JP2023014062A - Anti-SARS-CoV-2 antibody - Google Patents

Abstract

To provide a technique to neutralize SARS-CoV-2.SOLUTION: The present invention provides an isolated antibody binding to SARS-CoV-2 or an antibody fragment comprising an antigen-binding region thereof, comprising amino acid sequences of predetermined heavy and light chains; or an antibody or an antibody fragment comprising an antigen-binding region thereof, which compete with the antibody or the antibody fragment comprising an antigen-binding region thereof in binding to the SARS-CoV-2.SELECTED DRAWING: Figure 5

Description

The present invention relates to anti-SARS-CoV-2 antibodies.

Novel coronavirus infectious disease (COVID-19) is an infectious disease that occurred in 2020. A novel coronavirus belonging to the genus Betacoronavirus of the family Coronavirus (a coronavirus belonging to the genus Betacoronavirus (introduced in China in January 2020) Limited to those newly reported to the World Health Organization as having the ability to infect humans.
tory syndrome coronavirus 2)”. ) due to acute respiratory syndrome.

Antibodies that bind to and neutralize the virus are termed "neutralizing antibodies." If a neutralizing antibody against SARS-CoV-2 is developed, it is expected to be used as a silver bullet for COVID-19. SARS
Several neutralizing antibodies against CoV-2 have been developed so far (Non-Patent Documents 1 to 4).

The _IC50 value, which is an index of the ability to neutralize SARS-CoV-2, and the epitope characteristics that bind to mutant strains differ greatly among neutralizing antibodies. A useful level of _IC50 value for the treatment of COVID-19 is 20 ng/
To date, no antibodies have been reported in Japan that have been confirmed to have effective neutralizing ability against SARS-CoV-2 at a volume of 1 ml or less. On the other hand, outside Japan, REGN10933 and REGN10987 (both from Regeneron) and LY3819253 (from Eli Lilly) have already been developed and are on the market.

Seydoux et al., Immunity, 53, 98-105 (2020) Hansen et al., Science, 369, 1010-1014 (2020) Liu et al., Nature, 584, 450-456 (2020) Kreer et al., Cell, 182, 843-854 (2020)

An object of the present invention is to provide a technique for neutralizing SARS-CoV-2.

As a result of intensive studies to solve the above problems, the present inventors found that an antibody containing a given amino acid sequence has the ability to neutralize SARS-CoV-2.

The present invention can provide the following aspects.
<1> containing the amino acid sequences of the heavy and light chains of any one of (IV), (II), (III), and (I) below,
isolated,
An antibody or antibody fragment comprising an antigen-binding region thereof that binds to SARS-CoV-2 or an antibody or antigen-binding thereof that competes with said antibody or antibody fragment comprising an antigen-binding region thereof for binding to said SARS-CoV-2 An antibody fragment containing the region.
(IV):
the heavy chain CDR1 amino acid sequence is the amino acid sequence of SEQ ID NO: 16;
the heavy chain CDR2 amino acid sequence is the amino acid sequence of SEQ ID NO: 17;
the heavy chain CDR3 amino acid sequence is the amino acid sequence of SEQ ID NO: 18;
the amino acid sequence of the CDR1 of the light chain is the amino acid sequence of SEQ ID NO: 19;
The amino acid sequence of CDR2 of the light chain is AAS, and the amino acid sequence of CDR3 of the light chain is the amino acid sequence of SEQ ID NO:20.
(II):
the heavy chain CDR1 amino acid sequence is the amino acid sequence of SEQ ID NO: 6;
the heavy chain CDR2 amino acid sequence is the amino acid sequence of SEQ ID NO: 7;
the heavy chain CDR3 amino acid sequence is the amino acid sequence of SEQ ID NO:8;
the amino acid sequence of the CDR1 of the light chain is the amino acid sequence of SEQ ID NO:9;
The amino acid sequence of CDR2 of the light chain is AAS, and the amino acid sequence of CDR3 of the light chain is the amino acid sequence of SEQ ID NO:10.
(III):
the heavy chain CDR1 amino acid sequence is the amino acid sequence of SEQ ID NO: 11;
the heavy chain CDR2 amino acid sequence is the amino acid sequence of SEQ ID NO: 12;
the heavy chain CDR3 amino acid sequence is the amino acid sequence of SEQ ID NO: 13;
the light chain CDR1 amino acid sequence is the amino acid sequence of SEQ ID NO: 14;
The amino acid sequence of CDR2 of the light chain is GNN, and the amino acid sequence of CDR3 of the light chain is the amino acid sequence of SEQ ID NO:15.
(I):
the heavy chain CDR1 amino acid sequence is the amino acid sequence of SEQ ID NO: 1;
the heavy chain CDR2 amino acid sequence is the amino acid sequence of SEQ ID NO: 2;
the heavy chain CDR3 amino acid sequence is the amino acid sequence of SEQ ID NO: 3;
the amino acid sequence of the CDR1 of the light chain is the amino acid sequence of SEQ ID NO: 4;
The amino acid sequence of CDR2 of the light chain is GKN, and the amino acid sequence of CDR3 of the light chain is the amino acid sequence of SEQ ID NO:5.

<2> The antibody that binds to SARS-CoV-2 comprises the following heavy and light chain amino acid sequences, <
1>, the isolated antibody that binds to SARS-CoV-2 or an antibody fragment that comprises an antigen-binding region thereof, or an antibody fragment that binds to SARS-CoV-2, or comprises the antibody or antigen-binding region thereof An antibody fragment comprising an antibody or antigen-binding region thereof that competes with an antibody fragment.
In the case of (IV) above:
the amino acid sequence of FR1 of the heavy chain is the amino acid sequence of SEQ ID NO: 45;
the amino acid sequence of FR2 of the heavy chain is the amino acid sequence of SEQ ID NO: 46;
the amino acid sequence of FR3 of the heavy chain is the amino acid sequence of SEQ ID NO: 47;
the heavy chain FR4 amino acid sequence is the amino acid sequence of SEQ ID NO: 48;
the amino acid sequence of FR1 of the light chain is the amino acid sequence of SEQ ID NO: 49;
the amino acid sequence of FR2 of the light chain is the amino acid sequence of SEQ ID NO: 50;
The amino acid sequence of FR3 of the light chain is the amino acid sequence of SEQ ID NO:51, and the amino acid sequence of FR4 of the light chain is the amino acid sequence of SEQ ID NO:52.
In the case of (II) above:
the amino acid sequence of FR1 of the heavy chain is the amino acid sequence of SEQ ID NO: 29;
the amino acid sequence of FR2 of the heavy chain is the amino acid sequence of SEQ ID NO: 30;
the amino acid sequence of FR3 of the heavy chain is the amino acid sequence of SEQ ID NO: 31;
the heavy chain FR4 amino acid sequence is the amino acid sequence of SEQ ID NO: 32;
the amino acid sequence of FR1 of the light chain is the amino acid sequence of SEQ ID NO: 33;
the amino acid sequence of FR2 of the light chain is the amino acid sequence of SEQ ID NO: 34;
the amino acid sequence of FR3 of the light chain is the amino acid sequence of SEQ ID NO:35; and the amino acid sequence of FR4 of the light chain is the amino acid sequence of SEQ ID NO:36;
In the case of (III) above:
the amino acid sequence of FR1 of the heavy chain is the amino acid sequence of SEQ ID NO: 37;
the amino acid sequence of FR2 of the heavy chain is the amino acid sequence of SEQ ID NO: 38;
the amino acid sequence of FR3 of the heavy chain is the amino acid sequence of SEQ ID NO: 39;
the heavy chain FR4 amino acid sequence is the amino acid sequence of SEQ ID NO: 40;
the amino acid sequence of FR1 of the light chain is the amino acid sequence of SEQ ID NO: 41;
the amino acid sequence of FR2 of the light chain is the amino acid sequence of SEQ ID NO: 42;
the amino acid sequence of FR3 of the light chain is the amino acid sequence of SEQ ID NO:43; and the amino acid sequence of FR4 of the light chain is the amino acid sequence of SEQ ID NO:44;
In the case of (I) above:
the amino acid sequence of FR1 of the heavy chain is the amino acid sequence of SEQ ID NO: 21;
the amino acid sequence of FR2 of the heavy chain is the amino acid sequence of SEQ ID NO: 22;
the amino acid sequence of FR3 of the heavy chain is the amino acid sequence of SEQ ID NO: 23;
the heavy chain FR4 amino acid sequence is the amino acid sequence of SEQ ID NO: 24;
the amino acid sequence of FR1 of the light chain is the amino acid sequence of SEQ ID NO: 25;
the amino acid sequence of FR2 of the light chain is the amino acid sequence of SEQ ID NO: 26;
the amino acid sequence of FR3 of the light chain is the amino acid sequence of SEQ ID NO:27; and the amino acid sequence of FR4 of the light chain is the amino acid sequence of SEQ ID NO:28;

<3><1> or <2>, the isolated antibody that binds to SARS-CoV-2 or an antibody fragment comprising an antigen-binding region thereof, or in binding to the SARS-CoV-2 A pharmaceutical composition comprising an antibody or antibody fragment comprising an antigen binding region thereof that competes with an antibody or antibody fragment comprising an antigen binding region thereof.
<4> The pharmaceutical composition according to <3> for preventing or treating COVID-19.
<5> According to <1> or <2>, the isolated antibody that binds to SARS-CoV-2 or an antibody fragment containing an antigen-binding region thereof, or in binding to the SARS-CoV-2 An isolated DNA encoding a protein portion of an antibody or antibody fragment comprising an antigen binding region thereof that competes with an antibody or antibody fragment comprising an antigen binding region thereof.

According to the present invention, techniques for neutralizing SARS-CoV-2 can be provided.

4 is a graph showing results of examples according to one aspect of the present invention. Specifically, it is a graph showing the relationship between the specimen and the concentration of IgG antibody that specifically binds to the extracellular domain trimeric S protein of SARS-CoV-2 (conventional strain (Wuhan strain)). 4 is a graph showing results of examples according to one aspect of the present invention. More specifically, it is a graph showing the relationship between specimens and virus-neutralizing ability. FIG. 4 shows results of examples according to one aspect of the present invention. Specifically, it is a diagram showing the results of FACS. 4 is a photograph showing the results of an example according to one aspect of the present invention (drawing substitute photograph). More specifically, it is a photograph showing the results of agarose gel electrophoresis of the κ and λ light chain PCR amplification products (approximately 750 bp base length). 4 is a photograph showing the results of an example according to one aspect of the present invention (drawing substitute photograph). More specifically, it is a photograph showing the results of agarose gel electrophoresis of an IgG heavy chain PCR amplification product (base length of 1,400 bp). 4 is a graph showing results of examples according to one aspect of the present invention. Specifically, the S protein of the B.1.1.7 mutant strain (alpha strain or British strain) when the ability of the S protein of the conventional strain (Wuhan strain) to bind to the RBD (receptor binding domain) protein is 100%. is a graph showing the relative binding ability (%) of antibodies to the RBD protein of B.1.351 mutant strain (beta strain or South Africa strain) of the S protein of B.1.351. 4 is a graph showing results of examples according to one aspect of the present invention. Specifically, it is a graph showing the results of measuring the ability of an antibody to bind to the RBD (receptor binding domain) protein of the S protein of the conventional strain (Wuhan strain). FIG. 4 shows results of examples according to one aspect of the present invention. Specifically, (a) a diagram showing the binding site of the antibody NCV2SG48 in the RBD protein surface model, and (b) a diagram showing the mutation sites of the B.1.1.529 mutant strain (Omicron strain). FIG. 4 shows results of examples according to one aspect of the present invention. Specifically, the binding sites of the antibody NCV2SG48 in a model of the RBD protein surface, with hydrogen bonds added by somatic mutations. FIG. 4 shows results of examples according to one aspect of the present invention. Specifically, it shows the binding sites of the antibody NCV2SG53 in a model of the RBD protein surface. FIG. 4 shows results of examples according to one aspect of the present invention. Specifically, it shows that the interaction between the E484 residue of the RBD protein and the antibody NCV2SG53 is mediated by multiple hydrogen bonds.

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below. However, the present invention is not limited to the following preferred embodiments, and can be freely modified within the scope of the present invention.

One aspect of the present invention is
comprising the amino acid sequences of the heavy and light chains of any one of (I) to (IV) below,
isolated,
An antibody or antibody fragment comprising an antigen-binding region thereof that binds to SARS-CoV-2 or an antibody or antigen-binding thereof that competes with said antibody or antibody fragment comprising an antigen-binding region thereof for binding to said SARS-CoV-2 An antibody fragment containing the region.
(I):
the heavy chain CDR1 amino acid sequence is the amino acid sequence of SEQ ID NO: 1;
the heavy chain CDR2 amino acid sequence is the amino acid sequence of SEQ ID NO: 2;
the heavy chain CDR3 amino acid sequence is the amino acid sequence of SEQ ID NO: 3;
the amino acid sequence of the CDR1 of the light chain is the amino acid sequence of SEQ ID NO: 4;
The amino acid sequence of CDR2 of the light chain is GKN, and the amino acid sequence of CDR3 of the light chain is the amino acid sequence of SEQ ID NO:5.
(II):
the heavy chain CDR1 amino acid sequence is the amino acid sequence of SEQ ID NO: 6;
the heavy chain CDR2 amino acid sequence is the amino acid sequence of SEQ ID NO: 7;
the heavy chain CDR3 amino acid sequence is the amino acid sequence of SEQ ID NO:8;
the amino acid sequence of the CDR1 of the light chain is the amino acid sequence of SEQ ID NO:9;
The amino acid sequence of CDR2 of the light chain is AAS, and the amino acid sequence of CDR3 of the light chain is the amino acid sequence of SEQ ID NO:10.
(III):
the heavy chain CDR1 amino acid sequence is the amino acid sequence of SEQ ID NO: 11;
the heavy chain CDR2 amino acid sequence is the amino acid sequence of SEQ ID NO: 12;
the heavy chain CDR3 amino acid sequence is the amino acid sequence of SEQ ID NO: 13;
the light chain CDR1 amino acid sequence is the amino acid sequence of SEQ ID NO: 14;
The amino acid sequence of CDR2 of the light chain is GNN, and the amino acid sequence of CDR3 of the light chain is the amino acid sequence of SEQ ID NO:15.
(IV):
the heavy chain CDR1 amino acid sequence is the amino acid sequence of SEQ ID NO: 16;
the heavy chain CDR2 amino acid sequence is the amino acid sequence of SEQ ID NO: 17;
the heavy chain CDR3 amino acid sequence is the amino acid sequence of SEQ ID NO: 18;
the amino acid sequence of the CDR1 of the light chain is the amino acid sequence of SEQ ID NO: 19;
The amino acid sequence of CDR2 of the light chain is AAS, and the amino acid sequence of CDR3 of the light chain is the amino acid sequence of SEQ ID NO:20.

