Single domain antibodies against SARS-CoV-2 and their uses

Abstract

The present invention relates to the fields of immunology and molecular virology, in particular to the diagnosis, prevention and treatment of coronaviruses (e.g., SARS-CoV-2). Specifically, the present invention relates to single-domain antibodies against coronaviruses, and uses of the single-domain antibodies. The antibodies of the present invention can be used to diagnose, prevent and / or treat coronavirus infections and / or diseases caused by the infections.

Description

Technical Field

[0001] The present invention relates to the fields of immunology and molecular virology, in particular to the diagnosis, prevention and treatment of coronaviruses (e.g., SARS-CoV-2). Specifically, the present invention relates to single-domain antibodies against coronaviruses, and uses of the single-domain antibodies. The antibodies of the present invention can be used to diagnose, prevent and / or treat coronavirus infections and / or diseases caused by the infections. Background Art

[0002] Coronavirus infection can cause respiratory diseases in humans. Mild coronavirus infection can cause flu-like symptoms, while severe infection can develop into severe viral pneumonia, threatening human life and health. Coronavirus can infect both humans and animals. If some animal-derived coronaviruses break through the host barrier and infect humans, they may spread rapidly among the population and cause serious diseases.

[0003] SARS-CoV-2 is a type of virus that is spherical, has protrusions on the surface, and looks like a crown under an electron microscope. The viral gene is a continuous linear single-stranded RNA with a diameter of 75-160nm. SARS-CoV-2 is the seventh member of the coronavirus family that has been discovered to infect humans. The other six members are: HCoV 229E, HCoV NL63, HCoV OC43, HCoV HKU1, SARS-CoV, and MERS-CoV. SARS-CoV-2 belongs to the same beta coronavirus as SARS-CoV and MERS-CoV. SARS-CoV-2 contains spike glycoprotein (Spike protein, S protein). Spike protein is a large trimeric transmembrane glycoprotein with a large number of glycosylation modifications, which forms a special crown structure on the surface of the virus. It first binds to the receptor on the cell surface, then "deforms", and takes advantage of the situation to integrate the viral envelope with the cell membrane, thereby injecting the genetic material in the virus into the cell to achieve the purpose of infecting the cell. Spike protein is the most important surface membrane protein of coronavirus, containing two subunits S1 and S2. S1 mainly contains the receptor binding domain (RBD), which is responsible for recognizing the cell's receptors, while S2 contains the basic elements required for the membrane fusion process.

[0004] At present, there is no specific drug approved for the prevention or treatment of SARS-CoV-2 infection. Therefore, if an antibody that can specifically neutralize SARS-CoV-2 is developed for short-term prevention and effective treatment of COVID-19, it will be of great significance for the prevention and treatment of COVID-19 in my country and even globally. Summary of the invention

[0005] The inventors of the present application have obtained camel-derived single-domain antibodies that can specifically neutralize SARS-CoV-2 through in-depth research and creative work. The antibodies of the present invention have the potential to be used to prevent and / or treat coronavirus infection or diseases caused by coronavirus infection, and have significant clinical value.

[0006] In a first aspect, the present application provides a single-domain antibody or an antigen-binding fragment thereof that specifically binds to the receptor binding region (RBD) of the S protein of SARS-CoV-2, wherein the single-domain antibody or the antigen-binding fragment thereof comprises:

[0007] (1) The following three complementarity determining regions (CDRs):

[0008] A CDR1 having a sequence of SEQ ID NO: 1 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a CDR2 having a sequence of SEQ ID NO: 2 having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a CDR3 having a sequence of SEQ ID NO: 3 having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom; or,

[0009] (2) The following three complementarity determining regions (CDRs):

[0010] a CDR1 having a sequence of SEQ ID NO: 10 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a CDR2 having a sequence of SEQ ID NO: 11 having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, a CDR3 having a sequence of SEQ ID NO: 12 having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom; or,

[0011] (3) The following three complementarity determining regions (CDRs):

[0012] The CDR1 of SEQ ID NO: 19 or a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, the CDR2 of SEQ ID NO: 20 having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom, and the CDR3 of SEQ ID NO: 21 having one or several amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) therefrom.

[0013] In certain embodiments, the substitutions described in any one of (1)-(3) are conservative substitutions.

[0014] As described above, the single domain antibody or antigen binding fragment thereof further comprises a framework region (FRs). In certain embodiments, the single domain antibody or antigen binding fragment thereof comprises FRs from humans or camels.

