CN113388030A - Monoclonal antibody 32C7, and preparation method and application thereof - Google Patents

Abstract

The invention discloses a monoclonal antibody 32C7, a preparation method and application thereof. According to the invention, through preparing the neutralizing antibody 32C7 aiming at the RBD domain of the new coronavirus, the antibody 32C7 can be effectively combined with the RBD domain of the S protein of the new coronavirus through surface plasmon resonance detection in vitro, the neutralizing capacity of the antibody 32C7 is verified through a transgenic mouse infection model, the inhibiting effect of the neutralizing antibody 32C7 on the pulmonary viral titer and related inflammatory factors after the new coronavirus infection is determined, and the result shows that the neutralizing antibody can obviously inhibit the virus replication in vivo and reduce the generation of the inflammatory factors and the pulmonary inflammatory infiltration. The monoclonal neutralizing antibody 32C7 inhibits the new coronavirus from entering host cells, achieves the therapeutic effect of the new coronavirus neutralizing antibody, and can be effectively used for treating or preventing respiratory system injury caused by infection of the new coronavirus.

Description

Monoclonal antibody 32C7, and preparation method and application thereof

Technical Field

The invention relates to the field of biological medicines, in particular to an antibody for preventing and treating novel coronavirus infection and a preparation method thereof, and especially relates to a monoclonal antibody 32C7 and a preparation method and application thereof.

Background

The most significant target for the surface-mediated antibody response of SARS-CoV-2 virions is the homologous trimeric spike (S) protein. The spike protein facilitates viral entry into cells through the interaction of the receptor domain (RBD) with angiotensin converting enzyme 2(ACE 2). Thus, antibodies targeting the RBD domain of the spike protein are particularly important in combating current pandemics.

For example, Cheolmin Kim et al screened human monoclonal antibodies mAb directed to the receptor binding domain of the viral spike protein from a pool of antibodies constructed from peripheral blood mononuclear cells of convalescent patients. The results show that CT-P59 monoclonal antibody can effectively neutralize SARS-CoV-2 isolate, including D614G variant, without antibody-dependent enhancement effect, and block the interaction region of receptor binding domain and angiotensin converting enzyme 2(ACE2) receptor. Furthermore, the therapeutic effect of CT-P59 was evaluated in three animal models (ferret, hamster and rhesus monkey), which was able to reduce viral titers.

For another example, CN113024640A discloses an epitope peptide antigen detection neutralizing antibody kit based on screening of binding domain of the receptor of the new coronavirus RBD and ACE2, which contains epitope peptide antigen based on screening of binding domain of the receptor of the new coronavirus RBD and ACE2, the amino acid sequence of which is shown in SEQ ID nos. 1-4.

There remains a need for antibodies targeting the RBD domain of the spike protein, their preparation and use in therapeutic drugs or formulations thereof.

Disclosure of Invention

The invention aims to make up the defects of the prior art and provides a novel neutralizing antibody 32C7, a preparation method and application thereof, so that a safe and effective antibody is provided for clinical treatment of COVID-19, and the antibody or the antigen binding fragment thereof has wide application prospects. The details are as follows.

In a first aspect of the invention, there is provided an antibody 32C7 or an antigen binding fragment thereof, which is capable of specifically binding to a spike protein and which is capable of binding an epitope selected from amino acid residues at positions 345, 346, 347, 348, 349, 351, 352, 354, 440, 441, 442, 443, 444, 445, 448, 450, 451, 452, 466, 499, 509 of a spike protein or which is selected from T345, R346, F347, S349, N440, L441, K444, N450, Y351 and Y451 of a spike protein, to inhibit or block binding of said spike protein to a receptor.

In a second aspect of the invention, there is provided an antibody 32C7, or an antigen-binding fragment thereof, that comprises a heavy chain and a light chain and that exhibits a Kd for the receptor domain of spike protein of less than 120 nM.

In a third aspect of the invention, there is provided an antibody 32C7 or an antigen binding fragment thereof, comprising the antigen complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region, the amino acid sequences of which are SEQ ID NOs: 2. SEQ ID NO: 4. SEQ ID NO: 6; and

the variable region of light chain has the antigen complementarity determining regions CDR1, CDR2 and CDR3, whose amino acid sequences are respectively SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12.

the antibody 32C7 or antigen-binding fragment thereof according to the third aspect of the invention preferably comprises a heavy chain variable region and a light chain variable region having an amino acid sequence selected from the group consisting of those amino acid sequences having at least 80% identity.

The antibody 32C7 or antigen binding fragment thereof according to one aspect of the invention, preferably wherein the antibody 32C7 or antigen binding fragment thereof is a monoclonal antibody; the antigen binding fragments include Fab fragments, Fab ', F (ab')₂A fragment, a single chain variable fragment scFv, an scFv-Fc fragment or a single chain antibody ScAb.

In a fourth aspect of the invention, there is provided a nucleic acid molecule encoding the antibody 32C7 or antigen binding fragment thereof according to the first, second or third aspects of the invention.

In a fifth aspect of the invention, there is provided a vector comprising a nucleic acid molecule according to the fourth aspect of the invention.

In a sixth aspect of the invention, there is provided a method of making the antibody 32C7 or an antigen binding fragment thereof, the method comprising producing the monoclonal antibody by culturing a cell under conditions suitable for expression of the antibody 32C7 or an antigen binding fragment thereof, wherein the cell comprises the vector according to the fifth aspect of the invention.

In a seventh aspect of the invention, there is provided a composition comprising an antibody 32C7, or an antigen-binding fragment thereof, according to the first, second or third aspects of the invention.

In an eighth aspect, the invention provides the use of an antibody 32C7 or antigen-binding fragment thereof according to the first, second or third aspects of the invention in the manufacture of a medicament or reagent for the treatment or prophylaxis of infection by a novel coronavirus, preferably, but not limited to, the Original strain Original, british mutant strain b.1.1.7, south africa mutant strains b.1.351 and b.1.617.2 (indian Delta strain) in a human.