In (I) above, the amino acid sequences of CDR1 to CDR3 of the heavy chain and CDR1 to CDR3 of the light chain are, as long as they have the ability to neutralize SARS-CoV-2, respectively, the SEQ ID NOS: 1 to
4, GKN, it may be an amino acid sequence having a high degree of identity with the amino acid sequence represented by SEQ ID NO:5.
This also applies to the above (II) to (IV).

The "amino acid sequence having high identity" with the predetermined amino acid sequence is 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% in order of preference. The above means an amino acid sequence having 99% or more identity.
The identity can be determined using the BLAST (Basic Local Alignment Search Tool at the National
Center for Biological Information (basic local alignment search tool of the US National Center for Biological Information)), etc. (eg, default or default parameters).
Moreover, the "highly identical amino acid sequence" may be an amino acid sequence in which one or more amino acid sequences are substituted, deleted, inserted or added to the predetermined amino acid sequence. The plural number is, for example, two, three, or four.

Such substitutions are preferably conservative substitutions. A "conservative substitution" is the replacement of an amino acid residue with another, chemically similar amino acid residue that does not substantially alter the activity of the peptide. For example, replacing a hydrophobic residue with another hydrophobic residue, replacing a polar residue with another polar residue having the same charge, and the like. Examples of functionally similar amino acids that may undergo such substitutions include non-polar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, and the like. Polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine and the like. Examples of positively charged (basic) amino acids include arginine, histidine, and lysine. Moreover, aspartic acid, glutamic acid, etc. are mentioned as an (acidic) amino acid with a negative charge.

The aforementioned "isolated" means not existing in vivo. For example, it does not include antibodies produced in and present in an individual.

The antibody that binds to SARS-CoV-2 may be a chimeric antibody, a humanized antibody, or a human antibody. Any antibody can be produced using known methods.

In addition, the heavy chain constant region of the antibody that binds to SARS-CoV-2 is IgG (e.g., IgG1,
IgG2a, IgG2b, IgG3, IgG4), IgA (e.g., IgA1, IgA2
), IgE, IgM, and IgD constant regions.
The light chain constant region of the antibody that binds SARS-CoV-2 is κ or λ.

The antibody that binds to SARS-CoV-2 has the ability to bind to SARS-CoV-2.
The SARS-CoV-2-binding ability of the antibody that binds to SARS-CoV-2 is not particularly limited in its aspect, but can be, for example, a dissociation constant. When the dissociation constant is used as an index, for example, the dissociation constant (K _D ) between the antibody that binds to SARS-CoV-2 and SARS-CoV-2 is 175 nM or less and 50 nM or less in order of increasing preference. , 10 nM or less, 5 nM or less, 3 nM or less, 2.60 nM or less, 2.50 nM or less.
In addition, the association rate constant (k _a ) is set to 5.0×10 ⁴ Ms ^-1 or more, 8
0×10 ⁴ Ms ⁻¹ or more, 1.0×10 ⁵ Ms ⁻¹ or more, 3.0×10 ⁵ Ms ⁻¹ or more.
In addition, the dissociation rate constant (k _d ) is 1.0 × 10 ^-3 s ^-1 or less, 9
0×10 ⁻⁴ s ⁻¹ or less, 8.0×10 ⁻⁴ s ⁻¹ or less, 5.0×10 ⁻⁴ s ⁻¹ or less, 2.0×10 ⁻⁴ s ⁻¹ or less.
The dissociation constant, the association rate constant, and the dissociation rate constant can be measured by a conventional method such as a method using surface plasmon resonance, for example.

The binding ability to SARS-CoV-2 may be the ability to bind to the extracellular domain trimeric S protein of SARS-CoV-2, or the receptor binding domain (recep
tor binding domain (RBD)) protein.
The "S protein" is a spike protein.

The SARS-CoV-2 extracellular domain trimeric S protein is a trimer of the extracellular domain of the SARS-CoV-2 S protein, and the antibody that binds to the SARS-CoV-2 is not particularly limited as long as it has binding ability. For example, the SARS-CoV-2 is SARS-CoV-2 (conventional strain (
Wuhan strain)), for example, surface glycoprotein (NCBI Reference Sequence: YP_009724
390.1) trimers of the extracellular domain of the S protein (amino acids: 12-1213).

The RBD protein of the SARS-CoV-2 S protein is a receptor binding domain, and the SARS
-Antibody that binds to CoV-2 is not particularly limited as long as it has binding ability. For example, the SA
If RS-CoV-2 is SARS-CoV-2 (conventional strain (Wuhan strain)), for example, surface glycoprotein (NCBI
Reference Sequence: YP_009724390.1)
: 322-536).

The antibody that binds to SARS-CoV-2 has the ability to neutralize SARS-CoV-2.
The SARS-CoV-2-neutralizing ability of the antibody that binds to SARS-CoV-2 is not particularly limited in its embodiment, but for example, the half-inhibitory concentration (IC ₅₀ value) can be used as an indicator. When the _IC50 value is used as an index, the _IC50 value is 10,000 ng/ml or less, 1,100 ng/ml or less, 1,000 ng/ml or less, 850 ng/ml or less, and 800 ng/ml or less in order of increasing preference. , ≤750 ng/ml, ≤600 ng/ml, 5
00 ng/ml or less, 400 ng/ml or less, 300 ng/ml or less, 100 ng/ml or less, 60 ng/ml or less, 55 ng/m
l or less, 52 ng/ml or less, 50 ng/ml or less, 40 ng/ml or less, 35 ng/ml or less, 34 ng/ml or less, 30
≤27 ng/ml, ≤26 ng/ml, ≤25 ng/ml, ≤20 ng/ml, ≤19 ng/ml, ≤15 ng/ml, ≤11 ng/ml, 10 ng/ml ≤5.0 ng/ml, ≤4.0 ng/ml, 3.8 ng
/ml or less, 3.0 ng/ml or less, 2.9 ng/ml or less, 2.5 ng/ml or less, 2.4 ng/ml or less, 2.3 ng/ml or less, 2.0 ng/ml or less, 1.7 ng/ml or less, 1.5 ng/ml 1.4 ng/ml or less, 1.2 ng/ml or less, 1
.1 ng/ml or less, 1.0 ng/ml or less, 0.9 ng/ml or less, 0.8 ng/ml or less, 0.7 ng/ml or less, 0.5 ng
/ml or less.
Said IC ₅₀ value is, for example, binding to host cells infected with SARS-CoV-2, SARS-CoV-2 and serially diluted said SARS-CoV-2, as described in the Examples below. It can be calculated according to a conventional method, such as by applying a mixed solution with an antibody that causes cytopathic effect and culturing until cytopathic effect (CPE) appears.

The SARS-CoV-2 includes SARS-CoV-2 neutralized by an antibody that binds to the SARS-CoV-2
As long as it is not particularly limited, for example,
Conventional strains (sometimes referred to as "Wuhan strain", "Wuhan-Hu-1 strain", etc. at the time of filing the application),
B.1.1.7 Mutant strain (sometimes referred to as "alpha strain", "British strain", etc. at the time of filing this application. The mutation of RBD (amino acids: 328-533) is N501Y),
B.1.351 mutant strain (sometimes referred to as "beta strain", "South Africa strain", etc. at the time of filing of the application. The RBD mutation is K417N/E484K/N501Y),
B.1.1.248 mutant strain (sometimes referred to as "gamma strain", "Brazilian strain", etc. at the time of filing the application. The RBD mutation is K417T/E484K/N501Y),
B.1.525 mutant strain (sometimes referred to as "Eta strain", "Nigeria strain", etc. at the time of filing the application. The RBD mutation is E484K),
B.1.429 mutant strain (sometimes referred to as "Epsilon strain", "California strain", etc. at the time of filing the application; the RBD mutation is L452R),
P.2 mutant strain (sometimes referred to as "zeta strain" etc. at the time of filing the application. RBD mutation is
It is E484K. ),
P.3 mutant (sometimes referred to as "Theta strain", "Philippine strain", etc. at the time of filing the application. The RBD mutation is E484K/N501Y),
B.1.617.1 mutant strain (sometimes referred to as "kappa strain", "Indian strain", etc. at the time of filing the application. RBD mutation is L452R/E484Q),
B.1.617.2 mutant strain (sometimes referred to as "Delta strain", "Indian strain", etc. at the time of filing the application; the RBD mutation is L452R/T478K),
delta plus mutant strain mutated from the B.1.617.2 mutant strain (sometimes referred to as "delta strain", "Indian strain", etc. at the time of filing the application; the RBD mutation is L452R/T478K),
B.1.526 mutant strain (sometimes referred to as "iota strain" etc. at the time of filing the application. RBD mutation is S477N/E484K),
C.37 mutant strain (sometimes referred to as "lambda strain" etc. at the time of filing the application. RBD mutation is L452Q/F490S),
B.1.1.114 strain (sometimes referred to as "European strain (first wave)" at the time of filing the application).
),
B.1.1.284 strain (sometimes referred to as "European strain (second wave)" at the time of filing the application).
),
B.1.1.214 strain (sometimes referred to as "European strain (third wave)" at the time of filing the application).
)
B.1.1.529 mutant strain (sometimes referred to as "Omicron strain" at the time of filing the application. R
BD mutations are G339D/S371L/S373P/S375F/K417N/N440K/G446S/S477N/T478K/E484A/Q493R/G496
They are S/Q498R/N501Y/Y505H. ),
B.1.1 mutant strain (herein sometimes referred to as "B.1.1 strain". SARS-CoV-2/JP/Hiroshima-46059T/2020 strain isolated at National University Corporation Hiroshima University, Its complete genome sequence is G
"Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/h
human/JPN/JP_Hiro46059c/2020, complete genome”. The mutation in S protein is D614G, and there is no mutation in RBD. Based on the base sequence of the conventional strain, a total of 9 bases are mutated. ),
etc.
The SARS-CoV-2 may be a SARS-CoV-2 pseudovirus (sometimes referred to as a “pseudovirus” or “pseudotype virus”). Pseudovirus
Using SARS-CoV-2, for example, it may be prepared by a conventional method as described in the examples below, and for example, B.1 mutant strain (B.1.1.114 strain (European strain ( The spike protein of the first wave)) is expressed only, the mutation of the S protein is D614G, and the RBD has no mutation.), and the like.
In this embodiment, the SARS-CoV-2 may be one or plural.

The variable region of the antibody that binds to SARS-CoV-2 preferably comprises the following amino acid sequences.
In the case of (I) above:
the amino acid sequence of FR1 of the heavy chain is the amino acid sequence of SEQ ID NO: 21;
the amino acid sequence of FR2 of the heavy chain is the amino acid sequence of SEQ ID NO: 22;
the amino acid sequence of FR3 of the heavy chain is the amino acid sequence of SEQ ID NO: 23;
the heavy chain FR4 amino acid sequence is the amino acid sequence of SEQ ID NO: 24;
the amino acid sequence of FR1 of the light chain is the amino acid sequence of SEQ ID NO: 25;
the amino acid sequence of FR2 of the light chain is the amino acid sequence of SEQ ID NO: 26;
the amino acid sequence of FR3 of the light chain is the amino acid sequence of SEQ ID NO:27; and the amino acid sequence of FR4 of the light chain is the amino acid sequence of SEQ ID NO:28;
In the case of (II) above:
the amino acid sequence of FR1 of the heavy chain is the amino acid sequence of SEQ ID NO: 29;
the amino acid sequence of FR2 of the heavy chain is the amino acid sequence of SEQ ID NO: 30;
the amino acid sequence of FR3 of the heavy chain is the amino acid sequence of SEQ ID NO: 31;
the heavy chain FR4 amino acid sequence is the amino acid sequence of SEQ ID NO: 32;
the amino acid sequence of FR1 of the light chain is the amino acid sequence of SEQ ID NO: 33;
the amino acid sequence of FR2 of the light chain is the amino acid sequence of SEQ ID NO: 34;
the amino acid sequence of FR3 of the light chain is the amino acid sequence of SEQ ID NO:35; and the amino acid sequence of FR4 of the light chain is the amino acid sequence of SEQ ID NO:36;
In the case of (III) above:
the amino acid sequence of FR1 of the heavy chain is the amino acid sequence of SEQ ID NO: 37;
the amino acid sequence of FR2 of the heavy chain is the amino acid sequence of SEQ ID NO: 38;
the amino acid sequence of FR3 of the heavy chain is the amino acid sequence of SEQ ID NO: 39;
the heavy chain FR4 amino acid sequence is the amino acid sequence of SEQ ID NO: 40;
the amino acid sequence of FR1 of the light chain is the amino acid sequence of SEQ ID NO: 41;
the amino acid sequence of FR2 of the light chain is the amino acid sequence of SEQ ID NO: 42;
the amino acid sequence of FR3 of the light chain is the amino acid sequence of SEQ ID NO:43; and the amino acid sequence of FR4 of the light chain is the amino acid sequence of SEQ ID NO:44;
In the case of (IV) above:
the amino acid sequence of FR1 of the heavy chain is the amino acid sequence of SEQ ID NO: 45;
the amino acid sequence of FR2 of the heavy chain is the amino acid sequence of SEQ ID NO: 46;
the amino acid sequence of FR3 of the heavy chain is the amino acid sequence of SEQ ID NO: 47;
the heavy chain FR4 amino acid sequence is the amino acid sequence of SEQ ID NO: 48;
the amino acid sequence of FR1 of the light chain is the amino acid sequence of SEQ ID NO: 49;
the amino acid sequence of FR2 of the light chain is the amino acid sequence of SEQ ID NO: 50;
The amino acid sequence of FR3 of the light chain is the amino acid sequence of SEQ ID NO:51, and the amino acid sequence of FR4 of the light chain is the amino acid sequence of SEQ ID NO:52.

