WO2023008463A1

WO2023008463A1 - Neutralizing human antibody against wild type strain and mutant strain of sars-cov-2 and antigen-binding fragment thereof

Info

Publication number: WO2023008463A1
Application number: PCT/JP2022/028885
Authority: WO
Inventors: りゅう三浦; 幸人石坂; 美華子上野; 賢志忽那; 翔齋藤; 基木村
Original assignee: 株式会社イーベック; 国立研究開発法人国立国際医療研究センター
Priority date: 2021-07-28
Filing date: 2022-07-27
Publication date: 2023-02-02

Abstract

The present invention provides a human-derived monoclonal antibody against SARS-CoV-2 coronavirus, said antibody binding to the S protein of SARS-CoV-2 coronavirus and neutralizing the viral activity, an antigen-binding fragment thereof, a nucleic acid encoding the antibody or the antigen-binding fragment thereof, and a pharmaceutical composition for treating or preventing SARS-CoV-2 coronavirus infection, said composition containing the antibody or the antigen-binding fragment thereof.

Description

Neutralizing human antibodies and antigen-binding fragments thereof against SARS-CoV-2 wild-type and mutant strains

The present invention generally binds to the spike (S) protein of Severe acute respiratory syndrome coronavirus 2 (hereinafter referred to as "SARS-CoV-2"), including mutant strains, of SARS - Monoclonal antibodies that neutralize the biological activity of CoV-2, and antigen-binding fragments thereof, and their use as medicaments.

The emerging coronavirus SARS-CoV-2, which first emerged in Wuhan, China in 2019, has become a global pandemic known as severe acute respiratory disease novel coronavirus infection (hereinafter referred to as "COVID-19"). sparked an epidemic. Clinical features of COVID-19 include fever, dry cough, and malaise, which can lead to respiratory failure and death. As of June 9, 2021, the World Health Organization has reported 173,331,478 confirmed cases and 3,735,571 deaths. there is

SARS-CoV-2 has a typical betacoronavirus morphology. Similar to other coronaviruses, spherical virus particles with a diameter of about 100 nm are surrounded by a lipid bilayer envelope (outer membrane), and crown-like projections (spikes) are present on the envelope surface.

The spike is composed of a spike (S) protein trimer and binds to the angiotensin-converting enzyme 2 (ACE2) receptor (receptor) on the human cell membrane (Non-Patent Documents 1 and 2). After binding, the spike is activated by host cell proteases (Non-Patent Documents 3, 4), the viral envelope and the host cell membrane fuse, and the virus enters the cytoplasm. In other words, the S protein can suppress infection by inhibiting the binding of the spike and ACE2, which is the first step in virus infection of cells, and is therefore a target for antibody therapy including vaccines (Non-Patent Document 5). ).

SARS-CoV-2 has about 30,000 bases of positive single-stranded RNA as its genome. RNA is known to be less stable and more susceptible to mutation than DNA. SARS-CoV-2 also has mutations in the S protein that can affect increased transmissibility, virulence, antigenic changes, and the efficacy of acquired immunity in previously infected individuals and vaccinated individuals. Several novel mutant strains are emerging.

As a novel mutant strain with a mutation in the S protein of SARS-CoV-2, the British type (B.1.1.7: alpha strain ), the South African type (B.1.351: termed 'beta strain'), and the Brazilian type (P1: termed 'gamma strain'). Especially for alpha strains, epidemiological data suggest that there is a possibility of increased secondary infection rate and increased mortality risk.

In Japan, as of May 24, 2021, 11,235 people were confirmed to be infected with the alpha strain, but the Ministry of Health, Labor and Welfare said, ``The proportion of the British variant is about 80% nationwide, and some areas are It is estimated that it has almost replaced the conventional strain, except for the

A new mutant strain, with a rapidly increasing number of infected people in India, is considered to be a threat following the alpha strain. On May 11, 2021, WHO designated this mutant virus (B.1.617: Indotype) as a VOC (Variant of Concern). There are three subgroups (B.1.617.1, B.1.617.2, B.1.617.3) of the same mutant virus, and WHO calls B.1.617.1 "kappa strain", B.1.617 .2 was named the "delta strain". In particular, according to the WHO announcement dated June 15, 2021, the delta strain has spread to more than 80 countries and regions, is highly contagious, and is becoming a global mainstream. In the UK, which was attacked by the second wave of infection due to the alpha strain, the spread of vaccination has succeeded in converging the alpha strain, but it is said that the main cause of the current infection has been replaced by the delta strain.

Infection with the delta strain is spreading in Japan as well. According to the Ministry of Health, Labor and Welfare, as of June 7, 2021, 87 people in 12 prefectures have been confirmed to be infected, and the pace of increase is accelerating. It is estimated that by mid-July, the new infections will account for the majority of the cases, and experts are calling for the strengthening of the monitoring system to prevent the spread of the disease.

The characteristic of the Indian type is a mutation called "L452R". It shows that the 452nd amino acid of the S protein, which is used by viruses to invade cells, was mutated from L (leucine) to R (arginine). Although the B.1.617 variant's infectivity and pathogenicity are still unclear, the UK Public Health Agency announced on June 9, 2021 that the delta strain is about 60% more infectious than the alpha strain. Regarding the effectiveness of vaccines, Pfizer and AstraZeneca's vaccines are said to be less protective with only one dose, but more effective with two doses.

However, it has been pointed out that there may be individual differences in the effectiveness of the vaccine, and some people have health problems that prevent vaccination. The fact is that there is also a feeling of “vaccine avoidance”. Given that vaccines alone cannot completely control infections, therapeutic drugs are an important complement to vaccines.

Among therapeutic drugs, antibody drugs that utilize neutralizing monoclonal antibodies have been proven to have not only “therapeutic” effects but also “preventive” effects, so at present antibody drugs can counter SARS-CoV-2. It is expected to be the only therapeutic agent. In addition, it has the advantage of being able to manufacture pharmaceuticals of the same quality in large quantities.

Under these circumstances, treat SARS-CoV-2 infection containing human monoclonal antibodies, or antigen-binding fragments thereof, that bind to the SARS-CoV-2 S protein and neutralize the viral activity of SARS-CoV-2. Or development of the novel antibody drug for prevention is called for.

The present inventors aim to develop novel antibody drugs that correspond to wild type and / or mutant strains of SARS-CoV-2, from B lymphocytes isolated from the peripheral blood of SARS-CoV-2 infected recoverers attempted to obtain antibodies for As a result, we discovered a human-derived monoclonal antibody that binds to the SARS-CoV-2 S protein and neutralizes the viral activity of SARS-CoV-2 (wild type and/or mutant strains).