In a ninth aspect of the invention, there is provided a method of preventing and/or treating a coronavirus infection in a subject, comprising administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof of the invention, a composition thereof, or a medicament or agent of the invention.

The antibody 32C7 of the present invention has high affinity with the RBD domain of the novel coronavirus and dissociates very slowly. Experiments prove that the antibody can effectively inhibit infection of SARS-CoV-2 live virus, not only has obvious inhibition effect on an original strain, but also has good neutralization inhibition effect on a mutant virus strain, which shows that the antibody has excellent effect and application prospect compared with the prior representative antibody. In addition, animal model experiments further prove that the antibody of the invention can greatly reduce the virus titer of SARS-CoV-2, and related inflammatory factors are obviously reduced, and the inflammatory infiltration condition is obviously improved. The inventor further characterizes the structure of the combination of the antibody and RBD, and surprisingly finds that the antibody avoids the mutation sites of the current novel coronavirus mutant strains, so that the antibody has good inhibition effect on various mutant strains.

Drawings

FIG. 1 is an in vitro anti-neocoronavirus effect of antibody 32C7 according to example 2 of the present invention, wherein part A of FIG. 1 shows the interaction of antibody 32C7 with the SPR domain of a neocoronavirus capable of gradient dependence, at concentrations of 120, 60, 30, 15 and 7.5 in nM from top to bottom; part B of FIG. 1 shows that antibody 32C7 can dose-dependently inhibit infection by multiple subtypes of the SARS-CoV-2 mutant.

FIG. 2 is the effect of antibody 32C7 in transgenic mice new coronavirus infection model according to example 3 of the present invention, wherein part A of FIG. 2 is the new coronavirus titer in mouse lung slurry; FIG. 2B shows the expression level of the inflammatory factor in the lung polishing solution of mice; part C of fig. 2 is the mouse lung pathology.

FIG. 3 is an electron microscope observation result of the binding of the antibody 32C7 to the novel coronavirus S protein according to example 4 of the present invention, wherein part A of FIG. 3 is a structural diagram of the binding of the antibody 32C7 to the novel coronavirus S protein; part B of figure 3 is the epitope to which antibody 32C7 binds to the S protein of neocoronavirus.

Detailed Description

The following examples of the present invention will be described in detail in connection with specific examples which are intended to be illustrative of the invention only and should not be taken as limiting the scope of the invention but rather as a more detailed description of certain aspects, features and embodiments of the invention. The examples do not specify particular techniques or conditions, and are carried out according to techniques or conditions described in literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruke et al, Huang Petang et al) or according to product instructions. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Unless defined otherwise, all 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. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range. The terms "about," "approximately," or "approximately" when used in connection with a numerical value are intended to include collections or ranges of numerical values. For example, "about X" includes a numerical range that is ± 20%, ± 10%, ± 5%, ± 2%, ± 1%, ± 0.5%, ± 0.2% or ± 0.1% of X, where X is a numerical value. In one embodiment, the term "about" refers to a range of values that is 5% more or less than a fixed value. In another embodiment, the term "about" refers to a range of values that is 2% more or less than a fixed value. In another embodiment, the term "about" refers to a range of values that is 1% more or less than the stated value.

In the present invention, the term "spike protein" refers to a protein of a trimeric structure located at the outermost layer of the novel coronavirus, and is also referred to as spike glycoprotein or S protein. It is a structural protein derived from coronaviruses including SARS-CoV and 2019-nCoV. Thus, the terms "antibody against coronavirus S protein," "anti-coronavirus S protein antibody," "anti-S protein antibody," "coronavirus S protein antibody," "S protein antibody," or "antibody that binds to S protein" are used interchangeably herein to refer to an antibody herein that is capable of binding coronavirus S protein (e.g., 2019-nCoV S protein, SARS-CoV S protein) with sufficient affinity such that the antibody can be used as a diagnostic, prophylactic, and/or therapeutic agent that targets coronavirus S protein.

As used herein, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or fragment thereof or a T-cell receptor. Epitopic determinants generally consist of chemically active surface groupings of molecules (e.g., amino acids or sugar side chains) and generally have specific three-dimensional structural characteristics as well as specific charge characteristics. The epitope to which the antibody of the present invention specifically binds is preferably an amino acid residue selected from the group consisting of amino acid residues at positions 345, 346, 347, 348, 349, 351, 352, 354, 440, 441, 442, 443, 444, 445, 448, 450, 451, 452, 466, 499, 509 of the spike protein. Preferably, it specifically binds to at least one epitope of T345, R346, F347, S349, Y351, N450, Y451, N440, L441 and K444 of the spike protein.

As used herein, the term "antibody" refers to an immunologically active portion of an immunoglobulin molecule (Ig) molecule, i.e., a molecule that contains an antigen binding site that specifically binds to (immunoreacts with) an antigen. By "specifically binds" or "immunoreactive with" is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides or binds with much lower affinity (Kd). The antibodies of the invention exhibit a Kd of less than 120 nM for the receptor domain of the spike protein. Preferably, it has a Kd for the receptor domain of the spike protein of less than 80 nM, e.g., 70nM, 60nM, 50nM, 40nM, 30nM, 20nM, 10nM, even less than 8nM, e.g., 7nM, even less than 5 nM. Antibodies include, but are not limited to, monoclonal antibodies, chimeric antibodies, dAbs (domain antibodies), single chain antibodies, Fab 'and F (ab')₂Fragment, scFvs. Preferably, the antibody of the invention is a monoclonal antibody or an antigen-binding fragment thereof.

The term "antibody" as used herein also includes humanized antibodies, recombinant antibodies, human antibodies produced from transgenic non-human animals, and antibodies selected from libraries using enrichment techniques available to those skilled in the art.