[0015] In certain embodiments, the single domain antibody consists of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

[0016] In certain embodiments, the single domain antibody or antigen-binding fragment thereof is a camelid VHH or a humanized VHH.

[0017] The single-domain antibody or antigen-binding fragment thereof as described above, wherein the single-domain antibody or antigen-binding fragment thereof comprises a sequence selected from the following:

[0018] (1) the sequence set forth in SEQ ID NO:8, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto;

[0019] (2) the sequence set forth in SEQ ID NO:17, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto; or,

[0020] (3) the sequence set forth in SEQ ID NO:26, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.

[0021] In another aspect, the present application provides a multispecific antibody comprising the single domain antibody or antigen-binding fragment thereof as described above, and another antibody or antigen-binding fragment thereof, or antibody analog.

[0022] In certain embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody or a tetraspecific antibody.

[0023] In certain embodiments, the additional antibody or fragment thereof, or antibody analog has binding specificity to at least one molecule or antigen among a tumor-specific antigen (TSA), a tumor-associated antigen (TAA), a T cell engager molecule, a tumor immunity-related molecule or an autoimmune regulation-related molecule.

[0024] In certain embodiments, the additional antibody or fragment thereof, or antibody analog has antigen binding specificity to at least one of PD-1, PDL-1, CD19, CD20, CTLA4, IL6, IL6R or TNFα.

[0025] In another aspect, the present application provides a fusion protein comprising the single domain antibody or antigen-binding fragment thereof as described above, and another biologically active polypeptide.

[0026] In certain embodiments, the additional biologically active polypeptide is a polypeptide or protein having therapeutic activity, binding activity, or enzymatic activity.

[0027] In certain embodiments, the additional biologically active polypeptide is linked to the N-terminus and / or C-terminus of the single domain antibody or antigen-binding fragment thereof, optionally via a linker.

[0028] In certain embodiments, the fusion protein comprises one or more (e.g., two) of the single domain antibodies or antigen binding fragments thereof and one or more (e.g., two) of the additional biologically active polypeptides; wherein the multiple single domain antibodies or antigen binding fragments thereof are the same or different, and the multiple additional biologically active polypeptides are the same or different.

[0029] In certain embodiments, the fusion protein comprises one of the single domain antibodies or antigen binding fragments thereof and one of the additional biologically active polypeptides.

[0030] In certain embodiments, the fusion protein comprises one of the single domain antibodies or antigen binding fragments thereof and two of the additional biologically active polypeptides.

[0031] In certain embodiments, the two additional biologically active polypeptides are optionally linked to the N-terminus and / or C-terminus of the single domain antibody or antigen-binding fragment thereof, respectively, via a linker.

[0032] In another aspect, the present application provides a conjugate comprising the single domain antibody or antigen binding fragment thereof as described above and a chemotherapeutic drug, a radiopharmaceutical or an imaging agent linked to the single domain antibody or antigen binding fragment thereof.

[0033] On the other hand, the present application provides an isolated nucleic acid molecule encoding a single domain antibody or antigen binding fragment thereof as described above, a multispecific antibody as described above, or a fusion protein as described above. According to codon degeneracy known in the art, in certain embodiments, the nucleotide sequence of the nucleic acid molecule can be replaced according to codon degeneracy. In certain embodiments, the nucleotide sequence of the nucleic acid molecule is codon optimized.

[0034] On the other hand, the present application provides a vector comprising a nucleic acid molecule encoding an isolation as described above. In certain embodiments, the vector is a cloning vector or an expression vector. In certain embodiments, the vector of the present invention is, for example, a plasmid, a cosmid, a phage, a lentivirus, etc. In certain embodiments, the vector is capable of expressing a single domain antibody or an antigen-binding fragment thereof, a multispecific antibody or a fusion protein of the present invention in a subject (e.g., a mammal, such as a human).

[0035] On the other hand, the application provides a kind of host cell, it comprises the nucleic acid molecule of separation as mentioned above and / or the carrier as mentioned above.Host cell can be eukaryotic cell (for example mammalian cell, insect cell, yeast cell) or prokaryotic cell (for example Escherichia coli).Suitable eukaryotic cell includes but is not limited to NS0 cell, Vero cell, Hela cell, COS cell, CHO cell, HEK293 cell, BHK cell and MDCKII cell.