Accordingly, the present disclosure includes the following [1] to [14].
[1] A monoclonal antibody against the SARS-CoV-2 coronavirus that binds to the spike protein of the SARS-CoV-2 coronavirus and neutralizes its viral activity, and an antigen-binding fragment thereof.
[2] a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 1;
The antibody or antigen-binding fragment thereof according to [1] above, which comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:5 or an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO:5.
[3] the amino acid sequence of SEQ ID NO: 9 or a heavy chain containing amino acids having 90% or more identity with the amino acid sequence of SEQ ID NO: 9, and the amino acid sequence of SEQ ID NO: 10 or 90% or more with the amino acid sequence of SEQ ID NO: 10 The antibody or antigen-binding fragment thereof according to [1] or [2] above, comprising a light chain comprising identical amino acids.
[4] (i) the variable region of the heavy chain is
(a) a heavy chain CDR1 amino acid sequence comprising the amino acid sequence of SEQ ID NO:2;
(b) a heavy chain CDR2 amino acid sequence comprising the amino acid sequence of SEQ ID NO:3; and (c) a heavy chain CDR3 amino acid sequence comprising the amino acid sequence of SEQ ID NO:4;
contains
(ii) the variable region of the light chain is
(a) a light chain CDR1 amino acid sequence comprising the amino acid sequence of SEQ ID NO:6;
(b) a light chain CDR2 amino acid sequence comprising the amino acid sequence of SEQ ID NO:7; and (c) a light chain CDR3 amino acid sequence comprising the amino acid sequence of SEQ ID NO:8;
The antibody or antigen-binding fragment thereof according to any one of [1] to [3] above, containing
[5] The antibody or antigen-binding fragment thereof according to any one of [1] to [4] above, which has neutralizing activity against either or both of a SARS-CoV-2 wild type and a mutant strain An antibody or antigen-binding fragment thereof having an (IC50) of about 20 ng/mL or less.
[6] The antibody or antigen-binding fragment thereof according to any one of [1] to [5] above, wherein K against S protein trimer, alpha-type receptor binding site (RBD), delta-type RBD An antibody or antigen-binding fragment thereof having a _D value of 1×10 ⁻⁹ M or less and a K _D value of 1×10 ⁻¹⁰ M or less against kappa-type RBD.
[7] Treatment or prevention of SARS-CoV-2 coronavirus infection, comprising the antibody or antigen-binding fragment thereof according to any one of [1] to [6] above and a pharmaceutically acceptable carrier A pharmaceutical composition for
[8] (1) Use of the antibody or antigen-binding fragment thereof according to any one of [1] to [6] above in the treatment or prevention of SARS-CoV-2 coronavirus infection,
(2) Use of the antibody or antigen-binding fragment thereof according to any one of [1] to [6] above in the manufacture of a medicament for the treatment or prevention of SARS-CoV-2 coronavirus infection, or (3) A method for treating or preventing SARS-CoV-2 coronavirus infection, wherein the subject or patient in need thereof is subjected to any one of [1] to [6] above. A method comprising administering an antibody or antigen-binding fragment thereof.
[9] The pharmaceutical composition according to [7] above, or the use or method according to [8] above, wherein the SARS-CoV-2 is a mutant strain.
[10] The pharmaceutical composition, use, or method of [9] above, wherein the mutant strain is an alpha strain, a delta strain, or a kappa strain.
[11] An isolated nucleic acid molecule encoding the amino acid sequence of the antibody or antigen-binding fragment thereof according to any one of [1] to [6] above, or any of these nucleic acid molecules and highly stringent An isolated nucleic acid molecule that hybridizes under suitable conditions.
[12] A recombinant expression vector incorporating the isolated nucleic acid molecule of [11] above.
[13] An isolated host cell into which the recombinant expression vector of [12] above has been introduced.
[14] The antibody of any one of [1] to [6] above, which comprises culturing the host cell of [13] above and purifying the antibody from the cultured host cell, or the antibody thereof A method for producing an antigen-binding fragment.

The monoclonal antibodies, or antigen-binding fragments thereof, that neutralize the biological activity of SARS-CoV-2 according to the present invention bind to the S protein of wild-type and/or mutant SARS-CoV-2. , loses (neutralizes) its biological activity and is therefore useful in the prevention or treatment of SARS-CoV-2 infection. In particular, it is useful as a neutralizing antibody against SARS-CoV-2 mutant strains with increased transmissibility and pathogenicity. Further, the fully human monoclonal antibodies or antigen-binding fragments thereof against the S protein of SARS-CoV-2 according to the present invention have no or at least reduced immunogenicity in humans compared to antibodies that are not fully human antibodies. They have the advantage of having fewer side effects when administered to a human subject or patient because they have no or at least a reduced immune response compared to antibodies that are not fully human antibodies.

FIG. 2 is a flow chart showing the procedure for producing an anti-SARS-CoV-2-S antibody according to the present invention. FIG. 2 shows the nucleotide and corresponding amino acid sequences of the genes encoding the heavy and light chains of an anti-SARS-CoV-2-S antibody (EV053264b) according to the invention. In the figure, amino acids are represented by single-letter codes. Dark shaded amino acid sequences are signal sequences, light shaded amino acid sequences are variable region amino acid sequences, and underlined amino acid sequences are CDRs (Kabat). The affinity (or binding activity) of the anti-SARS-CoV-2-S antibody (EV053264b) according to the present invention to the wild-type spike trimer, alpha strain RBD, delta strain RBD, and kappa strain RBD was measured using a Biacore T-200 It is a figure which shows the result analyzed using. Fig. 10 shows the results of measuring the neutralizing activity of the anti-SARS-CoV-2-S antibody (EV053264b) according to the present invention against wild strain, alpha strain, delta strain, and kappa strain.

Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings, but the embodiments are solely for the purpose of facilitating understanding of the principles of the present invention, and the scope of the present invention is , is not limited to the following embodiments. Other embodiments in which a person skilled in the art replaces each element of the embodiments of the present invention shown below with equivalents obvious to a person skilled in the art based on the disclosure of the present specification and the common general technical knowledge in the field are also within the scope of the present invention It will be understood to be included in Each of the documents cited below for the description of the present invention is hereby incorporated by reference in its entirety.

1. Explanation of Terms As used herein, scientific and technical terms used in connection with the present invention have the meanings that are commonly understood by those of ordinary skill in the art, unless otherwise specified. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In general, the nomenclature used in connection with the techniques of cell culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry described herein are well known in the art and It is commonly used.

1) Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
"Severe acute respiratory syndrome coronavirus 2" (abbreviated as "SARS-CoV-2") is a virus belonging to the family Coronaviridae and belonging to the genus Betacoronavirus. The genome of SARS-CoV-2 consists of 30 kb of single-stranded RNA, and its gene shares 80% homology with the SARS coronavirus (SARS-CoV) that caused a pandemic in 2002-2003 ( Zhou P. et al., Nature, 579:270-273, 2020 (Non-Patent Document 2)). Coronavirus particles are composed of three proteins: the spike (S) protein, the envelope (E) protein, and the membrane (M) protein. Crown-like projections (spikes) on the virus surface, which are morphological features, are formed by S protein trimers.

2) SARS-CoV-2 spike (S) protein “SARS-CoV-2 S protein” is one of the glycoproteins present on the surface of the SARS-CoV-2 virus and consists of 1181 amino acid residues. (GenBank Protein Accesion # QHD43416.1). Strains of SARS-CoV-2 with the S protein identified in this GenBank Protein Accesion # QHD43416.1 are referred to herein as "wild strains." The S protein contains two functional domains, S1, which contains the N-terminal domain (NTD) and receptor binding domain (RBD), and S2, which mediates the fusion of the viral and host cell membranes. Spike first binds to host cell ACE2 via the RBD. The ACE2-bound spike is activated by being cleaved by two host cell proteases (TMPRSS2 and furin), fuses the viral membrane with the cell membrane, and delivers viral genes into the cell. After that, it forms a structure surrounded by a lipid bilayer membrane in the cell and replicates the viral genes therein (Non-Patent Document 4). Therefore, infection can be suppressed by inhibiting the binding of spike and ACE2, which is an early stage of infection.

