High affinity monoclonal antibodies to angiotensin-converting enzyme ii (ACE2)

Abstract

To provide monoclonal antibodies having high affinity to angiotensin-converting enzyme II (ACE2) or antigen binding fragments thereof.SOLUTION: Provided is a monoclonal antibody having high affinity to angiotensin-converting enzyme II (ACE2) or an antigen binding fragment thereof, the monoclonal antibody recognizing amino acids corresponding to threonine at position 27, phenylalanine at position 28, lysine at position 31, histidine at position 34, leucine at position 79, methionine at position 82, and tyrosine at position 83 of a specific sequence as a minimum epitope.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to high-affinity monoclonal antibodies against angiotensin-converting enzyme II (ACE2). [Background technology]

[0002] Recently, the novel coronavirus disease known as COVID-19 has spread worldwide and become a problem. There are multiple spike proteins on the surface of this novel coronavirus (SARS-CoV-2). It is known that these spike proteins enter human cells by binding to receptors present on the surface of the cells, causing infection in humans. This human receptor is called angiotensin-converting enzyme II (ACE2: Angiotensin II-Converting Enzyme).

[0003] Coronaviruses are RNA viruses, and it is extremely difficult to prevent the emergence of mutant strains, so new mutant strains are appearing one after another. In particular, it has been reported that when multiple mutations occur in the ACE2 binding region of the spike protein, the effectiveness of the vaccine is weakened and the prevention of infection is reduced. Therefore, there is a problem that the existing treatments and vaccines for COVID-19, such as those published by the National Institute of Health Sciences in Japan, cannot be guaranteed to be effective against new mutant strains, and efforts are currently being made to establish new treatments and develop new treatments and vaccines as a countermeasure against coronaviruses that mutate one after another.

[0004] For example, Non-Patent Documents 1 to 3 report on a treatment for COVID-19 in which ACE2 is artificially modified and produced, and then administered from the outside to concentrate the binding of SARS-CoV-2 on the artificially modified ACE2 (i.e., a decoy). In addition, Non-Patent Documents 4 to 7 describe neutralizing antibodies that inhibit the binding of SARS-CoV-2 to ACE2.

[0005] However, the artificially modified ACE2 and the treatment using it in Non-Patent Documents 1-3 are still under development, and their therapeutic effects have not been fully verified. In addition, the antibody in Non-Patent Document 4 has only confirmed the binding inhibition using a recombinant purified product of the spike protein to which ACE2 binds, and the antibodies in Non-Patent Documents 5-7 are still under development in terms of practical application. Therefore, there is still a demand for the establishment of treatments, therapeutic drugs, vaccines, etc. that are more effective than the artificially modified ACE2 in Non-Patent Documents 1-3 and the antibodies in Non-Patent Documents 4-7. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Anum Glasgow et al.,Proc.Natl.Acad.Sci.USA,117(45),28046-28055(2020). [Non-Patent Document 2] Kui K.Chan et al.,Science.,369(6508),1261-1265(2020). [Non-Patent Document 3] Yusuke Higuchi et al.,Nat Commun.,12,3802,(2021) [Non-Patent Document 4] Latest Insight on COVID-19 Research, Dec 2020, Adipogen Life Sciences website, Internet <URL:https: / / adipogen.com / pub / media / wysiwyg / Catalogs / PDFs-URLs / COVID-19_INSIGHTS_ACE2_CoV2_UPDATE_Dec_2020_final_URLs.pdf> [Non-Patent Document 5] Yanyun Du et al.,Nat Commun.,12,5000,(2021) [Non-Patent Document 6] Yuning Chen et al.,Signal Transduction and Targeted Therapy.,6,315,(2021) [Non-Patent Document 7] Fengwen Zhang et al.,Nat Microbiol.,8,1051-1063(2023) Summary of the Invention

[0007] The present invention provides a high-affinity monoclonal antibody or antigen-binding fragment thereof against angiotensin-converting enzyme II (ACE2).

[0008] The present inventors have discovered a high-affinity monoclonal antibody against ACE2 and that such an antibody has a good ability to inhibit infection with the new coronavirus (SARS-CoV-2). The present invention is based on these findings.

