Nanobodies targeting sars-cov-2 spike protein and uses thereof

Abstract

The application discloses a nanobody targeting SARS-CoV-2 spike protein and application thereof. The application provides a nanobody targeting SARS-CoV-2 spike protein, which comprises HCDR1 shown in any one of SEQ ID NO: 6, 9, 12, 15 and 18; HCDR2 shown in any one of SEQ ID NO: 7, 10, 13, 16 and 19; and HCDR3 shown in any one of SEQ ID NO: 8, 11, 14, 17 and 20. The nanobody provided by the application can specifically recognize different subtypes of coronavirus spike proteins, and can be applied to the fields of biological and pharmaceutical research and development, clinical in vitro diagnosis, immunological research and the like.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine or biotechnology, and more specifically to nanobodies targeting the SARS-CoV-2 spike protein and their applications. Background Technology

[0002] Coronaviruses (CoVs) are a highly diverse family of enveloped, positive-sense, single-stranded RNA viruses capable of infecting humans, other mammals, birds, and livestock. Their widespread transmission poses a significant challenge to global public health security and also impacts global economic and cultural exchange. Over the past 20 years, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have repeatedly spread in human populations, exhibiting high pathogenicity. By infecting human bronchial epithelial cells, lung cells, and upper respiratory tract cells, SARS-CoV, MERS-CoV, and SARS-CoV-2 infections can develop into severe, life-threatening respiratory lesions and lung damage; to date, no specific preventative or treatment methods have been approved. Given the long development cycle, high difficulty, and high risk associated with small molecule drug development, neutralizing antibodies are a key tool in combating epidemic viral diseases and can help simplify the early discovery of antibody-based drug therapies. 1–3 Antibodies against SARS-CoV-2 can resist viral escape, may be active against different subtypes of SARS virus, and neutralize the virus. 4,5 Sum effector function 6 It has a protective effect on the human body.

[0003] The spike protein (S protein) of coronaviruses is a key mediator for viral attachment, infection, and entry into cells. It belongs to class I fusion glycoproteins and forms a homotrimer containing two functionally distinct S1 and S2 subunits. The S1 subunit can be further divided into two relatively independent domains: the N-terminal domain (NTD) and the C-terminal domain (CTD). The receptor-binding domain (RBD) contained in the exposed S1 subunit is responsible for binding to host cell receptors, thereby determining virus-cell selectivity and pathogenicity. Most coronaviruses, such as SARS-CoV and MERS-CoV, have their RBD located in the CTD. Only a small number of beta coronaviruses have their RBD located in the NTD. The S2 subunit is anchored to the cell membrane via a transmembrane region. This transmembrane S2 subunit contains a heptapeptide repeat region and a fusion peptide, which, after conformational rearrangement, mediates the fusion of the viral membrane and the cell membrane, allowing the virus to enter the cell. 7 All viruses, including SARS-CoV-2, are constantly mutating. Some mutations do not affect the characteristics of the virus, but others may influence these characteristics, such as its transmissibility, the severity of illness after infection, or the effectiveness of vaccines, treatments, and diagnostic tools. To date, the SARS-CoV-2 protein has produced several mutant strains, including alpha, beta, delta, and omega-3. 8 A key aspect of any antiviral treatment regimen is how to avoid drug resistance to therapeutic agents due to the rapid mutation of the viral pathogen. 9 This resistance becomes particularly pronounced when selective pressure is applied in a drug-based treatment setting. For example, when HIV drugs are used alone, mutations in this drug-selective approach lead to widespread resistance. The subsequent success of combination therapies for HIV suggests that requiring simultaneous mutations at multiple gene locations in the virus may be the most effective way to avoid resistance.

[0004] In 1989, Professor Hamers Casterman, an immunologist at the Free University of Brussels in Belgium, and his colleagues discovered a novel antibody in camel serum with a structure different from traditional antibodies: this antibody naturally lacks a light chain and consists only of two heavy chains, and is called a heavy-chain antibody (HCAb). The variable domain of the heavy-chain antibody (VHH) obtained through in vitro recombinant synthesis possesses structural stability and antigen-binding activity comparable to the original heavy-chain antibody. It is the smallest known unit capable of binding target antigens, with a crystal diameter of 2.5 nanometers, a length of 4 nm, and a molecular mass of only 15 kDa. Its mass is only about one-tenth of the molecular mass of traditional antibodies and about half that of antigen-binding fragments; it is called a single-domain antibody, also known as a nanobody (Nb).

[0005] Compared to traditional antibody molecules, nanobodies offer certain advantages in antigen detection, in vitro clinical diagnostics, and immunological research. Due to their smaller molecular weight, higher stability, better solubility, better permeability, ability to be expressed by microorganisms, and capacity to recognize hidden epitopes, nanobodies have gradually emerged as a new force in next-generation therapeutic biopharmaceuticals and clinical diagnostic reagents. Because of their smaller molecular weight, nanobodies also exhibit significant advantages in the combination of multiple epitopes with antibodies. For example, researchers have found that tandemly linking multiple identical nanobodies can significantly enhance the affinity of antibodies for spike protein trimers. 10 Fusing bispecific nanobodies into the conventional IgG1 Fc domain has also been shown to improve neutralizing efficacy. 11 The targeted development of multivalent nanobodies has been applied not only to the SARS-CoV-2 protein... 12 It has also been applied to other targets with clinical therapeutic value. 13–15 The multivalent nanobody Ozoralizumab, co-developed by Taisho Pharmaceutical Co., Ltd., has been used in clinical treatment of rheumatoid arthritis. 16 . Summary of the Invention

[0006] The problem the invention aims to solve

[0007] In view of the above-mentioned problems existing in the prior art, the purpose of the present invention is to provide a nanobody that targets the SARS-CoV-2 spike protein.

[0008] Solution for solving the problem

[0009] A first aspect of the present invention provides a nanobody targeting the SARS-CoV-2 spike protein, comprising:

[0010] HCDR1 as shown in any one of SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15 and SEQ ID NO: 18;

[0011] HCDR2 represented by any one of SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16 and SEQ ID NO: 19; and,

[0012] HCDR3 shown in any one of SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17 and SEQ ID NO: 20.

[0013] In some embodiments, the nanobody targeting the SARS-CoV-2 spike protein comprises any one or more of the following (i) to (v):

[0014] (i) HCDR1 shown in SEQ ID NO: 12, HCDR2 shown in SEQ ID NO: 13 and HCDR3 shown in SEQ ID NO: 14;

[0015] (ii) HCDR1 shown in SEQ ID NO: 6, HCDR2 shown in SEQ ID NO: 7 and HCDR3 shown in SEQ ID NO: 8;

[0016] (iii) HCDR1 shown in SEQ ID NO: 15, HCDR2 shown in SEQ ID NO: 16 and HCDR3 shown in SEQ ID NO: 17;

[0017] (iv)HCDR1 shown in SEQ ID NO: 18, HCDR2 shown in SEQ ID NO: 19 and HCDR3 shown in SEQ ID NO: 20;

[0018] (v) HCDR1 shown in SEQ ID NO: 9, HCDR2 shown in SEQ ID NO: 10 and HCDR3 shown in SEQ ID NO: 11.