In the above (I), the amino acid sequences of FR1 to FR4 of the heavy chain and FR1 to FR4 of the light chain are represented by SEQ ID NOS: 21 to 28, respectively, as long as they have the ability to neutralize SARS-CoV-2. It may be an amino acid sequence that has a high degree of identity with the amino acid sequence to be identified.
This also applies to the above (II) to (IV).

The definition of the "highly identical amino acid sequence" and the aspect of the substitution refer to the explanations given above.
In addition, the above-mentioned "amino acid sequence having high identity" is, in the above-mentioned predetermined amino acid sequence,
It may be an amino acid sequence in which one to multiple amino acid sequences are substituted, deleted, inserted or added. The plural numbers are, for example, 2, 3, 4, 5, 6, and 7.

The amino acid sequence of the heavy chain variable region of (I) (consisting of the amino acid sequence of CDR1-3 of the heavy chain of (I) and FR1-4 of the heavy chain) is represented by SEQ ID NO:61. The amino acid sequence of the heavy chain variable region of (I) is the SEQ ID NO: 61, as long as it has the ability to neutralize SARS-CoV-2.
It may be an amino acid sequence having a high identity with the amino acid sequence represented by.
The amino acid sequence of the light chain variable region of (I) (consisting of the amino acid sequence of CDR1-3 of the light chain of (I) and FR1-4 of the light chain) is represented by SEQ ID NO:62. The amino acid sequence of the light chain variable region of (I) is the SEQ ID NO: 62 as long as it has the ability to neutralize SARS-CoV-2.
It may be an amino acid sequence having a high identity with the amino acid sequence represented by.
The amino acid sequence of the heavy chain variable region of (II) (consisting of the amino acid sequence of CDR1-3 of the heavy chain of (II) and FR1-4 of the heavy chain) is represented by SEQ ID NO:63. (II)
may be an amino acid sequence highly identical to the amino acid sequence represented by SEQ ID NO: 63, as long as it has the ability to neutralize SARS-CoV-2.
The amino acid sequence of the light chain variable region of (II) (consisting of the amino acid sequence of CDR1-3 of the light chain of (II) and FR1-4 of the light chain) is represented by SEQ ID NO:64. (II)
may be an amino acid sequence highly identical to the amino acid sequence represented by SEQ ID NO: 64, as long as it has the ability to neutralize SARS-CoV-2.
The amino acid sequence of the heavy chain variable region of (III) (consisting of the amino acid sequence of CDR1-3 of the heavy chain of (III) and FR1-4 of the heavy chain) is represented by SEQ ID NO:65. (I
The amino acid sequence of the heavy chain variable region of II) may be an amino acid sequence highly identical to the amino acid sequence represented by SEQ ID NO: 65 as long as it has the ability to neutralize SARS-CoV-2. .
The amino acid sequence of the light chain variable region of (III) (consisting of the amino acid sequence of CDR1-3 of the light chain of (III) and FR1-4 of the light chain) is represented by SEQ ID NO:66. (I
The amino acid sequence of the light chain variable region of II) may be an amino acid sequence highly identical to the amino acid sequence represented by SEQ ID NO: 66 as long as it has the ability to neutralize SARS-CoV-2. .
The amino acid sequence of the heavy chain variable region of (IV) (consisting of the amino acid sequence of CDR1-3 of the heavy chain of (IV) and FR1-4 of the heavy chain) is represented by SEQ ID NO:67. (IV)
may be an amino acid sequence highly identical to the amino acid sequence represented by SEQ ID NO: 67, as long as it has the ability to neutralize SARS-CoV-2.
The amino acid sequence of the light chain variable region of (IV) (consisting of the amino acid sequence of CDR1-3 of the light chain of (IV) and FR1-4 of the light chain) is represented by SEQ ID NO:68. (IV)
may be an amino acid sequence highly identical to the amino acid sequence represented by SEQ ID NO: 68, as long as it has the ability to neutralize SARS-CoV-2.
The definition of the "highly identical amino acid sequence" and the aspect of the substitution refer to the explanations given above.
In addition, the above-mentioned "amino acid sequence having high identity" is, in the above-mentioned predetermined amino acid sequence,
It may be an amino acid sequence in which one to multiple amino acid sequences are substituted, deleted, inserted or added. The plurality is a natural number selected from 2 to 25, for example.

Antibodies that bind to SARS-CoV-2 can be obtained using techniques commonly used in the art, such as chemical synthesis and genetic recombination, based on sequence information.
In addition, the production thereof may include a step of recovering the obtained antibody that binds to SARS-CoV-2. The recovery process may be a purification process, a concentration process, or the like. A known purification technique can be used in the purification step, and the antibody that binds to SARS-CoV-2 may be purified to homogeneity.

Next, antibody fragments containing the antigen-binding region of the antibody that binds to SARS-CoV-2 are described.
The term “antigen-binding region” refers to a region present in an antibody and capable of recognizing an antigen.
An "antibody fragment containing an antigen-binding region" refers to a protein containing a portion of an antibody and capable of binding to an antigen. Examples of antibody fragments include F(ab') ₂ , Fab', Fab, Fv (variable f
fragment of antibody), disulfide-bonded Fv, single-chain antibody (scFv), and polymers thereof. Such antibody fragments are chemically or genetically engineered functional molecules such as non-peptidic polymers such as polyethylene glycol (PEG), radioactive substances, toxins, low-molecular-weight compounds, cytokines, growth factors, albumin, enzymes, and other antibodies. may be combined.

Next, an antibody or an antibody fragment comprising an antigen-binding region thereof that competes with an antibody that binds to SARS-CoV-2 or an antibody fragment comprising the antigen-binding region thereof for binding to SARS-CoV-2 will be described.
For example, such an "antibody fragment containing an antibody or an antigen-binding region thereof" is referred to as an "antibody or the like according to one embodiment of this embodiment", and "an antibody that binds to SARS-CoV-2 or an antigen-binding region thereof. If an "antibody fragment" is referred to as a "reference antibody or the like", the phrase "the antibody or the like according to one embodiment of this embodiment "competes with the reference antibody or the like" in binding to the SARS-CoV-2" refers to one embodiment of this embodiment The site on SARS-CoV-2 to which the antibody etc. related to binds and SARS to which the reference antibody etc. binds
-The site on CoV-2 is the same, or the antibody or the like according to one embodiment in this embodiment sterically hinders the binding of the reference antibody or the like to SARS-CoV-2 SARS-CoV- It means that it binds to the site on 2. That is, the antibody or the like according to one aspect of this aspect recognizes the same epitope completely or partially as the epitope of the reference antibody or the like.

A competitive assay commonly used in the art may be used to identify an antibody or the like according to one aspect of this embodiment. For example, when the antibody or the like according to one form of this embodiment is present in excess, the binding of the reference antibody or the like to SARS-CoV-2 is at least 10% or more, 15%
20% or more 25% or more 30% or more 35% or more 40% or more 45% or more 5
Block (eg, reduce) 0% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, or more.
Antibody-antigen binding of antibodies and the like to SARS-CoV-2 in such competitive assays can be performed by a person skilled in the art by appropriately selecting binding measurement in a solid-phase or liquid-phase system. Such methods include, but are not limited to, ELISA, EIA, surface plasmon resonance, FRET, and LRET methods.
In addition, when measuring such antigen-antibody binding, antibodies, etc. and / or antigens (SARS-CoV-2)
is labeled with an enzyme, fluorescent substance, luminescent substance, radioactive isotope, etc., and the antigen-antibody reaction can be detected using a measurement method suitable for the physical and/or chemical properties of the labeled substance. .

Another aspect of the present invention is the "antibody fragment comprising an antibody that binds to SARS-CoV-2 or an antigen-binding region thereof, or the antibody or antigen-binding thereof in binding to SARS-CoV-2" according to the above aspect. an antibody or an antibody fragment containing an antigen-binding region thereof that competes with an antibody fragment containing the region"
1 is an isolated DNA encoding a protein portion of .

Based on the amino acid sequence of the antibody that binds to SARS-CoV-2, a person skilled in the art can readily design a DNA sequence encoding the protein portion thereof. In addition, using this, known manipulations can be performed by known genetic engineering techniques.

For example, the antibody that binds to SARS-CoV-2 is obtained by inserting DNA encoding the heavy chain and DNA encoding the light chain of the antibody into an expression vector, transforming a host cell using the vector, and Cells can be cultured for production. In this case, the DNA encoding the heavy chain and the DNA encoding the light chain may be inserted into the same expression vector and used to transform the host cell, or the DNA encoding the heavy chain and the light chain may be transformed. inserting the coding DNA into a separate vector,
The two vectors may be used to transform host cells. In this case, the DNAs encoding the heavy and light chain variable regions may be inserted into a vector into which the DNAs encoding the heavy and light chain constant regions of a specific isotype have been previously inserted. The vector may also contain DNA encoding a signal peptide that facilitates secretion of the antibody from the host cell.
In this case, the DNA encoding the signal peptide and the DNA encoding the antibody are ligated in-frame. The antibody can be obtained as a mature protein because the signal peptide is removed when the antibody is produced.

In addition, as the DNA encoding each amino acid sequence, the DNA sequence and BLAST (Basic Local
80 in order of decreasing preference when calculated using the Alignment Search Tool at the National Center for Biological Information, etc. (e.g., default or default parameters) % or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99%
It may be a DNA consisting of a nucleotide sequence having the above identity and encoding a protein having the ability to neutralize SARS-CoV-2.

In addition, the DNA encoding each amino acid sequence is a DNA that can hybridize under stringent conditions with a DNA consisting of a sequence complementary to the DNA encoding each amino acid sequence, It may be a DNA that encodes a protein that has the ability to neutralize -2. Those skilled in the art can appropriately determine stringent conditions. For example, Southern hybridization is followed by washing at 68° C., 0.1×SSC, and a salt concentration corresponding to 0.1% SDS.

Another aspect of the present invention is
SARS-CoV-2 (conventional strain (Wuhan strain)) surface glycoprotein (NCBI Reference Sequence: YP_0
09724390.1) in the S protein in
An epitope contained in the amino acid sequence (SEQ ID NO: 69) consisting of the 403rd amino acid (R) to the 505th amino acid (Y), or
Recognizing an epitope contained in an amino acid sequence (SEQ ID NO: 70) consisting of the 445th amino acid (V) to the 501st amino acid (N),
An isolated antibody or antibody fragment comprising an antigen binding region thereof, or an antibody fragment comprising an antibody or antigen binding region thereof that competes with said antibody or antibody fragment comprising an antigen binding region thereof in recognizing said epitope.

The amino acid sequence (SEQ ID NO: 69) consisting of the 403rd amino acid (R) to the 505th amino acid (Y) also includes the 445th amino acid (V) to the 501st amino acid (N
) (SEQ ID NO: 70) is also an amino acid sequence in the RBD protein (amino acids: 322-536).

In the above, the epitope is an epitope contained in the predetermined amino acid sequence in the S protein in the surface glycoprotein (NCBI Reference Sequence: YP_009724390.1) of SARS-CoV-2 (conventional strain (Wuhan strain)). As described above, as long as the S protein in the surface glycoprotein contains the predetermined amino acid sequence, SARS-CoV-2 is not limited to SARS-CoV-2 (conventional strain (Wuhan strain)), and specific examples thereof is as already mentioned.

The "epitope contained in the amino acid sequence consisting of the 403rd amino acid (R) to the 505th amino acid (Y)" is preferably "the 403rd amino acid (R), the 406th amino acid (E), the 409th amino acid (Q), 415th amino acid (T), 417
th amino acid (K), 420th amino acid (D), 421st amino acid (Y), 4
53rd amino acid (Y), 455th amino acid (L), 457th amino acid (R)
, 458th amino acid (K), 460th amino acid (N), 473rd amino acid (
Y), 474th amino acid (Q), 475th amino acid (A), 487th amino acid (N), 489th amino acid (Y), 493rd amino acid (Q), 494th amino acid (S) , 496th amino acid (G), 498th amino acid (Q), 501st amino acid (N), and 502nd amino acid (G)".

The "epitope contained in the amino acid sequence consisting of the 445th amino acid (V) to the 501st amino acid (N)" is preferably "the 449th amino acid (Y), the 478th amino acid (T), the 479th amino acid (P), 484th amino acid (E), 48
6th amino acid (F), 487th amino acid (N), 489th amino acid (Y),
490th amino acid (F), 492nd amino acid (L), 493rd amino acid (Q
), the 494th amino acid (S), and the 498th amino acid (Q)”.

Specifically for this embodiment, an isolated SARS-CoV-2 comprising the amino acid sequences of the heavy and light chains of any one of (I) to (IV) above, as previously described. Details of an antibody or antibody fragment comprising an antigen binding region thereof, or an antibody fragment comprising an antibody or antigen binding region thereof that competes with said antibody or antibody fragment comprising an antigen binding region thereof for binding to said SARS-CoV-2 to invoke.