In the present specification, a "mutant strain" is defined as a SAR-CoV-2 strain that has a mutation in the S protein of wild-type SARS-CoV-2. Non-limiting examples of mutant strains include the aforementioned three strains of alpha strain (British type), beta strain (South African type), and gamma strain (Brazilian type) having the N501Y mutation, as well as the Indian type mutant virus ( named "B.1.617"). The Indotype includes three subgroups (B.1.617.1 (“kappa strains”), B.1.617.2 (“delta strains”), B.1.617.3), which are has a mutation called L452R in which the 452nd amino acid located within the RBD of is changed from L (leucine) to R (arginine). In addition to this mutation, the kappa strain has a mutation called E484Q in the RBD and the delta strain has a mutation called T478K.

　In this specification, simply referring to "SARS-CoV-2" includes its wild strains and mutant strains unless otherwise specified.

3) Antibodies that bind to the S protein of SARS-CoV-2 As used herein, the term "antibody that binds to the S protein of SARS-CoV-2" binds to any site of the S protein of SARS-CoV-2 The epitope can be, for example, a linear epitope, a discontinuous sequence epitope, a conformational epitope, or a fragment of the S protein, or the like. It is not limited to RBD. As used herein, the terms "anti-SARS-CoV-2 antibody", "antibody capable of neutralizing SARS-CoV-2", "anti-SARS-CoV-2 S protein antibody", "SARS-CoV-2 S The terms "antibody that binds to a protein", "antibody capable of neutralizing the biological activity of SARS-CoV-2", etc. are used interchangeably to identify SARS-CoV-2 by binding to the S protein of SARS-CoV-2. is intended to refer to an antibody that inhibits the biological activity of As mentioned above, spikes that play an important role in the early stages of infection are formed by S protein trimers. In addition, the S protein trimer was used as an antigen in the examples described later. A specific example of the human monoclonal antibody according to the present invention described in the Examples is herein referred to as "EV053264b".

4) Antibody As used herein, the term "antibody" refers to four polypeptide chains, namely two heavy (H) chains and two light (L) chains, which are interconnected by disulfide bonds. It is intended to refer to an immunoglobulin molecule consisting of conjugates. Monoclonal antibodies in the present invention also consist of immunoglobulin molecules each containing two heavy (H) and light (L) chains. Each H chain consists of an H chain variable region (sometimes referred to as “HCVR” or “VH”) and an H chain constant region (the H chain constant region consists of three domains, “CH1”, “CH2 ”, and “CH3” (generic name: CH)). Each L chain consists of an L chain variable region (sometimes referred to as "LCVR" or "VL") and an L chain constant region (the L chain constant region consists of one domain and is sometimes referred to as "C"). The region up to the beginning of each constant region (also called constant region or constant region) is called a variable region (or variable region).

In particular, VH and VL are important in that they are involved in antibody binding specificity. Because antibodies interact with target antigens primarily through the amino acid residues of VH and VL, the amino acid sequences within the variable regions vary more between individual antibodies than sequences outside of the variable regions. In addition, VH and VL can be further subdivided into regions called framework regions (FR) that are more constant among different antibodies and hypervariable regions called complementarity determining regions (CDR). VH and VL consist of three CDRs and four FRs, respectively, which are arranged from amino-terminus to carboxy-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

Based on the amino acid sequence representing the variable region and the amino acid sequence of the CDRs, binding to the SARS-CoV-2 S protein and exhibiting the biological activity of SARS-CoV-2 can be performed by techniques well known in the art. Neutralizing human monoclonal antibodies, monoclonal antibodies (including chimeric antibodies and humanized antibodies), or antigen-binding fragments thereof can be obtained. These antibodies are within the scope of the invention.

5) Antibody "antigen-binding fragments" (or simply "antibody fragments")
The term "antigen-binding fragment" of an antibody (or simply "antibody fragment") as used herein refers to one or more antibodies capable of binding to an antigen (the S protein of SARS-CoV-2). refers to a fragment (e.g., VH) of The fragment also includes a peptide having the minimum amino acid sequence that binds to the antigen. Examples of binding moieties included within the term "antigen-binding fragment" of an antibody include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments consisting of the VH and CH1 domains, (iv) an Fv fragment consisting of the VL and VH domains of a single arm of the antibody, (v) a dAb fragment consisting of the VH domain (Ward ES. et al., Nature, 341:544-546, 1989) (vi) binds (vii) bispecific antibodies, and (viii) multispecific antibodies. In the present specification, when the term "antibody" is used without particular distinction, it includes not only full-length antibodies but also such "antigen-binding fragments."

6) Isotype Heavy chains are divided into γ chain, μ chain, α chain, δ chain, and ε chain, depending on the difference in the constant region. ) immunoglobulins are formed. Furthermore, in humans, IgG has four subclasses, IgG1 to IgG4. On the other hand, it is divided into κ chain and λ chain depending on the difference in the light chain constant region. The class (subclass) of the antibody EV053264b shown in Examples below is IgG1(λ).

2. Antibodies of the present invention or antigen-binding fragments thereof (or "viral activity" or "infectivity") (the terms are used interchangeably herein), or at least inhibit infection. (hereinafter referred to as "the antibody of the present invention"). Specifically, the antibodies of the present invention bind to the S protein of either or both wild-type and mutant SARS-CoV-2 and are capable of neutralizing the biological activity of said protein or antigen binding thereof. It is a sex fragment. More particularly, the antibodies of the present invention are antibodies or antigen-binding fragments thereof that can bind to the S protein of both wild-type and mutant SARS-CoV-2 and neutralize the biological activity of the protein. be. In particular, the antibodies of the present invention are antibodies or antigen-binding fragments thereof that are capable of binding to the S protein of mutant SARS-CoV-2 and neutralizing the biological activity of said protein.

As methods for producing these antibodies, known methods can be used (Riechmann L. et al., Nature, 332: 323-327, 1988). As a non-limiting example of such a method, in the present invention, a method for producing a fully human antibody, including the procedures shown in the examples below, can be used.

Table 1 shows the correspondence between human-derived anti-SARS-CoV-2 antibodies and antigen-binding fragments thereof according to one embodiment of the antibody of the present invention and the SEQ ID NOs of their amino acid sequences in the sequence listing.

The monoclonal antibody EV053264b according to one embodiment of the antibody of the present invention comprises a heavy chain (HC) consisting of the amino acid sequence of SEQ ID NO:9 and a light chain (LC) consisting of the amino acid sequence of SEQ ID NO:10.

In each of the heavy and light chains of EV053264b, the heavy chain variable region consists of the amino acid sequence of SEQ ID NO:1, and the light chain variable region consists of the amino acid sequence of SEQ ID NO:5.