[0019] In some implementations, the nanobody targeting the SARS-CoV-2 spike protein also includes a framework region.

[0020] In some optional implementations, the frame region includes:

[0021] FR1 shown in any one of SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 30 and SEQ ID NO: 36, or FR1 having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with any one of SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 30 and SEQ ID NO: 36;

[0022] FR2 shown in any one of SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 31 and SEQ ID NO: 33, or FR2 having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with any one of SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 31 and SEQ ID NO: 33;

[0023] FR3 represented by any one of SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 32 and SEQ ID NO: 34, or FR3 having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with any one of SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 32 and SEQ ID NO: 34; and,

[0024] FR4 shown in either SEQ ID NO: 24 or SEQ ID NO: 35, or FR4 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with either SEQ ID NO: 24 or SEQ ID NO: 35.

[0025] In some specific implementations, the frame region includes any one or more of the following (i) to (v):

[0026] (i) FR1 shown in SEQ ID NO: 21, FR2 shown in SEQ ID NO: 28, FR3 shown in SEQ ID NO: 29 and FR4 shown in SEQ ID NO: 24;

[0027] (ii) FR1 shown in SEQ ID NO: 21, FR2 shown in SEQ ID NO: 22, FR3 shown in SEQ ID NO: 23 and FR4 shown in SEQ ID NO: 24;

[0028] (iii) FR1 shown in SEQ ID NO: 30, FR2 shown in SEQ ID NO: 31, FR3 shown in SEQ ID NO: 32 and FR4 shown in SEQ ID NO: 24;

[0029] (iv) FR1 shown in SEQ ID NO: 36, FR2 shown in SEQ ID NO: 33, FR3 shown in SEQ ID NO: 34 and FR4 shown in SEQ ID NO: 35;

[0030] (v) FR1 shown in SEQ ID NO: 25, FR2 shown in SEQ ID NO: 26, FR3 shown in SEQ ID NO: 27 and FR4 shown in SEQ ID NO: 24.

[0031] In some more specific embodiments, the nanobody targeting the SARS-CoV-2 spike protein comprises the sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 5, or the sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with any one of SEQ ID NO: 1 to SEQ ID NO: 5.

[0032] In some embodiments, the nanobody targeting the SARS-CoV-2 spike protein further comprises a tag, a signal peptide, a linker sequence, or any combination thereof.

[0033] In some specific implementations, the nanobody targeting the SARS-CoV-2 spike protein includes a tag at its N-terminus and / or C-terminus; and / or, includes a signal peptide at its N-terminus.

[0034] In some preferred embodiments, the nanobody targeting the SARS-CoV-2 spike protein comprises the sequence shown in any one of SEQ ID NO: 39 to SEQ ID NO: 43, or the sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with any one of SEQ ID NO: 39 to SEQ ID NO: 43.

[0035] A second aspect of the present invention provides a polynucleotide encoding a nanobody targeting the SARS-CoV-2 spike protein as described in the first aspect of the present invention.

[0036] A third aspect of the present invention provides an expression vector containing the polynucleotides described in the second aspect of the present invention.

[0037] A fourth aspect of the present invention provides a host cell in which the expression vector described in the third aspect of the present invention is introduced or contained.

[0038] A fifth aspect of the present invention provides a method for producing nanobodies targeting the SARS-CoV-2 spike protein, comprising the steps of: culturing the host cells described in the fourth aspect of the present invention, isolating the nanobodies from the culture, and, optionally, purifying the nanobodies.

[0039] A sixth aspect of the present invention provides a pharmaceutical composition comprising the nanobody targeting the SARS-CoV-2 spike protein as described in the first aspect of the present invention, and a pharmaceutically acceptable excipient, diluent or carrier.

[0040] A seventh aspect of the present invention provides a detection or diagnostic kit containing the nanobody targeting the SARS-CoV-2 spike protein as described in the first aspect of the present invention.

[0041] The eighth aspect of the present invention provides the use of nanobodies targeting the SARS-CoV-2 spike protein as described in the first aspect of the present invention in the preparation of a medicament, wherein the medicament is used to treat or prevent diseases or symptoms caused by SARS-CoV-2 infection.

[0042] In some preferred embodiments, the disease or condition caused by SARS-CoV-2 infection includes infection with the novel coronavirus.

[0043] The ninth aspect of the present invention provides the use of nanobodies targeting the SARS-CoV-2 spike protein as described in the first aspect of the present invention in the preparation of reagents, wherein the reagents are used to detect or diagnose diseases or symptoms caused by SARS-CoV-2 infection.

[0044] In some preferred embodiments, the disease or condition caused by SARS-CoV-2 infection includes infection with the novel coronavirus.

[0045] The effects of the invention

[0046] This invention develops various nanobodies capable of specifically recognizing different subtypes of coronavirus spike proteins. The nanobodies disclosed in this invention can be applied independently in biomedical research and development (gene-engineered drug development, ADC drug development); clinical in vitro diagnostics (colloidal gold method, enzyme-linked immunosorbent assay, electrochemiluminescence method); and basic research such as immunology. Furthermore, based on the antigenic epitope verification and comparison of these nanobodies, they can be covalently designed with existing nanobodies that recognize different antigenic epitopes to develop bispecific or multispecific multivalent nanobodies for application in drug development or clinical in vitro diagnostics. Attached Figure Description

[0047] Figure 1 This shows an SDS-PAGE gel image of purified Delta-type spike protein antigen.

[0048] Figure 2 Construction of expression vectors for positive clonal antibodies. Figure 2 'a' in the figure shows the PCR product purified using a PCR purification kit. Figure 2 b in the figure shows the recovery of the vector pCDNA3.1 using a gel recovery kit. Figure 2 The 'c' in the figure represents the bacterial culture PCR verification. Samples that have been verified correctly by PCR are sent for sequencing.

[0049] Figure 3 The following are the SDS-PAGE results for the purification of five different nanobodies. In each group: Lane 1: whole cell sample; Lane 2: expression medium supernatant sample; Lane 3: flow-through sample from nickel affinity chromatography column; Lane 4: Buffer C washed sample; Lane 5: Buffer D washed sample; Lane 6: Buffer E washed sample; Lane 7: Buffer F eluted sample.

[0050] Figure 4 The final samples consist of five different nanobodies. Lane 1: purified SM-JSN-B1-2 antibody; Lane 2: purified SM-JSN-B1-3 antibody; Lane 3: purified SM-JSN-B1-11 antibody; Lane 4: purified SM-JSN-B1-22 antibody; Lane 5: purified SM-JSN-B1-24 antibody.

