embodiment of the invention, the recombinant vectors may specifically be pET-28B-B3A3 and pET-28B-I3A10. Wherein:
the recombinant vector pET-28B-B3A3 is a recombinant prokaryotic expression vector pET-28B-B3A3 which expresses a single-domain antibody B3A3 (fused with a histidine tag) by adopting a DNA molecule 1 to replace a DNA molecule between NcoI and XhoI of a prokaryotic expression vector pET-28B (Novagen company, cat# 69865-3). The DNA molecule 1 is obtained by adding an XhoI recognition site (coded amino acid sequence is LE) and a coding sequence (ctcgagcaccaccaccaccaccac) of 6 histidine tags to the 3' -end of SEQ ID No.3 (372 nd position of SEQ ID No. 3) and keeping other nucleotide sequences of SEQ ID No.3 unchanged;
the recombinant vector pET-28b-I3A10 is obtained by replacing a DNA molecule between NcoI and XhoI of a prokaryotic expression vector pET-28b with a DNA molecule 2, and obtaining the recombinant prokaryotic expression vector pET-28b-I3A10 for expressing the single-domain antibody I3A10 (fused with a histidine tag). DNA molecule 2 is obtained by adding XhoI recognition site (encoded amino acid sequence LE) and 6 histidine tag encoding sequence (ctcgagcaccaccaccaccaccac) to the 3' -end of SEQ ID No.4 (378 th site of SEQ ID No. 4) and keeping the other nucleotide sequence of SEQ ID No.4 unchanged.
In one embodiment of the invention, the recombinant microorganism (recombinant bacterium) may specifically be BL21/pET-28B-B3A3 and BL21/pET-28B-I3A10. Wherein:
recombinant microorganism BL21/pET-28B-B3A3 contains a DNA molecule shown as SEQ ID No.3, and expresses a single-domain antibody B3A3 with the amino acid sequence of SEQ ID No. 1. The recombinant microorganism BL21/pET-28B-B3A3 is a recombinant bacterium obtained by introducing the recombinant vector pET-28B-B3A3 into escherichia coli BL21 (DE 3).
Recombinant microorganism BL21/pET-28b-I3A10 contains DNA molecule shown as SEQ ID No.4, and expressed amino acid sequence is SEQIDNo.2 single domain antibody I3A10. The recombinant microorganism BL21/pET-28b-I3A10 is a recombinant bacterium obtained by introducing the recombinant vector pET-28b-I3A10 into escherichia coli BL21 (DE 3).
The invention also provides a pharmaceutical composition comprising the single domain antibody and a pharmaceutically acceptable carrier.
The pharmaceutically acceptable carrier may be a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, adsorption carrier, surfactant, or lubricant.
Wherein the pharmaceutical composition has a neutralizing antiviral effect of inhibiting or neutralizing SARS-CoV-2 activity. The pharmaceutical composition is used for improving, preventing or treating diseases caused by SARS-CoV-2 infection and/or inhibiting SARS-CoV-2 infection.
Further, the pharmaceutical composition of the invention comprises a first antibody and a second antibody or antigen-binding fragment thereof, wherein the first antibody is a single domain antibody of the invention and the second antibody is any antibody or antigen-binding fragment thereof that neutralizes infection by the SARS-CoV-2 virus.
The invention also provides the application of the single domain antibody and/or the biological material in preparing medicines for inhibiting or neutralizing SARS-CoV-2 activity.
The inhibition or neutralization of SARS-CoV-2 activity includes specific binding to the RBD region of SARS-CoV-2 spike protein (S protein), thereby disabling SARS-CoV-2 from binding to the receptor ACE 2.
In the above application, the medicament for inhibiting or neutralizing SARS-CoV-2 activity can be used for improving, preventing or treating diseases caused by SARS-CoV-2 infection and/or for inhibiting SARS-CoV-2 infection.
The invention also provides the use of said single domain antibodies and/or said biological material in the preparation of a product for detecting SARS-CoV-2 levels and/or the spike protein of SARS-CoV-2.
The use of the single domain antibodies of the invention to monitor the quality of the anti-SARS-CoV-2 vaccine by detecting whether the antigen contains a specific epitope with the correct conformation is also contemplated to be within the scope of the invention.
