Engineered human antibodies having neutralizing activity against human coronavirus

Abstract

The present invention relates to engineered human antibodies having neutralizing activity against human coronavirus and use thereof. The novel human antibodies against coronavirus, of the present invention, specifically bind to SARS-CoV-1, SARS-CoV-2, or a variant thereof, exhibit neutralizing activity against SARS-CoV-1, SARS-CoV-2, or a variant thereof, and have cross-reactivity, and thus can be used for the prevention or treatment of coronavirus infection. In addition, the antibodies and fragments having immunological activities thereof can be used to rapidly detect various types of coronaviruses, and thus can be used for the immunodiagnosis of human coronaviruses, particularly SARS-CoV-2 with high infectivity, and variants thereof.

Description

Engineered human antibodies with neutralizing activity against human coronaviruses

[0001] The present invention relates to engineered human antibodies having neutralizing activity against human coronaviruses and their uses.

[0002] Coronaviruses are enveloped viruses with a single-stranded, positive-sense RNA genome. Since their discovery in 1937, they have been isolated from various animals, including humans. Coronaviruses can be divided into four groups. Alpha-coronaviruses and beta-coronaviruses primarily infect mammals, while gamma-coronaviruses and delta-coronaviruses infect birds, although recent cases of delta-coronavirus infection have been confirmed in pigs. Coronaviruses can transmit between species. Representative examples include the SARS coronavirus, which caused the global outbreak of severe acute respiratory syndrome in 2003, and the MERS coronavirus, which spread in Korea in 2015. Both of these viruses are known to have originated from bats. Bats are mammals capable of flight, allowing them to inhabit wide areas. Furthermore, because they often live in groups in confined spaces like caves, if one individual is infected with a virus, the infection can spread to the entire group and spread to other animals through flight over wide areas. Furthermore, bats, unlike other mammals, have high body temperatures, making them resistant to viruses. This allows bats, the hosts of coronaviruses, to continue transmitting the infection without being affected. As described above, coronaviruses can spread between species, and it has been confirmed that a novel coronavirus originating from bats can spread to humans and cause significant problems. Therefore, the development of effective diagnostic methods and therapeutics for the detection of novel bat-derived coronaviruses is urgent.Research on antiviral agents is actively underway both domestically and internationally. Non-specific antiviral agents that have shown clinical efficacy include type I interferon and ribavirin. However, their viral suppression effect has been reported only in the early stages of infection. Treatments for MERS-CoV and SARS-CoV have recently been attracting attention due to the increasing need for development. However, there are currently no approved treatments or vaccines domestically or internationally, and an evaluation system for substances being developed has not been established. Therefore, the development of a system for evaluating efficacy is urgently needed.

[0003] SARS-CoV-2 virus infection (novel coronavirus infection, COVID-19) is a new coronavirus that was reported to have originated in Wuhan, China in December 2019. After an incubation period of approximately 2 to 14 days, the main symptoms include fever, respiratory symptoms such as cough or difficulty breathing, and pneumonia, but asymptomatic cases of infection have also been reported. The World Health Organization (WHO) declared COVID-19 a "pandemic," the highest alert level, and as of November 11, 2023, there have been 690,240,112 confirmed cases and 6,906,402 deaths in 230 countries around the world, causing enormous damage to global health, economy, and society. The SARS-CoV-2 virus binds to the ACE2 (Angiotensin converting enzyme 2) receptor present on host cells, just like the SARS-CoV-1 virus that was prevalent in 2003. The spike protein of the SARS-CoV-2 virus is 76% similar to the spike protein of the SARS-CoV-1 virus, and the RBD (Receptor Binding Domain) shows 74% protein sequence similarity, but three known antibodies that bind to the SARS-CoV-1 virus-RBD, S230 (PDB:6NB6), m396 (PDB:2G75, 2DD8), and 80R (PDB:2GHW), do not bind to the SARS-CoV-2 RBD.

[0004] The SARS-CoV-2 virus genome is an RNA virus, and it has a protein shell (capsid) containing the genome surrounded by an outer membrane (envelope), and on the surface, there are spike proteins in the form of membrane glycoproteins that protrude like protrusions. Functionally, it is divided into an S1 subunit that binds to the host cell receptor and an S2 subunit that fuses with the membrane. The S1 subunit contains an RBD (receptor binding domain) that binds to the ACE2 (angiotensin converting enzyme 2) receptor on the host cell. The spike protein plays a crucial role in infecting the host with the virus by binding to the ACE2 receptor present on the host and penetrating into the host cell. Therefore, most vaccines currently being administered or under development are designed to induce neutralizing antibodies that neutralize the spike protein. However, various mutations have occurred in the viral spike protein, causing various variants to break through and infect, and countermeasures are needed.

[0005] The purpose of the present invention is to provide an antibody or a fragment thereof having immunological activity specific for a human coronavirus or a variant thereof.

[0006] In addition, it is an object of the present invention to provide a nucleic acid molecule encoding the antibody or a fragment thereof having immunological activity, a vector comprising the same, and a host cell comprising the same.

[0007] In addition, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating human coronavirus infection.

[0008] In addition, it is an object of the present invention to provide a composition for detecting human coronavirus or its variants.

[0009] In addition, it is an object of the present invention to provide a kit for detecting human coronavirus or its variants.

[0010] It is also an object of the present invention to provide a method for detecting a human coronavirus or a variant thereof.

[0011] In addition, it is an object of the present invention to provide a method for providing information necessary for diagnosing human coronavirus infection.

[0012] In addition, it is an object of the present invention to provide a method for preventing or treating human coronavirus infection.

[0013] In addition, it is an object of the present invention to provide a method for producing an antibody specific for human coronavirus or a variant thereof or a fragment thereof having immunological activity.

[0014] To solve the above problem, the present invention provides an antibody specific for a novel human coronavirus or a variant thereof or a fragment thereof having immunological activity.

[0015] In addition, the present invention provides a nucleic acid molecule encoding the antibody or a fragment thereof having immunological activity, a vector comprising the same, and a host cell comprising the same.

[0016] In addition, the present invention provides a pharmaceutical composition for preventing or treating human coronavirus infection, comprising an antibody specific for the human coronavirus or a variant thereof or a fragment thereof having immunological activity.

[0017] In addition, the present invention provides a composition for detecting a human coronavirus or a variant thereof, comprising an antibody specific for the human coronavirus or a variant thereof or a fragment having immunological activity thereof.

[0018] In addition, the present invention provides a kit for detecting human coronavirus or a variant thereof comprising the composition.

[0019] The present invention also provides a method for detecting a human coronavirus or a variant thereof.

[0020] Additionally, the present invention provides a method for providing information necessary for diagnosing human coronavirus infection.

[0021] In addition, the present invention provides a method for preventing or treating human coronavirus infection, comprising administering to a subject an antibody or a fragment having immunological activity thereof specific to the human coronavirus or a variant thereof.

[0022] In addition, the present invention provides a method for producing an antibody or a fragment having immunological activity specific to a human coronavirus or a variant thereof, comprising the steps of: a) culturing a host cell comprising a vector including a nucleic acid molecule encoding an antibody or a fragment having immunological activity specific to the human coronavirus or a variant thereof; and b) recovering the antibody or fragment expressed by the host cell.

[0023] The novel human antibodies against coronaviruses of the present invention specifically bind to SARS-CoV-1, SARS-CoV-2 or variants thereof, exhibit neutralizing activity therefor, and have cross-reactivity, and thus can be used for the prevention or treatment of coronavirus infection. In addition, since various types of coronaviruses can be rapidly detected using the antibodies and fragments thereof having immunological activity, they can be used for the immune diagnosis of human coronaviruses, particularly, SARS-CoV-2 and variants thereof with high infectivity.

[0024] Figure 1 is a diagram showing an expression vector for an animal cell of a dimeric SARS-CoV-2 RBD region antigen protein and the results of analyzing the size and purity using an SDS-PAGE gel after expressing and purifying it in animal cells.

[0025] Figure 2 is a diagram illustrating an iterative screening process to amplify human antibody clones with enhanced binding affinity to the SARS-CoV-2 RBD region.

[0026] Figure 3 is a diagram showing the results of an enrichment test of a SARS-CoV-2 affinity maturation library using a flow cytometer.

[0027] Figure 4 is a diagram showing the amino acid sequence analysis results of four scFv human antibody variants (IJ427, IJ452, IJ2416, and IJ2483) that exhibit improved binding affinity to SARS-CoV-2 RBD.

[0028] Figure 5 shows the results of analyzing the binding affinity of four scFv antibody variants (IJ427, IJ452, IJ2416, and IJ2483) to SARS-CoV-2 RBD using a flow cytometer.

[0029] Figure 6 is a diagram showing the results of SDS-PAGE gel analysis of four antibodies that bind to SARS-CoV-2 RBD expressed and purified in animal cells.

[0030] Figure 7 is a diagram showing the binding affinity of four antibodies (IJ427, IJ2416, IJ4G, and IJ24G) to SARS-CoV-2 RBD confirmed by ELISA analysis.

[0031] Figure 8 is a diagram showing the results of SDS-PAGE gel analysis of three antibodies (N57Y, F107Y, and YY) that have only common CDR mutations of SARS-CoV-2 RBD binding antibodies, expressed and purified in animal cells.

[0032] Figure 9 is a diagram showing the binding affinity of variants, including three antibodies (N57Y, F107Y, and YY) that have only common CDR mutations of SARS-CoV-2 RBD binding antibodies, confirmed by ELISA analysis.

[0033] Figure 10 is a diagram showing the results of analyzing the binding affinity of two SARS-CoV-2 RBD binding antibodies (IJ4G and YY).

[0034] Figure 11 is a diagram showing an expression vector for animal cells of dimeric and tetrameric antigen proteins of the RBD region of the SARS-CoV-2 B.1.617.2 variant, and the results of analyzing the size and purity using an SDS-PAGE gel after expressing and purifying the same in animal cells.

[0035] Figure 12 is a diagram illustrating an iterative screening process to amplify human antibody clones that specifically bind to the SARS-CoV-2 B.1.617.2 variant RBD region.

[0036] Figure 13 is a diagram showing the results of an enrichment test of the SARS-CoV-2 B.1.617.2 library using a flow cytometer.

[0037] Figure 14 is a diagram showing the amino acid sequence analysis results of four scFv human antibody variants (IJ30, IJ37, IJ64, and IJ73) that show high binding affinity to SARS-CoV-2 B.1.617.2 RBD.

[0038] Figure 15 shows the results of analyzing the binding affinity of four scFv antibody variants (IJ30, IJ37, IJ64, and IJ73) to SARS-CoV-2 B.1.617.2 RBD using a flow cytometer.

[0039] Figure 16 is a diagram showing the expression vectors for animal cells of five SARS-CoV-2 variant B.1.617.2 RBD-binding antibodies (IJ2, IJ30, IJ37, IJ64, and IJ73) and the results of SDS-PAGE gel analysis after expressing and purifying them in animal cells.

