KR20220145614A - Novel antibody specifically binding to SARS-CoV-2 or antigen-binding fragment thereof, and a composition for diagnosis of SARS-CoV-2 - Google Patents

Abstract

The present invention relates to a novel antibody that specifically binds to the receptor-binding domain of SARS-CoV-2, or antigen-binding fragment thereof, and a composition for diagnosing SARS-CoV-2. Since the antibody or antigen-binding fragment thereof of the present invention binds to SARS-CoV-2 with high specificity and affinity, it can be usefully used for diagnosis of SARS-CoV-2.

Description

A novel antibody or antigen-binding fragment thereof that specifically binds to the receptor binding domain of SARS-CoV-2, and a composition for diagnosis of SARS-CoV-2 {Novel antibody specifically binding to SARS-CoV-2 or antigen-binding fragment thereof, and a composition for diagnosis of SARS-CoV-2}

The present invention relates to a novel antibody or antigen-binding fragment thereof and a diagnostic composition that specifically binds to coronavirus, and more particularly, to a receptor binding domain of SARS-CoV-2. A novel antibody or antigen-binding fragment thereof and a composition for diagnosis of SARS-CoV-2 are provided.

Coronavirus is an RNA virus that causes respiratory diseases such as rhinorrhea in mammals and birds. The shape of the surface of the virus resembles the corona that makes up the outer layer of the sun, so it is called a coronavirus.

SARS-CoV, which causes Severe Acute Respiratory Syndrome (SARS), is also one of the coronaviruses. In 2003, about 8,000 people worldwide were infected with SARS-CoV, of which about 10% died. did.

In addition, 'MERS-CoV', which causes Middle East Respiratory Syndrome (MERS), is also one of the coronaviruses. About 200 people were reported to have died, and after the first case was discovered in May 2015 in Korea, about 200 people were infected until July 2015, and it is known that about 20% of them, or about 40, died.

In December 2019, there was an outbreak of coronavirus (SARS-CoV-2 or COVID-19), also called novel coronavirus (2019-nCoV), in Wuhan, China. It has been declared a pandemic.

Research continues through the published genome to develop a vaccine against SARS-CoV-2, and efforts are being made to find antibodies or antigen-binding fragments having binding ability to SARS-CoV-2.

Although it is predicted that COVID-19 will end soon, there is a possibility that new coronavirus strains will occur in the future, and in order to prepare for this, various antibodies for diagnosis or neutralization of COVID-19 need to be developed.

An object according to an embodiment of the present invention is to provide a novel antibody or antigen-binding fragment thereof that specifically binds to coronavirus.

Another object of the present invention is to provide a novel antibody or antigen-binding fragment thereof that specifically binds to the receptor binding domain of SARS-CoV-2.

Objects of the present invention are not limited to those mentioned above, and other objects not mentioned will be clearly understood by those of ordinary skill in the art to which the present invention belongs from the following description.

To solve the above technical problem, a novel antibody or antigen-binding fragment thereof that specifically binds to the receptor binding domain of SARS-CoV-2 according to an aspect of the present invention is provided.

In the present invention, the heavy chain variable region and sequence comprising the heavy chain CDR1 set forth in SEQ ID NO: 1, the heavy chain CDR2 set forth in SEQ ID NO: 2, and the heavy chain CDR3 set forth in SEQ ID NO: 3 that specifically bind to the receptor binding domain of SARS-CoV-2 An antibody or antigen-binding fragment thereof comprising a light chain variable region comprising the light chain CDR1 set forth in SEQ ID NO: 4, the light chain CDR2 set forth in SEQ ID NO: 5 and the light chain CDR3 set forth in SEQ ID NO: 6 was developed, which has high affinity for SARS-CoV-2 The figure and specificity are shown, and it was confirmed that SARS-CoV-2 can be diagnosed through this.

In addition, a heavy chain variable region comprising SEQ ID NO: 7; And a novel antibody or antigen-binding fragment thereof that specifically binds to the receptor binding domain of SARS-CoV-2 comprising a light chain variable region comprising SEQ ID NO: 8 may be provided.

Here, the heavy chain variable region and the light chain variable region may be linked by a linker of SEQ ID NO: 9.

In the present invention, “antibody” may be interpreted in the broadest sense, and specifically, an intact monoclonal antibody, a polyclonal antibody, and a multispecific antibody formed from two or more intact antibodies (eg, a bispecific antibody). ), and antibody fragments exhibiting the desired biological activity. An antibody is a protein produced by the immune system capable of recognizing and binding to a specific antigen, and the antibody typically has a Y-shaped protein consisting of four amino acid chains (two heavy chains and two light chains). Each antibody mainly has two regions: a variable region and a constant region. The variable region located in the distal portion of the arm of Y binds and interacts with the target antigen. The variable region comprises a complementarity determining region (CDR) that recognizes and binds a specific binding site on a specific antigen. The constant region located at the tail of Y is recognized and interacted with by the immune system. Target antigens have multiple binding sites, called epitopes, that are generally recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, an antigen may have more than one corresponding antibody.

The whole antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected to the heavy chain by a disulfide bond. The whole antibody includes IgA, IgD, IgE, IgM and IgG, and IgG is a subtype, including IgG1, IgG2, IgG3 and IgG4.

The antibody fragment refers to a fragment having an antigen-binding function, and includes Fc, Fd, Fab, Fab', Fv and F(ab')2. The Fc refers to a tail portion of an antibody that interacts with a cell surface receptor called an Fc receptor. The Fd refers to the heavy chain portion included in the Fab fragment, and F(ab′)2 refers to a fragment including Fd, Fab, Fab′ and Fv. The Fab has a structure having a light chain and heavy chain variable regions, a light chain constant region and a first constant region (CH1 domain) of the heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region comprising one or more cysteine residues at the C terminus of the heavy chain CH1 domain. The F(ab')2 antibody is produced by forming a disulfide bond between a cysteine residue in the hinge region of Fab'. Fv (variable fragment) refers to a minimal antibody fragment having only a heavy chain variable region and a light chain variable region. A disulfide-stabilized Fv antibody fragment (dsFv) has a heavy chain variable region and a light chain variable region linked by a disulfide bond. The variable regions of the light chain are linked by covalent bonds. Such antibody fragments can be obtained using proteolytic enzymes (for example, by restriction digestion of the entire antibody with papain to obtain Fab, and by digestion with pepsin to obtain F(ab')2 fragments), preferably It can be produced through genetic recombination technology.

In general, an immunoglobulin has a heavy chain and a light chain, each heavy and light chain comprising a constant region and a variable region. The variable regions of the light and heavy chains include three complementarity-determining regions (hereinafter referred to as "CDRs") and four framework regions (hereinafter referred to as "FRs") called complementarity determining regions. . The CDRs of each chain mainly play a role in binding to an epitope of an antigen, and are typically named CDR1, CDR2, and CDR3 sequentially, starting from the N-terminus. In addition, the FR of each chain may be sequentially named FR1, FR2, FR3, FR4, starting from the N-terminus.