[0051] Figure 5 ELISA analysis of five different nanobodies with the coronavirus spike protein. ELISA data of five nanobodies with the Delta type of the coronavirus spike protein (…). Figure 5 a) and affinity ( Figure 5 (b) or ELISA data with wild-type coronavirus spike protein ( Figure 5 c) and affinity ( Figure 5 (d) Detailed Implementation

[0052] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0053] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0054] In this specification, the terms "substantially" or "truly" are used to indicate that the standard deviation from the theoretical model or theoretical data is within 5%, preferably 3%, and more preferably 1%.

[0055] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0056] In this specification, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes both the scenario in which the event occurs and the scenario in which the event does not occur.

[0057] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0058] According to the present invention, the terms “polypeptide,” “protein,” and “peptide” are used interchangeably herein to refer to a polymeric form of amino acids of any length, including encoded and non-coding amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having a similar peptide backbone.

[0059] According to the present invention, the terms "nucleic acid molecule," "polynucleotide," "polynucleotide," and "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acid molecules can be linear or circular.

[0060] According to the present invention, the three-letter codes and single-letter codes for amino acids used are as described in J.biol.chem, 243, p3558 (1968).

[0061] The term "antibody" used in this invention refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the heavy chain of immunoglobulins differ, thus their antigenicity also differs. Accordingly, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, based on differences in the amino acid composition of its hinge region and the number and position of disulfide bonds in its heavy chain, it can be further divided into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ chains or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either a κ chain or a λ chain.

[0062] In this invention, the antibody light chain may further include a light chain constant region, which includes human or mouse κ, λ chains or variants thereof.

[0063] In this invention, the antibody heavy chain may further include a heavy chain constant region, which includes human or mouse IgG1, IgG2, IgG3, IgG4 or variants thereof.

[0064] The approximately 110 amino acids near the N-terminus of both the antibody heavy and light chains exhibit significant sequence variation, forming the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable, forming the constant region (C region). The variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. The number and position of CDR amino acid residues in the VL and VH regions of the antibody or antigen-binding fragment conform to the known Kabat numbering rules and the Kabat or AbM or IMGT definition rules (http: / / bioinf.org.uk / abs / ).

[0065] The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and antibody analogues, which typically includes at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody. The antibody fragment retains at least some of the binding specificity of the parent antibody. Typically, when activity is expressed on a molar basis, the antibody fragment retains at least 10% of the parent antibody's binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the parent antibody's binding affinity to the target. Examples of antigen-binding fragments include, but are not limited to: Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single-chain antibodies, domain antibodies, single-domain antibodies or nanobodies, and multispecific antibodies. A review of engineered antibody variants is available in Holliger and Hudson, 2005, Nat. Biotechnol. 23:1126-1136.

[0066] In this invention, the terms "single-domain antibody" (sdAb), "VHH", and "V" are used interchangeably. H "H" and "nanobody" have the same meaning and can be used interchangeably. They refer to the variable region of the heavy chain of a cloned antibody. A single-domain antibody consisting of only one variable region of the heavy chain is constructed. It is the smallest antigen-binding fragment with complete function.

[0067] The term "antigen binding site" in this invention refers to the three-dimensional spatial site recognized by the antibody or antigen binding fragment of this invention.

[0068] The term "epitope" refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed from adjacent amino acids or from non-adjacent amino acids arranged side-by-side through the ternary folding of a protein. Epitopes formed from adjacent amino acids are generally retained after exposure to denaturing solvents, while epitopes formed through ternary folding are generally lost after treatment with denaturing solvents. Epitopes typically comprise at least 3-15 amino acids in a unique spatial conformation. Methods for determining which epitopes bind to a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of epitopes include techniques in the art, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0069] The terms "specific binding" and "selective binding" used in this invention refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, when measured in an instrument using surface plasmon resonance (SPR) technology, the antibody binds at approximately less than 10... -7 M or even smaller equilibrium dissociation constant (K) DAn antibody that recognizes a predetermined antigen binds to it, and its affinity for the predetermined antigen is at least twice that for a nonspecific antigen other than the predetermined antigen or closely related antigens (such as BSA). The term "antibody that recognizes an antigen" may be used interchangeably with the term "antibody that specifically binds."

[0070] According to the present invention, an amino acid "addition" refers to the addition of an amino acid to the C-terminus or N-terminus of an amino acid sequence. According to the present invention, an amino acid "deletion" refers to the deletion of one, two, or three or more amino acids from an amino acid sequence. According to the present invention, an amino acid "insertion" refers to the insertion of an amino acid residue at an appropriate position in an amino acid sequence, wherein the inserted amino acid residues may be all or partly adjacent to each other, or the inserted amino acids may not be adjacent to each other.

[0071] According to the present invention, an amino acid "substitution" refers to the replacement of an amino acid residue at a certain position in an amino acid sequence by another amino acid residue; wherein, "substitution" can be a conserved amino acid substitution.

[0072] According to the present invention, “conservative modification,” “conservative substitution,” or “conservative replacement” refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and rigidity), such that changes can be made frequently without altering the protein’s biological activity. Those skilled in the art will appreciate that, in general, the substitution of a single amino acid in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al. (1987), Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). Furthermore, the substitution of structurally or functionally similar amino acids is unlikely to impair biological activity. Exemplary conserved substitutions are described in the following “Exemplary Conservative Amino Acid Substitutions.”

[0073] Exemplary amino acid conservative substitution

[0074] Original residues Conservative replacement Ala(A) Gly;Ser Arg(R) Lys;His Asn(N) Gln; His; Asp Asp(D) Glu;Asn Cys(C) Ser;Ala;Val Gln(Q) Asn; Glu Glu(E) Asp; Gln Gly(G) Ala His(H) Asn;Gln Ile(I) Leu; Val Leu(L) Ile; Val Lys(K) Arg; His Met(M) Leu; Ile; Tyr Phe(F) Tyr; Met; Leu Pro(P) Ala Ser(S) Thr Thr(T) Ser Trp(W) Tyr; Phe Tyr(Y) Trp; Phe Val(V) Ile; Leu

[0075] "Identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same base or amino acid monomer subunit—for example, if every position in two DNA molecules is occupied by adenine—then the molecules are homologous at that position. The percentage of identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared, multiplied by 100%. For example, at optimal sequence alignment, if six out of ten positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparisons are made when the highest percentage of identity is obtained by aligning the two sequences.

[0076] According to the present invention, the term "codon optimization" refers to the configuration of the nucleotide sequence encoding a polypeptide to contain codons preferred by the host cell or organism in order to improve gene expression and increase translation efficiency in the host cell or organism.

[0077] According to the present invention, the term "tag" refers to a short peptide that is fused or linked to a target protein (e.g., the antibody of the present invention), thereby facilitating the soluble expression, detection, and / or purification of the recombinant protein. The tag may be fused to or linked to the N-terminus and / or C-terminus of the target protein (optionally via a linker or protease cleavage site).