[0040] Figure 17 is a diagram showing the binding affinity of five antibodies (IJ2, IJ30, IJ37, IJ64, and IJ73) to SARS-CoV-2 B.1.617.2 RBD confirmed by ELISA analysis.

[0041] Figure 18 is a diagram illustrating an iterative screening process to amplify human antibody clones with enhanced binding affinity to the RBD region of SARS-CoV-2 variant B.1.617.2.

[0042] Figure 19 is a diagram showing the results of an enrichment test of a SARS-CoV-2 B.1.617.2 affinity maturation library using a flow cytometer.

[0043] Figure 20 shows the results of analyzing the binding affinity of six scFv antibody variants (IJ203, IJ225, IJ228, IJ232, IJ235, and IJ274) to SARS-CoV-2 B.1.617.2 RBD using a flow cytometer.

[0044] Figure 21 is a diagram showing the amino acid sequence analysis results of scFv human antibody variant IJ225 showing improved binding affinity to SARS-CoV-2 B.1.617.2 RBD of IJ225.

[0045] Figure 22 is a diagram showing the results of SDS-PAGE gel analysis of the SARS-CoV-2 B.1.617.2 RBD-binding IJ225 antibody expressed and purified in animal cells.

[0046] Figure 23 is a diagram confirming the enhanced binding affinity of the IJ225 antibody to SARS-CoV-2 B.1.617.2 RBD by ELISA analysis.

[0047] Figure 24 is a diagram showing the results of analyzing the binding affinity of the IJ225 antibody for SARS-CoV-2 B.1.617.2 RBD.

[0048] Figure 25 is a diagram showing the cross-RBD binding affinity of antibodies IJ4G and YY, which bind to SARS-CoV-2 RBD, and antibody IJ225, which binds to SARS-CoV-2 B.1.617.2 RBD, confirmed by ELISA analysis.

[0049] Figure 26 is a diagram showing the results of analyzing the cross-RBD binding affinity of antibodies IJ4G and YY that bind to SARS-CoV-2 RBD, and antibody IJ225 that binds to SARS-CoV-2 B.1.617.2 RBD.

[0050] Figure 27 is a diagram showing the results of analyzing the neutralizing ability of three antibodies (IJ225, IJ4G, and YY) against the SARS-CoV-2 pseudovirus.

[0051] Figure 28 is a diagram showing the binding affinity of three antibodies (IJ225, IJ4G, and YY) to SARS-CoV-1 confirmed by ELISA analysis.

[0052] Figure 29 is a diagram showing a sequence list of the present invention.

[0053] Figure 30 is a diagram showing the sequences of antibodies of the present invention.

[0054] Hereinafter, the present invention will be described in detail with reference to the attached drawings, using exemplary embodiments of the present invention. However, the following exemplary embodiments are provided as examples of the present invention. If a detailed description of a technology or configuration well known to those skilled in the art is judged to unnecessarily obscure the gist of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

[0055] Additionally, the terminology used in this specification is intended to appropriately express preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the customs of the field to which the present invention pertains. Therefore, the definitions of these terms should be determined based on the contents throughout this specification. Throughout this specification, when a part is said to "include" a certain component, unless specifically stated otherwise, this does not mean that other components are excluded, but rather that other components may be included.

[0056] Unless otherwise defined, all technical terms used in this invention have the same meaning as commonly understood by those skilled in the art. While preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of the present invention. The contents of all publications cited herein as references are incorporated herein by reference.

[0057] Throughout this specification, the conventional one-letter and three-letter codes for naturally occurring amino acids are used, as well as generally accepted three-letter codes for other amino acids, such as Aib (α-aminoisobutyric acid) and Sar (N-methylglycine). Furthermore, amino acids referred to herein by abbreviations are described according to the IUPAC-IUB nomenclature as follows:

[0058] 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.

[0059]

[0060] In one aspect, the present invention relates to an antibody or a fragment thereof having immunological activity specific for a human coronavirus or a variant thereof.

[0061] In one embodiment, the human coronavirus may be Severe Acute Respiratory Syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), or SARS-CoV-2, more preferably SARS-CoV-1 or SARS-CoV-2.

[0062] In one embodiment, the variant may be an alpha variant (B.1.1.7), a beta variant (B.1.351), a gamma variant (P.1), a delta variant (B.1.617.2), a kappa variant (B.1.617.1) or a mu variant (B.1.1.529) of SARS-CoV-2, more preferably a delta variant (B.1.617.2).

[0063] In one embodiment, the fragment having immunological activity can be any one selected from the group consisting of Fab, Fd, Fab', dAb, F(ab'), F(ab')2, scFv (single chain fragment variable), Fv, single chain antibody, Fv dimer, complementarity determining region fragment, humanized antibody, chimeric antibody and diabody.

[0064] In one embodiment, the antibody of the present invention or a fragment thereof having immunological activity may comprise a VH domain comprising a CDRH (Complementarity determining regions Heavy chain) 1 comprising an amino acid sequence of SEQ ID NO: 1 or 2, a CDRH2 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NO: 3 to 10, and a CDRH3 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NO: 11 to 16.

[0065] In one embodiment, the antibody of the present invention or an immunologically active fragment thereof may comprise a VH domain comprising an FR1 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 25 to 27, an FR2 comprising an amino acid sequence of SEQ ID NOs: 28 or 29, an FR3 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 30 to 35, and an FR4 comprising an amino acid sequence of SEQ ID NOs: 36 or 37.

[0066] In one embodiment, the antibody of the present invention or a fragment thereof having immunological activity may comprise a VL domain comprising CDRL1 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 17 to 19, CDRL2 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 20 or 21, and CDRL3 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 22 to 24.

[0067] In one embodiment, the antibody of the present invention or an immunologically active fragment thereof may comprise a VL domain comprising an FR1 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 38 to 40, an FR2 comprising an amino acid sequence of SEQ ID NO: 41, an FR3 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 42 to 47, and an FR4 comprising an amino acid sequence of SEQ ID NO: 48.

[0068] In one embodiment, the antibody of the present invention or a fragment thereof having immunological activity may comprise a VH domain comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 49 to 61.

[0069] In one embodiment, the antibody of the present invention or a fragment thereof having immunological activity may comprise a VL domain comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 62 to 72.

[0070] In one embodiment, the antibody of the present invention or a fragment thereof having immunological activity may be IJ4G comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 49 and a VL domain comprising the amino acid sequence of SEQ ID NO: 62.

[0071] In one embodiment, the antibody of the present invention or a fragment thereof having immunological activity may be IJ427 comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 50 and a VL domain comprising the amino acid sequence of SEQ ID NO: 62.

[0072] In one embodiment, the antibody of the present invention or a fragment thereof having immunological activity may be IJ452 comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 51 and a VL domain comprising the amino acid sequence of SEQ ID NO: 63.

[0073] In one embodiment, the antibody of the present invention or a fragment thereof having immunological activity may be IJ24G comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 52 and a VL domain comprising the amino acid sequence of SEQ ID NO: 64.

[0074] In one embodiment, the antibody of the present invention or a fragment thereof having immunological activity may be IJ2416 comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 53 and a VL domain comprising the amino acid sequence of SEQ ID NO: 65.

[0075] In one embodiment, the antibody of the present invention or a fragment thereof having immunological activity may be IJ2483 comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 54 and a VL domain comprising the amino acid sequence of SEQ ID NO: 66.

[0076] In one embodiment, the antibody of the present invention or a fragment thereof having immunological activity may be IJ30 comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 55 and a VL domain comprising the amino acid sequence of SEQ ID NO: 67.

[0077] In one embodiment, the antibody of the present invention or a fragment thereof having immunological activity may be IJ37 comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 56 and a VL domain comprising the amino acid sequence of SEQ ID NO: 68.

[0078] In one embodiment, the antibody of the present invention or a fragment thereof having immunological activity may be IJ64 comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 57 and a VL domain comprising the amino acid sequence of SEQ ID NO: 69.

[0079] In one embodiment, the antibody of the present invention or a fragment thereof having immunological activity may be IJ73 comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 57 and a VL domain comprising the amino acid sequence of SEQ ID NO: 70.

[0080] In one embodiment, the antibody of the present invention or a fragment thereof having immunological activity may be IJ2 comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 57 and a VL domain comprising the amino acid sequence of SEQ ID NO: 71.

[0081] In one embodiment, the antibody of the present invention or a fragment thereof having immunological activity may be IJ225 comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 58 and a VL domain comprising the amino acid sequence of SEQ ID NO: 72.

[0082] In one embodiment, the antibody of the present invention or a fragment thereof having immunological activity may be N57Y comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 59 and a VL domain comprising the amino acid sequence of SEQ ID NO: 71.

[0083] In one embodiment, the antibody of the present invention or a fragment thereof having immunological activity may be F107Y comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 60 and a VL domain comprising the amino acid sequence of SEQ ID NO: 71.

[0084] In one embodiment, the antibody of the present invention or a fragment thereof having immunological activity may be YY comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 61 and a VL domain comprising the amino acid sequence of SEQ ID NO: 71.

[0085] The sequences of the antibodies of the present invention are shown in Tables 1 and 2 below.

[0086]

[0087]

[0088]

[0089] In one embodiment, the antibody of the present invention or an immunologically active fragment thereof may comprise a heavy or light chain variable region, a heavy or light chain constant region, a framework region, or any portion thereof of human origin.

[0090] In one embodiment, the antibody of the present invention may be a monoclonal antibody.

[0091] In one embodiment, the antibody of the present invention or a fragment thereof having immunological activity can specifically bind to the receptor binding domain (RBD) of a human coronavirus, and can specifically bind to the receptor binding domain (RBD) of SARS-CoV-1, SARS-CoV-2, or a variant thereof.

[0092] In one embodiment, the antibody or immunologically active fragment thereof of the present invention can specifically bind to an antigen, virus particle or virus-infected cell of SARS-CoV-1, SARS-CoV-2 or a variant thereof.

[0093] In one embodiment, the antibody or immunologically active fragment thereof of the present invention may be cross-reactive to human coronaviruses, and may be cross-reactive with the RBD of SARS-CoV-1 and the RBD of SARS-CoV-2, or may be cross-reactive with the RBD of SARS-CoV-1, the RBD of SARS-CoV-2, and the RBD of variants thereof.

[0094] In one embodiment, the antibody of the invention may be a neutralizing antibody and is capable of neutralizing SARS-CoV-2 or a variant thereof.

