Human antibody against coronavirus mutant strain, or antigen-binding fragment thereof

Abstract

The purpose of the present invention is to provide an antibody against a coronavirus (SARS-CoV-2) mutant strain, in particular, an omicron substrain. Moreover, another purpose of the present invention is to provide a pharmaceutical composition against coronavirus infections, the pharmaceutical composition using said antibody. The present invention provides: an antibody that binds to a spike protein of coronavirus and has the ability to neutralize coronavirus including an omicron substrain, or an antigen-binding fragment thereof; and a pharmaceutical composition for preventing or treating coronavirus infections, the pharmaceutical composition comprising said antibody or antigen-binding fragment thereof.

Description

Human antibodies or antigen-binding fragments thereof against coronavirus mutant strains

[0001] The present invention relates to an antibody or an antigen-binding fragment thereof against a novel coronavirus (SARS-CoV-2) mutant strain, and a therapeutic or prophylactic agent for coronavirus using the antibody.

[0002] Although the novel coronavirus disease (COVID-19) has been classified as Category 5 under the Infectious Diseases Control Act, the Omicron sublineage strain epidemic continues with the 9th wave (XBB.1.5, E.G.5.1, HK.3) and 10th wave (BA.2.86, JN.1). This is related to the fact that the successively emerging Omicron substrains, such as the XBB and BA.2.86 strains, are resistant to existing neutralizing antibodies and vaccines, posing a risk of reinfection. A new Omicron substrain, the JN.1 strain, has now spread to a global pandemic.

[0003] The effectiveness of neutralizing antibodies against SARS-CoV-2 in preventing the disease from becoming severe has been proven in the United States, and in Japan, LONAPREVE (a combination of casirivimab and imdevimab, Roche) and ZEVUDI (sotrovimab, GSK) were granted special approval in 2021. Furthermore, Evasheld (a combination of ticagevimab and silgavimab, AstraZeneca) was granted special approval in 2022, but due to a global supply shortage, it was only available to certain immunocompromised cases.

[0004] Neutralizing antibody therapy was expected to be a reliable treatment, but these antibodies were found to be ineffective against the Omicron substrain (Non-Patent Document 1). In light of this situation, new neutralizing antibodies have been developed, including the S2K146 antibody (Non-Patent Document 2) and EliLilly's LY-CoV1404 (bebtelovimab) (Non-Patent Document 3). However, these antibodies have been reported to not neutralize recent Omicron substrains such as XBB.1.5, EG.5.1, and BA.2.86 (Non-Patent Document 4). Furthermore, bebtelovimab was granted emergency approval in the United States without its clinical efficacy being known, but it has been reported to be ineffective against BQ.1 and BQ.1.1. On the other hand, only the SA55 antibody maintained neutralizing activity against the Omicron substrain (Non-Patent Document 5). However, further neutralizing antibodies against SARS-CoV-2 are needed.

[0005] In 2020, the present inventors also discovered and reported antibodies (9-105 antibody, 10-121 antibody) that exhibit strong neutralizing activity against SARS-CoV-2 wild-type strains and mutant strains (B.1.1.7 strain, mink cluster 5 strain, B.1.351 strain, P.1 strain, B.1.617.1 strain, and B.1.617.2 strain) (Patent Document 1, Non-Patent Document 6). The present inventors have also discovered and reported antibodies (1-58 antibody, 3-1 antibody, 1-44 antibody, 4-66 antibody) that exhibit strong neutralizing activity against the Omicron strain (Patent Document 2).

[0006] Thus, the development of antibodies against SARS-CoV-2 mutant strains and treatments using such antibodies is a global priority, and there is a continuing need to develop antibodies that neutralize SARS-CoV-2 mutant strains, including the Omicron substrain.

[0007] WO2022 / 044573WO2024 / 053719

[0008] Touret et al., iScience 26, 106413, 2023Park YJ et al., Vol.375, pp.449-454, Science, 2022Iketani S. et al., Vol.694, pp.553-556, Nature. 2022Wang Q, et. al., Nature, 2023 Dec;624(7992):639-644Yang S, et. al., Lancet Infect Dis. 2024 Feb;24(2):e70-e72.Kaku Y. et al., Cell Report Vol 36, 2, 109385, 2021.Hansen et al. Science 2020 Aug 21;369(6506):1010-1014.Jones et al. Sci Transl Med. 2021 May 12;13(593):eabf1906.Pinto et al. Nature 2020 Jul;583(7815):290-295.

[0009] An object of the present invention is to provide an antibody or antigen-binding fragment thereof that broadly cross-neutralizes SARS-CoV-2 mutant strains. Another object of the present invention is to provide a pharmaceutical composition for treating coronavirus infection using the antibody or antigen-binding fragment thereof.

[0010] The present inventors have conducted extensive research with the aim of developing a treatment for coronavirus infection using a neutralizing antibody that neutralizes not only the Omicron strain, a SARS-CoV-2 mutant, but a wide range of SARS-CoV-2 mutant strains as well. As a result, they identified cases of breakthrough infection with the Omicron strain after vaccination against SARS-CoV-2, and by screening peripheral blood B cells from these cases using the receptor binding domain (RBD) or spike trimer, they discovered antibodies that broadly neutralize SARS-CoV-2 mutant strains, thereby completing the present invention. The present invention includes the following aspects.

[0011] [1] An antibody or antigen-binding fragment thereof capable of neutralizing SARS-CoV-2 virus (particularly the Omicron substrain), comprising the following heavy chain CDR1-3 and light chain CDR1-3: (1) a heavy chain CDR1 selected from the group consisting of a heavy chain CDR1 represented by SEQ ID NO: 5 (GIMFSDFA), a heavy chain CDR1 represented by SEQ ID NO: 11 (DGSLSSHDW), a heavy chain CDR1 consisting of a sequence in which one amino acid is deleted, substituted, or added in any of the heavy chain CDR1 represented by SEQ ID NO: 5 or 11, and a heavy chain CDR1 having 85% or more substantial identity to any of the heavy chain CDR1 represented by SEQ ID NO: 5 or 11; (2) A heavy chain CDR2 selected from the group consisting of a heavy chain CDR2 represented by SEQ ID NO: 6 (ISGDGDET), a heavy chain CDR2 represented by SEQ ID NO: 12 (IFHSGST), a heavy chain CDR2 consisting of a sequence in which one amino acid is deleted, substituted, or added in any of the heavy chain CDR2 represented by SEQ ID NO: 6 or 12, and a heavy chain CDR2 having 85% or more substantial identity with any of the heavy chain CDR2 represented by SEQ ID NO: 6 or 12; (3) A heavy chain CDR3 selected from the group consisting of a heavy chain CDR3 represented by SEQ ID NO: 7 (VKAETKYFGAHQTFHD), a heavy chain CDR3 represented by SEQ ID NO: 13 (ARGRGLIDS), a heavy chain CDR3 consisting of a sequence in which one or two amino acids are deleted, substituted, or added in any of the heavy chain CDR3s represented by SEQ ID NO: 7 or 13, and a heavy chain CDR3 having 80% or more substantial identity to any of the heavy chain CDR3s represented by SEQ ID NO: 7 or 13; (4) A light chain CDR1 selected from the group consisting of a light chain CDR1 represented by SEQ ID NO: 8 (QSLSIW), a light chain CDR1 represented by SEQ ID NO: 14 (QGISNS), a light chain CDR1 consisting of a sequence in which one amino acid is deleted, substituted, or added in any of the light chain CDR1s represented by SEQ ID NO: 8 or 14, and a light chain CDR1 having 80% or more substantial identity with any of the light chain CDR1s represented by SEQ ID NO: 8 or 14; (5) a light chain CDR2 represented by SEQ ID NO: 9 (RAS) or SEQ ID NO: 15 (SAS); and(6) An antibody or antigen-binding fragment thereof comprising a light chain CDR3 selected from the group consisting of a light chain CDR3 represented by SEQ ID NO: 10 (QQYSTFPYT), a light chain CDR3 represented by SEQ ID NO: 16 (QQYFSVRT), a light chain CDR3 consisting of a sequence in which one amino acid is deleted, substituted, or added in either of the light chain CDR3 represented by SEQ ID NO: 10 or 16, and a light chain CDR3 having 80% or more substantial identity to either of the light chain CDR3 represented by SEQ ID NO: 10 or 16.

[0012] [2] The antibody or antigen-binding fragment thereof according to [1] above, wherein the heavy chain CDR1 is the heavy chain CDR1 represented by SEQ ID NO: 5 or SEQ ID NO: 11, the heavy chain CDR2 is the heavy chain CDR2 represented by SEQ ID NO: 6 or SEQ ID NO: 12, the heavy chain CDR3 is the heavy chain CDR3 represented by SEQ ID NO: 7 or SEQ ID NO: 13, the light chain CDR1 is the light chain CDR1 represented by SEQ ID NO: 8 or SEQ ID NO: 14, the light chain CDR2 is the light chain CDR2 represented by SEQ ID NO: 9 or SEQ ID NO: 15, and the light chain CDR3 is the light chain CDR3 represented by SEQ ID NO: 10 or SEQ ID NO: 16. [3] The antibody or antigen-binding fragment thereof according to [1] above, wherein the combination of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 is any of the following: (1) heavy chain CDR1 represented by SEQ ID NO: 5, heavy chain CDR2 represented by SEQ ID NO: 6, heavy chain CDR3 represented by SEQ ID NO: 7, light chain CDR1 represented by SEQ ID NO: 8, light chain CDR2 represented by SEQ ID NO: 9, and light chain CDR3 represented by SEQ ID NO: 10, (2) heavy chain CDR1 represented by SEQ ID NO: 11, heavy chain CDR2 represented by SEQ ID NO: 12, heavy chain CDR3 represented by SEQ ID NO: 13, light chain CDR1 represented by SEQ ID NO: 14, light chain CDR2 represented by SEQ ID NO: 15, and light chain CDR3 represented by SEQ ID NO: 16. [4] The antibody or antigen-binding fragment thereof according to [1] above, having a heavy chain variable region represented by SEQ ID NO: 1 and a light chain variable region represented by SEQ ID NO: 2. [5] The antibody or antigen-binding fragment thereof according to [1] above, which has a heavy chain variable region represented by SEQ ID NO: 3 and a light chain variable region represented by SEQ ID NO: 4.

[0013] [6] In a neutralization assay using a virus (pseudovirus or authentic virus), the neutralizing activity of an antibody against the JN.1 strain, an Omicron lineage strain of SARS-CoV-2, is at a 50% inhibitory concentration (IC) of about 1 μg / mL or less (preferably about 0.8 μg / mL or less, more preferably about 0.5 μg / mL or less). 50 [7] The antibody or antigen-binding fragment thereof according to any one of [1] to [5] above, having a neutralizing activity expressed in terms of μg / mL (50% inhibitory concentration) of about 1 μg / mL or less (preferably about 0.8 μg / mL or less, more preferably about 0.5 μg / mL or less, and even more preferably about 0.2 μg / mL or less) against at least four mutant strains (preferably at least five mutant strains, more preferably at least six mutant strains, even more preferably at least seven mutant strains, and particularly preferably eight mutant strains) selected from the group consisting of Omicron lineage strains of SARS-CoV-2, BA.1, BA.2, BA.5, BQ.1.1, XBB, XBB.1.5, EG.5.1, and JN.1, in a neutralization assay using a virus (pseudovirus or authentic virus). 50 [8] The antibody or antigen-binding fragment thereof according to any one of [1] to [7] above, which is selected from the group consisting of immunoglobulin molecules, monoclonal antibodies, human antibodies, chimeric antibodies, CDR-grafted antibodies, Fab, Fab', F(ab')2, Fd, Fv, disulfide-linked Fv, scFv, single-domain antibodies, nanobody antibodies, diabody antibodies, bispecific antibodies, and multispecific antibodies.

[0014] [9] A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of [1] to [8] above.

[10] A vector comprising the nucleic acid according to [9] above.

[11] A host cell comprising the nucleic acid according to [9] above or the vector according to

[10] above.

[12] A method for producing the antibody or antigen-binding fragment thereof according to any one of [1] to [8] above, the method comprising a step of culturing the host cell according to

[11] above under conditions suitable for expression of the antibody or antigen-binding fragment thereof.

[0015]

[13] A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of [1] to [8] above, and a pharmacologically acceptable carrier.

[14] The pharmaceutical composition according to

[13] above, further comprising at least one other neutralizing antibody or antigen-binding fragment thereof against SARS-CoV-2 and / or a SARS-CoV-2 mutant strain.

[15] The pharmaceutical composition according to

[13] above, characterized in that it is administered in combination with other anti-coronavirus drugs.

[16] The pharmaceutical composition according to

[15] above, wherein the anti-coronavirus drug is at least one anti-coronavirus drug selected from antiviral drugs (preferably remdesivir, molnupiravir, nilmatrervir / ritonavir, ensitrervir, ensitrervir fumarate) and neutralizing antibody drugs (preferably casirivimab / imdevimab, sotrovimab, tixagevimab / silgavimab).

[17] Use of the antibody or antigen-binding fragment thereof according to any one of [1] to [8] above in the manufacture of a pharmaceutical composition for the prevention or treatment of SARS-CoV-2 virus infection (preferably infection with a SARS-CoV-2 mutant virus, more preferably infection with the Omicron strain of SARS-CoV-2 virus, and even more preferably infection with the Omicron substrain of SARS-CoV-2 virus).

[0016] The present invention provides novel antibodies that broadly neutralize SARS-CoV-2 mutant strains, particularly the Omicron substrain.

