KR20220143255A - HUMAN ANTIBODIES TARGETING SARS-CoV-2 - Google Patents

Abstract

The present invention relates to a human antibody against a novel coronavirus (COVID-19 virus: SARS-CoV-2) or fragments having immunological activities thereof. A human antibody binding to a COVID-19 virus-RBD region, which is the cause of a COVID-19 disease, and fragments with immunological activities thereof, according to the present invention, bind with high affinity to the RBD region of a spike protein of a COVID-19 virus. Thus, it is possible to effectively prevent a COVID-19 virus from infecting a host, and the present invention can be used for prevention or treatment of COVID-19 virus infection. Also, the antibody and fragments having immunological activities thereof can be used for quick immunodiagnosis (detection), thereby being useful for the diagnosis and epidemiological investigation of a highly infectious COVID-19 virus.

Description

COVID-19 virus target human antibody {HUMAN ANTIBODIES TARGETING SARS-CoV-2}

The present invention relates to a human antibody or a fragment having immunological activity against novel coronavirus (COVID-19 virus: SARS-CoV-2).

Coronaviruses are enveloped viruses with single-stranded positive RNA as their genome. Coronaviruses can be divided into four groups. Among them, alpha-coronavirus and beta-coronavirus mainly infect mammals, while gamma-coronavirus and delta-coronavirus infect birds. Recently, there has been a confirmed case of infection with a delta-coronavirus in pigs. Coronaviruses can be transmitted heterogeneously. Representative examples include the SARS coronavirus that caused severe acute respiratory syndrome that spread worldwide in 2003 and the MERS coronavirus that spread and spread in Korea in 2015. known to have originated. Bats have the ability to fly among mammals, so they can live in a wide area, and due to the nature of a large number of individuals living in a group in a narrow space such as a cave, if one individual becomes infected with the virus, the infection is transmitted to the group and flight is not possible. It can spread the infection to other animals through widespread movement. In addition, bats, unlike other mammals, have a high body temperature, which makes them resistant to viruses, so bats, the coronavirus hosts, are not affected and can transmit persistent infections.

As described above, it has been confirmed that coronaviruses can spread between different species and that novel coronaviruses derived from bats can spread to humans and cause great problems. development is urgent. Currently, research on antiviral drugs is being actively conducted at home and abroad, and non-specific antiviral agents that have shown efficacy in clinical trials include type I interferon and ribavirin, but it has been reported that they have antiviral effects only at the initial stage of infection, and MERS. Coronavirus therapeutics and therapeutics for SARS coronavirus have recently attracted attention due to the growing need for development (A. Zumla et.al, Nature Reviews Drug Discovery, 15:327-347,2016). However, there are currently no approved therapeutic agents or vaccines at home and abroad, and there is an urgent need to develop a system for efficacy evaluation because an evaluation system for developing substances has not been established.

Corona-19 virus infection (COVID-19), a new coronavirus reported to have originated in Wuhan, China, in December 2019, has an incubation period of about 2 to 14 days after infection, followed by fever, coughing or shortness of breath. Respiratory symptoms and pneumonia are the main symptoms, but asymptomatic infections are rare. The World Health Organization (WHO) has declared a 'pandemic', the highest alert level for COVID-19, and as of 21:00 on April 11, 2020, 1,715,224 people in 215 countries around the world, and 103,827 deaths. It is a disease with a fatality rate of 6.05%. The genome of the Corona-19 virus is one of the RNA viruses. The envelope surrounds the protein capsid containing the genome, and on the surface, spike proteins in the form of membrane glycoproteins protrude like projections. Among them, the spike protein plays an important role in infecting the host with a virus by binding to the surface of a receptor that protrudes like a nail and penetrating into the host cell. The spike protein is functionally divided into an S1 subunit that binds to a receptor in the host cell and an S2 subunit that fuses to the membrane. domain) exists. The RBD region of the spike protein is very suitable as a target for the development of a human antibody with virus neutralizing ability to prevent the virus from infecting host cells. All human antibodies with the ability to neutralize were antibodies that bind to the RBD region of coronavirus.

Accordingly, a novel human antibody having virus neutralizing ability was discovered by binding to the RBD region of the Corona-19 virus using a high-speed screening system.

An object of the present invention is to provide an antibody or a fragment having immunological activity thereof for use in the detection of COVID-19 virus, prevention or treatment of COVID-19 virus infection.

In order to achieve the above object, the present invention provides an antibody or fragment having immunological activity specific for the Corona-19 virus.

In addition, the present invention provides an isolated nucleic acid molecule encoding an antibody or fragment having immunological activity specific for COVID-19 virus, a vector comprising the same, and a host cell transformed with the vector.

In addition, the present invention provides a method for producing an antibody or fragment having immunological activity specific to the Corona-19 virus.

In addition, the present invention provides a pharmaceutical composition for preventing or treating COVID-19 virus infection (novel coronavirus infection, COVID-19).

In addition, the present invention provides a composition and a detection kit for detecting the Corona-19 virus.

In addition, the present invention provides a method for detecting the Corona-19 virus.

In addition, the present invention provides a method for providing information on the diagnosis of COVID-19 virus infection.

According to the present invention, the human antibody and fragments having immunological activity that bind to the COVID-19 virus-RBD region, which are the cause of the COVID-19 disease, bind to the RBD region of the spike protein of the Corona-19 virus with high affinity to prevent COVID-19. Since it can effectively prevent the virus from infecting the host, it can be used for the prevention or treatment of COVID-19 virus infection, and immunodiagnosis (detection) is possible quickly using the antibody and its immunologically active fragments Therefore, it can be usefully used for the diagnosis and epidemiological investigation of the high-infectivity Corona-19 virus.

1 is a diagram illustrating the expression and purification of the dimeric Corona-19 virus-RBD region antigen protein expression vector and the tetrameric Corona-19 virus-RBD region antigen protein expression vector prepared in the present invention in animal cells and confirmed by SDS-PAGE gel. .
2 is a schematic diagram of antibody screening using a flow cytometer.
3 is a diagram illustrating an iterative screening process for amplifying human antibody clones showing specific binding to the Corona-19 virus-RBD region.
Figure 4 is a diagram showing the amino acid sequences of six kinds of scFv human antibody variants showing high binding affinity to the Corona-19 virus-RBD region.
5 is a diagram showing the amino acid sequences of four VH human antibody variants showing high binding affinity to the Corona-19 virus-RBD region.
6 is a diagram showing the results of analysis of the Corona-19 virus-RBD binding force through flow cytometry of six scFv antibody variants.
7 is a diagram showing the results of analysis of the Corona-19 virus-RBD binding force through flow cytometry of four VH antibody variants.
8 is a diagram illustrating the expression and purification of IgG-type antibodies of scFv variants JS 1, JS 2, JS 3, JS 4, JS 5 and JS 7 and confirmed by SDS-PAGE gel.
9 is a diagram confirming the binding affinity of the IgG-type antibodies of scFv variants JS 1, JS 2, JS 3 and JS 4 by ELISA.
10 is an expression vector for producing an IgG-type antibody prepared by introducing the VH mutant JS-VH 4 as a dimer/tetramer (top), and the expression and purification thereof, confirmed by SDS-PAGE gel (bottom).
11 is a diagram confirming the binding force of an IgG-type antibody prepared by introducing the VH variant JS-VH 4 as a dimer/tetramer by ELISA.
12 is a diagram showing the results of analysis of the Corona-19 virus-RBD binding force of five JS-VH 4-derived scFv mutants.
13 is a diagram showing the amino acid sequence analysis results of five JS-VH 4-derived scFv antibody variants.
14 is a JS-VH4-derived antibody mutant by substituting the mutations of four types (BM 13, BM 18, BM 57 and BM 73) in which amino acid mutations have occurred in the VH region of the JS-VH4-derived antibody variants with the sequence of JS-VH 4 (BM 13-1) , BM 18-1, BM 57-1 and BM 73-1) is a diagram comparing the binding force to the Corona-19 virus-RBD.

Hereinafter, the present invention will be described in detail by way of embodiments of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples for the present invention, and when it is determined that detailed descriptions of well-known techniques or configurations known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted, and , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of equivalents interpreted therefrom and the description of the claims to be described later.

In addition, the terms used in this specification are terms used to properly express preferred embodiments of the present invention, which may vary depending on the intention of a user or operator or customs in the field to which the present invention belongs. Accordingly, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

All technical terms used in the present invention, unless otherwise defined, have the meaning as commonly understood by one of ordinary skill in the art of the present invention. In addition, although preferred methods and samples are described herein, similar or equivalent ones are also included in the scope of the present invention. The contents of all publications herein incorporated by reference are incorporated herein by reference.

Throughout this specification, common one-letter and three-letter codes for naturally occurring amino acids are used as well as generally accepted for other amino acids such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), etc. A three-character code is used. Amino acids referred to by abbreviation in the present invention are also described according to the IUPAC-IUB nomenclature as follows:

Alanine: A, Arginine: R, Asparagine: N, Aspartic Acid: D, Cysteine: C, Glutamic Acid: E, Glutamine: Q, Glycine: G, Histidine: H, Isoleucine: I, Leucine: L, Lysine: K, Methionine : M, phenylalanine: F, proline: P, serine: S, threonine: T, tryptophan: W, tyrosine: Y and valine: V.

In one aspect, the present invention relates to an antibody or fragment having immunological activity specific for Corona-19 virus (SARS-CoV-2).

In one embodiment, it may be an Fc variant or a Fab variant of an antibody specific for COVID-19 virus.

In one embodiment, the fragment having immunological activity is Fab, Fd, Fab', dAb, F(ab'), F(ab') ₂ , single chain fragment variable (scFv), Fv, single chain antibody, Fv dimer It may be any one selected from the group consisting of a sieve, a complementarity determining region fragment, a humanized antibody, a chimeric antibody, and a diabody, more preferably scFv or Fab.

In one embodiment, the antibody may be a monoclonal antibody.

In one embodiment, the antibody may be an IgG.

Such antibodies are not only in the form of whole antibodies, but also include functional fragments of antibody molecules. 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. A functional fragment of an antibody molecule refers to a fragment having an antigen-binding function, and examples of antibody fragments include (i) a light chain variable region (VL), a heavy chain variable region (VH), and a light chain constant region (CL) and a Fab fragment consisting of the first constant region of the heavy chain (CH1); (ii) an Fd fragment consisting of the VH and CH1 domains; (iii) an Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of a VH domain (Ward ES et al., Nature 341:544-546 (1989)); (v) an isolated CDR region; (vi) a bivalent fragment comprising two linked Fab fragments F(ab')2 fragments: (vii) single chain Fv molecules (scFv) joined by a peptide linker joining the VH and VL domains to form an antigen binding site (viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix) diabody WO94/13804, which is a multivalent or multispecific fragment produced by gene fusion), and the like.