[0095] In one embodiment, the antibody of the present invention or a fragment thereof having immunological activity can specifically bind to any one RBD (Receptor Binding Domain) selected from the group consisting of SARS-CoV-1 wild type, SARS-CoV-2 wild type, SARS-CoV-2 alpha variant, SARS-CoV-2 beta variant, SARS-CoV-2 gamma variant, SARS-CoV-2 delta variant, SARS-CoV-2 kappa variant and SARS-CoV-2 mu variant, and the RBD of the SARS-CoV-1 wild type can comprise the amino acid sequence of SEQ ID NO: 79, the RBD of the SARS-CoV-2 wild type can comprise the amino acid sequence of SEQ ID NO: 77, and the RBD of the SARS-CoV-2 delta variant can comprise the amino acid sequence of SEQ ID NO: 78.

[0096] In one embodiment, the antibody of the present invention may comprise a light chain constant region (CL) comprising the amino acid sequence of SEQ ID NO: 73, a heavy chain constant region domain 1 (CH1) comprising the amino acid sequence of SEQ ID NO: 74, a heavy chain constant region domain 2 (CH2) comprising the amino acid sequence of SEQ ID NO: 75, and a heavy chain constant region domain 3 (CH3) comprising the amino acid sequence of SEQ ID NO: 76.

[0097] The above antibody is not only in the form of a whole antibody, but also includes functional fragments of an antibody molecule. A whole antibody has a structure having two full-length light chains and two full-length heavy chains, each light chain being linked to a heavy chain by a disulfide bond. A functional fragment of an antibody molecule means a fragment that possesses an antigen-binding function, and examples of antibody fragments include (i) a Fab fragment consisting of a variable region (VL) of a light chain and a variable region (VH) of a heavy chain and a constant region (CL) of a light chain and a heavy chain constant region 1 (CH1) of a heavy chain; (ii) a Fd fragment consisting of the VH and CH1 domains; (iii) a Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of a VH domain; (v) separate CDR regions; (vi) a F(ab')2 fragment, which is a bivalent fragment comprising two linked Fab fragments; (vii) single-chain Fv molecules (scFv) joined by a peptide linker that joins the VH domain and the VL domain to form an antigen-binding site; (viii) bispecific single-chain Fv dimers; and (ix) diabodies, which are multivalent or multispecific fragments produced by genetic fusion.

[0098] In the present invention, the antibody or a fragment thereof having immunological activity may be selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, human antibodies, and fragments thereof having immunological activity. The antibody may be produced recombinantly or synthetically.

[0099] Animal-derived antibodies, produced by immunizing an animal with a desired antigen, can generally elicit immune rejection when administered to humans for therapeutic purposes. Chimeric antibodies have been developed to suppress this immune rejection. Chimeric antibodies utilize genetic engineering to replace the constant region of an animal-derived antibody, which causes anti-isotype reactions, with the constant region of a human antibody. While chimeric antibodies offer significant improvements in anti-isotype reactions compared to animal-derived antibodies, they still contain animal-derived amino acids in their variable regions, posing a risk of potential anti-idiotypic reactions. Humanized antibodies were developed to improve this potential. These antibodies are produced by grafting the complementarity determining regions (CDRs), which play a crucial role in antigen binding, from the variable region of a chimeric antibody onto a human antibody framework.

[0100] The most important aspect of CDR grafting technology for producing humanized antibodies is selecting an optimized human antibody that can best accommodate the CDR regions of an animal-derived antibody. To this end, antibody databases, crystal structure analysis, and molecular modeling techniques are utilized. However, even when the CDR regions of an animal-derived antibody are grafted onto an optimized human antibody framework, there are many cases where antigen binding ability is not preserved because amino acids located in the animal-derived antibody framework may affect antigen binding. Therefore, the application of additional antibody engineering techniques to restore antigen binding ability is essential.

[0101] The above antibody or fragment having immunological activity may be isolated from a living organism (not existing in a living organism) or non-naturally occurring, for example, may be synthetically or recombinantly produced.

[0102] In the present invention, "antibody" refers to a substance produced by antigen stimulation within the immune system, and its type is not particularly limited, and can be obtained naturally or non-naturally (e.g., synthetically or recombinantly). Antibodies are highly stable both in vitro and in vivo and have a long half-life, making them advantageous for mass expression and production. In addition, antibodies inherently have a dimer structure, and thus have very high avidity. A complete antibody has a structure with two full-length light chains and two full-length heavy chains, each light chain being linked to a heavy chain by a disulfide bond. The constant region of antibodies is divided into the heavy chain constant region and the light chain constant region. The heavy chain constant region has the gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and the subclasses are 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 the kappa (κ) and lambda (λ) types.

[0103] In the present invention, the term "heavy chain" is interpreted to mean a full-length heavy chain and fragments thereof, which comprises a variable region domain VH comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen and three constant region domains CH1, CH2 and CH3 and a hinge. In addition, the term "light chain" is interpreted to mean a full-length light chain and fragments thereof, which comprises a variable region domain VL comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen and a constant region domain CL.

[0104] In the present invention, the term "variable region or variable domain" refers to a portion of an antibody molecule that exhibits many sequence variations while performing the function of specifically binding to an antigen, and the variable region includes complementarity determining regions (CDR1, CDR2, and CDR3). Between the CDRs, a framework region (FR) portion exists, which serves 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.

[0105] The term "scFv (single chain fragment variable)" used in the present invention refers to a single-chain antibody produced by expressing only the variable region of an antibody through genetic recombination, and refers to an antibody in the form of a single chain in which the VH region and VL region of an antibody are linked by a short peptide chain. The term "scFv" is intended to include scFv fragments, including antigen-binding fragments, unless otherwise specified or otherwise understood from the context. This will be apparent to those skilled in the art.

[0106] In the present invention, the term "complementarity determining region (CDR)" refers to the amino acid sequence of the hypervariable region of the heavy and light chains of an immunoglobulin. The heavy and light chains may each include three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs may provide key contact residues for antibody binding to an antigen or epitope.

[0107] In the present invention, the terms “specifically bind” or “specifically recognize” have the same meaning as commonly known to those skilled in the art, and mean that an antigen and an antibody specifically interact to cause an immunological reaction.

[0108] In the present invention, the term "antigen-binding fragment" refers to a fragment of the entire immunoglobulin structure, and a part of a polypeptide that includes a portion capable of binding to an antigen. 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 the light and heavy chains, a constant region of the light chain, and the first constant region (CH1) of the heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibodies are produced when the cysteine ​​residues in the hinge region of Fab' form a disulfide bond. Fv is the minimum antibody fragment that has only a heavy chain variable region and a light chain variable region, and recombinant techniques for producing Fv fragments are widely known in the art. A two-chain Fv has a heavy chain variable region and a light chain variable region linked non-covalently, and a single-chain Fv generally has a heavy chain variable region and a single chain variable region linked covalently via a peptide linker or directly at the C-terminus, so that they can form a dimer-like structure like a two-chain Fv. The linker may be a peptide linker composed of any amino acid of 1 to 100 or 2 to 50, and suitable sequences are known in the art. The antigen-binding fragment can be obtained using a proteolytic enzyme (for example, a Fab can be obtained by restriction digestion of a whole antibody with papain, and a F(ab')2 fragment can be obtained by digestion with pepsin), or can be produced through genetic recombination technology.

[0109] The term "hinge region" as used herein refers to a region contained in the heavy chain of an antibody, which exists between the CH1 and CH2 regions and functions to provide flexibility to the antigen-binding site within the antibody. 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.

[0110] In one aspect, the present invention relates to an isolated nucleic acid molecule encoding an antibody of the present invention or a fragment thereof having immunological activity, a vector comprising the same, and a host cell transformed with the vector.

[0111] The nucleic acid molecules of the present invention may be isolated or recombinant, and include DNA and RNA in single-stranded and double-stranded forms, as well as corresponding complementary sequences. An isolated nucleic acid is a nucleic acid that has been separated from the surrounding genetic sequence present in the genome of the organism 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, the nucleic acid resulting from such a procedure may be understood as an isolated nucleic acid molecule. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of a separate fragment or as a component of a larger nucleic acid construct. A nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For example, the DNA of a pre-sequence or secretory leader is operably linked to the DNA of a polypeptide if the polypeptide is expressed as a preprotein, i.e., the form in which the polypeptide is secreted; a promoter or enhancer is operably linked to a coding sequence if it influences the transcription of the polypeptide sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Operably linked generally means that the DNA sequences to be linked are contiguous, and in the case of a secretory leader, contiguous and within the same reading frame. However, enhancers need not be contiguous. Linkage is accomplished by ligation at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in a conventional manner.

[0112] The isolated nucleic acid molecule encoding the antibody of the present invention or a fragment thereof having immunological activity may be modified in various ways in the coding region without changing the amino acid sequence of the antibody expressed from the coding region due to codon degeneracy or in consideration of the codons preferred in the organism to which the antibody is to be expressed. It will be readily understood by those skilled in the art that various modifications or alterations may be made in a portion other than the coding region without affecting the expression of the gene, and that such modified genes are also included in the scope of the present invention. That is, the nucleic acid molecule of the present invention may be modified by substitution, deletion, insertion, or a combination of one or more nucleic acid bases, as long as it encodes a protein having an activity equivalent thereto, 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.

[0113] An isolated nucleic acid molecule encoding an antibody of the present invention or a fragment thereof having immunological activity according to the present invention can be inserted into an expression vector for protein expression. The expression vector typically comprises the protein operably linked, i.e., in a functional relationship, with regulatory sequences, a selectable marker, an optional fusion partner, and / or additional elements. Under appropriate conditions, the antibody of the present invention or a fragment thereof having immunological activity can be produced by culturing a host cell transformed with the nucleic acid, preferably an expression vector containing an isolated nucleic acid molecule encoding an antibody of the present invention or a fragment thereof having immunological activity, to induce protein expression. Various suitable host cells can be used, including, but not limited to, mammalian cells, bacteria, insect cells, and yeast. Methods for introducing exogenous nucleic acids into host cells are well known in the art and will vary depending on the host cell used. Preferably, Escherichia coli, which has low production costs and high industrial value, can be used as the host cell for production.

[0114] The vector of the present invention includes, but is not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, a viral vector, and the like. A suitable vector may include, in addition to expression control elements such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, and an enhancer, a signal sequence or a leader sequence for membrane targeting or secretion, and may be manufactured in various ways depending on the purpose. The promoter of the vector may be constitutive or inducible. The signal sequence may include, but is not limited to, a PhoA signal sequence, an OmpA signal sequence, etc. when the host is an Escherichia sp. fungus; an α-amylase signal sequence, a subtilisin signal sequence, etc. when the host is a Bacillus sp. fungus; an MFα signal sequence, a SUC2 signal sequence, etc. when the host is a yeast; and an insulin signal sequence, an α-interferon signal sequence, an antibody molecule signal sequence, etc. when the host is an animal cell. Additionally, the vector may include a selection marker for selecting host cells containing the vector, and, if it is a replicable expression vector, an origin of replication.