[0017] Figure 1 shows the amino acid sequences of the heavy and light chain variable regions of the 1-94 and 2-66 antibodies of the present invention. Each column indicates the positions of CDR1-3 of the heavy and light chains, and FR1-4 of the heavy and light chains. Figure 2 shows the results of a neutralization test of 26 obtained antibodies against the Omicron strain. Figure 3 shows the amino acid mutations in the Spike protein of SARS-CoV-2 mutant strains. Figure 4 shows the results of measuring the neutralizing activity of various antibodies against various Omicron substrains. Figure 5 shows the results of measuring the neutralizing activity of the 1-94 and 2-66 antibodies, as well as the comparative antibody VIR-7831, against the Omicron substrain. Figure 6 shows the results of measuring the neutralizing activity of 27 obtained monoclonal antibodies against Omicron substrains and conventional mutant strains. Figure 7 shows the results of measuring the binding activity of the 1-94 and 2-66 antibodies of the present invention to the spike protein of wild-type and mutant strains. 8 shows the results of measuring the RBD-ACE2 binding inhibitory activity using the 1-94 and 2-66 antibodies of the present invention. The lower graph shows the IC ５０ FIG. 9 shows the results of measuring the binding activity of the 1-94 and 2-66 antibodies of the present invention to the RBD of the Omicron substrain using surface plasmon resonance (SPR). FIG. 10 shows the experimental protocol for evaluating the therapeutic effect of the 1-94 and 2-66 antibodies of the present invention against in vivo infection with the Omicron substrain (JN.1) using hamsters. FIG. 11 shows the results of evaluating the therapeutic effect of the 1-94 and 2-66 antibodies of the present invention against in vivo infection with the Omicron substrain (JN.1).

[0018] The present invention will be described in more detail below, along with preferred methods and materials that can be used in carrying out the present invention, but the present invention is not limited to the embodiments described below. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Furthermore, any materials and methods equivalent or similar to those described herein can also be used in carrying out the present invention. Furthermore, all publications and patents cited in this specification in connection with the present invention are incorporated herein by reference and constitute a part of this specification, for example, to describe methods, materials, and the like that can be used in the present invention.

[0019] In this specification, the expression "A to B" indicating a numerical range means a numerical range including the endpoints A and B. The same applies to "A to B." In this specification, "about" is used to mean an allowance of ±10%. In this specification, "one or more" means "one, two, three, four or more," unless the meaning is clearly indicated or the meaning is clear from the surrounding context.

[0020] "SARS-CoV-2," also known as the "novel coronavirus," refers to a newly emerged coronavirus that was first isolated in Wuhan, China, in 2020 and identified as the cause of an outbreak of severe acute respiratory disease. It binds to the human host cell receptor angiotensin-converting enzyme 2 (ACE2) via the viral spike protein. Various SARS-CoV-2 variants, classified as SARS coronaviruses, have been isolated and identified. SARS-CoV-2 has repeatedly mutated since the initial Wuhan strain, and new mutant strains such as the prototype (614G), alpha strain (B.1.1.7), beta strain (B.1.351), gamma strain (P.1), delta strain (B.1.617.2), kappa strain (B.1.617.1), lambda strain (C.37), and mu strain (B.1.621) have been reported one after another, causing major epidemics. Subsequently, highly infectious omicron strains (BA.1, BA.1.1, BA.2, BA.2.12.1, BA.3, BA.4 / 5) have been reported and are becoming a global problem. Furthermore, sublineages of the Omicron strain (BQ.1.1, CH.1.1, XBB.1.5, EG.5.1, BA.2.86, JN.1) continue to be reported. It is possible that as yet undetected mutant strains and new mutant strains may emerge in the future. As used herein, the terms "SARS-CoV-2," "SARS-CoV-2 virus," "SARS-CoV-2 mutant strain," or "SARS-CoV-2 virus mutant strain" are not limited to the SARS-CoV-2 strains described above, but also include other SARS-CoV-2 mutant strains that may be newly detected in the future. In this specification, unless otherwise specified or clear from the context, the expression "spike protein of SARS-CoV-2" is used to mean any type of spike protein, including not only the SARS-CoV-2 strains mentioned above, but also other SARS-CoV-2 variants that may be newly detected in the future.

[0021] "SARS-CoV-2 spike protein," "spike protein," or "SARS-CoV-2(S)" refers to the spike protein of the SARS-CoV-2 coronavirus. The SARS-CoV-2 spike protein is a 1,273 amino acid type I membrane glycoprotein that forms a trimer and exists as spikes (peplomers) protruding from the surface of SARS-CoV-2 virus particles. The spike protein is composed of an S1 subunit and an S2 subunit and functions as host receptor binding and membrane fusion. It binds to ACE2 via an approximately 215 amino acid receptor binding domain (RBD) present in the S1 subunit. The amino acid sequence of the full-length SARS-CoV-2 spike protein (SEQ ID NO: 17) has been deposited in GeneBank under accession number YP_009724390. As used herein, reference to the spike protein of SARS-CoV-2 includes recombinant SARS-CoV-2 spike protein or fragments thereof.

[0022] "ACE2" is angiotensin-converting enzyme 2, a receptor to which SARS-CoV-2 binds. ACE2 is a type I transmembrane glycoprotein of 805 amino acids present on the cell surface that splits angiotensin II into angiotensin 1-7 polypeptides. The split polypeptides have functions such as cardioprotection and vasodilation. Unless otherwise specified, ACE2 as used herein refers to human ACE2. ACE2 acts as a functional receptor for the SARS-CoV-2 virus. The virus binds to ACE2 via its RBD. It has been reported that TMPRSS2, a serine transmembrane protease present on the host cell membrane, is required for virus binding to ACE2 and intracellular entry via membrane fusion.

[0023] Embodiments of the present invention are described below, but the present invention is not limited thereto. One embodiment of the present invention is an antibody or an antigen-binding fragment thereof that binds to the spike protein of SARS-CoV-2 and has a specific amino acid sequence, and is useful for binding to the SARS-CoV-2 spike protein and neutralizing SARS-CoV-2 mutant strains, particularly the Omicron strain and Omicron substrain. Hereinafter, in this specification, antibodies and antigen-binding fragments thereof that bind to the spike protein of SARS-CoV-2 will be collectively referred to as "antibodies against SARS-CoV-2, etc.", "anti-SARS-CoV-2 antibodies, etc.", "antibodies against the spike protein of SARS-CoV-2, etc.", "anti-SARS-CoV-2(S) antibodies, etc.", or simply as "antibodies, etc." or "antibodies" of the present invention. However, when the text clearly indicates either an antibody or an antigen-binding fragment thereof, or when it is clearly understood from the context that it refers to either an antibody that binds to the spike protein of SARS-CoV-2 or a fragment that binds to the spike protein of SARS-CoV-2.

[0024] As used herein, the term "antibody" is used to encompass both intact antibodies and fragments thereof. However, when the context clearly indicates either an intact antibody or an antibody fragment, or when the context clearly indicates either, it is understood to mean either an antibody or an antibody fragment. It is clear to those skilled in the art that an antigen-binding fragment (an antibody fragment that binds to an antigen) can compete with the specific binding of an intact antibody to an antigen and exhibit the same effect as an antibody. See Fundamental Immunology, Chapter 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989)).

[0025] In certain embodiments, the antibodies of the present invention inhibit binding of the virus to its host cell receptor, angiotensin-converting enzyme 2 (ACE2), and are useful for preventing SARS coronavirus entry into host cells. In certain embodiments, the antibodies of the present invention exhibit antiviral activity by inhibiting cell fusion mediated by the spike protein of SARS-CoV-2. In certain other embodiments, the antibodies of the present invention prevent, treat, or ameliorate at least one symptom of SARS-CoV-2 viral infection in a subject (preferably a human). In certain other embodiments, the antibodies of the present invention are administered therapeutically or prophylactically to a subject (preferably a human) infected with or at risk of infection with SARS-CoV-2 virus.

[0026] The antibodies of the invention can be full-length (e.g., IgG1, IgG2, or IgG4) or can contain antigen-binding sites thereof (e.g., Fab, Fab', F(ab')2, Fd, Fv, disulfide Fv, scFv, single domain, nanobody, diabody), and can be modified to improve functionality or to remove non-essential effector functions. Also, in certain embodiments, the antibodies of the invention can be bispecific or multispecific.

[0027] In one aspect, the present invention provides an isolated recombinant monoclonal antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2. In some embodiments, the antibody of the present invention is a fully human monoclonal antibody. In one aspect, the antibody or the like of the present invention binds to an epitope within the receptor binding domain (RBD) of the spike protein of SARS-CoV-2. In some embodiments, the antibody or the like of the present invention binds to one or more amino acids selected from amino acids 322 to 536 (corresponding to the RBD sequence) of the spike protein of SARS-CoV-2 (registered in GenBank under Accession Number YP_009724390) (SEQ ID NO: 17). In addition, the complete genome information of the SARS-CoV-2 virus is registered in GeneBank as Genome Reference Sequence: NC_045512. In certain embodiments, the antibodies etc. of the present invention bind to the spike protein of various mutant strains, preferably the Omicron strain and its substrains, and are particularly useful against these mutant strains.

[0028] Representative anti-SARS-CoV-2 antibodies of the present invention include, for example, antibodies having a heavy chain variable region represented by the amino acid sequence set forth in SEQ ID NO: 1 or 3. Representative anti-SARS-CoV-2 antibodies of the present invention include, for example, antibodies having a light chain variable region represented by the amino acid sequence set forth in SEQ ID NO: 2 or 4. More representative anti-SARS-CoV-2 antibodies of the present invention include, for example, antibodies having a combination of SEQ ID NOs: 1 and 2, or SEQ ID NOs: 3 and 4, as the set of amino acid sequences of the heavy chain variable region and the light chain variable region.

[0029] The amino acid sequences of the heavy chain variable region (HCVR), light chain variable region (LCVR), heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) of two representative anti-SARS-CoV-2 antibodies of the invention are set forth in the table below.

[0030] The respective sequences are as follows:

[0031]

[0032] The antibodies of the present invention provide antibodies or antigen-binding fragments thereof comprising HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a sequence having at least 70%, 75%, or 80% substantial identity thereto, preferably at least 90%, 92%, or 95% substantial identity thereto, and more preferably at least 98% or 99% substantial identity thereto.

[0033] The antibodies of the present invention also provide antibodies or antigen-binding fragments thereof comprising an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or a sequence having at least 70%, 75%, or 80% substantial identity thereto, preferably at least 90%, 92%, or 95% substantial identity thereto, and more preferably at least 98% or 99% substantial identity thereto.

[0034] The present invention also provides an antibody or antigen-binding fragment thereof comprising an HCVR and LCVR amino acid sequence pair (HCVR / LCVR), including any of the HCVR amino acid sequences listed in Table 1 and any of the LCVR amino acid sequences listed in Table 1 paired therewith, preferably SEQ ID NOs: 1 and 2, or SEQ ID NOs: 3 and 4.

[0035] The present invention also provides an antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from either of the two HCDR1 amino acid sequences listed in Table 1, or a sequence having at least 85%, preferably at least 90%, substantial identity thereto.

[0036] The present invention also provides an antibody or antigen-binding fragment thereof comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from either of the two HCDR2 amino acid sequences listed in Table 1, or a sequence having at least 85%, preferably at least 90%, substantial identity thereto.

[0037] The present invention also provides an antibody or antigen-binding fragment thereof comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from either of the two HCDR3 amino acid sequences listed in Table 1, or a sequence having at least 80%, preferably at least 90%, substantial identity thereto.

[0038] The present invention also provides an antibody or antigen-binding fragment thereof comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from either of the two LCDR1 amino acid sequences listed in Table 1, or a sequence having at least 80%, preferably at least 90%, substantial identity thereto.

[0039] The present invention also provides an antibody or antigen-binding fragment thereof comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from a sequence having any of the three LCDR2 amino acid sequences listed in Table 1.

[0040] The present invention also provides an antibody or antigen-binding fragment thereof comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the four LCDR3 amino acid sequences listed in Table 1, or a sequence having at least 80%, preferably at least 90%, substantial identity thereto.

[0041] The present invention also provides an antibody or antigen-binding fragment thereof comprising an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) including any of the two HCDR3 amino acid sequences listed in Table 1 and any of the two LCDR3 amino acid sequences listed in Table 1 paired with any of them. According to certain embodiments, the present invention provides an antibody or antigen-binding fragment thereof comprising the HCDR3 / LCDR3 amino acid sequence pair contained in any of the two representative anti-SARS-CoV-2 antibodies listed in Table 1. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair is selected from SEQ ID NOs: 7 and 10 (1-94) or 13 and 16 (2-66).

[0042] The present invention also provides an antibody or antigen-binding fragment thereof comprising a set of six CDRs (i.e., the set of HCDR1-HCDR2-HCDR3+LCDR1-LCDR2-LCDR3) contained in any of the representative anti-SARS-CoV-2 antibodies listed in Table 1. In certain embodiments, the set of amino acid sequences of HCDR1-HCDR2-HCDR3+LCDR1-LCDR2-LCDR is SEQ ID NO:5-SEQ ID NO:6-SEQ ID NO:7+SEQ ID NO:8-SEQ ID NO:9-SEQ ID NO:10 (1-94), or SEQ ID NO:11-SEQ ID NO:12-SEQ ID NO:13+SEQ ID NO:14-SEQ ID NO:15-SEQ ID NO:16 (2-66).