The product for detecting SARS-CoV-2 level and/or SARS-CoV-2 spike protein comprises a product for detecting antigen-antibody binding by enzyme-linked immunosorbent assay, immunofluorescence assay, radioimmunoassay, luminescent immunoassay, colloidal gold immunochromatography, agglutination method or turbidimetry.
The invention also provides the application of the single domain antibody and/or the biological material in preparing products for diagnosing or assisting in diagnosing diseases caused by SARS-CoV-2 infection.
In the above application, the disease caused by SARS-CoV-2 infection can be respiratory system infection and/or digestive system infection. The respiratory infection may be a respiratory infection and/or a pulmonary infection, which may be nasopharyngitis, rhinitis, pharyngolaryngitis, tracheitis and/or bronchitis, and the pulmonary infection may be pneumonia, such as new coronavirus pneumonia (Corona Virus Disease 2019, covd-19, abbreviated as new coronavirus pneumonia). The digestive system infection may be diarrhea.
In the above application, the product may be a reagent or a kit.
The reagent or kit contains any one of the single domain antibodies or a combination thereof. The kit may be a chemiluminescent immunoassay kit, an enzyme-linked immunoassay kit, a colloidal gold immunoassay kit, or a fluorescent immunoassay kit, but is not limited thereto.
Herein, the terms "single domain antibody that specifically binds SARS-CoV-2 spike protein" and "anti-RBD single domain antibody" have the same meaning and are used interchangeably.
Herein, the term "neutralizing antibody" refers to an antibody that is capable of neutralizing, i.e., preventing, inhibiting, reducing, impeding or interfering with the ability of a pathogen to initiate and/or maintain an infection in a host. As described herein, these antibodies, alone or in combination, can be used as a prophylactic or therapeutic agent, in combination with active vaccination, as a diagnostic tool or as a production tool after appropriate formulation.
In one embodiment, the single domain antibodies of the invention are capable of neutralizing SARS-CoV-2 pseudotype virus and various mutants thereof or combinations thereof. Exemplary SARS-CoV-2 mutants include, but are not limited to, alpha mutants, beta mutants, gamma mutants, delta mutants and Kappa mutants.
The invention utilizes phage antibody library technology, and obtains positive clone through multiple rounds of screening and enrichment of phage with RBD protein antigen specificity, obtains corresponding coding sequence through sequencing, expresses and purifies in escherichia coli BL21 (DE 3), and successfully obtains broad-spectrum neutralization single-domain antibodies B3A3 and I3A10 which specifically bind SARS-CoV-2 spike protein (S protein) receptor binding region (receptor binding domain, RBD). The single domain antibodies B3A3 and I3A10 of the invention have high affinity for SARS-CoV-2 antigen (RBD protein), and can inhibit or neutralize SARS-CoV-2 activity by specifically binding to the RBD region of SARS-CoV-2 spike protein (S protein), so that SARS-CoV-2 loses the ability of binding to receptor ACE 2. Meanwhile, the single domain antibody of the invention has obvious inhibition effect on SARS-CoV-2 main epidemic strains (Alpha mutant strain, beta mutant strain, gamma mutant strain, delta mutant strain and Kappa mutant strain), has stronger inhibition activity and neutralization activity on SARS-CoV-2 pseudotype virus, has the capability of broad-spectrum inhibition on SARS-CoV-2 pseudotype virus infection of various mutant strains, and is a neutralization antibody with broad-spectrum effect. Has good broad spectrum and neutralization activity. The single domain antibody of the present invention may be expressed and produced in prokaryotic cell, yeast cell, eukaryotic cell and other recombination system to obtain antibody product with neutralizing SARS-CoV-2 infection, and may be prepared into specific antibody medicine for preventing and treating novel coronavirus pneumonia (COVID-19), diagnosis reagent or diagnosis kit for SARS-CoV-2, etc. and has wide application foreground and important significance in medicine application, clinical diagnosis, etc.
Drawings
FIG. 1 is a statistical result of neutralization activity of single domain antibody B3A3 in inhibiting invasion of various SARS-CoV-2 pseudotyped viruses.