[0009] According to the present invention, the following inventions are provided. (1) A monoclonal antibody or an antigen-binding fragment thereof against angiotensin-converting enzyme II (ACE2), A monoclonal antibody or antigen-binding fragment thereof, characterized by recognizing the amino acids corresponding to threonine at position 27, phenylalanine at position 28, lysine at position 31, histidine at position 34, leucine at position 79, methionine at position 82, and tyrosine at position 83 of SEQ ID NO: 1 as minimal epitopes. (2) A monoclonal antibody or an antigen-binding fragment thereof described in (1), characterized in that it has the activity of inhibiting the binding of the RBD (receptor binding domain) of the spike protein of SARS-CoV-2 to ACE2. (3) A monoclonal antibody or an antigen-binding fragment thereof according to (1) or (2), having an equilibrium dissociation constant (KD) of interaction with ACE2 of 250 nM or less. (4) A monoclonal antibody or an antigen-binding fragment thereof according to any one of (1) to (3), which has an inhibitory effect on infection with wild-type, alpha, beta, gamma, delta, or omicron strains (BA.1) of SARS-CoV-2. (5) A human monoclonal antibody or an antigen-binding fragment thereof according to any one of (1) to (4), The heavy chain variable region is comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in GYTFTRYV (SEQ ID NO: 6), ISPYNDAI (SEQ ID NO: 7) and ARNYGNSYFDY (SEQ ID NO: 8), respectively; The light chain variable region is A monoclonal antibody or antigen-binding fragment thereof, comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in QDISNY (SEQ ID NO: 9), YTS and HQDSKHPWT (SEQ ID NO: 10), respectively. (6) The monoclonal antibody or antigen-binding fragment thereof according to any one of (1) to (5), which is an IgG. (7) The monoclonal antibody or antigen-binding fragment thereof according to any one of (1) to (6), wherein the monoclonal antibody or antigen-binding fragment thereof is selected from a humanized antibody, a chimeric antibody, Fab, Fab', F(ab')2, Fab'-SH, Fv, diabody (dibodies), dsFv, and scFv (single chain Fv). (8) The monoclonal antibody or antigen-binding fragment thereof according to any one of (1) to (6), which is produced by a hybridoma cell having the accession number NITE AP-03950 deposited at the National Institute of Technology and Evaluation on July 20, 2023. (9) A pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of (1) to (8). (10) The pharmaceutical composition according to (9) for the prevention or treatment of novel coronavirus infection. (11) A composition for testing, comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of (1) to (8). (12) A nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of (1) to (8). (13) A host cell into which the nucleic acid according to (12) has been introduced in an expressible manner. (14) The host cell according to (13), which is a lactic acid bacterium. (15) A pharmaceutical composition comprising a host cell according to either (13) or (14). (16) A food composition comprising a host cell according to either (13) or (14).

[0010] According to the present invention, a high-affinity monoclonal antibody or an antigen-binding fragment thereof against ACE2 can be provided. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows the results of Western blotting using anti-ACE2 antibody as the primary antibody. [Diagram 2] Figure 2 shows the results of evaluating the effect of anti-ACE2 antibodies on the enzymatic activity of ACE2. [Diagram 3] Figure 3 shows the results of evaluating the affinity of anti-ACE2 antibodies for each site of the epitope. [Figure 4] Figure 4 shows the results of evaluating the ability of each anti-ACE2 antibody clone to inhibit infection with SARS-CoV-2 pseudoparticles. [Diagram 5] Figure 5 shows the results of evaluating the ability of each anti-ACE2 antibody clone to inhibit infection with SARS-CoV-2 pseudoparticles. [Figure 6] Figure 6 shows the results of evaluating the ability of anti-ACE2 antibodies to inhibit infection with each mutant SARS-CoV-2 virus. [Figure 7] Figure 7 shows the results of evaluating the ability of anti-ACE2 antibodies or their antigen-binding fragments to inhibit infection with each mutant virus of SARS-CoV-2, SARS-CoV, and MERS-CoV. Description of the Invention

[0012] According to one embodiment of the present invention, there is provided a monoclonal antibody or an antigen-binding fragment thereof against angiotensin-converting enzyme II (ACE2), characterized in that the monoclonal antibody or an antigen-binding fragment thereof recognizes the amino acids corresponding to threonine at position 27, phenylalanine at position 28, lysine at position 31, histidine at position 34, leucine at position 79, methionine at position 82, and tyrosine at position 83 of SEQ ID NO:1 as minimal epitopes.

[0013] The monoclonal antibody of the present invention can be obtained by a known method. Such a method is not limited to, but includes, for example, immunizing a non-human mammal with an ACE2 antigen protein or a partial peptide of an ACE2 protein, collecting antibody-producing cells (e.g., B cells) from the immunized non-human mammal, fusing the antibody-producing cells with myeloma cells to prepare a hybridoma (fusion cell line), and collecting the antibody produced by the hybridoma to obtain the desired monoclonal antibody. In this specification, when simply described as ACE2, it refers to the ACE2 protein unless otherwise specified.

[0014] In order to obtain the monoclonal antibody of the present invention, the full-length protein of ACE2 can be used as an immunogen. The full-length protein of ACE2 can be obtained by a known method, but is not limited to such a method. For example, the desired ACE2 can be expressed intracellularly using cultured cells such as Expi293F, and purified, or a commercially available product can be used. In addition, in order to obtain the monoclonal antibody of the present invention, a partial peptide of ACE2 (e.g., the extracellular region of ACE2) can be synthesized and used as an immunogen. ACE2 or a partial peptide thereof used as an immunogen may be modified, for example, by adding an avidin tag (Avi-tag), a histidine tag (His-tag), or an Fc region of an antibody (e.g., IgG, etc.) as long as it can be used as an antigen.