In the present invention, the variable region of the heavy chain may be designated as 'VH' and the variable region of the light chain as 'VL', and the CDRs of the heavy chain may be designated as 'VH-CDR1', 'VH-CDR2', and 'VH-CDR3', respectively. , the CDRs of the light chain are 'VL-CDR1', 'VL-CDR2', 'VL-CDR3', respectively, and the FRs of the heavy chain are 'VH-FR1', 'VH-FR2', 'VH-FR3', 'VH', respectively -FR4', the FR of the light chain may be named 'VL-FR1', 'VL-FR2', 'VL-FR3', 'VL-FR4'.

In addition, when the antibody of the present invention includes a constant region as described above, it may include a constant region derived from IgG, IgA, IgD, IgE, IgM or a combination thereof or a hybrid thereof. .

As used herein, the term "combination" means that, when forming dimers or multimers, polypeptides encoding single-chain immunoglobulin constant regions of the same origin form bonds with single-chain polypeptides of different origin. For example, dimers or multimers may be formed from two or more constant regions selected from the group consisting of constant regions of IgG, IgA, IgD, IgE and IgM.

In addition, the present invention includes functional variants of the antibody or antigen-binding fragment. If these variants can compete with the antibody or antigen-binding fragment of the present invention for specific binding to SARS-CoV-2 and retain the ability to bind SARS-CoV-2, then the functional antibody or antigen-binding fragment of the present invention is considered a variant. Functional variants include, but are not limited to, derivatives that are substantially similar in primary structural sequence, and include, for example, in vitro or in vivo modifications, chemicals and/or biochemicals. and they are not found in the parental monoclonal antibodies of the present invention. Such modifications include, for example, acetylation, acylation, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, crosslinking, disulfide bond formation, glycosylation, hydroxylation, methylation, oxidation, pegylation, proteolysis. and phosphorylation. A functional variant may be an antibody comprising an amino acid sequence optionally containing one or more amino acid substitutions, insertions, deletions or combinations thereof compared to the amino acid sequence of the parent antibody. Furthermore, functional variants may include truncated forms of the amino acid sequence at either or both the amino terminus or the carboxy terminus.

Functional variants of the invention may have the same, different, higher or lower binding affinity compared to the parent antibody of the invention, but still be able to bind SARS-CoV-2. As an example, the amino acid sequence of a variable region including, but not limited to, a framework structure, a hypervariable region, in particular a complementarity-determining region (CDR) of a light or heavy chain may be modified. Generally a light or heavy chain region comprises three hypervariable regions, comprising three CDR regions, and a more conserved region, namely a framework region (FR). A hypervariable region comprises amino acid residues from the CDRs and amino acid residues from the hypervariable loops.

Functional variants within the scope of the present invention may have about 95%-99%, or about 97%-99% amino acid sequence identity to the parent antibody herein.

In addition, the present invention provides an immunoconjugate in which one or more tags are additionally bound to the antibody or antigen-binding fragment. In one embodiment, a drug may be further attached to the antibody or antigen-binding fragment. That is, the antibody or antigen-binding fragment according to the present invention may be used in the form of an antibody-drug conjugate to which a drug is bound. The use of antibody-drug conjugates (ADCs), i.e., immunoconjugates, for local delivery of drugs allows for targeted delivery of the drug moiety to infected cells, which when administered unconjugated to normal cells as well. This is because unacceptable levels of toxicity can result. Maximal efficacy and minimal toxicity of ADCs can be improved by increasing drug-connectivity and drug-releasing properties, as well as selectivity of polyclonal and monoclonal antibodies (mAbs).

As used herein, the term "hybrid" means that sequences corresponding to the constant regions of immunoglobulin heavy chains of two or more different origins exist in the constant regions of single-chain immunoglobulin heavy chains, for example, IgG, IgA, IgD, IgE and 1 to 4 domains selected from the group consisting of CH1, CH2, CH3 and CH4 of IgM.

In addition, in the present invention, the antibody may have the same or different origins of the variable and constant regions, and the CDRs, except for the variable and constant regions, may have the same or different origins.

As used herein, the term "antibody or antigen-binding fragment thereof that specifically binds to the receptor binding domain of SARS-CoV-2" refers to an antibody or antigen thereof capable of inhibiting activity by specifically binding to SARS-CoV-2. binding fragments.

Specifically, the antibody binding to SARS-CoV-2 may be a mouse antibody, a chimeric antibody, or a humanized antibody.

In addition, the antibody of the present invention comprises a heavy chain variable region comprising SEQ ID NO: 7; and a light chain variable region comprising SEQ ID NO: 8.

According to an embodiment of the present invention, a novel antibody or antigen-binding fragment thereof that specifically binds to coronavirus may be provided.

According to another embodiment of the present invention, a novel antibody or antigen-binding fragment thereof that specifically binds to the receptor binding domain of SARS-CoV-2 may be provided.

Effects of the present invention are not limited to those mentioned above, and other effects not mentioned will be clearly understood by those of ordinary skill in the art to which the present invention belongs from the following description.

1 is after obtaining phagemid DNA of a monoclonal antibody, sequencing is performed using 5'-, 3'-oligonucleotides of single chain Fv (scFv), and the common region of the sequences read in both directions This is a schematic diagram of a method for identifying the amino acid sequence of an antibody produced through a monoclonal antibody production service to synthesize the final scFv sequence (ABI3730XL, Sanger-method sequencing, Phred Score ≥20).
2 is a chicken-derived single chain fragment variable (scFv) of a light chain variable region--Linker--Heavy chain variable region structure according to an embodiment of the present invention; The structure is diagrammed.
3 is a result of cross-testing of CEA8 scFv according to an embodiment of the present invention using CoV1 (SARS-CoV-1), CoV2 (SARS-CoV-2) antigen protein (RBD) (antigen: 1 ug/ ml, antibody: 2ug/ml).
4 is a graph showing the results of confirming the binding difference between the CoV1 antigen protein, the CoV2 antigen protein, and the CEA8 scFv (antigen: 8 - 0.315 ug/ml, antibody: 2 ug/ml).
5 is a graph showing the degree of binding of the antibody to the antigen according to the amount of antigen applied to the CoV2 antigen protein (antigen: 400 - 20 ng/well, antibody: 2ug/ml).
6 shows the titer of the CEA8 antibody measured using the CoV2 antigen protein.
7 is a graph showing the degree of SARS-CoV-2 inhibition (Inhibition %) by concentration of the mini-antibody prepared using the CEA8 antibody.

Objects and effects of the present invention, and technical configurations for achieving them will become clear with reference to the embodiments described below in detail in conjunction with the accompanying drawings. In describing the present invention, if it is determined that a detailed description of a well-known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description thereof will be omitted. And the terms described below are terms defined in consideration of donation in the present invention, which may vary depending on the intention or custom of the user or operator.