In one embodiment, the antibody or fragment having immunological activity specific for the Corona-19 virus of the present invention is CDRH (Complementarity determining regions Heavy) comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 1 to 7 chain) 1, a VH comprising a CDRH2 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 8 to 13, and a CDRH3 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 14 to 21 It can contain domains.

In one embodiment, the antibody or fragment having immunological activity specific for the Corona-19 virus of the present invention is FR1 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 45 to 48, SEQ ID NOs: 49 to FR2 comprising any one selected from the group consisting of the amino acid sequence of 53, FR3 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 54 to 58, and the amino acid sequence of SEQ ID NOs: 59 or 62 a VH domain comprising FR4.

In one embodiment, the antibody or fragment having immunological activity specific for the Corona-19 virus of the present invention is CDRL (Complementarity Determining Regions Light) comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 22 to 30. chain) 1, a CDRL2 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 31 to 36, and a CDRL3 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 37 to 44; It can contain domains.

In one embodiment, the antibody or fragment having immunological activity specific for the Corona-19 virus of the present invention is FR1 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 63 to 71, SEQ ID NOs: 72 to From the group consisting of FR2 containing any one selected from the group consisting of the amino acid sequence of 81, FR3 containing any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 82 to 90, and the amino acid sequence of SEQ ID NOs: 91 to 98 It may include a VL domain comprising FR4 comprising any one selected.

In one embodiment, the antibody or fragment having immunological activity specific for the Corona-19 virus of the present invention is (i) CDRH1 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 1 to 7, the sequence a VH domain comprising a CDRH2 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 8 to 13, and a CDRH3 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 14 to 21; and/or

(ii) Complementarity determining regions light chain (CDRL) 1 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 22 to 30, and any one selected from the group consisting of the amino acid sequences of SEQ ID NOs: 31 to 36 and a V domain comprising a VL domain comprising CDRL2 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 37 to 44.

In one embodiment, the antibody or fragment having immunological activity specific for the Corona-19 virus of the present invention comprises a VH domain comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 99 to 112; And it may include a VL domain comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 113 to 123.

In one embodiment, the antibody or fragment having immunological activity specific for the Corona-19 virus of the present invention is CL comprising the amino acid sequence of SEQ ID NO: 124, CH1 comprising the amino acid sequence of SEQ ID NO: 125, SEQ ID NO: 125 It may include a CH2 sequence comprising the amino acid sequence of SEQ ID NO: 125 or a CH3 sequence comprising the amino acid sequence of SEQ ID NO: 125.

In one embodiment, the antibody or fragment having immunological activity specific for the Corona-19 virus of the present invention is i) FR1 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 45 to 48, SEQ ID NO: CDRH1 comprising any one selected from the group consisting of the amino acid sequence of 1 to 7, FR2 comprising any one selected from the group consisting of SEQ ID NOs: 49 to 53 amino acid sequence, the group consisting of the amino acid sequence of SEQ ID NOs: 8 to 13 CDRH2 comprising any one selected from, FR3 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 54 to 58, comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 14 to 21 a VH domain comprising CDRH3 and FR4 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NO: 59 or 62; And / or ii) FR1 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 63 to 71, CDRL1 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 22 to 30, SEQ ID NO: 72 FR2 comprising any one selected from the group consisting of the amino acid sequence of to 81, CDRL2 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 31 to 36, the group consisting of the amino acid sequence of SEQ ID NOs: 82 to 90 FR3 comprising any one selected from, CDRL3 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 37 to 44, and any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 91 to 98 a V domain comprising a VL domain comprising FR4.

In one embodiment, the antibody or fragment having immunological activity specific for the Corona-19 virus of the present invention comprises scFv variants JS 1, JS 2, JS 3, JS 4, JS 5 and JS 7; VH variants JS-VH 4, JS-VH 9, JS-VH 19 and JS-VH 21; and BM 13, BM 18, BM 57 and BM 73, which are scFv variants derived from JS-VH 4, may be any one variant selected from the group consisting of.

The fragment having the immunological activity of the antibody specific for the Corona-19 virus of the present invention may be manufactured to be included as a dimer or tetramer when the antibody is in the form of an IgG.

In the present invention, the antibody or fragment having immunological activity thereof may be selected from the group consisting of an animal-derived antibody, a chimeric antibody, a humanized antibody, a human antibody, and a fragment having immunological activity thereof. The antibody may be recombinantly or synthetically produced.

An animal-derived antibody produced by immunizing an animal to be immunized with a desired antigen may cause an immune rejection reaction when administered to a human for therapeutic purposes. A chimeric antibody is one in which the constant region of an animal-derived antibody causing an anti-isotype reaction is substituted with that of a human antibody using a genetic engineering method. Chimeric antibodies have significantly improved anti-isotype response compared to animal-derived antibodies, but still have animal-derived amino acids in the variable region, potentially causing side effects on anti-idiotypic responses. are doing A humanized antibody was developed to improve these side effects. This is produced by grafting complementarity determining regions (CDRs), which play an important role in antigen binding, into a human antibody framework among variable regions of a chimeric antibody.

The most important thing in the CDR grafting technology for producing a humanized antibody is to select an optimized human antibody that can best accept the CDR region of an animal-derived antibody. structure analysis, molecular modeling technology, etc. are used. However, even when the CDR regions of an animal-derived antibody are transplanted into the optimized human antibody framework, there are cases where amino acids that affect antigen binding are present while being located in the framework of the animal-derived antibody. Therefore, it can be said that the application of additional antibody engineering technology to restore antigen binding force is essential.

The antibody or fragment having immunological activity thereof may be isolated from a living body (not present in the living body) or may be non-naturally occurring, for example, synthetically or recombinantly produced. can

As used herein, the term "antibody" refers to a substance produced by stimulation of an antigen in the immune system, the type is not particularly limited, and can be obtained naturally or non-naturally (eg, synthetically or recombinantly). can Antibodies are advantageous for mass expression and production because they are very stable and have a long half-life not only in vitro but also in vivo. In addition, since the antibody essentially has a dimer structure, the adhesion (avidity) is very high. A complete antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to a heavy chain by a disulfide bond. The constant region of an antibody is divided into a heavy chain constant region and a light chain constant region, and the heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) types.

As used herein, the term "heavy chain" refers to a variable region domain V _H and three constant region domains C _H1 , C _H2 and C _H3 comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen. and a full-length heavy chain including a hinge and a fragment thereof. In addition, the term "light chain" refers to both full-length light chains and fragments thereof comprising a variable region domain _VL and a constant region domain _CL comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. interpreted as including

As used herein, the term “variable region or variable domain” refers to a portion of an antibody molecule that exhibits many variations in sequence while performing a function of specifically binding to an antigen, and the variable region has complementarity There are determining regions CDR1, CDR2 and CDR3. Between the CDRs, a framework region (FR) portion exists to support the CDR ring. The "complementarity determining region" is a ring-shaped region involved in antigen recognition, and the specificity of the antibody for the antigen is determined as the sequence of this region changes.

As used herein, the term "scFv (single chain fragment variable)" refers to a single-chain antibody made by expressing only the variable region of an antibody through genetic recombination, and a single-chain form in which the VH region and the VL region of the antibody are connected by a short peptide chain. refers to the antibody of The term "scFv" is intended to include scFv fragments, including antigen-binding fragments, unless otherwise specified or understood otherwise by context. This is obvious to those skilled in the art.

As used herein, the term "complementarity determining region (CDR)" refers to the amino acid sequence of the hypervariable region of the heavy chain and light chain of an immunoglobulin. The heavy and light chains may each comprise three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs may provide key contact residues for the binding of an antibody to an antigen or epitope.

In the present invention, the terms "specifically binding" or "specifically recognized" have the same meaning as commonly known to those skilled in the art, and mean that an antigen and an antibody specifically interact to undergo an immunological reaction. .

As used herein, the term "antigen-binding fragment" refers to a fragment of the entire immunoglobulin structure, and refers to a portion of a polypeptide including an antigen-binding portion. For example, it may be scFv, (scFv) 2 , scFv-Fc, Fab, Fab' or F(ab') 2 , but is not limited thereto. Among the antigen-binding fragments, Fab has a structure having variable regions of light and heavy chains, constant regions of light chain and first constant region of heavy chain (C _H1 ), 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 C _H1 domain. The F(ab') 2 antibody is produced by forming a disulfide bond with a cysteine residue in the hinge region of Fab'. Fv is a minimal antibody fragment having only a heavy chain variable region and a light chain variable region, and a recombinant technique for generating an Fv fragment is well known in the art. In a double-chain Fv (two-chain Fv), the heavy chain variable region and the light chain variable region are connected by a non-covalent bond, and in single-chain Fv (single-chain Fv), the heavy chain variable region and the single chain variable region are generally shared through a peptide linker. They are linked by a bond or are linked directly at the C-terminus to form a dimer-like structure like a double-stranded Fv. The linker may be a peptide linker consisting of any amino acids from 1 to 100 or 2 to 50 amino acids, and an appropriate sequence is known in the art. The antigen-binding fragment can be obtained using a proteolytic enzyme (for example, Fab can be obtained by restriction digestion of the entire antibody with papain, and F(ab') 2 fragment can be obtained by digestion with pepsin), It can be produced through genetic recombination technology.

In the present invention, the term "hinge region" is a region included in the heavy chain of an antibody, which exists between the C _H1 and C _H2 regions, and functions to provide flexibility of the antigen-binding site in the antibody. means area. For example, the hinge may be derived from a human antibody, and specifically, may be derived from IgA, IgE, or IgG, such as IgG1, IgG2, IgG3, or IgG4.

In one aspect, the present invention relates to an isolated nucleic acid molecule encoding an antibody of the invention or an immunologically active fragment thereof, a vector comprising the same, and a host cell transformed with the vector.

The nucleic acid molecules of the present invention may be isolated or recombinant, and include single-stranded and double-stranded forms of DNA and RNA as well as corresponding complementary sequences. An isolated nucleic acid is a nucleic acid that has been separated from surrounding genetic sequences present in the genome of the individual from which the nucleic acid was isolated, in the case of a nucleic acid isolated from a naturally occurring source. In the case of a nucleic acid synthesized enzymatically or chemically from a template, such as a PCR product, a cDNA molecule, or an oligonucleotide, a nucleic acid resulting from such a procedure can be understood as an isolated nucleic acid molecule. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of separate fragments or as a component of a larger nucleic acid construct. Nucleic acids are operably linked when placed into a functional relationship with another nucleic acid sequence. For example, the DNA of the presequence or secretion leader is operably linked to the DNA of the polypeptide when it is expressed as a preprotein in the form before the polypeptide is secreted, and the promoter or enhancer is the polypeptide sequence is operably linked to a coding sequence when it affects the transcription of, or a ribosome binding site is operably linked to a coding sequence when positioned to facilitate translation. In general, operably linked means that the DNA sequences to be linked are located contiguously, and in the case of a secretory leader it is contiguous and in the same reading frame. However, enhancers need not be located adjacent to each other. Linkage is achieved by ligation at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to conventional methods.