As understood in the art, an antibody is a glycoprotein or antigen-binding portion thereof comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH1, CH2, and CH 3). The light chain comprises a light chain variable region (VL) and a light chain constant region (CL). The variable regions of the heavy and light chains comprise Framework Regions (FR) and Complementarity Determining Regions (CDR). The four FRs are relatively conserved, while the CDR regions (CDR1, CDR2, and CDR3) comprise hypervariable regions.Antibody building blocks are known to generally comprise tetramers. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain. The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. FR and CDR from NH₂End to COOH end arranged as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. In addition, the constant region may mediate binding of the immunoglobulin to host tissues or factors. Antibody constant regions are not directly involved in binding of an antibody to an antigen, but they exhibit different effector functions, such as participation in antibody-dependent cellular cytotoxicity (ADCC) or complement-mediated cytotoxicity (CDC) of an antibody. In general, antibody molecules are directed to IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule.

In the present invention, the antigen complementarity determining regions CDR1, CDR2 and CDR3 of the antibody heavy chain variable region are SEQ ID NO: 2. SEQ ID NO: 4 and SEQ ID NO: 6; the antigen complementarity determining regions CDR1, CDR2 and CDR3 of the antibody light chain variable region are SEQ ID NOs: 8. SEQ ID NO: 10 and SEQ ID NO: 12.

The term "antibody fragment" as used herein encompasses a portion of an intact antibody, such as the antigen binding or variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab ', F (ab')₂Fv fragment, scFV fragment, diabody, or linear antibody. Papain digestion of antibodies produces two identical "Fab" or antigen-binding fragments (each with a single antigen-binding site) and a residual "Fc" fragment (the name of which reflects its ability to crystallize readily). Pepsin treatment of antibodies produces F (ab')₂A fragment which has two antigen binding sites and which retains its ability to cross-link an antigen.

The term "Fv" as used herein means the smallest antibody fragment that contains the complete antigen recognition and antigen binding site. The fragment contains a dimer of a heavy chain variable region domain and a light chain variable region domain in tight, non-covalent association. The folding of these two domains results in the formation of six hypervariable loops (three loops each from the H and L chains) which facilitate the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable region (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, albeit with lower affinity. "Single-chain Fv" ("sFv" or "scFv") are antibody fragments comprising VH and VL antibody domains joined into a single polypeptide chain. The sFv polypeptide may also comprise a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding.

The term "Fab" fragment as used herein contains the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of several residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region.

The term "monoclonal antibody", also sometimes referred to as "monoclonal antibody" or mAb, as used herein, refers to an immunoglobulin derived from a pure line of cells, having the same structural and chemical properties, and being specific for a single antigenic determinant. Monoclonal antibodies differ from conventional polyclonal antibody preparations (typically having different antibodies directed against different determinants), each monoclonal antibody being directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are also advantageous in that they are obtained by hybridoma or recombinant engineered cell culture, and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a homogeneous population of antibodies, but this is not to be construed as requiring production of the antibody by any particular method.

The antibodies of the invention may comprise an Fc region from an IgG, for example IgG1, IgG2, IgG3 or IgG 4.

Unless otherwise specified, an antibody or antigen-binding fragment thereof as described herein is an isolated antibody or antigen-binding fragment thereof. The term "isolated" as used herein refers to a nucleic acid or antibody that has been extracted from its natural environment. Nucleic acids, peptides and proteins that have been "isolated" thus include nucleic acids and proteins purified by standard purification methods. The term also includes nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids and/or polypeptides.

In the present invention, the term "receptor binding domain" refers to the region of the S protein that binds or binds to ACE2, also known as RBD.

The term "antigen binding site" or "binding portion" refers to the part of an immunoglobulin molecule that is involved in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable ("V") region of the heavy ("H") chain and the light ("L") chain. Three highly divergent segments (called "hypervariable regions") in the V regions of the heavy and light chains are inserted between more conserved flanking segments called "framework regions" or "FRs". Thus, the term "FR" refers to the naturally found amino acid sequences between and adjacent to hypervariable regions of an immunoglobulin. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity determining regions" or "CDRs".

In the present invention, the terms "binding" and "immunological binding" are used interchangeably and refer to a non-covalent interaction that occurs between an immunoglobulin molecule and an antigen specific for said immunoglobulin. The strength or affinity of the immunological binding interaction may be expressed as a dissociation constant (Kd), with smaller Kd representing higher affinity. "affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to an intrinsic binding affinity that reflects a 1: 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the binding dissociation equilibrium constant (Kd). Affinity can be measured by common methods known in the art, including those known in the art and described herein.

In the present invention, the term "specifically binds" generally means that an antibody binds to an epitope through its antigen binding domain, and that the binding requires complementarity between the antigen binding domain and the epitope. Thus, when an antibody binds to the epitope through its antigen binding domain, it binds to the epitope more readily than it binds to a random, unrelated epitope, which is said to "specifically bind" to the epitope. The term "specificity" is used herein to define the relative affinity of an antibody for binding to an epitope. For example, antibody "a" can be considered to have a higher specificity for a particular epitope than antibody "B", or antibody "a" can be considered to bind epitope "C" with a higher specificity than to bind the relevant epitope "D".

Unless otherwise indicated, the terms "fragment," "antibody fragment," "antigen-binding fragment," and "antigen-binding fragment" are used interchangeably.

Variant antibodies are also included within the scope of the invention. Thus, variants of the sequences recited in the present invention are also included within the scope of the present invention. Other variants of antibody sequences with improved affinity may be obtained using methods known in the art and are included within the scope of the present invention. One skilled in the art can modify the amino acid sequence of a polypeptide using recombinant methods and/or synthetic chemical techniques for producing variant polypeptides. For example, amino acid substitutions may be used to obtain antibodies with further improved affinity. Alternatively, codon optimization of the nucleotide sequence may be used to increase translation efficiency in an expression system for the production of antibodies. Such variant antibody sequences have 80% or greater (i.e., 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater) sequence identity to the sequences recited in the present invention. The sequence identity is calculated relative to the sequences recited in the present invention. Or best aligned, such as by the program GAP or BESTFIT using default GAP weights.

In this context, the percent homology between two amino acid sequences is equal to the percent identity between the two sequences. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e.,% homology is the number of identical positions/total number of positions x 100), taking into account the number of gaps that need to be introduced and the length of each gap to produce an optimal alignment of the two sequences. Comparison of sequences and determination of percent identity between two sequences can be accomplished using mathematical algorithms. The percent identity between two amino acid sequences can be determined using the algorithms of e.meyers and w.miller (comput.appl.biosci.,4:11-17 (1988)). In addition, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J.mol.biol.48:444-453 (1970)).