FIG. 2 is a statistical result of neutralization activity of the single domain antibody I3A10 to inhibit invasion of various pseudotyped viruses of SARS-CoV-2.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
EXAMPLE 1 screening and preparation of Single-domain antibodies that specifically bind to the RBD region of SARS-CoV-2 spike protein
1. Phage library screening
Basic principles and basic protocols for phage library screening reference Shao Ningsheng et al, "biological library technology-phage display and SELEX technology", the screening procedure is briefly described as follows: firstly, 50 mug SARS-CoV-2 spike protein RBD region recombinant protein (product numbers: 40592-V08H86, 40592-V08H88, RBD protein) antigen from Brazil and India respectively is utilized to make use of commercial kit EZ-Link of Simer Feishier technology company TM The Sulfo NHS-LC-LC-Biotin was labeled with Biotin and desalted and purified (desalting column was purchased from Sesameiser technologies). The first round of screening was a liquid phase selection by first combining 12 μg of biotin-labeled RBD protein with 1mL (10 12 -10 13 ) Incubating the phage library mixture at room temperature for 1 hour; subsequently, 100. Mu.L of streptavidin magnetic beads (GE life sciences) were added, and the turntable was rotated and incubated for 30 minutes at room temperature; the magnetic beads and RBD protein antigens are linked together by adsorption of biotin and streptavidin to capture phage antibodies having RBD protein binding activity in the liquid phase. Then washing the magnetic beads with PBS-T and PBS for 10-15 times, and adding 400 mu L of eluent into the magnetic beads for eluting for 5min; 200. Mu.L of the eluent was added to 5mL of a logarithmic growth phase TG1 E.coli culture and incubated at 37℃for 1 hour; after the above culture was infected with helper phage CM13, the culture was expanded overnight and the phage in the supernatant was purified by PEG6000 (Sigma Co.) on the next day, 10 was obtained 12 Phage VHH antibody libraries were used for the next round of screening. The second round of screening was solid phase selection by first coating 50 μg of RBD protein on 5mL immunoadsorption tube overnight at 4deg.C, then washing the tube 3 times with PBS and adding 1mL of 10 harvested from the first round of screening 12 Phage VHH antibody library, rotating the turntable at room temperature for 30 min, and standingIncubating for 1.5 hours; then PBS-T and PBS are used for washing the immune tube for 15-20 times, and eluent is added for eluting; half of the eluent was added to 5mL of a TG1 E.coli culture in the logarithmic growth phase and incubated at 37℃for 1 hour; after infection of the helper phage CM13 with the above culture, the culture was expanded overnight and the phage in the supernatant was purified with PEG the next day for the next round of screening. A third round of screening, which is a liquid phase screening, and a fourth round of screening, which is a solid phase screening, were performed using a similar method. After the last round of screening, 1000 single clones are selected for culture and auxiliary phage rescue, so that rescue phage are obtained and used for identification of subsequent positive clones.
2. Phage ELISA assays identified positive clones that bound to RBD protein.
Basic principles and basic protocols of enzyme-linked immunosorbent assay (ELISA) are described in Cao Xuetao et al, immunology techniques and uses thereof. The identification procedure is briefly described as follows: RBD protein 25 ng/well was coated overnight in 96 well immunoplates. After blocking the multiwell plates with BSA, 100. Mu.L of rescue phage per well in step one was added and incubated at room temperature for 1 hour. After washing the plate 5 times with PBS-T, anti-phage antibody Anti-M13 Anti-body [ B62-FE2] (HRP) (Abcam company #ab50370) was added, and incubated at room temperature for 1 hour. After washing the plate with PBS-T6 times to remove unbound antibody, 100. Mu.L of TMB substrate (Beijing Soy Bao Co.) was added to each well, incubated at 37℃in the dark for 5-30 minutes, stop solution (50. Mu.L) was added to each well to terminate the reaction, the experimental results were measured within 20 minutes, and absorbance was read at 450 nm. A group of negative control measurement holes are arranged in the experiment, and positive results can be judged when the ratio of the sample holes to the control holes is greater than 5.