An isolated nucleic acid molecule encoding an antibody or immunologically active fragment thereof of the present invention is expressed from a coding region due to codon degeneracy or in consideration of codons preferred in the organism in which the antibody is to be expressed. Various modifications may be made to the coding region within the range that does not change the amino acid sequence of the antibody, and various modifications or modifications may be made within the range that does not affect the expression of the gene even in parts other than the coding region, such modifications It will be well understood by those skilled in the art that genes are also included within the scope of the present invention. That is, as long as the nucleic acid molecule of the present invention encodes a protein having an equivalent activity, one or more nucleic acid bases may be mutated by substitution, deletion, insertion, or a combination thereof, and these are also included in the scope of the present invention. The sequence of such a nucleic acid molecule may be single-stranded or double-stranded, and may be a DNA molecule or an RNA (mRNA) molecule.

An isolated nucleic acid molecule encoding an antibody of the present invention or an immunologically active fragment thereof according to the present invention can be inserted into an expression vector for protein expression. Expression vectors usually comprise a protein operably linked, ie, placed in a functional relationship, with regulatory or regulatory sequences, selectable markers, optional fusion partners, and/or additional elements. In appropriate conditions, by culturing a host cell transformed with a nucleic acid, preferably an expression vector containing an isolated nucleic acid molecule encoding an antibody according to the present invention or an immunologically active fragment thereof, to induce protein expression by a method Antibodies or fragments having immunological activity thereof according to the present invention can be produced. A variety of suitable host cells can be used, including, but not limited to, mammalian cells, bacteria, insect cells, and yeast. Methods for introducing an exogenous nucleic acid into a host cell are known in the art and will vary depending on the host cell used. Preferably, E. coli, which has a high industrial use value due to low production cost, can be produced as a host cell.

The vector of the present invention includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector and a viral vector. Suitable vectors include a signal sequence or leader sequence for membrane targeting or secretion in addition to expression control elements such as promoter, operator, start codon, stop codon, polyadenylation signal and enhancer, and may be prepared in various ways depending on the purpose. The promoter of the vector may be constitutive or inducible. The signal sequence includes a PhoA signal sequence and an OmpA signal sequence when the host is Escherichia sp., and an α-amylase signal sequence and a subtilisin signal when the host is a Bacillus sp. When the host is yeast, the MFα signal sequence, SUC2 signal sequence, etc., and when the host is an animal cell, an insulin signal sequence, α-interferon signal sequence, antibody molecule signal sequence, etc. can be used, It is not limited thereto. The vector may also include a selection marker for selecting a host cell containing the vector, and in the case of a replicable expression vector, an origin of replication.

As used herein, the term "vector" refers to a carrier capable of inserting a nucleic acid sequence for introduction into a cell capable of replicating the nucleic acid sequence. Nucleic acid sequences may be exogenous or heterologous. Vectors include, but are not limited to, plasmids, cosmids, and viruses (eg, bacteriophages). One skilled in the art can construct vectors by standard recombinant techniques (Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1988; and Ausubel et al., In: Current Protocols in Molecular Biology, John, Wiley & Sons, Inc, NY, 1994 et al.).

In one embodiment, when constructing the vector, expression control sequences such as promoter, terminator, enhancer, etc., membrane targeting or secretion according to the type of host cell to produce the antibody. Sequences and the like can be appropriately selected and combined in various ways depending on the purpose.

As used herein, the term "expression vector" refers to a vector comprising a nucleic acid sequence encoding at least a portion of a transcribed gene product. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. The expression vector may include various regulatory sequences. In addition to regulatory sequences that control transcription and translation, vectors and expression vectors may contain nucleic acid sequences that also serve other functions.

In the present invention, the term "host cell" includes eukaryotes and prokaryotes, and refers to any transformable organism capable of replicating the vector or expressing a gene encoded by the vector. A host cell may be transfected or transformed by the vector, which refers to a process in which an exogenous nucleic acid molecule is delivered or introduced into a host cell.

In one embodiment, the host cell may be a bacterium or an animal cell, the animal cell line may be a CHO cell, a HEK cell or an NSO cell, and the bacterium may be E. coli.

In one aspect, the invention provides a method comprising: culturing a host cell comprising a vector comprising the isolated nucleic acid molecule of the invention; And it relates to a method for producing an antibody or a fragment having an immunological activity specific to the Corona-19 virus, comprising the step of recovering the antibody or a fragment having an immunological activity thereof from a host cell culture.

In one embodiment, after recovering the antibody or the immunologically active fragment from the host cell culture, the method may further comprise purifying the antibody or the immunologically active fragment.

The antibody or fragment having immunological activity of the present invention may be isolated or purified by various methods known in the art. Standard purification methods include chromatography techniques, electrophoresis, immunization, sedimentation, dialysis, filtration, concentration, and chromatofocusing techniques. As is known in the art, various native proteins, such as, for example, bacterial proteins A, G, and L, bind antibodies and these proteins can be used for purification. Often, purification by specific fusion partners may be possible.

After transforming the vector according to the present invention into an appropriate host cell, for example, E. coli or yeast cell, and then culturing the transformed host cell, the antibody according to the present invention or a fragment having immunological activity thereof can be mass-produced. have. Appropriate culture methods and medium conditions according to the type of host cells can be easily selected by those skilled in the art from known techniques known to those skilled in the art. The host cell may be a prokaryotic organism such as E. coli or Bacillus subtilis . It may also be a eukaryotic cell derived from yeast, such as Saccharomyces cerevisiae , an insect cell, a plant cell, or an animal cell. More preferably, the animal cells may be autologous or allogeneic animal cells. The transformant prepared by introducing autologous or allogeneic animal cells can be administered to an individual and used for cell therapy to treat cancer. Any method known to those skilled in the art may be used for the vector introduction method into the host cell. Other organisms, including transgenic (eg, genetically engineered) mice, or other mammals, can be used to produce antibodies of the invention or fragments having immunological activity thereof (see, eg, US 6,300,129). For example, mice engineered by replacing only the variable regions (heavy chain V, D, and J segments, and light chain V and J segments) of a mouse immune gene with the corresponding human variable sequences can generate high-affinity antibodies with human variable sequences. It is known that it can be used for mass production (see, for example, US Pat. No. 6,586,251; US Pat. No. 6,596,541, and US Pat. No. 7,105,348).

In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating COVID-19 virus infection (novel coronavirus infection, COVID-19) containing an antibody or fragment having immunological activity specific to the Corona-19 virus. it's about

In the present invention, the term "prevention" refers to any action that suppresses or delays the occurrence, spread, and recurrence of COVID-19 virus infection by administration of the composition according to the present invention.

As used herein, the term "treatment" refers to any action that improves or advantageously changes the symptoms of COVID-19 virus infection and its complications by administration of the composition according to the present invention. Those of ordinary skill in the art to which the present invention pertains, with reference to the data presented by the Korean Medical Association, etc., know the exact standard of the disease for which the composition of the present application is effective, and can determine the degree of improvement, improvement and treatment will be.

The term "therapeutically effective amount" used in combination with an active ingredient in the present invention means an amount effective to prevent or treat COVID-19 virus infection, and the therapeutically effective amount of the composition of the present invention includes several factors, such as For example, it may vary depending on the method of administration, the target site, and the condition of the patient. Therefore, when used in the human body, the dosage should be determined as an appropriate amount in consideration of both safety and efficiency. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. These considerations in determining effective amounts are, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

The pharmaceutical 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 not to cause side effects, and the effective dose level is determined by the patient's Health status, severity of COVID-19 virus infection, drug activity, drug sensitivity, administration method, administration time, administration route and excretion rate, treatment period, factors including drugs used in combination or concurrently, and other medical fields It may be determined according to known factors. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. 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, which can be easily determined by those skilled in the art.

The pharmaceutical composition of the present invention may include a carrier, diluent, excipient, or a combination of two or more commonly used in biological agents. As used herein, the term “pharmaceutically acceptable” refers to exhibiting properties that are not toxic to cells or humans exposed to the composition. The carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. Compounds described in , saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components can be mixed and used, and if necessary, other antioxidants, buffers, bacteriostats, etc. Conventional additives may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable dosage form such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets. Furthermore, it can be preferably formulated according to each disease or component using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group including oral formulations, topical formulations, suppositories, sterile injection solutions and sprays, and oral or injection formulations are more preferred.

As used herein, the term "administration" means providing a predetermined substance to a subject or patient by any suitable method, and parenteral administration (eg, intravenous, subcutaneous, intraperitoneal) according to a desired method. Alternatively, it can be applied locally as an injection formulation) or orally administered, and the dosage varies according to the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease. Liquid formulations for oral administration of the composition of the present invention include suspensions, internal solutions, emulsions, syrups, etc., and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. and the like may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. The pharmaceutical composition of the present invention may be administered by any device capable of transporting an active substance to a target cell. Preferred administration methods and formulations are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, and the like. For injections, aqueous solvents such as physiological saline solution and Ringer's solution, vegetable oil, higher fatty acid esters (eg, ethyl oleate), and non-aqueous solvents such as alcohols (eg, ethanol, benzyl alcohol, propylene glycol, glycerin, etc.) Stabilizers for preventing deterioration (e.g., ascorbic acid, sodium hydrogen sulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH control, to inhibit microbial growth and a pharmaceutical carrier such as a preservative (eg, phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

As used herein, the term "individual" refers to monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, It refers to any animal, including bat, camel, rat, rabbit or guinea pig, and "sample" may be droplet, sputum, whole blood, plasma, serum, urine or saliva isolated therefrom.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additive includes starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate. , lactose, mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, Calcium stearate, sucrose, dextrose, sorbitol and talc and the like may be used. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

In one aspect, the present invention relates to a composition for detecting Corona-19 virus, comprising an antibody specific for the Corona-19 virus of the present invention or a fragment having immunological activity thereof.

Preferably, the antibody or fragment having immunological activity thereof of the present invention used in the detection (diagnostic) composition is detectably labeled. The various methods available for labeling biomolecules are well known to those skilled in the art and are contemplated within the scope of the present invention. The methods are described in Tijssen, 'Practice and theory of enzyme immunoassays', Burden, RHand von Knippenburg (Eds), Volume 15 (1985), 'Basic methods in molecular biology'; Davis LG, Dibmer MD; Battey Elsevier (1990), Mayer et al., (Eds) 'Immunochemical methods in cell and molecular biology' Academic Press, London (1987), or in the series 'Methods in Enzymology', Academic Press, Inc.

There are labeling methods and many other labels known to those skilled in the art. Examples of the types of labels that can be used in the present invention include enzymes, radioactive isotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds and bioluminescent compounds.