The antibodies of the invention also include amino acid sequence modified antibodies, preferably, the antibodies of the invention comprise a heavy chain variable domain comprising CDR1, CDR2 and CDR3 sequences and a light chain variable domain comprising CDR1, CDR2 and CDR3 sequences, wherein one or more of these CDR sequences comprise a specific amino acid sequence or modification thereof based on the antibodies described herein, and the modified antibodies retain the desired functional properties of the anti-novel coronavirus antibodies of the invention.

The term "modified" as used herein means that amino acid modifications do not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence. Such modifications include substitutions, additions and deletions of amino acids. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. Antibodies of the invention may include glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modifications, and the like.

Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, the group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; the amino acids having aliphatic-hydroxy side chains are serine and threonine; the group of amino acids having amide-containing side chains is asparagine and glutamine; the group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids having basic side chains is lysine, arginine and histidine; and the amino acid group having a sulfur-containing side chain is cysteine and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine valine, glutamic-aspartic acid, and asparagine-glutamine. Thus, one or more amino acid residues in a CDR region of an antibody of the invention can be replaced with other amino acid residues from the same side chain family.

As described herein, the invention encompasses minor variations in the amino acid sequence of an antibody or immunoglobulin molecule, provided that the amino acid sequence maintains at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% variation. In particular, conservative amino acid substitutions are contemplated. Conservative substitutions are those occurring in the amino acid family, which are related to their side chains. Genetically encoded amino acids are generally classified into the following families: (1) the acidic amino acid is aspartic acid or glutamic acid; (2) the basic amino acid is lysine, arginine and histidine; (3) the nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan and (4) the amino acids without electric polarity are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other families of amino acids include (i) serine and threonine, which are the aliphatic-hydroxy family; (ii) asparagine and glutamine, which are a family of amides; (iii) alanine, valine, leucine, and isoleucine, which are aliphatic families; and (iv) phenylalanine, tryptophan, and tyrosine, which are aromatic families. For example, it is reasonable to expect that an isolated substitution of an isoleucine or valine for a leucine, an isolated substitution of a glutamic acid for an aspartic acid, an isolated substitution of a serine for a threonine, or a similar substitution of a structurally related amino acid will not have a significant effect on the binding or properties of the resulting molecule, particularly if the substitution does not involve an amino acid in a framework position. Whether an amino acid change results in a functional peptide can be readily determined by determining the specific activity of the polypeptide derivative. The measurement method is not particularly limited, and measurement can be performed by a method known in the art. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by one skilled in the art. Preferably, the amino-and carboxy-termini of the fragment or analog occur near the boundaries of the functional domains. Domains and functional domains can be identified by comparing nucleotide and/or amino acid sequence data to public or private sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains (which occur in other proteins of known structure and/or function). Methods for identifying protein sequences that fold into known three-dimensional structures are known.

Preferred amino acid substitutions are as follows: (1) reducing susceptibility to proteolysis; (2) reduced susceptibility to oxidation; (3) altering the binding affinity for forming a protein complex; (4) altering binding affinity and (4) imparting or modifying a physicochemical or functional property of the analog. Analogs can include various muteins of sequences other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally occurring sequence (preferably in the portion of the polypeptide outside the domains that form intermolecular contacts). Conservative amino acid substitutions should not substantially alter the structural characteristics of the parent sequence (e.g., a substituted amino acid should not tend to disrupt the helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence).

Another class of variable region modifications that may be present are mutation of amino acid residues in the VH and/or VK CDR1, CDR2, and/or CDR3 regions to improve one or more binding properties (e.g., affinity) of the antibody of interest. Mutations can be introduced by site-directed mutagenesis or PCR-mediated mutagenesis. Preferably, conservative modifications (as described above) are introduced. The mutation may be a substitution, addition or deletion of an amino acid, but is preferably a substitution. In addition, the residues in the CDR regions typically vary by no more than one, two, three, four or five.

Nucleic acid molecules

The present invention provides nucleic acid molecules that encode the antibodies of the invention. The nucleic acid may be present in an intact cell, in a cell lysate, or in a partially purified or substantially pure form. Nucleic acids are "isolated" when purified of other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques, including alkali/SDS treatment, CsCl banding (banding), column chromatography, agarose gel electrophoresis, and other techniques well known in the art. The nucleic acids of the invention may be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

The nucleic acid of the invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID No. 2, 4, 6, 8, 10, 12 or a nucleic acid encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from any one of SEQ ID No. 2, 4, 6, 8, 10, 12. Preferably, the coding sequence consists of the sequence shown in SEQ ID No. 1, 3, 5, 7, 9, 11.

The nucleic acids of the invention can be obtained using standard molecular biology techniques. Once the DNA fragments encoding the VH and VL segments are obtained, these are further manipulated by standard recombinant DNA techniques to, for example, convert the variable region genes to full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, a DNA fragment encoding a VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" as used herein is intended to mean that two DNA fragments are linked such that the amino acid sequences encoded by the two DNA fragments remain in frame (in-frame).

Isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operably linking the DNA encoding the VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH 3). The sequence of the human heavy chain constant region gene is known in the art.

The isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as the Fab light chain gene) by operably linking the DNA encoding the VL to another DNA molecule encoding the light chain constant region CL. The sequence of the human light chain constant region gene is known in the art.

The invention also provides polynucleotide variants encoding the peptide sequences of the heavy and light chains of the antibodies of the invention or antigen-binding fragments thereof. These polynucleotide variants may have at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% or more sequence identity compared to the polynucleotide sequences of the present invention. Such contiguous sequences may encode a CDR sequence, or may encode the entire variable region. The variable region sequence may be fused to any suitable constant region sequence as is known in the art. As will be appreciated by those skilled in the art, these values can be suitably adjusted to determine the corresponding identity of the proteins encoded by the two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like.