Through the above screening, sequencing and homology alignment analysis were performed on all positive clones (phage containing single domain antibodies capable of binding to RBD protein), and the results showed that all positive clones could be summarized as 2 independent clones, representative clones of which were: b3A3 and I3a10.
3. Obtaining anti-RBD Single-Domain antibodies
Through the screening, positive clones are amplified by high-fidelity PCR to complete the frame of the single domain antibody, and the encoding nucleic acid molecule of the RBD-resistant single domain antibody and the encoded amino acid sequence thereof are obtained.
Co-screening to obtain 2 single-domain antibodies (named B3A3 and I3A10 respectively), wherein the single-domain antibodies sequentially comprise a framework region FR1, a complementarity determining region CDR1, a framework region FR2, a complementarity determining region CDR2, a framework region FR3, a complementarity determining region CDR3 and a framework region FR4, and the specific information is as follows:
the amino acid sequence of the single domain antibody B3A3 is shown as SEQ ID No. 1. Positions 1 to 25 of SEQ ID No.1 are the framework region FR1, positions 26 to 34 are the complementarity determining region CDR1, positions 35 to 50 are the framework region FR2, positions 51 to 57 are the complementarity determining region CDR2, positions 58 to 95 are the framework region FR3, positions 96 to 113 are the complementarity determining region CDR3, and positions 114 to 124 are the framework region FR4. The nucleic acid molecule encoding the single domain antibody B3A3 is shown in SEQ ID No. 3.
The amino acid sequence of the single domain antibody I3A10 is shown as SEQ ID No.2. Positions 1 to 25 of SEQ ID No.2 are the framework region FR1, positions 26 to 34 are the complementarity determining region CDR1, positions 35 to 50 are the framework region FR2, positions 51 to 57 are the complementarity determining region CDR2, positions 58 to 95 are the framework region FR3, positions 96 to 115 are the complementarity determining region CDR3, and positions 116 to 126 are the framework region FR4. The nucleic acid molecule encoding the single domain antibody I3A10 is shown in SEQ ID No. 4.
B3A3 and I3A10 are anti-RBD single domain antibodies (single domain antibodies that specifically bind SARS-CoV-2 spike protein) and are broad-spectrum neutralizing single domain antibodies that specifically bind SARS-CoV-2 spike protein (S protein) receptor binding domain (receptor binding domain, RBD).
4. Prokaryotic expression vector construction for expressing anti-RBD single domain antibody
1. Prokaryotic expression vector for expressing single domain antibody B3A3
The DNA molecule 1 was used to replace the DNA molecule between NcoI and XhoI of the prokaryotic expression vector pET-28B (Novagen, cat# 69865-3) to obtain the recombinant prokaryotic expression vector pET-28B-B3A3 expressing the single-domain antibody B3A3 (fused with a histidine tag). DNA molecule 1 is a DNA molecule with a coding sequence (ctcgagcaccaccaccaccaccac) comprising an XhoI recognition site (encoded amino acid sequence LE) and 6 histidine tags added to the 3' -end of SEQ ID No.3 (372 nd position of SEQ ID No. 3) to maintain SEQIDDNA fraction obtained without changing other nucleotide sequence of No.3And (5) a seed.
2. Prokaryotic expression vector for expressing single domain antibody I3A10
And (3) replacing the DNA molecule between NcoI and XhoI of the prokaryotic expression vector pET-28b by using the DNA molecule 2 to obtain a recombinant prokaryotic expression vector pET-28b-I3A10 for expressing the single-domain antibody I3A10 (fused with a histidine tag). DNA molecule 2 is obtained by adding XhoI recognition site (encoded amino acid sequence LE) and 6 histidine tag encoding sequence (ctcgagcaccaccaccaccaccac) to the 3' -end of SEQ ID No.4 (378 th site of SEQ ID No. 4) and keeping the other nucleotide sequence of SEQ ID No.4 unchanged.