Commonly used labels include fluorescent substances (eg, fluorescein, rhodamine, Texas red, etc.), enzymes (eg, horseradish peroxidase, β-galactosidase, alkaline phosphatase), radioactive isotopes (eg, ³² P or ¹²⁵ I), biotin, digoxigenin, colloidal metals, chemiluminescent or bioluminescent compounds (eg dioxetane, luminol or acridinium). Labeling methods such as covalent bonding of enzymes or biotinyl groups, iodination, phosphorylation, and biotinylation are well known in the art.

Detection methods include, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzymatic reactions, and the like. Commonly used detection assays include radioactive isotope or non-radioactive isotope methods. These include, among others, Western blotting, overlay-assay, RIA (Radioimmuno Assay) and IRMA (Immune Radioimmunometric Assay), EIA (Enzyme Immuno Assay), ELISA (Enzyme Linked Immuno Sorbent Assay), FIA (Fluorescent Immuno Assay) and CLIA (Chemioluminescent Immune). Assay).

In one aspect, the present invention relates to a corona-19 virus detection kit comprising the composition for detecting the corona-19 virus of the present invention.

In one embodiment, the kit may include the composition for detecting COVID-19 virus according to the present invention and a reagent for detecting the antigen-antibody complex. The reagent for detecting the antigen-antibody complex includes a reagent for radial immunoassay, ELISA (Enzyme linked immunosorbent assay), or immunofluorescence analysis.

In one embodiment, antigen-antibody complex detection is immunoelectrophoresis such as Ouchterlony plate, Western blot, Crossed IE, Rocket IE, Fused Rocket IE, Affinity IE, which can simply detect the antibody and/or antigen through antigen-antibody binding. (Immuno Electrophoresis) can be achieved. The reagents or substances used in these methods are known and include, for example, antigen-antibody reactions, substrates that specifically bind antigen, nucleic acid or peptide aptamers, receptors that interact with complexes or reactions with ligands or cofactors. can be detected through, or a mass spectrometer can be used. The reagent or material that specifically interacts with or binds to the antigen-antibody complex of the present application may be used in a chip method or in combination with nanoparticles. The immunoassay or immunostaining method is described in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J. M. ed., Humana Press, NJ, 1984, et al. By analyzing the intensity of the final signal by the above-described immunoassay process, that is, by performing signal comparison with a normal sample, it is possible to diagnose whether or not the corona-19 virus is infected by detecting the corona-19 virus in the subject.

In one aspect, the present invention provides a method comprising: contacting a sample isolated from a specimen with an antibody specific for the Corona-19 virus of the present invention or a fragment having immunological activity thereof; and detecting antigen-antibody complex formation.

In one aspect, the present invention comprises the steps of contacting a sample isolated from a specimen with an antibody specific for the Corona-19 virus of the present invention or a fragment having immunological activity thereof to form an antigen-antibody complex; And it relates to a method for providing information on the diagnosis of COVID-19 virus infection, comprising the step of detecting the formation of the complex.

The present invention will be described in more detail through the following examples. However, the following examples are only intended to embody the contents of the present invention, and the present invention is not limited thereto.

Example 1. Corona-19 virus RBD (Receptor Binding Domain) antigen production

In order to make an antibody that binds to the Receptor Binding Domain (RBD) region, which is known to be important for the virus to infect the host by binding to the surface of the ACE2 (Angiotensin converting enzyme 2) receptor present in the host cell, the RBD region (sequence No. 126) was prepared as an antigen. In addition, in order to prepare an antigen more suitable for antibody screening using a flow cytometer, a dimeric antigen fused with a GST tag to the antigen and a tetramer fused with a Streptavidin tag ( A vector for expression of animal cells to produce an antigen in the form of tetrameric) was prepared, respectively (FIG. 1). Freestyle 293 expression culture medium (Gibco, 12338-018) 30 ml+ After mixing each vector, PEI (Polyethylenimine) (Polyscience, 23966) and a solution of each vector in a 4:1 ratio were left at room temperature for 20 minutes, and the previous day, 2x10 ⁶ cells/ml Expi293F animal cells subcultured at a density of 300 ml were treated and transfected, and then incubated for 7 days at 37° C., 125 rpm and 8% CO ₂ in a shaking CO ₂ incubator, followed by centrifugation to take only the supernatant. After that, the equilibrium (equilibrium) was adjusted using 25x PBS. Thereafter, it was filtered with a 0.2 μm filter (Merck Millipore) using a bottle top filter, and 1 ml of GSH resin and Ni-NTA resin was added to the filtered culture solution, respectively, and stirred at 4° C. for 16 hours. Thereafter, the resin was recovered through the column, washed with 5ml PBS, and eluted with 4ml of 50mM Tris-HCl+10mM reduced glutation (SIGMA) at pH8.0 and 250mM imidazole. After buffer change with PBS using Centrifugal filter units 3K (Merck Millipore), two antigenic proteins (Corona of SEQ ID NO: 127) each purified under non-reducing conditions (NR) and reducing conditions (R) through SDS-PAGE The size and purity of -19-RBD-GST and Corona-19-RBD-Streptavidin of SEQ ID NO: 128 were analyzed (FIG. 1).

Example 2. Screening of human antibodies using dimeric form of COVID-19 virus-RBD

In order to use the antigen protein fused with GST among the antigen proteins prepared in Example above for screening using a flow cytometer, Alexa488 fluorescent molecule was labeled. Thereafter, a screening of human antibodies binding to the Corona-19 virus-RBD region was performed using the bacterial-displayed human scFv antibody library constructed by the present inventors. Specifically, 1 ml of library cells were cultured in 25 ml TB medium containing 2% glucose and 40 μg/ml of chloramphenicol at 37° C. and 250 rpm for 4 hours, and the cultured cells were cultured in 100 ml of 40 μg/ml of chloramphenicol. After incubation in TB medium at a ratio of 1:100 to OD600 = 0.5, and then cooled at 25°C and 250 rpm for 20 minutes, 1 mM IPTG was added to induction for 5 hours at 25°C and 250 rpm conditions, and E. coli overexpressed with protein was harvested based on OD600=8. Thereafter, the cells were resuspended using 1 ml of 10 Mm Tris-HCl (pH 8.0) to make spheroplasts suitable for screening using a flow cytometer, washed twice to remove residual medium, In 1 ml of STE [0.5 M sucrose, 10 Mm Tris-HCl, 10 Mm EDTA (pH 8.0)], the outer membrane was removed by rotation at 37° C. for 30 minutes to give an osmotic shock. After that, the peptidoglycan layer was removed by rotation in a solution of 1ml of Solution A and 20 μl of 50mg/ml lysozyme solution at 37°C for 15 minutes and washed with 1ml of PBS. In 300 μl of medium, 700 μl of PBS and 200 nM (monomer basis) of Corona-19-RBD-GST-Alexa488 probe were added and rotated at room temperature for 1 hour to label the spheroplast with the fluorescent probe. Thereafter, clones showing high fluorescence due to increased antigen binding strength were recovered using a flow cytometer (FIG. 2), and scFv genes were amplified from the recovered clones by PCR, cultured, expression induction, extracellular membrane and peptidoglycan. Selecting spheroplasts showing high affinity for the Corona 19 virus-RBD region through the spheroplasting process to remove the compartment layer, antigen labeling, and selective gating of flow cytometry The process was repeated ( FIG. 3 ). After the selection process using a flow cytometer and the reselection process to increase the selection purity, the desired clones are amplified through the enrichment process, the process of securing the selected scFv variant gene through PCR amplification, the subcloning process, and the transformation process. The next round of selection was performed to enrich. ScFv mutant clones with excellent binding affinity to the Corona-19 virus-RBD region were obtained through a search process of several repeated rounds as described above.

Example 3. Confirmation of gene sequence of COVID-19 binding antibody variant

In order to confirm the gene sequence of the scFv mutant clones obtained in Example 2, the DNA base sequence was analyzed using Sanger sequencing. Six scFv antibody variants with antibody sequences were selected through analysis. In this process, only the VH of the scFv was additionally selected four VH antibody variants with increased binding affinity to the Corona-19 virus-RBD region. In addition, the VH amino acid sequences (SEQ ID NOs: 99-104) and VL amino acid sequences (SEQ ID NOs: 113-118) of six scFv variants (FIG. 4) and the VH amino acid sequences of four VH variants (SEQ ID NOs: 105-108) (FIG. 5) was confirmed.

Example 4. Confirmation of binding affinity to the Corona-19 virus-RBD region of antibody variants

In order to confirm the binding affinity of the six scFv antibody variants and four VH antibody variants selected in Example 3 to the Corona-19 virus-RBD region, avidity analysis using a flow cytometer was performed. To this end, each mutant and Fc (wild type) to be used as a control were prepared as spheroplasts in the manner performed in the above Examples. Corona-19 RBD-Alexa 488 antigen used for screening was bound to the prepared spheroplasts at a concentration of 50 nM and incubated for 1 hour at room temperature. After washing twice with PBS to remove non-specific binding, the binding ability of the antibody variants to the Corona-19 virus-RBD region was analyzed using a flow cytometer. Here, as a positive control, the CR3022 antibody developed as a SARS RBD-binding antibody by Crucell (merged with Johnson & Johnson) was used (recently known to show excellent binding to SARS-CoV-2 RBD) (References: US Patent US8,106,176 B2, Tian et al BioRxiv 2020, Joyce et al BioRxiv 2020).

As a result, all of the six scFv variants and four VH variants exhibited significantly increased fluorescence signals compared to the control Fc (wild-type) ( FIGS. 6 and 7 ), and through this, the scFv antibody variants of the present invention and It was confirmed that the VH antibody variants had a high affinity for binding to the COVID-19 virus-RBD region.

Example 5. IgG form antibody production and purification of 6 scFv variants

After producing the six scFv antibody variants selected in the above-mentioned silsing in the form of IgG, the binding ability with Corona-19 virus-RBD was confirmed. To this end, each of the six scFv heavy and light chain expression vectors was prepared, the heavy and light chain genes of the variants were mixed in a ratio of 1:1, and PEI and the mutant gene were mixed in a ratio of 4:1 and used in Expi293F animal cells. transfected. Thereafter, in a CO ₂ stirred incubator, 37° C., 125 rpm, and 8% CO ₂ conditions were cultured for 7 days and centrifuged to take only the supernatant. Equilibrated with 25xPBS and filtered through a 0.2 μm filter (Merck Millipore). 500 μl of Protein A resin was added to the filtered culture solution, stirred at 4° C. for 16 hours, and then passed through a column to recover the resin. The recovered resin was washed with 5 ml PBS, eluted with 3 ml 100 mM glycine buffer (pH 2.7), and neutralized with 1M Tris-HCl (pH 8.0). After changing the buffer to PBS using centrifugal filter units 3K (Merck Millipore), the size and purity of each of the six purified antibodies were analyzed by SDS-PAGE (FIG. 8).

