CN116143910A

CN116143910A - Nano antibody for targeting SARS-CoV-2 spike protein and application thereof

Info

Publication number: CN116143910A
Application number: CN202211248766.2A
Authority: CN
Inventors: 赵爽; 张佳星; 肖景景; 吕子霖; 何东; 衡杰; 李京; 刘玉洁; 郭春龙
Original assignee: Shuimu Future Beijing Technology Co ltd
Current assignee: Shuimu Future Beijing Technology Co ltd
Priority date: 2022-10-12
Filing date: 2022-10-12
Publication date: 2023-05-23

Abstract

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

Description

Nano antibody for targeting SARS-CoV-2 spike protein and application thereof

Technical Field

The invention relates to the biomedical or biotechnological field, in particular to a nano antibody targeting SARS-CoV-2 spike protein and application thereof.

Background

Coronaviruses (CoVs) are a highly diverse family of enveloped positive-sense single-stranded RNA viruses that can infect humans, other mammals, birds, livestock, etc., and their widespread transmission not only is a great challenge to global public health safety, but also has a significant impact on global economic and cultural exchanges. During the past 20 years, severe acute respiratory syndrome coronavirus (Severe Acute Respiratory Syndrome Corona Virus, SARS-CoV), middle east respiratory syndrome coronavirus (Middle East Respiratory Syndrome Corona Virus, MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (Severe Acute Respiratory Syndrome Corona Virus 2, SARS-CoV-2) were transmitted multiple times in the human population, with a high degree of pathogenicity. SARS-CoV by infection of human bronchial epithelial cells, lung cells and upper respiratory tract cells MERS-CoV and SARS-CoV-2 infections can progress to severe, life-threatening respiratory lesions and lung injuries, for which no specific preventive or therapeutic methods have been approved to date. In view of the defects of long period, great difficulty and high risk of development of small molecular medicines, the neutralizing antibody is a key means for resisting epidemic viral diseases, and helps to simplify early drug treatment discovery based on antibodies ^1–3 . Antibodies against SARS-CoV-2 are resistant to viral escape, and may be active against different subtypes of SARS virus, and are neutralized by the virus ^4,5 Sum effector function ⁶ Has the effect of protecting human body.

Coronavirus Spike protein (also called S protein) is a key mediator of viral attachment, infection and entry into cells, and it belongs to class I fusion glycoproteins that form homotrimers, comprising two functionally distinct S1 and S2 subunits. The S1 subunit can be further divided into two relatively independent regions (domains), an N-terminal domain (NTD) and a C-terminal domain (CTD), respectively. Wherein the surface exposed S1 subunit comprises a receptor binding domain (Receptor binding domain, RBD) responsible for binding to host cell receptors, thereby determining the virus-cell selectivity and pathogenicity. Most of the RBDs of the S protein of coronaviruses are located in CTDs, such as SARS-CoV and MERS-CoV. Only a small fraction of RBDs of beta coronaviruses are located in NTDs. The S2 subunit is anchored to the membrane by a transmembrane region, and the transmembrane S2 subunit comprises a heptad repeat region and a fusion peptide, which mediates fusion of the viral membrane and cell membrane following conformational rearrangement to allow entry of the virus into the cell ⁷ . All viruses including SARS-CoV-2 are constantly mutated, some mutations do not affect the characteristics of the virus, but some changes may affect the characteristics of the virus, such as its extent of transmission, the severity of the disease after infection, or the therapeutic effects of vaccines, therapeutic drugs, diagnostic tools, etc. So far, SARS-CoV-2 protein produced various mutants of alpha, beta, delta, and omimetic, respectively ⁸ . One key point of any antiviral treatment regimen is how to avoid the creation of therapeutic drugs due to rapid variation of viral pathogensResistance to drugs of (2) ⁹ . Such resistance becomes more pronounced, especially when selective pressure is applied in the context of drug treatment. For example, when HIV drugs are used alone, such drug-selective mutations result in extensive drug resistance. Subsequent success of combination therapy with HIV suggests that the requirement for simultaneous mutation of the virus at multiple gene locations may be the most effective way to avoid drug resistance.

In 1989, the university of brussel freedom immunologist, hamers masterman professor and his colleagues found a novel antibody in camel serum that was structurally different from the traditional antibody: the antibody naturally lacks a light chain and consists of only two heavy chains, which is called a heavy chain antibody (HCAb). The variable region VHH (variable domain of heavy chain of heavy-chain antibody) structure of the heavy chain antibody, which is prepared by in vitro recombination, has the equivalent structural stability and the binding activity with antigen as the original heavy chain antibody, is the known minimum unit capable of binding the target antigen, has the crystal diameter of 2.5 nanometers and the length of 4nm, has the molecular mass of only 15kDa, has the mass of only about one tenth of the molecular mass of the traditional antibody, is about one half of the molecular mass of an antigen binding fragment, and is also called as a Nanobody (Nb).

Compared with the traditional antibody molecules, the nano antibody has certain advantages in the aspects of antigen detection, clinical in-vitro diagnosis, immunological research and the like. Also, nanobodies have been developed as an emerging force in new generation therapeutic biological medicine and clinical diagnostic reagents, due to their smaller molecular weight, good stability, good solubility, good permeability, microbial expression, and ability to recognize hidden epitopes. Because nanobodies have smaller molecular weights, nanobodies also have significant advantages in multi-epitope antibody combinations. For example, after a plurality of identical nano antibodies are connected in series, the researcher can obviously improve the affinity of the antibodies to spike protein trimer ¹⁰ . Fusing bispecific nanobodies to conventional IgG1 Fc domains has also been shown to increase neutralization ¹¹ . The development of multivalent nano antibody is aimed at, and is not only applied to SARS-CoV-2 protein ¹² Is also applied to other targets with clinical treatment value ^13–15 . Multivalent nanobody Ozoralizumab developed by the company Taisho has been used clinically to treat rheumatoid arthritis ¹⁶ 。

Disclosure of Invention

Problems to be solved by the invention

Based on the above problems in the prior art, it is an object of the present invention to provide a nanobody targeting SARS-CoV-2 spike protein.