5. Expression and purification of anti-RBD single domain antibodies
1. Expression of anti-RBD single domain antibodies
And (3) respectively and independently transforming the two prokaryotic expression vectors (pET-28B-B3A 3 and pET-28B-I3A 10) obtained in the step four into BL21 (DE 3) escherichia coli competent cells (purchased from Beijing full-scale gold company) to obtain recombinant BL21/pET-28B-B3A3 (transformed into pET-28B-B3A 3) and recombinant BL21/pET-28B-I3A10 (transformed into pET-28B-I3A 10). Inoculating recombinant BL21/pET-28B-B3A3 and recombinant BL21/pET-28B-I3A 10) to LB liquid culture medium, culturing at 37deg.C and 230rpm to OD 600 =0.5, then IPTG was added to the culture system at a final concentration of 0.3mM and the induction culture was continued at 18 ℃ at 230rpm for 16 hours, after which the cells were harvested by centrifugation and stored at-80 ℃ for use.
2. Purification of anti-RBD single domain antibodies
(1) Lysing bacteria
The cells obtained in step 1 were resuspended in lysis buffer (50 mM Tris-Cl pH 8.0, 100mM NaCl,5mM imidazole), sonicated, and centrifuged at 12000 Xg for 15 min, and the supernatant was transferred to a new tube.
(2) Affinity chromatography
The supernatant obtained in the above (1) was passed through a Ni-NTA agarose column (purchased from GE life sciences) on an AKTA Purifier (GE life sciences), unbound proteins were washed off with 20 column volumes of washing solution (50 mM Tris-Cl pH 8.0, 300mM NaCl,10mM imidazole), and finally, gradient elution of 0-500mM imidazole was performed by using the apparatus from the band procedure and fractional collection of protein peaks was performed, 1 tube was collected for each 1mL of eluted proteins, and purified anti-RBD single domain antibodies B3A3 and I3A10 were identified by SDS-PAGE.
And (3) loading the purified anti-RBD single domain antibody into a dialysis bag, dialyzing overnight to replace a PBS buffer system, and completing the steps of concentration and the like.
Example 2 affinity assay for anti-RBD Single-Domain antibodies
Antibody to be tested: example 1 two RBD single domain antibodies B3A3 and I3a10 were purified.
The affinity determination of antibodies was performed using a BIAcore T-200 biomolecular interaction apparatus (GE life sciences), which was developed based on Surface Plasmon Resonance (SPR) technology, and enables label-free, rapid, real-time, automated manipulation and detection of interactions between biological macromolecules.
When the interaction between the recombinant RBD protein and the corresponding single-domain antibody is measured, antigen RBD protein (Gamma strain RBD product number: 40592-V08H86, kappa strain RBD product number: 40592-V08H 88) is coated on a sensor chip, and then the single-domain antibody is used as a mobile phase to measure the binding constant, dissociation constant and affinity constant.
1. RBD protein coupled CM5 chip: the RBD protein coupling temperature was 25℃and the buffer was HBS-P (10mM HEPES,150mM NaCl,3mM EDTA and 0.05% P20, pH 7.4). The program template is Immobilisation, CM5 chip channel 2 amino coupling is selected, ligand is RBD-His protein with 5 mug/ml, protein buffer system is sodium acetate with pH of 5.5 (NaAC 5.5), target coupling amount is 300RU, and eluent is 50mM NaOH. Chip activator 50mmol/L N-hydroxysuccinimide (NHS) and 200 mmol/L3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) activated chip, and blocking agent 1mol/L ethanolamine hydrochloride.
2. anti-RBD single domain antibodies with RBD protein antigen affinity and kinetic assay: a multi-cycle kinetic template was selected, the assay temperature was 25 degrees Celsius, the buffer was HBS-P, the sample flow path was 2-1, the sample binding time was 180s, the flow rate was 30ul/min, the dissociation time was 300s, the regeneration eluent was Glycine-HCL 2.5, the regeneration liquid binding time was 30s, the flow rate was 30ul/min, the stabilization time was 0s, and the single domain antibody concentration was serially diluted (2, 4,8,16,32,64 nM). The resulting data were analyzed by Biacore Evaluation Software software to calculate the binding constant (ka), dissociation constant (KD) and affinity constant (KD).
The chips, reagents and buffers used in the Biacore assays described above were all purchased from GE life sciences.