However, the present invention is not limited to the embodiments disclosed below and may be implemented in various different forms. Only the present embodiments are provided so that the disclosure of the present invention is complete, and to fully inform those of ordinary skill in the art to which the present invention belongs, the scope of the invention, the present invention is defined by the scope of the claims it will only be Therefore, the definition should be made based on the content throughout this specification.

As used herein, the term "antibody" refers to a protein molecule serving as a receptor that specifically recognizes an antigen, including immunoglobulin molecules having immunological reactivity with a specific antigen, polyclonal antibody, monoclonal antibody, Includes both whole antibodies and antibody fragments. The term also includes chimeric antibodies, such as humanized antibodies and bivalent or bispecific molecules (eg, bispecific antibodies), diabodies, triabodies and tetrabodies. includes The term further includes single-chain antibodies having a binding function to FcRn, scaps, derivatives of antibody constant regions, and artificial antibodies based on protein scaffolds. The whole antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected to the heavy chain by a disulfide bond. The whole antibody includes IgA, IgD, IgE, IgM and IgG, and IgG is a subtype, including IgG1, IgG2, IgG3 and IgG4. The antibody fragment refers to a fragment having an antigen-binding function, and includes Fd, Fab, Fab', F(ab')2 and Fv. The Fd refers to the heavy chain portion included in the Fab fragment. The Fab has a structure having a light chain and heavy chain variable regions, a light chain constant region and a first constant region (CH1 domain) of the heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region comprising one or more cysteine residues at the C terminus of the heavy chain CH1 domain. The F(ab')2 antibody is produced by forming a disulfide bond with a cysteine residue in the hinge region of Fab'. Fv (variable fragment) refers to a minimal antibody fragment having only a heavy chain variable region and a light chain variable region. In a double disulfide Fv (dsFv), the heavy chain variable region and the light chain variable region are linked by a disulfide bond, and in a single chain Fv (scFv), the heavy chain variable region and the light chain variable region are covalently linked via a peptide linker. Such antibody fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restriction digestion of the whole antibody with papain, and F(ab')2 fragments can be obtained by digestion with pepsin), preferably It can be produced through genetic recombination technology.

As used herein, the term "monoclonal antibody" refers to an antibody molecule of a single molecular composition obtained from substantially the same antibody population, and such monoclonal antibody exhibits single binding specificity and affinity for a specific epitope.

Typically, immunoglobulins have heavy and light chains, each heavy and light chain comprising a constant region and a variable region (these regions are also known as domains). The variable regions of light and heavy chains include three variable regions called complementarity-determining regions (hereinafter referred to as "CDRs") and four framework regions (FRs). The CDRs mainly serve to bind to an epitope of an antigen. The CDRs of each chain are typically called CDR1, CDR2, CDR3 sequentially, starting from the N-terminus, and are also identified by the chain in which the specific CDR is located.

Meanwhile, the antibody may be in the form of a chimeric antibody or a humanized antibody with reduced immunogenicity for application to the human body as described above.

In addition, when the antibody of the present invention includes a constant region as described above, it may include a constant region derived from IgG, IgA, IgD, IgE, IgM or a combination thereof or a hybrid thereof. .

As used herein, the term "combination" means that, when forming a dimer or multimer, a polypeptide encoding a single-chain immunoglobulin constant region of the same origin forms a bond with a single-chain polypeptide of a different origin. For example, dimers or multimers may be formed from two or more constant regions selected from the group consisting of constant regions of IgG, IgA, IgD, IgE and IgM.

As used herein, the term "hybrid" means that sequences corresponding to immunoglobulin heavy chain constant regions of two or more different origins exist in the single-chain immunoglobulin heavy chain constant region, for example, IgG, IgA, IgD, IgE and 1 to 4 domains selected from the group consisting of CH1, CH2, CH3 and CH4 of IgM.

Hereinafter, the antibody or antigen-binding fragment of the present invention described above will be described in more detail.

According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof that specifically binds to the receptor binding domain of SARS-CoV-2 is heavy chain CDR1 set forth in SEQ ID NO: 1, heavy chain CDR2 set forth in SEQ ID NO: 2, and SEQ ID NO: 3 and a light chain variable region comprising a heavy chain variable region comprising the heavy chain CDR3 described in

The antibody or antigen-binding fragment thereof that specifically binds to the receptor binding domain of SARS-CoV-2 according to some embodiments of the present invention includes a heavy chain variable region comprising SEQ ID NO: 7; and a novel antibody or antigen-binding fragment thereof that specifically binds to a receptor binding domain of SARS-CoV-2 comprising a light chain variable region comprising SEQ ID NO: 8.

The antibody of the present invention can be easily prepared by known antibody production techniques. For example, the method for preparing a monoclonal antibody may be prepared through a hybridoma using B lymphocytes obtained from an immunized animal, or may be performed by using a phage display technology or the like.

Antibody library using phage display technology is a method of expressing antibody on the surface of phage by obtaining an antibody gene directly from B lymphocytes without producing a hybridoma. Monoclonal antibody is generated by immortalization of B-cells. Existing problems related to this can be overcome. In general, phage display technology includes the steps of 1) inserting a random sequence of oligonucleotides into the gene region corresponding to the phage coat protein pIII (or pIV N-terminus; 2) a part of the native coat protein and expressing a fusion protein of a polypeptide encoded by an oligonucleotide of the random sequence; 3) treating a receptor material capable of binding to the polypeptide encoded by the oligonucleotide; 4) eluting the peptide-phage particles bound to the receptor using a low pH or binding competitive molecule; 5) amplifying the eluted phage in a host cell by panning; 6) repeating the method to obtain the desired amount; and 7) determining the sequence of the active peptide from the DNA sequence of the phage clones selected by panning.

The method for producing a monoclonal antibody according to an embodiment of the present invention may be performed using phage display technology. One of ordinary skill in the art is skilled in the art of known phage display techniques, for example, Barbas et al. (METHODS: A Companion to Methods in Enzymology 2:119, 1991 and J. Virol. 2001 Jul; 75(14):6692-9) and Winter et al. (Ann. Rev. Immunol. 12:433, 1994), each step of the manufacturing method of the present invention can be easily performed with reference to the disclosure. Phage that can be used to construct the antibody library include, but are not limited to, for example, filamentous phage, fd, M13, f1, If1, Ike, Zj/Z, Ff, Xf, Pf1 or Pf3 phage. In addition, vectors that can be used for expression of heterologous genes on the surface of the filamentous phage include, for example, phage vectors such as fUSE5, fAFF1, fd-CAT1 or fdtetDOG or pHEN1, pComb3, pComb8 or pSEX, etc. There is a phagemid vector of, but is not limited thereto. In addition, the helper phage that can be used to provide the wild-type envelope protein required for successful re-infection of the recombinant phage for amplification includes, for example, M13K07 or VSCM13, but is not limited thereto.

