Antibodies or fragments thereof that specifically bind to the nucleocapsid protein of severe acute respiratory syndrome coronavirus 2, and their uses

Abstract

This invention provides an antibody or fragment thereof that specifically binds to the NTD or CTD of the SARS-CoV-2 N protein, and its applications. [Solution] An antibody or antigen-binding fragment thereof that specifically binds to the NTD or CTD of the SARS CoV2 N protein, which consists of a specific amino acid sequence, is provided.

Description

[Technical Field]

[0001] Antibodies or fragments thereof that specifically bind to the nucleocapsid protein of Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), and technologies related to their applications are provided. [Background technology]

[0002] SARS-CoV-2 is the virus that causes COVID-19, and it has been spreading rapidly worldwide since the beginning of 2020. Like common coronaviruses, SARS-CoV-2 consists of a nucleocapsid and an envelope surrounding the nucleocapsid. The nucleocapsid contains the viral genome (RNA) and a nucleocapsid protein (N protein) that binds to the viral genome, and the envelope contains lipids and spike proteins (S protein), membrane proteins (M protein), and envelope proteins (E protein) that bind to these lipids.

[0003] The N protein is a protein involved in viral core formation, as well as viral genome packaging and transcription. It has a structure in which the N-terminal domain (NTD) and C-terminal domain (CTD) are linked via a linker containing a serine (S) / arginine (R) rich region. Non-patent document 1 describes that the N protein is phosphorylated in large quantities and shows a mapping of phosphorylation sites. Since the amino acid sequence of the N protein is conserved among strains, the N protein is used as a diagnostic marker, etc. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Klann et al., Molecular Cell, 80(1), 2020, pp. 164-174 [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] The inventors have found that the linker of the SARS-CoV-2 N protein, which has a S / R-rich region (a large amount of phosphorylation sites), can be cleaved by apoptosis-inducing cysteine ​​proteases (e.g., caspase 3, caspase 6) or by autolysis. Therefore, even when an antibody that specifically binds to the SARS-CoV-2 N protein is used, the SARS-CoV-2 N protein may not be detected depending on the site to which the antibody binds.

[0006] Therefore, the first object of the present invention is to provide an antibody or fragment thereof that specifically binds to the NTD or CTD of the SARS-CoV-2 N protein, and an application thereof.

[0007] Furthermore, the present inventors have found that in a method for detecting the SARS-CoV-2 N protein by forming a sandwich structure with two types of antibodies that specifically bind to the N protein, for example, when an antibody that specifically binds to NTDs and an antibody that specifically binds to CTDs are used as the two types of antibodies, the sandwich structure cannot be formed when the linker having an S / R-rich region is cleaved, thus reducing the detection sensitivity of the SARS-CoV-2 N protein.

[0008] Therefore, a second object of the present invention is to provide a kit and method for detecting the N protein of SARS-CoV-2 with high sensitivity. [Means for solving the problem]

[0009] As a result of intensive studies to achieve the above object, the present inventors have found various antibodies or fragments thereof that specifically bind to the NTD or CTD of the N protein of SARS CoV2. In addition, the present inventors have found that by using two or more antibodies or fragments thereof that specifically bind to the NTD and recognize different epitopes in the NTD, and / or two or more antibodies or fragments thereof that specifically bind to the CTD and recognize different epitopes in the CTD, the N protein can be detected with high sensitivity. The present invention has been completed as a result of further intensive studies based on these findings.