[0043] In another embodiment, the present invention provides an antibody or antigen-binding fragment thereof comprising a combination of any of the three CDR sets (i.e., HCDR1-HCDR2-HCDR3) contained in the HCVR amino acid sequence and any of the three CDR sets (i.e., LCDR1-LCDR2-LCDR3) contained in the LCVR amino acid sequence pair defined by any of the representative anti-SARS-CoV-2 antibodies listed in Table 1. In certain embodiments, the set of HCDR1-HCDR2-HCDR3 is SEQ ID NO:5-SEQ ID NO:6-SEQ ID NO:7, or SEQ ID NO:11-SEQ ID NO:12-SEQ ID NO:13, and the set of LCDR1-LCDR2-LCDR3 is SEQ ID NO:8-SEQ ID NO:9-SEQ ID NO:10, or SEQ ID NO:14-SEQ ID NO:15-SEQ ID NO:16.

[0044] Methods for identifying CDRs within the amino acid sequences of HCVRs and LCVRs are well known in the art and can be used to identify CDRs. Typical examples used to identify CDR boundaries include, for example, the IMGT definition, Kabat definition, Chothia definition, AbM definition, Martin definition, Gelfand definition, and Honneger definition (Aho's definition). Public databases are also available for identifying CDR sequences within antibodies.

[0045] The present invention includes anti-SARS-CoV-2(S) antibodies and the like having modified glycosylation patterns. In certain embodiments, modifications can be made to remove undesired glycosylation sites; for example, antibodies lacking fucose moieties have enhanced antibody-dependent cellular cytotoxicity (ADCC). In certain embodiments, galactosylation modifications can improve complement-dependent cytotoxicity (CDC). Antibodies and the like having such modifications on their sugar chains are also included in the present invention.

[0046] In another aspect, the present invention provides antibodies that compete with antibodies comprising HCVR CDRs and LCVR CDRs having an amino acid sequence selected from the sequences listed in Table 1 for specific binding to the spike protein of SARS-CoV-2.

[0047] In another aspect, the present invention provides antibodies that block or reduce (preferably completely block) binding of the spike protein of SARS-CoV-2 to ACE2. Antibodies that block or reduce (preferably completely block) binding of the spike protein of SARS-CoV-2 to ACE2 may bind to or near the binding site of the spike protein of SARS-CoV-2 to ACE2. In one embodiment, the present invention provides antibodies that block or reduce (preferably completely block) binding of the spike protein of SARS-CoV-2 to mammalian, preferably human, ACE2.

[0048] In another aspect, the present invention provides nucleic acid molecules encoding anti-SARS-CoV-2 antibodies, etc. For example, the present invention provides nucleic acid molecules encoding any of the HCVR amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the nucleic acid sequences encoding the HCVR amino acid sequences listed in Table 1, or a sequence having at least 90%, 92%, or 95% sequence identity thereto, and at least 98% or 99% sequence identity thereto. The present invention also provides nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the nucleic acid sequences encoding the LCVR amino acid sequences listed in Table 1, or a sequence having at least 90%, 92%, or 95% sequence identity thereto, and at least 98% or 99% sequence identity thereto.

[0049] The present invention also provides nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 1, wherein the nucleic acid molecules comprise a polynucleotide sequence selected from any of the nucleic acid sequences encoding the HCDR1 amino acid sequences listed in Table 1, or sequences having at least 90%, 92%, or 95% sequence identity thereto, and at least 98% or 99% sequence identity thereto. The present invention also provides nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1, wherein the nucleic acid molecules comprise a polynucleotide sequence selected from any of the nucleic acid sequences encoding the HCDR2 amino acid sequences listed in Table 1, or sequences having at least 90%, 92%, or 95% sequence identity thereto, and at least 98% or 99% sequence identity thereto. The present invention also provides nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 1, wherein the nucleic acid molecules comprise a polynucleotide sequence selected from any of the nucleic acid sequences encoding the HCDR3 amino acid sequences listed in Table 1, or sequences having at least 90%, 92%, or 95% sequence identity thereto, and at least 98% or 99% sequence identity thereto.

[0050] The present invention also provides nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 1, wherein the nucleic acid molecule comprises a polynucleotide sequence selected from any of the nucleic acid sequences encoding the LCDR1 amino acid sequences listed in Table 1, or a sequence having at least 90%, 92%, or 95% sequence identity, or at least 98% or 99% sequence identity thereto. The present invention also provides nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 1, wherein the nucleic acid molecule comprises a polynucleotide sequence selected from any of the nucleic acid sequences encoding the LCDR2 amino acid sequences listed in Table 1, or a sequence having at least 90%, 92%, or 95% sequence identity thereto, and at least 98% or 99% sequence identity thereto. The present invention also provides nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1, wherein the nucleic acid molecule comprises a polynucleotide sequence selected from any of the nucleic acid sequences encoding the LCDR3 amino acid sequences listed in Table 1, or a sequence having at least 90%, 92%, or 95% sequence identity thereto, and at least 98% or 99% sequence identity thereto.

[0051] The present invention also provides nucleic acid molecules encoding an HCVR amino acid sequence, wherein the HCVR amino acid sequence comprises a set of three CDR amino acid sequences (i.e., HCDR1-HCDR2-HCDR3), where the HCDR1-HCDR2-HCDR3 amino acid sequence set is defined by any of the anti-SARS-CoV-2 antibodies listed in Table 1. The present invention also provides nucleic acid molecules encoding an LCVR amino acid sequence, wherein the LCVR amino acid sequence comprises a set of three CDR amino acid sequences (i.e., LCDR1-LCDR2-LCDR3), where the LCDR1-LCDR2-LCDR3 amino acid sequence set is defined by any of the anti-SARS-CoV-2 antibodies listed in Table 1.

[0052] The present invention also provides nucleic acid molecules encoding both HCVR and LCVR, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding any of the HCVR amino acid sequences listed in Table 1 and a nucleic acid sequence encoding any of the LCVR amino acid sequences listed in Table 1, preferably a nucleic acid molecule encoding a set of HCVR and LCVR defined by any of the anti-SARS-CoV-2 antibodies listed in Table 1. The nucleic acid molecule comprises a polynucleotide sequence selected from any of the nucleic acid sequences encoding the HCVR amino acid sequence listed in Table 1, or a sequence having at least 90%, 92%, or 95% sequence identity thereto, and at least 98% or 99% sequence identity thereto, and a polynucleotide sequence selected from any of the nucleic acid sequences encoding the LCVR amino acid sequence listed in Table 1, or a sequence having at least 90%, 92%, or 95% sequence identity thereto, and at least 98% or 99% sequence identity thereto.

[0053] The antibodies of the present invention include anti-SARS-CoV-2(S) antibodies that bind to the same epitope or at least a portion of the same epitope as any of the antibodies having the amino acid sequences of HCVR and LCVR listed in Table 1 that specifically bind to the spike protein of SARS-CoV-2. Such antibodies include antibodies against the spike protein of SARS-CoV-2 that cross-compete with any of the antibodies having the amino acid sequences of HCVR and LCVR listed in Table 1, and antigen-binding fragments thereof.

[0054] Using methods known in the art, one can readily determine whether an antibody (including a complete antibody and a fragment thereof) binds to the same epitope as a reference anti-SARS-CoV-2(S) antibody, etc., or competes for binding with a reference anti-SARS-CoV-2(S) antibody, etc., of the present invention. For example, to determine whether a test antibody binds to the same epitope as a reference anti-SARS-CoV-2(S) antibody, etc., of the present invention, the reference antibody, etc., is subjected to conditions that allow binding to the spike protein of SARS-CoV-2 under saturating conditions. The ability of the test antibody to bind to the spike protein of SARS-CoV-2 is then evaluated. If the test antibody is able to bind to the spike protein of SARS-CoV-2 after saturation binding with the reference anti-SARS-CoV-2(S) antibody, etc., it can be concluded that the test antibody binds to a different epitope than the reference anti-SARS-CoV-2(S) antibody, etc. On the other hand, if the test antibody is unable to bind to the SARS-CoV spike protein after saturation binding with the reference anti-SARS-CoV-2(S) antibody, it is concluded that the test antibody binds to the same epitope as the epitope bound by the reference anti-SARS-CoV-2(S) antibody. To determine whether a test antibody competes with the reference anti-SARS-CoV-2(S) antibody for binding, they can be tested for competition with each other. In a first test, the reference antibody is allowed to bind to the SARS-CoV-2 spike protein under saturating conditions, followed by evaluation of the binding of the test antibody to the SARS-CoV-2 spike protein. In a second test, the test antibody is allowed to bind to the SARS-CoV-2 spike protein under saturating conditions, followed by evaluation of the binding of the reference antibody to the SARS-CoV-2 spike protein. If in both tests, only the (saturating) antibody in the first test binds to the spike protein of SARS-CoV-2, it can be concluded that the test antibody and the reference antibody, etc. compete for binding to the spike protein of SARS-CoV-2. As will be understood by those skilled in the art, an antibody that competes with the reference antibody, etc. for binding will not necessarily bind to the same epitope as the reference antibody, but will sterically block binding of the reference antibody, etc. by binding to an overlapping or adjacent epitope.Therefore, two antibodies can be concluded to bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other to the antigen. Antibodies that compete with the antibodies of the present invention are also included in the present invention.

[0055] In one aspect, the present invention provides a recombinant expression vector capable of expressing the anti-SARS-CoV-2(S) antibody of the present invention, etc. In another aspect, the present invention provides a recombinant expression vector capable of expressing a polypeptide comprising the heavy chain variable region and / or light chain variable region of the anti-SARS-CoV-2(S) antibody of the present invention. For example, the present invention includes a recombinant expression vector comprising the above-mentioned nucleic acid molecule, i.e., a nucleic acid molecule encoding any one or a combination of the HCVR, LCVR, and CDR amino acid sequences listed in Table 1. Expression vectors can be any vector used in the technical field without limitation, and such vectors are known to those skilled in the art. The antibody of the present invention, etc. can be produced by culturing a host cell into which such a vector has been introduced under conditions that allow the production of the antibody or antibody fragment, and recovering the produced antibody and antibody fragment. Such methods are also within the scope of the present invention.

[0056] In yet another embodiment, the antibody or antigen-binding fragment thereof of the present invention can be used in combination with another antibody that specifically binds to the spike protein of SARS-CoV-2 (so-called cocktail antibody). When used in combination with another antibody, it is preferable to use the other antibody with a spectrum different from the spectrum of the antibody or antigen-binding fragment thereof against SARS-CoV-2 mutant strains. For example, but not limited to, the antibody or antigen-binding fragment thereof can be used in combination with the 9-105 antibody, 10-121 antibody, 1-58 antibody, 3-1 antibody, 1-44 antibody, and 4-66 antibody reported by the present inventors (Patent Document 1, Patent Document 2, Non-Patent Document 6), or existing antibodies such as REGN-10933 and REGN-10987 (Regeneron / Roche) (Non-Patent Document 7), LY-CoV555 (Eli Lilly) (Non-Patent Document 8), and VIR-7831 and VIR-7832 (GSK) (Non-Patent Document 9). The combination of antibodies is not limited to two types, and may be three or more types. Taking into consideration the spectrum of antibodies against SARS-CoV-2 mutant strains, the antibody or antigen-binding fragment thereof of the present invention may be combined with another antibody or antigen-binding fragment thereof, or two or more types of antibodies or antigen-binding fragments thereof of the present invention may be combined. Preferably, taking into consideration the spectrum of antibodies against SARS-CoV-2 mutant strains, the antibody or antigen-binding fragment thereof of the present invention can be used together with any one of the 9-105 antibody, 10-121 antibody, 1-58 antibody, 3-1 antibody, 1-44 antibody, and 4-66 antibody reported by the present inventors, or two or more of these antibodies. Another example of a combination of two antibodies is the 1-94 antibody of the present invention and the 2-66 antibody.

[0057] In another aspect, the present invention provides a method for treating or preventing a disease associated with SARS-CoV-2 in a subject (preferably a human) using an anti-SARS-CoV-2(S) antibody or antigen-binding portion thereof of the present invention. The therapeutic or prophylactic method comprises administering a therapeutically or prophylactically effective amount of a pharmaceutical composition comprising an antibody or the like of the present invention to a subject (preferably a human) in need thereof. In one embodiment, the present invention provides a method for preventing, treating, or ameliorating at least one symptom of SARS-CoV-2 infection, comprising administering a therapeutically or prophylactically effective amount of an anti-SARS-CoV-2(S) antibody or the like of the present invention to a subject (preferably a human) in need thereof. In one embodiment, the present invention provides a method for ameliorating or reducing the severity of at least one symptom or sign of SARS infection in a subject by administering an anti-SARS-CoV-2(S) antibody or the like of the present invention. The at least one symptom or sign is selected from the group consisting of lung inflammation, alveolar damage, fever, respiratory problems (nasal congestion, runny nose, sore throat, cough, shortness of breath), headache, fatigue, dysgeusia, dysosmia, diarrhea, vomiting, increased blood clotting, thrombosis, Kawasaki disease-like vasculitis, renal dysfunction (e.g., nephritis), organ failure, pneumonia, septic shock, and death. In another embodiment, the antibodies of the present invention are administered therapeutically or prophylactically to a subject infected with or at risk of infection with SARS-CoV-2. At-risk subjects (patients) include, but are not limited to, immunocompromised individuals, elderly people (age 65 or older), those with underlying conditions such as lung infection, heart disease, respiratory disease, diabetes, or dialysis patients, healthcare workers, and those who have had close contact with or are expected to have close contact with a person with confirmed or suspected SARS infection.

[0058] The invention also includes the use of an anti-SARS-CoV-2(S) antibody or antigen-binding fragment thereof of the invention in the manufacture of a medicament.