It will be appreciated in the art that sequences engineered for host codon bias based on the antibody coding sequences disclosed herein are suitable for use in the present invention. The base sequence of the present invention can be modified with preference according to degenerate codons in order to adapt to the needs of different hosts. Codon bias engineering does not generally alter the sequence of the product protein or polypeptide.

The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to single-or double-stranded RNA, DNA, or mixed polymers.

For recombinant production of the antibody, the nucleic acid encoding it is inserted into a vector for further cloning (amplification of the DNA) or for expression. DNA encoding the antibody of the invention was isolated according to the methods set forth in the examples. As will be appreciated by those skilled in the art, a number of vectors are available for use in the recombinant production of antibodies. Carrier components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter and a transcription termination sequence.

In the present invention, the vector refers to an expression vector including, but not limited to, a plasmid, retrovirus, YAC, EBV-derived episome, and the like. Suitable vectors are those encoding fully functional human CH or CL immunoglobulin sequences with suitable restriction sites engineered so that any VH or VL sequence can be readily inserted and expressed. The resulting chimeric antibody can bind to any strong promoter, including retroviral LTRs, e.g., the SV-40 early promoter, Rous sarcoma virus LTR, and Moloney murine leukemia virus LTR. Likewise, native Ig promoters and the like may be used.

Preferred vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include murine Moloney leukemia virus. DNA viral vectors are preferred. These vectors include pox vectors such as: smallpox or fowlpox vectors, herpes virus vectors such as: herpes simplex I virus (HSV) vectors. The choice of a particular vector will depend on the target cell and the conditions being treated. Introduction can be by standard techniques such as infection, transfection, transduction or transformation. Examples of gene transfer modes include, for example, naked DNA, CaPO4 precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

Suitable host cells for cloning or expressing DNA are prokaryotic cells, yeast cells or higher eukaryotic cells. Examples of commonly used prokaryotic host cells include E.coli, Bacillus subtilis, and the like. Commonly used eukaryotic host cells include yeast cells, insect cells, mammalian cells, and the like. Host cells for antibody production of the invention transformed with the above-described expression or cloning vectors are cultured in conventional nutrient media, suitably modified, for inducing promoters, selecting transformants or amplifying genes encoding the desired sequences. Antibodies prepared from the cells can be purified using purification techniques known to those of ordinary skill in the art.

The terms "subject" and "patient" are used interchangeably herein to refer to any animal that may be in need of an antibody-related formulation or a medicament, treatment, and vaccine described herein. Subjects and patients thus include, but are not limited to, primates (including humans), canines, felines, rodents, and other mammalian subjects. Preferably, the subject is a human. As is apparent from the context in which the terms are used, subjects and patients refer to subjects or patients susceptible to infection by a coronavirus and/or subjects or patients infected by a coronavirus.

In the present invention, the term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, the object of which is to prevent or slow down (lessen) the progression of an undesired physiological change or disorder, such as an autoimmune disease. Beneficial or desired clinical results include, but are not limited to, results, whether detectable or undetectable, including alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total). "treatment" also means an extended life span compared to the life span expected when not receiving treatment. Those in need of treatment include those already with the condition or disorder, as well as those susceptible to the condition or disorder, or those in need of prevention of the condition or disorder.

Composition comprising a metal oxide and a metal oxide

The present invention provides a composition comprising the antibody 32C7 of the present invention or an antigen-binding fragment thereof. As an example of a composition, the composition may be a conjugate of the monoclonal antibody or antigen-binding portion thereof described above and other substances, including cytotoxins, drugs, radiotoxins.

A cytotoxin or cytotoxic agent includes any agent that is detrimental to a cell (e.g., kills a cell). Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthrax dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof.

The compositions may also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine and lomustine, cyclophosphamide, busulfan, dibromomannitol, streptozocin, mitomycin C, and cis-dichlorodiammineplatinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., actinomycin D), bleomycin, mithramycin, and Anthranilamycin (AMC)), and antimitotics (e.g., vincristine and vinblastine), duocarmycin, calicheamicin, maytansine and risutatin, and derivatives thereof.

Medicine

In one embodiment, the antibodies, fragments thereof, of the invention may be used as therapeutic agents. Such drugs will generally be used to diagnose, prognose, monitor, treat, ameliorate, prevent and/or delay the progression of a disease or pathology associated with the spike protein. In certain embodiments, it is capable of treating or preventing respiratory damage caused by infection with a new coronavirus. An antibody preparation, preferably an antibody with high specificity and high affinity for its target antigen, is administered to a subject and will generally have an effect as it binds to the target. Administration of the antibody can reduce, antagonize, neutralize, eliminate, or inhibit or interfere with the binding of the endogenous ligand to which the target naturally binds.

The medicament of the present invention comprises the antibody of the present invention, or an antigen-binding portion thereof, and a pharmaceutically acceptable carrier. "pharmaceutically acceptable carrier" includes any and all physiologically compatible carriers of solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The medicaments of the invention may also be administered in combination therapy, i.e. in combination with other therapeutic agents and agents thereof. Such therapeutic agents include any anti-infective active agent, small molecule drugs including, but not limited to, redciclovir, ribavirin, oseltamivir, zanamivir, hydroxychloroquine, interferon, analgesics, azithromycin, and corticosteroids.

The term "effective amount" as used herein means an amount of a drug or pharmaceutical agent that elicits the biological or pharmacological response in a tissue, system, animal or human that is being sought, for example, by a researcher or clinician. Furthermore, the term "therapeutically effective amount" means an amount that causes improved treatment, cure, prevention, or alleviation of a disease, disorder, or side effect, or a decrease in the rate of progression of a disease or condition, as compared to a corresponding subject not receiving that amount. The term also includes within its scope an amount effective to enhance normal physiological function. In general, an effective amount herein will vary depending on various factors, such as the given drug or compound, pharmaceutical formulation, route of administration, type of disease or disorder, subject being treated, and the like, but can nevertheless be routinely determined by one of skill in the art. An effective amount of a compound of the present invention can be readily determined by one skilled in the art by conventional methods known in the art.