[0115] 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. The nucleic acid sequence may be exogenous or heterologous. Examples of vectors include, but are not limited to, plasmids, cosmids, and viruses (e.g., bacteriophages). Those skilled in the art can construct vectors using standard recombinant techniques.

[0116] In one embodiment, when producing the vector, expression control sequences such as promoters, terminators, enhancers, etc., sequences for membrane targeting or secretion, etc. may be appropriately selected and combined in various ways according to the purpose, depending on the type of host cell to be used to produce the antibody.

[0117] As used herein, the term "expression vector" refers to a vector containing 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. Expression vectors may contain various regulatory sequences. In addition to regulatory sequences that regulate transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that provide additional functions.

[0118] 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. The host cell may be transfected or transformed by the vector, which refers to the process by which an exogenous nucleic acid molecule is transferred or introduced into the host cell.

[0119] In one embodiment, the host cell may be a bacterial or an animal cell, the animal cell line may be a CHO cell, a HEK cell, or a NSO cell, and the bacteria may be E. coli.

[0120] In one aspect, the present invention relates to a method for producing an antibody or a fragment having immunological activity thereof specific for a human coronavirus or a variant thereof, comprising the steps of: a) culturing a host cell comprising a vector comprising an antibody or a fragment having immunological activity thereof specific for the isolated human coronavirus or a variant thereof; and b) recovering the antibody or a fragment having immunological activity thereof from the host cell culture.

[0121] In one embodiment, the method may further comprise a step of purifying the antibody or a fragment thereof having immunological activity after the step of recovering the antibody or a fragment thereof having immunological activity from the host cell culture.

[0122] The antibodies of the present invention or fragments thereof having immunological activity can be isolated or purified using various methods known in the art. Standard purification methods include chromatography, electrophoresis, immunoassays, precipitation, dialysis, filtration, concentration, and chromatofocusing techniques. As is known in the art, various natural proteins, such as bacterial proteins A, G, and L, bind to antibodies and can be used for purification. Often, purification using specific fusion partners may be possible.

[0123] The antibody according to the present invention or a fragment thereof having immunological activity can be mass-produced by transforming the vector according to the present invention into an appropriate host cell, such as E. coli or yeast cells, and then culturing the transformed host cell. Appropriate culture methods and medium conditions depending on the type of host cell can be easily selected by those skilled in the art from known techniques. The host cell may be a prokaryotic organism such as E. coli or Bacillus subtilis. In addition, it may be a eukaryotic cell derived from yeast such as Saccharomyces cerevisiae, insect cells, plant cells, or animal cells. More preferably, the animal cell may be an autologous or allogeneic animal cell. A transformant produced by introducing the vector into an autologous or allogeneic animal cell may be administered to a subject and used for cell therapy to treat cancer, etc. Any method known to those skilled in the art may be used for introducing the vector into the host cell. Transgenic (e.g., genetically engineered) mice, or other organisms, including other mammals, can be used to produce antibodies of the invention or immunologically active fragments thereof (see, e.g., US 6,300,129). For example, it is known that engineered mice in which only the variable regions of mouse immune genes (heavy chain V, D, and J segments, and light chain V and J segments) are replaced with corresponding human variable sequences can be used to mass-produce high-affinity antibodies having human variable sequences.

[0124] In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating human coronavirus infection, comprising an antibody or a fragment thereof having immunological activity specific to the human coronavirus or a variant thereof of the present invention.

[0125] In one embodiment, the human coronavirus infection may be Middle East respiratory syndrome (MERS), severe acute respiratory syndrome (SARS), or coronavirus disease 2019 (COVID-19).

[0126] In one embodiment, COVID-19 may be caused by infection with SARS-CoV-2 wild type, SARS-CoV-2 alpha variant, SARS-CoV-2 beta variant, SARS-CoV-2 gamma variant, SARS-CoV-2 delta variant, SARS-CoV-2 kappa variant, or SARS-CoV-2 mu variant.

[0127] In the present invention, the term “prevention” means any act of inhibiting or delaying the occurrence, spread, and recurrence of human coronavirus infection by administering a composition according to the present invention.

[0128] The term "treatment" as used herein refers to any action that improves or beneficially alters the symptoms of human coronavirus infection and its complications through administration of a composition according to the present invention. Those skilled in the art will be able to reference materials provided by the Korean Medical Association and other sources to determine the precise criteria for diseases for which the composition of the present invention is effective, and to determine the degree of improvement, enhancement, and treatment.

[0129] The term "therapeutically effective amount" used in combination with the active ingredient in the present invention refers to an amount effective in preventing or treating human coronavirus infection. The therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as the administration method, target site, and patient condition. Therefore, the dosage for human use should be determined as an appropriate amount, taking both safety and efficacy into consideration. It is also possible to estimate the amount for human use from the effective amount determined through animal testing.

[0130] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects. The effective dosage level may be determined based on factors including the patient's health condition, the severity of the human coronavirus infection, the activity and sensitivity of the drug, the method of administration, the time of administration, the route of administration and excretion rate, the duration of treatment, drugs used in combination or simultaneously, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that achieves the maximum effect with the minimum amount without side effects, and this can be easily determined by those skilled in the art.

[0131] The pharmaceutical composition of the present invention may include a carrier, diluent, excipient, or a combination of two or more thereof commonly used in biological preparations. The term "pharmaceutically acceptable" as used herein means that the composition exhibits a property of not being toxic to cells or humans exposed to the composition. The carrier is not particularly limited as long as it is suitable for delivering the composition in vivo, and for example, compounds described in Merck Index, 13th ed., Merck & Co. Inc., saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed and used. If necessary, other common additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into a main use form such as an aqueous solution, suspension, or emulsion, pills, capsules, granules, or tablets. Furthermore, it can be formulated preferably according to each disease or ingredient using an appropriate method in the field.

[0132] In one embodiment, the pharmaceutical composition may be in one or more dosage forms selected from the group consisting of oral dosage forms, topical preparations, suppositories, sterile injectable solutions and sprays, with oral or injectable dosage forms being more preferred.

[0133] The term "administration" used in the present invention means providing a predetermined substance to an individual or patient by any appropriate method, and may be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally, or locally in the form of an injection) or orally depending on the intended method, and the dosage range varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease. Liquid preparations for oral administration of the composition of the present invention include suspensions, oral solutions, emulsions, syrups, etc., and may include various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc. The pharmaceutical composition of the present invention may be administered by any device that allows the active substance to move to target cells. Preferred administration methods and formulations include intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, and drip injection. Injections can be manufactured using aqueous solvents such as saline solution and Ringer's solution, non-aqueous solvents such as vegetable oil, higher fatty acid ester (e.g., ethyl oleate, etc.), alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.), and pharmaceutical carriers such as stabilizers to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers to adjust pH, and preservatives to inhibit microbial growth (e.g., phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

[0134] The term "subject" used in the present invention means any animal, including a monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, bat, camel, rat, rabbit or guinea pig, including a human, that has developed or may develop the human coronavirus infection, and the "specimen" may be droplets, sputum, whole blood, plasma, serum, urine or saliva isolated therefrom.

[0135] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable additive. At this time, the pharmaceutically acceptable additive may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, maltose, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharmaceutically acceptable additive according to the present invention is preferably included in the composition in an amount of 0.1 to 90 parts by weight, but is not limited thereto.

[0136] In one aspect, the present invention relates to a composition for detecting a human coronavirus or a variant thereof, comprising an antibody specific for a human coronavirus or a variant thereof or a fragment thereof having immunological activity.

[0137] The antibodies or immunologically active fragments thereof used in the detection (diagnostic) compositions of the present invention are preferably detectably labeled. Various methods for labeling biomolecules are well known to those skilled in the art and are contemplated within the scope of the present invention. Labeling methods and many other labels are known to those skilled in the art. Examples of labels that can be used in the present invention include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds, and bioluminescent compounds.

[0138] Commonly used labels include fluorescent substances (e.g., fluorescein, rhodamine, Texas Red, etc.), enzymes (e.g., horseradish peroxidase, β-galactosidase, alkaline phosphatase), radioisotopes (e.g., 32P or 125I), biotin, digoxigenin, colloidal metals, chemiluminescent or bioluminescent compounds (e.g., dioxetane, luminol, or acridinium). Labeling methods such as covalent attachment of enzymes or biotinyl groups, iodination, phosphorylation, and biotinylation are well known in the art.

[0139] Detection methods include, but are not limited to, autoradiography, fluorescence microscopy, and direct and indirect enzymatic reactions. Commonly used detection assays include radioisotope or non-radioisotope methods. These include Western blotting, overlay assays, radioimmuno assays (RIAs) and immune radioimmunometric assays (IRMAs), enzyme immunoassays (EIAs), enzyme-linked immunosorbent assays (ELISAs), fluorescent immunoassays (FIAs), and chemioluminescent immune assays (CLIAs).

[0140] In one aspect, the present invention relates to a kit for detecting human coronavirus or variants thereof comprising the composition.

[0141] In one embodiment, the kit may include a composition 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.

[0142] In one embodiment, detection of the antigen-antibody complex can be achieved by immunoelectrophoresis such as Ouchterlony plate, Western blot, Crossed IE, Rocket IE, Fused Rocket IE, Affinity IE, which can simply detect antibodies and / or antigens through antigen-antibody binding. The reagents or substances used in these methods are known, and can be detected, for example, through antigen-antibody reaction, reaction with a substrate that specifically binds to the antigen, a nucleic acid or peptide aptamer, a receptor or ligand that interacts with the complex, or reaction with a cofactor, or using a mass spectrometer. The reagents or substances that specifically interact or bind with the antigen-antibody complex of the present disclosure can be used in a chip format or with nanoparticles. By analyzing the intensity of the final signal through the above-described immunoassay process, i.e., performing a signal comparison with a normal sample, the presence or absence of coronavirus infection can be diagnosed by detecting the coronavirus in the subject.

[0143] In one aspect, the present invention relates to a method for detecting a human coronavirus or variant thereof, comprising the steps of: contacting a sample isolated from a specimen with an antibody of the present invention or a fragment thereof having immunological activity; and detecting the formation of an antigen-antibody complex.

[0144] In one embodiment, the human coronavirus may be Severe Acute Respiratory Syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), or SARS-CoV-2, more preferably SARS-CoV-1 or SARS-CoV-2.

[0145] In one embodiment, the variant may be an alpha variant (B.1.1.7), a beta variant (B.1.351), a gamma variant (P.1), a delta variant (B.1.617.2), a kappa variant (B.1.617.1) or a mu variant (B.1.1.529) of SARS-CoV-2, more preferably a delta variant (B.1.617.2).

[0146] In one aspect, the present invention relates to a method for providing information necessary for diagnosing human coronavirus infection, comprising the steps of: contacting a sample isolated from a specimen with an antibody of the present invention or a fragment thereof having immunological activity to form an antigen-antibody complex; and detecting the formation of the complex.