[0036] On the other hand, the present application provides a method for preparing a single-domain antibody or an antigen-binding fragment thereof as described above, a multi-specific antibody as described above, or a fusion protein as described above, which comprises culturing a host cell as described above under conditions allowing protein expression, and recovering the single-domain antibody or its antigen-binding fragment, multi-specific antibody, or fusion protein from the cultured host cell culture.

[0037] The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81 (1985)). In addition, these antigen-binding fragments can also be directly produced by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000)).

[0038] On the other hand, the present application provides a pharmaceutical composition comprising a single-domain antibody or an antigen-binding fragment thereof as described above, a multispecific antibody as described above, a fusion protein as described above, a conjugate as described above, an isolated nucleic acid molecule as described above, a vector as described above, or a host cell as described above, and a pharmaceutically acceptable carrier and / or excipient.

[0039] On the other hand, the present application provides a kit comprising a single-domain antibody or an antigen-binding fragment thereof as described above, a multispecific antibody as described above, a fusion protein as described above, a conjugate as described above, an isolated nucleic acid molecule as described above, a vector as described above, a host cell as described above, or a pharmaceutical composition as described above, and optionally instructions for use.

[0040] On the other hand, the present application provides a method for neutralizing the virulence of coronavirus in a sample, comprising contacting a sample containing coronavirus with a single domain antibody or an antigen-binding fragment thereof as described above, or a multispecific antibody as described above.

[0041] In certain embodiments, the coronavirus is SARS-CoV-2.

[0042] On the other hand, the present application provides a method for detecting the presence or level of coronavirus in a sample, which comprises using the antibody or antigen-binding fragment thereof as described above or the conjugate as described above.

[0043] In certain embodiments, the method is an immunological assay, such as an enzyme immunoassay (eg, ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.

[0044] In certain embodiments, the coronavirus is SARS-CoV-2, and the method comprises: using an antibody or antigen-binding fragment thereof as described above, wherein the antibody or antigen-binding fragment thereof optionally comprises a detectable label.

[0045] The bispecific antibody as described above, or the use of the immunoconjugate as described above in a kit, wherein the kit is used for a method for neutralizing the virulence of a coronavirus in a sample, or a method for detecting the presence or level of a coronavirus in a sample.

[0046] Use of the bispecific antibody as described above, or the immunoconjugate as described above, in the preparation of a medicament for preventing and / or treating a disease caused by SARS-CoV-2 in a subject.

[0047] In certain embodiments, the medicament further comprises an additional pharmaceutically active agent.

[0048] In certain embodiments, the subject is a mammal, such as a human.

[0049] Definition of terms

[0050] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA procedures used herein are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0051] As used herein, "severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)", formerly known as "new coronavirus" or "2019-nCov", belongs to the genus Beta coronavirus and is an enveloped single-stranded positive-sense RNA virus. The genome sequence of SARS-CoV-2 is known to those skilled in the art, and can be found, for example, in GenBank: MN908947. SARS-CoV-2 contains at least three membrane proteins, including surface spike protein (S), integral membrane protein (M) and membrane protein (E). The receptor of SARS-CoV-2, like SARS-CoV, is specifically bound to angiotensin-converting enzyme 2 (ACE2) on the host cell through the receptor binding domain (RBD) on the S protein, which is connected to the membrane fusion and cell entry of the virus, and plays a vital role in the process of viral infection of cells.

[0052] As used herein, the terms "new coronavirus pneumonia" and "COVID-19" refer to pneumonia caused by infection with SARS-CoV-2. The two have the same meaning and can be used interchangeably.

[0053] The term "antibody" is used in the broadest sense herein. "Antibodies" include complete monoclonal antibodies, single domain antibodies, heavy chain antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), and antibody fragments (e.g., antigen binding fragments) with the desired biological activity. Basic four-chain antibodies generally refer to four heteromeric glycoproteins composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain and light chain has regularly spaced intrachain disulfide bridges. Each heavy chain and light chain has a variable domain (VH and VL) at its respective N-terminus. The variable domains of each heavy chain / light chain pair form antigen binding sites, respectively. The term "antibody" is not limited to any specific method for producing antibodies. For the types, structures, biological activities, and preparation methods of antibodies, see Fundamental Immunology (William E. Paul, 1976). th Edition, LWW, 2012); Antibody Engineering Springer Lab Manual (Roland Kontermann, Stefan Dübel, ISBN978-3-662-04605-0 (eBook)); Schiel JE, Davis DL, Borisov OV, State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody CHaracteriazation Volume 1. Monoclonal Antibody Therapeutics: Structure, Function, and Regulatory Space (American Chemical Society, 2014). Antigen-binding fragments are antibody fragments that retain the ability to bind to specific antigens, non-limiting examples of which include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementary determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, linear antibodies, nanobodies, domain antibodies or single-domain antibody antigen-binding fragments, which contain at least a portion of an antibody sufficient to confer specific antigen binding ability.