In addition, yeast display technology is a technology for expressing proteins on the surface of yeast cells. Since yeast uses a eukaryotic protein expression system, post-translational modification of the expressed protein occurs when this technology is used, so that a recombinant protein closer to a human protein can be obtained than a phage or ribosome display method. have. In addition, yeast display technology has the advantage of enabling quantitative analysis simultaneously with the selection process because clones are selected through the fluorescense-activated cell sorting (FACS) method. In general, yeast display technology for scFv antibodies comprises the steps of 1) inserting a scFv library linked by a short amino acid linker to the C-terminus of Aga2, a cell wall protein of yeast, and a sequence of a tag; 2) Aga2 and scFv antibody genes are ligated to Aga1, a yeast cell wall protein, by disulfide bonds so that they are expressed on the yeast cell surface; 3) binding an antigen to the scFv antibody library expressed on the yeast surface; 4) treating a secondary antibody to which a fluorescent material specifically recognizing an antigen and a tag is bound; 5) selecting yeasts expressing the scFv antibody that specifically binds to the antigen through FACS; 6) obtaining DNA sequence information of clones selected by FACS and determining the antibody variable region sequence having high antigen-binding ability.

The antibody according to an embodiment of the present invention is a hybridoma-derived monoclonal antibody or a polynucleotide encoding a display clone can be easily isolated and sequenced using conventional procedures. For example, oligonucleotide primers designed to specifically amplify the heavy and light chain coding regions from hybridoma or phage template DNA may be used. Once the polynucleotide is isolated, it can be placed into an expression vector, which can then be introduced into an appropriate host cell to produce the desired antibody from a transformed host cell (ie, a transformant). Therefore, the method for producing a monoclonal antibody of the present invention may be a method for producing a monoclonal antibody comprising amplifying a polynucleotide encoding a monoclonal antibody in an expression vector containing a polynucleotide encoding the monoclonal antibody. Not limited.

One or more amino acid substitutions in the CDR sequences provided in Table 1 or Table 2 to provide improved biological activity, such as improved binding affinity for SARS-CoV-2, those of ordinary skill in the art to which the present invention pertains can be modified to contain

For example, a library of antibody variants (eg, Fab or scFv variants) can be generated, expressed by phage display technology, and then screened for binding affinity to SARS-CoV-2. Alternatively, computer software can be used to actually simulate binding of an antibody to SARS-CoV-2 and identify amino acid residues on the antibody that form the binding region. Such residues can be avoided upon substitution to prevent a decrease in binding affinity, or targeted for substitution to provide stronger binding. In certain embodiments, at least one or all substitutions in a CDR sequence may correspond to conservative substitutions.

In certain embodiments, antibodies and antigen-binding fragments thereof are listed in Table 5 (or those) and at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99%) of one or more CDR sequences having sequence identity, wherein the corresponding CDR sequences are listed in Table 1 or Table 2 (or those ) and retains a binding affinity to SARS-CoV-2 at a level similar to or much higher than its parent antibody having a substantially identical sequence except for having 100% sequence identity.

antibody division order SEQ ID NO: CEA8 heavy chain variable-region: VH) QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEYVSAISSNGGSTYYADSVKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYCVKGYYNDNSGYYTLAFDYWGQGTLVTVSSASTQSPSVTS 7 VH-CDR1 SYAM One VH-CDR2 AISSNGGST 2 VH-CDR3 KGYYNDNSGYYTLAFDY 3 linker GGGSFRSSSSGGGGSGGGG 9 light chain variable-region: VL) ELVLTQPPSVSGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGPVFGGGTKLTVL 8 VL-CDR1 SGSSSNIGSNTVN 4 VL-CDR2 SNNQ 5 VL-CDR3 AAWDDSLNG 6

[Antibody of SARS-CoV-2, amino acid sequence of 'CEA8']

antibody DNA sequence SEQ ID NO: CEA8 gagctcgtgctgactcagccaccctcagtgtctgggacccccgggcagagggtcaccatcTcttgttctggaagcagctccaacatcggaagtaatactgtaaactggtaccagcagctcccaggaacggcccccaaactcctcatctatagtaataatcagcggccctcaggggtccctgaccgattctctggctccaagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattattactgtgcagcatgggatgacagcctgaatggtccggtgttcggcggagggaccaagctgaccgtcctaggcggtggttcctttagatcttcctcctctggtggcggtggctcgggcggtggtgggcaggtgcagctggtgcagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcctctggattcaccttcagtagctatgctatgcactgggtccgccaggctccagggaagggactggaatatgtttcagctattagtagtaatgggggtagcacatactacgcagactcagtgaagggcagattcaccatctccagagacaattccaagaacacgctgtatcttcaaatgagcagtctgagagctgaggacacggctgtgtattactgtgtgaagggatattacaatgataatagtggttattacaccctagcttttgactactggggccagggaaccctggtcaccgtctcctcagcctccacacagagcccatcggtcactagt 10

[SARS-CoV-2 antibody, DNA sequence of 'CEA8']

In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to the receptor binding domain of SARS-CoV-2 provided herein is a fully human antibody.

In certain embodiments, fully human antibodies are produced using recombinant methods. For example, a transgenic animal, such as a mouse, can be made to carry a transgene or transchromosome of human immunoglobulin genes, and thus can generate fully human antibodies after immunization with the appropriate antigen. Fully human antibodies can be isolated from such transgenic animals, or alternatively by hybridoma technology by fusing spleen cells of the transgenic animal with an immortal cell line to produce hybridoma cells that secrete fully human antibodies. can be manufactured.

Exemplary transgenic animals include, but are not limited to, the OmniRat (OmniRat, engineered to contain an endogenous expression of a rat immunoglobulin gene with inactivated, concomitantly functional recombinant human immunoglobulin loci); OmniMouse (engineered to contain a recombinant human immunoglobulin locus with inactivated endogenous expression of mouse immunoglobulin genes, while concurrently with a J-locus deletion and a C-kappa mutation); OmniFlic (transgenic rat engineered to contain a recombinant human immunoglobulin locus with endogenous expression of a rat immunoglobulin gene inactivated and concurrently with a single common, rearranged VkJk light chain and functional heavy chain) (Osborn M. et al., Journal of Immunology, 2013, 190: 1481-90; Ma B. et al., Journal of Immunological Methods 400-401 (2013) 78-86; Geurts A. et al. , Science, 2009, 325:433).

Other suitable transgenic animals, such as HuMab mice (Lonberg, N. et al. Nature 368(6474): 856 859 (1994)), Xeno-Mouse (Mendez et al. Nat Genet) ., 1997, 15:146-156), TransChromo mice (Ishida et al. Cloning Stem Cells, 2002, 4:91-102) and VelocImmune mice (Murphy et al. Proc Natl) Acad Sci USA, 2014, 111:5153-5158), Kymouse (Lee et al. Nat Biotechnol, 2014, 32:356-363), and transgenic rabbit (Flisikowska et al. PLoS One, 2011, 6) :e21045) can also be used.