[0147] In one embodiment, the human coronavirus may be Severe Acute Respiratory Syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), or SARS-CoV-2, more preferably SARS-CoV-1 or SARS-CoV-2.

[0148] In one embodiment, the variant may be an alpha variant (B.1.1.7), a beta variant (B.1.351), a gamma variant (P.1), a delta variant (B.1.617.2), a kappa variant (B.1.617.1) or a mu variant (B.1.1.529) of SARS-CoV-2, more preferably a delta variant (B.1.617.2).

[0149] In addition, the present invention provides a method for preventing or treating human coronavirus infection, comprising the step of administering to a subject an antibody or a fragment thereof having immunological activity specific to the human coronavirus or a variant thereof.

[0150] In one embodiment, the human coronavirus may be Severe Acute Respiratory Syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), or SARS-CoV-2, more preferably SARS-CoV-1 or SARS-CoV-2.

[0151] In the method for preventing or treating human coronavirus infection of the present invention, the "subject" may be an individual requiring treatment for human coronavirus infection, or an individual at risk of developing human coronavirus infection. The "subject" may refer to any animal, including humans.

[0152] In the method for preventing or treating human coronavirus infection of the present invention, the specific description is the same as above.

[0153] The present invention is described in more detail through the following examples. However, the following examples are intended only to concretize the content of the present invention and are not intended to limit the present invention.

[0154]

[0155] Example 1. Production of antibodies against SARS-CoV-2 RBD

[0156] 1-1. Production of RBD (Receptor Binding Domain) Dimeric Antigen of SARS-CoV-2 Virus

[0157] In order to create an antibody that binds to the RBD (Receptor Binding Domain) region known to bind to the surface of the ACE2 (Angiotensin converting enzyme 2) receptor present in the host cell to infect the host cell, the RBD region of the SARS-CoV-2 virus was prepared as an antigen. In addition, in order to create an antigen more suitable for antibody screening using a flow cytometer, vectors for animal cell expression were created to create a dimeric antigen by fusing a GST tag to the antigen. (Gibco, 12338-018) After mixing the vector for animal cell expression in 30 ml, PEI (Polyethylenimine) (Polyscience, 23966) and each vector were mixed in a 4:1 ratio, left at room temperature for 20 minutes, and then incubated with 2 × 10 6After transfection into Expi293F animal cells subcultured at a density of 300 ml cells / ml, the cells were incubated in a shaking CO2 incubator at 37°C, 125 rpm, and 8% CO2 for 7 days, centrifuged, and only the supernatant was collected. After equilibration with 25x PBS, the medium was filtered through a 0.2 μm filter (Merck Millipore) using a bottle top filter, and 1 ml of GSH resin was added to the filtered culture medium, which was stirred at 4°C for 16 hours. Afterwards, the resin was recovered through a column, washed with 5 ml of PBS, and eluted with 2.5 ml of 50 mM Tris-HCl (pH 8.0) and 10 mM reduced glutathione (SIGMA). After buffer exchange with PBS using 3K centrifugal filter units (Merck Millipore), the purified SARS-CoV-2 RBD-GST antigen protein was analyzed for size and purity under non-reducing (NR) and reducing (R) conditions using SDS-PAGE (Fig. 1). In addition, the GST-fused dimeric antigen protein SARS-CoV-2 RBD-GST was labeled with an Alexa488 fluorescent molecule for use in screening using a flow cytometer.

[0158]

[0159] 1-2. Construction of a large error-prone library of anti-SARS-CoV-2 scFv antibodies.

[0160] To further enhance the binding affinity of antibodies S.IJ4, S.IJ24, and S.IJ39, which were previously evolved by the present inventors to bind to SARS-CoV-2 using the anti-SARS-CoV-1 antibody S230, to the SARS-CoV-2 RBD through affinity maturation, the genomes of each scFv antibody were amplified using Error Prone PCR technique with TaqPolymerase (TaKaRa), dNTPs (Invitrogen), MgCl2, and MnCl2 (SIGMA) to generate 0.3% mutations. The amplified genomes were treated with SfiI (New England BioLab) restriction enzyme, inserted into the SfiI-treated pMopac12-NlpA-FLAG vector using T4 DNA Ligase (enzynomics), and then transformed into JudeI cells. After spreading the transformed E. coli on solid medium, they were cultured at 37°C for 16 hours, recovered in TB medium containing 2% glucose, and each 2×10 8 We have secured an initial library of size.

[0161]

[0162] 1-3. Screening of human antibody variants

[0163] 1 ml each of S.IJ4, S.IJ24 and S.IJ39 library cells prepared in Example 1-2 were cultured in 25 ml TB medium containing 2% glucose and 40 μg / ml chloramphenicol at 37°C and 250 rpm for 4 hours, and the cultured cells were dispensed into 100 ml TB medium containing 40 μg / ml chloramphenicol at a ratio of 1:100 to determine the OD 600 = After culturing to 0.5, cooling was performed at 25℃ and 250 rpm for 20 minutes, and 1 mM IPTG was added and induction was performed at 25℃ and 250 rpm for 5 hours to produce E. coli with overexpressed protein. 600= 8 was obtained as the standard. After that, the cells were resuspended using 1 ml of 10 mM Tris-HCl (pH 8.0) to make them into spheroplasts suitable for screening using a flow cytometer, washed twice to remove the remaining medium, and the outer cell membrane was removed by applying osmotic shock through rotation in 1 ml of STE [0.5 M sucrose, 10 mM Tris-HCl, 10 mM EDTA (pH 8.0)] solution at 37°C for 30 minutes. The peptidoglycan layer was removed by rotation in a solution containing 1 ml of Solution A and 20 μl of 50 mg / ml lysozyme solution at 37°C for 15 minutes. After this, 1 ml of PBS was washed, and 300 ㎕ of this was added to 700 ㎕ of PBS and 200 nM (based on monomer) of antigen protein (SARS-CoV-2 RBD-GST-Alexa488 produced in Example 1-1 above) and rotated at room temperature for 1 hour to label the spheroplasts with a fluorescent probe. Clones showing high fluorescence due to increased antigen binding were recovered using flow cytometry, and scFv genes were amplified by PCR method from the recovered clones, and the process of culturing, expression induction, spheroplasting to remove the outer membrane and peptidoglycan layer, antigen labeling, and selective gating of the flow cytometer was repeated to select spheroplasts showing improved affinity for the RBD region of SARS-CoV-2 than before. After the selection process using a flow cytometer and the re-selection process to increase purity, the selection process was followed by an enrichment process, a process to secure the selected scFv variant gene through PCR amplification, a subcloning process, and a transformation process, and the next round of selection was performed to enrich the desired clones.Through this repeated multiple rounds of exploration, scFv variant clones with improved affinity for the ARS-CoV-2 RBD region were obtained (Fig. 2).

[0164]

[0165] 1-4. Confirmation of amplification of scFv variants

[0166] Each round library prepared in the manner of Example 1-3 and the scFv-type S230 and parent antibodies S.IJ4, S.IJ24, and S.IJ39 as controls were prepared as spheroplasts, and the SARS-CoV-2 RBD-GST-Alexa488 antigen used for screening was bound at a concentration of 100 nM (based on monomer) and incubated at room temperature for 1 hour. After washing twice with PBS to remove non-specific binding to the antigen, flow cytometry analysis was performed, and it was confirmed that as the rounds progressed, variants with enhanced binding affinity to the SARS-CoV-2 RBD region were amplified in the library (Fig. 3).

[0167]

[0168] 1-5. Gene sequence analysis of scFv antibody variants

[0169] In order to confirm the gene sequence of the scFv variant clones obtained in Example 1-3, the DNA base sequence was analyzed using Sanger sequencing, and through the analysis, four scFv antibody variants having antibody sequences, IJ427, IJ452, IJ2416, and IJ2483, were selected (Fig. 4).

[0170]

[0171] 1-6. Binding analysis of the SARS-CoV-2 RBD region

[0172] To confirm the binding affinity of the four scFv antibody variants selected in Example 1-5 to the SARS-CoV-2 RBD region, a flow cytometry-based binding analysis was performed. To this end, each variant, the scFv form S230 to be used as a control, and the parent antibodies S.IJ4 and S.IJ24 were prepared as spheroplasts, and the SARS-CoV-2 RBD-GST-Alexa488 antigen was incubated at a concentration of 34 nM (based on monomer) at room temperature for 1 hour. After washing twice with PBS to remove non-specific binding to the antigen, the binding affinity to the SARS-CoV-2 RBD region was analyzed using a flow cytometer. As a result of the analysis, the four scFv variants showed significantly increased fluorescence signals compared to the control group S230 and the parent antibodies S.IJ4 and S.IJ24, confirming that scFv antibody variants with enhanced binding affinity to the SARS-CoV-2 RBD region were selected (Fig. 5).

[0173]

[0174] 1-7. Production and purification of IgG type antibodies

[0175] To determine whether the four selected scFv antibody variants exhibit enhanced binding affinity to SARS-CoV-2 RBD even in the IgG form, heavy and light chain expression vectors for the four scFvs (IJ427, IJ452, IJ2416, and IJ2483) were constructed, respectively. Furthermore, IJ427, IJ452, IJ2416, and IJ2483 were divided into two groups based on their sequences, and IJ4G and IJ24G variants containing only common mutations in each group were derived, and heavy and light chain expression vectors for these were constructed, respectively. The heavy chain genes and light chain genes of IJ427, IJ452, IJ2416, IJ2483, IJ4G and IJ24G mutants were mixed in a 1:1 ratio, and PEI and mutant genes were mixed in a 4:1 ratio, left at room temperature for 20 minutes, and then 2×10 6Transfected Expi293F animal cells subcultured at a density of 10 cells / ml. After culturing for 7 days in a CO2 incubator at 37°C, 125 rpm, and 8% CO2, the supernatant was centrifuged and collected. Equilibrated with 25xPBS, the supernatant was filtered using a 0.2 μm syringe filter (Merck Millipore). 150 μl of Protein A resin was added to the filtered culture medium, stirred at room temperature for 1 hour, and then centrifuged to recover the protein-bound resin. The recovered resin was washed with 1 ml of PBS, eluted with 600 μl of 100 mM glycine (pH 2.7), neutralized with 1 M Tris-HCl (pH 8.0), and buffer exchanged with PBS was performed using centrifugal filter units 3K (Merck Millipore). The size and purity of the four proteins purified under non-reducing and reducing conditions were analyzed using SDS-PAGE (Fig. 6) (IJ452 and IJ2483 mutants were not expressed).