Example 6. Confirmation of binding affinity to the Corona-19 virus-RBD region of the IgG-type antibody of the scFv variant

ELISA experiments were performed on the antibodies of JS1, JS2, JS3, and JS4 among the six types of antibodies that were expressed and purified in Example 5. Specifically, a dimeric RBD fused with a monomeric RBD and a GST tag or a tetrameric RBD fused with a streptavidin tag were mixed with 50 μl each of different Flat Bottom Polystyrene High Bind 96 wells. Plate (costar) in 0.05 M Na ₂ CO ₃ pH 9.6 at 4 μg/ml at 4 ° C., after immobilization for 16 hours, 100 μl of 4% skim milk (GenomicBase) (in 0.05% PBST pH 6.0/pH 7.4) Blocking was carried out at room temperature for 2 hours. Thereafter, four JS 1, JS 2, JS 3 and JS 4 antibodies were washed 4 times with 180 μl of 0.05% PBST and serially diluted with 1% skim milk (in 0.05% PBST) and Trastuzumab as a control. was dispensed into each well of 50 μl and reacted at room temperature for 1 hour. After the washing process, 50 μl of anti-human HRP conjugate (Jackson Immunoresearch) was reacted with each antibody at room temperature for 1 hour and washed. After 50 μl of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added to develop color, 50 μl of 2 MH ₂ SO ₄ was added to terminate the reaction and analyzed using an Epoch Microplate Spectrophotometer (BioTek). As a result, it was confirmed that all of the IgG antibodies of JS1, JS2, JS3 and JS4 had stronger binding to the Corona-19 virus-RBD region than the control, Trastuzumab (FIG. 9).

Example 7. VH variants IgG type antibody production and purification

As a result of the flow cytometry analysis of Example 4 among the four VH antibody variants selected in Example 3, the JS-VH 4 mutant, which showed the highest binding affinity to the Corona-19 virus-RBD, was also found in the form of IgG, In order to confirm whether it has strong binding to RBD, expression vectors in which JS-VH 4 variants are introduced as dimeric and tetrameric, respectively, in the constant region of the antibody, Fc (Crystallization Fragment), were prepared, respectively, and Expi293F animals The cells were transfected. Thereafter, in a CO ₂ stirred incubator, 37° C., 125 rpm, and 8% CO ₂ conditions were cultured for 7 days and centrifuged to take only the supernatant. Equilibrated with 25xPBS and filtered through a 0.2 μm filter (Merck Millipore). 500 μl of Protein A resin was added to the filtered culture solution, stirred at 4° C. for 16 hours, and then passed through a column to recover the resin. The recovered resin was washed with 5 ml PBS, eluted with 3 ml 100 mM glycine buffer (pH 2.7), and neutralized with 1M Tris-HCl (pH 8.0). After changing the buffer to PBS using centrifugal filter units 3K (Merck Millipore), the size and purity of the two purified antibodies (dimer and tetramer) were analyzed through SDS-PAGE ( FIG. 10 ).

Example 8. VH variants Confirmation of binding ability of IgG-type antibody to Corona-19 virus-RBD region

An ELISA experiment was performed on the antibody of the dimeric or tetrameric JS-VH 4 mutant, which was expressed and purified in Example 7 above. Specifically, a dimeric RBD fused with a monomeric RBD and a GST tag or a tetrameric RBD fused with a streptavidin tag were mixed with 50 μl each of different Flat Bottom Polystyrene High Bind 96 wells. Plate (costar) in 0.05 M Na ₂ CO ₃ pH 9.6 at 4 μg/ml at 4 ° C., after immobilization for 16 hours, 100 μl of 4% skim milk (GenomicBase) (in 0.05% PBST pH 6.0/pH 7.4) Blocking was carried out at room temperature for 2 hours. Thereafter, two JS-VH 4 antibodies (dimeric JS_VH 4_Fc and tetrameric JS_VH 4_Fc) were washed 4 times with 180 μl of 0.05% PBST and serially diluted with 1% skim milk (in 0.05% PBST). As a control, 50 μl of Trastuzumab and JS 3 antibody were dispensed into each well and reacted at room temperature for 1 hour. After the washing process, 50 μl of Protein HRP conjugate (GenScript) was reacted with each antibody at room temperature for 1 hour and washed. After 50 μl of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added to develop color, 50 μl of 2 MH ₂ SO ₄ was added to terminate the reaction and analyzed using an Epoch Microplate Spectrophotometer (BioTek). As a result, it was confirmed that both the dimeric JS_VH 4_Fc and the tetrameric JS_VH 4_Fc (IgG antibody) had significantly stronger binding to the Corona-19 virus-RBD region than the control, Trastuzumab, and, in particular, showed a higher binding force than JS 3 (FIG. 11) .

Example 9. Screening of antibodies derived from JS-VH 4 that bind to the COVID-19 RBD region

Bacterial-displayed human scFv constructed by the present inventors to discover JS-VH 4 that specifically binds to COVID-19 RBD and discover JS-VH 4 derived antibodies having binding affinity to Corona-19 RBD A VL shuffling library derived from JS-VH 4 in which JS-VH 4 was collectively introduced into the VH region of the antibody library was prepared. In addition, screening of human antibodies that bind to Corona-19 RBD using the corona-19-RBD-GST-Alexa488 probe, an antigen used for primary antibody discovery, and the VL shuffling library derived from the bacteria-displayed JS-VH 4 prepared above. proceeded. Specifically, 1 ml of library cells were cultured in 25 ml TB medium containing 2% glucose and 40 μg/ml of chloramphenicol at 37° C. and 250 rpm for 4 hours, and the cultured cells were cultured in 100 ml of 40 μg/ml of chloramphenicol. After incubation in TB medium at a ratio of 1:100 to OD600=0.5, and then cooled at 25°C and 250 rpm for 20 minutes, 1 mM IPTG was added to induction for 5 hours at 25°C and 250 rpm conditions and protein This overexpressed E. coli was harvested based on OD600=8. Thereafter, the cells were resuspended using 1 ml of 10 Mm Tris-HCl (pH 8.0) to make spheroplasts suitable for screening using a flow cytometer, washed twice to remove residual medium, In 1 ml of STE [0.5 M sucrose, 10 Mm Tris-HCl, 10 Mm EDTA (pH 8.0)], the outer membrane was removed by rotation at 37° C. for 30 minutes to give an osmotic shock. After that, the peptidoglycan layer was removed by rotation in a solution of 1ml of Solution A and 20 μl of 50mg/ml lysozyme solution at 37°C for 15 minutes and washed with 1ml of PBS, Of these, 700 μl of PBS and 200 nM (monomer basis) of Corona-19-RBD-GST-Alexa488 probe were added to 300 μl, and the spheroplast was labeled with the fluorescent probe by rotation at room temperature for 1 hour. Thereafter, clones showing high fluorescence due to increased antigen binding strength were recovered using a flow cytometer (FIG. 2), and scFv genes were amplified from the recovered clones by PCR, cultured, expression induction, extracellular membrane and peptidoglycan. Selecting spheroplasts showing high affinity for the Corona 19 virus-RBD region through the spheroplasting process to remove the compartment layer, antigen labeling, and selective gating of flow cytometry The process was repeated ( FIG. 3 ). After the selection process using a flow cytometer and the reselection process to increase the selection purity, the desired clones are amplified through the enrichment process, the process of securing the selected scFv variant gene through PCR amplification, the subcloning process, and the transformation process. The next round of selection was performed to enrich. Five kinds of scFv mutant clones (BM 13, BM 18, BM 23, BM 57 and BM 73) with improved binding affinity to the Corona-19 virus-RBD region were obtained through this repeated several rounds of screening process. .

Example 10. Confirmation of JS-VH 4-derived scFv variants for Corona-19 virus-RBD region binding affinity

In order to confirm the binding affinity of the JS-VH 4 derived scFv mutants obtained in Example 9 to the Corona-19 virus-RBD region, avidity analysis using a flow cytometer was performed. To this end, each mutant and JS-VH 4 to be used as a control were prepared as spheroplasts in the manner performed in the above Examples. 25 Corona 19 RBD-Alexa 488 antigen used for screening on the produced spheroplasts It was bound to a concentration of nM and incubated for 1 hour at room temperature. After washing twice with PBS to remove non-specific binding, the binding ability of each scFv variant to the Corona-19 virus-RBD region was analyzed using a flow cytometer. As a result of the analysis, it was confirmed that five kinds of scFv mutants exhibited fluorescence signals similar to those of JS-VH 4 ( FIG. 12 ).

Example 11. Gene sequence identification of scFv variants derived from JS-VH 4

To confirm the gene sequences of the five scFv mutant clones obtained in Example 9, DNA nucleotide sequences were analyzed using Sanger sequencing. Of the five variants, four scFv variants except BM 23 were screened using the VL shuffling library derived from JS-VH 4 to which JS-VH 4 was introduced, but amino acid substitutions were also made in the VH region during PCR amplification. It was confirmed that this occurred (FIG. 13). In addition, the VH amino acid sequences (SEQ ID NOs: 109 to 112) of four JS-VH 4 derived scFv variants (BM 13, BM 18, BM 57 and BM 73) and five JS-VH 4 derived scFv variants ( VL amino acid sequences (SEQ ID NOs: 119 to 123) of BM 13, BM 18, BM23, BM 57 and BM 73) were identified.

Example 12. Confirmation of increase in Corona-19 virus-RBD region binding force by substitution of VH region amino acids

As a result of confirming the sequence in Example 11, the amino acid substitution mutations of the four scFv variants (BM 13, BM 18, BM 57, and BM 73) in which amino acid substitutions occurred in the VH region were substituted again with JS-VH 4 to the VH region. It was confirmed how the newly introduced amino acid had an effect on the binding of the Corona-19 virus-RBD. As a result, when all four variants were substituted again with JS-VH 4, it was shown that the binding to the Corona-19 virus-RBD was reduced (FIG. 14), and the amino acids of the VH region newly discovered through this It was confirmed that the binding force to -RBD was improved.