In the heavy chain variable region of EV053264b, CDR1 consists of the amino acid sequence of SEQ ID NO:2, CDR2 consists of the amino acid sequence of SEQ ID NO:3, and CDR3 consists of the amino acid sequence of SEQ ID NO:4.

In the light chain variable region of EV053264b, CDR1 consists of the amino acid sequence of SEQ ID NO:6, CDR2 consists of the amino acid sequence of SEQ ID NO:7, and CDR3 consists of the amino acid sequence of SEQ ID NO:8.

Antibodies of the present invention include not only those containing amino acid sequences such as the specific heavy chains or light chains shown in SEQ ID NOS: 1 to 10, their variable regions, or their CDRs, but also Included are anti-SARS-CoV-2 antibodies that comprise substantially identical amino acid sequences and are capable of binding to the SARS-CoV-2 S protein and neutralizing the biological activity of SARS-CoV-2. Here, the term "substantially identical" means that one or more amino acid residues are deleted, substituted, inserted, or added in the amino acid sequence of the antibody of the present invention, or any two or more of these When the combination is made, it means that there is a deletion, substitution, insertion, or addition of one or more amino acid residues at any position in the same sequence and one or more amino acid sequences. Two or more of deletions, substitutions, insertions and additions may occur simultaneously.

Typically, amino acid sequences substantially identical to the amino acid sequences of specific heavy or light chains, variable regions thereof, or CDRs thereof shown in each of SEQ ID NOS: 1-10 are represented by SEQ ID NOS: 1-10. 20, at least, for example, about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more It includes amino acid sequences that have an identity (or sequence identity) greater than.

For example, about 20% or less of the number of amino acid residues in the above amino acid sequence of EV053264b (or about 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% or less) of amino acid residues deletion, substitution, insertion, addition, or a monoclonal antibody having an amino acid sequence with a combination of any two or more thereof, Antibodies of the invention as long as they retain activity equivalent to that of the limiting monoclonal antibody (EV053264b) (i.e., binds to the SARS-CoV-2 S protein and neutralizes the viral activity of SARS-CoV-2) can be included in the range of Antibodies of the present invention may include antibodies whose amino acid sequences other than the CDRs are also derived from humans. can be included in the antibody of 1 to several (specifically, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9) in the framework region (FR) as necessary , 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1) amino acid residue deletion, substitution, insertion , additions, or combinations of any two or more of these.

Amino acids that make up proteins in nature can be grouped according to the properties of their side chains. For example, amino acids with similar biochemical properties include aromatic amino acids (tyrosine, phenylalanine, tryptophan), Basic amino acids (lysine, arginine, histidine), acidic amino acids (aspartic acid, glutamic acid), neutral amino acids (serine, threonine, asparagine, glutamine), amino acids with hydrocarbon chains (alanine, valine, leucine, isoleucine, proline) , and others (glycine, methionine, cysteine). Substitutions of amino acid residues between amino acid residues having side chains with similar biochemical properties can be made while retaining the biological activity of the original protein.

For example, amino acid residues (including unnatural amino acids) included in the same group of the following groupings can be substituted for each other (while retaining the biological activity of the original protein). Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine; Group B: aspartic acid, glutamic acid, isoaspartic acid , isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid; Group C: asparagine, glutamine; Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid; Group E : proline, 3-hydroxyproline, 4-hydroxyproline; Group F: serine, threonine, homoserine; Group G: phenylalanine, tyrosine, tryptophan.

The identity of amino acid sequences and nucleotide sequences is determined using the BLAST algorithm by Karlin and Altschu (Karlin S and Altschu SF, PNAS, 87:2264-2268, 1990; PNAS, 90:5873-5877, 1993). can. Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul SF. et al., J Mol Biol, 215:403-410, 1990). When analyzing the base sequence using BLASTN, the parameters are, for example, score=100 and wordlength=12. When analyzing an amino acid sequence using BLASTX, the parameters are, for example, score=50 and word length=3. When using BLAST and Gapped BLAST programs, use the default parameters for each program. Alternatively, when determining the identity of the amino acid sequences of proteins, the amino acid sequences of the two types of proteins to be compared are aligned and the number of portions with the same amino acid residues is counted. number of amino acid residues) x 100 (%)".

Accordingly, typically, in one aspect, the present disclosure provides monoclonal antibodies and antigen-binding fragments thereof characterized by the constitutions of [1] to [6] below.
[1] A monoclonal antibody against the SARS-CoV-2 coronavirus that binds to the spike protein of the SARS-CoV-2 coronavirus and neutralizes its viral activity, and an antigen-binding fragment thereof.
[2] a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80% (or at least 90% or 95%) identity with the amino acid sequence of SEQ ID NO: 1;
[1] above, which contains the amino acid sequence of SEQ ID NO:5 or a light chain variable region comprising an amino acid sequence having at least 80% (or at least 90% or 95%) identity with the amino acid sequence of SEQ ID NO:5 or an antigen-binding fragment thereof.
[3] a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 or amino acids having at least 80% (or at least 90% or 95%) identity with the amino acid sequence of SEQ ID NO: 9, and the amino acid sequence or sequence of SEQ ID NO: 10 The antibody or antigen-binding fragment thereof of [1] or [2] above, comprising a light chain comprising amino acids having at least 80% (or at least 90% or 95%) identity with the amino acid sequence of number 10 .
[4] (i) the variable region of the heavy chain is
(a) a heavy chain CDR1 amino acid sequence comprising the amino acid sequence of SEQ ID NO:2;
(b) a heavy chain CDR2 amino acid sequence comprising the amino acid sequence of SEQ ID NO:3; and (c) a heavy chain CDR3 amino acid sequence comprising the amino acid sequence of SEQ ID NO:4;
contains
(ii) the variable region of the light chain is
(a) a light chain CDR1 amino acid sequence comprising the amino acid sequence of SEQ ID NO:6;
(b) a light chain CDR2 amino acid sequence comprising the amino acid sequence of SEQ ID NO:7; and (c) a light chain CDR3 amino acid sequence comprising the amino acid sequence of SEQ ID NO:8;
The antibody or antigen-binding fragment thereof according to any one of [1] to [3] above, containing
[5] The antibody or antigen-binding fragment thereof according to any one of [1] to [4] above, which has neutralizing activity against either or both of a SARS-CoV-2 wild type and a mutant strain An antibody or antigen-binding fragment thereof having an (IC50) of about 20 ng/mL or less.
[6] The antibody or antigen-binding fragment thereof according to any one of [1] to [5] above, wherein the _K value for S protein trimer, alpha-type RBD, and delta-type RBD is 1 × An antibody or antigen-binding fragment thereof that is 10 ⁻⁹ M or less and has a K _D value of 1×10 ⁻¹⁰ M or less for kappa-type RBD.

3. Nucleic Acids Encoding Antibodies of the Invention In another aspect, the invention also provides nucleic acid molecules that encode the amino acid sequences of the antibodies of the invention. Typically, isolated nucleic acid molecules are provided that encode the amino acid sequences of monoclonal antibodies and antigen-binding fragments thereof characterized by each of [1] to [6] above.