[0078] According to the present invention, the terms "signal peptide," "secretory peptide," "signal sequence," or "signal peptide sequence" refer to short peptides that, when fused with a target protein (e.g., the antibody of the present invention), promote the secretion of the target protein expressed by the cell onto the cell membrane or extracellularly. The signal peptide is typically located at the N-terminus of the target protein, and various signal peptides are known to those skilled in the art, such as, but not limited to, erythropoietin signal sequences, human insulin signal sequences, human interleukin-2 signal sequences, albumin signal sequences, etc.

[0079] According to the present invention, the term "protease cleavage site" refers to a site that can be specifically recognized and cleaved by a protease. Various specific proteases and their recognition sites are well known to those skilled in the art and are described in numerous prior art documents. Those skilled in the art can use appropriate protease cleavage sites in fusion proteins and perform cleavage with corresponding proteases, depending on the specific circumstances. The use of protease cleavage sites can be advantageous, for example, they can be used to cleave signal peptides and / or tags from fusion proteins to obtain mature proteins with the desired activity.

[0080] According to the present invention, the terms "peptide linker", "linking sequence" or "artificial linking sequence" refer to a short peptide used to link two molecules (e.g., protein, polypeptide).

[0081] According to the present invention, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector enables the expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, cosmids, etc.

[0082] According to the present invention, the terms "cell," "cell line," and "cell culture" are used interchangeably, and all such names include progeny. Therefore, the terms "transformer" and "transformed cell" include primary test cells and cultures derived therefrom, regardless of the number of transfections. It should also be understood that, due to intentional or unintentional mutations, all progeny cannot be exactly identical in terms of DNA content. This includes mutant progeny with the same function or biological activity as those screened from the original transformed cells.

[0083] "Administration," "giving," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration," "giving," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Administration," "giving," and "treatment" also mean the treatment of, for example, cells, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo. "Treatment," when applied to humans, veterinary, or research subjects, refers to therapeutic, preventative, or prophylactic measures, research, and diagnostic applications.

[0084] "Treatment" means administering an oral or topical therapeutic agent, such as an antibody comprising any of the present invention, to a patient who has one or more symptoms of a disease, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves one or more symptoms of a disease, whether by inducing the regression of such symptoms or inhibiting their progression to any clinically measurable degree. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been relieved can be evaluated using any clinical testing method commonly used by a physician or other healthcare professional to assess the severity or progression of the symptoms.

[0085] An "effective dose" includes a dose sufficient to improve or prevent the symptoms or condition of a medical condition. An effective dose also means a dose sufficient to allow or facilitate diagnosis. The effective dose for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective dose can be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0086] "Pharmaceutical composition" means containing one or more antibodies as described herein, as well as other components such as physiological / pharmaceutical-grade carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity.

[0087] As used in this article, the terms “novel coronavirus,” “2019-nCoV,” and “SARS-CoV-2” refer to a new strain of coronavirus, and its official classification name, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has been announced by the International Committee on Taxonomy of Viruses (ICTV).

[0088] The technical solution of the present invention will be described in detail below:

[0089] Nanobodies targeting the SARS-CoV-2 spike protein

[0090] In some embodiments of the present invention, nanobodies targeting the SARS-CoV-2 spike protein are provided, the nanobodies being able to specifically bind to the SARS-CoV-2 spike protein.

[0091] In some embodiments of the present invention, the nanobody targeting the SARS-CoV-2 spike protein comprises:

[0092] HCDR1 as shown in any one of SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15 and SEQ ID NO: 18;

[0093] HCDR2 represented by any one of SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16 and SEQ ID NO: 19; and,

[0094] HCDR3 shown in any one of SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17 and SEQ ID NO: 20.

[0095] In some specific embodiments of the present invention, the nanobody targeting the SARS-CoV-2 spike protein comprises:

[0096] HCDR1 as shown in SEQ ID NO: 6;

[0097] HCDR2 as shown in SEQ ID NO: 7; and,

[0098] HCDR3 as shown in SEQ ID NO: 8.

[0099] In some specific embodiments of the present invention, the nanobody targeting the SARS-CoV-2 spike protein comprises:

[0100] HCDR1 as shown in SEQ ID NO: 9;

[0101] HCDR2 as shown in SEQ ID NO: 10; and,

[0102] HCDR3 as shown in SEQ ID NO: 11.

[0103] In some specific embodiments of the present invention, the nanobody targeting the SARS-CoV-2 spike protein comprises:

[0104] HCDR1 as shown in SEQ ID NO: 12;

[0105] HCDR2 as shown in SEQ ID NO: 13; and,

[0106] HCDR3 as shown in SEQ ID NO: 14.

[0107] In some specific embodiments of the present invention, the nanobody targeting the SARS-CoV-2 spike protein comprises:

[0108] HCDR1 as shown in SEQ ID NO: 15;

[0109] HCDR2 as shown in SEQ ID NO: 16; and,

[0110] HCDR3 as shown in SEQ ID NO: 17.

[0111] In some specific embodiments of the present invention, the nanobody targeting the SARS-CoV-2 spike protein comprises:

[0112] HCDR1 as shown in SEQ ID NO: 18;

[0113] HCDR2 as shown in SEQ ID NO: 19; and,

[0114] HCDR3 as shown in SEQ ID NO: 20.

[0115] In some embodiments of the present invention, the nanobody targeting the SARS-CoV-2 spike protein further comprises a framework region; said framework region comprises:

[0116] FR1 shown in any one of SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 30 and SEQ ID NO: 36, or FR1 having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with any one of SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 30 and SEQ ID NO: 36;

[0117] FR2 shown in any one of SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 31 and SEQ ID NO: 33, or FR2 having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with any one of SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 31 and SEQ ID NO: 33;

[0118] FR3 represented by any one of SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 32 and SEQ ID NO: 34, or FR3 having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity with any one of SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 32 and SEQ ID NO: 34; and,

[0119] FR4 shown in either SEQ ID NO: 24 or SEQ ID NO: 35, or FR4 having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with either SEQ ID NO: 24 or SEQ ID NO: 35.

[0120] In some specific embodiments of the present invention, the frame region includes:

[0121] FR1 as shown in SEQ ID NO: 21;

[0122] FR2 as shown in SEQ ID NO: 22;

[0123] FR3 as shown in SEQ ID NO: 23; and

[0124] FR4 as shown in SEQ ID NO: 24.

[0125] In some specific embodiments of the present invention, the frame region includes:

[0126] FR1 as shown in SEQ ID NO: 25;

[0127] FR2 as shown in SEQ ID NO: 26;

[0128] FR3 as shown in SEQ ID NO: 27; and

[0129] FR4 as shown in SEQ ID NO: 24.

[0130] In some specific embodiments of the present invention, the frame region includes:

[0131] FR1 as shown in SEQ ID NO: 21;

[0132] FR2 as shown in SEQ ID NO: 28;

[0133] FR3 as shown in SEQ ID NO: 29; and

[0134] FR4 as shown in SEQ ID NO: 24.