[0054] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883) or the IMGT numbering system (Lefrance et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, those skilled in the art will easily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0055] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0056] As used herein, the term "camel-derived antibody" refers to an antibody against an antigen produced by Camelidae animals (including camels, alpacas and llamas) after immunization or antigen invasion. It is known to those skilled in the art that among the antibodies produced by Camelidae animals, there are "heavy-chain antibodies" (HCAb) lacking light chains. Such antibodies contain only one variable domain of heavy chain of HCAb (VHH) and two conventional CH2 and CH3 regions, and the VHH region cloned and expressed separately has good structural stability and antigen binding activity. VHH is the smallest unit currently known to bind to the target antigen.

[0057] As used herein, the terms "single domain antibody (sdAb)", "domain antibody" and "nanobody" are used interchangeably and refer to an antibody fragment consisting of a single variable domain (e.g., heavy chain variable region) in an antibody. Typically, a single domain antibody, a domain antibody or a nanobody consists of four framework regions and three complementarity determining regions, wherein the four framework regions are FR1-FR4, and the three complementarity determining regions are CDR1-CDR3.

[0058] In certain embodiments, the single domain antibody of the present invention may have a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In certain embodiments, the single domain antibody is a camel-derived VHH, a humanized VHH, or a camel-derived VH (e.g., a camel-derived VH). The camel-derived VHH, the humanized VHH, or the camel-derived VH comprises a domain or protein that is advantageous for use as a functional antigen binding. These antibodies do not require a light chain variable region, that is, they can bind to the antigen with high affinity and specificity. Compared with antibodies composed of heavy chains and light chains, nano antibodies have high solubility, high stability to heat, pH, proteases, and other deforming agents, and only require single chain expression for large-scale production.

[0059] As used herein, the term "multispecific antibody" refers to an antibody with multiple different antigen binding specificities, including, for example, bispecific antibodies, trispecific antibodies, and tetraspecific antibodies. "Bispecific antibodies" refer to antibodies with two different antigen binding specificities, which are formed by coupling arms of a first antibody (or a fragment thereof) and a second antibody (or a fragment thereof) or an antibody analog, wherein the coupling method includes, but is not limited to, chemical reaction, gene fusion, and enzymatic. A trispecific antibody is an antibody with three different antigen binding specificities, and a tetraspecific antibody is an antibody with four different antigen binding specificities.

[0060] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen to which it is directed. The strength or affinity of a specific binding interaction can be expressed as the equilibrium dissociation constant (KD) or half-maximal effect concentration (EC50) of the interaction.

[0061] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0062] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with a subject and an active ingredient, which is well known in the art and includes, but is not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH regulators include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc.

[0063] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or illness or symptom in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical result. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, alleviating symptoms (whether partially or completely), alleviating or improving prognosis, reducing or inhibiting disease recurrence, etc., whether detectable or undetectable. In addition, "treatment" can also refer to, compared with the expected survival period (if not treated), prolonging the survival period.

[0064] Advantageous Effects of the Invention

[0065] The present invention provides single-domain antibodies that neutralize SARS-CoV-2. Specifically, these single-domain antibodies bind to epitopes on the RBD region of the S protein of SARS-CoV-2 and neutralize SARS-CoV-2. The antibodies of the present invention can inhibit the binding of the RBD protein of SARS-CoV-2 to the receptor ACE2. Therefore, the antibodies of the present invention can be used to diagnose, prevent and / or treat coronavirus infections and / or diseases caused by the infections.

[0066] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 The SDS-PAGE electrophoresis of the recombinant protein RBD-mFc is shown.

[0068] Figure 2 The gene fishing electropherogram of anti-SARS-CoV-2 nanoantibodies is shown.

[0069] Figure 3 The results of the library capacity determination of the phage-displayed SARS-CoV-2 nanobody library are shown.

[0070] Figure 4 The amino acid sequence of the target gene VHH of the SARS-CoV-2 nanoantibody library successfully inserted as shown by the sequencing method.

[0071] Figure 5 The results of phage display technology screening for SARS-CoV-2 specific nanoantibody phage enrichment are shown.