[0059] The term "antibody" can refer to an immunoglobulin molecule (i.e., a "complete antibody molecule") comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, a multimer thereof (e.g., IgA or IgM), or an antigen-binding fragment thereof. Each heavy chain of an antibody comprises a heavy chain variable region (HCVR or VH) and a heavy chain constant region (comprising domains CH1, CH2, and CH3). Each light chain of an antibody comprises a light chain variable region (LCVR or VL) and a light chain constant region (CL). HCVRs and LCVRs are further subdivided into framework regions (FRs) and complementarity-determining regions (CDRs). Each HCVR and LCVR comprises three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In embodiments such as antibodies of the present invention, the FRs of the antibody (or antigen-binding fragment thereof) can be identical to human germline sequences or can be naturally or artificially modified.

[0060] An antibody can have one or more CDR residues substituted, or one or more CDR residues omitted or added, without losing its binding properties. Antibodies that bind even when one or two CDR residues are omitted have also been reported. CDR residues that do not contact the antigen can be identified by molecular modeling or experimentally. Substitutions of CDR residues, if any, are preferably made at residues that do not contact the antigen. The amino acid residue to be substituted and the position for substitution within the CDR are selected experimentally based on various factors. Substitutions can be conservative or non-conservative. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein (e.g., antigen-binding activity or binding properties). Examples of groups of amino acids having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate; and 7) sulfur-containing side chains: cysteine ​​and methionine. Conservative amino acid substitutions refer to substitutions between amino acids within the same class, while non-conservative amino acid substitutions refer to the exchange of a member of one of these classes for a member of another class. Preferred conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.

[0061] The human anti-SARS-CoV-2(S) antibodies and the like disclosed herein include those in which one or more amino acid substitutions, additions, and / or deletions occur in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding sequences. The present invention includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, in which one or more amino acids within one or more framework and / or CDR regions may be conservatively substituted or mutated to the corresponding residue in the germline sequence from which the antibody is derived or to the corresponding residue in another human germline sequence (such sequence alterations are sometimes referred to herein as "conservative mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one skilled in the art can generate numerous antibodies and antigen-binding fragments containing one or more individual mutations or combinations thereof. The resulting antibodies and antigen-binding fragments can be readily identified and selected for one or more desired properties, such as improved binding specificity, increased binding affinity, or reduced immunogenicity. Antibodies and antigen-binding fragments obtained by such general methods are also encompassed by the present invention.

[0062] In some embodiments, amino acid substitutions in the anti-SARS-CoV-2(S) antibodies of the present invention confer one or more of the following: (1) reduced susceptibility to proteolysis; (2) reduced susceptibility to oxidation; (3) altered binding affinity for forming protein complexes; and (4) conferring or modifying other physicochemical or functional properties, while retaining specific binding to the spike protein of SARS-CoV-2. For example, one or more amino acid substitutions, preferably conservative amino acid substitutions, may be made in a portion of the polypeptide, preferably outside the domain that forms intermolecular contacts between the antigen and the antibody. Conservative amino acid substitutions should not substantially alter the structural features of the parent sequence; for example, the substituted amino acid should not alter the antiparallel β-sheet that constitutes the immunoglobulin-binding domain occurring in the parent sequence or disrupt other secondary structures that characterize the parent sequence. Examples of polypeptide secondary and tertiary structures known to those skilled in the art are described, for example, in Proteins, Structures and Molecular Principles (Creighton, Ed., W.H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al., Nature 354:105 (1991), which are incorporated herein by reference.

[0063] The present invention also includes human anti-SARS-CoV-2(S) antibodies, etc., comprising variants of any of the HCVR, LCVR, and CDR amino acid sequences disclosed herein with one or more conservative substitutions, such as anti-SARS-CoV-2(S) antibodies, etc., with no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 conservative amino acid substitution relative to any of the HCVR, LCVR, and CDR amino acid sequences disclosed herein.

[0064] The antibodies of the present invention are preferably human antibodies. A human antibody refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences. The antibodies exemplified herein are human antibodies produced by in vitro activation of B cells collected from patient donors who were infected with SARS-CoV-2 and recovered. As used herein, the term "human antibody" is not intended to include monoclonal antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) are grafted onto human FR sequences, but is intended to include antibodies recombinantly produced in non-human mammals or in the cells of non-human mammals. The antibodies described in the examples herein are human antibodies.

[0065] As used herein, "recombinant" refers to an antibody or antigen-binding fragment of the present invention that is produced, expressed, isolated, or obtained by techniques or methods known in the art as recombinant DNA technology, including, for example, transgenic expression, and includes antibodies or antigen-binding fragments thereof that are expressed in a non-human mammal (including a transgenic non-human mammal, e.g., a transgenic mouse) or mammalian cell (e.g., CHO cell) expression system, or that are isolated from a recombinant combinatorial human antibody library.

[0066] As used herein, the phrase "specifically binds" and the like means that an antibody or an antigen-binding fragment thereof binds to an antigen under physiological conditions to form a complex. Specific binding is at least about 1 x 10 -8 The specific binding between two molecules is characterized by an equilibrium dissociation constant of M or less. Methods for determining whether two molecules specifically bind to each other are well known in the art, and include, for example, equilibrium dialysis, surface plasmon resonance, etc., and these methods can be used appropriately to confirm the binding. Surface plasmon resonance can be determined, for example, by BIACORE™.

[0067] As used herein, the terms "antigen-binding fragment" of an antibody, "antigen-binding fragment thereof," and "fragment" of an antibody include any naturally occurring, enzymatically or biochemically obtained, synthetically produced, or genetically engineered polypeptide or glycoprotein that specifically binds to and forms a complex with the antigen, spike protein of SARS-CoV-2. As used herein, the term refers to one or more fragments of an antibody that retain the ability to bind to the spike protein of SARS-CoV-2, and may be produced, for example, by recombinant DNA techniques or by enzymatic or chemical cleavage of an intact antibody. As used herein, the term includes, but is not limited to, Fab, Fab', F(ab')2, Fd, Fv, disulfide Fv, dAb fragments, fragments containing the complementarity-determining regions (CDRs), single domains such as isolated CDR fragments, defined FR3-CDR3-FR4 peptides, single-chain antibodies (scFv), minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), diabodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, bispecific antibodies, multispecific antibodies (e.g., triabodies, tetrabodies), polypeptides comprising at least a portion of an antibody with specific antigen-binding ability, and other engineered molecules such as small modular immunopharmaceuticals.

[0068] Antigen-binding fragments of antibodies can be obtained from intact antibody molecules or their amino acid or genetic information using any suitable standard technique, such as, for example, proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the variable and (optionally) constant domains of the antibody. The DNA can be sequenced and manipulated using chemical or molecular biological techniques, for example, to appropriately position one or more variable and / or constant domains, or to create any antigen-binding fragment, for example, by introducing optional codons to create cysteine ​​residues or modify, add, or delete amino acids. Without limitation, Fab, Fv, and scFv antibody fragments can all be expressed in and secreted from E. coli, allowing for the facile production of large amounts of these fragments. Alternatively, Fab'-SH fragments can be expressed in E. coli, recovered, and chemically coupled to produce F(ab')2 fragments. F(ab')2 fragments can also be expressed in recombinant host cells and isolated from the culture. Other reported techniques can also be used to produce Fv fragments and single-chain Fvs (scFvs). Fvs and scFvs are antibody fragments lacking constant regions and possessing an intact antigen-binding site. Furthermore, scFv fusion proteins can be produced by fusing an effector protein to either the amino or carboxy terminus of an scFv. Various techniques for producing antibody fragments are known, and these techniques can be used without limitation in the present invention.

[0069] Antigen-binding fragments of antibodies typically contain at least one variable domain. Variable domains may be of any size or amino acid composition and generally contain at least one CDR, which is flanked or in-frame by one or more framework sequences. In antigen-binding fragments having a VH domain connected to a VL domain, the VH and VL domains are positioned relative to one another in any suitable configuration. Antigen-binding fragments of antibodies include, for example, dimeric variable regions, such as VH-VH, VH-VL, or VL-VL dimers. Alternatively, antigen-binding fragments of antibodies contain monomeric VH or VL domains.

[0070] In certain embodiments, an antigen-binding fragment of an antibody contains at least one variable domain covalently linked to at least one constant domain. Exemplary arrangements of variable and constant domains within an antigen-binding fragment of an antibody of the invention include, but are not limited to, (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any arrangement of variable and constant domains, including any of the exemplary arrangements described above, the variable and constant domains are either directly linked to each other or linked by a linker region. The linker region consists of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids and connects adjacent variable and / or constant domains. Furthermore, antigen-binding fragments of antibodies of the present invention include homo- or hetero-dimers (or other multimers) of any of the above variable and constant domain arrangements, in which one or more monomeric VH or VL domains are non-covalently associated.

[0071] Like intact antibody molecules, antigen-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically contain at least two different variable domains, each capable of specifically binding to a different antigen or a different epitope on the same antigen. Any multispecific antibody, including the bispecific antibody herein, can be produced using conventional techniques available in the art and can be used as an antigen-binding fragment of an antibody for the purposes of the present invention.

[0072] The antibody of the present invention is preferably an isolated antibody. As used herein, the term "isolated antibody" refers to an antibody that is substantially free from other antibodies with different antigen specificities.

[0073] As used herein, the terms "neutralizing antibody," "neutralizing antibody," "antibody exhibiting neutralizing activity," "antibody having neutralizing activity," and the like refer to an antibody whose binding to the spike protein of SARS-CoV-2 results in inhibition of at least one biological activity of SARS-CoV-2, and the antibodies, etc. of the present invention include those that prevent (reduce) or block the binding of SARS-CoV-2 to ACE2, or prevent (reduce) or block the infection of cells via SARS-CoV-2 binding to ACE2 (i.e., inhibit cell membrane fusion). Preferably, the antibodies, etc. of the present invention completely block the binding of SARS-CoV-2 to ACE2.

[0074] The antibodies, etc. of the present invention immunospecifically bind to the spike protein of SARS-CoV-2 and have the ability to neutralize SARS-CoV-2 viral infection. The activity of the antibodies, etc. of the present invention is determined by in vitro or in vivo assays. The activity can be confirmed and measured based on the binding activity to the spike protein of SARS-CoV-2, preferably the RBD of the spike protein. The activity can also be confirmed and measured by the neutralizing activity against the SARS-CoV-2 virus. Preferably, the activity is measured by the neutralizing activity. The ability of the antibodies, etc. of the present invention to bind to SARS-CoV-2 and neutralize the activity of SARS-CoV-2 can be measured using any standard method known to those skilled in the art, including the binding assays or neutralization assays described herein. Representative in vitro assays for measuring binding and blocking activity are described in the Examples section of the present specification. The term "inhibitory concentration 50%" ("IC 50 ") represents the concentration of an antibody of the invention required for 50% neutralization of the SARS-CoV-2 virus. 50 The higher the value, the stronger the neutralizing activity.

[0075] "TCID 50 The term "TCID" refers to the amount of virus required to infect 50% of the cells in tissue culture. 100x and 200x are TCID 50 This refers to a 100 or 200 times higher concentration of virus compared to the original concentration.

[0076] In certain embodiments, the antibodies of the present invention exhibit a 50% inhibitory concentration (IC) of about 0.01 μg / mL to about 1.0 μg / mL, or about 0.01 μg / mL to about 0.5 μg / mL, preferably about 0.1 μg / mL or less, in neutralizing the Omicron substrain, a mutant strain of the SARS-CoV-2 virus, in a pseudovirus neutralization assay. 50 The neutralizing activity is expressed as a neutralizing activity expressed as a concentration of 50 μg / mL (μg / mL). The type of target cells used in the neutralization assay using pseudoviruses is not particularly limited, and any cells can be used as long as they can detect viral infection. Examples include, but are not limited to, artificially created cells (293F / ACE2 / TMPRSS2 cells), human lung cancer cells (alveolar epithelial cells, Calu-3), and gastrointestinal mucosal epithelial cells (Caco-2). Since neutralizing activity may vary depending on the target cell, for example, the 50% inhibitory concentration can be defined as the neutralizing activity when using 293F / ACE2 / TMPRSS2 cells. The antibody used in the neutralization assay described in the Examples herein exhibited a 50% inhibitory concentration of approximately 0.01 μg / mL against the SARS-CoV-2 prototype (614G). This demonstrates that the antibody of the present invention has potent neutralizing activity.

[0077] In certain embodiments, the antibodies, etc. of the present invention have the ability to neutralize Omicron substrains, which are mutant strains of the SARS-CoV-2 virus. Examples of Omicron substrains include, but are not limited to, XBB.1.16 strain, EG.5.1 strain, BA.2.86 strain, and JN.1 strain. The antibodies, etc. of the present invention preferably exhibit a 50% inhibitory concentration (IC) of about 0.01 μg / mL to about 1 μg / mL, or about 0.01 μg / mL to about 0.6 μg / mL against multiple Omicron substrains in a neutralization assay using pseudoviruses. 50 The neutralizing activity was expressed as a function of the number of Omicron substrains (μg / mL), indicating that the antibodies of the present invention have potent neutralizing activity against a wide range of Omicron substrains.

[0078] As used herein, "epitope" refers to a site or region on an antigen that is an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and include residues directly involved in the affinity of the interaction. Epitopes can also be conformational, including nonlinear amino acid relationships. In certain embodiments, epitopes include determinants that are chemically active surface groups of molecules, such as amino acids, sugar side chains, phosphate groups, or sulfonyl groups, and have, for example, specific three-dimensional structural features and / or specific residue modifications. When amino acid substitutions are made in the antibodies of the present invention, substitutions that do not affect or have minimal impact on the binding activity of the antibody or its antigen-binding fragment to the epitope are preferred.