The results are shown in Table 1. The results show that the affinity constants of the obtained single domain antibodies (B3A 3 and I3A 10) and the two SARS-CoV-2 RBD mutant proteins (Gamma strain and Kappa strain) are between 0.023nMol/L and 45.1 nMol/L. The lower KD value indicates stronger affinity, and the obtained single domain antibodies have ideal affinity with RBD proteins as a whole. As can be seen from the results in Table 1, the affinity of the single domain antibody B3A3 was better, and the affinity of the single domain antibody I3A10 was inferior.
TABLE 1 affinity constants for interaction of Single Domain antibodies with SARS-CoV-2 RBD region
EXAMPLE 3 determination of SARS-CoV-2 pseudotyped Virus neutralization Activity of anti-RBD Single-Domain antibodies
Antibody to be tested: example 1 two single domain antibodies B3A3 and I3a10 were purified.
The SARS-CoV-2 pseudotyped virus is a novel virus particle formed by assembling the replication core element of a retrovirus with the envelope spike glycoprotein (i.e., S protein) of the SARS-CoV-2 virus. Compared with the true virus, the pseudovirus can only infect cells once, has wide host range and high titer, is not easy to be inactivated by serum complement, and can replace the true virus to carry out neutralization detection. The ability of pseudoviruses to infect cells depends on the type and nature of the glycoprotein it coats and is an ideal tool for studying the neutralizing antibody inhibition efficiency, receptor utilization and invasion infection mechanism of SARS-CoV-2.
1. Packaging preparation of SARS-CoV-2 pseudotype virus
The construction method of SARS-CoV-2S gene expression plasmid comprises:
according to the sequence information of S protein in GenBank (GenBank number: QHD 43416.1), the 21563-25330 th nucleotide of SARS-CoV-2S gene (nucleotide sequence is GenBank Accession No. MN908947.3 (Update Date 18-MAR-2020) gene) is synthesized, the fragment (small fragment) between EcoRI and XhoI recognition sites of plasmid vector pCAGGS is replaced by SARS-CoV-2S gene, other sequences of pCAGGS vector are kept unchanged, and the obtained recombinant expression vector is SARS-CoV-2S gene expression plasmid pSARS-CoV-2-wildtype type S (wild type S gene expression plasmid).
On the basis of the wild type novel coronavirus S gene (GenBank: QHD 43416.1), each mutant S gene was synthesized for packaging of mutant pseudotyped viruses. Comprising the following steps: alpha mutant (GenBank number: QQX 99439.1); beta mutant (GenBank number: QUN 71013.1); gamma mutant (GenBank: QVQ 47339.1); delta mutant (GenBank: QVI 56963.1); kappa mutant (GenBank accession number QXP 08802.1).
According to the construction method of SARS-CoV-2S gene expression plasmid, each mutant S gene expression plasmid is constructed by using the following mutant S gene:
the SARS-CoV-2Alpha mutant (GenBank number: QQX 99439.1) S gene (nucleotide sequence GenBank Accession No. MW531680.1 (Update Date 27-JAN-2021) gene at nucleotides 21503-25261); the SARS-CoV-2Alpha mutant S gene was used to replace the fragment (small fragment) between EcoRI and XhoI recognition sites of the plasmid vector pCAGGSS, and the other sequences of the pCAGGSS vector were kept unchanged, thereby obtaining a recombinant expression vector named pSARS-CoV-2 Alpha-S (SARS-CoV-2 Alpha mutant S gene expression plasmid).
SARS-CoV-2Beta mutant (GenBank number: QUN 71013.1) S gene (nucleotide sequence GenBank Accession No. MZ068155.1 (Update Date 11-MAY-2021) gene at nucleotides 21516-25274); the SARS-CoV-2Beta mutant S gene was used to replace the fragment (small fragment) between EcoRI and XhoI recognition sites of the plasmid vector pCAGGSS, and the other sequences of the pCAGGSS vector were kept unchanged, thereby obtaining a recombinant expression vector named pSARS-CoV-2-Beta-S (SARS-CoV-2 Beta mutant S gene expression plasmid).