[0072] Figure 6 The results of the binding ability test of positive clones of VHH identified at the phage level to RBD-mFc and mFC are shown.

[0073] Figure 7 The SDS-PAGE electropherogram of the Nanobody is shown.

[0074] Figure 8 The ELISA test results of the nanobody binding activity to RBD are shown.

[0075] Fig. 9 The results of the detection of the reactivity of the nanoantibody with the SARS-CoV-2 receptor binding region protein RBD trimer strimer are shown.

[0076] Fig.10 The results of the neutralization activity assay of the nanoantibody in the SARS-CoV2 VSVpp pseudovirus infection model are shown.

[0077] Sequence information

[0078] The information of the partial sequences involved in the present invention is provided in Table 1 below.

[0079] Table 1: Description of sequences

[0080]

[0081] DETAILED DESCRIPTION

[0082] The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.

[0083] Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references. For example, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention can be found in Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al., eds., (1987)); METHODS IN ENZYMOLOGY series (Academic Press): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames and GR Taylor, eds. (1995)); and ANIMAL CELL CULTURE (Animal Cell Culture). CELL CULTURE) (RI Freshney, ed. (1987)).

[0084] In addition, if the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the present invention. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.

[0085] Example 1. Preparation of camel-derived nanobodies against SARS-CoV-2 RBD protein

[0086] 1.1 Preparation and activity identification of SARS-CoV-2 receptor binding region RBD protein:

[0087] The SARS-CoV-2 receptor binding region RBD-mFc protein was prepared by self-expression. With reference to the full gene sequence of SARS-CoV2-2 (MN908947.3), the full-length amino acid sequence of the extracellular region of the SARS-CoV2-2 spike protein was optimized according to the human codon preference to obtain the encoding nucleic acid sequence. The nucleotide sequence encoding the RBD protein of SARS-CoV2 was connected to the eukaryotic expression plasmid vector ptt5 mFc (Shengong, GN201916320), which contains the nucleotide sequence encoding the mFc tag. The constructed recombinant plasmid was transfected into ExpiCHO cells (purchased from Thermofisher) for expression and purification. The results of SDS-PAGE electrophoresis showed that the RBD-mFc protein was successfully expressed and purified ( Figure 1 ), with a size of approximately 60KDa.

[0088] 1.2 Construction and screening of SARS-CoV-2-nanoantibody immune library

[0089] A healthy adult alpaca was immunized with RBD-mFc protein by multiple subcutaneous injections. After immunization, peripheral blood was drawn from the alpaca, total RNA was isolated, and the nanoantibody (VHH) gene was amplified by reverse transcription and PCR ( Figure 2 ), the VHH gene was cloned into the phage vector Pcgmt (retained in the laboratory), and transformed into ER2738 host cells (purchased from Lucigen) to construct a phage display library. After testing, the constructed library capacity was 2.67×10 7 CFU( Figure 3 ). 30 monoclonal clones were randomly selected for sequencing, and the results showed that 29 were successfully inserted ( Figure 4), the library insertion rate was 96.7%. Subsequently, the library was screened using phage display technology. After one round of "adsorption-washing-enrichment", a total of 2359 single clones were picked for Elisa detection. The results showed that 128 positive clones were obtained, and the total positive clone rate of VHH fragments was about 5%. The positive rate of P0 generation was 1%, and the positive rate of P1 generation was 9% ( Figure 5 Among them, the phage-positive monoclonal Nb-5-cov, Nb-4-cov, and Nb-6-cov phage levels have good binding ability with RBD-mFC ( Figure 6 ).

[0090] The plasmids of phage-positive monoclones Nb-5-cov, Nb-4-cov and Nb-6-cov were extracted and sequenced to obtain the CDR sequences and variable region sequences of the three antibodies Nb-5-cov, Nb-4-cov and Nb-6-cov, as shown in Table 1.

[0091] 1.3 Eukaryotic expression of camel-derived nanobodies against SARS-CoV-2 receptor binding region RBD protein

[0092] The above three nanoantibody genes with different sequences were cloned into the ptt5 hIgG1 vector, and then the recombinant plasmid was mixed with the transfection reagent PEI 1:2 and transfected into HEK293F cells; cultured in a shaking incubator at 37°C and 5% CO2 for 6 days; then the cell supernatant was collected, purified using Protein A, and the concentration was measured by ultraviolet spectrometry. The purity of the expressed and purified nanoantibody was greater than 90% ( Figure 7 ), which can be used for subsequent functional activity studies and stored at -80°C for future use.