<110> Korea University Research and Business Foundation <120> HUMAN ANTIBODIES TARGETING SARS-CoV-2 <130> DP-2020-0213 <160> 128 <170> KoPatentIn 3.0 <210> 1 <211> 8 <212> PRT < 213> Artificial Sequence <220> <223> CDRH1 (JS 1, JS 2, JS 4) <400> 1 Gly Phe Ala Ile Gly Asp Asp Asn Ala 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDRH1 (JS 3) <400> 2 Gly Phe Ala Ile Gly Gly Asn Ala 1 5 <210> 3 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDRH1 (JS 5) <400> 3 Gly Asp Ser Ile Ser Gly Asp Tyr 1 5 <210> 4 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDRH1 (JS 7) <400> 4 Gly Tyr Ala Leu Thr His Tyr Gly 1 5 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDRH1 (JS_VH 4, JS_VH 19, JS_VH 21, BM 13, BM 18 , BM 73) <400> 5 Gly Phe Thr Phe Glu Asp Tyr Ala 1 5 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDRH1 (JS_VH 9) <400> 6 Gly Phe Thr Phe Gly Asp Tyr Ala 1 5 <210> 7 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDRH1 (BM 57) <400> 7 Gly Phe Thr Phe Glu Gly Tyr Ala 1 5 <210> 8 <211 > 10 <212> PRT <213> Artificial Sequence <220> <223> CDRH2 (JS 1, JS 2, JS 3, JS 4) <400> 8 Ile Arg Ser Asn Ser Tyr Gly Gly Thr Ala 1 5 10 <210 > 9 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDRH2 (JS 5) <400> 9 Ile Tyr Tyr Thr Gly Arg Val 1 5 <210> 10 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDRH2 (JS 7) <400> 10 Ile Ser Ala Tyr Asn Gly Asp Thr 1 5 <210> 11 <211> 8 <212> PRT <213> Artificial Sequence <220 > <223> CDRH2 (JS_VH 4, JS_VH 9, JS_VH 19, BM 13, BM 73) <400> 11 Ile Ser Gly Asp Gly Phe Phe Thr 1 5 <210> 12 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDRH2 (JS_VH 21) <400> 12 Ile Ser Gly Asp Gly Phe Tyr Thr 1 5 <210> 13 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDRH2 (BM 18, BM 57) <400> 13 Ile Ser Gly Asp Gly Ser Phe Thr 1 5 <210> 14 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> CDRH3 (JS 1) <400> 14 Ala Arg Arg Ala His Tyr Tyr Gly Asp Ser Thr Tyr Asn Pro Ser Trp 1 5 10 15 Tyr Phe Asp Leu 20 <210> 15 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> CDRH3 (JS 2, JS 3, JS 4) <400> 15 Ala Arg Arg Ala His Tyr Tyr Gly Gly Ser Thr Tyr Asn Pro Ser Trp 1 5 10 15 Tyr Phe Asp Leu 20 <210> 16 <211> 17 <212> PRT <213> Artificial Sequence <220> < 223> CDRH3 (JS 5) <400> 16 Ala Lys Asp Pro Pro Trp Thr Met Pro Gly Asn Tyr Trp Tyr Phe Asp 1 5 10 15 Leu <210> 17 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> CDRH3 (JS 7) <400> 17 Thr Thr Gly Leu Thr Gly Cys Gly Gly Asp Cys Tyr Tyr Trp Tyr Phe 1 5 10 15 Asp Leu <210> 18 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDRH3 (JS_VH 4, JS_VH 9, JS_VH 21, BM 57) <400> 18 Ala Arg Asp Ser Phe Gly Gly His Leu Asp Leu 1 5 10 <210> 19 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDRH3 (JS_VH 19) <400> 19 Val Arg Asp Ser Phe Gly Gly His Leu Asp Leu 1 5 10 <210> 20 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDRH3 (BM 13) <400> 20 Val Arg Asp Ser Leu Gly Gly His Leu Asp Leu 1 5 10 <210> 21 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDRH3 (BM 18, BM 73) <400> 21 Ala Arg Gly Ser Phe Gly Gly His Leu Asp Leu 1 5 10 <210> 22 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CDRL1 (JS 1) <400> 22 Gln Asp Val Ser Arg Arg 1 5 <210> 23 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CDRL1 (JS 2, JS 3) <400> 23 Gln Asp Ile Ser Arg Arg 1 5 <210> 24 <211> 6 <212 > PRT <213> Artificial Sequence <220> <223> CDRL1 (JS 4) <400> 24 Gln Asp Ile Ser Gly Arg 1 5 <210> 25 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CDRL1 (JS 5) <400> 25 Gln Tyr Ile Gly Thr Tyr 1 5 <210> 26 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDRL1 (JS 7) <400 > 26 Ser Ser Asp Ile Gly Asn Tyr Asn Arg 1 5 <210> 27 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDRL1 (BM 13, BM 57) <400> 27 Gly Ser Asn Leu Gly Ala Gly Tyr Asp Val His Trp Tyr 1 5 10 <210> 28 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDRL1 (BM 18) <400> 28 Asn Ser Asn Ile Gly Ala Gly Tyr Asp Val His Trp Tyr 1 5 10 <210> 29 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDRL1 (BM 73) <400> 29 Ser Ser Asn Ile Gly Ala Gly Ser Asp Val Tyr Trp Tyr 1 5 10 <210> 30 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDRL1 (BM 23) <400> 30 Ser Ser Asn Ile Gly Ala Gly Tyr Asp Val His Trp Tyr 1 5 10 <210> 31 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CDRL2 (JS 1) <400> 31 Gly Val Ser 1 <210> 32 <211> 3 <212> PRT < 213> Artificial Sequence <220> <223> CDRL2 (JS 2, JS 3, JS 4) <400> 32 Gly Ala Ser 1 <210> 33 <211> 3 <212> PRT <213> Artificial Sequence <220> < 223> CDRL2 (JS 5) <400> 33 Ala Ala Ser 1 <210> 34 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> CDRL2 (JS 7) <400> 34 Glu Val Ser 1 <210> 35 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDRL2 (BM 13, BM 18, BM 57, BM 73) <400> 35 Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 1 5 10 <210> 36 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDRL2 (BM 23) <400> 36 Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 1 5 10 <210> 37 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDRL3 (JS 1, J S 2, JS 4) <400> 37 Gln Gln Gly Tyr Ser Ser Pro Arg Thr 1 5 <210> 38 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDRL3 (JS 3) < 400> 38 Gln Gln Gly Tyr Gly Ser Pro Arg Thr 1 5 <210> 39 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDRL3 (JS 5) <400> 39 Gln Gln Ala Asp Ser Phe Pro Leu Thr 1 5 <210> 40 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDRL3 (JS 7) <400> 40 Ser Ser Tyr Thr Ser Ser Asp Ala Tyr Val 1 5 10 <210> 41 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDRL3 (BM 13, BM 57) <400> 41 Ala Ser Trp Asp Asp Ser Leu Asn Ala His Val Phe Gly 1 5 10 <210> 42 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDRL3 (BM 18) <400> 42 Gln Ser Tyr Asp Asn Ser Leu Ser Asp Trp Val Phe Gly 1 5 10 <210> 43 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDRL3 (BM 73) <400> 43 Gln Ser Tyr Asp Ser Ser Leu Ser Gly Trp Ala Phe Gly 1 5 10 < 2 10> 44 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDRL3 (BM 23) <400> 44 Gln Ser Tyr Asp Ser Asn Leu Ser Gly Phe Val Ser Gly 1 5 10 <210> 45 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> VH-FR1 (JS 1, JS 2, JS 3, JS 4) <400> 45 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Ser 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser 20 25 <210> 46 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> VH-FR1 (JS 5 ) <400> 46 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Ser Pro Glu 1 5 10 15 Thr Leu Ser Leu Ser Cys Ala Val Ser 20 25 <210> 47 <211> 26 <212> PRT <213 > Artificial Sequence <220> <223> VH-FR1 (JS 7) <400> 47 Gln Val Asn Leu Arg Glu Ser Gly Ala Ser Glu Val Lys Lys Pro Gly 1 5 10 15 Ala Ser Val Arg Val Ser Cys Lys Ala Ser 20 25 <210> 48 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> VH-FR1 (JS_VH 4, JS_VH 9, JS_VH 19, JS_VH 21, BM 13, BM 18, BM 57, BM 73) <400> 48 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 49 <211> 17 <212> PRT <213> Artificial Sequence <220> <223 > VH-FR2 (JS 1, JS 3, JS 4) <400> 49 Leu Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly 1 5 10 15 Tyr <210> 50 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VH-FR2 (JS 2) <400> 50 Leu Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Asp 1 5 10 15 Tyr <210> 51 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VH-FR2 (JS 5) <400> 51 Trp Asn Trp Leu Arg Gln Ser Pro Gly Lys Ala Pro Glu Trp Ile Gly 1 5 10 15 Tyr <210> 52 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VH-FR2 (JS 7) <400> 52 Phe Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val Gly 1 5 10 15 Trp <210> 53 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VH-FR2 (JS_VH 4, JS_VH 9, JS_VH 19 , JS_VH 21, BM 13, BM 18, BM 57, BM 73) <400> 53 Met His Trp Val Arg Gln Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 15 Leu <210> 54 <211> 38 < 212> PRT <213> Artificial Sequence <220> <223> VH-FR3 (JS 1, JS 2, JS 3) <400> 54 Glu Tyr Ala Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu 1 5 10 15 Ser Lys Asn Ile Ala Tyr Leu Glu Met Asn Ser Leu Arg Ala Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 55 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> VH-FR3 (JS 4) <400> 55 Glu Tyr Ala Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu 1 5 10 15 Ser Lys Asn Ile Ala Cys Leu Glu Met Asn Ser Leu Arg Ala Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 56 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> VH-FR3 (JS 5) <400> 56 Thr Tyr Asn Pro Ser Phe Lys Ser Arg Val Thr Ile Gln Leu Asp Thr 1 5 10 15 Ser Lys Thr Gln Phe Ser Leu Glu Leu Thr Ser Val Thr Ala Val Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 57 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> VH-FR3 (JS 7) <400> 57 Glu Tyr Ala Gln Lys Phe Gln Asp Arg Val Thr Leu Thr Thr Asp Thr 1 5 10 15 Ser Thr Ser Thr Gly Tyr Met Asp Leu Arg Ser Leu Gly Ser Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 58 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> VH-FR3 (JS_VH 4, JS_VH 9, JS_VH 19, JS_VH 21, BM 13, BM 18, BM 57, BM 73) <400> 58 His Tyr Ala Asp Ser Val Glu Gly Arg Phe Thr Val Ser Arg Asp Asn 1 5 10 15 Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 59 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VH-FR4 ( JS 1, JS 3, JS 4) <400> 59 Trp Gly Arg Gly Thr Pro Val Thr Val Ser Ser 1 5 10 <210> 60 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VH-FR4 (JS 2) <400> 60 Trp Gly His Gly Thr Pro Val Thr Val Ser Ser 1 5 10 <210> 61 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VH- FR4 (JS 5, JS 7) <400> 61 Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 62 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VH -FR4 (JS_VH 4, JS_VH 9, JS_VH 19, JS_VH 21, BM 13, BM 18, BM 57, BM 73) <400> 62 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 63 < 211> 26 <212> PRT <213> Artificial Sequence <220> <223> VL-FR1 (JS 1, JS 3) <400> 63 Glu Ile Val Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 20 25 <210> 64 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> VL-FR1 (JS 2) <400> 64 Glu Ile Val Leu Thr Gln Ser Pro Ser Phe Leu Phe Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 20 25 <210> 65 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> VL-FR1 (JS 4) < 400> 65 Glu Ile Val Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Phe Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 20 25 <210> 66 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> VL-FR1 (JS 5) <400> 66 Asp Ile Val Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 20 25 <210> 67 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> VL-FR1 (JS 7) <400> 67 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Val Thr Ile Ser Cys Thr Gly Thr 20 25 <210> 68 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> VL-FR1 (BM 13, BM 57) < 400> 68 His Val Ile Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Ile Ile Ser Cys Thr Gly Ser 20 25 <210> 69 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> VL-FR1 (BM 18) <400> 69 His Val Ile Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser 20 25 <210> 70 < 211> 25 <212> PRT <213> Artificial Sequence <220> <223> VL-FR1 (BM 73) <400> 70 His Val Ile Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Thr Val Ile Ile Ser Cys Thr Gly Ser 20 25 <210> 71 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> VL-FR1 (BM 23) <400> 71 His Val Ile Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Tyr 20 25 <210> 72 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VL- FR2 (JS 1, JS 2) <400> 72 Leu Ala Trp Tyr Gln Arg Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210> 73 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VL-FR2 (JS 3) <400> 73 Leu Ala Trp Tyr Gln Arg Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 1 5 10 15 Phe <210> 74 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VL-FR2 (JS 4) <400> 74 Leu Ala Trp Tyr Gln Arg Lys Leu Gly Lys Ala Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210 > 75 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VL-FR2 (JS 5) <400> 75 Leu Ala Trp Tyr Gln Gln Gln Pro Gly Lys Ala Pro Arg Leu Leu Ile 1 5 10 15 Phe <210> 76 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VL-FR2 (JS 7) <400> 76 Val Ser Trp Tyr Gln Gln Ser Pro Gly Thr Ala Pro Gln Leu Ile Ile 1 5 10 15 Phe <210> 77 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VL-FR2 (BM 13) <400> 77 Gln Gln Pro Pro Gly Thr Ala Pro Lys Leu Val Ile Tyr Asp Asn Thr 1 5 10 15 Asn <210> 78 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VL-FR2 (BM 18) <400> 78 Gln Gln Val Pro Gly Thr Ala Pro Arg Leu Leu Ile Tyr Gly Tyr Asn 1 5 10 15 Gln <210> 79 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VL-FR2 (BM 57) <400> 79 Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Val Ile Tyr Asp Asn Thr 1 5 10 15 Asn <210> 80 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VL-FR2 (BM 73) <400 > 80 His Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Gly Asp Asn 1 5 10 15 Asn <210> 81 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VL-FR2 ( BM 23) <400> 81 Gln Gln Leu Pro Gly Ala Ala Pro Lys Leu Leu Ile Tyr Gly Asp Asn 1 5 10 15 Asn <210> 82 <211> 36 <212> PRT <213> Artificial Sequence <220> <223 > VL-FR3 (JS 1, JS 2, JS 4) <400> 82 Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Thr Gly Ser Gly Ser Gly 1 5 10 15 Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser Asp Asp Phe Ala 20 25 30 Thr Tyr Tyr Cys 35 <210> 83 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> VL-FR3 (JS 3) <400> 83 Asn Leu Ala Ser Arg Val Pro Ser Arg Phe Thr Gly Ser Gly Ser Gly 1 5 10 15 Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser Asp Asp Phe Ala 20 25 30 Thr Tyr Tyr Cys 35 <210> 84 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> VL-FR3 (JS 5) <400> 84 Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Ser Ala 20 25 30 Thr Tyr Tyr Cys 35 <210> 85 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> VL-FR3 (JS 7) <400> 85 Asp Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly 1 5 10 15 Asn Thr Ala Ser Leu Thr Val Ser Gly Leu Gln Ala Glu Asp Glu Ala 20 25 30 Asp Tyr Tyr Cys 35 <210> 86 <211 > 25 <212> PRT <213> Artificial Sequence <220> <223> VL-FR3 (BM 13) <400> 86 Gly Ser Thr Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln 1 5 10 15 Ala Asp Asp Glu Ala Asp Tyr Tyr Cys 20 25 <210> 87 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> VL-FR3 (BM 18) <400 > 87 Gly Ser Arg Ser Gly Pro Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 1 5 10 15 Ser Glu Asp Glu Ala Gly Tyr Tyr Cys 20 25 <210> 88 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> VL-FR3 (BM 57) <400> 88 Gly Ser Thr Ser Ala Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln 1 5 10 15 Ala Asp Asp Glu Ala Asp Tyr Tyr Cys 20 25 <210> 89 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> VL-FR3 (BM 73) <400> 89 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu Gln 1 5 10 15 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys 20 25 <210> 90 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> VL-FR3 (BM 23) <400> 90 Ala Ser Lys Ser Ala Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 1 5 10 15 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys 20 25 <210> 91 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VL-FR4 (JS 1, JS 2, JS 3 , JS 4) <400> 91 Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg 1 5 10 <210> 92 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VL-FR4 (JS 5) <400> 92 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 1 5 10 <210> 93 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VL-FR4 (JS 7) <400> 93 Phe Gl y Thr Gly Thr Lys Leu Thr Val Leu Gly 1 5 10 <210> 94 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VL-FR4 (BM 13) <400> 94 Thr Gly Thr Gln Leu Thr Ala Leu Gly 1 5 <210> 95 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VL-FR4 (BM 18) <400> 95 Gly Gly Thr Lys Val Thr Val Leu Gly 1 5 <210> 96 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VL-FR4 (BM 57) <400> 96 Thr Gly Thr Gln Leu Thr Val Leu