Solution for solving the problem

In a first aspect, the invention provides a nanobody targeting SARS-CoV-2 spike protein comprising:

SEQ ID NO: 6. SEQ ID NO: 9. SEQ ID NO: 12. SEQ ID NO:15 and SEQ ID NO:18, HCDR1 as set forth in any one of claims;

SEQ ID NO: 7. SEQ ID NO: 10. SEQ ID NO: 13. SEQ ID NO:16 and SEQ ID NO:19 or HCDR2 as set forth in any one of claims; and, a step of, in the first embodiment,

SEQ ID NO: 8. SEQ ID NO: 11. SEQ ID NO: 14. SEQ ID NO:17 and SEQ ID NO:20, and HCDR3 as set forth in any one of claims.

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

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

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

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

(iv) SEQ ID NO:18, HCDR1, SEQ ID NO:19 and HCDR2 and SEQ ID NO: HCDR3 as shown at 20;

(v) SEQ ID NO:9, HCDR1, SEQ ID NO:10 and HCDR2 and SEQ ID NO:11, HCDR3.

In some embodiments, the nanobody targeting SARS-CoV-2 spike protein further comprises a framework region.

In some optional embodiments, the framework region comprises:

SEQ ID NO: 21. SEQ ID NO: 25. SEQ ID NO:30 and SEQ ID NO:36, or FR1 as set forth in any one of SEQ ID NOs: 21. SEQ ID NO: 25. SEQ ID NO:30 and SEQ ID NO:36, FR1 having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to any one of claims;

SEQ ID NO: 22. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO:31 and SEQ ID NO:33, or FR2 as set forth in any one of SEQ ID NOs: 22. SEQ ID NO: 26. SEQ ID NO: 28. SEQ ID NO:31 and SEQ ID NO:33, FR2 having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to any one of the claims;

SEQ ID NO: 23. SEQ ID NO: 27. SEQ ID NO: 29. SEQ ID NO:32 and SEQ ID NO:34, or FR3 as set forth in any one of SEQ ID NOs: 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 to any one of the above; and, a step of, in the first embodiment,

SEQ ID NO:24 and SEQ ID NO:35, or FR4 as set forth in any one of SEQ ID NOs: 24 and SEQ ID NO:35, or an FR4 having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity.

In some specific embodiments, the framework region comprises any one or more of the following (i) - (v):

(i) SEQ ID NO:21, FR1, SEQ ID NO:28, FR2, SEQ ID NO:29 and FR3 and SEQ ID NO:24, FR4;

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

(iii) SEQ ID NO:30, FR1, SEQ ID NO:31, FR2, SEQ ID NO:32 and FR3 and SEQ ID NO:24, FR4;

(iv) SEQ ID NO:36, FR1, SEQ ID NO:33, FR2, SEQ ID NO:34 and FR3 and SEQ ID NO: FR4 indicated by 35;

(v) SEQ ID NO:25, FR1, SEQ ID NO:26, FR2, SEQ ID NO:27 and FR3 and SEQ ID NO:24, FR4.

In some more specific embodiments, the nanobody targeting SARS-CoV-2 spike protein comprises SEQ ID NO: 1-SEQ ID NO:5, or a sequence as set forth in any one of SEQ ID NOs: 1-SEQ ID NO:5, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity.

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

In some specific embodiments, the nanobody targeting SARS-CoV-2 spike protein comprises a tag at its N-terminus and/or C-terminus; and/or comprises a signal peptide at its N-terminus.

In some preferred embodiments, the nanobody targeting SARS-CoV-2 spike protein comprises SEQ ID NO: 39-SEQ ID NO:43, or a sequence as set forth in any one of SEQ ID NOs: 39-SEQ ID NO:43, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity.

In a second aspect, the invention provides a polynucleotide encoding a nanobody of the invention that targets SARS-CoV-2 spike protein.

In a third aspect the invention provides an expression vector comprising a polynucleotide according to the second aspect of the invention.

In a fourth aspect, the invention provides a host cell into which or containing an expression vector according to the third aspect of the invention is introduced.

In a fifth aspect, the invention provides a method for producing a nanobody targeting SARS-CoV-2 spike protein, comprising the steps of: culturing the host cell of the fourth aspect of the invention, isolating nanobodies from the culture, and, optionally, purifying the nanobodies.

In a sixth aspect, the invention provides a pharmaceutical composition comprising a nanobody targeting SARS-CoV-2 spike protein according to the first aspect of the invention, and a pharmaceutically acceptable excipient, diluent or carrier.

In a seventh aspect, the invention provides a detection or diagnostic kit comprising a nanobody of the first aspect of the invention that targets SARS-CoV-2 spike protein.

In an eighth aspect, the invention provides the use of a nanobody targeting SARS-CoV-2 spike protein according to the first aspect of the invention in the manufacture of a medicament for the treatment or prophylaxis of a disease or condition caused by SARS-CoV-2 infection.

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

In a ninth aspect, the invention provides the use of a nanobody targeting SARS-CoV-2 spike protein according to the first aspect of the invention in the preparation of a reagent for detecting or diagnosing a disease or condition caused by SARS-CoV-2 infection.

ADVANTAGEOUS EFFECTS OF INVENTION

The present invention developed various nanobodies that are capable of specifically recognizing different subtypes of coronavirus spike proteins. The nano antibody disclosed by the invention can be independently applied to the field of biological medicine research and development (genetic engineering medicine research and development and ADC medicine research and development); clinical in vitro diagnosis (colloidal gold method, enzyme-linked immunosorbent assay, electrochemiluminescence method); basic research such as immunology research. In addition, based on the antigen epitope verification and comparison of the nano antibody, the nano antibody can be subjected to covalent design with the nano antibodies which are used for recognizing different antigen epitopes and are available in the market, and bispecific or multispecific multivalent nano antibodies can be developed and applied to drug development or clinical in-vitro diagnosis.

Drawings

FIG. 1 shows SDS-PAGE gel of spike protein Delta type antigen purification.

FIG. 2 shows the construction of the expression vector for positive cloned antibodies. A in fig. 2 shows that the PCR purification kit purifies the PCR product. B in fig. 2 shows the recovery of vector pcdna3.1 using the gel recovery kit. FIG. 2 c shows the bacterial liquid PCR verification, and the correct sample for PCR verification is sent for sequencing.

FIG. 3 shows the result of SDS-PAGE of purified 5 different nanobodies. In each group: lane 1: a whole cell sample; lane 2: expression medium supernatant samples; lane 3: a flow-through sample of a nickel affinity chromatography column; lane 4: washing the impurity sample by using Buffer C; lane 5: buffer D washes the miscellaneous sample; lane 6: washing the impurity sample by Buffer E; lane 7: buffer F eluted the sample.