[0176]

[0177] 1-8. Analysis of the binding affinity of IgG variants to the SARS-CoV-2 RBD

[0178] To confirm the binding affinity of antibodies to SARS-CoV-2 RBD on protein, ELISA experiments were performed on four antibodies (IJ427, IJ2416, IJ4G, and IJ24G) expressed and purified in Examples 1-7, S230, and the parent antibodies S.IJ4 and S.IJ24. A monomeric form of RBD fused with a His tag was diluted to 4 μg / ml in 0.05 M Na2CO3, pH 9.6, and 50 μl each was immobilized in a Flat Bottom Polystyrene High Bind 96-well microplate (costar) at 4 °C for 16 h, and then blocked with 100 μl of 4% skim milk (GenomicBase) (in 0.05% PBST, pH 7.4) at room temperature for 2 h. After washing four times with 150 μl of 0.05% PBST, 50 μl of seven antibodies (IJ427, IJ2416, IJ4G, IJ24G, S230, S.IJ4, and S.IJ24) serially diluted from a concentration of 1 μM were dispensed into each well and reacted for 1 hour at room temperature. After washing, antibody reaction was performed with 50 μl of Anti-Protein A-HRP (GensScript) at room temperature for 1 hour and washed again. After adding 50 μl of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) to develop the color, the reaction was terminated by adding 50 μl of 2 M H2SO4, and the results of analysis using an Epoch Microplate Spectrophotometer (BioTek) confirmed that the four newly discovered antibodies had binding affinity to SARS-CoV-2 RBD that was up to 179 times higher than that of the existing S.IJ4 and S.IJ24 antibodies (Fig. 7).

[0179]

[0180] Example 2. Production of antibody variants against SARS-CoV-2 RBD

[0181] 2-1. Production and production of mutant antibodies with common mutations in the CDR region

[0182] Sequence analysis revealed that six antibodies, IJ4G, IJ427, IJ452, IJ24G, IJ2416, and IJ2483, which had enhanced binding affinity to the SARS-CoV-2 RBD region selected in Example 1, all had mutations at positions 57N in CDR2 and 107F in CDR3 (Fig. 4). It was expected that mutations at these two positions would induce enhanced binding affinity to the SARS-CoV-2 RBD region. To confirm this, heavy and light chain animal cell expression vectors of five antibodies having N57Y, N57H, F107Y, YY (N57Y / F107Y), or HY (N57H / F107Y) mutations at S230 were each constructed using the QuikChangeTM site-directed mutagenesis method. The animal cell expression vector constructed in Expi293F cells using the method described in Example 1-7 was transfected using PEI (Polyethylenimine) and cultured for 7 days, and then purified using 150 μl of Protein A resin. The size and purity of the three proteins purified under non-reducing and reducing conditions, excluding the non-expressed N57H and HY (N57H / F107Y) mutants, were analyzed using SDS-PAGE (Fig. 8).

[0183]

[0184] 2-2. Binding Affinity Analysis for the SARS-CoV-2 RBD Region

[0185] To measure the binding affinity of antibodies to SARS-CoV-2 RBD on protein, ELISA experiments were performed on three antibodies (N57Y, F107Y, and YY) expressed and purified in Example 2-1, and S230, S.IJ4, S.IJ24, IJ4G, IJ427, IJ24G, and IJ2416. RBD in monomeric form fused with a His tag was diluted to 4 μg / ml in 0.05 M Na2CO3, pH 9.6, and 50 μl each was immobilized in a Flat Bottom Polystyrene High Bind 96-well microplate (costar) at 4 °C for 16 hours, and then blocked with 100 μl of 4% skim milk (GenomicBase) (in 0.05% PBST, pH 7.4) at room temperature for 2 hours. After washing four times with 150 μl of 0.05% PBST, 50 μl of 10 antibodies (N57Y, F107Y, YY, S230, S.IJ4, S.IJ24, IJ4G, IJ427, IJ24G, and IJ2416) serially diluted from a concentration of 1 μM were dispensed into each well and reacted for 1 hour at room temperature. After washing, antibody reaction was performed with 50 μl of Anti-Protein A-HRP (GensScript) at room temperature for 1 hour and washed again. After adding 50 μl of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) to develop the color, the reaction was terminated by adding 50 μl of 2 M H2SO4, and the result was analyzed using an Epoch Microplate Spectrophotometer (BioTek). As a result, N57Y and F107Y showed a similar level of binding affinity to S.IJ4 and S.IJ24 antibodies before affinity maturation for the SARS-CoV-2 RBD region, but in the case of the YY variant with both mutations introduced, the binding affinity was similar to that of S.IJ4 and S.IJ24 antibodies before affinity maturation.It showed a significantly improved binding affinity than IJ24 (Fig. 9). In addition, IJ4G, IJ427, and YY mutants with YY introduced showed higher binding affinity than IJ24G and IJ2416 mutants with HY mutations introduced, confirming that the mutations N57Y and F107Y are important for binding to the RBD region (Fig. 9).

[0186]

[0187] 2-3. Binding affinity analysis for SARS-CoV-2 RBD

[0188] To measure the binding affinity of IJ4G and YY selected in the above examples to SARS-CoV-2 RBD, the Octet BLI system R8 (Sartorius) was used. Specifically, the NTA biosensor was equilibrated using 1× Kinetic Buffer, and 250 μl of SARS-CoV-2 RBD-His antigen at a concentration of 50 nM diluted in 1× Kinetic Buffer was immobilized for 180 s. Then, 250 μl of serially diluted antibodies were flowed for 600 s to bind to SARS-CoV-2 RBD, and the binding was dissociated for 600 s using 1× Kinetic Buffer. Fitting was performed using the standard equation with Octet BLI Analysis 12.2 software, and R 2 The kon, koff, and KD values ​​were calculated so that the values ​​were greater than 0.99. The analysis results confirmed that IJ4G and YY bound very strongly to SARS-CoV-2 RBD with binding affinities of 0.447 nM and 1.77 nM, respectively (Fig. 10).

[0189]

[0190] Example 3. Production of antibodies against the RBD of the SARS-CoV-2 delta variant virus.

[0191] 3-1. Production of RBD antigen of B.1.617.2 virus

[0192] In order to produce antigens in the dimeric form with a GST tag fused to the RBD region of the SARS-CoV-2 virus delta variant (B.1.617.2) virus and in the tetrameric form with a streptavidin tag fused to the RBD region, vectors for animal cell expression were constructed. The constructed animal cell expression vectors were transfected into Expi293F animal cells according to the method described in Example 1-1 and cultured for 7 days. 1 ml of GSH resin and Ni-NTA resin were added to the filtered culture medium, stirred at 4 °C for 16 hours, and then passed through a column to recover the resin, which was washed with 5 ml of PBS and 5 ml of 10 mM and 20 mM imidazole, respectively. After washing, the mixture was eluted with 2.5 ml of 50 mM Tris-HCl (pH 8.0) + 10 mM reduced glutathione (SIGMA) and 250 mM imidazole, respectively. After buffer exchange with PBS using centrifugal filter units 3K (Merck Millipore), the size and purity of each purified antigen protein under non-reducing (NR) and reducing (R) conditions were analyzed using SDS-PAGE (Fig. 11).

[0193]

[0194] 3-2. Human Antibody Variant Screening

[0195] In Example 3-1, the streptavidin-fused tetrameric antigen protein SARS-CoV-2 B.1.617.2 RBD-Streptavidin produced was labeled with an Alexa488 fluorescent molecule, and the SARS-CoV-1 S230, m396, and 80R library cells previously produced by the present inventors (Korean Patent Publication No. 10-2022-0163625) were made into spheroplasts. Then, 200 nM (monomer basis) of the antigen protein SARS-CoV-2 B.1.617.2 RBD-Streptavidin-Alexa488 was added, and the spheroplasts were rotated for 1 hour at room temperature to label the fluorescent probe. Clones showing high fluorescence due to increased antigen binding were recovered using a flow cytometer, and multiple rounds of selection were performed to enrich the desired clones. Through repeated rounds of screening, scFv variant clones with affinity for the ARS-CoV-2 B.1.617.2 RBD region were obtained (Fig. 12).

[0196]

[0197] 3-3. Confirmation of amplification of scFv variants

[0198] Each round library constructed in Example 3-2 and the parent antibodies S230, m396, and 80R in scFv form were prepared as spheroplasts. The SARS-CoV-2 B.1.617.2 RBD-Streptavidin-Alexa488 antigen used for screening was conjugated to the prepared spheroplasts at a concentration of 100 nM (based on monomers) and incubated at room temperature for 1 hour. After washing twice with PBS to remove non-specific binding to the antigen, it was confirmed that as the flow cytometry rounds progressed, variants with enhanced binding affinity to the SARS-CoV-2 B.1.617.2 RBD region were amplified from the library (Fig. 13).

[0199]

[0200] 3-4. Gene sequence analysis of scFv variants

[0201] In order to confirm the gene sequence of the scFv variant clones obtained in Example 3-2, the DNA base sequence was analyzed using Sanger sequencing, and through the analysis, four scFv antibody variants (IJ30, IJ37, IJ64, and IJ73) having antibody sequences were selected (Fig. 14).

[0202]

[0203] 3-5. Binding Affinity Analysis for the RBD Region of SARS-CoV-2 B.1.617.2

[0204] To confirm the binding affinity of the four scFv antibody variants selected in Examples 3-4 to the SARS-CoV-2 B.1.617.2 RBD region, a flow cytometry-based binding analysis was performed. To this end, the four variants and the scFv form S230 to be used as a control were prepared as spheroplasts, and the SARS-CoV-2 B.1.617.2 RBD-Streptavidin-Alexa488 antigen used for screening was incubated at a concentration of 70 nM (based on monomers) at room temperature for 1 hour. After washing twice with PBS to remove non-specific binding to the antigen, the binding affinity to the SARS-CoV-2 B.1.617.2 RBD region was analyzed using a flow cytometer. As a result of the analysis, the four scFv variants showed a significantly increased fluorescence signal compared to the control scFv form S230, confirming that scFv antibody variants with binding affinity to the SARS-CoV-2 B.1.617.2 RBD region were selected (Fig. 15).

[0205]

[0206] 3-6. Production and purification of IgG type antibodies

[0207] To confirm whether the four scFv antibody variants selected in Examples 3-4 also have enhanced binding affinity to SARS-CoV-2 B.1.617.2 RBD in the IgG form, heavy and light chain expression vectors were constructed, respectively. In addition, heavy and light chain expression vectors were constructed to additionally express the IJ2 variant containing only common mutations in the sequence of the four selected antibodies in the IgG form. The animal cell expression vectors constructed by the method described in Examples 1-7 were transfected into Expi293F cells using PEI (Polyethylenimine), cultured for 7 days, and then purified using 150 μl of Protein A resin. The size and purity of the five proteins (IJ30, IJ37, IJ64, IJ73, and IJ2) purified under non-reducing (NR) and reducing (R) conditions were analyzed by SDS-PAGE (Fig. 16).

[0208]

[0209] 3-7. Analysis of the binding affinity of IgG variants to the RBD region of SARS-CoV-2 B.1.617.2.