Gly 1 5 <210 > 97 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VL-FR4 (BM 73) <400> 97 Gly Gly Thr Arg Val Thr Val Leu Gly 1 5 <210> 98 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VL-FR4 (BM 23) <400> 98 Thr Gly Thr Lys Val Thr Val Leu Gly 1 5 <210> 99 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> VH-JS 1 <400> 99 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Ser 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Ala Ile Gl y Asp Asn 20 25 30 Ala Leu Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Tyr Ile Arg Ser Asn Ser Tyr Gly Gly Thr Ala Glu Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Ile 65 70 75 80 Ala Tyr Leu Glu Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Arg Ala His Tyr Tyr Tyr Gly Asp Ser Thr Tyr Asn Pro 100 105 110 Ser Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Pro Val Thr Val Ser 115 120 125 Ser <210> 100 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> VH-JS 2 < 400> 100 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Ser 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Ala Ile Gly Asp Asn 20 25 30 Ala Leu Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Asp Tyr Ile Arg Ser Asn Ser Tyr Gly Gly Thr Ala Glu Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Ile 65 70 75 80 Ala Tyr Leu Glu Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Arg Ala His Tyr Tyr Gly Gly Ser Thr Tyr Asn Pro 100 105 110 Ser Trp Tyr Phe Asp Leu Trp Gly His Gly Thr Pro Val Thr Val Ser 115 120 125 Ser <210> 101 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> VH-JS 3 <400> 101 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Ser 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Ala Ile Gly Gly Asn 20 25 30 Ala Leu Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Tyr Ile Arg Ser Asn Ser Tyr Gly Gly Thr Ala Glu Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Ile 65 70 75 80 Ala Tyr Leu Glu Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Arg Ala His Tyr Tyr Gly Gly Ser Thr Tyr Asn Pro 100 105 110 Ser Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Pro Val Th r Val Ser 115 120 125 Ser <210> 102 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> VH-JS 4 <400> 102 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Ser 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Ala Ile Gly Asp Asn 20 25 30 Ala Leu Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Tyr Ile Arg Ser Asn Ser Tyr Gly Gly Thr Ala Glu Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Glu Ser Lys Asn Ile 65 70 75 80 Ala Cys Leu Glu Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Arg Ala His Tyr Tyr Gly Gly Ser Thr Tyr Asn Pro 100 105 110 Ser Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Pro Val Thr Val Ser 115 120 125 Ser <210> 103 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> VH-JS 5 <400> 103 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Ser Pro Glu 1 5 10 15 Thr Le u Ser Leu Ser Cys Ala Val Ser Gly Asp Ser Ile Ser Gly Asp 20 25 30 Tyr Trp Asn Trp Leu Arg Gln Ser Pro Gly Lys Ala Pro Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Thr Gly Arg Val Thr Tyr Asn Pro Ser Phe Lys 50 55 60 Ser Arg Val Thr Ile Gln Leu Asp Thr Ser Lys Thr Gln Phe Ser Leu 65 70 75 80 Glu Leu Thr Ser Val Thr Ala Val Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Asp Pro Pro Trp Thr Met Pro Gly Asn Tyr Trp Tyr Phe Asp Leu 100 105 110 Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 104 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> VH-JS 7 <400> 104 Gln Val Asn Leu Arg Glu Ser Gly Ala Ser Glu Val Lys Lys Pro Gly 1 5 10 15 Ala Ser Val Arg Val Ser Cys Lys Ala Ser Gly Tyr Ala Leu Thr His 20 25 30 Tyr Gly Phe Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp 35 40 45 Val Gly Trp Ile Ser Ala Tyr Asn Gly Asp Thr Glu Tyr Ala Gln Lys 50 55 60 Phe Gln Asp Arg Val Thr Leu Thr Thr Asp Thr Ser Thr Ser Thr Gly 65 70 75 80 Tyr Met Asp Leu Arg Ser Leu Gly Ser Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Thr Thr Gly Leu Thr Gly Cys Gly Gly Asp Cys Tyr Tyr Trp Tyr 100 105 110 Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 105 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> VH-JS_VH 4 <400> 105 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Ser Gly Asp Gly Phe Phe Thr His Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Ser 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 Ser Phe Gly Gly His Leu Asp Leu Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 106 <211> 118 <212> PRT < 213> Artificial Sequence <220> <223> VH-JS_VH 9 <400> 106 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Ser Gly Asp Gly Phe Phe Thr His Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Ser 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 Ser Phe Gly Gly His Leu Asp Leu Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 107 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> VH-JS_VH 19 <400> 107 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Ser Gly Asp Gl y Phe Phe Thr His Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Ser 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 Val Arg Asp Ser Phe Gly Gly His Leu Asp Leu Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 108 <211> 118 <212> PRT <213> Artificial Sequence <220> <223 > VH-JS_VH 21 <400> 108 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Ser Gly Asp Gly Phe Tyr Thr His Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Ser 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 Ser Phe Gly Gly His Leu Asp Leu Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Va l Ser Ser 115 <210> 109 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> VH-BM 13 <400> 109 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Ser Gly Asp Gly Phe Phe Thr His Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Ser 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 Val Arg Asp Ser Leu Gly Gly His Leu Asp Leu Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 110 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> VH-BM 18 <400> 110 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Ser Gly Asp Gly Ser Phe Thr His Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Ser 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 Gly Ser Phe Gly Gly His Leu Asp Leu Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 111 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> VH-BM 57 <400> 111 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Gly Tyr 20 25 30 Ala Met His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Ser Gly Asp Gly Ser Phe Thr His Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Ser 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 Ser Phe Gly Gly His Leu Asp Leu Trp Gly Gln Gly Thr 100 1 05 110 Leu Val Thr Val Ser Ser 115 <210> 112 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> VH-BM 73 <400> 112 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Glu 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Glu Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Ser Gly Asp Gly Phe Phe Thr His Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Ser 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 Gly Ser Phe Gly Gly His Leu Asp Leu Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 113 <211> 108 <212> PRT <213> Artificial Sequence < 220> <223> VL-JS 1 <400> 113 Glu Ile Val Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Arg Arg 20 25 30 Leu Ala Trp Tyr Gln Arg Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Val Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Ser Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg 100 105 <210> 114 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> VL-JS 2 <400> 114 Glu Ile Val Leu Thr Gln Ser Pro Ser Phe Leu Phe Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Arg Arg 20 25 30 Leu Ala Trp Tyr Gln Arg Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Ser Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg 100 105 <210> 115 <211> 108 <212> PRT <213> Artifi cial Sequence <220> <223> VL-JS 3 <400> 115 Glu Ile Val Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Arg Arg 20 25 30 Leu Ala Trp Tyr Gln Arg Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Phe Gly Ala Ser Asn Leu Ala Ser Arg Val Pro Ser Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Gly Ser Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg 100 105 <210> 116 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> VL-JS 4 <400> 116 Glu Ile Val Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Phe Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Gly Arg 20 25 30 Leu Ala Trp Tyr Gln Arg Lys Leu Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Ser Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys Arg 100 105 <210> 117 <211 > 108 <212> PRT <213> Artificial Sequence <220> <223> VL-JS 5 <400> 117 Asp Ile Val Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Tyr Ile Gly Thr Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Gln Pro Gly Lys Ala Pro Arg Leu Leu Ile 35 40 45 Phe Ala Ala Ser Glu Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Ala Asp Ser Phe Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 118 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> VL-JS 7 <400> 118 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Val Thr Ile Ser Cys Th r Gly Thr Ser Ser Asp Ile Gly Asn Tyr 20 25 30 Asn Arg Val Ser Trp Tyr Gln Gln Ser Pro Gly Thr Ala Pro Gln Leu 35 40 45 Ile Ile Phe Glu Val Ser Asp Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Asp Ala Tyr Val Phe Gly Thr Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 119 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> VL-BM 13 <400> 119 His Val Ile Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Ile Ile Ser Cys Thr Gly Ser Gly Ser Asn Leu Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Pro Pro Gly Thr Ala Pro Lys Leu 35 40 45 Val Ile Tyr Asp Asn Thr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Thr Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp Asp Asp Ser 85 90 95 Leu Asn Ala His Val Phe Gly Thr Gly Thr Gln Leu Thr Ala Leu Gly 100 105 110 <210> 120 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> VL-BM 18 <400> 120 His Val Ile Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Asn Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Val Pro Gly Thr Ala Pro Arg Leu 35 40 45 Leu Ile Tyr Gly Tyr Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Arg Ser Gly Pro Ser Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Gln Ser Glu Asp Glu Ala Gly Tyr Tyr Cys Gln Ser Tyr Asp Asn Ser 85 90 95 Leu Ser Asp Trp Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly 100 105 110 <210> 121 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> VL-BM 57 <400> 121 His Val Ile Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gly Gln 1 5 10 15 Arg Val Ile Ile Ser Cys Thr Gly Ser Gly Ser Asn Leu Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Val Ile Tyr Asp Asn Thr Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Thr Ser Ala Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Asp Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp As p Asp Ser 85 90 95 Leu Asn Ala His Val Phe Gly Thr Gly Thr Gln Leu Thr Val Leu Gly 100 105 110 <210> 122 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> VL- BM 73 <400> 122 His Val Ile Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Thr Val Ile Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Ser Asp Val Tyr Trp Tyr His Gln Leu Pro Gly Thr Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Asp Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser 85 90 95 Leu Ser Gly Trp Ala Phe Gly Gly Gly Thr Arg Val Thr Val Leu Gly 100 105 110 <210> 123 <211> 112 < 212> PRT <213> Artificial Sequence <220> <223> VL-BM 23 <400> 123 His Val Ile Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Tyr Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Ala Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Gly Asp Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Ala Ser Lys Ser Ala Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu 65 70 75 80 Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Asn 85 90 95 Leu Ser Gly Phe Val Ser Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 110 <210> 124 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> CL <400> 124 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 Gl u Cys 100 105 <210> 125 <211> 331 <212> PRT <213> Artificial Sequence <220> <223> CH1, CH2, CH3 <400> 125 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 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 100 105 110 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 115 120 125 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 130 135 140 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 145 150 155 160 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 165 170 175 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 180 185 190 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 195 200 205 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 210 215 220 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 225 230 235 240 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 245 250 255 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 260 265 270 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 275 280 285 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 290 29 5 300 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 305 310 315 320 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 126 <211> 218 <212> PRT <213> Artificial Sequence <220> <223> SARS-CoV-2-RBD <400> 126 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn T yr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn 210 215 <210> 127 <211> 440 <212> PRT <213> Artificial Sequence <220> <223> SARS-CoV-2-RBD-GST <400> 127 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Va l 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Gly Ser Met Ser Pro Ile 210 215 220 Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro Thr Arg Leu Leu 225 230 235 240 Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu Tyr Glu Arg Asp 245 250 255 Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu Gly Leu Glu Phe 260 265 270 Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys Leu Thr Gln Ser 275 280 285 Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn Met Leu Gly Gly 290 295 300 Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu Gly Ala Val Leu 305 310 315 320 Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser Lys Asp Phe Glu 325 330 335 Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys Met 340 345 350 Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn Gly Asp His Val 355 360 365 Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr 370 375 380 Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu Val Cys Phe Lys 385 390 395 400 Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys Ser Ser 405 410 415 Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe Gly Gly 420 425 430 Gly Asp His Pro Pro Lys Ser Asp 435 440 <210> 128 <211> 379 <212> PRT <213> Artificial Sequence <220> <223> SARS-CoV-2-RBD-Streptavidin <400> 128 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Gly Ser Asp Pro Ser Lys 210 215 220 Asp Ser Lys Ala Gln Val Ser Ala Ala Glu Ala Gly Ile Thr Gly Thr 225 230 235 240 Trp Tyr Asn Gln Leu Gly Ser Thr Phe Ile Val Thr Ala Gly Ala Asp 245 250 255 Gly Ala Leu Thr Gly Thr Tyr Glu Ser Ala Val Gly Asn Ala Glu Ser 260 265 270 Arg Tyr Val Leu Thr Gly Arg Tyr Asp Ser Ala Pro Ala Thr Asp Gly 275 280 285 Ser Gly Thr Ala Leu Gly Trp Thr Val Ala Trp Lys Asn Asn Tyr Arg 290 295 300 Asn Ala His Ser Ala Thr Thr Trp Ser Gly Gln Tyr Val Gly Gly Ala 305 310 315 320 Glu Ala Arg Ile Asn Thr Gln Trp Leu Leu Thr Ser Gly Thr Thr Glu 325 330 335 Ala Asn Ala Trp Lys Ser Thr Leu Val Gly His Asp Thr Phe Thr Lys 340 345 350 Val Lys Pro Ser Ala Ala Ser Ile Asp Ala Ala Lys Lys Ala Gly Val 355 36 0 365Asn Asn Gly Asn Pro Leu Asp Ala Val Gln Gln 370 375