[0079] As used herein, "cross-compete" means that an antibody or antigen-binding fragment thereof binds to an antigen and inhibits or blocks the binding of another antibody or antigen-binding fragment thereof. In some embodiments, a first and a second antibody bind to the same epitope, thereby inhibiting or blocking the binding of the other antibody. Alternatively, the first and second antibodies bind to different but overlapping epitopes, such that binding of one antibody inhibits or blocks binding of the other antibody, e.g., through steric hindrance. Cross-competition between antibodies can be measured by methods known in the art, including, for example, surface plasmon resonance and biolayer interferometry assays.

[0080] As used herein, "substantial identity" or "identity" of the amino acid sequence of an antibody or antigen-binding fragment thereof refers to the identity when two amino acid sequences are optimally aligned, for example, using the programs GAP or BESTFIT with default gap weights. Non-identical amino acid residues in the compared sequences preferably differ between the sequences by conservative amino acid substitutions. Amino acid sequence identity can be measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, analysis can be performed using GCG software (e.g., GCG Version 6.1), FASTA (e.g., FASTA2 or FASTA3), BLASTP, or TBLASTN.

[0081] When "sequence identity" or "identity" is referred to in the context of a nucleic acid sequence, it is compared to an optimally aligned sequence of another nucleic acid (or its complementary strand), with appropriate nucleotide insertions or deletions, and measured by any well-known algorithm of sequence identity, such as, for example, FASTA, BLAST, or GAP.

[0082] As used herein, the term "subject" refers to an animal, preferably a mammal, and more preferably a human, infected with or at risk of being infected with a virus and in need of amelioration, prevention, and / or treatment of a disease or condition, such as a viral infection. In one embodiment, the subject is a human infected with or at risk of being infected with SARS-CoV-2.

[0083] The antibodies of the present invention specifically bind to the spike protein of SARS-CoV-2 and can therefore be used to detect the SARS-CoV-2 virus or spike protein of SARS-CoV-2, which are antigens.

[0084] "Binding affinity," which refers to the binding activity of an antibody and an antigen, generally refers to the combined strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). "Binding affinity" is expressed as a unique numerical value reflecting a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X with its partner Y can generally be expressed by the equilibrium dissociation constant (Kd), calculated as the ratio koff / kon. See, for example, Chen, et al. (1999) J. Mol Biol 293:865-881. Affinity can be measured by common methods known in the art. Low-affinity antibodies generally bind to antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind to antigens more rapidly and tend to remain bound for longer. Exemplary measurement methods include, but are not limited to, radiolabeled antigen binding assays (RIA), ELISA-based immunoassays, and surface plasmon resonance assays using Biacore®. Methods and reagents suitable for determining the binding properties of the antibodies of the present invention, or their antigen-binding fragments, or modified / mutated derivatives thereof, are known in the art and are commercially available. Furthermore, instruments and software designed for such analyses are also commercially available (e.g., Biacore® A100 and Biacore® 2000).

[0085] In certain embodiments, antibody-antigen binding assays can be performed as either direct binding assays or competitive binding assays. Binding can be detected using standard ELISA or standard flow cytometry assays. In direct binding assays, a test antibody is tested for binding to its antigen, while in competitive binding assays, the test antibody's ability to compete with a known antibody for binding to the spike protein of SARS-CoV-2 is assessed. These methods are used to screen for those that offer desired properties.

[0086] Antibodies specific for the spike protein of SARS-CoV-2 can also be further labeled. The label can be attached to the N-terminus or C-terminus of the antibody or to the side chain of any amino acid residue. The label can be, for example, avidin-biotin, a radionuclide, a fluorescent dye, or an MRI-detectable label. In certain embodiments, the labeled antibodies are used in diagnostic assays, including imaging assays.

[0087] In addition to the amino acid sequences of the antibodies, etc. of the present invention described herein, the present invention also provides nucleotide sequences corresponding to and encoding the amino acid sequences. Thus, the present invention also includes isolated nucleic acids having nucleotide sequences encoding the antibodies, etc. of the present invention. The isolated nucleic acids are preferably cDNAs. These nucleic acid sequences include nucleic acid sequences encoding the light and heavy chains and part or all of the CDRs of the antibodies of the present invention, and are also encompassed by the present invention. Thus, the present invention also includes polynucleotide sequences encoding the VH and VL framework regions, including the CDRs and FRs, of the antibodies described herein, as well as expression vectors for efficient expression thereof in cells (e.g., mammalian cells). Methods for producing antibodies using polynucleotides are known in the art, and these methods can be used as appropriate. Methods for producing the antibodies of the present invention using these methods are also encompassed by the present invention.

[0088] Exemplary techniques for producing antibodies that can be used in the present invention are described below. The antibodies of the present invention can be prepared using a wide range of techniques known in the art, including genetic recombination techniques, phage display techniques, or a combination thereof.

[0089] Methods for producing and screening for specific antibodies using genetic recombination techniques are well known and commonly used in the art. DNA encoding the amino acid sequences of the antibodies of the present invention can be easily prepared using known techniques. For example, but not limited to, DNA sequences encoding the amino acids of each CDR can be chemically synthesized with reference to the sequences shown in Figure 1, and these can be combined as desired. In this case, they can also be combined with any DNA encoding the amino acid sequences of the VH or LV FRs.

[0090] To express an antibody, its antigen-binding fragment, or a variant thereof, an expression vector containing a polynucleotide encoding the antibody or the like is constructed. Thus, replicable vectors containing a nucleotide sequence encoding an antibody molecule, the heavy or light chain of the antibody, the variable domain of the heavy or light chain of the antibody or a portion thereof, or any CDR of the heavy or light chain, operably linked to a promoter, are provided and are also included in the present invention. Such vectors can contain nucleotide sequences encoding the constant region of the antibody molecule, and variable domains of the antibody or the like may also be cloned into the vector for expression of the entire heavy chain, the entire light chain, or both the entire heavy and light chains. The expression vector is transfected into host cells, and the transfected cells are then cultured to produce the antibody. Thus, the present invention includes host cells containing a polynucleotide encoding the amino acid sequence of the antibody or the like of the present invention. When expressing a double-chain antibody, vectors encoding both the heavy and light chains may be simultaneously expressed in host cells for expression of the entire immunoglobulin molecule.

[0091] Mammalian cell lines available as hosts for expression of recombinant antibodies include many immortalized cell lines known in the art and available from the ATCC, including, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells (e.g., HepG2), human renal epithelial cells (HREpC), human embryonic kidney (HEK) cells, and numerous other cell lines. Appropriate cell lines or host systems can be selected to ensure the correct modification and processing of the expressed antibody or portion thereof. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product are preferably used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, HeLa, COS, MDCK, HEK293, 3T3, W138, BT483, Hs578T, HTB2, BT2O, T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any functional immunoglobulin chains), SP20, CRL7O3O, and HsS78Bst cells. Antibodies and the like can also be produced using human cell lines developed by immortalizing human lymphocytes using genetic recombination techniques, and the human cell line PER.C6. can also be used to produce antibodies and the like.

[0092] Other cell lines that can be used as hosts for the expression of recombinant antibodies also include, for example, insect cells (e.g., Sf21 / Sf9, Trichoplusiani Bti-Tn5b1-4) or yeast cells (e.g., S. cerevisiae, Pichia, etc.), plant cells, and chicken cells.

[0093] The transfected cell line is maintained in cell culture media and conditions known in the art that result in the expression and production of the antibody. Media and culture techniques known in the art can be used without limitation. Antibody production can be carried out on a large scale by a bioreactor process using fed-batch, batch, perfusion, or continuous-feed bioreactor methods known in the art, and such techniques allow for the large-scale production of antibodies.

[0094] The present invention also relates to methods for producing the antibodies of the present invention. The production methods of the present invention include a step of culturing a host cell of the present invention under conditions suitable for expression of the antibody or antigen-binding fragment thereof. Such methods may further include a step of isolating the antibody or antigen-binding fragment thereof from the host cell culture and, optionally, a step of incorporating the isolated antibody or antigen-binding fragment thereof into a composition.

[0095] Antibodies, antigen-binding fragments thereof, or amino acid substitutions thereof can also be produced using the so-called phage display technology. A phage display library can be created with reference to the sequences described herein, and antibodies, or antigen-binding fragments thereof, of the present invention can be produced using binding to the spike protein of SARS-CoV-2 as an indicator. In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequence encoding them. Phages expressing antigen-binding domains that bind to the antigen of interest can be selected or identified, for example, using labeled antigen or antigen bound or captured to a solid surface or bead.

[0096] Antibody purification and isolation: After production by expression using recombinant techniques, antibody molecules can be purified by any method known in the art for purifying immunoglobulin molecules, such as chromatography (e.g., ion exchange, affinity, particularly affinity for specific antigens Protein A or Protein G, and size exclusion column chromatography), centrifugation, differential solubility, or any other standard protein purification technique. Furthermore, antibodies of the present invention may be fused to heterologous polypeptide sequences (commonly referred to as "tags") known to facilitate purification.

[0097] Using recombinant technology, antibodies can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If antibodies are produced intracellularly, the first step is to remove particulate debris, which is fragments of lysed host cells, for example, by centrifugation or ultrafiltration. If antibodies are secreted into the medium, the supernatant from the expression system is concentrated using a protein concentration filter, for example, an ultrafiltration unit.

[0098] Antibody-containing preparations prepared from cells can be purified using, for example, hydroxylapatite chromatography, hydrophobic interaction chromatography, ion exchange chromatography, gel electrophoresis, dialysis, and / or affinity chromatography, alone or in combination with other purification steps. When the antibody contains a CH3 domain, Bakerbond ABX resin is useful for purification. Other protein purification techniques, such as fractionation on an ion exchange column, ethanol precipitation, reverse-phase HPLC, silica chromatography, heparin chromatography, anion or cation exchange resins (such as polyaspartic acid columns), Sepharose chromatography, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available, depending on the antibody to be recovered.

[0099] In certain embodiments, substantially purified or isolated antibodies of the invention are provided, and in certain embodiments, such purified or isolated recombinantly expressed antibodies can be administered to a patient to achieve a prophylactic or therapeutic effect.

[0100] Human antibodies can be produced using methods well known in the art. Human antibodies can also be obtained by in vitro methods, such as phage display, ribosome display, and yeast display. Phage display technology (see, for example, U.S. Pat. No. 5,969,108) can be used to produce human antibodies or antibody fragments in vitro from immunoglobulin variable (V) domain gene repertoires derived from unimmunized donors. Alternatively, human antibodies can also be produced by in vitro activated B cells (see, for example, U.S. Pat. Nos. 5,567,610 and 5,229,275).

[0101] Immunoglobulin genes undergo various modifications during the maturation of immune responses, including recombination between V, D, and J gene segments in the variable regions, isotype switching, and hypermutation. While recombination and somatic hypermutation generate antibody diversity and affinity maturation, they can also increase the immunogenicity risk of antibodies. Generally, mutations in CDR regions are thought to contribute to improved affinity and function, while mutations in framework regions can increase the immunogenicity risk. Therefore, risk can be reduced by maintaining framework sequences similar to germline sequences. It is desirable to remove potential structural factors that could lead to instability, aggregation, heterogeneity, or increased immunogenicity of the resulting antibodies. Examples of undesirable structural factors include unpaired cysteines (which can lead to unwanted disulfide bond formation or variable sulfhydryl adduct formation), N-linked glycosylation sites (leading to heterogeneity in structure and activity), and deamidation (e.g., NG, NS), isomerization (DG), oxidation (exposed methionine), and hydrolysis (DP) sites. Therefore, to reduce the risk of immunogenicity and improve pharmaceutical properties, it is desirable to restore the framework sequences to the germline, restore the CDRs to the germline, and / or remove structural elements. The antibodies of the present invention can be mutated or substituted with amino acids from this perspective, and these are also included in the present invention.

[0102] In one embodiment, the antibody of the present invention is an antibody fragment or an antibody comprising such a fragment. An antibody fragment comprises a portion of an intact antibody, such as its antigen-binding region or variable region. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fd, Fv fragments, disulfide Fv, single-chain antibodies (ScFv), single-domain antibodies, nanobodies, and diabodies.

[0103] In another embodiment, the antibody of the present invention is a domain antibody, e.g., an antibody comprising a small functional binding unit of an antibody corresponding to the variable heavy (VH) or variable light (VL) region of a human antibody. Examples of domain antibodies include, but are not limited to, antibodies produced using Domantis technology (see, e.g., WO 04 / 058821; WO 04 / 081026; WO 04 / 003019; WO 03 / 002609).

[0104] In certain embodiments, the antibodies of the present invention are linear antibodies. Linear antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions.

[0105] In some embodiments, antibodies of the present invention may be monospecific, bispecific, or multispecific. Multispecific antibodies contain antigen-binding domains specific for different epitopes of a single target polypeptide or specific for multiple target polypeptides. Any of the multispecific antigen-binding molecules of the present invention, or variants thereof, can be constructed using molecular biology techniques known to those skilled in the art (e.g., recombinant DNA and protein expression techniques).

[0106] In certain embodiments, bispecific SARS-CoV-2 spike protein-specific antibodies can be generated by combining variable regions that bind to distinct domains of the SARS-CoV-2 spike protein into a single binding molecule with dual-domain specificity. Properly designed and engineered bispecific antibodies can enhance overall SARS-CoV-2 viral inhibitory activity through increased specificity and avidity. In one example of bispecificity, a single VL segment (e.g., VL1) is combined with two different VH domains (e.g., VH1 and VH2) to generate a bispecific antibody containing two binding "arms" (VH1-VL1 and VH2-VL1). The use of a single VL segment can reduce the complexity of the system and increase the efficiency of cloning, expression, and purification methods.