In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to the receptor binding domain of SARS-CoV-2 is a diabody, scFv, scFv dimer, BsFv, dsFv, (dsFv)2, dsFv-dsFv', Fv fragment, Fab, Fab', F(ab')2, ds diabody, nanobody, domain antibody, or bivalent domain antibody.

In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to the receptor binding domain of SARS-CoV-2 further comprises an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region comprises a heavy and/or light chain constant region. The heavy chain constant region comprises a CH1, CH1-CH2, or CH1-CH3 domain. In some embodiments, the constant region may further comprise one or more modifications conferring desirable properties. For example, the constant region can be modified to reduce or lack one or more effector functions, to improve FcRn receptor binding, or to introduce one or more cysteine residues.

In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to the receptor binding domain of SARS-CoV-2 further comprises a conjugate. A variety of conjugates can be linked to the antibodies or antigen-binding fragments provided herein ("Conjugate Vaccines", Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr. (eds.), Carger Press, New York, (1989)) ). These conjugates may be linked to the antibody or antigen-binding fragment by covalent binding, affinity binding, intercalation, coordination binding, complexation, association, blending or addition, among other methods. In certain embodiments, the antibodies and antigen-binding fragments disclosed herein can be engineered to contain specific sites outside of the epitope binding moiety, which can be used for binding to one or more conjugates. For example, such sites may include one or more reactive amino acid residues, such as cysteine or histidine residues, to facilitate covalent linkage with the conjugate.

-D-갈락토시다제), 방사성 동위원소 (예를 들어 123I, 124I, 125I, 131I, 35S, 3H, 111In, 112In, 14C, 64Cu, 67Cu, 86Y, 88Y, 90Y, 177Lu, 211At, 186Re, 188Re, 153Sm, 212Bi, 및 32P, 다른 란탄 계열 원소, 발광 표지), 발색단 모이어티, 디곡시게닌, 비오틴/아비딘, DNA 분자 또는 검출용 금이 포함될 수 있다. In certain embodiments, the antibody may be linked indirectly to a conjugate, or linked via another conjugate. For example, the antibody or antigen-binding fragment may be conjugated to biotin and then indirectly conjugated to a second conjugate conjugated to avidin. The conjugate may be a detectable label, a pharmacokinetic modifying moiety, a purification moiety, or a cytotoxic moiety. Examples of detectable labels include fluorescent labels (eg, fluorescein, rhodamine, dansyl, phycoerythrin, or Texas red), enzyme-substrate labels (eg, horseradish peroxidase, alkaline phosphatase, Luceriferase, glucoamylase, lysozyme, sugar oxidase or

-D-galactosidase), radioactive isotopes (eg 123I, 124I, 125I, 131I, 35S, 3H, 111In, 112In, 14C, 64Cu, 67Cu, 86Y, 88Y, 90Y, 177Lu, 211At, 186Re, 188Re, 153Sm, 212Bi, and 32P, other lanthanide elements, luminescent labels), chromophore moieties, digoxigenin, biotin/avidin, DNA molecules or gold for detection.

In certain embodiments, the conjugate may be a pharmacokinetic modifying moiety, such as PEG, which helps to increase the half-life of the antibody. Other suitable polymers include, for example, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, copolymers of ethylene glycol/propylene glycol, and the like. In certain embodiments, the conjugate may be a purification moiety, such as a magnetic bead. A “cytotoxic moiety” can be any agent that is detrimental to or capable of damaging or killing a cell.

Examples of cytotoxic moieties include, but are not limited to, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin Bicin, dihydroxy anthracyndione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogues, antimetabolites ( e.g. methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g. mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclotosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-chlorodiamine platinum (II) (DDP) cisplatin), anthra Cyclins (eg, daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (eg, dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)) ) and anti-mitotic agents (eg, vincristine and vinblastine).

As another embodiment, the present invention may provide a polynucleotide encoding the antibody, an expression vector containing the polynucleotide, and a transformant into which the vector is introduced.

The expression vector comprising the polynucleotide encoding the antibody provided in the present invention is not particularly limited thereto, but mammalian cells (eg, human, monkey, rabbit, rat, hamster, mouse cell, etc.), plant cell, yeast The vector can be a vector capable of replicating and/or expressing the polynucleotide in a eukaryotic or prokaryotic cell, including a cell, insect cell or bacterial cell (eg, E. coli, etc.), preferably in a host cell, the nucleotide is It may be a vector that is operably linked to an appropriate promoter for expression and includes at least one selectable marker. For example, the polynucleotide may be introduced into a phage, a plasmid, a cosmid, a mini-chromosome, a virus, or a retroviral vector.

The expression vector comprising the polynucleotide encoding the antibody may be an expression vector comprising a polynucleotide encoding a heavy chain or a light chain of the antibody, respectively, or an expression vector comprising both a polynucleotide encoding a heavy chain or a light chain.

The transformant introduced with the expression vector provided in the present invention is not particularly limited thereto, but bacterial cells such as E. coli, Streptomyces, Salmonella typhimurium, etc. transformed by introducing the expression vector; yeast cells; Fungal cells such as Pichia pastoris; insect cells such as Drosophila and Spodoptera Sf9 cells; CHO (chinese hamster ovary cells), SP2/0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T, Bow melanoma cells, HT-1080, BHK ( animal cells such as baby hamster kidney cells, HEK (human embryonic kidney cells), and PERC.6 (human retinal cells); or plant cells.

As used herein, the term “introduction” refers to a method of delivering a vector including a polynucleotide encoding the antibody to a host cell. Such introduction is a calcium phosphate-DNA co-precipitation method, DEAE-dextran-mediated transfection method, polybrene-mediated transfection method, electroshock method, microinjection method, liposome fusion method, lipofectamine and protoplast fusion method, etc. It can be carried out by several methods known in the art. In addition, transduction means the delivery of a target object into a cell using virus particles by means of infection. In addition, the vector can be introduced into the host cell by gene bambadment or the like. In the present invention, introduction may be used in combination with transformation.

As another embodiment, the present invention provides an antibody-drug conjugate in which a drug is bound to the antibody.

As used herein, the term "antibody-drug conjugate" refers to a substance in which a drug is bound to an antibody using the target specificity of the antibody, non-toxicity during blood circulation, and pharmacokinetic advantages. Such an antibody-drug conjugate usually includes three components, a monoclonal antibody-linker-drug, and can enhance the therapeutic effect by the antibody by delivering the drug to the bacteria targeted by the antibody, especially SARS-CoV-2. have.

As used herein, the term “drug” refers to a substance that can effectively treat a disease targeted by the antibody by binding directly or indirectly to an antibody. Drugs capable of binding an antibody include radionuclides, drugs, lymphokines, toxins, bispecific antibodies, and the like. In addition, the aforementioned radionuclides include 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, 186Re, and the like, but is not limited thereto. In addition, examples of the drug or toxin include etoposide, teniposide, adriamycin, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycins, cis-platinum and cis-platinum homologues, bleo mycins, esperamicins, 5-fluorouracil, melphalan and other nitrogen mustards may be mentioned, but the above examples are not limited to drugs or toxins capable of binding to the antibody of the present invention.