[0015] To obtain the monoclonal antibody of the present invention, the type of non-human mammal to be immunized is not limited, but examples thereof include mice, rats, guinea pigs, rabbits, dogs, goats, etc., and is preferably a mouse. The non-human mammal to be immunized may also be an animal with an autoimmune disease. Immunization may be performed, but is not limited, for example, by intravenous, subcutaneous, intraperitoneal, intramuscular, or subcutaneous injection into the footpad. The interval between immunizations is not limited, but may be at intervals of several days to several weeks, preferably 1 to 2 weeks, and immunization may be performed about 1 to 10 times.

[0016] The antibody-producing cells for obtaining the monoclonal antibody of the present invention are not particularly limited, and may be prepared from spleen cells or lymph nodes of an immunized non-human mammal, and are preferably prepared from spleen cells. It is not necessary to carry out a special procedure for separating antibody-producing cells from the collected cell population, but preferably, only antibody-producing cells are separated from the cell population.

[0017] By fusing the above-mentioned antibody-producing cells with myeloma cells, cells (hybridomas) that proliferate semi-permanently while producing the monoclonal antibody of the present invention can be produced. This fusion may be performed by a known cell fusion method, and is not limited to, for example, fusion in the presence of a cell fusion promoter, fusion using a commercially available cell fusion device that utilizes electrical stimulation (e.g., electroporation), etc. As the myeloma cells to be fused with the antibody-producing cells, generally available cell lines of animals such as mice can be used. As the cell lines to be used, but are not limited to, those that cannot survive in HAT selection medium (medium containing hypoxanthine, aminopterin, and thymidine) and can survive only in a state fused with the antibody-producing cells may be used. As the myeloma cells, but are not limited to, for example, SP2 / 0, P3U1, NSI, P3-X63-Ag8.653, etc., are included, and P3U1 is preferred.

[0018] Hybridomas that produce the monoclonal antibody of the present invention can be selected from cells after cell fusion treatment. The method of selecting hybridomas may be performed by known methods, and is not limited to, for example, the cells after cell fusion treatment are cultured in an appropriate medium such as HAT medium for a certain period of time to form colonies, and the culture supernatant of each well of the colony-positive culture plate is collected to confirm the antibody titer against ACE2. The method of confirming the antibody titer against ACE2 includes, but is not limited to, flow cytometry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), etc., and is preferably flow cytometry.

[0019] The selected cells in the well may be cloned to make them single cells. Cloning may be performed by a known method, and is not limited thereto. For example, the cell suspension is appropriately diluted with an appropriate medium (e.g., RPMI1640 medium containing 10 to 20% FCS), and then the cells are seeded in each well of a culture plate (the number of cells to be placed in each well of the culture plate is preferably one cell per well, in order to increase the probability that one cell will be placed in one well). After a certain period of time (e.g., 7 to 10 days. During this time, it is preferable to confirm that the cells are single colonies), the culture supernatant of the colony-positive well is collected, and the antibody titer of the collected culture supernatant is confirmed. Cells that produce antibodies that show high affinity to ACE2 may be selected, and the cells in the selected well may be increased to some extent to establish a hybridoma strain. Cloning may also be performed several times as necessary. These procedures yielded, for example, a hybridoma having accession number NITE AP-03950, which was deposited at the National Institute of Technology and Evaluation on July 20, 2023.

[0020] From the established hybridoma line, the monoclonal antibody against ACE2 of the present invention can be purified and collected by an appropriate method. This may be performed by a known method, and is not limited to, for example, a method of preparing an antibody from a culture supernatant obtained by culturing in a medium with a reduced serum concentration, a method of preparing an antibody from a culture supernatant obtained by culturing in a commercially available serum-free medium, a method of injecting a hybridoma into the abdominal cavity of an animal, collecting ascites, and preparing an antibody from the ascites, etc.

[0021] Methods for culturing the established hybridoma strain include, but are not limited to, for example, a method using a culture flask, a method using a spinner flask, a method using a shaker flask, a method using a bioreactor, and the like.

[0022] The monoclonal antibody of the present invention may be purified by a known method, and examples of such methods include, but are not limited to, a method of purification using an ACE2 affinity column, a method of purification using ion exchange chromatography, and a method of purification using an affinity column for immunoglobulin-binding proteins such as protein A or protein G. The monoclonal antibody of the present invention can be purified by appropriately selecting or combining these known methods.