[0010] The present invention includes the following aspects. Item 1. An antibody or a fragment thereof (antigen-binding fragment) that specifically binds to the amino acid sequence represented by SEQ ID NO: 1 or 2. Item 2. The following formula (A-1) or (A-2): GFX A31 X A41 X A51 X A61 X A71 X A81 (A-1) [In the formula, X A31 is T or S, X A41 is F, L, or I, X A51 is D, S, N, or T, X A61 is D, N, S, I, or T, X A71 is Y, F, or N, X A81 is G, W, Y, S or T] GFX A33 X A43 X A53 X A63 X A73 X A83 X A93 X A103 (A-2) [In the formula, X A33 is T or S, XA43 is F, L, or I, X A53 is D, S, N, or T, X A63 is D, N, S, or T, X A73 is N or S, X A83 is G or Y, X A93 is Y, F, or N, X A103 [is T, G, W, or Y] A heavy chain CDR1 consisting of an amino acid sequence represented by, The following formula (B-1) or (B-2): IX B21 X B31 X B41 X B51 X B61 X B71 X B81 (B-1) [In the formula, X B21 is S, N, D, W, or H, X B31 is Y, G, P, or S, X B41 is S, D, or G, X B51 is G, S, T, or A, X B61 is G, D, T, N, or R, X B71 is R, K, T, S, N, G, Y, or D, X B81 [is T, I, or M] IX B23 X B33 X B43 X B53 X B63 X B73 (B-2) [In the formula, X B23 is S, N, D, W, or H, X B33 is Y, G, P, or S, X B43 is S, D, or G, X B53 is G, S, T, or A, X B63 is R, K, T, S, N, G, Y, or D, X B73 [is T, I, or M] A heavy chain CDR2 consisting of the amino acid sequence represented by, The following equations (C-1) to (C-8): X C11 X C21 X C31 X C41 X C51 X C61 X C71 X C81 X C91 X C101 X C111 X C121 X C131 X C141 X C151 (C-1) [In the formula, X C11 is A or S, X C21 is R, T, or K, X C31 is D, N, S, or Y, X C41 is G, S, or Y, X C51 is G, I, or D, X C61 is S or I, X C71 is Y, V, or A, X C81 is Y, H, S, T, K, or E, X C91 is S, I, or D, X C101 is P or Y, X C111 is I, Y, G, P, T, A, or V, X C121 is N, S, Y, P, G, or F, X C131 It is F, C, M, or L. X C141 is Q or D, X C151 is F, Y, V, or S] X C13 X C23 X C33 X C43 X C53 X C63 X C73 X C83 (C-2) [wherein, X C13 is A or S, X C23 is R, T, or K, X C33 is D, N, S, or Y, X C43 is I, Y, G, P, T, A, or V, X C53 is N, S, Y, P, G, or F, X C63 is F, C, M, or L, X C73 is Q or D, X C83 is F, Y, V, or S] X C15 X C25 X C35 X C45 X C55 [[ID=QQ]]X C65 X C75 X C85 X C95 TIFTFX C155 X C165 X C175 X C185 X C195 (C-3) [wherein, X C15 is A or S, X C25 is R, T, or K, X C35 is G, S, or Y, X C45 is G, I, or D, X C55 is S or I, X C65 is Y, V, or A, X C75is Y, H, S, T, K, or E, X C85 is S, I, or D, X C95 is P or Y, X C155 is I, Y, G, P, T, A, or V, X C165 is N, S, Y, P, G, or F, X C175 is F, C, M, or L, X C185 is Q or D, X C195 is F, Y, V, or S] ARLWSGYALDI (C-4) X C16 X C26 X C36 X C46 X C56 X C66 X C76 X C86 X C96 X C106 X C116 X C126 X C136 (C-5) [wherein, X C16 is A or S, X C26 is R, T, or K, X C36 is D, N, S, or Y, X C46 is G, S, or Y, X C56 is G, I, or D, X C66 is Y, H, S, T, K, or E, X C76 is S, I, or D, X C86 is P or Y, X C96 is I, Y, G, P, T, A, or V, X C106 is N, S, Y, P, G, or F, X C116 is F, C, M, or L, X C126 is Q or D, X C136 is F, Y, V, or S] VX C27 PLLVX C77 HGX C107 X C117 X C127 X C137 X C147 (C-6) [wherein, X C27 is S or K, X C77 is V, Y, or L, X C107 is I, Y, G, P, T, A, or V, X C117 is N, S, Y, P, G, or F, X C127 is F, C, M, or L, X C137 is Q or D, X C147 is F, Y, V, or S] VSPSX C58 X C68 X C78 X C88 X C98 X C108 (C-7) [wherein, X C58 is G or A, X C68 is I, Y, G, P, T, A, or V, X C78 is N, S, Y, P, G, or F, X C88 is F, C, M, or L, X C98 is Q or D, X C108 is F, Y, V, or S] SPSX C49 X C59 X C69 X C79 X C89 X C99 (C-8) [wherein, X C49 is G or A, X C59 is I, Y, G, P, T, A, or V, X C69 is N, S, Y, P, G, or F, X C79 It is F, C, M, or L. X C89 is Q or D, X C99 [is F, Y, V, or S] A heavy chain