Such antibody-drug conjugates can be prepared through various methods for preparing antibody-drug conjugates known in the art.

As another embodiment, the present invention provides a cosmetic composition for preventing or alleviating coronavirus-19, comprising the antibody.

Since the antibody binds to the RBD of SARS-CoV-2 with high affinity, the SARS-CoV-2 activity is inhibited, neutralized, and/or cytotoxic, thereby preventing or preventing a disease in which SARS-CoV-2 is a cluster. May bring symptom relief. The antibody is as described above.

As used herein, the term "prevention" may refer to any action that inhibits or delays the onset of coronavirus-19 by administration of the composition. In addition, in the present invention, the term "alleviation" may refer to any action in which the symptoms of COVID-19 are improved or advantageously changed by administration of the cosmetic composition.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound. Examples of acceptable pharmaceutical carriers for compositions formulated as liquid solutions are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostats may be added as needed. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets.

The pharmaceutical composition may be in various oral or parenteral formulations. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient in one or more compounds, for example, starch, calcium carbonate, sucrose or lactose ( lactose), gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, etc. can be used.

The pharmaceutical composition is any selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories It may have one formulation.

The composition of the present invention is administered in a pharmaceutically effective amount.

As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is dependent on the subject's type and severity, age, sex, and drug. Activity, sensitivity to drug, administration time, administration route and rate of excretion, duration of treatment, factors including concomitant drugs and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. and may be administered single or multiple. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, and can be easily determined by those skilled in the art.

As another embodiment, the present invention provides a method of treating coronavirus-19 using the antibody.

The antibody and coronavirus-19 are the same as described above.

In one embodiment, the present invention includes the step of detecting SARS-CoV-2 in an isolated biological sample of an individual suspected of coronavirus-19 using the antibody through an antigen-antibody reaction, coronavirus- 19 provides a method of providing information for diagnosis. Also, this method may be a method of diagnosing COVID-19.

The method of treating COVID-19 is COVID-19 comprising administering a pharmaceutical composition further comprising an antibody and a pharmaceutically acceptable carrier to an individual suffering from or suspected of having COVID-19. may be a method of treating , and the pharmaceutically acceptable carrier is the same as described above. The method of treating COVID-19 may preferably be a method of treating COVID-19 comprising administering a composition comprising an antibody.

The subject includes mammals, birds, etc., including cattle, pigs, sheep, dogs, humans, and the like, and subjects for which coronavirus-19 is treated by administration of the composition of the present invention include without limitation.

In this case, the composition may be administered singly or multiple times in a pharmaceutically effective amount. In this case, the composition may be administered in the form of a liquid, powder, aerosol, capsule, enteric-coated tablet or capsule or suppository. Routes of administration include, but are not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, endothelial administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, and the like. However, when administered orally, the peptides are digestible, so oral compositions must be formulated to coat the active agent or to protect it from degradation in the stomach. In addition, the pharmaceutical composition may be administered by any device capable of transporting the active agent to a target cell.

The antibody, individual, and SARS-CoV-2 are as described above. Since SARS-CoV-2 is known as the main causative virus in coronavirus-19, the antibody of the present invention can be usefully used for diagnosis of coronavirus-19, which is known to overexpress SARS-CoV-2.

The method of providing information for the diagnosis of COVID-19 involves reacting the human monoclonal antibody specific for the RBD of SARS-CoV-2 of the present invention with an isolated biological sample of an individual suspected of coronavirus-19, and reacting the antigen- By detecting the formation of the antibody complex, SARS-CoV-2 can be detected, which can provide information for the diagnosis of COVID-19.

As used herein, the term "biological sample" includes tissue, cells, whole blood, serum, plasma, tissue autopsy samples (brain, skin, lymph node, spinal cord, etc.), cell culture supernatant, ruptured eukaryotic cells and bacterial expression systems. However, the present invention is not limited thereto. The presence or absence of SARS-CoV-2 can be confirmed by reacting these biological samples with the antibody of the present invention in a manipulated or unmanipulated state.

As used herein, the term "antigen-antibody complex" refers to a combination of the SARS-CoV-2 antigen in a sample and the monoclonal antibody according to the present invention that recognizes it, and the formation of the antigen-antibody complex is performed by a colormetric method. ), electrochemical method, fluorimetric method, luminometry, particle counting method, visual assessment and scintillation counting method selected from the group consisting of It can be detected by any method. However, it is not necessarily limited to these, and various applications and applications are possible.

In the present invention, various markers can be used to detect the antigen-antibody complex. Specific examples may be selected from the group consisting of enzymes, fluorescent substances, ligands, luminescent substances, microparticles, and radioactive isotopes, but are not necessarily limited thereto.

-D-갈락토시다제, 호스래디쉬 퍼옥시다제, β-라타마제 등을 포함하며, 형광물로는 플루오레세인, Eu3+, Eu3+ 킬레이트 또는 크립테이트 등을 포함하며, 리간드로는 바이오틴 유도체 등을 포함하며, 발광물로는 아크리디늄 에스테르, 이소루미놀 유도체 등을 포함하며, 미소입자로는 콜로이드 금, 착색된 라텍스 등을 포함하며, 방사성 동위원소로는 57Co, 3H, 125I, 125I-볼톤(Bonton) 헌터(Hunter) 시약 등을 포함한다.Enzymes used as detection markers include acetylcholinesterase, alkaline phosphatase,

-D-galactosidase, horseradish peroxidase, β-latamase, etc., and the fluorescent substance includes fluorescein, Eu3+, Eu3+ chelate or cryptate, and the ligand is a biotin derivative. The luminescent material includes acridinium ester, isoluminol derivative, etc., the microparticle includes colloidal gold and colored latex, and the radioactive isotope includes 57Co, 3H, 125I, 125I- Bolton's Hunter's reagent, and the like.

Preferably, the antigen-antibody complex can be detected using enzyme immunosorbent assay (ELISA). The enzyme immunosorbent assay (ELISA) includes a direct ELISA using a labeled antibody recognizing an antigen attached to a solid support, and an indirect ELISA using a labeled secondary antibody recognizing a capture antibody in a complex of an antibody recognizing an antigen attached to a solid support. , direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of an antibody attached to a solid support and another antibody that recognizes an antigen in a complex of an antibody attached to a solid support and another antibody that recognizes an antigen in a complex Various ELISA methods are included, such as indirect sandwich ELISA using a labeled secondary antibody that recognizes the antibody.