Fig. 4 is a final sample of 5 different nanobodies. Wherein, lane 1: purifying the SM-JSN-B1-2 antibody; lane 2: purifying the SM-JSN-B1-3 antibody; lane 3: purifying the SM-JSN-B1-11 antibody; lane 4: purifying the SM-JSN-B1-22 antibody; lane 5: the SM-JSN-B1-24 antibody was purified.

FIG. 5 is an ELISA analysis of 5 different nanobodies against coronavirus spike protein. The 5 nanobodies were either associated with ELISA data of the coronavirus spike Delta type (a in FIG. 5) and with affinity (b in FIG. 5) or with ELISA data of the coronavirus spike wild type (c in FIG. 5) and with affinity (d in FIG. 5).

Detailed Description

In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In the present specification, the numerical range indicated by the term "numerical value a to numerical value B" means a range including the end point numerical value A, B.

In the present specification, the use of "substantially" or "substantially" means that the standard deviation from the theoretical model or theoretical data is within 5%, preferably 3%, more preferably 1%.

In the present specification, the meaning of "can" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

In this specification, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

Reference throughout this specification to "some specific/preferred embodiments," "other specific/preferred embodiments," "an embodiment," and so forth, means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the elements may be combined in any suitable manner in the various embodiments.

According to the present invention, the terms "polypeptide", "protein", "peptide" are used interchangeably herein to refer to polymeric forms of amino acids of any length, and may include encoded and non-encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a similar peptide backbone.

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

According to the invention, the amino acid three-letter codes and the one-letter codes used are as described in J.biol. Chem,243, p3558 (1968).

The term "antibody" as used herein refers to an immunoglobulin that is a tetrapeptide chain structure formed by two identical heavy chains and two identical light chains joined by interchain disulfide bonds. The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Accordingly, immunoglobulins can be classified into five classes, or isotypes of immunoglobulins, i.e., igM, igD, igG, igA and IgE, with their respective heavy chains being the μ, δ, γ, α and ε chains, respectively. The same class of Ig can be further classified into different subclasses according to the amino acid composition of the hinge region and the number and position of disulfide bonds of the heavy chain, e.g., igG can be classified into IgG1, igG2, igG3, and IgG4. Light chains are classified by the difference in constant regions as either kappa chains or lambda chains. Each class Ig of the five classes of Igs may have either a kappa chain or a lambda chain.

In the present invention, the antibody light chain may further comprise a light chain constant region comprising a kappa, lambda chain of human or murine origin or variants thereof.

In the present invention, the antibody heavy chain may further comprise a heavy chain constant region comprising an IgG1, igG2, igG3, igG4 or variant thereof of human or murine origin.

The sequences of the heavy and light chains of the antibody near the N-terminus vary widely, being the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C-region). The variable region includes 3 hypervariable regions (HVRs) and 4 Framework Regions (FR) that are relatively conserved in sequence. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the light chain variable region (VL) and heavy chain variable region (VH) consists of 3 CDR regions and 4 FR regions, arranged in the order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR3. CDR amino acid residues of the VL and VH regions of an antibody or antigen binding fragment conform to the known Kabat numbering convention and the Kabat or AbM or IMGT definition convention (http:// bioinf org uk/abs /).

The term "antigen-binding fragment" refers to antigen-binding fragments of antibodies and antibody analogs, which generally include at least a portion of the antigen-binding or variable regions (e.g., one or more CDRs) of the parent antibody (parental antibody). The antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, an antibody fragment retains at least 10% of the parent binding activity when expressed on a molar basis. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody 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. Engineered antibody variants are reviewed in Holliger and Hudson,2005, nat. Biotechnol.23:1126-1136.

In the present invention, the term "single domain antibody" (single domain antibody, sdAb), "VHH", "V _H H "," nanobody "have the same meaning and are used interchangeably to refer to the variable region of a cloned antibody heavy chain, constructing a single domain antibody consisting of only one heavy chain variable region, which is the smallest antigen binding fragment with complete function.

The term "antigen binding site" according to the invention refers to a three-dimensional spatial site recognized by an antibody or antigen binding fragment of the invention.

The term "epitope" refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed by contiguous amino acids, or non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are typically maintained after exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost after treatment with denaturing solvents. Epitopes typically comprise at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation detection assays, among others. Methods for determining the spatial conformation of an epitope include techniques in the art such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

The terms "specifically bind", "selectively bind" and "selectively bind" as used herein refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, antibodies are present at about less than 10 as measured in an instrument by Surface Plasmon Resonance (SPR) techniques ^-7 M or even smaller equilibrium dissociation constant (K _D ) Binds to the predetermined antigen and has an affinity to bind to the predetermined antigen that is at least twice as great as its affinity to bind to non-specific antigens other than the predetermined antigen or closely related antigens (e.g., BSA, etc.). The term "antibody that recognizes an antigen" may be used interchangeably herein with the term "antibody that specifically binds".

According to the invention, amino acid "addition" refers to the addition of an amino acid at the C-or N-terminus of an amino acid sequence. According to the invention, an amino acid "deletion" refers to the deletion of 1, 2 or 3 or more amino acids from the amino acid sequence. According to the present invention, amino acid "insertions" refer to insertions of amino acid residues at appropriate positions in the amino acid sequence, which may be adjacent to each other in whole or in part, or which may not be adjacent to each other.

According to the present invention, an amino acid "substitution" refers to the replacement of a certain amino acid residue at a certain position in the amino acid sequence by another amino acid residue; wherein a "substitution" may be a conservative amino acid substitution.

According to the present invention, "conservative modification", "conservative substitution" or "conservative substitution" refers to substitution of an amino acid in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), such that changes can be made frequently without altering the biological activity of the protein. Those skilled in The art know that in general, single amino acid substitutions in The non-essential region of a polypeptide do not substantially alter biological activity (see, e.g., watson et al (1987) Molecular Biology of The Gene, the Benjamin/Cummings pub. Co., page 224, (4 th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to destroy biological activity. Exemplary conservative substitutions are set forth below in "exemplary amino acid conservative substitutions".