CDR3 consisting of an amino acid sequence represented by one of the following, The following equations (D-1) to (D-3): SX D21 X D31 X D41 X D51 X D61 X D71 (D-1) [In the formula, X D21 is E, D, Q, or G, X D31 is H, Y, G, or S, X D41 is R, S, I, K, or L, X D51 is S, H, N, or F, X D61 is Y or K, X D71 [is Y, S, W, or N] SX D23 X D33 X D43 X D53 X D63 X D73 GKNKDL (D-2) [In the formula, X D23 is E, D, Q, or G, X D33 is H, Y, G, or S, X D43 is R, S, I, K, or L, X D53 is S, H, N, or F, X D63is Y or K, X D73 is Y, S, W, or N] NIGGKT (D-3) a light chain CDR1 consisting of an amino acid sequence represented by any of the following, the following formulas (E-1) to (E-3): X E11 X E21 SX E41 GSX E71 (E-1) [wherein, X E11 is L, V, or I, X E21 is K, N, or E, X E41 is D, Y, or K, X E71 is H or L] VGTSDIVG (E-2) X E12 X E22 X E32 (E-3) [wherein, X E12 is K, S, or W, X E22 is A or H, X E32 is N or S] a light chain CDR2 consisting of an amino acid sequence represented by any of the following, the following formulas (F-1) to (F-7): GX F21 X F31 X F41 X F51 X F61 X F71 X F81 X F91 X F101 (F-1) [wherein, X F21 is V or A, X F31 is S, D, N, G, or K, X F41 is Y, F, V, L, or H, X F51 is S, K, or N, X F61 is G or N, X F71 is G, A, E, or K, X F81 is H, S, Y, or V, X F91 is N, G, or S, X F101 [is I, V, L, or Y] GX F23 X F33 X F43 X F53 X F63 X F73 X F83 X F93 YX F113 (F-2) [In the formula, X F23 is V or A, X F33 is S, D, N, or G, X F43 is Y, F, V, L, or H, X F53 is S, K, or N, X F63 is G or N, X F73 is G, A, E, or K, X F83 is H, S, or Y, X F93 is N, G, or S, X F113 [is I, V, L, or Y] GX F24 X F34 X F44 X F54 IX F74 X F84 X F94 YX F114 YX F134 (F-3) [In the formula, X F24 is V or A, X F34 is S, D, N, or G, X F44 is Y, F, V, L, or H, X F54 is S, K, or N, X F74 is G or N, X F84 is G, A, E, or K, X F94 is H, S, or Y, X F114 is N, G, or S, X F134 [is I, V, L, or Y] GX F25 X F35 X F45 X F55 X F65 X F75 X F85 X F95 (F-4) [In the formula, X F25 is V or A, X F35 is S, D, N, G, or K, X F45 is Y, F, V, L, or H, X F55 is S, K, or N, X F65 is G or N, X F75 is G, A, E, or K, X F85 is H, S, Y, or V, X F105 [is I, V, L, or Y] QYDSTPPLT (F-5) QVWDSYTYV (F-6) QFINGPLT (F-7) A light chain CDR3 consisting of an amino acid sequence represented by one of the following: An antibody or a fragment thereof (antigen-binding fragment) containing [the specified substance]. Section 3. A heavy chain CDR1 consisting of an amino acid sequence represented by formula (A-1), A heavy chain CDR2 consisting of an amino acid sequence represented by formula (B-1) or (B-2), A heavy chain CDR3 consisting of an amino acid sequence represented by any of the formulas (C-1), (C-2), (C-4), and (C-6)~(C-8), A light chain CDR1 consisting of an amino acid sequence represented by formula (D-1), A light chain CDR2 consisting of an amino acid sequence represented by any of the formulas (E-1) to (E-3), A light chain CDR3 consisting of an amino acid sequence represented by any of the formulas (F-1) to (F-5) and The antibody or fragment thereof as described in item 2, including the antibody described in item 2. Section 4. The following formula (A-1-1): G FX A32 X A42 X A52 X A62 X A72 X A82 (A-1-1) [In the formula, X A32 is T or S, X A42 is F or L, X A52 is D, S, N, or T, X A62 is D, S, or N, X A72 is Y or F, X A82 [is G, W, S, or Y] A heavy chain CDR1 consisting of an amino acid sequence represented by, The following equations (B-1-1) and (B-2-1): IX B22 X B32 X B42 X B52 X B62 X B72 X B82 (B-1-1) [In the formula, X B22 is S, N, D, or H, X B32 is Y, G, or P, X B42 is S or D, X B52 is G or S, X B62 is G, D, T, or N, X B72 is R, K, T, S, G, or