In addition, according to this aspect, an isolated antibody that recognizes the epitope, or an antibody fragment that includes an antigen-binding region thereof, or an antibody fragment that includes the antibody or an antigen-binding region thereof in recognition of the epitope. A specific example of an antibody or an antibody fragment comprising an antigen-binding region thereof is isolated SARS comprising the amino acid sequences of the heavy and light chains of any one of (I) to (IV) above. - an antibody or an antibody fragment comprising an antigen binding region thereof that binds to CoV-2 or an antibody or an antigen binding region thereof that competes with said antibody or antibody fragment comprising an antigen binding region thereof in binding to said SARS-CoV-2 is an antibody fragment comprising

Another aspect of the present invention is the "isolated antibody that binds to SARS-CoV-2 or an antibody fragment comprising an antigen-binding region thereof, or an antibody fragment thereof that binds to SARS-CoV-2, or the An antibody or an antibody fragment containing an antigen-binding region thereof that competes with an antibody or an antibody fragment containing an antigen-binding region thereof” (In this embodiment, it may be described as “an antibody that binds to SARS-CoV-2, etc.”) A pharmaceutical composition comprising

The pharmaceutical composition according to this aspect can be used as a pharmaceutical composition for diseases that can be prevented or treated by administration of the antibody or the like that binds to SARS-CoV-2 according to the aspect. Such diseases include, for example, COVID-19.
The antibody or the like that binds to SARS-CoV-2 may be one or more.

The pharmaceutical composition according to this aspect can be produced according to a conventional method in the pharmaceutical field. The pharmaceutical composition according to this aspect may be used in combination with an active ingredient other than the SARS-CoV-2-binding antibody or the like according to the above aspect, as long as the effect is not impaired.
The pharmaceutical composition may contain carriers, diluents, and excipients that are commonly used in the pharmaceutical field, in addition to the antibody that binds to SARS-CoV-2, etc., according to the above embodiment.
For example, aqueous solutions for injection include physiological saline, PBS, and isotonic solutions containing glucose and other adjuvants. It may be used in combination with an ionic surfactant or the like. Sesame oil, soybean oil and the like are used as the oily liquid, and benzyl benzoate, benzyl alcohol and the like may be used together as the solubilizer.
The pharmaceutical composition according to this aspect can be administered in various forms, including parenteral administration such as injections and infusions. An injection is preferred.

The dosage for injections varies depending on symptoms, age, body weight, etc. In general, parenteral administration for adults is, for example, about 50 mg to 5000 mg at a time, and these are administered once, or It can be administered subcutaneously or intravenously in several doses.

The subject (preferably a mammal, more preferably a human) for prevention or treatment in this embodiment is preferably a subject diagnosed as likely to develop COVID-19 or a subject who has developed COVID-19. can be administered to these subjects.

Another aspect of the present invention is
(a) obtaining B cells that bind to the extracellular domain trimeric spike protein (S protein) of SARS-CoV-2 from a human biological sample recovered from COVID-19;
(b) the step of synthesizing a first cDNA from the mRNA of the gene encoding the amino acid sequence of the antibody heavy chain contained in the obtained B cells;
(c) synthesizing a second cDNA from the mRNA of the gene encoding the amino acid sequence of the light chain of the antibody contained in the obtained B cells;
(d) using a primer capable of amplifying the first cDNA, the synthesized first cDNA
Amplifying NA;
(e) using a primer capable of amplifying the second cDNA, the synthesized second cDNA
Amplifying NA;
(f) producing an antibody using the amplified first cDNA and second cDNA;
(g) measuring the neutralizing ability of the produced antibodies against SARS-CoV-2; and
(h) A method for obtaining an antibody, comprising the step of selecting an antibody that has the ability to neutralize SARS-CoV-2.

The method according to this aspect includes the following step (a).
(a) Obtaining B cells that bind to the extracellular domain trimeric spike protein (S protein) of SARS-CoV-2 from a human biological sample recovered from COVID-19.

Humans who have recovered from COVID-19 are not particularly limited as long as they have B cells that bind to the extracellular domain trimeric spike protein (S protein) of SARS-CoV-2, but the COVID- It is preferable that the person is a person who has recovered from COVID-19 and has passed 12 days or more from the date of positive test for COVID-19. A conventional method can be used as a positive determination method for COVID-19. Examples include PCR test, antigen test, LAMP method, immunochromatographic method, ELISA method, virus isolation test method, and the like.

In addition, the human who has recovered from COVID-19 is preferably a human who received oxygen inhalation treatment at the time of COVID-19. Such humans are also those who were severely ill at the time of contracting said COVID-19.

In addition, humans who have recovered from the COVID-19 disease have the extracellular domain trimer of SARS-CoV-2
It may be a human having a biological sample containing an antibody that specifically binds to the S protein.
A human having a biological sample containing an antibody that specifically binds to the extracellular domain trimeric S protein of SARS-CoV-2 can be determined by a conventional method. Examples include the ELISA method, immunochromatography method, IFA method, and the like described.
The antibody that specifically binds to the SARS-CoV-2 extracellular domain trimeric S protein has a concentration in the biological sample of 1 μg/ml or more, 10 μg/ml or more, in descending order of preference, 2
It is 0 μg/ml or more, 50 μg/ml or more, or 90 μg/ml or more.

Although the subclass of the antibody that specifically binds to the extracellular domain trimeric S protein of SARS-CoV-2 is not particularly limited, in general, humans who have recovered from COVID-19 have IgM antigen receptors, IgG There are B cells with antigen receptors or IgA antigen receptors on their surface, and normally no B cells expressing IgE antigen receptors on their surface are detected. Therefore, the antibody is usually an IgM antibody,
Although it is an IgG antibody or an IgA antibody, it may be an IgE antibody. In addition, since IgM antibodies do not have high neutralizing ability and IgE antibodies are antibodies involved in allergic reactions that do not have neutralizing ability,
IgG antibodies or IgA antibodies are preferred.

In addition, the human who has recovered from COVID-19 may be a human having a biological sample containing an antibody capable of neutralizing SARS-CoV-2.
A human having a biological sample containing an antibody having the ability to neutralize SARS-CoV-2 can be determined by a conventional method.
Examples include methods using oV-2 and host cells (eg, Vero cells).
The neutralization ability against SARS-CoV-2 is, for example, as described in the examples below,
Using the biological sample, SARS-CoV-2, and host cells that are first diluted 10-fold and then serially diluted 2-fold, the maximum dilution ratio that can prevent SARS-CoV-2 infection of the host cells ( For example, when 2-fold dilution is carried out n times, it becomes “10×2 ⁿ ”) can be used as an index. At this time, COVI
When using a biological sample obtained from a human not affected by D-19, the dilution ratio is set to 10 (no or very weak neutralization ability).
At this time, the maximum dilution rate is 20 or more, 40 or more, 100 or more, and 600 or more in order of preference. On the other hand, the higher the upper limit, the better, but it is, for example, 10,000 or less, or 1,000 or less.

Although the subclass of antibodies with neutralizing ability against SARS-CoV-2 is not particularly limited, in general, humans who have recovered from COVID-19 have IgM antigen receptors, IgG antigen receptors or IgA antigen receptors. B cells with surface-bearing B cells and usually no B cells expressing IgE antigen receptors on their surface are detected. Therefore, the antibody is usually an IgM, IgG or IgA antibody, but IgE
It may be an antibody. In addition, since IgM antibodies do not have high neutralizing ability and IgE antibodies do not have high neutralizing ability and are involved in allergic reactions, IgG antibodies or IgA antibodies are preferable.

As a method to obtain B cells that bind to the extracellular domain trimeric spike protein (S protein) of SARS-CoV-2 from humans who have recovered from COVID-19, such B cells can be obtained. There is no particular limitation as long as it is a method. It can be obtained according to a conventional method, for example, by the method described in Examples below.

Since CD19 is a marker molecule for B cells, said B cells are CD19 positive.
In addition, for example, macrophages and neutrophils express Fc receptors, and may have antibodies on their surface via the Fc receptors. Since the cells that bind to the extracellular domain trimer spike protein (S protein) are B cells, the cells obtained in this step (a) contain macrophages and neutrophils that can become noise in the method according to this aspect is not included.

Said B cells are preferably IgD negative.
Humans who have recovered from COVID-19 have B cells that express both IgM and IgD antigen receptors, B cells that express IgG antigen receptors, and IgA antigen receptors on their surface. B cells that express the IgE antigen receptor on their surface are usually not detected. In addition, IgM antibodies do not have a high neutralizing ability, and IgE antibodies are antibodies involved in allergic reactions that do not have a neutralizing ability. IgG antibody or IgA antibody.
For these reasons, in the method according to this aspect, the B cells obtained in this step (a) are preferably IgD-negative B cells in order to efficiently obtain IgG antibodies or IgA antibodies.

The "CD19-positive" cells express CD19 at a detectable level on the cell surface, and refer to cells that are sorted by flow cytometry or the like using an anti-CD19 antibody, for example.
The aforementioned “IgD-negative” cells refer to cells that do not express IgD at a detectable level on the cell surface and, for example, are not sorted by flow cytometry or the like using an anti-IgD antibody.
The cells that "bind to the extracellular domain trimer spike protein (S protein) of SARS-CoV-2" are, for example, the labeled extracellular domain trimer spike protein of SARS-CoV-2 ( S protein) refers to cells sorted by flow cytometry or the like.

For example, for the CD19-positive, IgD-negative B cells, a fluorescently labeled anti-CD19 antibody,
Using a fluorescently labeled anti-IgD antibody, and for B cells that bind to the SARS-CoV-2 extracellular domain trimeric spike protein (S protein), the SARS-CoV-2 extracellular domain trimeric It can be obtained by FACS analysis using a spike protein (S protein) (fluorescently labeled). At this time, dead cells may be excluded by a conventional method.

The fluorescently-labeled anti-CD19 antibody and the fluorescently-labeled anti-IgD antibody may be commercially available or appropriately prepared.

The SARS-CoV-2 extracellular domain trimer spike protein (S protein) may be commercially available or appropriately prepared. It may be prepared by such a method. For example, said SARS-CoV-2 is SARS-CoV-2
(Conventional strain (Wuhan strain)), for example, surface glycoprotein (NCBI Reference Sequence:
YP_009724390.1) trimer of extracellular domain of S protein (amino acids: 12-1213).

In addition, the labeling of the SARS-CoV-2 extracellular domain trimeric spike protein (S protein) binds to the SARS-CoV-2 extracellular domain trimeric spike protein (S protein) B cells There is no time limit as long as it is a sign that can be obtained. The method of labeling the extracellular domain trimeric spike protein (S protein) of SARS-CoV-2, wherein the SARS-C
Conventional methods can be used, depending on the oV-2 extracellular domain trimeric spike protein (S protein) and the nature of the label. In addition, commercially available products that have already been labeled may also be used.

The B cells obtained as described above are B cells having IgG antigen receptors, IgA antigen receptors, or IgE antigen receptors on their surfaces, preferably IgG antigen receptors or IgA antigen receptors. It is a B cell that had on the surface.
Specifically, humans who have recovered from COVID-19 have B cells that express both IgM and IgD antigen receptors, B cells that express IgG antigen receptors, and IgA antigen receptors. Since we have B cells that express the receptor on their surface and we usually do not detect B cells that express the IgE antigen receptor on their surface, sorting IgD-negative cells usually reveals IgG antigen receptors or IgA antigens. This is based on the fact that only B cells with receptors on their surface can be obtained.

The SARS-CoV-2 is not particularly limited as long as it is a virus that causes COVID-19 and has an extracellular domain trimeric spike protein (S protein).
As a specific example of SARS-CoV-2, the description given above is used.

The biological sample is not particularly limited as long as it contains B cells that bind to the extracellular domain trimer spike protein (S protein) of SARS-CoV-2. For example, blood, cerebrospinal fluid,
Lymph fluid etc. are mentioned.
When the biological sample is blood, it may be whole blood, serum, plasma, leukocyte fraction, or the like.
When the biological sample is blood, it may be peripheral blood, heart blood, umbilical cord blood, or the like.
In any case, the biological sample may be obtained by a conventional method.

In addition, this step (a) may include a step of obtaining a lymphocyte fraction from the biological sample, as described in Examples below. It may also include a step of collecting cells of B-lineage (a step of excluding non-B-lineage cells).

Also, the human may be a non-human mammal. For mammals other than humans,
For example, cows, goats, sheep, pigs, monkeys, dogs, cats, rats, mice, hamsters,
Examples include guinea pigs.

The method according to this aspect includes the following step (b).
(b) a step of synthesizing a first cDNA from the mRNA of the gene encoding the antibody heavy chain amino acid sequence contained in the obtained B cells;

mR of the gene encoding the amino acid sequence of the heavy chain of the antibody contained in the obtained B cells
The method for synthesizing the first cDNA from NA is not particularly limited as long as it is a method for synthesizing the first cDNA. It can be synthesized by a conventional method, for example, by reverse transcription reaction as described in the examples below.

The reaction conditions of the reverse transcription reaction and the primer sequences to be used are particularly as long as the target first cDNA is synthesized and the synthesized first cDNA is amplified in the step (d) described later. There are no restrictions, but conventional methods can be followed.
Since the cDNA to be synthesized differs depending on the primer sequence to be used, by appropriately selecting the primer sequence to be used, m of the gene encoding the desired heavy chain amino acid sequence
A first cDNA can be synthesized from RNA.
In addition, the reverse transcription reaction primer may be a dT primer or a primer specific to the constant region of the antibody gene. sell.

The method according to this aspect includes the following step (c).
(c) a step of synthesizing a second cDNA from the mRNA of the gene encoding the amino acid sequence of the light chain of the antibody contained in the obtained B cells;

The details are the same as those described for the step (b).

The method according to this aspect includes the following step (d).
(d) using a primer capable of amplifying the first cDNA, the synthesized first cDNA
Amplifying NA.

The reaction conditions for the amplification reaction are not particularly limited as long as the first cDNA is amplified, but conventional methods can be followed according to the primer sequences and the like to be used.

The primer sequences to be used can be designed, for example, with reference to International Publication 2015/182749.