[0093] Example 2. Reactivity of Nanobodies Against SARS-CoV-2 RBD Protein with SARS-CoV-2 RBD Protein

[0094] 2.1 Preparation of reaction plates

[0095] RBD-mFc protein, RBD-his protein, and mFc protein were diluted with 50mM CB buffer to a final concentration of 2μg / mL; 100μL of coating solution was added to each well of the 96-well ELISA plate, and the plates were coated at 2-8℃ for 16-24h and then at 37℃ for 2h; the plates were washed once with PBST washing solution; 200μL of blocking solution was then added to each well, and the plates were blocked at 37℃ for 2h; the blocking solution was discarded. After drying, the plates were stored in an aluminum foil bag at 2-8℃ for future use.

[0096] 2.2 ELISA detection of RBD nanoantibodies Nb-4-cov, Nb-5-cov, and Nb-6-cov

[0097] Take nanoantibodies Nb-4-cov, Nb-5-cov, and Nb-6-cov and dilute them with 20mM PBS buffer to a final concentration of 1μg / mL. Take the ELISA plate coated with RBD-mFc protein, RBD-his protein, and mFc protein, add 100μL of diluted sample to each well, and place it in a 37℃ incubator for reaction for 60min. Wash the ELISA plate with PBST washing solution 5 times, add 100μL of HRP-labeled goat anti-human IgG reaction solution to each well, and place it in a 37℃ incubator for reaction for 30min. After completing the enzyme marker reaction step, wash the ELISA plate with PBST washing solution 5 times, add 50μL of TMB color developer to each well, and place it in a 37℃ incubator for reaction for 15min. After completing the color development reaction step, add 50μL of stop solution to each well of the ELISA plate after the reaction, and detect the OD450 / 630 value of each well on the microplate reader. The results showed that the nanobodies Nb-4-cov, Nb-5-cov and Nb-6-cov had RBD-specific binding activity ( Figure 8 ).

[0098] Example 3. Nanobodies against SARS-CoV-2 RBD protein and SARS-CoV-2 receptor binding region RBD trimer Reactivity of strimer

[0099] 3.1 Preparation of reaction plates

[0100] Dilute the strimer protein with 50mM CB buffer to a final concentration of 2μg / mL; add 100μL of coating solution to each well of the 96-well ELISA plate, coat at 2-8℃ for 16-24h, and then coat at 37℃ for 2h; wash once with PBST washing solution; then add 200μL of blocking solution to each well, block at 37℃ for 2h; discard the blocking solution. After drying, put it into an aluminum foil bag and store it at 2-8℃ for later use.

[0101] 3.2 ELISA test

[0102] Take the nanoantibodies Nb-4-cov, Nb-5-cov, and Nb-6-cov, and dilute them 10 times in a gradient starting from 1 μg / mL with 20mM PBS buffer, with a total of 6 gradient dilutions. Take the ELISA plate coated with strimer protein, add 100 μL of diluted sample to each well, and place it in a 37°C incubator for reaction for 60 minutes. Wash the ELISA plate 5 times with PBST washing solution, add 100 μL of HRP-labeled sheep anti-human IgG reaction solution to each well, and place it in a 37°C incubator for reaction for 30 minutes. After completing the enzyme marker reaction step, wash the ELISA plate 5 times with PBST washing solution, add 50 μL of TMB color developer to each well, and place it in a 37°C incubator for reaction for 15 minutes. After completing the color development reaction step, add 50 μL of stop solution to each well of the ELISA plate after the reaction, and detect the OD450 / 630 value of each well on the microplate reader. The results show ( Fig. 9 ), the EC50 value of the nanobody Nb-6-cov and strimer is 0.02972ug / ml, which has good binding activity.

[0103] Example 4. Anti-SARS-CoV-2 RBD protein nanoantibodies in SARS-CoV2 VSVpp pseudovirus infection model and capability analysis