SARS-CoV-2Gamma mutant (GenBank number: QVQ 47339.1) S gene (nucleotide sequence GenBank Accession No. MZ264787.1 (Update Date 21-MAY-2021) gene at nucleotides 21555-25322); the SARS-CoV-2Gamma mutant S gene was used to replace the fragment (small fragment) between EcoRI and XhoI recognition sites of the plasmid vector pCAGGSS, and the other sequences of the pCAGGSS vector were kept unchanged, thereby obtaining a recombinant expression vector named pSARS-CoV-2 Gamma-S (SARS-CoV-2 Beta mutant S gene expression plasmid).
SARS-CoV-2 Delta mutant (GenBank: QVI 56963.1) S gene (nucleotide sequence is GenBank Accession No. MZ208926.1 (Update Date 17-MAY-2021) gene at nucleotides 21443-25204); the SARS-CoV-2 Delta mutant S gene was used to replace the fragment (small fragment) between EcoRI and XhoI recognition sites of the plasmid vector pCAGGSS, and the other sequences of the pCAGGSS vector were kept unchanged, thereby obtaining a recombinant expression vector named pSARS-CoV-2-Delta-S (SARS-CoV-2 Beta mutant S gene expression plasmid).
SARS-CoV-2Kappa mutant (GenBank number: QXP 08802.1) S gene (nucleotide sequence: genBank Accession No. MZI571142.1 (Update Date 16-JUL-2021) gene of nucleotides 21538-25359); the SARS-CoV-2Kappa mutant S gene was used to replace the fragment (small fragment) between EcoRI and XhoI recognition sites of the plasmid vector pCAGGSS, and the other sequences of the pCAGGSS vector were kept unchanged, thereby obtaining a recombinant expression vector named pSARS-CoV-2-Kappa-S (SARS-CoV-2 Beta mutant S gene expression plasmid).
The wild-type S gene expression plasmid and the mutant S gene expression plasmid (collectively referred to as SARS-CoV-2S gene expression plasmid) prepared as described above were used to prepare the following pseudoviruses:
293T cells (from basic medical institute of China medical sciences) were inoculated into 10 cm dishes, cultured to 80% cell confluency using DMEM medium (from Gibco corporation) containing 10% fetal bovine serum (from Semer Feisher), co-transfected with 15. Mu.g SARS-CoV-2S gene expression plasmid and 15. Mu.g PNL 4.3-Luc-R-E-plasmid (BioVector NTCC Inc.), the DMEM medium containing 2% fetal bovine serum was replaced 6 hours after transfection, culturing was continued for 36 hours and the culture supernatant containing SARS-CoV-2 pseudotype virus was harvested, sub-packaged and frozen for long-term storage at-80 ℃.
The pseudoviruses obtained by co-transfecting pSARS-CoV-2-wildtype type-S and PNL 4.3-Luc-R-E-were designated wild-type pseudoviruses, the pseudoviruses obtained by co-transfecting pSARS-CoV-2-Alpha-S and PNL 4.3-Luc-R-E-were designated Alpha mutant pseudoviruses, the pseudoviruses obtained by co-transfecting pSARS-CoV-2-Beta-S and PNL 4.3-Luc-R-E-were designated Beta mutant pseudoviruses, the pseudoviruses obtained by co-transfecting pSARS-CoV-2-Gamma-S and PNL 4.3-Luc-R-E-were designated Gamma mutant pseudoviruses, the pseudoviruses obtained by co-transfecting pSARS-CoV-2-DeltaS and PNL 4.3-Luc-R-E-were designated Delta mutant pseudoviruses, and the pseudoviruses obtained by co-transfecting pSARS-CoV-2-Gamma-S and PNL 4.3-Luc-R-E-were designated Beta mutant pseudoviruses.