[0023] The epitope (antigenic determinant) recognized by the monoclonal antibody of the present invention is a part of the ACE2 polypeptide that is recognized when the monoclonal antibody of the present invention shows high affinity for ACE2. Specifically, the minimum epitopes are the threonine residue at position 27 of SEQ ID NO: 1 (corresponding to, for example, position 29 in SEQ ID NO: 2 in the case of a mutant), the phenylalanine residue at position 28 (corresponding to, for example, position 30 in SEQ ID NO: 2 in the case of a mutant), the lysine residue at position 31 (corresponding to, for example, position 33 in SEQ ID NO: 2 in the case of a mutant), the histidine residue at position 34 (corresponding to, for example, position 36 in SEQ ID NO: 2 in the case of a mutant), the leucine residue at position 79 (corresponding to, for example, position 81 in SEQ ID NO: 2 in the case of a mutant), the methionine residue at position 82 (corresponding to, for example, position 84 in SEQ ID NO: 2 in the case of a mutant), and the tyrosine residue at position 83 (corresponding to, for example, position 85 in SEQ ID NO: 2 in the case of a mutant). In the epitope recognized by the monoclonal antibody of the present invention, other amino acid residues than the minimal epitope may be the same as or different from those in the ACE2 polypeptide; for example, in the amino acid sequence shown in SEQ ID NO: 1, the residue at position 19 (in the case of a mutant, for example, corresponding to position 21 in SEQ ID NO: 2) may be serine or may be different, the residue at position 24 (in the case of a mutant, for example, corresponding to position 26 in SEQ ID NO: 2) may be glutamine or may be different, and the residue at position 35 (in the case of a mutant, for example, corresponding to position 37 in SEQ ID NO: 2) may be glutamic acid or may be different.

[0024] The monoclonal antibodies of the present invention are immunoglobulin molecules that, like common antibodies, consist of two pairs of polypeptide chains (each pair has a light chain and a heavy chain). The light chains of the antibodies can be classified as kappa and lambda light chains. The heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and the antibody isotypes can be IgM, IgD, IgG, IgA, and IgE, respectively. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH), which consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL), which consists of one domain, CL. The constant region of the antibody can mediate the binding of the immunoglobulin to factors, including host tissues or various immune system cells (e.g., effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can also be subdivided into highly variable regions called complementarity determining regions (CDRs) interspersed with highly conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs in the present invention are identified according to the IMGT method.

[0025] According to a preferred embodiment of the present invention, there is provided a monoclonal antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region comprises HCDR1 having the amino acid sequence represented by GYTFTRYV (sequence number 6), HCDR2 having the amino acid sequence represented by ISPYNDAI (sequence number 7), and HCDR3 having the amino acid sequence represented by ARNYGNSYFDY (sequence number 8).

[0026] According to another preferred embodiment of the present invention, there is provided a monoclonal antibody or an antigen-binding fragment thereof, wherein the light chain variable region comprises an LCDR1 having the amino acid sequence represented by QDISNY (sequence number 9), an LCDR2 having the amino acid sequence represented by YTS, and an LCDR3 having the amino acid sequence represented by HQDSKHPWT (sequence number 10).

[0027] According to a more preferred embodiment of the present invention, there is provided a monoclonal antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising an HCDR1 having the amino acid sequence represented by GYTFTRYV (sequence number 6), an HCDR2 having the amino acid sequence represented by ISPYNDAI (sequence number 7), and an HCDR3 having the amino acid sequence represented by ARNYGNSYFDY (sequence number 8), and a light chain variable region comprising an LCDR1 having the amino acid sequence represented by QDISNY (sequence number 9), an LCDR2 having the amino acid sequence represented by YTS, and an LCDR3 having the amino acid sequence represented by HQDSKHPWT (sequence number 10).

[0028] The monoclonal antibodies of the present invention may be recombinant antibodies or antigen-binding fragments prepared and expressed by recombinant means, e.g., chimeric, humanized, or fully human antibodies, preferably chimeric or humanized antibodies. These antibodies of the present invention may be prepared according to known methods, e.g., by recombinant expression of heavy and light chains, without being limited thereto.

[0029] The antigen-binding fragment of the monoclonal antibody of the present invention means a partial region of the monoclonal antibody of the present invention, and includes, but is not limited to, Fab, Fab', F(ab')2, Fab'-SH, Fv, diabody (dibodies), dsFv, scFv (single chain Fv), etc. The above-mentioned antigen-binding fragment can be obtained by, but is not limited to, cleaving the monoclonal antibody of the present invention with various proteases according to the purpose, or by isolating the V of the monoclonal antibody of the present invention. H and V L The antibody can be obtained by, for example, preparing a recombinant gene from a cDNA encoding the antibody. The antigen-binding fragment of the monoclonal antibody of the present invention binds to the same antigen (particularly the epitope) as that bound by the monoclonal antibody of the present invention.

[0030] The above Fab can be obtained, for example, by treating the antibody molecule with papain, and the above F(ab')2 can be obtained, for example, by treating the antibody molecule with pepsin. The above Fab' can be obtained, for example, by cleaving the disulfide bond in the hinge region of the above F(ab')2.

[0031] The above scFv can be, for example, the V H and V L The scFv can be obtained by obtaining a cDNA encoding the above, constructing a DNA encoding the scFv, inserting this DNA into an expression vector, and introducing the expression vector into a host organism for expression.

[0032] The above diabody is, for example, the V H and V L The scFv can be obtained by obtaining a cDNA encoding the above, constructing a DNA encoding the scFv such that the length of the amino acid sequence of the peptide linker is 8 residues or less, inserting this DNA into an expression vector, and introducing the expression vector into a host organism for expression.

[0033] The above dsFv can be, for example, the V H and V L The antibody can be obtained by obtaining a cDNA encoding the above-mentioned antibody, constructing a DNA encoding a dsFv, inserting this DNA into an expression vector, and introducing the expression vector into a host organism for expression.