Further, as a non-limiting example, an anti-SARS-CoV-2 antibody or antigen-binding fragment thereof capable of binding to the S protein of SARS-CoV-2 and neutralizing its biological activity (i.e., typically An isolated nucleic acid (molecule) (polynucleotide) that has a high degree of identity with the amino acid sequence of a monoclonal antibody and an antigen-binding fragment thereof characterized by the construction of each of [1] to [6] above. A nucleic acid (ie, an isolated nucleic acid selected from those that hybridize to the nucleic acid under highly stringent conditions) is provided. Here, "having high identity" means a degree of sequence identity that allows hybridization to a given nucleic acid sequence under highly stringent conditions, e.g., at least about 60%, 70% %, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity do. For example, the nucleic acid is DNA or RNA, typically DNA.

"Highly stringent conditions" are, for example, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 50% formamide, and 50°C. (See, e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press (1989), especially Section 11.45 "Conditions for Hybridization of Oligonucleotide Probes"). Under these conditions, it can be expected that polynucleotides (eg, DNA) having higher identity can be efficiently obtained as the temperature is raised. However, multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration can be considered as factors that affect the stringency of hybridization, and those skilled in the art can select these factors as appropriate. It is possible to achieve similar stringency in .

Nucleic acids that hybridize under highly stringent conditions include nucleic acids encoding amino acid sequences, for example, 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% Nucleic acids having greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99% identity are included.

The identity of nucleotide sequences can be determined using the identity search algorithm described above (Karlin S and Altschu SF, PNAS, 87:2264-2268, 1990; PNAS, 90:5873-5877, 1993). .

4. Vector, Host Cell, and Antibody Production Method According to the Present Invention In still another aspect, the present invention relates to a vector incorporating the nucleic acid molecule, a host cell into which the vector has been introduced, and a method for producing an antibody using these vectors.

Antibodies of the present invention can also be produced as recombinant human antibodies using known methods (Boulianne GL et al., Nature, 312:643-646, 1984, Jones PT et al., Nature, 321:522-525 , 1986, etc.). For example, the antibody of the present invention can be produced by culturing host cells introduced with the vector of the present invention and purifying the produced antibody from the culture supernatant. More specifically, cDNAs encoding VH and VL are inserted into animal cell expression vectors containing genes encoding human antibody CH and/or human antibody CL produced from the same cell or different human cells, respectively. It can be produced by constructing a human antibody expression vector and introducing it into animal cells for expression.

The vector that incorporates the nucleic acid encoding the VH or VL of the antibody of the present invention is not necessarily limited, but it is possible to use a vector that is commonly used for expression of protein genes and the like and that is particularly suitable for expression of antibody genes or a vector for high expression. can. Non-limiting examples include vectors containing FE promoters and/or CMV enhancers. In addition, expression vectors incorporating nucleic acids encoding VH or VL are usually prepared and co-transfected into host cells, but they may be incorporated into a single expression vector.

The host cells into which the expression vector is introduced are not necessarily limited, but include cells that are commonly used for expression of protein genes, etc., and are particularly suitable for expression of antibody genes. Examples thereof include bacteria (E. coli, etc.), actinomycetes, yeast, insect cells (SF9, etc.), and mammalian cells (COS-1, CHO, myeloma cells, etc.).

For the industrial production of recombinant antibodies, recombinant animal cell lines that stably produce the antibodies at high levels, such as CHO cell lines, are generally used. Known methods can be used for generating such recombinant cell lines, cloning, gene amplification and screening for high expression (for example, Omasa T., J. Biosci. Bioeng., 94: 600-605 , 2002, etc.).

In addition to antibodies consisting of two heavy chains and two light chains, the above recombinant antibodies include antigen-binding fragments such as Fab (Fragment of antigen binding), Fab', F(ab') ₂ , antibody An active fragment bound by a linker or the like (for example, single chain antibody (single chain Fv: scFv) or disulfide stabilized antibody (disulfide stabilized Fv: dsFv)), a peptide containing an active antibody fragment (for example, a peptide containing CDRs) ) is included. These can be produced by known methods such as treating the antibody of the present invention with an appropriate proteolytic enzyme or genetic recombination techniques.

Antibody purification can be performed using known purification means such as salting out, gel filtration, ion exchange chromatography, or affinity chromatography.

In addition, scFv (single chain Fragment of variable region) diversified by artificially shuffling VH and VL genes using phage display antibody technology, which uses genetic engineering technology to express recombinant antibodies on the surface of phages. Antibodies can also be expressed as phage fusion proteins to obtain specific antibodies. This technology is highly evaluated as a humanized antibody production technology that can avoid immunity and can replace the cell fusion method. Specific antibodies or antigen-binding fragments thereof produced using this technique with reference to, for example, the amino acid sequences of SEQ ID NOs: 2-4 and 6-8 are also within the scope of the present invention.

Furthermore, an antibody obtained by applying Potellegent technology, which significantly improves the ADCC activity of an antibody by modifying the sugar chain portion of the antibody, to the antibody of the present invention (Niwa R et al., Clin. Cancer Res. ., 10:6248-6255, 2004) and an antibody obtained by applying Complement technology that improves CDC activity to the antibody of the present invention (Kanda Y. et al., Glycobiology, 17:104 -118, 2007) are also within the scope of the present invention. Similarly, antibodies obtained by modifying the amino acid sequence of the constant region portion of the antibody to modify ADCC activity (WO2007/039682 and WO2007/100083) or CDC activity (WO2007/011041 and WO2011/091078). , are within the scope of the present invention.

Furthermore, an antibody or an antigen-binding fragment thereof obtained by applying a technique for partial substitution of the Fc region (see WO2006/071877) to add protease resistance ability to the antibody and enable oral administration, It is within the scope of the present invention.

As a technique for producing antibodies, laboratory animals such as mice, rabbits, and goats are usually used to obtain polyclonal antibodies and monoclonal antibodies. Since it has a sequence characteristic of animal species, if it is administered to humans as it is, it may be recognized as a foreign substance by the human immune system and cause a human anti-animal antibody response (that is, the production of antibodies against antibodies).

The anti-SARS-CoV-2 monoclonal antibody or antigen-binding fragment thereof according to the present invention can be obtained from antibody-producing cells derived from the blood of a person who has recovered from SARS-CoV-2 infection, and is a fully human antibody. This fully human antibody, even when administered to the human body as an antibody drug, does not have immunogenicity, or at least has reduced immunogenicity compared to an antibody that is not a fully human antibody, and no immune response is observed, or It has at least a reduced advantage compared to antibodies that are not fully human antibodies.

5. Pharmaceutical compositions containing an antibody of the invention, use of an antibody of the invention as a medicament, use of an antibody of the invention for the manufacture of a medicament, and treatment or prevention comprising administering an antibody of the invention to a patient Methods The present invention, in yet another aspect, includes the use of the antibody of the present invention as a medicament. In one embodiment, pharmaceutical compositions for preventing or treating SARS-CoV-2 infection are provided comprising an antibody of the invention and a pharmaceutically acceptable carrier. In one embodiment, use of the antibodies of the invention in treating or preventing SARS-CoV-2 coronavirus infection is provided. Furthermore, in one embodiment there is provided use of the antibody of the invention in the manufacture of a medicament for the treatment or prevention of SARS-CoV-2 coronavirus infection. Further, in one embodiment, a method of treating or preventing SARS-CoV-2 coronavirus infection is provided comprising administering an antibody of the invention to a subject or patient in need thereof.