[0107] Other exemplary bispecific formats include, but are not limited to, scFv-based bispecific formats. Bispecific antibody formats include, for example, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knobs-into-holes, common light chains (e.g., common light chains with knobs-into-holes), crossMab, crossFab, (SEED)body, leucine zipper, duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab2 bispecific formats. Bispecific antibodies can also be constructed using peptide / nucleic acid conjugates.

[0108] The present invention also encompasses further modifications of the antibodies of the present invention, as well as variants and fragments thereof, including substitutions, additions, and / or deletions of one or more amino acid residues and / or polypeptides in the variable light chain (VL) domain and / or variable heavy chain (VH) domain and Fc region, and post-translational modifications. Antibody conjugates in which other substances, such as proteins, peptides, drugs, or labels, are covalently bound to the antibody are also included as modifications. Such antibody modifications and alterations can be used to alter the biochemical and / or functional properties of the antibody so that it is suitable for the treatment and / or diagnosis of SARS-CoV-2 infection. Techniques for making such modifications and alterations are known in the art and can be used without limitation in the present invention.

[0109] In certain embodiments, antibody modifications can be made by introducing one or more amino acid modifications (e.g., substitutions, deletions, and / or additions) into one or more variable regions of the antibody. In another embodiment, amino acid modifications can be introduced into framework regions. Modified antibodies can be screened for their activity using the methods described herein. Amino acid substitutions can be conservative or non-conservative, as described above. Also, if desired, non-naturally occurring amino acids or amino acid analogs can be substituted or added into the antibody sequence. Examples of such amino acids include, but are not limited to, D-isomers of the naturally occurring amino acids, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, designer amino acids such as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and other amino acid analogs.

[0110] In another embodiment, any cysteine ​​residue in the antibodies of the present invention that is not involved in maintaining the proper conformation of the antibody or antigen-binding fragment thereof may be substituted, for example with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Adding one or more cysteine ​​bond(s) to an antibody can also improve its stability. This is particularly useful when the antibody is an antibody fragment such as an Fv fragment.

[0111] In certain embodiments, the antibodies and other substances of the present invention can be conjugated (conjugated or covalently bonded) to other substances using methods known in the art. Examples of substances to be conjugated include therapeutic agents, therapeutic adjuvants, detectable labels, and solid supports. Substances that can be conjugated to antibodies include, but are not limited to, amino acids, peptides, proteins, polysaccharides, nucleosides, nucleotides, oligonucleotides, nucleic acids, haptens, drugs, hormones, lipids, lipid assemblies, synthetic polymers, microparticles, biological cells, viruses, fluorophores, chromophores, dyes, toxins, haptens, enzymes, antibodies, antibody fragments, radioisotopes, solid matrices, semi-solid matrices, and combinations thereof. Methods for conjugating these other substances to antibodies are known in the art and can be used without limitation in the present invention.

[0112] In one embodiment, the antibodies of the present invention are bound to a solid support. Antibodies bound to a solid support can be used, for example, in screening, purification, as part of a manufacturing process, or as part of a diagnostic method or composition. Solid supports are generally substantially insoluble in liquid phases. Numerous supports are known to those skilled in the art and can be used in the present invention. Solid supports include, but are not limited to, solid and semi-solid matrices, such as aerogels and hydrogels, resins, beads, biochips (including thin film-coated biochips), microfluidic chips, silicon chips, multiwell plates (also called microtiter plates or microplates), membranes, conductive and non-conductive metals, glass (including microscope slides), and magnetic supports. More specific examples of solid supports include silica gel, polymer membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, alumina gel, polysaccharides such as Sepharose, polyacrylate, polystyrene, polyacrylamide, polyols, agarose, agar, cellulose, dextran, starch, Ficoll, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose, polyvinyl chloride, polypropylene, polyethylene (including polyethylene glycol), nylon, latex beads, magnetic beads, paramagnetic beads, and superparamagnetic beads.

[0113] In certain embodiments, the solid support may contain a reactive functional group for binding to an antibody of the present invention, such as a hydroxyl group, a carboxyl group, an amino group, a thiol group, an aldehyde group, a halogen group, a nitro group, a cyano group, an amide group, a urea group, a carbonate group, a carbamate group, an isocyanate group, a sulfone group, a sulfonate group, a sulfonamide group, or a sulfoxide group.

[0114] In one embodiment, the antibodies of the present invention can be conjugated to a label for the purpose of diagnosis and other measurements (e.g., detection of SARS-CoV-2 virus). Labels that can be conjugated to antibodies include, but are not limited to, chromophores, fluorophores, fluorescent proteins, phosphorescent dyes, tandem dyes, particles, haptens, enzymes, and radioisotopes. Techniques for conjugating antibodies to these labeling substances are well known in the art and can be used in the present invention. In addition, in diagnosis and other measurements using labeled antibodies, detection methods based on the type of label are also well known in the art, and such detection methods can also be used in the present invention.

[0115] The antibodies, etc. of the present invention can be used for treating SARS-CoV-2 virus infection, preventing SARS-CoV-2 virus infection, and / or detecting, diagnosing, and / or prognosing SARS-CoV-2 virus infection. The antibodies, etc. of the present invention can also be used for treating, preventing, or diagnosing any SARS-CoV-2 virus mutant strain, so long as they specifically bind to the antigen.

[0116] In one embodiment, the present invention is a method for treating a subject by administering an effective amount of an antibody, etc. of the present invention to the subject. In some embodiments, the antibody or antigen-binding fragment thereof is substantially purified. In some embodiments, the antibody, etc. of the present invention is administered to a subject infected with or suspected of being infected with SARS-CoV-2. In some embodiments, the antibody, etc. of the present invention is administered prophylactically to a subject at risk of infection with SARS-CoV-2. In another embodiment, the antibody, etc. of the present invention is administered to a subject after infection or after the onset of symptoms, or prophylactically before infection. In some embodiments, the antibody, etc. of the present invention is administered to a subject who is infected but asymptomatic.

[0117] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention, or pharmaceutical compositions comprising the same, and methods of the present invention of administering them to a subject treat SARS-CoV-2 virus infection or reduce viral infection in a subject. In other embodiments, the pharmaceutical compositions and methods of the present invention prevent, reduce the risk of, or delay SARS-CoV-2 virus infection in a subject.

[0118] The subject to which the antibody, etc. of the present invention is administered is a mammal, preferably a human. In a more specific embodiment, the subject is, for example, an immunocompromised subject, or a subject who is particularly at risk or susceptible to infection with the SARS-CoV-2 virus. At-risk subjects (patients) include, but are not limited to, immunocompromised individuals, elderly individuals (age 65 or older), individuals with underlying conditions such as lung infections, heart disease, respiratory disease, diabetes, or dialysis patients, healthcare workers, and individuals who have been in close contact with or are expected to be in contact with individuals with confirmed or suspected SARS-CoV-2 infection.

[0119] Therapeutic administration can be a single dose schedule or a multiple dose schedule, and the antibodies or antigen-binding fragments thereof of the invention can be used in passive immunization.

[0120] In certain embodiments, the antibodies, etc. of the present invention can be administered to a subject in combination with one or more other drugs, such as antiviral drugs. Examples of other drugs include, but are not limited to, antiviral drugs (remdesivir, molnupiravir, nilmatrervir / ritonavir, ensitrervir, ensitrervir fumarate) and neutralizing antibody drugs (casirivimab / imdevimab, sotrovimab, tixagevimab / silgavimab). In certain embodiments, the antibodies, etc. of the present invention can be administered to a subject in combination with a therapeutic agent that helps counter or reduce potential side effects associated with the antibodies, etc. of the present invention, if any. The antibodies, etc. of the present invention can also be administered to a subject in combination with a therapeutic agent for diseases that pose a risk of becoming severe or that cause complications in SARS-CoV-2 infection, such as diabetes, cardiovascular disease, kidney disease, and chronic respiratory diseases such as COPD. Such agents can include, for example, anti-inflammatory agents (e.g., corticosteroids and nonsteroidal anti-inflammatory agents), anti-infective agents, nutritional supplements such as antioxidants, and any other drugs known in the art that improve the physical or mental condition of a subject.

[0121] In another embodiment, the antibodies of the present invention can be administered to a subject in combination with one or more other neutralizing antibodies against SARS-CoV-2. Such other antibodies can include the neutralizing antibodies described above.

[0122] The present invention also provides a method for producing a pharmaceutical composition, which comprises the step of mixing an antibody or the like of the present invention with one or more pharmaceutically acceptable carriers.

[0123] The present invention also provides pharmaceutical compositions comprising the antibodies of the present invention as an active ingredient. Such pharmaceutical compositions contain a therapeutically effective amount of the antibodies of the present invention and further comprise a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" refers to a carrier approved for use by a regulatory agency or listed in a pharmacopoeia generally accepted for use in animals, particularly humans. A carrier includes a diluent, adjuvant, excipient, or vehicle with which an active ingredient is administered. Such carriers include sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Physiological saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. The pharmaceutical composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0124] The pharmaceutical composition of the present invention can take any form, for example, a solution, a suspension, an emulsion, a tablet, a pill, a capsule, a powder, a sustained-release preparation, etc. Techniques for formulating pharmaceutical compositions into each form are known in the art, and these can be used as reference in the production of the pharmaceutical composition of the present invention, and a formulation suitable for the administration method is carried out.

[0125] The dosage of the antibody, etc. of the present invention is selected appropriately depending on the symptoms, age, condition, administration route, etc. of the subject. When the antibody, etc. of the present invention is used for the treatment of a disease in an adult patient or for the prevention of such a disease, it is typically administered once or multiple times at a single dose of, but not limited to, about 0.01 mg per kg of body weight as a lower limit, preferably about 0.05 mg, more preferably about 0.1 mg, and about 100 mg per kg of body weight as an upper limit, preferably about 50 mg, more preferably about 20 mg, and even more preferably about 10 mg. When an antigen-binding fragment of the antibody of the present invention is used, it is typically administered at a higher dose than the above-mentioned dose, for example, but not limited to, about 1.5 to about 50 times, preferably about 1.5 to about 20 times, and more preferably about 1.5 to about 10 times the dose. The frequency and interval of administration are adjusted depending on the severity of the condition. In one embodiment, the antibody, etc. of the present invention is administered as a starting dose of at least about 1 mg to about 1000 mg. In certain embodiments, the initial dose is followed by second or multiple subsequent doses of the antibody or antigen-binding fragment thereof in an amount that is about the same as or less than that of the initial dose, where the subsequent doses are separated by at least 1 to 3 days; at least 1 week; at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; or at least 12 weeks.

[0126] The administration treatment plan, including the administration dose and administration schedule, can be determined by the physician who diagnosed the subject (patient) based on the patient's symptoms, age, condition, administration route, etc., and by referring to guidelines presented by various organizations, as necessary.

[0127] The administration route of the pharmaceutical composition of the present invention can be determined arbitrarily, taking into account the symptoms, condition, and other conditions of the subject. Administration routes include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The pharmaceutical composition can be administered by any convenient route, for example, by infusion or bolus injection, or by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, and intestinal mucosa), and may also be administered together with other biologically active agents. Administration can be systemic or local.

[0128] Various delivery systems are known for administering active pharmaceutical ingredients, and the pharmaceutical compositions of the present invention can be administered using these. Examples include encapsulation in liposomes, microparticles, nanoparticles, and microcapsules. In some embodiments, the pharmaceutical compositions can also be delivered in controlled release systems. Controlled release systems place the composition near the target, thus requiring only a small systemic dose.

[0129] When the pharmaceutical composition of the present invention is administered by injection, injectable preparations include intravenous, subcutaneous, intradermal, intracranial, intraperitoneal, and intramuscular injection forms, or drip infusion forms. These injectable preparations can be prepared with reference to known methods. For example, injectable preparations can be prepared by dissolving, suspending, or emulsifying the antibody or salt of the present invention in a sterile aqueous or oily medium commonly used for injections. Aqueous media for injection include, for example, physiological saline, an isotonic solution containing glucose, and other adjuvants. These media can also be used in combination with appropriate solubilizers such as alcohols (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Oily media include, for example, sesame oil and soybean oil, which can be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. The prepared injections are preferably filled into suitable ampoules.

[0130] When the pharmaceutical composition of the present invention is an injection, it can be prepared as an injection to be prepared at the time of use or as a pre-filled syringe.The pharmaceutical composition of the present invention can be administered subcutaneously or intravenously with a standard needle and syringe.Also, a pen delivery device can be used for subcutaneous administration.The pen delivery device can be reusable or disposable.

[0131] Pharmaceutical compositions, whether oral or parenteral, are prepared in dosage forms prepared in unit doses suitable for the desired dose of the active ingredient. Dosage forms containing a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of antibody contained in a unit dose is generally preferably about 5 to about 1,000 mg per dosage form.