Claims (17)

Antibody or fragment having immunological activity specific for COVID-19 virus (SARS-CoV-2). According to claim 1, wherein the fragment having immunological activity is Fab, Fd, Fab', dAb, F(ab'), F(ab') ₂ , scFv (single chain fragment variable), Fv, single chain antibody, Fv Any one selected from the group consisting of a dimer, a complementarity determining region fragment, a humanized antibody, a chimeric antibody, and a diabody, an antibody or fragment having immunological activity specific to the Corona-19 virus. According to claim 1, Complementarity determining regions heavy chain (CDRH) comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 1 to 7, any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 8 to 13 CDRH2 comprising one, and a VH domain comprising a CDRH3 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 14 to 21. Antibody or immunological activity thereof specific for Corona-19 virus fragment with. According to claim 1, FR1 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 45 to 48, FR2 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 49 to 53, SEQ ID NO: An antibody specific for COVID-19 virus, comprising a VH domain comprising FR3 comprising any one selected from the group consisting of the amino acid sequence of 54 to 58, and FR4 comprising the amino acid sequence of SEQ ID NO: 59 or 62; Fragments with immunological activity thereof. According to claim 1, Complementarity determining regions light chain (CDRL) 1 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 22 to 30, any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 31 to 36 CDRL2 comprising one, and a VL domain comprising a CDRL3 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 37 to 44, antibody specific for Corona-19 virus or immunological activity thereof fragment with. According to claim 1, FR1 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 63 to 71, FR2 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 72 to 81, SEQ ID NO: Corona- 19 A virus-specific antibody or fragment having immunological activity thereof. The method of claim 1,
(i) CDRH1 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 1 to 7, CDRH2 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 8 to 13, and SEQ ID NOs: 14 to a VH domain comprising CDRH3 comprising any one selected from the group consisting of the amino acid sequence of 21; and/or
(ii) Complementarity determining regions light chain (CDRL) 1 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 22 to 30, and any one selected from the group consisting of the amino acid sequences of SEQ ID NOs: 31 to 36 An antibody specific for Corona-19 virus or immunological thereof, comprising a V domain comprising a VL domain comprising CDRL2 comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 37 to 44. fragments with activity. According to claim 1, wherein the VH domain comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 99 to 112; And a VL domain comprising any one selected from the group consisting of the amino acid sequence of SEQ ID NOs: 113 to 123, an antibody or fragment having immunological activity specific for Corona-19 virus. An isolated nucleic acid molecule encoding the antibody of claim 1 or a fragment having immunological activity thereof. A vector comprising the isolated nucleic acid molecule of claim 9 . A host cell transformed with the vector of claim 10 . a) culturing a host cell comprising a vector comprising the isolated nucleic acid molecule of claim 9; and
b) a method for producing an antibody or fragment having immunological activity specific for Corona-19 virus, comprising the step of recovering the antibody or fragment having immunological activity thereof from host cell culture. A pharmaceutical composition for the prevention or treatment of COVID-19 virus infection (novel coronavirus infection, COVID-19) containing the antibody or fragment having immunological activity specific to the Corona-19 virus of claim 1. A composition for detecting the Corona-19 virus, comprising an antibody or a fragment having immunological activity thereof specific to the Corona-19 virus of claim 1. A Corona-19 virus detection kit comprising the composition for detecting the Corona-19 virus of claim 14. (a) contacting the sample isolated from the specimen with the antibody or fragment having immunological activity specific to the Corona-19 virus of claim 1; and
(b) detecting the antigen-antibody complex formation, comprising the step of detecting the corona-19 virus. (a) forming an antigen-antibody complex by contacting the sample isolated from the specimen with the antibody or fragment having immunological activity specific to the Corona-19 virus of claim 1; and
(b) a method for providing information on the diagnosis of COVID-19 virus infection, comprising the step of detecting the formation of the complex.

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