Since the cDNA to be amplified differs depending on the primer sequences used, the cDNA encoding the desired heavy chain amino acid sequence can be amplified by appropriately selecting the primer sequences to be used.

In addition, if a sufficient amount of the amplified product cannot be obtained in this step (d), or if contamination is to be prevented, multiple similar steps are performed using the amplified product obtained in step (d) as a template. You may perform twice (for example, twice).
In that case, the primers to be used can be designed, for example, with reference to International Publication 2015/182749.

In addition, after amplification, an electrophoresis step may be included as appropriate in order to confirm whether or not the cDNA of interest has been amplified. As a method of electrophoresis, a conventional method can be used.

The method according to this aspect includes the following step (e).
(e) using a primer capable of amplifying the second cDNA, the synthesized second cDNA
Amplifying NA.

The reaction conditions for the amplification reaction are not particularly limited as long as the second cDNA is amplified, but conventional methods can be followed according to the primer sequences and the like to be used.

The primer sequences to be used can be designed, for example, with reference to International Publication 2015/182749.

Since the cDNA to be amplified differs depending on the primer sequences used, the cDNA encoding the desired light chain amino acid sequence can be amplified by appropriately selecting the primer sequences to be used.

In addition, if a sufficient amount of the amplification product cannot be obtained in this step (e), or if contamination is to be prevented, etc., multiple similar steps are performed using the amplification product obtained in step (e) as a template. You may perform twice (for example, twice).
In that case, the primers to be used can be designed, for example, with reference to International Publication 2015/182749.

In addition, after amplification, an electrophoresis step may be included as appropriate in order to confirm whether or not the cDNA of interest has been amplified. As a method of electrophoresis, a conventional method can be used.

The method according to this aspect includes the following step (f).
(f) producing an antibody using the amplified first cDNA and second cDNA;

The method for producing an antibody using the amplified first cDNA and second cDNA is not particularly limited as long as the antibody is produced. Common law can be followed, e.g.
As described in Examples below, a method of incorporating the amplified first cDNA and second cDNA into an expression plasmid vector and producing an antibody using expression cells (eg, Expi293 cells) can be mentioned.

The method according to this aspect includes the following step (g).
(g) measuring the neutralizing ability of the produced antibodies against SARS-CoV-2;

The method for measuring the neutralizing ability of the produced antibody against SARS-CoV-2 is not particularly limited as long as the neutralizing ability can be measured. A conventional method can be followed, for example, a method using SARS-CoV-2 and host cells (eg, Vero cells) as described in the Examples below. In addition, as an index of neutralizing ability, for example, half-inhibitory concentration ( _IC50 value) can be used.

The SARS-CoV-2 is not particularly limited as long as it is a virus that causes COVID-19,
A specific example is as described above.

The method according to this aspect includes the following step (h).
(h) selecting antibodies capable of neutralizing SARS-CoV-2;

The method for selecting the antibody having the ability to neutralize SARS-CoV-2 is not particularly limited as long as the antibody can be selected.
For example, the IC ₅₀ value can be used as an index for the ability to neutralize SARS-CoV-2. At this time, the neutralizing ability is, in order of preference, 10,000 ng/ml or less, 1, 10
≤0 ng/ml, ≤1,000 ng/ml, ≤850 ng/ml, ≤800 ng/ml, ≤750 ng/ml, 600 n
≤500 ng/ml, ≤400 ng/ml, ≤300 ng/ml, ≤100 ng/ml, ≤60 ng/ml, ≤55 ng/ml, ≤52 ng/ml, ≤50 ng/ml ≤40 ng/ml, ≤35 ng/ml, 34 ng/ml
≤30 ng/ml, ≤27 ng/ml, ≤26 ng/ml, ≤25 ng/ml, ≤20 ng/ml, 19
ng/ml or less, 15 ng/ml or less, 11 ng/ml or less, 10 ng/ml or less, 5.0 ng/ml or less, 4.0 ng/ml or less, 3.8 ng/ml or less, 3.0 ng/ml or less, 2.9 ng/ml ≤2.5 ng/ml, ≤2.4 ng/ml, 2
≤.3 ng/ml, ≤2.0 ng/ml, ≤1.7 ng/ml, ≤1.5 ng/ml, ≤1.4 ng/ml, 1.2 ng
/ml, 1.1 ng/ml or less, 1.0 ng/ml or less, 0.9 ng/ml or less, 0.8 ng/ml or less, 0.7 ng/ml or less, 0.5 ng/ml or less.

The SARS-CoV-2 is not particularly limited as long as it is a virus that causes COVID-19,
A specific example is as described above.

The method according to this aspect may include the following step (i) after step (f) and before step (g).
(i) measuring the ability of the produced antibody to bind to the receptor binding domain (RBD) protein of the S protein of SARS-CoV-2;

The RBD protein of the SARS-CoV-2 S protein may be commercially available or appropriately prepared, for example, prepared by the method described in Examples below. can be For example, if the SARS-CoV-2 is SARS-CoV-2 (conventional strain (Wuhan strain)), for example, RBD protein (amino acids : 322-536).

As a method for measuring the binding ability of the produced antibody to the receptor binding domain (RBD) protein of the S protein of SARS-CoV-2, a conventional method can be followed, for example, ELISA described in the Examples below. In addition to the method, a surface plasmon resonance method and the like can be used.

When the method according to this aspect includes the step (i) after the step (f) and before the step (g), the step (g) comprises: measuring the ability of the antibody to neutralize SARS-CoV-2, if capable.
As the binding ability, for example, a dissociation constant (K _D ) can be used as an index. in this case,
The dissociation constants are, in descending order of preference, 175 nM or less, 50 nM or less, 10 nM or less, 5 nM or less,
3 nM or less, 2.60 nM or less, 2.50 nM or less.
In addition, the association rate constant (k _a ) is set to 5.0×10 ⁴ Ms ^-1 or more, 8
0×10 ⁴ Ms ⁻¹ or more, 1.0×10 ⁵ Ms ⁻¹ or more, 3.0×10 ⁵ Ms ⁻¹ or more.
In addition, the dissociation rate constant (k _d ) is 1.0 × 10 ^-3 s ^-1 or less, 9
0×10 ⁻⁴ s ⁻¹ or less, 8.0×10 ⁻⁴ s ⁻¹ or less, 5.0×10 ⁻⁴ s ⁻¹ or less, 2.0×10 ⁻⁴ s ⁻¹ or less.

SARS-CoV-2 is not particularly limited as long as it is a virus that causes COVID-19,
A specific example is as described above.

Another aspect of the present invention is
(AB) Weights of antibodies contained in B cells that bind to the extracellular domain trimeric spike protein (S protein) of SARS-CoV-2, obtained from biological samples of humans who have recovered from COVID-19. synthesizing a first cDNA from the mRNA of a gene encoding the amino acid sequence of the strand;
(C) the mRNA of the gene encoding the amino acid sequence of the light chain of the antibody, contained in the B cell;
synthesizing a second cDNA from
(D) using a primer capable of amplifying the first cDNA, the synthesized first cDNA
Amplifying NA;
(E) using a primer capable of amplifying the second cDNA, the synthesized second cDNA
Amplifying NA;
(F) producing an antibody using the amplified first cDNA and second cDNA;
(G) measuring the neutralizing ability of the produced antibody against SARS-CoV-2; and
(H) A method for obtaining an antibody, comprising the step of selecting an antibody that has the ability to neutralize SARS-CoV-2.

For the details of this aspect, the description of the above aspect is used.

Examples are set forth below, but none of the examples are to be construed in a limiting sense.

<Example 1>
(1) Specimen information of people who have recovered from COVID-19 5 healthy people living in Hiroshima Prefecture (donor ID: HD01-HD05 in Table 1 below), also living in Hiroshima Prefecture, SARS-
Blood was collected from 18 individuals (donor IDs in Table 1 below: NCV01-NCV18) who recovered after infection with CoV-2.
The blood sampling period was from April 14, 2020 to January 25, 2021. According to the summary of the National Institute of Infectious Diseases, the European strain B.1.1.114 was the predominant strain in Japan during this period.
(first wave), European lineage B.1.1.284 (second wave), European lineage B.1.1.214 (third wave), and the B.1.1.7 mutant strain (alpha strain) studied in this example Or it is also called the British strain.The mutation is N501Y.) and
Before the B.1.351 mutant strain (also known as the beta strain or the South African strain. The mutation is K417N/E484K/N501Y) entered and spread in Japan, blood donors had no history of infection with any of the mutant strains. I didn't. Table 1 shows the severity of COVID-19 in blood donors, the presence or absence of oxygen inhalation treatment, and the number of days elapsed from the date of positive PCR test to the date of blood collection.

(2) Quantitation of serum IgG antibodies that specifically bind to the extracellular domain trimeric S protein of SARS-CoV-2 (conventional strain (Wuhan strain))
Using serum prepared from the blood of a person who has recovered from COVID-19 or an uninfected person, SA
I in serum that binds to the extracellular domain trimeric S protein of RS-CoV-2 (conventional strain (Wuhan strain))
gG antibody was quantified by ELISA.
The extracellular domain trimeric S protein of SARS-CoV-2 (conventional strain (Wuhan strain)) was prepared by the following method. SARS-CoV-2 (conventional strain (Wuhan strain)) surface glycoprotein (NCBI Reference S
sequence: YP_009724390.1) in the extracellular domain trimeric S protein (amino acids: 12-12
13), the expression plasmid containing the gene sequence encoding Effectene Transfection Reagent
(QIAGEN) into Drosophila Schneider S2 cells (80% confluent) and 10% F
After culturing in Schneider's Drosophila Medium (LONZA) supplemented with BS, the medium was replaced with Insect Xpress medium (LONZA) supplemented with puromycin. After that, S2 cells were cultured in a 2L Erlenmeyer flask at 28°C and induced with 0.5 mM CuSO ₄ for protein expression. Seven days after induction, the culture supernatant was harvested and added to Strep-Tactin affinity column binding buffer (100 mM Tris-HCl, 150
mM NaCl, 1 mM EDTA, 15 μg/ml avidin, pH 8.0). This protein-containing mixture was applied to a Strep-Tactin affinity column (IBA) and Sepharose 6 Increase 10/300 G.
Purified by L gel filtration chromatography (Cytiva).

ELISA was performed as follows. Extracellular domain trimeric S protein of the conventional strain (Wuhan strain) at a concentration of 2 μg/ml in 1×BBS was added (40 μl per well) to an ELISA plate (MaxiSorp, cat#.442404), Fix at 4°C overnight. then per well
After washing three times with 200 µl of T-PBS, 200 µl of Blocking Buffer (1% BSA/PBS) was added and incubated at room temperature for 1 hour.
Then, the cells were washed three times with 200 µl of T-PBS per well, 40 µl of serially diluted serum was added, and incubated overnight at 4°C or at room temperature for 2 hours.
Next, wash 3 times with 200 μl of T-PBS per well, and add HRP-labeled anti-human IgG antibody to Reagent.
Dilute 2,000-fold with Diluent, add 40 μl per well, and incubate at 4° C. overnight or at room temperature for 2 hours.
Next, wash three times with 200 µl of T-PBS per well and add 50 µl of SureBlue TMB substrate.
was added and the reaction was stopped by adding 50 μl of 1N HCl. After that, the emission intensity (wavelength 450 nm) was measured. SARS-CoV-2 (conventional strain (Wuhan strain)
), the concentration (μg/ml) of IgG antibody that specifically binds to the extracellular domain trimeric S protein was calculated.

The results are shown in FIG. In addition, "PCR positive for 12 days or more" in the figure indicates those who have been diagnosed with COVID-19 by PCR test (positive judgment) and who have recovered for 12 days or more after the judgment date.
“Less than 12 days” means that the infection with COVID-19 was determined by PCR test (positive determination) and 12 days from the date of determination
Indicate those who have recovered less than a day.
Arrows also indicate the results of four donors whose IDs are NCV01, NCV02, NCV04, and NCV08. Detailed results for each donor were 18.144 μg/ml for NCV01, 75.541 μg/ml for NCV02 and 95.032 μg/ml for NCV04.
g/ml, NCV08 was 23.374 μg/ml.

(3) Evaluation of serum neutralization ability against SARS-CoV-2
Evaluate the ability (neutralizing ability) to prevent B.1.1 mutants from infecting Vero cells using sera prepared from the blood of people who have recovered from COVID-19 or uninfected people as specimens bottom.
Specifically, I did the following: SARS-CoV-2/JP/Hiroshi
The ma-46059T/2020 strain (1/13 seed, freeze-thaw once, 1.5×10 ⁸ TCID ₅₀ /ml) was used. VeroE6/TMPRSS2 cells (JCRB1819) (purchased from JCRB) were used as cells. Reagents include cell culture medium Du
lbecco's Modified Eagle Medium (DMEM) (Fujifilm Wako Pure Chemical Industries), fetal bovine serum (FB
S; Biosera) was used.
Serum prepared from the blood of a person who recovered from COVID-19 was diluted 10-fold with DMEM containing 10% FBS, and then serially diluted 2-fold (2 ⁰ to 2 ¹¹ -fold dilution), 50 μl each, 12 cells A dilution of was prepared. To this, 50 μl of virus solution diluted to 100 TCID _50/50 μl with DMEM was added and mixed, and reacted at 37° C. for 60 minutes. 100 μl of the virus-specimen mixture was added directly to VeroE6/TMPRSS2 cells in a 24-well plate and incubated for 3 days until cytopathic effect (CPE) appeared. As an index of virus neutralization ability, the "maximum dilution rate" that can prevent virus infection was used.