[0210] To measure the binding affinity of antibodies to SARS-CoV-2 B.1.617.2 RBD on protein, ELISA experiments were performed on the five antibodies expressed and purified in Examples 3-6 and the parent antibody S230. The monomeric form of B.1.617.2 RBD fused with a His tag was diluted to 4 μg / ml in 0.05 M Na2CO3, pH 9.6, and 50 μl was immobilized in a Flat Bottom Polystyrene High Bind 96-well microplate (costar) at 4 °C for 16 h, and then blocked with 100 μl of 4% skim milk (GenomicBase) (in 0.05% PBST, pH 7.4) at room temperature for 2 h. After washing four times with 150 μl of 0.05% PBST, 50 μl of five types of antibodies serially diluted from 1 μM concentration were dispensed into each well and reacted for 1 hour at room temperature. After washing, antibody reaction was performed for 1 hour at room temperature with 50 μl of Anti-Protein A-HRP (GensScript) and washed again. 50 μl of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added each time to develop color, and 50 μl of 2 M H2SO4 was added each time to terminate the reaction. The plate was then analyzed using an Epoch Microplate Spectrophotometer (BioTek). As a result, the five types of antibodies were found to have binding affinity for SARS-CoV-2 B.1.617.2 RBD, and among them, IJ2 antibody showed the highest binding affinity (Fig. 17).

[0211]

[0212] Example 4. Production of antibody variants against the RBD of the SARS-CoV-2 delta variant virus.

[0213] 4-1. Production of a large error-prone library of anti-SARS-CoV-2 B.1.617.2 scFv antibodies

[0214] In order to further enhance the binding affinity of the antibodies discovered in Example 3 to the RBD of the SARS-CoV-2 delta variant (B.1.617.2) through affinity maturation, IJ2 and IJ30, which had the highest binding affinity, were selected, and the genomes of each scFv antibody were amplified using the Error Prone PCR technique with TaqPolymerase (TaKaRa), dNTPs (Invitrogen), MgCl2, and MnCl2 (SIGMA) to generate 0.3% mutations. The amplified genomes were treated with SfiI (New England BioLab) restriction enzyme, inserted into the pMopac12-NlpA-FLAG vector treated with SfiI restriction enzyme using T4 DNA Ligase (enzynomics), and then transformed into JudeI cells. After spreading the transformed E. coli on solid medium, they were cultured at 37°C for 16 hours, recovered in TB medium containing 2% glucose, and each 2×10 8 We have secured an initial library of the size.

[0215]

[0216] 4-2. Human Antibody Variant Screening

[0217] In Example 3-1, the GST-fused dimeric antigen protein SARS-CoV-2 B.1.617.2 RBD-GST produced was labeled with an Alexa488 fluorescent molecule, and the IJ2 and IJ30 library cells produced in Example 4-1 were made into spheroplasts. Then, 100 nM (monomer basis) of the antigen protein SARS-CoV-2 B.1.617.2 RBD-GST-Alexa488 was added and rotated at room temperature for 1 hour to label the spheroplasts with a fluorescent probe. Clones showing high fluorescence due to increased antigen binding were recovered using a flow cytometer, and multiple rounds of a selection process were performed to amplify the desired clones. Through repeated multiple rounds of screening, scFv variant clones with improved affinity for the SARS-CoV-2 B.1.617.2 RBD region were secured (Fig. 18).

[0218]

[0219] 4-3. Confirmation of amplification of scFv variants

[0220] Each round library, prepared in the manner of Example 4-2, and S230 in scFv form, and parent antibodies IJ2 and IJ30 were prepared as spheroplasts. The SARS-CoV-2 B.1.617.2 RBD-GST-Alexa488 antigen used for screening was bound to the prepared spheroplasts at a concentration of 25 nM (based on monomers) and incubated at room temperature for 1 hour. After washing twice with PBS to remove non-specific binding to the antigen, flow cytometry analysis was performed. As a result, it was confirmed that variants with enhanced binding affinity to the SARS-CoV-2 B.1.617.2 RBD region were amplified in the library as the rounds progressed (Fig. 19).

[0221]

[0222] 4-4. Binding Affinity Analysis for the RBD Region of SARS-CoV-2 B.1.617.2

[0223] To confirm the binding affinity of the six scFv antibody variants (IJ203, IJ225, IJ228, IJ232, IJ235, and IJ274) selected in Example 4-3 to the SARS-CoV-2 B.1.617.2 RBD region, a binding affinity analysis was performed using a flow cytometer. For this purpose, each variant, the scFv form S230 to be used as a control, and the parent antibody S.IJ2 were prepared as spheroplasts. The SARS-CoV-2 B.1.617.2 RBD-GST-Alexa488 antigen used for screening was bound to the prepared spheroplasts at a concentration of 25 nM (based on monomer) and incubated at room temperature for 1 hour. After washing twice with PBS to remove non-specific binding to the antigen, the binding affinity to the SARS-CoV-2 B.1.617.2 RBD region was analyzed using a flow cytometer. The analysis results showed that the IJ225 variant exhibited a significantly increased fluorescence signal compared to the control scFv S230 and the parent antibody IJ2, and a much higher fluorescence signal than other newly discovered variants, confirming that it is an scFv antibody variant with greatly enhanced binding affinity to the SARS-CoV-2 B.1.617.2 RBD region (Fig. 20).

[0224]

[0225] 4-5. Gene sequence analysis of scFv IJ225

[0226] To confirm the gene sequence of the secured scFv variant IJ225, the DNA base sequence was analyzed using Sanger sequencing (Fig. 21).

[0227]

[0228] 4-6. Production and purification of IgG antibody of scFv IJ225

[0229] To determine whether the selected IJ255 scFv antibody variants also exhibit enhanced binding affinity to SARS-CoV-2 B.1.617.2 RBD in IgG form, heavy and light chain expression vectors were constructed, respectively. After antibody production in Expi293F cells using the method described in Example 1-7, the size and purity of purified IJ225 proteins under non-reducing (NR) and reducing (R) conditions were analyzed by SDS-PAGE (Fig. 22).

[0230]

[0231] 4-7. Analysis of the binding affinity of IgG antibodies to the RBD region of SARS-CoV-2 B.1.617.2.

[0232] To measure the binding affinity of antibodies to SARS-CoV-2 B.1.617.2 RBD on protein, ELISA experiments were performed on IJ225 expressed and purified in Examples 4-6, S230 as a control, and IJ2 as a parent antibody. B.1.617.2 RBD in a monomeric form fused with a His tag was diluted to 4 μg / ml in 0.05 M Na2CO3, pH 9.6, and 50 μl was immobilized in a Flat Bottom Polystyrene High Bind 96-well microplate (costar) at 4 °C for 16 h, and then blocked with 100 μl of 4% skim milk (GenomicBase) (in 0.05% PBST, pH 7.4) at room temperature for 2 h. After washing four times with 150 μl of 0.05% PBST, 50 μl of three types of antibodies (IJ225, S230, and IJ2) serially diluted from a concentration of 1 μM were dispensed into each well and reacted for 1 hour at room temperature. After washing, antibody reaction was performed with 50 μl of Anti-Protein A-HRP (GensScript) at room temperature for 1 hour and washed again. 50 μl of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added to develop color, and 50 μl of 2 M H2SO4 was added to terminate the reaction. The results of analysis using an Epoch Microplate Spectrophotometer (BioTek) confirmed that IJ225 had significantly improved binding affinity to SARS-CoV-2 B.1.617.2 RBD than IJ2 (Fig. 23).

[0233]

[0234] 4-8. Binding Affinity Analysis of IJ225 to SARS-CoV-2 B.1.617.2 RBD

[0235] To measure the binding affinity of IJ225 to SARS-CoV-2 B.1.617.2 RBD, an Octet BLI system R8 (Sartorius) was used. The NTA biosensor was equilibrated with 1× Kinetic Buffer, and 250 μl of 50 nM SARS-CoV-2 B.1.617.2 RBD-His antigen diluted in 1× Kinetic Buffer was immobilized for 180 s. Then, 250 μl of serially diluted antibody was flowed for 600 s to bind to SARS-CoV-2 B.1.617.2 RBD, and the binding was dissociated for 600 s using 1× Kinetic Buffer. Fitting was performed using the standard equation with Octet BLI Analysis 12.2 software, and R 2 The kon, koff, and KD values ​​were calculated such that the values ​​were greater than 0.99. The analysis results confirmed that IJ225 binds very strongly to SARS-CoV-2 B.1.617.2 RBD with a binding affinity of 1.23 nM (Fig. 24).

[0236]

[0237] Example 5. Confirmation of SARS-CoV-2 RBD cross-binding ability

[0238] 5-1. Cohesion analysis

[0239] In order to confirm the cross-binding ability of the IJ4G and YY antibodies evolved to bind to SARS-CoV-2 RBD in the present invention to bind to SARS-CoV-2 B.1.617.2 RBD, and to confirm the cross-binding ability of IJ225 evolved to bind to SARS-CoV-2 B.1.617.2 RBD to determine whether the antibodies bind to SARS-CoV-2 RBD, ELISA analysis was performed. The monomeric form of RBD fused to His tag was diluted to 4 μg / ml in 0.05 M Na2CO3 pH 9.6, and 50 μl was immobilized in a Flat Bottom Polystyrene High Bind 96-well microplate (costar) at 4 °C for 16 hours, and then blocked with 100 μl of 4% skim milk (GenomicBase) (in 0.05% PBST, pH 7.4) at room temperature for 2 hours. After washing four times with 150 μl of 0.05% PBST, 50 μl of three types of antibodies (IJ225, IJ4G, and YY) serially diluted from a concentration of 250 nM were dispensed into each well and reacted for 1 hour at room temperature. After washing, antibody reaction was performed with 50 μl of Anti-Protein A-HRP (GensScript) for 1 hour at room temperature and washed again. 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added at 50 μl each to develop color, and the reaction was terminated by adding 50 μl of 2 M H2SO4. The plate was then analyzed using an Epoch Microplate Spectrophotometer (BioTek). The analysis results confirmed that the IJ225 antibody binds strongly but weakly to SARS-CoV-2 RBD than YY, and that the IJ4G variant binds strongly to SARS-CoV-2 B.1.617.2 RBD than IJ225. However, the YY antibody was confirmed to bind very weakly to SARS-CoV-2 B.1.617.2 RBD (Fig. 25).