[0104] The SARS-CoV2-VSVpp pseudovirus neutralization method was carried out according to the reference method (doi: https: / / doi.org / 10.1101 / 2020.04.08.026948). The main experimental process is briefly described as follows: To construct a VSV pseudovirus carrying the SARS-CoV-2 spike protein, the SARS-CoV-2 spike gene (sequence source GenBank: MN908947.3) with 18 amino acids truncated at the C-terminus of the SARS-CoV-2 spike gene was cloned into the eukaryotic expression vector pCAG to obtain pCAG-nCoVSde18. The plasmid pCAG-nCoVSde18 was transfected into Vero-E6. After 48 hours of transfection, the VSVdG-EGFP-G (Addgene, 31842) virus was inoculated into cells expressing the SARS-CoV-2Sde18 truncated protein and incubated for 1 hour. The VSVdG-EGFP-G virus in the supernatant was then removed, and anti-VSV-G rat serum was added to block the infection of the residual VSVdG-EGFP-G. The progeny virus will carry the SARS-CoV-2 Sde18 truncated protein, and the pseudovirus VSV-SARS-CoV2 VSVpp will be obtained. 24 h after VSVdG-EGFP-G infection, the cell supernatant was collected, then centrifuged and filtered (pore size 0.45-μm, Millipore, SLHP033RB) to remove cell debris and stored at -80°C for later use. The virus titer was determined by the number of GFP-positive cells after the supernatant was infected with BHK21-hACE2 with gradient dilutions. The gene of hACE2 was integrated in BHK21 cells by the PiggyBac transposon system. The transposon vector (SBI system biosciences, PB514B-2) containing the hACE2 gene and the transposase plasmid were co-transfected into BHK21 cells, and the cells BHK21-hACE2 stably expressing hACE2 were obtained by screening with puromycin resistance and red fluorescence.

[0105] Antibody nanoantibodies Nb-4-cov, Nb-5-cov, and Nb-6-cov were diluted to 75 μg / mL as gradient 1, 4-fold gradient dilution, a total of 6 gradients, and the gradient diluted antibodies were mixed with diluted SARS-CoV2 VSVpp virus (MOI = 0.05) and incubated at 37 ° C for 1 h. All samples and viruses were diluted with 10% FBS-DMEM. 80 μL of the mixture was added to the pre-plated BHK21-hACE2 cells. After incubation for 12 hours, the infected cells were fluorescently imaged using a high-content imaging system based on a spinning disk confocal (Opera phenix or Operetta CLS, purchased from Perkinelmer). After completion, the obtained fluorescence images were quantitatively analyzed using Colμmbμs image management and analysis software to detect the number of green fluorescence-positive cells. The number of GFP-positive cells in the antibody-treated group was reduced (%) compared with the untreated control wells, and the inhibition rate was calculated.

[0106] The results are as follows Fig.10 As shown, the IC50 of the nanoantibody Nb-6-cov is 0.69 ng / mL, among which Nb-6-cov has a strong neutralizing effect.

[0107] In summary, the anti-SARS-CoV-2 nanoantibodies (Nb-4-cov nanoantibody, Nb-5-cov nanoantibody and Nb-6-cov nanoantibody) prepared by the present invention have high affinity binding to human RBD antigen, and Nb-6-cov has strong pseudovirus neutralizing activity.

[0108] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. A single domain antibody or antigen-binding fragment thereof that specifically binds to the receptor binding domain (RBD) of the S protein of SARS-CoV-2, wherein: The single domain antibody or antigen binding fragment thereof comprises a VHH, wherein the VHH comprises: (1) The following three complementarity determining regions (CDRs): The CDR1 sequence is SEQ ID NO: 1, the CDR2 sequence is SEQ ID NO: 2, and the CDR3 sequence is SEQ ID NO: 3; or, (2) The following three complementarity determining regions (CDRs): The CDR1 sequence is SEQ ID NO: 10, the CDR2 sequence is SEQ ID NO: 11, and the CDR3 sequence is SEQ ID NO: 12; or, (3) The following three complementarity determining regions (CDRs): The sequence of CDR1 is SEQ ID NO: 19, the sequence of CDR2 is SEQ ID NO: 20, and the sequence of CDR3 is SEQ ID NO:

21.

2. The single-domain antibody or antigen-binding fragment thereof of claim 1, further comprising a framework region (FRs).

3. The single-domain antibody or antigen-binding fragment thereof of claim 1, wherein the single-domain antibody or antigen-binding fragment thereof comprises FRs from humans or camels.

4. The single-domain antibody or antigen-binding fragment thereof of claim 1, wherein the single-domain antibody consists of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

5. The single-domain antibody or antigen-binding fragment thereof of claim 1, wherein the single-domain antibody or antigen-binding fragment thereof is a camelid-derived VHH or a humanized VHH.