[0135] In some specific embodiments of the present invention, the frame region includes:

[0136] FR1 as shown in SEQ ID NO: 30;

[0137] FR2 as shown in SEQ ID NO: 31;

[0138] FR3 as shown in SEQ ID NO: 32; and

[0139] FR4 as shown in SEQ ID NO: 24.

[0140] In some specific embodiments of the present invention, the frame region includes:

[0141] FR1 as shown in SEQ ID NO: 36;

[0142] FR2 as shown in SEQ ID NO: 33;

[0143] FR3 as shown in SEQ ID NO: 34; and

[0144] FR4 as shown in SEQ ID NO: 35.

[0145] In some more specific embodiments of the present invention, the nanobody targeting the SARS-CoV-2 spike protein comprises the sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 5, or the sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with any one of SEQ ID NO: 1 to SEQ ID NO: 5.

[0146] In some preferred embodiments of the present invention, the nanobody targeting the SARS-CoV-2 spike protein comprises the sequence shown in SEQ ID NO: 3.

[0147] In some preferred embodiments of the present invention, the nanobody targeting the SARS-CoV-2 spike protein comprises the sequence shown in SEQ ID NO: 1.

[0148] This invention includes not only complete nanobodies, but also fusion proteins formed by immunologically active nanobodies and other sequences. Therefore, this invention also includes derivatives and analogs of said nanobodies.

[0149] As used herein, the terms “derivative” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the nanobodies of the present invention. Derivatives or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide (nanobody) with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence or a sequence used to purify this polypeptide or a proteogenic sequence, or a fusion protein formed with a tag such as 6His). Based on the teachings herein, these derivatives and analogs are within the scope well known to those skilled in the art.

[0150] The nanobody of the present invention refers to a polypeptide containing the aforementioned CDR region that has SARS-CoV2 S protein binding activity. This term also includes variants of the polypeptide containing the aforementioned CDR region that have the same function as the nanobody of the present invention. These variants include (but are not limited to): deletion, insertion, and / or substitution of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and addition of one or more amino acids (typically less than 20, preferably less than 10, more preferably less than 5) at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, adding one or more amino acids at the C-terminus and / or N-terminus generally does not alter the function of the protein.

[0151] The variant forms of nanobodies include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the nanobodies of the present invention.

[0152] In some embodiments of the present invention, the nanobody targeting the SARS-CoV-2 spike protein further comprises a tag, a signal peptide, a linker sequence, or any combination thereof.

[0153] In some specific embodiments of the present invention, the nanobody targeting the SARS-CoV-2 spike protein includes a tag at its N-terminus and / or C-terminus; and / or, includes a signal peptide at its N-terminus.

[0154] In some more specific embodiments of the present invention, the nanobody targeting the SARS-CoV-2 spike protein comprises the sequence shown in any one of SEQ ID NO: 39 to SEQ ID NO: 43, or the sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with any one of SEQ ID NO: 39 to SEQ ID NO: 43.

[0155] In some preferred embodiments of the present invention, the nanobody targeting the SARS-CoV-2 spike protein comprises the sequence shown in SEQ ID NO: 41.

[0156] In some preferred embodiments of the present invention, the nanobody targeting the SARS-CoV-2 spike protein comprises the sequence shown in SEQ ID NO: 39.

[0157] <Polynucleotides, expression vectors, host cells, preparation methods>

[0158] In some embodiments of the present invention, a polynucleotide is provided that encodes the aforementioned nanobody targeting the SARS-CoV-2 spike protein.

[0159] The polynucleotides of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand.

[0160] The polynucleotides encoding the nanobodies of the present invention include: a coding sequence that encodes only the nanobodies; a coding sequence for the nanobodies and various additional coding sequences; a coding sequence for the nanobodies (and optional additional coding sequences) and a non-coding sequence.

[0161] The term "polynucleotide encoding nanobody" can include a polynucleotide encoding the nanobody, or it can include additional coding and / or non-coding sequences.

[0162] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as nanobodies.

[0163] In some embodiments of the present invention, an expression vector is provided containing the aforementioned polynucleotides. The expression vector further includes a suitable promoter or control sequence, which can be used to transform a suitable host cell to enable it to express the protein.

[0164] In some embodiments of the present invention, a host cell is provided which is introduced with or contains the expression vector described above.

[0165] In one specific implementation, the host cell is a bacterium, preferably Escherichia coli.

[0166] In another specific implementation, the host cell is yeast, preferably Pichia pastoris.

[0167] In another specific implementation, the host cell is a mammalian cell, preferably a CHO cell or a HEK293 cell.

[0168] In some embodiments of the present invention, a method for producing the above-described nanobody targeting the SARS-CoV-2 spike protein is provided, comprising the steps of: culturing host cells according to the present invention, isolating the antibody from the culture, and, optionally, purifying the antibody.

[0169] <Pharmaceutical Compositions, Kits, and Uses>

[0170] According to some embodiments of the present invention, a pharmaceutical composition is provided comprising a nanobody targeting the SARS-CoV-2 spike protein according to the present invention, as well as a pharmaceutically acceptable excipient, diluent, or carrier.

[0171] According to some embodiments of the present invention, a detection or diagnostic kit is provided, which contains nanobodies targeting the SARS-CoV-2 spike protein according to the present invention.

[0172] According to some embodiments of the present invention, the use of the SARS-CoV-2 spike protein-targeting nanobody according to the present invention in the preparation of a medicament for treating or preventing diseases or symptoms caused by SARS-CoV-2 infection is provided. According to some embodiments of the present invention, the SARS-CoV-2 spike protein-targeting nanobody according to the present invention is provided for treating or preventing diseases or symptoms caused by SARS-CoV-2 infection. According to some embodiments of the present invention, a treatment method for diseases or symptoms caused by SARS-CoV-2 infection is provided, comprising the step of administering an effective amount of the SARS-CoV-2 spike protein-targeting nanobody according to the present invention.

[0173] In some specific implementation plans, the disease or symptoms caused by SARS-CoV-2 infection are referred to as novel coronavirus infection (Corona Virus Disease 2019, COVID-19).

[0174] According to some embodiments of the present invention, the use of the SARS-CoV-2 spike protein-targeting nanobody according to the present invention in the preparation of reagents, wherein the reagents are used to detect or diagnose diseases or conditions caused by SARS-CoV-2 infection. According to some embodiments of the present invention, the SARS-CoV-2 spike protein-targeting nanobody according to the present invention is provided for the detection or diagnosis of diseases or conditions caused by SARS-CoV-2 infection. According to some embodiments of the present invention, a method for detecting or diagnosing diseases or conditions caused by SARS-CoV-2 infection is provided, comprising the step of contacting a sample to be tested with the SARS-CoV-2 spike protein-targeting nanobody according to the present invention.

[0175] In some specific implementation plans, the disease or symptoms caused by SARS-CoV-2 infection are referred to as novel coronavirus infection (Corona Virus Disease 2019, COVID-19).