Exemplary amino acid conservative substitutions

Original residue	Conservative substitutions
		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

"identity" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both comparison sequences is occupied by the same base or amino acid monomer subunit, for example if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent identity between two sequences is a function of the number of matched or homologous positions shared by the two sequences divided by the number of positions compared x 100%. For example, in the optimal alignment of sequences, if there are 6 matches or homologies at 10 positions in the two sequences, then the two sequences are 60% homologous. In general, a comparison is made when two sequences are aligned to give the maximum percent identity.

According to the present invention, the term "codon-optimized" means that the nucleotide sequence encoding the polypeptide has been configured to contain codons preferred by the host cell or organism to improve gene expression and to increase translation efficiency in the host cell or organism.

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

According to the present invention, the term "signal peptide", "secretory peptide", "signal sequence" or "signal peptide sequence" refers to a short peptide which, when fused to a protein of interest (e.g., an antibody of the present invention), is capable of promoting secretion of the protein of interest expressed by a cell onto a cell membrane or outside the cell. The signal peptide is typically located at the N-terminus of the protein of interest and various signal peptides are known to those skilled in the art, such as, but not limited to, a hemagglutinin signal sequence, a human insulin signal sequence, a human interleukin 2 signal sequence, an albumin signal sequence, and the like.

According to the present invention, the term "protease cleavage site" refers to a site that is 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 found in many prior art documents. The skilled person will be able to use suitable protease cleavage sites in the fusion protein and to cleave with the corresponding proteases, as the case may be. The use of protease cleavage sites may be advantageous, for example, in that they can be used to cleave signal peptides and/or tags from fusion proteins, thereby obtaining a mature protein with the activity of interest.

According to the present invention, the term "peptide linker", "linker sequence" or "artificial linker sequence" refers to a short peptide for linking two molecules (e.g. proteins, polypeptides).

According to the present invention, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids, phages, cosmids, and the like.

According to the present invention, the terms "cell", "cell line" and "cell culture" are used interchangeably and all such designations include offspring. Thus, the terms "transformant" and "transformed cell" include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that all offspring may not be exactly identical in terms of DNA content due to deliberate or unintentional mutations. Including mutant progeny having the same function or biological activity as screened in the original transformed cell.

"administering," "administering," and "treating," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to the contact of an exogenous drug, therapeutic, diagnostic, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration," "administration," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell includes contacting a reagent with the cell, and contacting the reagent with a fluid, wherein the fluid is in contact with the cell. "administration," "administration," and "treatment" also mean in vitro and ex vivo treatment of, for example, a cell by an agent, diagnostic agent, binding composition, or by another cell. "treatment" when applied to a human, veterinary or research subject refers to therapeutic, prophylactic or preventative measures, research and diagnostic applications.

"treatment" means administration of a therapeutic agent, such as an antibody comprising any of the present invention, for internal or external use to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the subject patient or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable extent. The amount of therapeutic agent (also referred to as a "therapeutically effective amount") effective to alleviate any particular disease symptom can vary depending on a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce a desired therapeutic effect in the patient. Whether a disease symptom has been reduced can be assessed by any clinical test method that a physician or other healthcare professional typically uses to assess the severity or progression of the symptom.

An "effective amount" comprises an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount is also meant to be an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on the following factors: such as the condition to be treated, the general health of the patient, the route of administration and the dosage and severity of the side effects. An effective amount may be the maximum dose or regimen that avoids significant side effects or toxic effects.

"pharmaceutical composition" means a composition comprising one or more of the antibodies described herein, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.

As used herein, the terms "novel coronavirus", "2019-nCoV", "SARS-CoV-2" refer to a novel strain of coronavirus, and its formal classification is declared by the international committee for classification of viruses (International Committee on Taxonomy of Viruses, ICTV) as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

The following describes the technical scheme of the invention in detail:

< nanobody targeting SARS-CoV-2 spike protein >

In some embodiments of the invention, nanobodies targeting SARS-CoV-2 spike protein are provided that are capable of specifically binding SARS-CoV-2 spike protein.

In some embodiments of the invention, a nanobody targeting SARS-CoV-2 spike protein comprises:

SEQ ID NO: HCDR1 as shown in fig. 6;

SEQ ID NO: HCDR2 as shown in fig. 7; and, a step of, in the first embodiment,

SEQ ID NO: HCDR3 as shown in fig. 8.

SEQ ID NO: HCDR1 as shown in 9;

SEQ ID NO: HCDR2 as shown in fig. 10; and, a step of, in the first embodiment,

SEQ ID NO:11, HCDR3.

SEQ ID NO:12, HCDR1;

SEQ ID NO: HCDR2 as shown in 13; and, a step of, in the first embodiment,

SEQ ID NO:14, HCDR3.

SEQ ID NO: HCDR1 as shown in 15;

SEQ ID NO:16, HCDR2; and, a step of, in the first embodiment,

SEQ ID NO:17, HCDR3.

SEQ ID NO: HCDR1 as shown at 18;

SEQ ID NO:19, HCDR2; and, a step of, in the first embodiment,

SEQ ID NO:20, and HCDR3.

In some embodiments of the invention, the nanobody targeting SARS-CoV-2 spike protein further comprises a framework region; the frame region includes:

In some embodiments of the invention, the framework regions comprise:

SEQ ID NO: FR1 indicated by 21;

SEQ ID NO: FR2 indicated by 22;

SEQ ID NO:23, FR3; and

SEQ ID NO:24, FR4.

In some embodiments of the invention, the framework regions comprise:

SEQ ID NO: FR1 indicated by 25;

SEQ ID NO: FR2 indicated by 26;

SEQ ID NO:27, FR3; and

SEQ ID NO:24, FR4.

In some embodiments of the invention, the framework regions comprise:

SEQ ID NO: FR1 indicated by 21;

SEQ ID NO: FR2 indicated by 28;

SEQ ID NO: FR3 indicated by 29; and

SEQ ID NO:24, FR4.

In some embodiments of the invention, the framework regions comprise:

SEQ ID NO:30, FR1 shown in fig. 30;

SEQ ID NO: FR2 indicated by 31;

SEQ ID NO:32, FR3; and

SEQ ID NO:24, FR4.

In some embodiments of the invention, the framework regions comprise:

SEQ ID NO: FR1 indicated by 36;

SEQ ID NO:33, FR2 shown in figure 33;

SEQ ID NO: FR3 indicated by 34; and

SEQ ID NO: FR4 indicated at 35.

In some more specific embodiments of the invention, the nanobody targeting SARS-CoV-2 spike protein comprises SEQ ID NO: 1-SEQ ID NO:5, or a sequence as set forth in any one of SEQ ID NOs: 1-SEQ ID NO:5, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity.