Through the following examples, the production of an antibody or antigen-binding fragment thereof that specifically binds to the receptor binding domain (RBD) of SARS-CoV-2 according to an embodiment of the present invention and its effects were examined. In addition, the neutralizing ability against SARS-CoV-2 virus was confirmed using a mini-antibody for antibody treatment.

Example 1

1) To prepare a single-chain antibody that specifically binds to the receptor binding domain of SARS-CoV-2, a group of candidate SARS-CoV-2 binding antibodies was prepared through phage display.

Specifically, by reacting 10 ¹³ chicken-derived single-chain antibody library phage (scFv phage library) with 3.5Х10 ⁹ diversity with SARS-CoV-2 for 1 hour, it specifically binds to the receptor binding domain of SARS-CoV-2. By eluting scFv-phage, poly-scFv-phage was obtained.

2) Using the obtained poly-scFv-phage, the process of step 1 was repeated a total of 3 times to selectively amplify only phage with good affinity (panning step: Panning). The obtained poly-scFv-phage was diluted, titration was analyzed, and the level of amplification was confirmed.

3) In order to confirm that the selected poly-scFv-phage exhibits affinity with SARS-CoV-2, ELISA analysis was performed on the poly-scFv-phage in the final panning step. For ELISA analysis, Corning, 3690 (Half well plate, High-binding) was used for the ELISA plate.

4) For ELISA analysis, 1 ul of poly-scFv-phage in the final panning step was added to 100 ul of medium and spread on an LB-ampicillin agar plate. A single colony was identified the next day.

5) Add 180ul each of medium containing ampicillin antibiotics to a 96-well culture plate, inoculate a single colony, incubate at 37°C in a shaker incubator, and then add 1M IPTG to each well so that a single scFv can be expressed.

6) Antigen coating:

After mixing the antigen (SARS-CoV-2 RBD-Fc Recombinant Protein) by concentration in the ELISA coating buffer, 50ul per well was dispensed in an ELISA plate and reacted overnight at about 4°C (overnight incubation). As the ELISA coating buffer, 0.1M sodium carbonate buffer (pH9.6) was used.

7) Blocking:

The next day, 3% skim milk in 1 x PBS-T was filled with antigen-coated wells and controls (background control, non-coated wells) to block the reaction at room temperature for 30 minutes. Skim milk corresponds to a blocking reagent.

8) Washing:

1x PBS-T was washed once at 150ul/well, a thick paper towel was laid, and the plate was shaken vigorously several times to remove all remaining small amounts of washing buffer.

9) Primary antibody treatment:

After washing is complete, 50ul of anti HA peroxidase diluted 1:2000 is added to each well and reacted at room temperature for 1 hour. Mix the primary antibody (Anti SARS-CoV-2 RBD scFv Antibody (CEA8)) in blocking buffer. Thus, 50ul per well was treated.

10) Washing:

After washing 3 times with 1x PBS-T 150ul/well, a thick paper towel was laid and the plate was shaken vigorously several times to remove all remaining small amounts of washing buffer. As the washing buffer, 1x PBS-T (1x PBS, pH7.4 + 0.05% Tween 20) was used.

11) Secondary antibody treatment:

Secondary antibody (Anti HA peroxidase conjugated antibody) was mixed with blocking buffer at a ratio of 1:2000 and treated at room temperature for 30 minutes at 50 μl per well.

12) Washing:

After washing 3 times with 1x PBS-T150ul/well, a thick paper towel was laid and the plate was shaken vigorously several times to remove all remaining washing buffer.

13) At room temperature, 50ul of TMB solution was added per well and color development was induced for 5 minutes.

14) Stop the reaction by adding 50ul per well of 1N sulfuric acid (1N sulfuric acid) as a stop solution to end the color reaction that turns blue. When the color of the reactant turns yellow, insert the microplate reader Absorbance (OD value) was measured at a wavelength of 450 nm using

3 is a result of cross-testing of CEA8 scFv according to an embodiment of the present invention using CoV1 (SARS-CoV-1), CoV2 (SARS-CoV-2) antigen protein (RBD) (antigen: 1 ug/ ml, antibody: 2ug/ml). Referring to Table 3 and Figure 3, it can be confirmed that the antibody according to the embodiment of the present invention specifically binds to the antigen protein (RBD) of CoV2.

Ag 3% skim CoV2 RBD 2.732 0.098 2.629 0.092 CoV1 RBD 0.088 0.104 0.095 0.092

4 is a graph showing the results of confirming the binding difference between the CoV1 antigen protein, the CoV2 antigen protein, and the CEA8 scFv (antigen: 8 - 0.315 ug/ml, antibody: 2 ug/ml). Referring to Table 4 and FIG. 4 , it can be confirmed that, in the case of the antibody according to an embodiment of the present invention, only CoV2 is specifically recognized even when the antigen is treated at a high concentration.

CoV2 CoV1 8 3.522 3.506 0.049 0.048 4 3.514 3.513 0.049 0.049 2 3.398 3.434 0.046 0.049 One 2.981 3.057 0.047 0.049 0.5 1.03 1.131 0.054 0.048 0.25 0.206 0.179 0.048 0.051 0.125 0.099 0.11 0.05 0.049 0.0625 0.076 0.074 0.048 0.05 0.03125 0.062 0.069 0.051 0.049 0 0.048 0.047 0.048 0.049

5 is a graph showing the degree of binding of the antibody to the antigen according to the amount of antigen applied to the CoV2 antigen protein (antigen: 400 - 20 ng/well, antibody: 2ug/ml). Antibody-antigen binding was confirmed at 40~400ng/well. Based on the results of this experiment, the amount of protein antigen application for antibody titer measurement was set to 50 ng/well.

PBS CEA8 scFv 400 0.052 0.047 3.188 3.09 200 0.053 0.045 3.164 3.177 150 0.046 0.149 3.034 2.991 100 0.047 0.159 3.111 3.04 50 0.046 0.188 2.322 2.507 40 0.047 0.062 2.225 1.905 20 0.048 0.063 0.983 0.723 0 0.047 0.051 0.054 0.055

6 shows the titer of the CEA8 antibody measured using the CoV2 antigen protein. Referring to FIG. 5, when the purified recombinant SARS-CoV-2 RBD protein was coated at 50 ng/well and the scFv antibody was serially dilution from 4 ug/ml to 0.0019 ug/ml, it was confirmed that the antigen was bound. (Antigen: 50ng/well, Antibody: 8 - 0.0002441ug/ml). Referring to Table 6 and Figure 6, it can be seen that the monoclonal antibody, CEA8 scFv, shows a linear response depending on the antigen concentration, so that the antibody can be used as a standard material.