[0176] The present invention will be further illustrated below with reference to the accompanying drawings and embodiments, but this is not intended to limit the invention. Specific materials used in the embodiments of the present invention and their sources are provided below. However, it should be understood that these are merely exemplary and not intended to limit the invention. Materials of the same or similar type, model, quality, properties, or functions as the reagents and instruments described below can be used to implement the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0177] Example 1: Expression and purification of coronavirus spike protein Delta antigen

[0178] 1. Expression and characterization of Delta-type spike protein antigens

[0179] 1.1 Preparation of recombinant expression plasmids

[0180] First, the Delta-type antigen encoding the spike protein (its corresponding amino acid sequence is shown in SEQ ID NO: 44) was cloned into the pcDNA3.1 vector (Invitrogen). The recombinant plasmid was then introduced into *E. coli* DH5α competent cells (Bomaide Biotechnology) via heat shock transformation and cultured overnight (37°C) in LB medium containing 50 μg / mL ampicillin. The bacterial culture was collected, and the plasmid was extracted.

[0181] 1.2 Cell transfection

[0182] Take 40 mL of cell medium (Cell Medium, Sinocare), add 3.2 mg of PEI transfection reagent and 0.8 mg of the recombinant expression plasmid prepared in step 1.1, and incubate at room temperature for 15 minutes. Add this mixture to 800 mL of a 2×10⁻⁶ m³ / mL solution. 6 HEK293F cells (Thermo Fisher) per mL were cultured at 37°C and 120 rpm for more than 72 hours.

[0183] 1.3 Purification of Delta-type spike protein antigen

[0184] After cell transfection and culture in step 1.2, the cells were removed by centrifugation at 7000 rpm for 40 minutes at 4°C. The supernatant was filtered through a 0.45 μm filter membrane, and the pH was adjusted to 8.0. The supernatant was then loaded into a StrepXT-1 mL (GE) pre-packed column overnight using a peristaltic pump. Impurities were eluted with Buffer A, and the target protein was eluted with Buffer B. The target protein was concentrated using a 100 kDa ultrafiltration tube to a volume of approximately 100 μL. Gel filtration chromatography was then performed using a Superose 6 Increase 5 / 150 GL (Cytiva) gel column with Buffer A. Protein samples from the UV-280 absorption peak were collected for SDS-PAGE gel electrophoresis to determine the content and purity of the target protein.

[0185] The specific components of the aforementioned buffer solution AB are as follows:

[0186] Buffer A: 25mM Tris, pH 8.0, 150mM NaCl;

[0187] Buffer B: 25mM Tris, pH 8.0, 150mM NaCl, 50mM Biotin.

[0188] Experimental results: such as Figure 1 As shown, the Delta-type spike protein antigen obtained after affinity chromatography and gel filtration chromatography purification was analyzed by SDS-PAGE gel electrophoresis, and its purity was approximately >95%.

[0189] The corresponding amino acid sequence of the Delta-type spike protein antigen constructed into the vector (SEQ ID NO: 44):

[0190]

[0191] Example 2: Screening and positive clone identification of natural nanobody libraries

[0192] 2.1 Screening of Phage Display Natural Nanobody Libraries

[0193] The applicant used its proprietary natural alpaca (Vicugna pacos) nanobody phage library to screen for spike protein Delta antigen-binding nanobodies. The steps are as follows:

[0194] (1) The spike protein Delta antigen (referred to as S-Delta protein) prepared in Example 1 was diluted in CBS coating solution (Na2CO3 3.03g, NaHCO3 6.0g, added to 1000ml sterile water) to a concentration of 30μg / mL. The antigen was coated on a 96-well microplate at a rate of 100μL / well (total of 10 wells) and incubated overnight at 4℃.

[0195] (2) Washing: Discard the antigen coated in step (1), wash 3 times with 250 μL / well PBST (1×PBS + 0.05% (v / v) Tween 20), and pat dry.

[0196] (3) Blocking: Add 200 μL / well blocking solution (1×PBS + 2% (m / v) skim milk powder) and block at room temperature for 2 hours. Block up to 10 wells and perform negative sieve.

[0197] (4) Shaking TG1 competent cells (HonorGene): Select one single clone and add it to 2YT bacterial culture solution (Sangon Biotech). Shake at 37℃ and 220rpm until the logarithmic growth phase (about 3h).

[0198] (5) Take 10 from the natural nanobody phage library 13 One phage (dissolved in 2% (m / v) skim milk powder in PBST) was bound to a milk-coated plate for 1 hour to remove antibodies from the phage library that non-specifically bind to milk. 100 μL / well, incubated at room temperature with shaking for 1 hour.

[0199] (6) Then aspirate the phage from step (5) and add it to the 96-well microplate coated with S-Delta protein prepared in steps (1) to (3) to bind with S-Delta antigen, 100 μL / well, and shake at room temperature for 2 hours.

[0200] (7) Washing: Discard the liquid in the microplate in step (6), pat dry, add 250 μL / well of PBST, and wash 10 times.

[0201] (8) Elution: Add 0.2M glycine-HCl (pH 2.2) to the microplate after washing in step (7), 100 μL / well, and shake for no more than 10 min.

[0202] (9) Neutralization: Add Tris-HCl at a ratio of 130 μL of 1M Tris-HCl (pH 9.1) to 1 mL of 0.2 M Glycine-HCl (pH 2.2).

[0203] (10) Infection: Take 550 μL of the elution solution neutralized in step (9) and infect 5 mL of TG1 bacterial solution (TG1 in logarithmic growth phase) at 37°C and 220 rpm for 30 min.

[0204] (11) Take 50 μL for output titration, and add M13KO7 helper phage (HonorGene) to the remaining bacterial culture (final concentration to 10). 10 (pcs / mL), 37℃, shaken at 220 rpm for 1 hour.

[0205] (12) Change the medium: Add all the bacterial culture obtained in step (11) to 50 mL of 2YT (containing kanamycin (kan, 50 μg / mL) and ampicillin (amp, 100 μg / mL)), change to 250 mL of shaking flask, and shake overnight at 37°C and 220 rpm.

[0206] (13) Repeat steps (1) to (12) for the second and third rounds of screening. The antigen coating amount for the second round of screening is 30 μg / mL and the number of washes is 15. The antigen coating amount for the third round of screening is 20 μg / mL and the number of washes is 20.

[0207] 2.2 Isolation and Identification of Positive Clones

[0208] (1) After three rounds of screening in step 2.1, the infection products from the first, second and third rounds were plated onto 2YT / Amp+ plates and incubated overnight at 37°C.

[0209] (2) The next day, pick bacteria to 2YT / Amp+ / helper phage(10) 10 Incubate overnight at 37°C and 220 rpm (number per mL).

[0210] (3) The binding strength of phages to S-Delta in the supernatant of monoclonal culture was identified by ELISA, and positive clones were selected for sequencing.