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

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

The invention not only comprises the complete nano antibody, but also comprises fusion proteins formed by the nano antibody with immunological activity and other sequences. Thus, the invention also includes derivatives and analogs of the nanobody.

As used herein, the terms "derivative" and "analog" refer to polypeptides that retain substantially the same biological function or activity of the nanobody of the invention. The derivatives or analogues of the invention may be (i) polypeptides having one or more (preferably conservative) amino acid residues substituted, which may or may not be encoded by the genetic code, or (ii) polypeptides having a substituent in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide (nanobody) with another compound, such as a compound that extends the half-life of the polypeptide, for example polyethylene glycol, or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or fusion proteins formed with a tag such as 6 His). These derivatives and analogs fall within the scope of the teachings herein, as known to those skilled in the art.

The nanobody of the present invention refers to a polypeptide having SARS-CoV 2S protein binding activity, comprising the above CDR region. The term also includes variants of polypeptides comprising the above-described CDR regions that have the same function as the nanobody of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminal and/or N-terminal end. For example, in the art, substitution with amino acids of similar or similar properties does not generally alter the function of the protein. As another example, the addition of one or more amino acids at the C-terminus and/or N-terminus typically does not alter the function of the protein.

Variant forms of nanobodies include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA capable of hybridizing with the DNA encoding the antibodies of the invention under high or low stringency conditions, and polypeptides or proteins obtained using antisera raised against nanobodies of the invention.

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

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

In some more specific embodiments of the invention, the nanobody targeting SARS-CoV-2 spike protein comprises SEQ ID NO: 39-SEQ ID NO:43, or a sequence as set forth in any one of SEQ ID NOs: 39-SEQ ID NO:43, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity.

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

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

< Polynucleotide, expression vector, host cell, and method for producing the same >

In some embodiments of the invention, a polynucleotide encoding a nanobody as described above that targets SARS-CoV-2 spike protein is provided.

The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

Polynucleotides encoding nanobodies of the invention include: a coding sequence encoding only nanobodies; the coding sequence of the nanobody and various additional coding sequences; the coding sequence (and optionally additional coding sequences) of the nanobody, and non-coding sequences.

The term "nanobody-encoding polynucleotide" may include polynucleotides encoding such nanobodies, as well as polynucleotides further comprising additional coding and/or non-coding sequences.

The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention relates in particular to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" means: (1) Hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturing agents such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42℃and the like during hybridization; or (3) hybridization only occurs when the identity between the two sequences is at least 90% or more, more preferably 95% or more. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the nanobody.

In some embodiments of the invention, there is provided an expression vector comprising a polynucleotide as described above. The expression vector also contains suitable promoters or control sequences, etc., which may be used to transform appropriate host cells to enable expression of the protein.

In some embodiments of the invention, a host cell is provided that incorporates or contains the expression vector described above.

In a specific embodiment, the host cell is a bacterium, preferably E.coli.

In another specific embodiment, the host cell is a yeast, preferably pichia pastoris.

In another specific embodiment, the host cell is a mammalian cell, preferably a CHO cell or HEK293 cell.

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

< pharmaceutical composition, kit and use >

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

According to some embodiments of the invention, there is provided a detection or diagnostic kit comprising a nanobody targeting SARS-CoV-2 spike protein according to the invention.

According to some embodiments of the present invention, there is provided the use of a nanobody targeting SARS-CoV-2 spike protein according to the invention in the manufacture of a medicament for the treatment or prevention of a disease or condition caused by SARS-CoV-2 infection. According to some embodiments of the present invention, there is provided nanobodies targeting SARS-CoV-2 spike protein according to the invention for use in the treatment or prevention of diseases or disorders caused by SARS-CoV-2 infection. According to some embodiments of the present invention, there is provided a method of treating a disease or disorder caused by SARS-CoV-2 infection comprising the step of administering an effective amount of a nanobody according to the invention that targets SARS-CoV-2 spike protein.

In some specific embodiments, the disease or disorder caused by SARS-CoV-2 infection is a novel coronavirus infection (Corona Virus Disease 2019, COVID-19).

According to some embodiments of the present invention, there is provided the use of a nanobody targeting SARS-CoV-2 spike protein according to the invention in the preparation of a reagent, wherein the reagent is for detecting or diagnosing a disease or disorder caused by SARS-CoV-2 infection. According to some embodiments of the present invention, there is provided nanobodies targeting SARS-CoV-2 spike protein according to the invention for use in the detection or diagnosis of diseases or conditions caused by SARS-CoV-2 infection. According to some embodiments of the present invention, there is provided a method for detecting or diagnosing a disease or disorder caused by SARS-CoV-2 infection, comprising the step of contacting a test sample with a nanobody according to the invention that targets SARS-CoV-2 spike protein.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention, in conjunction with the accompanying drawings. Specific materials and sources thereof used in embodiments of the present invention are provided below. However, it should be understood that these are merely exemplary and are not intended to limit the present invention, as materials that are the same as or similar to the type, model, quality, nature, or function of the reagents and instruments described below may be used in the practice of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1: expression and purification of coronavirus spike protein Delta antigen

1. Expression and characterization of spike protein Delta antigen

1.1 preparation of recombinant expression plasmids

First, a spike-protein-encoding Delta-type antigen (whose corresponding amino acid sequence is shown as SEQ ID NO: 44) was cloned into the pcDNA3.1 vector (Invitrogen). The recombinant plasmid was introduced into E.coli DH 5. Alpha. Competent cells (Bomaide organism) by heat shock transformation and cultured overnight (37 ℃) in LB medium containing 50. Mu.g/mL ampicillin. And collecting bacterial liquid and extracting plasmids.

1.2 cell transfection

Taking 40mL of Cell Medium (Cell Medium, yiqiao Shenzhou) respectively, adding 3.2mg of transfection reagent PEI and 0.8mg of recombinant expression plasmid prepared in step 1.1, incubating for 15 minutes at room temperature, adding 800mL of the mixture solution with the density of 2×10 ⁶ Each mL of HEK293F cells (Thermo Fisher, sieimer) was cultured at 37℃and 120rpm for 72 hours or more.