[0034] In the present invention, chimeric antibodies, humanized antibodies, and humanized antibodies may be prepared according to known methods using hybridomas or DNA or RNA extracted from the hybridomas as raw materials.

[0035] The equilibrium dissociation constant (KD) of the interaction between the monoclonal antibody or antigen-binding fragment of the present invention and ACE2 is, but is not limited to, preferably 250 nM or less, more preferably 200 nM or less.

[0036] In a preferred embodiment of the invention, there is provided a monoclonal antibody of the invention, or an antigen-binding fragment thereof, which is an IgG. Such an antigen-binding fragment may contain at least a portion of the constant region of an IgG.

[0037] The monoclonal antibody or antigen-binding fragment of the present invention can inhibit the binding of the novel coronavirus (SARS-CoV-2) to ACE2 and inhibit its entry into cells, thereby exerting the ability to inhibit infection with SARS-CoV-2, and can therefore be used for the prevention or treatment of novel coronavirus infection.

[0038] Therefore, according to a preferred embodiment of the present invention, there is provided a monoclonal antibody or an antigen-binding fragment thereof of the present invention, or a composition comprising the same, for preventing or treating COVID-19 infection. Such prevention or treatment of COVID-19 infection can be achieved by the monoclonal antibody or an antigen-binding fragment thereof of the present invention inhibiting the binding of the spike protein of SARS-CoV-2 to ACE2. In this case, the domain of the spike protein of SARS-CoV-2 that binds to ACE2 may be RBD (receptor_binding_domain). Therefore, according to another preferred embodiment of the present invention, there is provided a monoclonal antibody or an antigen-binding fragment thereof having an activity of inhibiting the binding of the RBD of the spike protein of SARS-CoV-2 to ACE2.

[0039] Examples of SARS-CoV-2 in the present invention include wild-type strains (Wuhan strains), alpha strains, beta strains, gamma strains, delta strains, and Omicron strains (BA.1).

[0040] Considering the chemical stability of the antibody, etc., it is also preferable from the standpoint of preventive or therapeutic effects, etc. to use the monoclonal antibody of the present invention or its antigen-binding fragment as a composition comprising the monoclonal antibody of the present invention or its antigen-binding fragment, or as a bacterium that biosynthesizes the monoclonal antibody of the present invention or its antigen-binding fragment in the body and secretes it outside the body, rather than directly using the monoclonal antibody of the present invention or its antigen-binding fragment for the prevention or treatment of COVID-19 infection.

[0041] Therefore, a preferred embodiment of the present invention provides a composition comprising the monoclonal antibody or antigen-binding fragment thereof of the present invention. Examples of such a composition include a pharmaceutical composition, a cosmetic composition, a food composition, a testing composition, etc., and the pharmaceutical composition or testing composition is preferred.

[0042] In addition, according to a preferred embodiment of the present invention, a nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention is provided. Such a nucleic acid may be endogenous, in which case the nucleic acid may be contained in the genome. In addition, such a nucleic acid may be exogenous, in which case the nucleic acid may be carried in an expressible manner by an appropriate vector or the like. Therefore, according to a preferred embodiment of the present invention, a host cell into which a nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention has been introduced in an expressible manner, and a composition comprising the host cell are provided. Examples of such compositions include pharmaceutical compositions, cosmetic compositions, food compositions, and test compositions, and are preferably pharmaceutical or food compositions.

[0043] The host cell in the present invention is not limited as long as it is capable of expressibly retaining a nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention, and may be derived from either a prokaryote (e.g., bacteria, etc.) or a eukaryote (e.g., mammals, etc.). Examples of the host cell include yeast, lactic acid bacteria, bifidobacteria, etc., and preferably lactic acid bacteria.

[0044] The compositions of the present invention (such as pharmaceutical compositions, cosmetic compositions, food compositions, and test compositions) may further contain known ingredients that are suitable for each composition and that are generally incorporated into compositions.

[0045] For example, when the composition of the present invention is used as a food composition, it may further contain foods such as sugars, fruits, fruit juices, vegetables, meats, etc., and food additives such as sweeteners, high-intensity sweeteners (aspartame, sucralose, glycyrrhizin, saccharin, stevia, dulcin, trichlorosucrose, thaumatin, acesulfame potassium, etc.), flavorings, colorings, acidulants, antioxidants, emulsifiers, preservatives, stabilizers, etc.

[0046] The recommended intake amount (amount of active ingredient) of the food composition of the present invention per meal or per day can be appropriately set based on the age, weight, sex, non-clinical or clinical test results, etc. of the subject to be prevented or treated for the novel coronavirus. The intake period of the composition of the present invention can also be appropriately set, and the composition may be taken repeatedly or daily over a long period of time.