[0211] Five different positive clone sequences were obtained (see Table 1).

[0212] Table 1. Binding activity and sequence of 5 positive clones

[0213]

[0214] In Table 1 above, for each nanobody sequence, the CDR1, CDR2 and CDR3 sequences are underlined in sequence (the CDR sequences are determined based on the IMGT definition rules).

[0215] The CDR and FR regions of the above-mentioned nanobodies are shown in Tables 2 to 6 below.

[0216] Table 2 Complementarity Determinant Region (CDR) and Frame Region (FR) Sequences of SM-JSN-B1-2

[0217] name sequence SEQ ID NO HCDR1 GRTDSSYV SEQ ID NO: 6 HCDR2 ISWSGGST SEQ ID NO: 7 HCDR3 AARRGNILISSGRSYDY SEQ ID NO: 8 FR1 DVQLQESGGGLVQAGGSLRLSCAAS SEQ ID NO: 21 FR2 IAWFRQAPGKDREFVGA SEQ ID NO: 22 FR3 HYGDSVQGRFTISRSNAENTGSLQMTSLKPEDTAVYYC SEQ ID NO: 23 FR4 WGQGTQVTVSS SEQ ID NO: 24

[0218] Table 3. Complementarity-determining region (CDR) and frame region (FR) sequences of SM-JSN-B1-3

[0219] name sequence SEQ ID NO HCDR1 GRSKYP SEQ ID NO: 9 HCDR2 ISYINNPF SEQ ID NO: 10 HCDR3 AARRTPPYSGNANYAGEGIYDL SEQ ID NO: 11 FR1 DVQLQESGGGLVETGDSLRLSCAAS SEQ ID NO: 25 FR2 MAWFRQTPGKERVIVAA SEQ ID NO: 26 FR3 YLDSVKGRFTISRDNVKNTVYLQMNNLIPEDTAVYTC SEQ ID NO: 27 FR4 WGQGTQVTVSS SEQ ID NO: 24

[0220] Table 4. Complementarity-determining region (CDR) and frame region (FR) sequences of SM-JSN-B1-11

[0221] name sequence SEQ ID NO HCDR1 TRSFSSAA SEQ ID NO: 12 HCDR2 ISGSSSIT SEQ ID NO: 13 HCDR3 AADYSPLARYGTSERSSRYAY SEQ ID NO: 14 FR1 DVQLQESGGGLVQAGGSLRLSCAAS SEQ ID NO: 21 FR2 MAWFRQAPGKEREFVAA SEQ ID NO: 28 FR3 STADSLKGRFTISRDNSKNTVYLQMNSLKPEDTAVYYC SEQ ID NO: 29 FR4 WGQGTQVTVSS SEQ ID NO: 24

[0222] Table 5. Complementarity-determining region (CDR) and frame region (FR) sequences of SM-JSN-B1-22

[0223]

[0224] Table 6. Complementary Determinant Region (CDR) and Frame Region (FR) Sequences of SM-JSN-B1-24:

[0225] name sequence SEQ ID NO HCDR1 GFAFDAYA SEQ ID NO: 18 HCDR2 ISAIDGSK SEQ ID NO: 19 HCDR3 ARTQDHFAGRDRCTDDWYSYNY SEQ ID NO: 20 FR1 DVQLQESGGGLVQAGGSLRLSCATS SEQ ID NO: 36 FR2 LGWFRQTPGKKREAVSC SEQ ID NO: 33 FR3 YYVDSVKGRFTISRDIAKSTVYLQMSDLRPEDTGVYYC SEQ ID NO: 34 FR4 ESQGTQVTVSS SEQ ID NO: 35

[0226] Example 3: Cloning, expression, and purification of positive clones

[0227] 3.1 PCR amplification of the gene fragment, in which a secretory peptide (i.e., a signal peptide) was added to the N-terminus of the antibody and 6*his-HA was added to the C-terminus. A pair of primers was designed as follows:

[0228] pCDNA3.1-SP-F-1

[0229] TGGATATCTGCAGAATTCGCCACCATGGGCTGGAGCTGTATTATCCTGTTCCTCGTGGCCACCGCCACCGGAGTGCACAGCGATGTGCAGCTG (SEQ ID NO: 37)

[0230] pCDNA3.1-HA-His-R

[0231] CGGTTTAAACTTAAGCTTCTAAGCGTAGTCCGGAACGTCGTACGGGTATGCGCCATGGTGATGGTGATGGTGGCGGCCGCTGGA (SEQ ID NO: 38)

[0232] Table 7 PCR amplification system

[0233] JSN-B1(2 / 3 / 11 / 22 / 24) 1μL Premstar MAX(2×)(TAKARA) 25μL pCDNA3.1-SP-F (10 μm) 1μL pCDNA3.1-HA-His-R (10 μm) 1μL <![CDATA[ddH2O]]> 22μL Total 50μL

[0234] 3.2 The pcDNA3.1 vector was recovered using two restriction endonucleases, EcoRI / HindIII-HF. The restriction enzyme digestion system is shown in the table below:

[0235] Table 8. pcDNA3.1 Vector Enzyme Digestion System

[0236] pcDNA3.1 vector 5μL EcoRI-HF(NEB) 7μL HindIII-HF(NEB) 7μL Cutsmart (NEB) 9μL <![CDATA[dd H2O]]> 62μL Total 90μL

[0237] 3.3 Homologous recombination of the target fragment and the vector pcDNA 3.1

[0238] The homologous recombination system is shown in the table below:

[0239] Table 9

[0240] ClonEXpressII (Vazyme) 2μL 5×CEII Buffer (Vazyme) 4μL pcDNA3.1 (EcoRI / HindIII-HF) (obtained in step 3.2) 3μL JSN-B1(2 / 3 / 11 / 22 / 24) (obtained via step 3.1) 1μL <![CDATA[dd H2O]]> 10μL Total 20μL

[0241] After successful cloning, the sample sequence was verified by sequencing. The final sample sequencing results are shown in the table below:

[0242] Table 10

[0243]

[0244] In Table 10 above, for each nanobody sequence, the dotted underlined part represents the secreted peptide (signal peptide (SP)); bold represents the 6*His tag; double underline represents the HA tag; and italic represents the linker sequence.

[0245] Example 4: Expression and purification of Delta-type nanobodies for coronavirus spike protein

[0246] 4.1 Expression of Delta-type nanobodies for coronavirus spike protein

[0247] 4.1.1 Preparation of expression plasmids

[0248] The recombinant plasmid containing the target gene (constructed in Example 3) was introduced into *E. coli* DH5α competent cells (Bomaide Biotechnology) via heat shock transformation and cultured overnight at 37°C in LB medium containing 50 μg / mL ampicillin. The bacterial culture was collected, and the plasmid was extracted.