1.3 spike protein Delta antigen purification

After the end of the cell transfection culture in step 1.2, cells were removed by centrifugation at 7000rpm at 4℃for 40 minutes, the supernatant was filtered through a 0.45 μm filter, the pH was adjusted to 8.0, and peristaltic pump was cycled overnight into strep XT-1mL (GE) pre-packed column. Eluting the hybrid protein by using Buffer A, and eluting the target protein by using Buffer B. Concentrating the target protein by using a 100kDa ultrafiltration tube, performing gel filtration chromatography when the concentration is about 100 mu L, wherein the type of a gel column is Superose 6 Increase 5/150 GL (cytova), the Buffer solution is Buffer A, and collecting a protein sample at a UV-280 ultraviolet absorption peak for SDS-PAGE gel electrophoresis to detect the content and purity of the target protein.

Wherein, the Buffer solution (Buffer) A-B comprises the following specific components:

Buffer A：25mM Tris，pH 8.0，150mM NaCl；

buffer B:25mM Tris,pH 8.0, 150mM NaCl,50mM Biotin (Biotin).

Experimental results: as shown in FIG. 1, the spike protein Delta type antigen obtained after affinity chromatography purification and gel filtration chromatography purification has a purity of about >95% by SDS-PAGE gel electrophoresis analysis.

Corresponding amino acid sequence of spike protein Delta type antigen constructed to vector (SEQ ID NO: 44):

MFVFLVLLPLVSSQCVNLRTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLDVYYHKNNKSWMESGVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSRGSASSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQNVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGHHHHHHHHSAWSHPQFEKGGGSGGGGSGGSAWSHPQFEK*

example 2: screening of natural nano antibody library and positive clone identification

2.1 screening of phage display Natural nanobody libraries

Screening of spike protein Delta antigen binding nanobodies was performed using applicant's own natural alpaca (Vicugna pacos) nanobody phage library. The method comprises the following steps:

(1) The spike protein Delta antigen (abbreviated as S-Delta protein) prepared in example 1 was diluted into CBS coating solution (Na ₂ CO ₃ 3.03g，NaHCO ₃ 6.0g, 1000mL of sterilized water) was added, the antigen concentration was 30. Mu.g/mL, and the mixture was coated on a 96-well ELISA plate at 100. Mu.L/well (total 10 wells), overnight at 4 ℃;

(2) Washing the plate: the antigen coated in step (1) was discarded, washed 3 times with 250. Mu.L/well of PBST (1 XPBS+0.05% (v/v) Tween 20), and dried.

(3) Closing: adding 200 μl/well of blocking solution (1 XPBS+2% (m/v) skimmed milk powder), blocking at room temperature for 2 hr, and blocking 10 wells more, and sieving.

(4) Shake TG1 competent cells (horgene, olog gene): one of the single clones was selected and added to a 2YT bacterial culture solution (bioengineering), and shaken at 37℃and 220rpm to logarithmic growth phase (approximately 3 h).

(5) 10 from natural nanobody phage library ¹³ Individual phages (PBST dissolved in 2% (m/v) skimmed milk powder (mill)) were bound to the plates coated with the mill for 1 hour in order to remove antibodies in the phage library that bind non-specifically to the mill. 100. Mu.L/well, and shaken at room temperature for 1 hour.

(6) And (3) sucking out phage in the step (5), adding the phage in the step (1) to the 96-well ELISA plate coated with the S-Delta protein and prepared in the step (3), combining with the S-Delta antigen, and oscillating for 2 hours at room temperature.

(7) Washing: the liquid in the ELISA plate in the step (6) is discarded, the plate is dried, 250 mu L/hole of PBST is added, and the plate is washed for 10 times.

(8) Eluting: 0.2M Glycine (Glycine) -HCl (pH 2.2) was added to the washed ELISA plate in step (7), 100. Mu.L/well, and the shaking was not more than 10min.

(9) And (3) neutralization: tris-HCl was added to neutralize at a rate of 130. Mu.L of 1M Tris-HCl (pH 9.1) per 1mL of 0.2M Glycine-HCl (pH 2.2).

(10) Infection: 550. Mu.L of the eluate neutralized in step (9) was infected with 5mL of TG1 strain (TG1 in logarithmic growth phase), and the mixture was shaken at 37℃and 220rpm for 30min.

(11) mu.L was used for output titration, M13KO7 helper phage (HororGene, orno gene) was added to the remaining broth (final concentration to 10) ¹⁰ and/mL), 37℃and 220rpm for 1 hour.

(12) Liquid replacement: the whole bacterial solution obtained in step (11) was added to 50mL of 2YT (containing kanamycin (kan, 50. Mu.g/mL) and ampicillin (amp, 100. Mu.g/mL)), and the flask was changed to 250mL shaking flask at 37℃and 220rpm and shaken overnight.

(13) And (3) repeating the steps (1) to (12) for a second round of screening, wherein the coating amount of the antigen for the second round of screening is 30 mug/mL, the washing times are 15 times, the coating amount of the antigen for the third round of screening is 20 mug/mL, and the washing times are 20 times.

2.2 isolation and identification of Positive clones

(1) After three rounds of screening in step 2.1, the first, second and third rounds of infection products are respectively coated on a 2YT/amp+ plate for overnight culture at 37 ℃;

(2) The following day was picked up to 2YT/amp+/helper phase (10) ¹⁰ Individual/mL), at 37℃at 220rpm, overnight.

(3) ELISA method is used for identifying the binding force between phage and S-Delta in the single clone culture supernatant, and positive clones are selected for sequencing.

A total of 5 different positive clone sequences were obtained (see Table 1).

Table 15 binding Activity and sequence of Positive clones

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

The CDR regions and FR regions of the nanobodies are specifically shown in tables 2 to 6 below.