When the same evaluation was performed using serum prepared from the blood of uninfected persons, the neutralizing ability was below the detection limit.
The results are shown in FIG. In addition, "PCR positive for 12 days or more" in the figure indicates those who have been diagnosed with COVID-19 by PCR test (positive judgment) and who have recovered for 12 days or more after the judgment date.
"Less than 12 days" means that the infection with COVID-19 was determined by PCR test (positive determination) and 12 days from the date of determination
Indicate those who have recovered less than a day.
In addition to four donors whose IDs are NCV01, NCV02, NCV04, and NCV08, the results for donor ID NCV07 are also indicated by arrows. Detailed results for each donor were 40 for NCV01, 640 for NCV02, 40 for NCV04, NC
V07 was 160 and NCV08 was less than 10.
In Figure 2, the dilution ratio of serum prepared from uninfected blood is 10 (no or very weak neutralizing ability), and the relative dilution ratio of sera with virus-neutralizing ability is expressed as a numerical value of 20 or more. . It means that the higher the number, the higher the virus neutralization ability even at a higher dilution ratio. still,
Effective relative dilutions for SARS-CoV-2 are 20-640.

(4) CD19-positive, IgD-negative, SARS-CoV-2 (conventional strain (Wuhan strain)) using FACS analysis
Peripheral blood B cells that bind to the extracellular domain trimeric S protein of 4 donors with donor IDs NCV01, NCV02, NCV04, and NCV08 were obtained using EDTA tubes. The leukocyte fraction was isolated by specific gravity centrifugation and stored frozen at -80°C. Specifically, 20 ml of peripheral blood was prepared and contained 1 mM to 2 mM EDTA. 5 ml of 1×PBS was placed in a 15 ml tube, 5 ml of blood was added, and diluted 2-fold. A long Pasteur pipette was inserted and 3 ml of Lymphocyte Separation Medium (Promo Cell) was added to the bottom of the tube with a Pasteur pipette. Centrifuged at 2,000 rpm x 30 minutes at room temperature (without brake). After centrifugation,
A human peripheral blood lymphocyte (PBL) layer was collected using a Pasteur pipette. in recovered PBL
An appropriate amount of PBS was added, the cells were centrifuged at 2,000 rpm for 5 minutes at room temperature (using a brake), the supernatant was discarded, and the cells were washed. This operation was repeated twice. The cells were preserved in a cell cryopreservation solution so that the cell count was 1×10 ⁷ cells/ml or less.

After cell lysis, the B cell fraction was enriched from leukocytes by negative selection of non-B cell lineages using magnetic beads. Specifically, the EasySep Human Pan-B Cell Enrichment Kit (
I followed the Veritas, cat#.19554) manual.
Next, APC/Cy7-labeled anti-CD19 antibody, FITC-labeled anti-IgD antibody, and the extracellular domain trimeric S protein of SARS-CoV-2 (conventional strain (Wuhan strain)) prepared by the method described below (Strep-T
actinXT DY-649 (fluorescently labeled with IBA-2-1568-050)) was used to label cells in the fractions. The viable lymphocyte fraction was gated on lymphocytes by FSC/SSC and dead cell staining with PI to exclude dead cells, and gated on CD19-positive B cells. Furthermore, among the class-switched B cells that become IgD negative, the extracellular domain trimer S of SARS-CoV-2 (conventional strain (Wuhan strain))
The protein-binding B cell fraction (fraction surrounded by a thick line indicated by an arrow in FIG. 3) was subjected to single-cell sorting on a 96-well PCR plate and then cryopreserved at -80°C. Note that FIG. 3 shows the cases where the donor ID is NCV01 and NCV02.
The SARS-CoV-2 (conventional strain (Wuhan strain)) extracellular domain trimeric S protein (Strep-Tac
tinXT DY-649 (fluorescently labeled with IBA-2-1568-050)) was prepared by the following method. SARS-Co
Surface glycoprotein of V-2 (conventional strain (Wuhan strain)) (NCBI Reference Sequence: YP_009724390
.1) The expression plasmid containing the gene sequence encoding the extracellular domain S protein (amino acids: 12-1213) was transfected with Effectene Transfection Reagent (QIAGEN) into Drosophil
a Schneider's D transduced into Schneider S2 cells (80% confluent) and supplemented with 10% FBS
Insect Xpress cultured in rosophila medium (LONZA) and added with puromycin
Replaced with medium (LONZA). After that, S2 cells were cultured in a 2L Erlenmeyer flask at 28°C, and 0.5 mM
The protein expression was induced with _CuSO4 . After 7 days of induction, the culture supernatant was harvested and added to the Strep-Tactin affinity column binding buffer (100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 15 μl
g/ml avidin, pH 8.0). The protein-containing mixture was purified using a Strep-Tactin affinity column (IBA) and Sepharose 6 Increase 10/300 GL gel filtration chromatography (Cytiva). Strep-TactinXT DY-649 (IBA-2-1568-05
0) for fluorescent labeling.

(5) RT-PCR and PCR
RT-PCR was performed according to a standard method using single-cell sorted cells.
Next, using 1 µl of each cDNA product as a template, the first PCR reaction was independently performed for the heavy chain and the light chain.
Next, the amplified product obtained in the first PCR reaction was diluted 10-fold with H ₂ O, and 1 μl of the diluted product was used as a template to perform the second PCR reaction independently for the heavy chain and the light chain.

The amplification product of the second PCR product was confirmed by agarose gel electrophoresis.
FIG. 4A shows PCR amplification products (base length of about 750 bp) of κ and λ light chains. FIG. 4B shows IgG
Heavy chain PCR amplification product (base length of 1,400 bp) is shown. In the following, a PCR amplification product of IgG heavy chain was used.
Numbers in the figure indicate cell IDs, and "N" indicates a negative control. In addition, numbers 08, 17, 22, 23, 30, 32, 37, 38, 42, 45, 52, 53, 60, 66, 67,
68, 69, 75, 82, 83, 90 and numbers 06, 07, 08, 10, 11, including "N" in Figure 4B;
17, 18, 20, 21, 22, 23, 24, 25, 30, 32, 35, 37, 38, 42, 45, 50, 52, 53, 59, 60,
61, 64, 66, 67, 68, 69, 70, 71, 72, 75, 78, 79, 82, 83, 86, 87, 90 are samples from which non-specific amplification products were obtained, or amplification products indicates samples for which no Others indicate samples from which amplification products of the desired size were obtained.

(6) Cloning of Antibody Gene into Expression Vector The PCR product obtained in (5) above was incorporated into a plasmid vector using the DNA assembling method, and the incorporated sequence was confirmed by Sanger sequencing.

(7) Antibody production and confirmation by non-denaturing PAGE After that, Expi293 cells were used to prepare antibody proteins. Using the cell culture supernatant, CBB staining was performed after non-denaturing PAGE, and it was confirmed that the heavy chain and light chain paired and maintained a normal structure.

(8) Evaluation of Specificity of Prepared Monoclonal IgG Antibodies (Human Antibodies) Through the above steps, 32 types of monoclonal IgG antibodies (human antibodies) were prepared.
In addition, the receptor binding domain (RBD) protein of the S protein of SARS-CoV-2 (conventional strain (Wuhan strain)) was prepared by the following method. SARS-CoV-2 (conventional strains (
An expression plasmid containing the gene sequence encoding the RBD protein (amino acids: 322-536) of the S protein in the surface glycoprotein (NCBI Reference Sequence: YP_009724390.1) of the Wuhan strain) was transfected into HEK293 cells using polyethyleneimine. (90% confluent) and 1
The cells were cultured in DMEM (Wako) supplemented with 0% FBS and non-essential amino acids (Gibco). The serum concentration was lowered to 2% immediately after gene transfer. Four days after transfection, these culture supernatants were harvested and His
-tag purified resin affinity column (Roche) and Superdex 200 Increase 10/300 GL gel filtration chromatography (Cytiva).
The RBD protein of the S protein of the B.1.1.7 mutant strain (alpha strain or British strain) and the RBD protein of the S protein of the B.1.351 mutant strain (beta strain or South Africa strain) were also prepared in the same manner according to a conventional method.
The prepared antibody, the extracellular domain trimeric S protein of SARS-CoV-2 (conventional strain (Wuhan strain)), the RBD protein of the S protein of the conventional strain (Wuhan strain), and the B.1.1.7 mutant strain (alpha RBD protein of S protein of B.1.351 mutant strain (beta strain or South African strain)
ELISA analysis was performed using the RBD protein of the protein, and the specificity (binding ability) was evaluated. Specifically, it was carried out as follows.

The following reagents were prepared.
・4×BBS: NaCl ₃₂ g, _H3BO3 42.5 g, NaOH 4.5 g dissolved in 1,000 ml of MQ water ・1×BBS: 4×BBS (125 ml) in MQ water (375 ml) Dilutions ・20% Tween: 50 ml Tween20 (Nacalai Tesque, #.28353-85) mixed with ₂₀₀ ml MQ water ・10x PBS: NaCl 160 g, KCl ₄ g, _Na2HPO4-12H2 58 g of O and 4 g of KH ₂ PO ₄ dissolved in 2,000 ml of MQ water T-PBS: 2.5 ml of 20% Tween, 100 ml of 10×PBS, and 897.5 ml of MQ water 1N HCl: 44 ml of HCl (Nacalai Tesque, #.18321-05, 11.3N) mixed with 456 ml of MQ water ・SureBlue TMB Microwell Peroxidase Substrate Kit (KPL, #.52-00-02) 4× 100ml
・Maxisorp ELISA plate (Nunc, #.442404, High-binding, 60/case)
・Blocking buffer: 1% BSA/PBS
・Reagent Diluent: Tris-buffered Saline containing 0.1% BSA and 0.05% Tween (20mM Tri
sbase, 150 nM NaCl, pH 7.2-7.4)

Extracellular domain trimer of SARS-CoV-2 (conventional strain (Wuhan strain)) at a concentration of 2 μg/ml in 1×BBS
S protein, RBD protein of S protein of SARS-CoV-2 (conventional strain (Wuhan strain)), B.1.1.7
RBD protein of the S protein of the mutant strain (alpha strain or UK strain), or the B.1.351 mutant strain (
Beta strain or South African strain) RBD protein of S protein was plated for ELISA (MaxiSorp
, cat#.442404) (40 μl per well) and fixed overnight at 4°C.
Then, wash 3 times with 200 μl of T-PBS per well and add 200 μl of Blocking Buffer (1% B
SA/PBS) was added and incubated for 1 hour at room temperature.
Thereafter, the wells were washed three times with 200 µl of T-PBS per well, 40 µl of the Expi293 culture supernatant obtained in (7) above was added, and incubated overnight at 4°C or at room temperature for 2 hours.
Next, wash 3 times with 200 μl of T-PBS per well, and add HRP-labeled anti-human IgG antibody to Reagent.
Dilute 2,000-fold with Diluent, add 40 μl per well and incubate at 4° C. overnight or at room temperature for 2 hours.
Next, wash three times with 200 µl of T-PBS per well and add 50 µl of SureBlue TMB substrate.
was added and the reaction was stopped by adding 50 μl of 1N HCl. After that, the emission intensity (wavelength 450 nm) was measured.

The results are shown in FIG. The values on the vertical axis are the RBD protein of the S protein of the B.1.1.7 mutant strain (alpha strain or British strain) when the binding ability of the S protein of the conventional strain (Wuhan strain) to the RBD protein is 100%. , represents the “relative binding capacity” of the S protein of the B.1.351 mutant strain (beta strain or South African strain) to the RBD protein.
Of the 32 antibodies prepared, 31 (96.8%) were found to bind to the RBD protein of the S protein of the B.1.1.7 mutant (alpha strain or British strain).
In addition, 21 species (65.6%) were found to bind to the RBD protein of the S protein of the B.1.351 mutant strain (beta strain or South African strain).
In addition, although not shown, it was confirmed that all of the 32 prepared antibodies bind to the extracellular domain trimeric S protein of SARS-CoV-2 (conventional strain (Wuhan strain)).

The antibody indicated by number 1 in the figure corresponds to "NCV1SG17" described later, the antibody indicated by number 2 corresponds to "NCV2SG53" to be described later, the antibody indicated by number 3 corresponds to "NCV1SG23" to be described later, and number 4. The antibody indicated by corresponds to "NCV2SG48" described later. Among them, the antibody "NCV2SG53" indicated by number 2 is a conventional strain (Wuhan strain), B.1.1.7 mutant strain (alpha strain or British strain), B.1.351 mutant strain (beta strain or South African strain). It had strong activity to bind to protein RBD.

(9) Summary In the same manner as in (3) above, the antibodies shown with numbers 1 to 4 were evaluated for their ability to prevent the B.1.1 mutant from infecting Vero cells (neutralizing ability), and IC ₅₀ values (N=3) were calculated. Also B.1.617
The ability to prevent .2 mutants from infecting Vero cells (neutralizing ability) was evaluated and an _IC50 value (N=1) was calculated. The results are shown in Table 2-1 below.
In addition, in the same manner as in (3) above, for the antibodies indicated by numbers 1 to 4, the conventional strain (Wuhan strain) is V
The ability to prevent ero cell infection (neutralizing ability) was evaluated, and the _IC50 value (N=1) was calculated. The results are shown in Table 2-2 below. The results of evaluation of specificity (binding ability) by ELISA obtained in (8) above are also shown in Table 2-2 below.

The results shown in Table 3 below have been obtained with conventional antibodies that have neutralizing activity against SARS-CoV-2.

(10) Evaluation of specificity of monoclonal IgG antibody (human antibody) produced (continued)
Using the antibodies indicated by numbers 1 to 4 and the RBD protein of the S protein of the conventional strain (Wuhan strain), specificity is determined by the Blitz system (Sartorius Japan Co., Ltd.) using a biosensor coated with protein A. (binding ability) was evaluated. Specifically, it was carried out as follows.
Each antibody was adjusted to a concentration of 10 μg/ml with a buffer solution (PBS containing 0.02% Tween and 0.1% BSA), which was captured by the biosensor and equilibrated, and then adjusted to each concentration with the same buffer solution. (Wuhan strain) S protein RBD protein was conjugated sequentially. The biosensor was then immersed in the same buffer for 900 seconds for dissociation. The results were analyzed with Blitz system software (Sartorius Japan Co., Ltd.).