[0132] Pharmaceutical compositions comprising the antibodies of the present invention are useful for treating and / or preventing diseases, disorders, or conditions associated with SARS coronavirus, such as SARS-CoV-2 infection, particularly SARS-CoV-2 infection. The pharmaceutical compositions of the present invention are also useful for ameliorating at least one symptom associated with SARS coronavirus infection, particularly SARS-CoV-2 virus infection. Such symptoms include, but are not limited to, pulmonary inflammation, alveolar damage, fever, respiratory disorders (nasal congestion, runny nose, sore throat, cough, shortness of breath), headache, fatigue, dysgeusia, dysosmia, diarrhea, vomiting, increased blood coagulation, thrombosis, Kawasaki disease-like vasculitis, renal dysfunction (e.g., nephritis), organ failure, pneumonia, septic shock, and death. In certain embodiments, the antibodies of the present invention are useful for treating subjects suffering from severe and acute respiratory infections caused by the SARS-CoV-2 virus. In certain embodiments, the antibodies of the present invention are useful for reducing viral titers in a host. In one embodiment, the antibodies of the invention are useful in preventing or reducing pulmonary inflammation in subjects infected with SARS-CoV-2. In one embodiment, the antibodies of the invention are useful in preventing or reducing interstitial, peribronchiolar, or perivascular inflammation, alveolar damage, and pleural changes in subjects infected with SARS-CoV-2.

[0133] Pharmaceutical compositions containing the antibodies, etc. of the present invention can be used in combination with other drugs or as a combination product with other drugs. Alternatively, two or more types of antibodies, etc. of the present invention can be used in combination in a single pharmaceutical composition. In such cases, it is preferable to combine antibodies or antigen-binding fragments thereof that recognize different epitopes. Examples of second therapeutic agents that can be used in combination with pharmaceutical compositions containing the antibodies, etc. of the present invention include those listed above. Synergistic effects are expected from these combinations.

[0134] Diagnostic methods using antibodies or the like of the present invention include a step of contacting an antibody or the like with a sample collected from a subject. Such a sample is not particularly limited as long as it is collected from a subject and is suspected of being contaminated with a virus or a portion of a virus, and includes, for example, samples collected from the nasal passages, sinus cavities, salivary glands, lungs, liver, pancreas, kidneys, ears, eyes, placenta, digestive tract, heart, ovaries, pituitary gland, adrenal gland, thyroid gland, brain, skin, or blood vessels (preferably peripheral blood vessels). Representative examples of such samples include saliva, blood, and nasal / pharyngeal swabs.

[0135] The anti-SARS-CoV-2(S) antibodies of the invention can be used to detect or measure SARS-CoV in a sample, e.g., for diagnostic purposes. In certain embodiments, one or more antibodies of the invention can be used in assays to detect diseases or disorders, such as viral infections. A typical diagnostic assay for SARS-CoV-2, for example, involves contacting a sample (preferably saliva, blood, or nasopharyngeal swab) obtained from a patient with an anti-SARS-CoV-2(S) antibody of the invention, where the anti-SARS-CoV-2(S) antibody is labeled with a detectable label or reporter molecule or is used as a capture ligand to selectively isolate SARS-CoV-2 from the patient sample. Alternatively, an unlabeled anti-SARS-CoV-2(S) antibody can be used in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule is a radioisotope; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Assay methods used to detect or measure SARS-CoV-2 in a sample include, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS). Antibody-based diagnostic and detection methods, including these, are known in the art, and such known methods can be used in the present invention.

[0136] The antibodies of the present invention can also be used as a kit for diagnosing SARS-CoV-2 virus infection. Furthermore, since the antibodies of the present invention specifically bind to the RBD of the spike protein of SARS-CoV-2, they can also be used in the quality control of vaccine production.

[0137] The present invention will be described in detail below using examples, but the present invention is not limited to the following examples. This research plan, which includes collaborators from the University of Tokyo, has undergone ethics review and been approved by the Graduate School of Life Sciences, Kumamoto University (Ethics No. 2013, Genome No. 461). Furthermore, research on post-vaccination cases has also undergone ethics review and been approved by the Graduate School of Life Sciences, Kumamoto University (Ethics No. 2643). Selection of cases infected with the Omicron strain of SARS-CoV-2 after vaccination and in the recovery phase, acquisition of consent, and collection of blood samples were performed by a physician in charge (Masayuki Amano) of the Endowed Department of Clinical Retrovirology at the Human Retrovirology Joint Research Center, and the samples were provided to the present inventors for study.

[0138] Example 1: Obtaining Neutralizing Antibodies Blood samples were collected from patients with subclinical (asymptomatic) SARS-CoV-2 infection after three doses of a monovalent vaccine, as described in our previous report (WO 2024 / 053719). 7-AAD-CD19+IgM-IgG+ cells that bound to NK56 (RBD) or NK48 (Spike trimer) were obtained by single-cell sorting. cDNA was synthesized from the RNA of the obtained cells using reverse transcriptase, and the variable regions of the antibody heavy and light chains were amplified by PCR. The PCR product was then cloned into an expression vector to create a plasmid, which was then transfected into 293T cells. The antibodies produced in the supernatant were assayed for their binding activity to Spike and their neutralizing activity against SARS-CoV-2 pseudovirus. A total of 181 antibodies were produced. Of these, 135 antibodies bound to the Spike protein, and 27 antibodies showed neutralizing activity.

[0139] B cells were isolated from the plasma of the selected cases, and antibodies were collected. 7AAD antibodies were isolated from peripheral blood mononuclear cells isolated from blood samples. - , CD19 + , IgG + , IgM - NK56 (RBD) or NK48 (Spike trimer) B cells were single-cell sorted.

[0140] Antibodies were then recovered as follows. mRNA was recovered from the resulting cells, and the IgG variable region was amplified from the recovered mRNA by RT-PCR, and paired heavy and light chain genes were selected. The heavy and light chain genes were each incorporated into an expression vector and transfected into HEK293T cells to express the antibodies, and the recombinant antibodies (recombinant IgG; rIgG) produced in the supernatant were recovered. 181 (types) of antibodies were obtained.

[0141] The binding of the 181 antibodies obtained to the SARS-CoV-2 spike protein was confirmed as follows. HEK293T cells were transfected with a plasmid co-expressing the SARS-CoV-2 spike protein and EGFP to express the spike protein on the cell surface. Two days after transfection, the cells were detached with trypsin and suspended in 0.2% BSA-PBS to serve as spike protein- and EGFP-expressing cells. 50 μL of the spike protein- and EGFP-expressing cell suspension was mixed with 50 μL of cell culture supernatant containing the obtained antibody or a sample of control plasma (infected patient plasma or normal patient plasma) (1:500 dilution, 50 μL) and allowed to stand at room temperature for 15 minutes. The cells were separated by centrifugation, the supernatant discarded, and then suspended and washed in 0.2% BSA-PBS. After suspending in 50 μL of 200-fold diluted APC-conjugated anti-human IgG (goat antibody) antibody, the cells were incubated at room temperature for 15 minutes. After washing with 0.2% BSA-PBS, the cells were fixed with 4% paraformaldehyde-PBS. The stained cells were analyzed by flow cytometry. EGFP-positive cells were gated as spike protein-expressing cells, and the spike protein-binding activity of the antibodies was determined by the APC positivity rate of the EGFP+ cell population. Flow cytometry analysis revealed that 135 of the 181 antibodies bound to the spike protein.

[0142] The 181 antibodies obtained were used to screen and identify antibodies that neutralize mutant viruses with the D614G spike mutation (prototype (614G) virus) as follows. Plasmids co-expressing the antibody heavy and light chains were transfected into HEK293T cells, and the SARS-CoV-2 neutralizing activity of the recombinant antibodies contained in the cell culture supernatant was examined in a pseudovirus neutralization test. Pseudoviruses were produced by transfecting HEK293T cells with the packaging plasmid psPAX2-IN / HiBiT, the transfer plasmid pWPI-Luc2, and the SARS-CoV-2 Spike expression plasmid pCMV3-SARS2-S at a ratio of 2:2:1. The SARS-CoV-2 Spike expression plasmid was prepared by modifying the wild-type plasmid obtained from Sino Biological Inc. and introducing the prototype (614G) mutation. Two days after transfection, the culture supernatant was collected, passed through a 0.22 μM filter, stored at -80°C, and used in the following tests. For the neutralization test, 293FT-DSP1-7-ACE2-TMPRSS2 cells (provided by Professor Inoue of the Institute of Medical Science, University of Tokyo) were used. The day before, 1 x 10 5 The cell suspension (100 μL / well) containing 100 cells / mL of recombinant antibodies was seeded onto a 96-well plate. The cell culture supernatant of each clone containing the recombinant antibody was diluted 2-fold with culture medium and added to another 96-well plate at 110 μL / well. Then, 200 TCID 50The pseudovirus was added at 110 μL / well and incubated for 1 hour at 37°C (hereinafter referred to as the "culture supernatant and virus mixture"). Experiments were performed on two wells of each sample. The cell culture supernatant prepared the day before was discarded, and the "culture supernatant and virus mixture" was added and incubated. After 5 hours, the "culture supernatant and virus mixture" was discarded, and 200 μL of fresh culture medium was added and further incubated. Two days after infection, the cell supernatant was discarded, and 30 μL of lysis buffer was added and stirred for 10 minutes to lyse the cells. 10 μL of the cell lysate was transferred to a white 96-well plate, and 50 μL of luciferase substrate was added, and fluorescence was measured. The RLU (relative light unit) of a sample containing only virus was defined as 100% infection, and the RLU of a sample containing no virus was defined as 0% infection. The neutralizing activity of cell culture supernatants containing each antibody was determined by the percentage reduction in RLU from 100% infection. Two tests were performed, and neutralizing antibodies were defined as those that showed neutralizing activity of approximately 40% or more from both tests. As a result, 27 antibodies showed neutralizing activity.

[0143] (Example 2) Neutralization test of Omicron strain using prepared purified antibodies The genes of the selected 27 antibodies were introduced into CHO cells, purified antibodies were prepared, and neutralization tests were performed using the purified antibodies. Introduction of the antibody genes into CHO cells and preparation of purified antibodies from cell culture supernatants were performed according to standard methods using an ExpiCHO Expression System (Thermo Fisher). Neutralizing activity was confirmed by neutralization tests using live viruses of the wild-type strain (Wuhan) and Omicron strains BA.2, BA.5, BQ.1.1, XBB, and XBB.1.5. Specifically, the tests were performed as follows, with reference to Amano et al. Sci Rep. 2022. 12(1):13524. The diluted antibody was incubated with 100 TCID50 of virus for 20 minutes, and the antibody-virus mixture was then spread onto VeroE6 TMPRSS2 cells (1x10 ４After 3 days of culture, virus-induced cell death was measured using Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) and determined as 50% inhibition concentration (IC ５０ The results are shown in Figure 2. The 1-94 antibody, 2-66 antibody, 2-6 antibody, and 1-38 antibody exhibited potent neutralizing activity against all virus strains tested, and the 2-38 antibody, 1-18 antibody, and 1-67 antibody also exhibited good neutralizing activity against a wide range of virus strains.

[0144] (Example 3) Neutralization test of purified antibodies against Omicron strain using pseudovirus Neutralization tests using pseudovirus against various mutant strains of the 27 selected antibodies were specifically carried out as follows. Neutralization tests were carried out against various SARS-CoV-2 mutant strains (prototype mutant strain (614G), XBB.1.16, EG.5.1, BA.2.86, JN.1). Expression plasmids expressing the Spike proteins of SARS-CoV-2 mutant strains were created by PCR-mediated mutation insertion based on the wild-type Spike expression plasmid. The amino acid mutations in the Spike protein of each strain are shown in Figure 3.

[0145] The SARS-CoV-2 mutant Spike expression plasmid, the lentiviral packaging plasmid psPAX2-IN / HiBiT, and the transfer plasmid pWPI-Luc2 were co-transfected into 293T cells, and the supernatant was collected after 2 days, frozen, and used as a pseudovirus.

[0146] The neutralizing activity was measured as follows, referring to Kaku et al. Cell Rep. 2021 Jul 13;36(2):109385. The monoclonal antibody was serially diluted 5-fold with medium in a 96-well plate and diluted to 200 TCID ５０The antibody-virus mixture was mixed with 1000 μg of virus / well and incubated at 37°C for 1 hour. The antibody-virus mixture was inoculated into 293FT-DSP1-7-ACE2-TMPRSS2 cells in a 96-well plate (3 wells per sample). After 2 hours of incubation at 37°C, the antibody-virus mixture was replaced with fresh medium and cultured for 2 days. Luciferase activity was measured using the britelite plus Reporter Gene Assay System (Revvity) to calculate the reduction in infection rate, and the IC ５０ It was decided that:

[0147] Seven antibodies expected to have cross-neutralizing activity were selected in Example 2, and their neutralizing activity against various Omicron substrains was compared with that of existing antibodies. Mutant strains tested were the prototype 614G, and pseudoviruses of the Omicron substrains XBB.1.16, EG.5.1, BA.2.86, and JN.1 strains, which are responsible for global pandemics. The existing antibodies used were REGN-10933 and REGN-10987 (Regeneron / Roche), LY-CoV555 (Eli Lilly), VIR-7831, and Bebtelovimab VIR-7832 (Eli Lilly). These existing antibodies were produced using a CHO cell expression system based on publicly known information about the antibodies. Comparison was also made with the 9-105 and 10-121 antibodies, as well as 4-66, 1-58, 3-1, 1-44, 4-66, and other antibodies already reported by the present inventors. ５０ ) is shown in Figure 4.

[0148] Existing antibodies showed neutralizing activity against the prototype variant (614G), but not against the Omicron substrains, except for VIR-7831. VIR-7831 also showed no activity against BA.2.86 and JN.1, which have caused recent global pandemics. The first antibodies reported by the inventors, 9-105 and 10-121, showed neutralizing activity against the prototype variant (614G), but not against the Omicron substrains. The next antibody reported by the inventors, 4-66, showed neutralizing activity against the Omicron substrains, but its neutralizing activity was weak against some of the Omicron substrains. In contrast, among the antibodies isolated from Omicron BTI cases, 1-94 and 2-66 antibodies showed neutralizing activity against the prototype variant, including XBB.L16, EG, 5.1, BA286, and even JN. It was found that the antibodies 1-94 and 2-66 of the present invention exhibited strong neutralizing activity against a wide range of Omicron mutant strains.