[0047] The use of the food composition of the present invention, "prevention or treatment of the new coronavirus," can be displayed directly or indirectly. Display acts include all acts for informing consumers of the above-mentioned use, and any expression that can recall or infer the above-mentioned use falls under the display act of the present invention, regardless of the purpose of the display, the content of the display, the object to be displayed, the medium, etc. Examples of direct display are descriptions on tangible objects such as the product itself, packaging, containers, labels, and tags, and examples of indirect display are advertising and promotional activities by places or means such as websites, storefronts, exhibitions, signs, bulletin boards, newspapers, magazines, television, radio, mailings, and e-mails. EXAMPLES

[0048] The present invention will be described in detail with reference to the following examples, but the present invention is not limited to these examples. Contents are expressed in mass % unless otherwise specified.

[0049] Preparation of purified proteins Four types of proteins, which are mutants of ACE2 (sequence number 1), namely, ACE2-Fc (sequence number 2: the extracellular domain of ACE2 fused with the Fc domain of mouse IgG, etc.), ACE2-His (sequence number 3: the extracellular domain of ACE2 fused with an 8xHis-tag, etc.), ACE2 (full length) (sequence number 4: ACE2 fused with an 8xHis-tag, etc.), and Sgp-RBD-His (sequence number 5: the receptor binding domain (RBD) of the S protein (S-glycoprotein: Sgp) of SARS-CoV-2 fused with an 8xHis-tag, etc.), were transiently expressed by introducing the genes corresponding to each protein into Expi293F (manufactured by Thermo) and culturing under appropriate conditions. After expression of each protein, ACE2-Fc was extracted from Expi293F and purified using protein G to a concentration of approximately 1 mg / L, ACE2-His was extracted from Expi293F and purified using an anti-His-tag antibody to a concentration of approximately 1 mg / L, and Sgp-RBD-His was extracted from Expi293F and purified using an anti-His-tag antibody to a concentration of approximately 3 mg / L by standard methods.

[0050] [Table 1] TIFF2025045908000002.tif254168TIFF2025045908000003.tif254151

[0051] Establishment, production, and purification of anti-human ACE2 antibody No. 648 Purified ACE2-Fc was immunized five times every seven days into three 4-week-old female autoimmune disease mice (MRL / MpJJmsSlc-lpr / lpr Japan SLC). Spleen cells were collected from the immunized mice and fused with myeloma cells (P3U1) by the PEG method to produce hybridomas. The hybridomas were seeded on 30 96-well plates (Thermo Fisher Scientific) and incubated at 37°C under a 5% CO2 atmosphere for 10 days. After incubation, the antibodies contained in the medium in which the hybridomas were cultured were reacted with Expi293F cells that transiently expressed ACE2 (full length), and 17 clones that were confirmed to react were selected by flow cytometry.

[0052] The antibodies (IgG) contained in the medium in which the selected hybridomas were cultured were purified using protein G sepharose, and a dilution series of the purified IgG (10 μg / mL, 5 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.2 μg / mL, 0.1 μg / mL) was prepared. 0.1 mL of the IgG prepared at each concentration was added to Expi293F cells transiently expressing ACE2 (full length), incubated at 4°C for 1 hour, and the reaction at each concentration was analyzed by flow cytometry. In addition, 0.1 mL of the IgG prepared at each concentration was added to Expi293F cells transiently expressing ACE2 (full length) and incubated at 4 ° C for 1 hour, and then 0.1 mL of 10 μg / mL Sgp-RBD-His was added and incubated at 4 ° C for 1 hour to confirm the ability of each antibody to inhibit the binding of Sgp-RBD-His to ACE2, thereby establishing anti-human ACE2 antibody No. 648. Analysis of the amino acid sequence of the established antibody No. 648 confirmed that its heavy chain variable region contains HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in GYTFTRYV (SEQ ID NO: 6), ISPYNDAI (SEQ ID NO: 7) and ARNYGNSYFDY (SEQ ID NO: 8), respectively, and its light chain variable region contains LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in QDISNY (SEQ ID NO: 9), YTS and HQDSKHPWT (SEQ ID NO: 10), respectively.

[0053] The binding specificity of the established antibody No. 648 was verified. The verification was performed by Western blotting using antibody No. 648 as the primary antibody. First, Expi293F cells that transiently expressed ACE2 (full length) and Calu-3 cells that endogenously expressed ACE2 were solubilized using RIPA buffer to prepare a cell solution. The polyacrylamide gel of this cell solution was subjected to SDS-PAGE and transferred to a PVDF membrane using a Tris-glycine buffer. Then, blocking was performed using Blocking One (manufactured by Nacalai Tesque). 2 μg / mL purified antibody IgG was used as the primary antibody. As the secondary antibody, a diluted HRP-labeled antibody of anti-mouse IgG (Fc specific) was used. In each step, the reaction was performed at 37 ° C for 1 hour, and then washed with PBST. After that, the color was developed using Chemi-Lumi One Ultra (manufactured by Nacalai Tesque), and the target protein was detected, and an ACE2 band was confirmed at around 140 kDa. Therefore, antibody No. 648 was shown to have affinity for both transiently expressed ACE2 (full-length) in Expi293F cells and endogenously expressed ACE2 in Calu-3 cells (Figure 1).