TABLE 2 SM-JSN-B1-2 complementarity determining region CDR and framework region FR sequences

Name of the name	Sequence(s)	Number (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

TABLE 3 SM-JSN-B1-3 complementarity determining region CDR and framework region FR sequences

Name of the name	Sequence(s)	Number (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

CDR and framework FR sequences of the complementarity determining regions of Table 4 SM-JSN-B1-11

Name of the name	Sequence(s)	Number (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

TABLE 5 SM-JSN-B1-22 complementarity determining region CDR and framework region FR sequences

The complementarity determining region CDR and framework region FR sequences of Table 6 SM-JSN-B1-24:

name of the name	Sequence(s)	Number (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

Example 3: cloning, expression and purification of positive clones

3.1PCR amplified gene fragment, wherein secretory peptide (i.e., signal peptide) was added to the N-terminus of the antibody, 6 x his-HA was added to the C-terminus, and a pair of primers was designed as follows:

pCDNA3.1-SP-F-1

TGGATATCTGCAGAATTCGCCACCATGGGCTGGAGCTGTATTATCCTGTTCCTCGTGGCCACCGCCACCGGAGTGCACAGCGATGTGCAGCTG(SEQ ID NO：37)

pCDNA3.1-HA-His-R

CGGTTTAAACTTAAGCTTCTAAGCGTAGTCCGGAACGTCGTACGGGTATGCGCCATGGTGATGGTGATGGTGGCGGCCGCTGGA(SEQ ID NO：38)

TABLE 7 PCR amplification System

JSN-B1(2/3/11/22/24)	1μL
		Premstar MAX(2×)(TAKARA)	25μL
pCDNA3.1-SP-F(10μm)	1μL
		pCDNA3.1-HA-His-R(10μm)	1μL
ddH ₂ O	22μL
		Aggregate (Total)	50μL

3.2 recovery of pcDNA3.1 vector with two restriction enzymes EcoRI/HindIII-HF, cleavage system is shown in the following Table:

TABLE 8 pcDNA3.1 vector cleavage System

pcDNA3.1 vector	5μL
		EcoRI-HF(NEB)	7μL
HindIII-HF(NEB)	7μL
		Cutsmart(NEB)	9μL
dd H ₂ O	62μL
		Aggregate (Total)	90μL

3.3 homologous recombination of the target fragment and the vector pcDNA3.1

Homologous recombination systems are shown in the following table:

TABLE 9

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 in step 3.1)	1μL
dd H ₂ O	10μL
		Aggregate (Total)	20μL

After cloning was successful, the sample sequence was verified by sequencing and the final sample sequencing results are shown in the following table:

table 10

In table 10 above, for each nanobody sequence, the underlined portion is a Secretory Peptide (SP); bold indicates a 6 x his tag; double underline indicates HA tag; italics indicates the linking sequence.

Example 4: expression and purification of coronavirus spike protein Delta type nanobody

4.1 expression of coronavirus spike protein Delta nanobody

4.1.1 preparation of expression plasmids

By heat shock transformation, the recombinant plasmid containing the target gene (constructed in example 3) was introduced into E.coli DH 5. Alpha. Competent cells (Bomaide organism) and cultured overnight at 37℃in LB medium containing 50. Mu.g/mL ampicillin. And collecting bacterial liquid and extracting plasmids.

4.1.2 cell transfection

1mL of Cell Medium (Yiqiao Shenzhou) was added with 0.08mg of the transfection reagent PEI and 0.02mg of the plasmid obtained in step 4.1.1) and incubated at room temperature for 15 minutes, and 20mL of the mixture solution was added with a density of 2X 10 ⁶ HEK293F cells were cultured at 37℃and 120rpm for 72 hours or more.

4.2 purification of coronavirus spike protein Delta nanobody

20mL of the Expi 293 (Thermofisher, sieimer) cells expressed the antibody for 5 days, and after centrifugation at 3000rpm for 20 minutes, the culture supernatant was collected. Adding nickel sulfate with the final concentration of 4mM into the culture medium supernatant, standing for 5 minutes, filtering by using a 0.22 mu M filter membrane, purifying the nano antibody by using a nickel ion affinity chromatography column, eluting the hetero protein by using Buffer C, buffer D and Buffer E respectively, eluting the target protein by using Buffer F, and then carrying out SDS-PAGE electrophoresis verification.

As shown in fig. 3 and 4, all nanobodies can obtain target proteins with high purity and high yield after expression and purification for further activity identification.

Wherein, the Buffer solution C-F comprises the following specific components:

Buffer C:25mM HEPEs,pH7.5,150mM NaCl；

buffer D25mM HEPEs,pH7.5,150mM NaCl,20mM imidazole;

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

Buffer F:25mM HEPEs,pH7.5,150mM NaCl,250mM imidazole。

example 5: ELISA (enzyme-Linked immuno sorbent assay) detection of affinity of coronavirus spike protein Delta type nano antibody

5.1 coating of coronavirus spike protein Delta antigen

High concentration Delta type antigen (amino acid sequence shown as SEQ ID NO: 44) was diluted with CBS diluent (pH 9.6) to adjust the concentration to 2. Mu.g/mL, and antigen was coated overnight in 96-well plates at 4℃according to the standard of coating volume of 100. Mu.L/well. The next day, the coating solution and antigen solution were discarded, washed 3 times with 250. Mu.L of PBST solution, and patted dry. 200. Mu.L/well of blocking solution (2% (m/v) skimmed milk powder+PBS) was added and blocked for 2 hours at room temperature with gentle shaking. Subsequently, the blocking solution was discarded, washed three times with 250. Mu.L of PBST, and patted dry.

5.2 nanobody dilution

Five nanobodies obtained by purification in example 4 were diluted 4-fold from high concentration (20. Mu.g/mL), diluted 8 gradients, 2% (m/v) skimmed milk powder+PBST. Subsequently, 100. Mu.L of the diluted antibody was transferred to a plate coated with coronavirus spike-protein Delta-type antigen and incubated for 1 hour at room temperature with gentle shaking. Washed three times with PBST and patted dry.

5.3 Secondary antibody treatment

HRP-labeled secondary antibody (0.5. Mu.g/mL, anti-HA tag murine monoclonal antibody (Anti-HA tag Mouse Monoclonal Antibody)) diluted in PBST was added to the sample plate at 100. Mu.L/well and incubated for 1 hour at room temperature with gentle shaking. Subsequently, the mixture was washed four times with PBST and then dried by patting.

5.4 color development and data analysis.

TMB single-component color-developing solution (Soy baby) was added at 100. Mu.L/well, the reaction was stopped with 50. Mu.L/Kong Zhongzhi solution (Soy baby), and the reading was performed at 450nm using an ELISA reader. Experimental data EC50 values were calculated by fitting a curve with GraphPad Prism 5.

The results are shown in fig. 5, a and B, where 5 different nanobodies exhibit different affinities for coronavirus spike protein Delta type, with the highest affinity for SM-JSN-B1-11 being 0.49nM, followed by SM-JSN-B1-2 with an affinity of 0.78nM.