[0149] The measurement results (neutralization curves) for the 1-94 and 2-66 antibodies of the present invention and the comparative antibody VIR-7831 are shown in FIG.

[0150] (Example 4) Neutralizing activity against Omicron substrains and conventional mutant strains Neutralization tests were performed using 27 monoclonal antibodies in the same manner as in Example 2, using live virus strains (authentic virus strains) of the wild-type strain (Wuhan), Beta, Gamma, Delta, and various SARS-CoV-2 mutant strains (BA.1, BA.2, BA.5, BQ.1.1, XBB, XBB.1.5, EG.5.1, JN.1). The results are shown in Figure 6. The 1-94 and 2-66 antibodies of the present invention exhibited the most potent and broadest neutralizing activity against the Omicron substrains.

[0151] (Example 5) Amino acid sequences and genetic characteristics of antibodies Sequence analysis was performed on the 1-94 antibody and 2-66 antibody obtained in the above Examples. The amino acid sequences of the heavy chain variable region and light chain variable region of the 1-94 antibody and 2-66 antibody are shown in Figure 1.

[0152] Example 6: Antibody Binding Activity to Mutant Strains The binding of the 1-94 and 2-66 antibodies obtained in the above Examples to spike proteins was measured as follows. HEK293T cells were transfected with plasmids expressing various SARS-CoV-2 spike proteins (spike proteins of wild-type (Wuhan), BA.4 / 5, XBB.1.5, BA.2.86, and EG.5.1 mutant strains) to express spike proteins on the cell surface. Two days after transfection, the cells were detached with trypsin and suspended in 0.2% BSA-PBS. 50 μL of the cell suspension was mixed with 50 μL of each antibody sample (concentration 2 μg / mL) and allowed to stand at room temperature for 15 minutes. The cells were separated by centrifugation, the supernatant discarded, and then suspended and washed in 0.2% BSA-PBS. After suspending in 50 μL of 200-fold diluted APC-conjugated anti-human IgG (goat antibody), the cells were allowed to stand at room temperature for 15 minutes. After washing with 0.2% BSA-PBS, the cells were fixed with 4% paraformaldehyde-PBS. The stained cells were analyzed by flow cytometry, and the spike protein binding activity of the antibodies was determined based on the APC positivity rate. The results are shown in Figure 7. "noAb" is a control without antibody. The 1-94 and 2-66 antibodies exhibited strong binding activity to spike-expressing cells of BA.4 / 5, XBB.1.5, BA.2.86, and EG.5.I mutant strains.

[0153] (Example 7) RBD-ACE2 Binding Inhibition Test Using a Cayman Chemical Company kit (#502050) according to the manufacturer's protocol, the inhibitory effect of RBD-ACE2 was examined. In addition to the two antibodies (1-94 and 2-66 antibodies) mentioned above, the inhibitory activity of antibodies already reported by the present inventors (9-105, 1-58, 3-1, and 10-121) was also measured in the same manner. The measured inhibitory activity data and calculated IC ５０ The results are shown in Figure 8. Both the 1-94 antibody and the 2-66 antibody inhibited the binding of wild-type RBD to the receptor ACE2.

[0154] (Example 8) SPR Analysis The binding activity of the 1-94 antibody and the 2-66 antibody to the RBD of XBB.1.5 was measured using surface plasmon resonance (SPR). Specifically, the procedure was as follows. The 1-94 and 2-66 antibodies were immobilized on a CM5 sensor chip using a Human Antibody Capture kit (cytiva). XBB.1.5 mutant RBD diluted with HBS-P buffer was passed through the sensor chip on which the antibodies were immobilized, and binding and dissociation were measured using a Biacore T200 to calculate the KD values. The results are shown in Figure 9. The binding constants (K Ｄ ) are 8.3 x 10 －１１ M, 2.3 x 10 －１１ M and showed strong binding activity.

[0155] Example 9 Therapeutic Effect of Antibodies on Coronavirus-Infected Animals Hamsters were infected with the JN.1 strain, an Omicron substrain of the coronavirus, and the therapeutic effect of the antibodies of the present invention (1-94 and 2-66 antibodies) was confirmed as follows. Six-week-old male Syrian hamsters (Japan SLC) were used. To evaluate the efficacy of the monoclonal antibody in hamsters, 1 x 10 Omicron substrain JN.1 strain (hCoV-19 / USA / CA-Stanford-165_S10 / 2023) was inoculated into each group of five hamsters under isoflurane anesthesia. ３ The monoclonal antibody was prepared at 30 μL per PFU (Plaque Forming Unit) and inoculated intranasally. One day after infection, hamsters were intraperitoneally injected with 1 mL of PBS-diluted monoclonal antibody (5 mg / kg). Four days after infection, the animals were euthanized, and virus titers in the nasal turbinates and lungs were measured by plaque assay using VeroE6 / TMPRSS2 cells. The experimental outline is shown in Figure 10, and the experimental results are shown in Figure 11. Administration of the 1-94 or 2-66 antibody significantly reduced the viral load in the lungs. The 1-94 and 2-66 antibodies were shown to have therapeutic effects in vivo against the new Omicron substrain JN.1.

[0156] In addition, blood antibody titers were measured using hamster serum. Briefly, serum samples treated at 56°C for 1 hour were serially diluted 5-fold and mixed with 100-400 FFU (focus forming units) of virus. The serum-virus mixture was inoculated into Vero E6-TMPRSS2-T2A-ACE2 cells. After incubation at 37°C for 1 hour, the cells were fixed with formalin. After removing the formalin, the cells were immunostained with an antibody against the SARS-CoV-2 nucleoprotein (N) protein, and the number of foci was quantified. The results were calculated as the 50% focus reduction neutralization titer (FRNT50). This confirmed that even low blood antibody titers had a significant inhibitory effect on virus in the lungs. These findings demonstrate that the 1-94 and 2-66 antibodies have potent therapeutic effects against in vivo infection with the Omicron substrain.

[0157] The above detailed description is merely illustrative of the objects and scope of the present invention and is not intended to limit the scope of the appended claims. Various modifications and substitutions to the described embodiments will be apparent to those skilled in the art from the teachings set forth herein, without departing from the scope of the appended claims.

[0158] The present invention provides antibodies against SARS-CoV-2 mutant strains. The antibodies of the present invention exhibit neutralizing activity against mutant strains such as the Omicron substrain and are useful as therapeutic agents for coronavirus infections.

Claims

1. An antibody or antigen-binding fragment thereof capable of neutralizing the SARS-CoV-2 virus, comprising the following heavy chain CDR1-3 and light chain CDR1-3: (1) a heavy chain CDR1 selected from the group consisting of a heavy chain CDR1 represented by SEQ ID NO: 5 (GIMFSDFA), a heavy chain CDR1 represented by SEQ ID NO: 11 (DGSLSSHDW), a heavy chain CDR1 consisting of a sequence in which one amino acid is deleted, substituted, or added in either of the heavy chain CDR1 represented by SEQ ID NO: 5 or 11, and a heavy chain CDR1 having 85% or more substantial identity to either of the heavy chain CDR1 represented by SEQ ID NO: 5 or 11; (2) A heavy chain CDR2 selected from the group consisting of a heavy chain CDR2 represented by SEQ ID NO: 6 (ISGDGDET), a heavy chain CDR2 represented by SEQ ID NO: 12 (IFHSGST), a heavy chain CDR2 consisting of a sequence in which one amino acid is deleted, substituted, or added in any of the heavy chain CDR2 represented by SEQ ID NO: 6 or 12, and a heavy chain CDR2 having 85% or more substantial identity with any of the heavy chain CDR2 represented by SEQ ID NO: 6 or 12; (3) A heavy chain CDR3 selected from the group consisting of a heavy chain CDR3 represented by SEQ ID NO: 7 (VKAETKYFGAHQTFHD), a heavy chain CDR3 represented by SEQ ID NO: 13 (ARGRGLIDS), a heavy chain CDR3 consisting of a sequence in which one or two amino acids are deleted, substituted, or added in any of the heavy chain CDR3s represented by SEQ ID NO: 7 or 13, and a heavy chain CDR3 having 80% or more substantial identity to any of the heavy chain CDR3s represented by SEQ ID NO: 7 or 13; (4) A light chain CDR1 selected from the group consisting of a light chain CDR1 represented by SEQ ID NO: 8 (QSLSIW), a light chain CDR1 represented by SEQ ID NO: 14 (QGISNS), a light chain CDR1 consisting of a sequence in which one amino acid is deleted, substituted, or added in any of the light chain CDR1s represented by SEQ ID NO: 8 or 14, and a light chain CDR1 having 80% or more substantial identity with any of the light chain CDR1s represented by SEQ ID NO: 8 or 14; (5) a light chain CDR2 represented by SEQ ID NO: 9 (RAS) or SEQ ID NO: 15 (SAS); and(6) An antibody or antigen-binding fragment thereof comprising a light chain CDR3 selected from the group consisting of a light chain CDR3 represented by SEQ ID NO: 10 (QQYSTFPYT), a light chain CDR3 represented by SEQ ID NO: 16 (QQYFSVRT), a light chain CDR3 consisting of a sequence in which one amino acid is deleted, substituted, or added in either of the light chain CDR3 represented by SEQ ID NO: 10 or 16, and a light chain CDR3 having 80% or more substantial identity to either of the light chain CDR3 represented by SEQ ID NO: 10 or 16.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain CDR1 is the heavy chain CDR1 represented by SEQ ID NO:5 or SEQ ID NO:11, the heavy chain CDR2 is the heavy chain CDR2 represented by SEQ ID NO:6 or SEQ ID NO:12, the heavy chain CDR3 is the heavy chain CDR3 represented by SEQ ID NO:7 or SEQ ID NO:13, the light chain CDR1 is the light chain CDR1 represented by SEQ ID NO:8 or SEQ ID NO:14, the light chain CDR2 is the light chain CDR2 represented by SEQ ID NO:9 or SEQ ID NO:15, and the light chain CDR3 is the light chain CDR3 represented by SEQ ID NO:10 or SEQ ID NO:

16.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the combination of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 is any of the following: (1) heavy chain CDR1 represented by SEQ ID NO:5, heavy chain CDR2 represented by SEQ ID NO:6, heavy chain CDR3 represented by SEQ ID NO:7, light chain CDR1 represented by SEQ ID NO:8, light chain CDR2 represented by SEQ ID NO:9, and light chain CDR3 represented by SEQ ID NO:10; (2) heavy chain CDR1 represented by SEQ ID NO:11, heavy chain CDR2 represented by SEQ ID NO:12, heavy chain CDR3 represented by SEQ ID NO:13, light chain CDR1 represented by SEQ ID NO:14, light chain CDR2 represented by SEQ ID NO:15, and light chain CDR3 represented by SEQ ID NO:

16.

4. The antibody or antigen-binding fragment thereof according to claim 1, having a heavy chain variable region represented by SEQ ID NO: 1 and a light chain variable region represented by SEQ ID NO:

2.

5. The antibody or antigen-binding fragment thereof according to claim 1, having a heavy chain variable region represented by SEQ ID NO: 3 and a light chain variable region represented by SEQ ID NO:

4.

6. In a neutralization assay using pseudovirus or authentic virus, the neutralizing activity of the antibody against the JN.1 strain, an Omicron lineage strain of SARS-CoV-2, is at a 50% inhibitory concentration (IC) of about 1 μg / mL or less. 50 The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, having a neutralizing activity expressed as a function of the antibody's activity (μg / mL).

7. In a neutralization assay using a pseudovirus or authentic virus, the antibody has a 50% inhibitory concentration (IC) of about 1 μg / mL or less against at least four mutant strains selected from the group consisting of BA.1, BA.2, BA.5, BQ.1.1, XBB, XBB.1.5, EG.5.1, and JN.1, which are Omicron lineage strains of SARS-CoV-2. 50 The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which exhibits neutralizing activity expressed in terms of IgG, IgG, and IgG antibodies (g / mL).

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, which is selected from the group consisting of an immunoglobulin molecule, a monoclonal antibody, a human antibody, a chimeric antibody, a CDR-grafted antibody, Fab, Fab', F(ab')2, Fd, Fv, a disulfide-linked Fv, scFv, a single domain antibody, a nanobody antibody, a diabody antibody, a bispecific antibody, and a multi-specific antibody.

9. A nucleic acid encoding an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

10. A vector comprising the nucleic acid of claim 9.

11. A host cell comprising the nucleic acid of claim 9 or the vector of claim 10.

12. A method for producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, comprising culturing a host cell according to claim 11 under conditions suitable for expression of said antibody or antigen-binding fragment thereof.

13. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 and a pharmacologically acceptable carrier.

14. The pharmaceutical composition of claim 13, further comprising at least one additional neutralizing antibody or antigen-binding fragment thereof against SARS-CoV-2 and / or a SARS-CoV-2 mutant strain.

15. The pharmaceutical composition according to claim 13, characterized in that it is administered in combination with other anti-coronavirus drugs.

16. The pharmaceutical composition according to claim 15, wherein the anti-coronavirus drug is at least one anti-coronavirus drug selected from antiviral drugs and neutralizing antibody drugs.

17. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 in the manufacture of a pharmaceutical composition for the prevention or treatment of SARS-CoV-2 virus infection.

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