[0054] Measurement of binding affinity of anti-human ACE2 antibodies using BLItz To perform more detailed binding affinity analysis, binding affinity was measured using BLItz (manufactured by Sartorius). ACE2-Fc purified by the above method was immobilized on the AMC (anti-mouse IgG-Fc capture) biosensor of BLItz, and the final concentration of purified IgG of anti-human ACE2 antibodies including antibody No. 648 was adjusted to 0.5 μM, and measurements were performed.

[0055] The results are shown in Table 2. Antibody No. 648 had an affinity of 1.973 × 10 for purified ACE2-Fc. -7 It was shown to have good affinity for M.

[0056] [Table 2]

[0057] Evaluation of the effect of antibody No. 648 on ACE2 enzyme activity The effect of antibody No. 648 on ACE2 enzyme activity was evaluated using a commercially available kit (Angiotensin II Converting Enzyme (ACE2) Activity Assay Kit (Fluorometric) (ab273297); Abcam). Purified ACE2-His and purified IgG of antibody No. 648 were used as measurement samples.

[0058] The results are shown in Figure 2. Figure 2 demonstrates that the binding of antibody No. 648 to ACE2-His does not affect its enzyme activity.

[0059] Determination of the epitope of antibody No. 648 Based on the amino acid residues that interact in the interaction region of ACE2 and S protein disclosed in the report by Han et al. (Han et al., Cell, 2022, 185, 630-640), alanine mutants were sequentially synthesized in which the amino acid residues that interact with S protein in the ACE2-Fc construct were replaced with alanine one by one, and the reactivity of antibody No. 648 to each alanine mutant was measured. This measurement was performed by Western blotting.

[0060] The measurement results are shown in Figure 3. From these results, it was shown that the reactivity of antibody No. 648 was reduced by substituting the 27th residue (corresponding to position 29 in SEQ ID NO: 2) threonine, the 28th residue (corresponding to position 30 in SEQ ID NO: 2) phenylalanine, the 31st residue (corresponding to position 33 in SEQ ID NO: 2) lysine, the 34th residue (corresponding to position 36 in SEQ ID NO: 2) histidine, the 79th residue (corresponding to position 81 in SEQ ID NO: 2) leucine, the 82nd residue (corresponding to position 84 in SEQ ID NO: 2) methionine, and the 83rd residue (corresponding to position 85 in SEQ ID NO: 2) tyrosine of ACE2 with alanine, and it was shown that the region in which these residues exist is particularly important for affinity. The region in which these residues with reduced reactivity exist corresponds to Patch1, which is the region in which SARS-CoV2_Spike-RBD and hACE2 interact as disclosed in the report by Han et al. (Han et al., Cell, 2022, 185, 630-640). On the other hand, the minimal epitope recognized by the antibodies disclosed in Non-Patent Documents 5 to 7, which are disclosed in the prior art, is different (see, for example, FIG. 5G of Non-Patent Document 7).

[0061] Preparation of ACE2-Fab complex Antibody No. 648 was digested with papain in the usual way to prepare Fab. The obtained Fab was purified using protein A sepharose and gel filtration. Next, the purified ACE2-His and the purified Fab were mixed in a molar ratio of about 1:1.5 and incubated at 4°C for about 1 hour, and then purified by gel filtration to obtain the ACE2-Fab complex.

[0062] Evaluation of the inhibitory effect of anti-human ACE2 antibodies on SARS-CoV-2 pseudoparticle infection Vesicular stomatitis virus pseudoparticles carrying the spike protein of SARS-CoV-2 (Wuhan strain) were produced according to the method of Tani et al. (Tani H et al. Virol J, 18, 16, 2021). 2 × 10 4TMPRSS2-expressing VeroE6 cells (hereinafter, VeroE6 / TMPRSS2 cells) were seeded at 1 / well and cultured overnight at 37°C and 5% CO2. Then, serially diluted antibody No. 648 or control antibody (Human / Mouse / Rat / Hamster ACE-2 Antibody, AF933, R&D Systems) was added and incubated at 37°C for 30 minutes. The cells were then infected with the prepared pseudoparticles and incubated at 37°C and 5% CO2 for 24 hours. The cells were washed with PBS and the luciferase luminescence value of each well was measured using the Blight-Glo Luciferase Assay System and GloMax Microplate Luminometer (Promega). The luminescence values ​​of the uninfected wells and the infected wells without antibody were set to 0 and 100%, respectively, and the pseudoparticle infection rate of each well was calculated from the luminescence value.

[0063] The evaluation results are shown in Figures 4 and 5. These results demonstrate that, among the selected anti-human ACE2 antibody clones, antibody No. 648 has the best binding inhibitory effect.