In particular, the anti-SARS-CoV-2 antibody or antigen-binding fragment thereof according to the present invention binds to the S protein of SARS-CoV-2 and has a high neutralizing ability. It is useful as a prophylactic or therapeutic drug for

As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents and the like that are physiologically compatible. be

Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof. When used as an injection or the like, the composition can contain pH adjusting agents and isotonic agents, such as sugars, polyalcohols such as mannitol and sorbitol, or sodium chloride. Pharmaceutically acceptable carriers can further contain minor amounts of auxiliary substances such as wetting and emulsifying agents, preservatives, buffers and stabilizers, which enhance the preservation or effectiveness of the antibody or antibody portion. .

The pharmaceutical composition of the present invention can be made into various dosage forms. Such compositions include, for example, solutions (eg, injectable and infusible solutions), dispersions, suspensions, tablets, capsules, troches, pills, powders, liposomes, suppositories, liquids, semisolids, and the like. , including solid dosage forms. Dosage forms may vary depending on the intended mode of administration and therapeutic application. Examples include those in the form of injectable or infusible solutions, such as compositions similar to those commonly used for passive immunization of humans with other antibodies. For example, the dosage form can be parenteral (eg, intravenously, subcutaneously, intraperitoneally, intramuscularly). Alternatively, antibody can be administered by intravenous infusion or injection. Alternatively, antibody can be administered by intramuscular or subcutaneous injection.

6. Methods for obtaining anti-SARS-CoV-2 antibodies and antigen-binding fragments thereof according to the present invention Next, methods for obtaining anti-SARS-CoV-2 monoclonal antibodies and antigen-binding fragments thereof according to the present invention will be described. , The method of obtaining the antibody, etc. of the present invention is not limited to these descriptions, and as described above, changes or modifications within the scope of ordinary creativity of those skilled in the art are possible.

Anti-SARS-CoV-2 antibodies and antigen-binding fragments thereof according to the present invention are obtained by cloning the DNA encoding the target antibody through various steps from the blood of a person who has recovered from SARS-CoV-2 infection. It can be obtained by performing production. FIG. 1 shows a flow chart of the steps for producing the anti-SARS-CoV-2-S antibody according to the present invention.

1) Proliferation induction of fully human antibody-producing cells against SARS-CoV-2 S protein B lymphocytes are separated from the blood of a person who has recovered from SARS-CoV-2 infection, and proliferation of the B lymphocytes is induced. The method for inducing proliferation is known per se, and for example, a transformation method (Kozbor D. and Roder JC., Immunol Today, 4:72-9, 1983).

2) Identification of fully human antibody-producing cells against the S protein of SARS-CoV-2 By ELISA with the S protein trimer as a solid phase and by measuring the neutralizing activity of the culture supernatant, proliferation was induced by the above-mentioned EBV infection. Antibody-producing cells are identified from the B lymphocyte group. In some cases, the B lymphocytes are seeded on a cell microarray, and cells producing S protein-binding antibodies are selected and isolated using glass slides or microbeads on which S protein trimers are immobilized.

3) Gene Acquisition and Antibody Production of Fully Human Antibody Against SARS-CoV-2 S Protein cDNA is synthesized from the S protein-binding antibody-producing B lymphocytes identified or isolated above, and the antibody gene is cloned. The acquired gene is introduced into CHO cells, antibody is expressed, and antigen-binding and neutralizing activity are measured.

Since the anti-SARS-CoV-2 antibody obtained as described above is a fully human antibody produced from B lymphocytes sensitized in the human body, it is possible to have an immune response to the antibody when administered to humans. It has the advantage of being less sensitive.

Another feature is that it uses the EB virus, which has the activity of infecting B lymphocytes and inducing their proliferation. The advantage of the EB virus method is that it can produce natural antibodies produced in the human body, and that antibodies can be obtained even from cells with a very low positive rate.

7. Method for Evaluating In Vitro Activity The antigen-binding property of an antibody or antibody composition can be evaluated by an ELISA method, a surface plasmon resonance (SPR) method, or the like. In addition, the ability of the antibody to suppress the biological activity of the S protein of SARS-CoV-2 in vitro can be evaluated by receptor binding inhibition assays such as ELISA, neutralization assays, and the like.

1) Antigen-binding activity A known method can be used to measure the binding activity between an antibody and the S protein of SARS-CoV-2. For example, ELISA using the S protein trimer or the RBD in the S protein as an antigen can be employed. Alternatively, binding affinity to S protein or RBD can be measured by a protein interaction analyzer such as Biacore T200 (registered trademark). The binding affinity (K _D value) is expressed as the ratio of kd (dissociation constant) to ka (binding constant) obtained by the measurement method (K _D =kd/ka). For example, the human anti-SARS-CoV-2 antibody or antigen-binding fragment thereof according to the present invention has a binding affinity (K _D value) to wild-type S protein, alpha-type RBD, and delta-type RBD of 1×10 ⁻ It can be ⁸ M or less, or it can be 1×10 ⁻⁹ M or less. Furthermore, the human anti-SARS-CoV-2 antibody or antigen-binding fragment thereof according to the present invention has a binding affinity (K _D value) for kappa-type RBD of 1×10 ⁻⁸ M or less and 1×10 ⁻⁹ M or less. , or 1×10 ⁻¹⁰ M or less. (See “5. Antigen binding (affinity) evaluation” and FIG. 3 in Examples)

2) In Vitro Neutralizing Activity As used herein, the terms “neutralizing”, “inhibitory effect”, “inhibition”, “suppression”, “capable of inhibiting”, etc. refer to antigens (SARS-CoV-2 ) by, for example, at least about 5-100%, 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100% %, or 80 to 100% reduction.

Evaluation of the in vitro neutralization ability of anti-SARS-CoV-2 antibodies can be performed using wild-type and/or mutant strains of SARS-CoV-2 (e.g., alpha, delta, kappa strains). . For example, the wild strain used is the JPN/TY/WK-521 strain, and mutant strains such as JPN/QK002/2020 as the alpha strain, JPN/TKYTK1734/2021 as the delta strain, and JPN/TKYTK5356/2021 as the kappa strain. was used to examine the ability to infect Vero E6 cells with TMPRSS2 cDNA-introduced cell lines (Matsuyama S et al., 2020) in the presence of anti-SARS-CoV-2 antibodies, thereby confirming the neutralizing ability of the antibodies. can be evaluated (see "6. Evaluation of neutralizing activity" in Examples and Fig. 4).

Typically, the anti-SARS-CoV-2 antibody or antigen-binding fragment thereof according to the present invention has a SARS-CoV-2 infectivity evaluation system using the Vero cells described above, and about 200 It has 50% infection inhibitory activity (IC50) at ng/mL or less, approximately 100 ng/mL or less, approximately 50 ng/mL or less, or approximately 20 ng/mL or less. A non-limiting example of the mutant strain described above includes the Delta strain.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

Fig. 1 shows the flowchart of the procedure for producing the anti-SARS-CoV-2-S antibody according to the present invention.