[0240]

[0241] 5-2. Binding affinity analysis

[0242] In the present invention, whether the IJ4G and YY antibodies evolved to bind to SARS-CoV-2 RBD bind to SARS-CoV-2 B.1.617.2 RBD (SEQ ID NO: 78) and whether the IJ225 antibodies evolved to bind to SARS-CoV-2 B.1.617.2 RBD bind to SARS-CoV-2 RBD (SEQ ID NO: 77) were confirmed by BLI analysis using the Octet BLI system R8 (Sartorius). The NTA biosensor was equilibrated using 1× Kinetic Buffer, and 250 μl of 50 nM RBD-His antigen diluted in 1× Kinetic Buffer was immobilized for 180 seconds. Afterwards, 250 μl of serially diluted antibody was flowed for 600 seconds to bind to SARS-CoV-2 B.1.617.2 RBD and the binding was dissociated for 600 seconds using 1× Kinetic Buffer. Fitting was performed using the standard equation with Octet BLI Analysis 12.2 software, and R 2 The kon, koff, and KD values ​​were calculated so that the values ​​were greater than 0.99. The analysis results showed that the IJ225 antibody bound strongly to SARS-CoV-2 B.1.617.2 RBD with a binding affinity of 1.30 nM, similar to that of SARS-CoV-2 RBD, and that the IJ4G antibody bound very strongly to B.1.617.2 RBD with a binding affinity of 0.231 nM. However, similar to the ELISA analysis results, the YY antibody was confirmed to have a weak binding affinity of 141 nM for SARS-CoV-2 B.1.617.2 RBD (Fig. 26).

[0243]

[0244] Example 6. Neutralization ability verification

[0245] The neutralizing ability of the antibodies discovered in the present invention was verified using the pseudovirus of SARS-CoV-2 and SARS-CoV-2 B.1.617.2 based on HIV-1 to determine whether they actually have neutralizing ability. After mixing the SARS-CoV-2 spike expression plasmid and the envelope and packaging plasmids in Opti-MEM culture medium, PEI (Polyethylenimine) (Polyscience, 23966) and plasmids were mixed in a 3:1 ratio and left at room temperature for 10 minutes, and then incubated with 8×10 5 After transfection into HEK293T cells cultured at a density of 10 cells / ml, the cells were cultured in a CO2 incubator at 37°C and 5% CO2 for 48 hours. After culture, only the supernatant was collected and filtered through a 0.45 μm syringe filter (Merck Millipore) to produce pseudoviruses. After mixing the ACE2 expression plasmid and TMPRSS2 expression plasmid in Opti-MEM culture medium, PEI and plasmid were mixed in a 3:1 ratio, left at room temperature for 10 minutes, and then incubated with 8 × 10 5 After transfection into HEK293T cells cultured at a density of 10 cells / ml, the cells were cultured in a CO2 incubator at 37°C and 5% CO2 for 48 hours. 100 μl of HEK293T cells expressing ACE2-TMPRSS2 were added to 2 × 10 5Cells / ml were dispensed into a white 96-well plate at a density of 10 cells / ml, and after 24 hours, 100 μl of a mixture of serially diluted antibodies and pseudoviruses incubated at 37°C for 1 hour was transferred to a 96-well plate containing ACE2-TMPRSS2-expressing HEK293T cells and incubated for 48 hours under 37°C and 5% CO2 conditions. After 48 hours, the supernatant was removed, and 20 μl of 1×CCLR (Cell Culture Lysis Reagent) (Promega) was added, followed by 100 μl luciferase assay system (Promega), and the luciferase luminescence was measured using a Micro plate reader. The measured luciferase signal was calculated by normalizing it to pseudovirus-positive wells (=100%) and ACE2-TMPRSS2 cell-negative wells (=0%) using the GraphPad Prism program. The analysis results showed that the IJ4G antibody had an IC of 0.77 nM against SARS-CoV-2. 50 , and has an IC of 3.832 nM against SARS-CoV-2 B.1.617.2. 50 It was confirmed that the YY antibody showed the highest neutralizing ability. The YY antibody neutralized only the SARS-CoV-2 pseudovirus, and IJ225 was confirmed to neutralize both SARS-CoV-2 and SARS-CoV-2 B.1.617.2 pseudoviruses (Fig. 27).

[0246]

[0247] Example 7. SARS-CoV-1 cross-binding assay

[0248] To determine whether anti-SARS-CoV-1 antibodies S230, IJ4G and YY, evolved to bind to SARS-CoV-2 RBD, and IJ225, evolved to bind to SARS-CoV-2 B.1.617.2 RBD, could bind to SARS-CoV-1 like the parent antibody, we performed an ELISA assay. The monomeric form of SARS-CoV-1 RBD (SEQ ID NO: 79) fused to a His tag was diluted to 4 μg / ml in 0.05 M Na2CO3, pH 9.6, and 50 μl each was immobilized in a Flat Bottom Polystyrene High Bind 96-well microplate (costar) at 4 °C for 16 h, and then blocked with 100 μl of 4% skim milk (GenomicBase) (in 0.05% PBST, pH 7.4) at room temperature for 2 h. After washing four times with 150 μl of 0.05% PBST, 50 μl of three types of antibodies (IJ225, IJ4G, and YY) serially diluted starting from a concentration of 250 nM were dispensed into each well and reacted for 1 hour at room temperature. After washing, antibody reaction was performed with 50 μl of Anti-Protein A-HRP (GensScript) for 1 hour at room temperature and washed again. 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added at 50 μl each to develop color, and the reaction was terminated by adding 50 μl of 2 M H2SO4. The plates were then analyzed using an Epoch Microplate Spectrophotometer (BioTek). The analysis results confirmed that all antibodies bound well to SARS-CoV-1 RBD, similar to the parent antibody, S230 (Fig. 28).

Claims

1. An antibody or a fragment thereof having immunological activity specific for human coronavirus or its variants.

2. In paragraph 1, the human coronavirus is SARS-CoV-1 (Severe Acute Respiratory Syndrome coronavirus), MERS-CoV (Middle East respiratory syndrome coronavirus) or SARS-CoV-2, an antibody or a fragment having immunological activity thereof.

3. In paragraph 1, the variant is an antibody or a fragment thereof having immunological activity, which is an alpha variant, a beta variant, a gamma variant, a delta variant, a kappa variant, or a mu variant of SARS-CoV-2.

4. In paragraph 1, the fragment having immunological activity is any one selected from the group consisting of Fab, Fd, Fab', dAb, F(ab'), F(ab')2, scFv (single chain fragment variable), Fv, single-chain antibody, Fv dimer, complementarity determining region fragment, humanized antibody, chimeric antibody, and diabody, an antibody or a fragment having immunological activity thereof.

5. An antibody or a fragment thereof having immunological activity, comprising a VH domain comprising CDRH (Complementarity determining regions Heavy chain) 1 comprising an amino acid sequence of SEQ ID NO: 1 or 2, CDRH2 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NO: 3 to 10, and CDRH3 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NO: 11 to 16, in the first paragraph.

6. An antibody or a fragment thereof having immunological activity, comprising a VH domain, wherein the VH domain comprises FR1 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 25 to 27, FR2 comprising the amino acid sequence of SEQ ID NOs: 28 or 29, FR3 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 30 to 31, and FR4 comprising the amino acid sequence of SEQ ID NOs: 36 or 37, in accordance with claim 1.

7. An antibody or a fragment thereof having immunological activity, comprising a VL domain comprising CDRL1 selected from the group consisting of amino acid sequences of SEQ ID NOs: 17 to 19, CDRL2 including the amino acid sequence of SEQ ID NOs: 20 or 21, and CDRL3 including the amino acid sequence of SEQ ID NOs: 22 to 24, in accordance with claim 1.

8. An antibody or a fragment thereof having immunological activity, comprising a VL domain, wherein the VL domain comprises FR1 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 38 to 40, FR2 comprising the amino acid sequence of SEQ ID NO: 41, FR3 comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 42 to 47, and FR4 comprising the amino acid sequence of SEQ ID NO: 48, in the first paragraph.

9. An antibody or a fragment thereof having immunological activity, comprising a VH domain selected from the group consisting of amino acid sequences of SEQ ID NOs: 49 to 61, in claim 1.

10. An antibody or a fragment thereof having immunological activity, comprising a VL domain selected from the group consisting of amino acid sequences of SEQ ID NOs: 62 to 72, in claim 1.

11. In paragraph 1, an antibody or a fragment thereof having immunological activity that specifically binds to the RBD (Receptor Binding Domain) of human coronavirus.

12. In claim 1, an antibody or a fragment thereof having immunological activity that is cross-reactive with human coronavirus.

13. In claim 12, an antibody or a fragment thereof having immunological activity that cross-reacts with the RBD of SARS-CoV-1 and the RBD of SARS-CoV-2.

14. In claim 13, an antibody or a fragment thereof having immunological activity, which is cross-reactive with the RBD of SARS-CoV-1, the RBD of SARS-CoV-2, and the RBD of a variant thereof.

15. In claim 1, an antibody or a fragment thereof having immunological activity, which is a neutralizing antibody.

16. An antibody or a fragment thereof having immunological activity that neutralizes SARS-CoV-2 or a variant thereof in accordance with claim 15.

17. An isolated nucleic acid molecule encoding the antibody of claim 1 or a fragment thereof having immunological activity.

18. A vector comprising the isolated nucleic acid molecule of claim 17.

19. A host cell transformed with the vector of clause 18.

20. A pharmaceutical composition for preventing or treating human coronavirus infection, comprising an antibody specific for the human coronavirus or a variant thereof of paragraph 1 or a fragment thereof having immunological activity.

21. A pharmaceutical composition for preventing or treating human coronavirus infection, wherein the human coronavirus infection in clause 20 is MERS (Middle East respiratory syndrome), SARS (Severe acute respiratory syndrome) or COVID-19 (Coronavirus disease 2019).

22. A composition for detecting human coronavirus or variants thereof, comprising an antibody specific for the human coronavirus or variants thereof of clause 1 or a fragment having immunological activity thereof.

23. A kit for detecting human coronavirus or its variants comprising the composition of claim 22. 24.(a) a step of contacting a sample separated from a specimen with the antibody of paragraph 1 or a fragment thereof having immunological activity; and (b) a method for detecting a human coronavirus or variant thereof, comprising the step of detecting antigen-antibody complex formation. 25.(a) a step of contacting a sample separated from a specimen with the antibody of paragraph 1 or a fragment thereof having immunological activity to form an antigen-antibody complex; and (b) A method for providing information necessary for diagnosing human coronavirus infection, comprising the step of detecting the formation of the complex.

26. A method for preventing or treating human coronavirus infection, comprising administering to a subject an antibody or a fragment thereof having immunological activity specific for the human coronavirus or a variant thereof of paragraph 1.

27. A method for preventing or treating human coronavirus infection in claim 26, wherein the human coronavirus infection is MERS (Middle East respiratory syndrome), SARS (Severe acute respiratory syndrome), or COVID-19 (Coronavirus disease 2019). 28.a) A step of culturing a host cell comprising a vector containing an antibody specific for the human coronavirus or a variant thereof of paragraph 1 or a fragment having immunological activity thereof; and b) A method for producing an antibody or a fragment thereof having immunological activity specific for a human coronavirus or a variant thereof, comprising the step of recovering the antibody or a fragment thereof having immunological activity from a host cell culture.

Images