2. Pseudovirus invasion inhibition assay
The single domain antibody B3A3 experimental group and the single domain antibody I3A10 experimental group are respectively set, and the invasion experiment of the pseudotyped virus is carried out according to the following steps:
the SARS-CoV-2 pseudotyped virus prepared in the first step is mixed with different dilutions of single domain antibodies (the final concentration of the single domain antibodies is 0, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100 and 1000nM respectively) and added into a pre-inoculated 96-well plate containing Calu-3 cells (human lung adenocarcinoma cells) for further incubation for 48 hours. SARS-CoV-2 pseudotype virus contains luciferase reporter gene, and pseudotype virus has the capability of infecting target cell of interest, and can be used for measuring its infectivity and level by means of detection of luciferase reporter gene. Cells were lysed according to the product instructions and the reporter activity in the cell lysate was detected using the luciferase reporter assay kit from Promega company (cat# E4550), and the raw readouts of luciferase were converted into percentage data for mapping.
The results are shown in fig. 1 and 2.
The result shows that the single domain antibodies B3A3 and I3A10 obtained by the invention have obvious inhibition effect on SARS-CoV-2 main epidemic strains (Alpha mutant strain, beta mutant strain, gamma mutant strain, delta mutant strain and Kappa mutant strain) and SARS-CoV-2 pseudotyped virusThe strong inhibition activity and neutralization activity show that the single domain antibody of the invention has the capability of broad spectrum inhibition of various mutant strains SARS-CoV-2 pseudotyped virus infection, is the neutralization antibody with broad spectrum effect, and particularly has half neutralization dose (ND 50 ) See table 2. The single domain antibody I3A10 has better broad spectrum and neutralization activity, and the single domain antibody B3A3 times.
Table 2 half-neutralizing dose of single domain antibody against SARS-CoV-2 pseudotype virus infection of various mutants
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (10)
1. A single domain antibody, characterized in that said single domain antibody consists of a heavy chain variable region comprising a complementarity determining region selected from the group consisting of A2):
a2 Amino acid sequences are complementarity determining region CDR1 at positions 26-34 of SEQ ID No.2, complementarity determining region CDR2 at positions 51-57 of SEQ ID No.2, and complementarity determining region CDR3 at positions 96-115 of SEQ ID No.2, respectively.
2. The single domain antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region is SEQ ID No.2.
3. Biomaterial related to a single domain antibody according to claim 1 or 2, characterized in that it is any one of the following C1) to C5):
c1 A nucleic acid molecule encoding the heavy chain variable region of the single domain antibody of claim 1 or 2;
c2 An expression cassette comprising C1) said nucleic acid molecule;
c3 A recombinant vector comprising C1) said nucleic acid molecule, or a recombinant vector comprising C2) said expression cassette;
c4 A recombinant microorganism comprising C1) said nucleic acid molecule, or a recombinant microorganism comprising C2) said expression cassette, or a recombinant microorganism comprising C3) said recombinant vector;
c5 A cell line containing the nucleic acid molecule of C1), or a cell line containing the expression cassette of C2), or a cell line containing the recombinant vector of C3).
4. A biological material according to claim 3, wherein the nucleic acid molecule is any one of the following:
d2 A DNA molecule having a nucleotide sequence of SEQ ID No. 4;
d3 A DNA molecule having 75% or more identity to the nucleotide sequence defined in D2) and encoding the heavy chain variable region of the single domain antibody of claim 1 or 2.
5. A pharmaceutical composition comprising the single domain antibody of claim 1 or 2 and a pharmaceutically acceptable carrier.
6. Use of a single domain antibody according to claim 1 or 2 and/or a biomaterial according to claim 3 or 4 for the manufacture of a medicament for inhibiting or neutralising SARS-CoV-2 activity.
7. The use according to claim 6, wherein the medicament for inhibiting or neutralizing SARS-CoV-2 activity is for ameliorating, preventing or treating a disease caused by SARS-CoV-2 infection and/or for inhibiting SARS-CoV-2 infection.
8. Use of a single domain antibody according to claim 1 or 2 and/or a biomaterial according to claim 3 or 4 for the preparation of a product for detecting SARS-CoV-2 and/or SARS-CoV-2 spike protein.
9. Use of a single domain antibody according to claim 1 or 2 and/or a biomaterial according to claim 3 or 4 for the manufacture of a product for the diagnosis or co-diagnosis of a disease caused by SARS-CoV-2 infection.
10. The use according to claim 7 and/or 9, wherein the disease caused by SARS-CoV-2 infection is a respiratory infection and/or a digestive system infection.
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