1. Gene cloning of fully human antibody against SARS-CoV-2 Gene cloning of the monoclonal antibody according to this example was performed by the following procedure.
1) Peripheral blood was collected from a person who recovered from SARS-CoV-2 infection.
2) Peripheral blood mononuclear cells (PBMC) were separated by centrifugation.
3) B lymphocytes were isolated from PBMC and infected with EBV. Infected cells were seeded on a 96-well plate and cultured for about 3 weeks.
4) Primary screening of anti-SARS-CoV-2 antibodies in the culture supernatant was performed. The screening targeted antibodies against the S protein trimer (AcroBiosystem), which is the major neutralizing target of SARS-CoV-2, and the wells containing target antibody-positive cells were identified by ELISA.
5) Neutralizing activity was measured in each well in which anti-SARS-CoV-2 antibody production was confirmed, and neutralizing antibody-positive wells were identified.
6) Using the culture supernatant of neutralizing antibody-positive wells, the isotype of the antibodies produced was confirmed by ELISA. That is, an anti-SARS-CoV-2 antibody screening plate on which the S protein trimer was immobilized was used, and antibodies specific to each isotype and subclass were used as secondary antibodies.
7) In some cases, the cells in the neutralizing antibody-positive well are seeded on a cell microarray, and a slide glass or microbeads on which the S protein trimer is immobilized are used to isolate single cells that produce the target antibody. released.
9) Antibody genes were amplified by PCR using the cDNA obtained by reverse transcription from total-RNA of antibody-producing cells using oligo-dT primer as a template. The primers used for PCR were designed based on a database of cDNAs encoding human IgG antibody H and L chains. A 5′-end primer and a 3′-end primer were designed to amplify the H chain from the translation initiation site to the constant region, and the L chain to amplify the full-length cDNA.

2. Confirmation that the obtained antibody gene encodes an anti-SARS-CoV-2 antibody The obtained H chain and L chain genes were each inserted into an expression vector and simultaneously introduced into CHO-K1 cells. Gene transfer was performed using Lipofectamine LTX and Plus Reagent (Thermo Fisher Scientific) under the manufacturer's recommended conditions. Two days later, the culture supernatant was collected, and the antibody in the culture supernatant bound to the S protein of SARS-CoV-2 by ELISA using a multiwell plate with S protein trimer immobilized. bottom. For clones confirmed to bind to the S protein, the neutralizing activity was measured using the culture supernatant.

3. Determination of Amino Acid Sequence of Antibody Based on Nucleotide Sequence The nucleotide sequences of each of the H chain and L chain were confirmed using an ABI sequencer (Thermo Fisher Scientific). From the obtained nucleotide sequences, the antibody signal sequence, H chain and L chain amino acid sequences, variable region amino acid sequence, and complementarity determining region (CDR) amino acid sequence were determined (Fig. 2). The Kabat method (www.bioinf.org.uk: Dr. Andrew CR Martin's Group, Antibodies: General Information) was used for CDR analysis. Table 1 shows the sequence numbers of the H chain and L chain amino acid sequences, the variable region amino acid sequence, and the complementarity determining region (CDR) amino acid sequence of the obtained antibody (EV053264b). Isotype and subclass were also confirmed.

4. Production and Purification of Antibody Protein The obtained anti-SARS-CoV-2 neutralizing antibody expression vector was introduced into ExpiCHO cells (Thermo Fisher Scientific), and the culture supernatant was collected. This culture supernatant was subjected to affinity purification using a Protein A column (Cytiva) to obtain a purified antibody. Gene introduction, culture, and purification were performed under the manufacturer's recommended conditions. After purification, antibody H chain (about 50 kDa) and antibody L chain (about 25 kDa) were confirmed by SDS-PAGE.

5. Evaluation of antigen binding (affinity) Anti-SARS-CoV-2 antibody antigen binding activity evaluation using S protein trimer, alpha-type RBD, delta-type RBD, and kappa-type RBD using Biacore T-200 affinity analysis was performed. Anti-human IgG was immobilized on a sensor chip using a human IgG capture kit (Cytiva), and an anti-SARS-CoV-2 antibody was captured and used as a ligand. Analytes used were S protein trimer, alpha-type RBD, delta-type RBD, and kappa-type RBD (AcroBiosystem), respectively. The results are shown in FIG. Anti-SARS-CoV-2 antibody (EV053264b) binds to S protein trimers, alpha-type RBDs, and delta-type RBDs with K _D values of 1.0 nM or less and to kappa-type RBDs with K _D values of 0.1 nM or less. showed activity.

6. Evaluation of neutralizing activity Neutralizing activity was confirmed as an evaluation of the efficacy of the anti-SARS-CoV-2 antibody. Neutralizing activity was evaluated by antibody inhibition rate of SARS-CoV-2 delta strain infection. Virus neutralization experiments using antibodies are based on the COVID19 serological test manual published by the National Institute of Infectious Diseases.
Conducted according to https://www.niid.go.jp/niid/images/pathol/pdf/COVID-19Serology_Ver1.pdf .

1) Cultured cells Vero E6 cells into which TMPRSS2 cDNA was introduced (Matsuyama S et al., PNAS, 117:7001-7003, 2020) were obtained from the Japanese Collection of Research Bioresources (JCRB) (JCRB1819 VeroE6 /TMPRSS2). . Cells were maintained in Dulbecco Modified Eagle's medium (DMEM, Nissui) supplemented with 10% fetal calf serum (FCS) and 400 μg/mL neomycin (G418, Sigma-Aldrich). liquid was used. Maintain at 37°C, 5% CO ₂ , rinse twice with phosphate buffer (Phosphate buffer saline (-), PBS, Nissui), then add 0.25% trypsin/EDTA solution (Lonza). was added at 1 mL per 10 cm plate. Cells were harvested with 10% FCS/DMEM and centrifuged at x120 g for 5 minutes. After removing the supernatant, the cells were suspended in 4% FCS/DMEM, counted, and seeded at 10 ⁴ cells/100 μL per well in a 96-well plate (Thermo Fisher Scientific). Viral infection experiments were performed the day after cell plate preparation.

2) Virus strain From the National Institute of Infectious Diseases, wild strain (JPN/TY/WK-521 strain), alpha strain (JPN/QK002/2020 strain), delta strain (JPN/TKYTK1734/2021 strain), and kappa strain ( JPN/TKYTK5356/2021 strain) was obtained. Virus stocks were serially diluted using Opti-MEM (Thermo Fisher Scientific) and added to 96-well plates. One dilution series was applied to eight wells as one set. Three to four days later, the virus concentration at which half of the eight wells showed CPE (Cytopathertic effects) was calculated as the 50% infectious dose (TCID50, Median tissue culture infectious dose).