Use of

The present invention provides the use of monoclonal antibody 32C7 or an antigen-binding fragment thereof in the manufacture of a medicament or agent for the treatment or prevention of a novel coronavirus infection in a human. The novel coronavirus includes but is not limited to Original strain, mutant strains such as B.1.351, B.1.1.7, P1 and B.1.617.2. More specifically, the mutant virus strain has a mutation selected from the group consisting of: b.1.1.7 (N501Y), B.1.351 (K417N, E484K, N501Y), P1 (K417T, E484K, N501Y), B.1.617.2 (L452R, T478K).

The term "original strain" as used herein refers to the SARS-CoV-2 (New crown) virus strain having the same sequence as the strain with GISAID number EPI _ ISL _403934, which is published in sequence in month 1 of 2020.

The term "mutant" as used herein refers to a new strain of coronavirus which is mutated in sequence as compared to the strain EPI ISL 403934.

Example 1

This example is the preparation of monoclonal antibody 32C7, as follows.

First, experimental material

1. Cells required for the experiment, viruses and transgenic mice, Vero E6 cells, SARS-CoV-2, humanized ACE2 transgenic mice (hACE 2-mie).

2. Reagents and materials required for the experiments

The S5 chip was purchased from GE, the filter from PALL, DMEM medium, and Fetal Bovine Serum (FBS) from GIBCO.

3. Instrument for experiment

SPR surface plasmon resonance small molecule interaction instrument, multifunctional enzyme labeling instrument, q-RT PCR instrument and CO₂Cell culture chambers were purchased from Thermo corporation.

Second, Experimental methods

1) Firstly, collecting peripheral blood of 26 novel coronavirus pneumonia rehabilitation patients, and separating serum and lymphocytes for storage, wherein the pathogen of the novel coronavirus pneumonia rehabilitation patients is a new crown original virus strain;

2) then, the activity of a specific neutralizing antibody of the novel coronavirus RBD protein in the serum of 26 recovered patients is screened through an in vitro ELISA test, and 3 recovered patients are found to have higher neutralizing antibody titer;

3) preparing single cell transcripts of the 3 patients with the specificity of the novel coronavirus RBD protein B cells by cell-related labeling and flow cytometry sorting technology, and amplifying by PCR technology to obtain corresponding antibody heavy chain genes and antibody light chain genes;

4) expressing the antibody heavy chain gene and the antibody light chain gene verified by sequencing in a eukaryotic cell, producing and purifying a novel coronavirus specific antibody, and finally obtaining 200 candidate antibodies;

5) finally, novel coronavirus neutralizing antibodies with high affinity are screened and combined through in vitro ELISA experiments, pseudovirus neutralizing experiments and animal experiments.

Third, experimental results

The present invention resulted in antibody 32C7 against the novel coronavirus. The heavy chain gene sequence and the light chain gene sequence corresponding to the antibody are obtained by performing antibody gene specific PCR amplification and PCR product sequencing on a single cell transcript of an RBD protein specific B cell, and variable region sequences corresponding to the heavy chain gene and the light chain gene are obtained by analyzing an IMGT website (http:// www.imgt.org). Antibody 32C7 has the amino acid sequence of SEQ ID NO: 13, the heavy chain variable region nucleic acid sequence set forth in SEQ ID NO: 14 and the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 15, a heavy chain constant region amino acid sequence set forth in seq id no; SEQ ID NO: 16, the variable light chain nucleic acid sequence of SEQ ID NO: 17 and the variable light chain region amino acid sequence shown in SEQ ID NO: 18, or a light chain constant region amino acid sequence as set forth in seq id no.

Antibody 32C7 has the amino acid sequence as set forth in SEQ ID NO:1, the nucleotide sequence of the heavy chain CDR1 region shown in SEQ ID NO: 2; SEQ ID NO: 3, the nucleotide sequence of the CDR2 region shown in SEQ ID NO: 4; SEQ ID NO: 5, the nucleotide sequence of the CDR3 region shown in SEQ ID NO: 6. Antibody 32C7 has the amino acid sequence as set forth in SEQ ID NO: 7, the light chain CDR1 region nucleotide sequence set forth in SEQ ID NO: 8; SEQ ID NO: 9, the nucleotide sequence of the CDR2 region of SEQ ID NO: 10; SEQ ID NO: 11, the nucleotide sequence of the CDR3 region shown in SEQ ID NO: 12.

Example 2

This example shows the in vitro anti-neocoronavirus effect of monoclonal antibody 32C7, as follows.

1. Experimental methods and results

1.1 detection of the interaction of monoclonal antibody 32C7 with the new coronavirus RBD Domain by plasmon resonance

1) The expressed and purified RBD protein was coupled to the chip and then stabilized with buffer overnight.

2) Monoclonal antibody 32C7 was diluted in buffer in a two-fold gradient starting at 120 nM, filtered using a 0.2 filter and then centrifuged to remove air bubbles.

3) The affinity constants and dissociation constants of monoclonal antibody 32C7 and the RBD of the novel coronavirus were determined as described above.

The results are shown in FIG. 1 as A: the monoclonal antibody 32C7 can be efficiently and rapidly combined with the RBD domain of the novel coronavirus, and the affinity constant reaches 1.00 multiplied by 10⁵. Its dissociation constant is 1.09X 10^-8The equilibrium dissociation constant between the two is 1.09X 10 by analysis and calculation^-8. Illustrates the monoclonal antibody 32C7 between the RBD domains of the novel coronavirusHas a high affinity and dissociates very slowly.

1.2 monoclonal antibody 32C7 antibody neutralization assay

1) Vero E6 cells were plated in 48-well plates and incubated at 37 ℃ for 24 hours.

2) The cells in (1) were infected with MOI =0.005, and monoclonal antibody 32C7 was diluted in a 5-fold gradient starting from 10 μ g/mL with a medium containing 2% fetal bovine serum and added to the cells in (1).

3) The supernatant was removed 48 hours after infection and the virus titer was determined by plaque assay.