The results are shown in Table 4 below and FIG.

<Example 2>
The amino acid sequences of the four monoclonal IgG antibodies (human antibodies) were determined according to a standard method. The results are shown below.

Antibody NCV1SG17 is a human Ig comprising the heavy and light chain variable region amino acid sequences shown below.
G antibody.
(I):
the heavy chain CDR1 amino acid sequence is the amino acid sequence of SEQ ID NO: 1;
the heavy chain CDR2 amino acid sequence is the amino acid sequence of SEQ ID NO: 2;
the heavy chain CDR3 amino acid sequence is the amino acid sequence of SEQ ID NO: 3;
the amino acid sequence of the CDR1 of the light chain is the amino acid sequence of SEQ ID NO: 4;
the amino acid sequence of the CDR2 of the light chain is GKN;
the amino acid sequence of the CDR3 of the light chain is the amino acid sequence of SEQ ID NO:5;
the amino acid sequence of FR1 of the heavy chain is the amino acid sequence of SEQ ID NO: 21;
the amino acid sequence of FR2 of the heavy chain is the amino acid sequence of SEQ ID NO: 22;
the amino acid sequence of FR3 of the heavy chain is the amino acid sequence of SEQ ID NO: 23;
the heavy chain FR4 amino acid sequence is the amino acid sequence of SEQ ID NO: 24;
the amino acid sequence of FR1 of the light chain is the amino acid sequence of SEQ ID NO: 25;
the amino acid sequence of FR2 of the light chain is the amino acid sequence of SEQ ID NO: 26;
The amino acid sequence of FR3 of the light chain is the amino acid sequence of SEQ ID NO:27, and the amino acid sequence of FR4 of the light chain is the amino acid sequence of SEQ ID NO:28.

Antibody NCV2SG53 is a human Ig comprising the heavy and light chain variable region amino acid sequences shown below.
G antibody.
(II):
the heavy chain CDR1 amino acid sequence is the amino acid sequence of SEQ ID NO: 6;
the heavy chain CDR2 amino acid sequence is the amino acid sequence of SEQ ID NO: 7;
the heavy chain CDR3 amino acid sequence is the amino acid sequence of SEQ ID NO:8;
the amino acid sequence of the CDR1 of the light chain is the amino acid sequence of SEQ ID NO:9;
the amino acid sequence of the CDR2 of the light chain is AAS;
the amino acid sequence of the CDR3 of the light chain is the amino acid sequence of SEQ ID NO: 10;
the amino acid sequence of FR1 of the heavy chain is the amino acid sequence of SEQ ID NO: 29;
the amino acid sequence of FR2 of the heavy chain is the amino acid sequence of SEQ ID NO: 30;
the amino acid sequence of FR3 of the heavy chain is the amino acid sequence of SEQ ID NO: 31;
the heavy chain FR4 amino acid sequence is the amino acid sequence of SEQ ID NO: 32;
the amino acid sequence of FR1 of the light chain is the amino acid sequence of SEQ ID NO: 33;
the amino acid sequence of FR2 of the light chain is the amino acid sequence of SEQ ID NO: 34;
The amino acid sequence of FR3 of the light chain is the amino acid sequence of SEQ ID NO:35, and the amino acid sequence of FR4 of the light chain is the amino acid sequence of SEQ ID NO:36.

Antibody NCV1SG23 is a human Ig comprising the heavy and light chain variable region amino acid sequences shown below.
G antibody.
(III):
the heavy chain CDR1 amino acid sequence is the amino acid sequence of SEQ ID NO: 11;
the heavy chain CDR2 amino acid sequence is the amino acid sequence of SEQ ID NO: 12;
the heavy chain CDR3 amino acid sequence is the amino acid sequence of SEQ ID NO: 13;
the light chain CDR1 amino acid sequence is the amino acid sequence of SEQ ID NO: 14;
the amino acid sequence of the CDR2 of the light chain is GNN;
the amino acid sequence of the CDR3 of the light chain is the amino acid sequence of SEQ ID NO: 15;
the amino acid sequence of FR1 of the heavy chain is the amino acid sequence of SEQ ID NO: 37;
the amino acid sequence of FR2 of the heavy chain is the amino acid sequence of SEQ ID NO: 38;
the amino acid sequence of FR3 of the heavy chain is the amino acid sequence of SEQ ID NO: 39;
the heavy chain FR4 amino acid sequence is the amino acid sequence of SEQ ID NO: 40;
the amino acid sequence of FR1 of the light chain is the amino acid sequence of SEQ ID NO: 41;
the amino acid sequence of FR2 of the light chain is the amino acid sequence of SEQ ID NO: 42;
The amino acid sequence of FR3 of the light chain is the amino acid sequence of SEQ ID NO:43, and the amino acid sequence of FR4 of the light chain is the amino acid sequence of SEQ ID NO:44.

Antibody NCV2SG48 is a human Ig comprising the heavy and light chain variable region amino acid sequences shown below.
G antibody.
(IV):
the heavy chain CDR1 amino acid sequence is the amino acid sequence of SEQ ID NO: 16;
the heavy chain CDR2 amino acid sequence is the amino acid sequence of SEQ ID NO: 17;
the heavy chain CDR3 amino acid sequence is the amino acid sequence of SEQ ID NO: 18;
the amino acid sequence of the CDR1 of the light chain is the amino acid sequence of SEQ ID NO: 19;
the amino acid sequence of the CDR2 of the light chain is AAS;
the amino acid sequence of the CDR3 of the light chain is the amino acid sequence of SEQ ID NO: 20;
the amino acid sequence of FR1 of the heavy chain is the amino acid sequence of SEQ ID NO: 45;
the amino acid sequence of FR2 of the heavy chain is the amino acid sequence of SEQ ID NO: 46;
the amino acid sequence of FR3 of the heavy chain is the amino acid sequence of SEQ ID NO: 47;
the heavy chain FR4 amino acid sequence is the amino acid sequence of SEQ ID NO: 48;
the amino acid sequence of FR1 of the light chain is the amino acid sequence of SEQ ID NO: 49;
the amino acid sequence of FR2 of the light chain is the amino acid sequence of SEQ ID NO: 50;
The amino acid sequence of FR3 of the light chain is the amino acid sequence of SEQ ID NO:51, and the amino acid sequence of FR4 of the light chain is the amino acid sequence of SEQ ID NO:52.

<Example 3>
The nucleotide sequences of the DNAs encoding the amino acid sequences of the heavy chain variable regions and the nucleotide sequences of the DNAs encoding the light chain variable regions of the four monoclonal IgG antibodies (human antibodies) were determined according to conventional methods.

The nucleotide sequence of the DNA encoding the amino acid sequence of the variable region of the heavy chain of the antibody NCV1SG17 is the nucleotide sequence of SEQ ID NO: 53, and the nucleotide sequence of the DNA encoding the amino acid sequence of the light chain variable region is the nucleotide sequence of SEQ ID NO: 54. A base sequence.
The nucleotide sequence of the DNA encoding the amino acid sequence of the heavy chain variable region of the antibody NCV2SG53 is the nucleotide sequence of SEQ ID NO: 55, and the nucleotide sequence of the DNA encoding the amino acid sequence of the light chain variable region is the nucleotide sequence of SEQ ID NO: 56. A base sequence.
The nucleotide sequence of the DNA encoding the amino acid sequence of the variable region of the heavy chain of antibody NCV1SG23 is the nucleotide sequence of SEQ ID NO: 57, and the nucleotide sequence of the DNA encoding the amino acid sequence of the light chain variable region is the nucleotide sequence of SEQ ID NO: 58. A base sequence.
The nucleotide sequence of the DNA encoding the amino acid sequence of the variable region of the heavy chain of antibody NCV2SG48 is the nucleotide sequence of SEQ ID NO: 59, and the nucleotide sequence of the DNA encoding the amino acid sequence of the light chain variable region is the nucleotide sequence of SEQ ID NO: 60. A base sequence.

<Example 4>
The effect of the four monoclonal IgG antibodies (human antibodies) to suppress the emergence of escape mutants from the B.1.1 mutant was evaluated. Specifically, it was carried out as follows.
The virus used was the SARS-CoV-2/JP/Hiroshima-46059T/2020 strain as the B.1.1 mutant, and the final concentrations of the antibodies NCV1SG17, NCV2SG53, NCV1SG23, and NCV2SG48 were 75 ng/ml and 178 ng/ml, respectively.
, 1100 ng/ml, and 825 ng/ml, and reacted with the virus at 37° C. for 60 minutes. twenty four
100 μl of the virus-specimen mixture was added directly to VeroE6/TMPRSS2 cells (JCRB1819) (purchased from JCRB) in a well plate and cultured for 3 days until cytopathic effect (CPE) appeared. It is suggested that escape mutant virus appeared in wells that produced CPE. Repeat this experiment 4 to 7 times
Repeatedly, the culture supernatant of the wells in which CPE occurred was collected, and virus sequence analysis was performed according to a conventional method.
Viral RNA was extracted from virus-infected media using a Maxwell RSC Instrument (Promega, AS4500). Preparation and amplification of cDNA is available on the ARTIC network (https://artic.ne
Integrated DNA Technologies, following the protocol published by twork/ncov-2019)
The V4 version of the ARTIC primer set was used to generate tiled amplicons across the viral genome. Sequencing libraries were NEB Next Ultra I for Illumina
I DNA Library Prep Kit (New England Biolabs, E7645) and paired-end 300bp sequencing was performed on MiSeq (Illumina) and MiSeq Reagent Kit v3 (Illumina, MS-102-
3003). Consensus sequences were generated using the DRAGEN COVID strain software (Illumi
na, ver. 3.5.6) using Nextcl
I did it using the ade website (https://clades.nextstrain.org).

The results are shown in Tables 5-1 and 5-2 below. It was confirmed that escape mutants appeared when the antibodies NCV1SG17 or NCV2SG53 were used.
Escape mutants (EM-17-1, EM-17-2, EM17-3) that appeared when using antibody NCV1SG17
both have the 'S494P' mutation, and the amino acid 'S
494" was expected to be important for recognition and neutralization by antibody NCV1SG17.
Escape mutant strains (EM-53-1, EM-53-2, EM-53-4
) has one of the “E484D”, “G485D”, and “G485R” mutations, and the S protein
It was predicted that the structure around amino acids 'E484' and 'G485' on the RBD protein is important for recognition and neutralization by antibody NCV2SG53.

<Example 5>
Two antibodies, antibody NCV2SG53 and antibody NCV2SG48, which were successfully crystallized for X-ray structure analysis, were subjected to epitope analysis. Specifically, it was carried out as follows.
Antibody NCV2SG48 (Fab) with a 6xHis tag added to the C-terminus was expressed in Expi293F cells and Ni-NTA
Purified using agarose resin (QIAGEN). Antibody NCV2SG53 (Fab) is antibody NCV2SG5
3 was papain-digested and purified using an HP Protein G column (Cytiva).
Each Fab fragment was mixed with an RBD protein with the same sequence as SARS-CoV-2 (conventional (Wuhan strain)) and B.1.1 mutant strains at a molar ratio of 1:1.2 and incubated on ice for 1 hour. excessive
Supe equilibrated with 20 mM Tris-HCl (pH 7.5)/150 mM NaCl to remove RBD protein
Loaded onto an rdex 200 Increase 10/300 GL column (Cytiva).
Fractions containing RBD protein and each Fab were collected and concentrated for crystallization. Crystallization is 20
°C by the sitting drop vapor diffusion method. RBD protein-Fab (antibody NCV2SG48
) was prepared by mixing 7.5 mg/ml RBD protein solution, 0.1 M Bis-Tris (pH 5.5), 0.5 M ammonium sulfate, 19% PEG3350 at 1:1 (v/v) in a 2 μl droplet. grown inside. Crystals of RBD protein-Fab (antibody NCV2SG53) were prepared in 7.5 mg/ml RBD protein solution and 0.1 M MES (pH 6.0).
, 0.25 M ammonium sulfate, 22.5% PEG3350 mixed 1:1 (v/v) in 2 μl droplets.
X-ray diffraction data collection was performed at SPring-8 beamline BL44XU using synchrotron radiation in a stream of nitrogen vapor at 90 K. Datasets were indexed and consolidated using the XDS package, scaled, and merged using the Aimless program. Phase determination was performed using the program Phaser from the PHENIIX package and the RBD structure (PDB ID: 7e
am) and Fab structures (PDB IDs: 7chb and 7chp) were carried out by molecular replacement using the program Molrep. Structural refinement consists of the program phenix.refine and the program coo
I ran it using t. Interactions between RBD proteins and Fabs were analyzed using the program PISA. Structural drawings were generated by the program pymol (PyMOL Molecular Graphics System, version 1.2r3pre, Schrodinger, LLC).

The results are shown in Table 6 below. The results for antibody NCV2SG48 are also shown in Figures 7A and 7B. Results for antibody NCV2SG53 are also shown in Figures 8A and 8B.

As shown in FIG. 7A, the antibody NCV2SG48 is characterized by a particularly large binding area with the RBD protein, and binds in a manner that almost completely covers the receptor binding motif (RBM) region responsible for binding to ACE2. It became clear that there is Previously known neutralizing antibodies (
It was found that the area bound to the RBD protein was significantly larger than that of casilibimab (REGN10933), imdevimab (REGN10987), and sotrovimab (S309)). As a result, even if many mutations occur in the RBD protein such as the B.1.1.529 mutant strain (Omicron strain), it is possible to maintain the binding to the RBD protein and acquire resistance to various mutations. It is assumed that
This increase in binding area includes somatic mutations, as shown in Figure 7-B.
plays a big role. That is, in the method for obtaining an antibody according to one embodiment of the present invention, in view of obtaining B cells that bind to S protein from "human biological samples recovered from COVID-19", COVID-19 It is presumed that mutations in the antibody accumulated during the period until recovery from the disease in humans who had recovered from the disease, resulting in such an increase in the binding area.