3) Calculation of IC50 The 50% inhibitory concentration (IC50) of the purified antibody (EV053264b) was evaluated using a 96-well plate (Thermo Fisher Scientific). That is, an antibody solution diluted with Opti-MEM to a final concentration of 250 ng/mL to 0.25 ng/mL was added to one column of a 96-well plate. Next, an equal amount of the virus solution adjusted to a final MOI of 0.01 was added to a 96-well plate, shaken and reacted at 37°C for 1 hour, and 100 µL each was added to the cell plate. After 2 days for the wild strain, after 3 days for the alpha and kappa strains, and after 4 days for the delta strain, 10 μL of Cell Proliferation and Toxicity Assay Kit (Fuji Film Wako Pure Chemical) solution was added to each well. After that, culture was continued at 37 degrees. After 2-3 hours, the absorbance at OD450nm was measured with a plate reader (BIO-RAD, Model 550), and the value of wells to which only the culture medium was added was subtracted from each value. The ratio (%) to the antibody was calculated, and the neutralizing activity (inhibition) rate at each antibody concentration was calculated (Fig. 4). IC50 was calculated from the following formula.
IC50=10^(LOG(A/B)*(50-C)/(DC)+LOG(B))
A: High concentration between 50%, B: Low concentration between 50%
C: Inhibition rate at B, D: Inhibition rate at A

The antibody of the present invention showed an infection-suppressing effect against wild strain, alpha strain, kappa strain, and delta strain, and the IC50 value was calculated to be 20 ng/mL or less.

The anti-SARS-CoV-2 antibody of the present invention is useful for applications in fields such as pharmaceutical compositions for preventing or treating diseases associated with SARS-CoV-2.

[Brief description of the array]
>EV053264b HCVR
QVQLQESGPGLVKPSETLSLTCTVSGGSVS SGSYYWS WIRQPPGKGLEWIG YIYYSGSTSYNPSLKS RVTMSVDTSKNQFSLKLSSVTAADTAVYFCAR GSGSGPRYYLDY (SEQ ID NO: 1)

>EV053264b HCDR1
SGSYYWS (SEQ ID NO: 2)

>EV053264b HCDR2
YIYYSGSTSYNPSLKS (SEQ ID NO: 3)

>EV053264b HCDR3
GSGSGPRYYLDY (SEQ ID NO: 4)

>EV053264b LCVR
SYVLTQPPSVVSVAPGQTASITC GGNNIGSKSVH WYRQKPGQAPVLVIY YDSDRPS GIPERFSGSNSGNTATLTISRVEAGDEADYFC QVWDSSSDHPNYV (SEQ ID NO: 5)

>EV053264b LCDR1
GGNNIGSKSVH (SEQ ID NO: 6)

>EV053264b LCDR2
YDSDRPS (SEQ ID NO: 7)

>EV053264b LCDR3
QVWDSSSDHPNYV (SEQ ID NO: 8)

>EV053264b HC
QVQLQESGPGLVKPSETLSLTCTVSGGSVS SGSYYWS WIRQPPGKGLEWIG YIYYSGSTSYNPSLKS RVTMSVDTSKNQFSLKLSSVTAADTAVYFCAR GSGSGPRYYLDY WGQGTLVTVSATSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK（配列番号９）

>EV053264b LC
SYVLTQPPSVVSVAPGQTASITC GGNNIGSKSVH WYRQKPGQAPVLVIY YDSDRPS GIPERFSGSNSGNTATLTISRVEAGDEADYFC QVWDSSSDHPNYV FGTGTRVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS)

>DNA encoding EV053264b HC (including signal sequence)
(SEQ ID NO: 11)

>EV053264b HC (includes signal sequence)
MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGGSVS SGSYYWS WIRQPPGKGLEWIG YIYYSGSTSYNPSLKS RVTMSVDTSKNQFSLKLSSVTAADTAVYFCAR GSGSGPRYYLDY WGQGTLVTVSATSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK（配列番号１２）

>DNA encoding EV053264b LC (including signal sequence)
(SEQ ID NO: 13)

>EV053264b LC (including signal sequence)
MAWTVLLLGLLSHCTGSVTSYVLTQPPSVVSVAPGQTASITC GGNNIGSKSVH WYRQKPGQAPVLVIY YDSDRPS GIPERFSGSNSGNTATLTISRVEAGDEADYFC QVWDSSSDHPNYV FGTGTRVTVLGQPKANPTVTLFPPSSEELQANKATLLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHREKSTVVTEC (sequence number 1)

Claims

A monoclonal antibody against the SARS-CoV-2 coronavirus that binds to the spike protein of the SARS-CoV-2 coronavirus and neutralizes its viral activity, and an antigen-binding fragment thereof.
a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 1; and
2. The antibody or antigen-binding fragment thereof according to claim 1, comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO:5 or an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO:5.
a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 or amino acids having 90% or more identity with the amino acid sequence of SEQ ID NO: 9, and an amino acid sequence of SEQ ID NO: 10 or having 90% or more identity with the amino acid sequence of SEQ ID NO: 10 3. The antibody or antigen-binding fragment thereof of claim 1 or 2, comprising a light chain comprising amino acids having
(i) the variable region of the heavy chain is
(a) a heavy chain CDR1 amino acid sequence comprising the amino acid sequence of SEQ ID NO:2;
(b) a heavy chain CDR2 amino acid sequence comprising the amino acid sequence of SEQ ID NO:3; and (c) a heavy chain CDR3 amino acid sequence comprising the amino acid sequence of SEQ ID NO:4;
contains
(ii) the variable region of the light chain is
(a) a light chain CDR1 amino acid sequence comprising the amino acid sequence of SEQ ID NO:6;
(b) a light chain CDR2 amino acid sequence comprising the amino acid sequence of SEQ ID NO:7; and (c) a light chain CDR3 amino acid sequence comprising the amino acid sequence of SEQ ID NO:8;
The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising
The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the neutralizing activity (IC50) against either or both of the SARS-CoV-2 wild type and mutant strain is about An antibody or antigen-binding fragment thereof that is 20 ng/mL or less.
The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which has a K D value of 1 × 10 -9 M or less for S protein trimer, alpha-type RBD, and delta-type RBD and a K D value for kappa-type RBD of 1×10 −10 M or less, or an antigen-binding fragment thereof.
A pharmaceutical composition for treating or preventing SARS-CoV-2 coronavirus infection, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier .
(1) use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 in the treatment or prevention of SARS-CoV-2 coronavirus infection;
(2) Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 in the manufacture of a medicament for treating or preventing SARS-CoV-2 coronavirus infection, or (3 ) A method for treating or preventing SARS-CoV-2 coronavirus infection, comprising administering the antibody of any one of claims 1 to 6 or its antigen-binding ability to a subject or patient in need thereof. A method comprising administering a fragment.
The pharmaceutical composition according to claim 7, or the use or method according to claim 8, wherein said SARS-CoV-2 is a mutant strain.
The pharmaceutical composition, use or method according to claim 9, wherein said mutant strain is an alpha strain, a delta strain, or a kappa strain.
An isolated nucleic acid molecule that encodes the amino acid sequence of the antibody or antigen-binding fragment thereof of any one of claims 1 to 6, or that hybridizes with any of these nucleic acid molecules under highly stringent conditions An isolated nucleic acid molecule.
A recombinant expression vector incorporating the isolated nucleic acid molecule according to claim 11.
An isolated host cell into which the recombinant expression vector according to claim 12 has been introduced.
A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which comprises culturing the host cell according to claim 13 and purifying the antibody from the cultured host cell. .