Example 6: ELISA evaluation of the selectivity of five nanobodies for coronavirus spike protein wild type and Delta mutant

The same antigen preparation protocol as in example 1 and the test protocol in example 5 were used, and the present example performed preparation of coronavirus spike-protein wild-type antigen and affinity data validation using ELISA experiments.

Amino acid sequence of spike protein wild-type antigen (SEQ ID NO: 45):

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPGSASSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSPIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGPALQIPFPMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGHHHHHHHHSAWSHPQFEKGGGSGGGGSGGSAWSHPQFEKGSDYKDDDDK*

As shown in FIGS. 5 c and d, the 5 nanobodies used in this patent exhibit similar affinity to the Delta type in wild-type antigen protein as compared to the Delta type of spike protein. According to this analysis, 5 nanobodies were able to bind to different subtypes of antigen proteins, suggesting that their binding epitopes to coronavirus spike proteins may be located in relatively conserved regions.

Reference to the literature

1.Pinto,D.et al.Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody.Nature 583,290–295(2020).

2.Baum,A.et al.Antibody cocktail to SARS-CoV-2spike protein prevents rapid mutational escape seen with individual antibodies.Sci New York N Y 369,eabd0831(2020).

3.Saunders,K.O.et al.Neutralizing antibody vaccine for pandemic and pre-emergent coronaviruses.Nature 594,553–559(2021).

4.Tortorici,M.A.et al.Ultrapotent human antibodies protect against SARS-CoV-2 challenge via multiple mechanisms.Sci New York N Y 370,950–957(2020).

5.Zost,S.J.et al.Potently neutralizing and protective human antibodies against SARS-CoV-2.Nature 584,443–449(2020).

6.Winkler,E.S.et al.Human neutralizing antibodies against SARS-CoV-2 require intact Fc effector functions for optimal therapeutic protection.Cell 184,1804-1820.e16(2021).

7.V’kovski,P.,Kratzel,A.,Steiner,S.,Stalder,H.&Thiel,V.Coronavirus biology and replication:implications for SARS-CoV-2.Nat Rev Microbiol 19,155–170(2021).

8.Harvey,W.T.et al.SARS-CoV-2variants,spike mutations and immune escape.Nat Rev Microbiol 19,409–424(2021).

9.Asdaq,S.M.B.et al.A Patent Review on the Therapeutic Application of Monoclonal Antibodies in COVID-19.Int J Mol Sci 22,11953(2021).

10.Xiang,Y.et al.Versatile and multivalent nanobodies efficiently neutralize SARS-CoV-2.Science 370,1479–1484(2020).

11.Chi,X.et al.An ultrapotent RBD-targeted biparatopic nanobody neutralizes broad SARS-CoV-2 variants.Signal Transduct Target Ther 7,44(2022).

12.Koenig,P.-A.et al.Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape.Science 371,eabe6230(2021).

13.Wade,J.et al.Generation of Multivalent Nanobody-Based Proteins with Improved Neutralization of Longα-Neurotoxins from Elapid Snakes.Bioconjugate Chem 33,1494–1504(2022).

14.Huet,H.A.et al.Multivalent nanobodies targeting death receptor 5 elicit superior tumor cell killing through efficient caspase induction.Mabs 6,1560–1570(2014).

15.Wang,J.et al.Research Progress and Applications of Multivalent,Multispecific and Modified Nanobodies for Disease Treatment.Front Immunol 12,838082(2022).

16.Takeuchi,T.,Kawanishi,M.,Nakanishi,M.,Yamasaki,H.&Tanaka,Y.Phase II/III results of the anti-TNF multivalent

compound‘ozoralizumab’in patient with rheumatoid arthritis(OHZORA trial).Arthritis Rheumatol(2022)doi:10.1002/art.42273./>

Claims

1. A nanobody targeting SARS-CoV-2 spike protein comprising:

2. The SARS-CoV-2 spike protein-targeted nanobody of claim 1 comprising any one or more of the following (i) - (v):

(v) SEQ ID NO:9, HCDR1, SEQ ID NO:10 and HCDR2 and SEQ ID NO:11, HCDR3.

3. The SARS-CoV-2 spike protein-targeting nanobody of claim 1 or 2, wherein the SARS-CoV-2 spike protein-targeting nanobody further comprises a framework region;

optionally, the framework region comprises:

4. A nanobody targeting SARS-CoV-2 spike protein according to claim 3, wherein said framework region comprises any one or more of the following (i) - (v):

5. The SARS-CoV-2 spike protein-targeting nanobody according to any one of claims 1 to 4, wherein said SARS-CoV-2 spike protein-targeting nanobody comprises the amino acid sequence of SEQ ID NO: 1-SEQ ID NO:5, or a sequence as set forth in any one of SEQ ID NOs: 1-SEQ ID NO:5, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity.

6. The SARS-CoV-2 spike protein-targeting nanobody according to any one of claims 1 to 5, wherein said SARS-CoV-2 spike protein-targeting nanobody further comprises a tag, a signal peptide, a linker sequence or any combination thereof;

optionally, the nanobody targeting SARS-CoV-2 spike protein comprises a tag at its N-terminus and/or C-terminus; and/or the number of the groups of groups,

comprising a signal peptide at its N-terminus;

preferably, the nanobody targeting SARS-CoV-2 spike protein comprises SEQ ID NO: 39-SEQ ID NO:43, or a sequence as set forth in any one of SEQ ID NOs: 39-SEQ ID NO:43, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity.

7. A polynucleotide encoding the nanobody of any one of claims 1-6 that targets SARS-CoV-2 spike protein.

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

9. A host cell into which or containing the expression vector of claim 8 has been introduced.

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

11. A pharmaceutical composition comprising a nanobody targeting SARS-CoV-2 spike protein according to any one of claims 1 to 6, and a pharmaceutically acceptable excipient, diluent or carrier.

12. A detection or diagnostic kit comprising a nanobody of any of claims 1-6 that targets SARS-CoV-2 spike protein.

13. Use of a nanobody targeting SARS-CoV-2 spike protein according to any one of claims 1 to 6 in the manufacture of a medicament for the treatment or prevention of a disease or condition caused by SARS-CoV-2 infection;

preferably, the disease or condition caused by SARS-CoV-2 infection includes a novel coronavirus infection.

14. Use of a nanobody targeting SARS-CoV-2 spike protein as claimed in any one of claims 1 to 6 in the preparation of a reagent for detecting or diagnosing a disease or condition caused by SARS-CoV-2 infection;