4) Vero E6 cells were plated in 96-well plates and incubated at 37 ℃ for 24 hours, then the above virus supernatants were diluted in a gradient to 96-well plates and incubated at 37 ℃ for 1 hour, and then the supernatants were discarded and 200. mu.L of medium containing 1.6% hydroxymethyl cellulose was added.

5) After 24 hours of culture, cells were fixed with 4% paraformaldehyde and 0.5% tween X-100 was added to permeabilize the cells. Followed by incubation with anti-SARS-CoV-2 nucleocapsid antibody and addition of HRP-labeled secondary antibody.

The result is shown as B in FIG. 1. The monoclonal neutralizing antibody 32C7 can effectively inhibit infection of SARS-CoV-2 live virus, has good neutralizing and inhibiting effects on original strain and SARS-CoV-2D 614G mutant strain (namely B.1.1.7 strain), and has IC₅₀4.588 ng/mL, and 127.6 ng/mL, respectively. For the novel coronavirus south Africa mutant strain 501Y.V2, the neutralizing antibody 32C7 still has certain neutralizing effect, and the IC is₅₀It was 1.42. mu.g/mL. However, representative antibodies CC12.1 and REGN10987 that have been published to date are directed against the IC of the D614G mutant₅₀22 ng/mL and 19.4 ng/mL, respectively, were found to be weak in neutralizing the south African mutant. IC of CV07-270₅₀It was 82.3 ng/mL.

Example 3

This example is the antiviral effect of antibody 32C7 in humanized mice, as follows.

1. The experimental process comprises the following steps:

1) human ACE2 transgenic (hACE) mice at 8 weeks of age were randomly assigned to drug evaluation groups, 4 mice. Negative control group 3 mice. Before formal experiments, the mice are adapted to the environment for 2-3 days.

2) On the day of challenge, mice were lightly anesthetized with 1% sodium pentobarbital (approximately 0.1 ml anesthetic per gram body weight) and then infected with 4X 10 by nasal drip using a pipette⁴SARS-CoV-2 virus fluid of PFU.

3) The antibody was injected intraperitoneally at 6 hours post-virus infection at 20 mg/kg. And weighing and recording the weight change of each group of animals every day at regular time, and observing the survival condition of the mice.

4) Mice were sacrificed the fifth day post infection, and lungs were removed and ground. And respectively detecting the virus titer of the lung grinding fluid and the expression condition of related inflammatory factors by using a qRT-PCR method. Simultaneously, the lungs of the mice were removed for histopathological examination.

5) And drawing a weight change curve and a survival rate curve according to the statistical result.

2. Results of the experiment

As shown in A in FIG. 2, antibody 32C7 was effective in reducing the virus titer of SARS-CoV-2 in a transgenic mouse model, and five days after virus infection, the virus titer in the lung of the antibody-treated mice was reduced by 90% relative to that in the lung of the virus-control mice. At the same time, as shown in fig. 2B, the expression of lung-associated inflammatory factors was also significantly reduced after antibody 32C7 treatment. The lung also demonstrated improved infiltration as shown by C in figure 2. In conclusion, the antibody 32C7 still has excellent protective effect on a SARS-CoV-2 transgenic mouse model and has obvious antiviral capability.

Example 4

In this example, the structure of the binding between antibody 32C7 and S protein was analyzed as follows.

1. Experimental procedure

1) Expression and purification of the protein: an S-6P mutant expressing SARS-CoV-2 (wherein the mutant is a mutant in which 6 serines of spike protein are mutated to 6 prolines). HEK293F cells in 8% CO₂In SMM 293T-I medium at 37 ℃. When the cell density reaches 2X 10⁶Transient transfection of S-2P and S-6P plasmids with 25-kDa Linear polyethyleneimine into HEK293F cells at PEI to DNA mass ratio of 3:1 at 1mg DNA per liter per mL. The cell culture supernatant was collected and centrifuged at 10000 Xg for 30 minutes. The secreted S-2P and S-6P proteins were purified using HisPur cobalt resin and Streptactin resin. Purification was performed using Superose 610/300 chromatography column in buffer containing 20 mM HEPES pH 7.2, 150 mM NaCl and 10% trehalose. The Fab region of antibody 32C7 was obtained after digestion of papain in a buffer containing 20 mM HEPES pH 7.2, 150 mM NaCl, 5 mM EDTA and 5 mM l-cysteine at 37 ℃ for 40 min. The resulting Fab was purified on a desalting column for l-cysteine and then on a HiTrap Q column for further purification. The purified Fabs were collected and concentrated to 0.6 mg/mL.

2) 2mL of S-6P (1.2 mg/mL) and 2mL of 32C7 Fab (0.6 mg/mL) were incubated at room temperature for 3min and then loaded onto a luminescent discharge gold grid. The grids were washed with buffer containing 20 mM HEPES, pH 7.2 and 150 mM NaCl. The grid was blotted using Mark IV Vitrobot at 100% humidity and 16 ℃ for 3 s, and then the grid was immersed in liquid ethane by immersion freezing. Micrographs of the S-6P-32C7 Fab complex were recorded on a 300 kV FEI Titan Krios electron microscope. On a K3 Summit direct electron detector, 3740 dynamic processes are recorded in a super-resolution mode with a nominal magnification of 81,000 times and a defocus range of 1.2-1.3 mm. The detector employs a GIF quantum energy filter with a slit width of 20 eV. The dose of the micrograph was divided into 32 frames and the total electron exposure was about 50 electrons per a.

2. Results of the experiment

As a result, as shown in FIG. 3A, it was found by structural analysis that the monoclonal antibody 32C7 could bind to RBD in "down" state of the novel coronavirus, resulting in the inability of the novel coronavirus to infect. Meanwhile, the 32C7 is found to be similar to the three types of epitopes reported at present. The epitope comprises amino acid residues 345, 346, 347, 348, 349, 351, 352, 354, 440, 441, 442, 443, 444, 445, 448, 450, 451, 452, 466, 499, 509 of the S protein (as shown in B in figure 3). 32C7 has 21 interacting amino acid residues with the RBD structure domain of the new coronavirus, and avoids the mutation sites of the current new coronavirus mutant strains, so that the mutant strains have good inhibitory effect on various mutant strains.