One 2 AVG 4 2.638 2.7 2.669 2 2.585 2.733 2.659 One 2.417 2.694 2.5555 0.5 2.425 2.499 2.462 0.25 2.387 2.344 2.3655 0.125 2.027 2.009 2.018 0.0625 1.635 1.817 1.726 0.03125 1.238 1.217 1.2275 0.015625 0.762 0.87 0.816 0.0078125 0.471 0.585 0.528 0.0039063 0.298 0.313 0.3055 0.0019531 0.188 0.186 0.187 0.0009766 0.128 0.13 0.129 0.0004883 0.091 0.095 0.093 0.0002441 0.069 0.081 0.075 0 0.051 0.054 0.0525

Example 2: Neutralizing ability test for SARS-CoV2 virus (PRNT50)

1) Prepare a mini-body for antibody treatment using CEA8 antibody, dilute the prepared mini-antibody (4-fold serial dilution), prepare by concentration, and react with the same amount of 100 PFU virus for 1 hour did.

2) Next, after infecting Vero E6 cells with the antibody and virus mixture for about 1 hour, 1 mL of 2X DMEM (10% FBS, 2% methyl cellulose) medium was dispensed and cultured for 5 days.

3) Next, cells were inactivated and immobilized using a mixture of acetone and methanol (1:1), and cells were stained with 1% crystal violet solution, and plaque formation was confirmed.

7 is a graph showing the degree of SARS-CoV-2 inhibition (Inhibition %) by concentration of the mini-antibody prepared using the CEA8 antibody. Referring to the graph, it can be seen that the number of plaques increased as the concentration of the mini-antibody increased. As a result of the neutralizing ability test of the IC ₅₀ mini-antibody against SARS-CoV2 virus, the IC ₅₀ was observed to be 4.94 ug/ml.

From the above results, it was confirmed that the novel antibody or antigen-binding fragment thereof according to an embodiment of the present invention has neutralizing ability against SARS-CoV-2.

In the present specification and drawings, preferred embodiments of the present invention have been disclosed, and although specific terms are used, these are only used in a general sense to easily explain the technical content of the present invention and help the understanding of the present invention. It is not intended to limit the scope. It will be apparent to those of ordinary skill in the art to which the present invention pertains that other modifications based on the technical spirit of the present invention can be implemented in addition to the embodiments disclosed herein.

<110> YI, Seo Jin LEE, Chae Eun Kim, Seung-Jae Kim, Min Jun EO, Ha Ryeong <120> Novel antibody specifically binding to SARS-CoV-2 or antigen-binding fragment thereof, and a composition for diagnosis of SARS-CoV-2 <130> GP210005KR <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> ANTIBODY VH <400> 1 Ser Tyr Ala Met His 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> ANTIBODY VH <400> 2 Ala Ile Ser Ser Asn Gly Gly Ser Thr 1 5 <210> 3 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> ANTIBODY VH <400> 3 Lys Gly Tyr Tyr Asn Asp Asn Ser Gly Tyr Tyr Thr Leu Ala Phe Asp 1 5 10 15 Tyr <210> 4 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> ANTIBODY VL <400> 4 Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Asn 1 5 10 <210> 5 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> ANTIBODY VL <400> 5 Ser Asn Asn Gln One <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> ANTIBODY VL <400> 6 Ala Ala Trp Asp Asp Ser Leu Asn Gly 1 5 <210> 7 <211> 135 <212> PRT <213> Artificial Sequence <220> <223> ANTIBODY VH <400> 7 Gln Val Gln Leu Val Gln 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 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val 35 40 45 Ser Ala Ile Ser Ser Asn Gly Gly Ser Thr 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 Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Lys Gly Tyr Tyr Asn Asp Asn Ser Gly Tyr Tyr Thr Leu Ala Phe 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Gln Ser Pro Ser Val Thr Ser 130 135 <210> 8 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> ANTIBODY VL <400> 8 Glu Leu Val Leu Thr Gln Pro Pro Ser Val Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 9 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> ANTIBODY LINKER <400> 9 Gly Gly Gly Ser Phe Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly <210> 10 <211> 792 <212> DNA <213> Artificial Sequence <220> <223> ANTIBODY DNA <400> 10 gagctcgtgc tgactcagcc accctcagtg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgttctg gaagcagctc caacatcgga agtaatactg taaactggta ccagcagctc 120 ccaggaacgg cccccaaact cctcatctat agtaataatc agcggccctc aggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag 240 tctgaggatg aggctgatta ttactgtgca gcatgggatg acagcctgaa tggtccggtg 300 ttcggcggag ggaccaagct gaccgtccta ggcggtggtt cctttagatc ttcctcctct 360 ggtggcggtg gctcgggcgg tggtgggcag gtgcagctgg tgcagtctgg gggaggcgtg 420 gtccagcctg ggaggtccct gagactctcc tgtgcagcct ctggattcac cttcagtagc 480 tatgctatgc actgggtccg ccaggctcca gggaagggac tggaatatgt ttcagctatt 540 agtagtaatg ggggtagcac atactacgca gactcagtga agggcagatt caccatctcc 600 agagacaatt ccaagaacac gctgtatctt caaatgagca gtctgagagc tgaggacacg 660 gctgtgtatt actgtgtgaa gggatattac aatgataata gtggttatta caccctagct 720 tttgactact ggggccaggg aaccctggtc accgtctcct cagcctccac acagagccca 780 tcggtcacta gt 792

Claims (10)

A heavy chain variable region comprising the heavy chain CDR1 set forth in SEQ ID NO: 1, the heavy chain CDR2 set forth in SEQ ID NO: 2, and the heavy chain CDR3 set forth in SEQ ID NO: 3, and the light chain CDR1 set forth in SEQ ID NO: 4, the light chain CDR2 set forth in SEQ ID NO: 5 and SEQ ID NO: 6 A novel antibody or antigen-binding fragment thereof that specifically binds to the receptor binding domain of SARS-CoV-2 comprising a light chain variable region comprising the light chain CDR3 described in .
a heavy chain variable region comprising SEQ ID NO:7; and a novel antibody or antigen-binding fragment thereof that specifically binds to a receptor binding domain of SARS-CoV-2 comprising a light chain variable region comprising SEQ ID NO: 8.
3. The method of claim 1 or 2,
A novel antibody or antigen-binding fragment thereof that specifically binds to the receptor binding domain of SARS-CoV-2, characterized in that the heavy chain variable region and the light chain variable region are linked by a linker of SEQ ID NO: 9.
The novel antibody or antigen-binding fragment thereof according to claim 1 or 2, which specifically binds to the receptor binding domain of SARS-CoV-2 further comprising an immunoglobulin constant region.
The novel antibody or antigen-binding fragment thereof according to claim 1 or 2, which specifically binds to the receptor binding domain of SARS-CoV-2, further comprising a conjugate.
An isolated polynucleotide encoding the antibody of claim 1 or 2 or an antigen-binding fragment thereof.
An expression vector comprising the polynucleotide of claim 6.
A transformant other than a human into which the vector of claim 7 is introduced.
A composition for diagnosis or treatment of SARS-CoV-2 comprising the antibody of claim 1 or 2 or an antigen-binding fragment thereof.
An antibody-drug conjugate, which is bound to the antibody of claim 1 or 2 or an antigen-binding fragment thereof.

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