[0249] 4.1.2 Cell transfection

[0250] Take 1 mL of Cell Medium (Essential Science), add 0.08 mg of PEI transfection reagent and 0.02 mg of the plasmid obtained in step 4.1.1), and incubate at room temperature for 15 minutes. Add this mixture to 20 mL of a solution with a density of 2 × 10⁻⁶. 6 HEK293F cells per mL were cultured at 37°C and 120 rpm for more than 72 hours.

[0251] 4.2 Purification of Coronavirus Spike Protein Delta-type Nanobodies

[0252] Five days after antibody expression in 20 mL Expi 293 (Thermofish) cells, the supernatant was collected by centrifugation at 3000 rpm for 20 minutes. Nickel sulfate to a final concentration of 4 mM was added to the supernatant, and after standing for 5 minutes, the mixture was filtered through a 0.22 μM filter and purified using a nickel affinity chromatography column. Impurities were eluted with Buffer C, Buffer D, and Buffer E, and the target protein was eluted with Buffer F. SDS-PAGE electrophoresis was then performed for verification.

[0253] like Figure 3 and Figure 4 As shown, all nanobody expressions and purifications yielded high-purity, high-yield target proteins for further activity identification.

[0254] The specific components of the aforementioned buffer CF are as follows:

[0255] Buffer C: 25mM HEPEs, pH7.5, 150mM NaCl;

[0256] Buffer D: 25mM HEPEs, pH7.5, 150mM NaCl, 20mM imidazole;

[0257] Buffer E: 25mM HEPEs, pH7.5, 150mM NaCl, 40mM imidazole;

[0258] Buffer F: 25mM HEPEs, pH7.5, 150mM NaCl, 250mM imidazole.

[0259] Example 5: ELISA detection of the affinity of Delta-type nanobodies for coronavirus spike protein

[0260] 5.1 Coating of the Delta-type antigen of the coronavirus spike protein

[0261] The high-concentration Delta antigen (amino acid sequence as shown in SEQ ID NO: 44) was diluted with CBS diluent (pH 9.6) to a concentration of 2 μg / mL. The antigen was coated overnight in 96-well plates at 4°C with a coating volume of 100 μL / well. The next day, the coating solution and antigen solution were discarded, and the plates were washed three times with 250 μL PBST solution and patted dry. 200 μL / well blocking buffer (2% (m / v) skim milk powder + PBS) was added, and the plates were gently mixed and blocked at room temperature for 2 hours. Subsequently, the blocking buffer was discarded, and the plates were washed three times with 250 μL PBST and patted dry.

[0262] 5.2 nanoparticle antibody dilution

[0263] The five nanobodies purified in Example 4 were diluted 4-fold starting from a high concentration (20 μg / mL), and then diluted eight times using 2% (m / v) skim milk powder + PBST as the diluent. Subsequently, 100 μL of the diluted antibody was transferred to a plate coated with the Delta type antigen of the coronavirus spike protein and incubated gently at room temperature for 1 hour. The plate was then washed three times with PBST and patted dry.

[0264] 5.3 Secondary Antibody Treatment

[0265] Add 100 μL of PBST-diluted HRP-labeled secondary antibody (0.5 μg / mL, anti-HA tag mouse monoclonal antibody) to each well of the sample plate and incubate gently at room temperature for 1 hour. Then wash four times with PBST and pat dry.

[0266] 5.4 Color development and data analysis.

[0267] Add 100 μL of TMB single-component chromogenic solution (Solepro) to each well, and terminate the reaction with 50 μL of stop solution (Solepro) per well. Read the values ​​at 450 nm using a microplate reader. Experimental data were fitted with a GraphPad Prism5 to obtain a curve, and the EC50 value was calculated.

[0268] The results are as follows Figure 5As shown in a and b, the five different nanobodies exhibited different affinities for the Delta type of coronavirus spike protein. Among them, SM-JSN-B1-11 had the highest affinity at 0.49 nM, followed by SM-JSN-B1-2 with an affinity of 0.78 nM.

[0269] Example 6: ELISA assessment of the selectivity of five nanobodies for wild-type and Delta mutant coronavirus spike protein

[0270] Using the same antigen preparation protocol as in Example 1 and the testing protocol in Example 5, this example prepares the wild-type antigen of the coronavirus spike protein and verifies the affinity data using an ELISA experiment.

[0271]

[0272] like Figure 5 As shown in c and d, compared to the Delta type of the spike protein, the five nanobodies used in this patent exhibit Delta-like affinity for the wild-type antigen protein. Based on this analysis, the fact that the five nanobodies can bind to different subtypes of the antigen protein suggests that their binding epitopes to the coronavirus spike protein may be located in relatively conserved regions.

[0273] References

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Claims

1. A nanobody targeting the SARS-CoV-2 spike protein, comprising HCDR1 with the amino acid sequence shown in SEQ ID NO: 12, HCDR2 with the amino acid sequence shown in SEQ ID NO: 13, and HCDR3 with the amino acid sequence shown in SEQ ID NO:

14.

2. The nanobody targeting the SARS-CoV-2 spike protein according to claim 1, wherein, The nanobody targeting the SARS-CoV-2 spike protein further comprises a framework region, which includes FR1 shown in SEQ ID NO: 21, FR2 shown in SEQ ID NO: 28, FR3 shown in SEQ ID NO: 29 and FR4 shown in SEQ ID NO:

24.

3. The nanobody targeting the SARS-CoV-2 spike protein according to claim 1 or 2, wherein, The nanobody targeting the SARS-CoV-2 spike protein contains the sequence shown in SEQ ID NO:

3.

4. The nanobody targeting the SARS-CoV-2 spike protein according to claim 3, wherein, The nanobody targeting the SARS-CoV-2 spike protein also includes a tag, a signal peptide, a linker sequence, or any combination thereof.

5. The nanobody targeting the SARS-CoV-2 spike protein according to any one of claims 1, 2, and 4, wherein, The nanobody targeting the SARS-CoV-2 spike protein contains a tag at its N-terminus and / or C-terminus; and / or, contains a signal peptide at its N-terminus.

6. The nanobody targeting the SARS-CoV-2 spike protein according to any one of claims 1, 2, and 4, wherein, The nanobody targeting the SARS-CoV-2 spike protein contains the sequence shown in SEQ ID NO:

41.

7. A polynucleotide encoding a nanobody targeting the SARS-CoV-2 spike protein according to any one of claims 1 to 6.

8. An expression vector containing the polynucleotide of claim 7.

9. A host cell containing the expression vector of claim 8.

10. A method for producing nanobodies targeting the SARS-CoV-2 spike protein, comprising the steps of: culturing the host cells of claim 9 and isolating the nanobodies from the culture.

11. The method of claim 10, wherein the method comprises purifying the nanobody.

12. A pharmaceutical composition comprising a nanobody targeting the SARS-CoV-2 spike protein as described in any one of claims 1 to 6, and a pharmaceutically acceptable carrier.

13. A diagnostic kit comprising a nanobody targeting the SARS-CoV-2 spike protein as described in any one of claims 1 to 6.

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