The mutation profile on RBD of the mutant strains known so far is summarized here: b.1.1.7 (N501Y), B.1.351 (K417N, E484K and N501Y), P1 (K417T, E484K and N501Y), B.1.617.2 (L452R, T478K) and 32C7 have good neutralizing effect on the new coronavirus mutant.

While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Various modifications or changes may be made to the exemplary embodiments without departing from the scope or spirit of the present invention. The scope of the invention should be accorded the broadest interpretation so as to encompass all modifications and equivalent structures and functions.

Sequence listing

<110> higher research institute of Shanghai Zhejiang university

<120> monoclonal antibody 32C7, and preparation method and application thereof

<130> BH2110331

<141> 2021-08-16

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ggattcacct tcagtagcta tcct 24

<210> 2

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Gly Phe Thr Phe Ser Ser Tyr Pro

1 5

<210> 3

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atatcatatg atggaagcaa taaa 24

<210> 4

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Ile Ser Tyr Asp Gly Ser Asn Lys

1 5

<210> 5

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gcgagagaca cggaagattg tagtagtacc acctgctatg ttgactat 48

<210> 6

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Ala Arg Asp Thr Glu Asp Cys Ser Ser Thr Thr Cys Tyr Val Asp Tyr

1 5 10 15

<210> 7

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cagagtgttt tatacagatc caacaataag aactac 36

<210> 8

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Gln Ser Val Leu Tyr Arg Ser Asn Asn Lys Asn Tyr

1 5 10

<210> 9

<211> 9

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tgggcatct 9

<210> 10

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Trp Ala Ser

1

<210> 11

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

caccaatatt atagtactcc tttcact 27

<210> 12

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

His Gln Tyr Tyr Ser Thr Pro Phe Thr

1 5

<210> 13

<211> 369

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gaggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcagt agctatccta tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtt atatcatatg atggaagcaa taaatactac 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgc gagagacacg 300

gaagattgta gtagtaccac ctgctatgtt gactattggg gccagggaac cctggtcacc 360

gtctcctca 369

<210> 14

<211> 123

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Pro Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Thr Glu Asp Cys Ser Ser Thr Thr Cys Tyr Val Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 15

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

100 105

<210> 16

<211> 339

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gatattgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60

atcaactgca agtccagcca gagtgtttta tacagatcca acaataagaa ctacttagct 120

tggtaccagc agaaaccagg acagcctcct aagctgctca tttactgggc atctacccgg 180

gaatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240

atcagcagcc tgcaggctga agatgtggca gtttattact gtcaccaata ttatagtact 300

cctttcactt tcggcggagg gaccaaggtg gatatcaaa 339

<210> 17

<211> 114

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Arg

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys His Gln

85 90 95

Tyr Tyr Ser Thr Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Asp Ile

100 105 110

Lys Arg

<210> 18

<211> 106

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

1 5 10 15

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

20 25 30

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

35 40 45

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

50 55 60

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

65 70 75 80

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

85 90 95

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

Claims (13)

1. Monoclonal antibody 32C7 or an antigen-binding fragment thereof, comprising the antigen complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region, the amino acid sequences of which are SEQ ID NOs: 2. SEQ ID NO: 4. SEQ ID NO: 6; and

the variable region of light chain has the antigen complementarity determining regions CDR1, CDR2 and CDR3, whose amino acid sequences are respectively SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12.

2. the monoclonal antibody 32C7 or an antigen-binding fragment thereof according to claim 1, which is capable of specifically binding to an epitope of spike protein, wherein the epitope is selected from the group consisting of amino acid residues at positions 345, 346, 347, 348, 349, 351, 352, 354, 440, 441, 442, 443, 444, 445, 448, 450, 451, 452, 466, 499, 509 of spike protein, or the epitope is selected from the group consisting of T345, R346, F347, S349, N440, L441, K444, N450, Y351 and Y451 of spike protein, to inhibit or block binding of the spike protein to a receptor.

3. The monoclonal antibody 32C7 or an antigen-binding fragment thereof according to claim 1, which exhibits a Kd of less than 120 nM for the receptor domain of spike protein.

4. The monoclonal antibody 32C7 or an antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region and a light chain variable region having an amino acid sequence with at least 80% identity to an amino acid sequence set forth in SEQ ID No. 2, 4, 6, 8, 10, and 12.

5. The monoclonal antibody 32C7 or antigen-binding fragment thereof of claim 1, wherein the antigen-binding fragment comprises a Fab fragment, a Fab ', a F (ab')₂A fragment, a single chain variable fragment scFv, an scFv-Fc fragment or a single chain antibody ScAb.

6. The monoclonal antibody 32C7 or antigen-binding fragment thereof according to claim 1, wherein the antibody has the amino acid sequence of SEQ ID NO: 14 and the variable heavy chain amino acid sequence of SEQ ID NO: 17, or a light chain variable region amino acid sequence as set forth in seq id no.

7. A composition comprising the monoclonal antibody 32C7 or antigen-binding fragment thereof according to any one of claims 1-6.

8. A nucleic acid molecule encoding the monoclonal antibody 32C7 or an antigen-binding fragment thereof according to any one of claims 1-6.

9. A vector comprising the nucleic acid molecule of claim 8.

10. A cell line capable of producing monoclonal antibody 32C7 or an antigen binding fragment thereof according to any one of claims 1-6.

11. A method of making monoclonal antibody 32C7, or an antigen-binding fragment thereof, comprising culturing the cell line of claim 10, thereby producing the monoclonal antibody.

12. Use of the antibody 32C7 or an antigen-binding fragment thereof according to any one of claims 1-6 in the manufacture of a medicament or agent for the treatment or prevention of a novel coronavirus infection.

13. The use according to claim 12, wherein the novel coronavirus comprises Original strain and mutant strain thereof, and the mutant strain comprises B.1.351, B.1.1.7, P1 and B.1.617.2.

Images