6. The single domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein: The single domain antibody or antigen binding fragment thereof comprises a sequence selected from the group consisting of: (1) the sequence shown in SEQ ID NO: 8; or, (2) the sequence shown in SEQ ID NO: 17; or, (3) The sequence shown in SEQ ID NO:

26.

7. A multispecific antibody comprising the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 6, and another antibody or antigen binding fragment thereof. The multispecific antibody of claim 7 , which is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

9. The multispecific antibody of claim 7, wherein the additional antibody or antigen-binding fragment thereof has binding specificity to at least one molecule or antigen among tumor-specific antigens, tumor-associated antigens, T cell engager molecules, tumor immunity-related molecules or autoimmune regulation-related molecules.

10. The multispecific antibody of claim 7, wherein the additional antibody or fragment thereof has antigen binding specificity to at least one of PD-1, PDL-1, CD19, CD20, CTLA4, IL6, IL6R or TNFα.

11. A fusion protein comprising the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 6, and another biologically active polypeptide.

12. The fusion protein of claim 11, wherein the additional biologically active polypeptide is a polypeptide or protein having therapeutic activity, binding activity or enzymatic activity.

13. The fusion protein of claim 11, wherein the additional biologically active polypeptide is linked to the N-terminus and / or C-terminus of the single domain antibody or antigen-binding fragment thereof via a linker.

14. The fusion protein of claim 11, comprising one or more of the single domain antibodies or antigen binding fragments thereof and one or more of the additional biologically active polypeptides; wherein, The plurality of single domain antibodies or antigen binding fragments thereof are the same or different, and the plurality of additional biologically active polypeptides are the same or different.

15. The fusion protein of claim 11, comprising one of the single domain antibodies or antigen binding fragments thereof and one of the additional biologically active polypeptides. The fusion protein of claim 11 , comprising one of the single domain antibodies or antigen binding fragments thereof and two of the additional biologically active polypeptides.

17. The fusion protein of claim 11, wherein the two additional biologically active polypeptides are respectively connected to the N-terminus and / or C-terminus of the single-domain antibody or the antigen-binding fragment thereof through a linker.

18. A conjugate comprising the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 6 and a chemotherapeutic drug, a radiopharmaceutical or an imaging agent linked to the single domain antibody or antigen binding fragment thereof.

19. An isolated nucleic acid molecule encoding the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 6, the multispecific antibody according to any one of claims 7 to 10, or the fusion protein according to any one of claims 11 to 17.

20. A vector comprising a nucleic acid molecule encoding the isolated nucleic acid of claim 19. The vector according to claim 20 , which is a cloning vector or an expression vector.

22. A host cell comprising the isolated nucleic acid molecule of claim 19 and / or the vector of claim 20.

23. A method for preparing the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 6, the multispecific antibody according to any one of claims 7 to 10, or the fusion protein according to any one of claims 11 to 17, comprising culturing the host cell according to claim 22 under conditions that allow protein expression, and recovering the single domain antibody or antigen binding fragment thereof, multispecific antibody, or fusion protein from the cultured host cell culture.

24. A pharmaceutical composition comprising the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 6, the multispecific antibody according to any one of claims 7 to 10, the fusion protein according to any one of claims 11 to 17, the conjugate according to claim 18, the isolated nucleic acid molecule according to claim 19, the vector according to claim 20 or the host cell according to claim 22, and a pharmaceutically acceptable carrier and / or excipient.

25. A kit comprising the single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 6, the multispecific antibody according to any one of claims 7 to 10, the fusion protein according to any one of claims 11 to 17, the conjugate according to claim 18, the isolated nucleic acid molecule according to claim 19, the vector according to claim 20, the host cell according to claim 22, or the pharmaceutical composition according to claim 24.

26. A method for neutralizing the virulence of a coronavirus in a sample in vitro, comprising contacting a sample containing the coronavirus with a single domain antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 6, or a multispecific antibody as described in any one of claims 7 to 10.

27. The method of claim 26, wherein the coronavirus is SARS-CoV-2.

28. A method for detecting the presence or level of coronavirus in a sample for non-diagnostic purposes, comprising using a single domain antibody or antigen binding fragment thereof as described in any one of claims 1 to 6 or a conjugate as described in claim 18.

29. The method of claim 28, which is an immunological assay.

30. The method of claim 28, which is an enzyme immunoassay, a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.

31. The method of claim 28, wherein the coronavirus is SARS-CoV-2, the method comprising: Use the single domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

32. The method of claim 31, wherein the single domain antibody or antigen-binding fragment thereof comprises a detectable label.

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