[0064] Evaluation of the inhibitory effect of anti-human ACE2 antibodies and their antibody fragments on infection with SARS-CoV-2 mutant viruses, SARS-CoV, and MERS-CoV 2 x 10 in a 96-well plate 4VeroE6 / TMPRSS2 cells were seeded at 1 / well and cultured overnight at 37°C and 5% CO2. Antibody No. 648 or its Fab fragment serially diluted with culture medium (DMEM containing 2% bovine serum albumin) was added and incubated at 37°C for 30 minutes. The antibody solution was then removed, and SARS-CoV-2 (Wuhan strain or various mutant strains) was mixed with the antibody dilution solution so that the multiplicity of infection was 0.003, inoculated into the cells, and incubated at 37°C and 5% CO2 for 1 hour. After incubation, the culture supernatant was removed, the cells were washed with culture medium, and 150 μL / well of culture medium was added and incubated at 37°C and 5% CO2 for 24 hours. The culture supernatant was then collected and RNA was extracted using the MagMax Viral / Pathogen Nucleic Acid Isolation kit (Thermo Fisher Scientific), after which quantitative PCR was performed using the THUNDERBIRD Probe One-step qRT-PCR kit (TOYOBO) and the following primers and probes as previously reported (Ohashi et al. Antiviral Res, 205, 105372, 2022). Forward primer S17: 5'-ACAGGTACGTTAATAGTTAATAGCGT (SEQ ID NO: 11)-3' Reverse primer R19: 5'-ATATTGCAGCAGTACGCACACA (SEQ ID NO: 12)-3' Probe R6-148 S21FT: 5'-FAM-ACACTAGCCATCCTTACTGCGCTTCG (SEQ ID NO: 13)-TAMRA-3' Viral RNA was quantified using the delta Ct method. Negative control wells were treated with culture medium alone, and the quantitative value of the negative control well was set as 100% to calculate the relative percentage of viral RNA in each well.

[0065] The evaluation results are shown in Figure 6. These results demonstrated that antibody No. 648 has an inhibitory effect on infection with all strains of SARS-CoV-2, including wild-type, alpha, beta, gamma, delta, and omicron (BA.1).

[0066] Next, the infection inhibitory activity of antibody No. 648 or its Fab fragment against SARS-CoV (Frankfurt strain) and MERS-CoV (EMC strain) was evaluated. Viral infection was performed as described above. SARS-CoV was quantified using the same primers and probe as SARS-CoV-2, and MERS-CoV was quantified using the following primers and probe. Forward primer upE-F: 5'-GCAACGCGCGATTCAGTT (SEQ ID NO: 14)-3' Reverse primer upE-R: 5'-GCCTCTACACGGGACCCATA (SEQ ID NO: 15)-3' Probe upE (FAM-TAM): 5'-FAM-CTCTTCACATAATCGCCCCGAGCTCG (SEQ ID NO: 16)-TAMRA-3'

[0067] The evaluation results are shown in Figure 7. The results showed that antibody No. 648 and its Fab had almost no inhibitory effect on infection with MERS-CoV, but had good inhibitory effect on the Wuhan strain and Omicron strain (BA.1) of SARS-CoV-2, and SARS-CoV.

Claims

1. A monoclonal antibody or an antigen-binding fragment thereof against angiotensin-converting enzyme II (ACE2), A monoclonal antibody or antigen-binding fragment thereof, characterized in that it recognizes the amino acids corresponding to threonine at position 27, phenylalanine at position 28, lysine at position 31, histidine at position 34, leucine at position 79, methionine at position 82, and tyrosine at position 83 of SEQ ID NO: 1 as minimal epitopes.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that it has activity of inhibiting the binding of the RBD (receptor_binding_domain) of the spike protein of SARS-CoV-2 to ACE2.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, having an equilibrium dissociation constant (KD) of interaction with ACE2 of 250 nM or less.

4. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, which has an inhibitory effect on infection with wild-type, alpha, beta, gamma, delta, or omicron strains (BA.1) of SARS-CoV-2.

5. 2. The human monoclonal antibody or antigen-binding fragment thereof of claim 1, The heavy chain variable region is comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in GYTFTRYV (SEQ ID NO: 6), ISPYNDAI (SEQ ID NO: 7) and ARNYGNSYFDY (SEQ ID NO: 8), respectively; The light chain variable region is A monoclonal antibody or antigen-binding fragment thereof, comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in QDISNY (SEQ ID NO: 9), YTS and HQDSKHPWT (SEQ ID NO: 10), respectively.

6. The monoclonal antibody or antigen-binding fragment thereof of claim 1 which is an IgG.

7. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof is selected from a humanized antibody, a chimeric antibody, Fab, Fab', F(ab')2, Fab'-SH, Fv, a diabody (dibodies), a dsFv, and a scFv (single chain Fv).

8. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, which is produced by a hybridoma cell having the accession number NITE AP-03950 deposited at the National Institute of Technology and Evaluation on July 20, 2023.

9. A pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

10. The pharmaceutical composition according to claim 9 for the prevention or treatment of novel coronavirus infection.

11. A composition for testing comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.

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

13. A host cell into which the nucleic acid according to claim 12 has been introduced in an expressible manner.

14. The host cell of claim 13 which is a lactic acid bacterium.

15. A pharmaceutical composition comprising a host cell according to any one of claims 13 or 14.

16. A food composition comprising a host cell according to any one of claims 13 or 14.

Images