From the results of confirming the amino acids forming hydrogen bonds between each of the antibodies and the RBD protein, the following was found about the epitopes of the two antibodies, antibody NCV2SG48 and antibody NCV2SG53.
First, for the antibody NCV2SG48, SARS-CoV-2 (conventional strain (Wuhan strain)) S protein (NCB
I Reference Sequence: YP_009724390.1), 403
amino acid sequence (SEQ ID NO: 6
9) recognize epitopes contained in The amino acid sequence is the RBD protein (amino
acids: 322-536).
The epitope is preferably "403rd amino acid (R), 406th amino acid (E), 409th amino acid (Q), 415th amino acid (T), 417th amino acid (K), 420th amino acid amino acid (D), 421st amino acid (Y), 453rd amino acid (Y), 455th amino acid (L), 457th amino acid (R), 458
th amino acid (K), 460th amino acid (N), 473rd amino acid (Y), 4
74th amino acid (Q), 475th amino acid (A), 487th amino acid (N)
, 489th amino acid (Y), 493rd amino acid (Q), 494th amino acid (
S), 496th amino acid (G), 498th amino acid (Q), 501st amino acid (N), and 502nd amino acid (G)".

Next, the antibody NCV2SG53 also recognizes an epitope contained in the amino acid sequence (SEQ ID NO: 70) consisting of the 445th amino acid (V) to the 501st amino acid (N) in the same manner as described above. The amino acid sequence is an amino acid sequence contained in RBD protein (amino acids: 322-536).
The epitope is preferably "449th amino acid (Y), 478th amino acid (T), 479th amino acid (P), 484th amino acid (E), 486th amino acid (F), 487th amino acid (N), 489th amino acid (Y), 490th amino acid (F), 492nd amino acid (L), 493rd amino acid (Q), 494
498th amino acid (S) and 498th amino acid (Q)".

<Example 6>
SARS-CoV-2 (conventional strain (Wuhan strain)) surface glycoprotein (NCBI Reference Sequence: YP_
009724390.1) were generated by the following method.
The following culture media were used for Lenti-X 293T cells (Clontech) and 293T/pTRC2puro_ACE2-P2A-TMPRSS2 cells. Dulbecco's Modified Eagle Medium (D
MEM) (Fuji Film Wako Pure Chemical), 10% fetal bovine serum (FBS; GIBCO), 25 mM HEPES buffer (Nacalai Tesque), and 1/100 volume penicillin-streptomycin mixture (Nacalai Tesque). .
Self-propagation defective HIV lentiviral vector (pWPI_ffLuc-P2A-EGFP) expressing luciferase gene and GFP gene simultaneously, and packaging plasmid (psPAX2) 1
μg and SARS-CoV-2 (conventional strain (Wuhan strain)) surface glycoprotein (NCBI Reference Sequence
: YP_009724390.1) and 1 μg of the plasmid expressing the extracellular domain S protein (amino acids: 12-1213) in 300 μl of Opti-MEM, followed by 12 μg of PEI-MAX (polyscience, 24765 -100) was added to form a mixed solution, which was allowed to stand at room temperature for 10 minutes.
The mixed solution was added dropwise to Lenti-X 293T cells (Clontech), which had been seeded at 1×10 ⁶ cells each in 3 mL of culture solution in a 6 cm dish the day before, and cultured for 6 to 16 hours. After culturing, remove the culture medium,
3 mL of the new culture medium was added and the cells were further cultured for 48 to 72 hours. After culturing, collect the culture medium and centrifuge (5
000 rpm for 2 minutes), the supernatant was stored at -80°C as a virus solution. A virus solution that had been freeze-thawed only once was used as the virus fluid for the evaluation of the neutralizing ability described below.
Neutralization ability evaluation was performed by the following method. Dispense 50 μl of virus solution diluted to 1-4×10 ⁶ RLU/ml with culture medium containing 10 μg/ml polybrene (Hexadimethrine bromide, sigma H9268) into 96-well round-bottom plates. and each of the antibodies numbered ¹ to ⁴ serially diluted from 2000 ng/mL (40 to 47 times diluted) from 2000 ng/mL to prepare 50 μl x 8 virus-antibody mixtures, The mixture was reacted at 37°C for 60 minutes. In addition, "both the antibody NCV2SG53 and the antibody NCV2SG48"
When using , 4 ^- fold serial dilution (40 to 47-fold dilution) was added to the virus solution so that the concentration of each antibody was 2000 ng/mL, and similarly, 50 μl ^x 8 pieces After preparing the virus-antibody mixture, it was allowed to react at 37°C for 60 minutes. In addition, the same was applied to the cases of using "cacilibimab (REGN10933)", "imdevimab (REGN10987)", and "both casilibimab (REGN10933) and imdevimab (REGN10987)".
Then, 10,000 cells of 293T/pTRC_ACE2+T were seeded in each well of a 96-well plate the day before.
50 μl of the virus-antibody mixture was added directly to MPRSS2 cells (50 μl) and cultured for 3 days. A sample of all experiments was performed with n=3. After culturing, the culture medium was removed and 5x passive lysi
s buffer (Promega, E6110) and luciferase substrate solution (One-Glo,
Promega, E1941) was added to each well and mixed well, then luciferase activity was measured with a luminometer (ARVO-X3, Perkin Elmer).
Assuming that the measured value in the maximum dilution well was 0% neutralizing activity, the neutralizing activity was calculated from the measured values in the other dilution wells, and the average value and deviation of n=3 were graphed. The antibody concentration exhibiting 50% neutralizing activity was calculated as the _IC50 value.

According to common practice, in the same manner as above,
B.1.1.7 Pseudoviruses with an extracellular domain S protein in the surface glycoproteins of mutant strains (alpha or UK strains),
B.1.351 mutant (Beta strain or South African strain) pseudovirus with extracellular domain S protein in the surface glycoprotein,
B.1.617.2 Pseudovirus with extracellular domain S protein in the surface glycoprotein of the 617.2 mutant strain (Delta strain or Indian strain),
B.1.1.529 mutant (Omicron strain) pseudovirus with extracellular domain S protein in the surface glycoprotein,
B.1 mutant strain (as mentioned above, it is a pseudovirus in the first place),
B.1. Pseudoviruses with extracellular domain S protein in surface glycoproteins of 617.1 mutants (Kappa strain or Indian strain),
Pseudovirus with extracellular domain S protein in the surface glycoprotein of the C.37 mutant (lambda strain),
a pseudovirus with an extracellular domain S protein in the surface glycoprotein of the delta plus mutant,
was prepared and the same test was performed.

The results are shown in Tables 7-1 and 7-2 below.

The present invention can be used to develop formulations for neutralizing SARS-CoV-2.

Claims (5)

comprising the amino acid sequences of the heavy and light chains of any one of (IV), (II), (III), and (I) below,
isolated,
An antibody or antibody fragment comprising an antigen-binding region thereof that binds to SARS-CoV-2 or an antibody or antigen-binding thereof that competes with said antibody or antibody fragment comprising an antigen-binding region thereof for binding to said SARS-CoV-2 An antibody fragment containing the region.
(IV):
the heavy chain CDR1 amino acid sequence is the amino acid sequence of SEQ ID NO: 16;
the heavy chain CDR2 amino acid sequence is the amino acid sequence of SEQ ID NO: 17;
the heavy chain CDR3 amino acid sequence is the amino acid sequence of SEQ ID NO: 18;
the amino acid sequence of the CDR1 of the light chain is the amino acid sequence of SEQ ID NO: 19;
The amino acid sequence of CDR2 of the light chain is AAS, and the amino acid sequence of CDR3 of the light chain is the amino acid sequence of SEQ ID NO:20.
(II):
the heavy chain CDR1 amino acid sequence is the amino acid sequence of SEQ ID NO: 6;
the heavy chain CDR2 amino acid sequence is the amino acid sequence of SEQ ID NO: 7;
the heavy chain CDR3 amino acid sequence is the amino acid sequence of SEQ ID NO:8;
the amino acid sequence of the CDR1 of the light chain is the amino acid sequence of SEQ ID NO:9;
The amino acid sequence of CDR2 of the light chain is AAS, and the amino acid sequence of CDR3 of the light chain is the amino acid sequence of SEQ ID NO:10.
(III):
the heavy chain CDR1 amino acid sequence is the amino acid sequence of SEQ ID NO: 11;
the heavy chain CDR2 amino acid sequence is the amino acid sequence of SEQ ID NO: 12;
the heavy chain CDR3 amino acid sequence is the amino acid sequence of SEQ ID NO: 13;
the light chain CDR1 amino acid sequence is the amino acid sequence of SEQ ID NO: 14;
The amino acid sequence of CDR2 of the light chain is GNN, and the amino acid sequence of CDR3 of the light chain is the amino acid sequence of SEQ ID NO:15.
(I):
the heavy chain CDR1 amino acid sequence is the amino acid sequence of SEQ ID NO: 1;
the heavy chain CDR2 amino acid sequence is the amino acid sequence of SEQ ID NO: 2;
the heavy chain CDR3 amino acid sequence is the amino acid sequence of SEQ ID NO: 3;
the amino acid sequence of the CDR1 of the light chain is the amino acid sequence of SEQ ID NO: 4;
The amino acid sequence of CDR2 of the light chain is GKN, and the amino acid sequence of CDR3 of the light chain is the amino acid sequence of SEQ ID NO:5. 2. The isolated antibody that binds SARS-CoV-2, or an antigen-binding region thereof, of claim 1, wherein said antibody that binds SARS-CoV-2 comprises the following heavy and light chain amino acid sequences: or an antibody fragment comprising an antibody or antigen binding region thereof that competes with said antibody or antibody fragment comprising an antigen binding region thereof for binding to said SARS-CoV-2.
In the case of (IV) above:
the amino acid sequence of FR1 of the heavy chain is the amino acid sequence of SEQ ID NO: 45;
the amino acid sequence of FR2 of the heavy chain is the amino acid sequence of SEQ ID NO: 46;
the amino acid sequence of FR3 of the heavy chain is the amino acid sequence of SEQ ID NO: 47;
the heavy chain FR4 amino acid sequence is the amino acid sequence of SEQ ID NO: 48;
the amino acid sequence of FR1 of the light chain is the amino acid sequence of SEQ ID NO: 49;
the amino acid sequence of FR2 of the light chain is the amino acid sequence of SEQ ID NO: 50;
The amino acid sequence of FR3 of the light chain is the amino acid sequence of SEQ ID NO:51, and the amino acid sequence of FR4 of the light chain is the amino acid sequence of SEQ ID NO:52.
In the case of (II) above:
the amino acid sequence of FR1 of the heavy chain is the amino acid sequence of SEQ ID NO: 29;
the amino acid sequence of FR2 of the heavy chain is the amino acid sequence of SEQ ID NO: 30;
the amino acid sequence of FR3 of the heavy chain is the amino acid sequence of SEQ ID NO: 31;
the heavy chain FR4 amino acid sequence is the amino acid sequence of SEQ ID NO: 32;
the amino acid sequence of FR1 of the light chain is the amino acid sequence of SEQ ID NO: 33;
the amino acid sequence of FR2 of the light chain is the amino acid sequence of SEQ ID NO: 34;
the amino acid sequence of FR3 of the light chain is the amino acid sequence of SEQ ID NO:35; and the amino acid sequence of FR4 of the light chain is the amino acid sequence of SEQ ID NO:36;
In the case of (III) above:
the amino acid sequence of FR1 of the heavy chain is the amino acid sequence of SEQ ID NO: 37;
the amino acid sequence of FR2 of the heavy chain is the amino acid sequence of SEQ ID NO: 38;
the amino acid sequence of FR3 of the heavy chain is the amino acid sequence of SEQ ID NO: 39;
the heavy chain FR4 amino acid sequence is the amino acid sequence of SEQ ID NO: 40;
the amino acid sequence of FR1 of the light chain is the amino acid sequence of SEQ ID NO: 41;
the amino acid sequence of FR2 of the light chain is the amino acid sequence of SEQ ID NO: 42;
the amino acid sequence of FR3 of the light chain is the amino acid sequence of SEQ ID NO:43; and the amino acid sequence of FR4 of the light chain is the amino acid sequence of SEQ ID NO:44;
In the case of (I) above:
the amino acid sequence of FR1 of the heavy chain is the amino acid sequence of SEQ ID NO: 21;
the amino acid sequence of FR2 of the heavy chain is the amino acid sequence of SEQ ID NO: 22;
the amino acid sequence of FR3 of the heavy chain is the amino acid sequence of SEQ ID NO: 23;
the heavy chain FR4 amino acid sequence is the amino acid sequence of SEQ ID NO: 24;
the amino acid sequence of FR1 of the light chain is the amino acid sequence of SEQ ID NO: 25;
the amino acid sequence of FR2 of the light chain is the amino acid sequence of SEQ ID NO: 26;
the amino acid sequence of FR3 of the light chain is the amino acid sequence of SEQ ID NO:27; and the amino acid sequence of FR4 of the light chain is the amino acid sequence of SEQ ID NO:28; 3. The isolated antibody of claim 1 or 2 that binds to SARS-CoV-2 or an antibody fragment thereof comprising an antigen binding region thereof, or said antibody or antigen binding thereof in binding to said SARS-CoV-2 A pharmaceutical composition comprising an antibody or an antibody fragment comprising an antigen binding region thereof that competes with an antibody fragment comprising the region. 4. A pharmaceutical composition according to claim 3 for the prevention or treatment of COVID-19. 3. The isolated antibody of claim 1 or 2 that binds to SARS-CoV-2 or an antibody fragment thereof comprising an antigen binding region thereof, or said antibody or antigen binding thereof in binding to said SARS-CoV-2 An isolated DNA encoding a protein portion of an antibody or antibody fragment comprising an antigen-binding region thereof that competes with an antibody fragment comprising the region.

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