Y, X B82 [is T or I] IWX B34 X B44 X B54 X B64 X B74 (B-2-1) [In the formula, X B34 is Y or S, X B44 is D or G, X B54 is G or A, X B64 is R or D, X B74 [is T or I] A heavy chain CDR2 consisting of an amino acid sequence represented by one of the following, The following equations (C-1-1), (C-2-1), (C-6-1), (C-7-1), and (C-8-1): X C12 RDGGSYX C82 SPIX C122 X C132 QX C152 (C-1-1) [In the formula, X C12 is A or S, X C82 is Y, H, or S, X C122 is N or S, X C132 is F or C, X C152 [is F or Y] ATX C34 X C44 X C54 X C64 X C74 X C84 (C-2-1) [In the formula, X C34 is N or S, X C44 is Y or G, X C54 is N or P, X C64 is F or M, X C74 is Q or D, X C84 [is Y or V] VSPLLVVHGPFFQY (C-6-1) VSPSGAGLDV (C-7-1) SPSAVGFDV (C-8-1) A heavy chain CDR3 consisting of an amino acid sequence represented by one of the following, The following equations (D-1-1) to (D-1-7): SEYKHYN (D-1-1) SEHRSYY (D-1-2) SDYSHYS (D-1-3) SQGINKW (D-1-4) SGHSSYY (D-1-5) SEHSSYY (D-1-6) SDHSSYY (D-1-7) A light chain CDR1 consisting of an amino acid sequence represented by one of the following, The following equations (E-1-1), (E-2), and (E-3-1): X E12 X E22 SDGSX E72 (E-1-1) [In the formula, X E12 is L, V, or I, X E22 is K or E, X E72 [is H or L] VGTSDIVG (E-2) KAN (E-3-1) A light chain CDR2 consisting of an amino acid sequence represented by one of the following, The following formulas (F-1-1), (F-2-1), (F-3-1), (F-4-1), and (F-5): GVSX F42 SGGHX F92 X F102 (F-1-1) [In the formula, X F42 is Y or F, X F92 is N or G, X F102 [is I or V] GADYSGASSYV (F-2-1) GANHNIGESYGYV (F-3-1) GVX F36 YX F56 X F66 X F76 X F86 X F96 (F-4-1) [In the formula, X F36 is S, G, or K, X F56 is S or N, X F66 is G or N, X F76 is G or K, X F86 is Y or V, X F96 [is V or Y] QYDSTPPLT (F-5) A light chain CDR3 consisting of an amino acid sequence represented by one of the following: An antibody or a fragment thereof (antigen-binding fragment) containing [the specified substance]. Section 5. A heavy chain CDR1 consisting of an amino acid sequence shown in any of sequence numbers 3 to 11, or an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences, A heavy chain CDR2 consisting of an amino acid sequence shown in any of sequence numbers 12-25 or an amino acid sequence in which 1-3 amino acids are mutated in these amino acid sequences, A heavy chain CDR3 consisting of an amino acid sequence shown in any of sequence numbers 26-41 or an amino acid sequence in which 1-3 amino acids are mutated in these amino acid sequences, A light chain CDR1 consisting of an amino acid sequence shown in any of sequence numbers 42-50 or an amino acid sequence in which 1-3 amino acids are mutated in these amino acid sequences, A light chain CDR2 consisting of an amino acid sequence shown in any of sequence numbers 51-58, or an amino acid sequence in which 1-3 amino acids are mutated in these amino acid sequences, or an amino acid sequence represented by KAN, SHN, or WAS, A light chain CDR3 consisting of an amino acid sequence shown in any of sequence numbers 59-73 or an amino acid sequence in which 1-3 amino acids are mutated in these amino acid sequences. The antibody or fragment thereof as described in item 2, including the antibody described in item 2. Section 5a. A heavy chain CDR1 consisting of an amino acid sequence shown in any of sequence numbers 3-9 or an amino acid sequence in which 1-3 amino acids are mutated in these amino acid sequences, A heavy chain CDR2 consisting of an amino acid sequence shown in any of sequence numbers 12-19 and 25, or an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences, A heavy chain CDR3 consisting of an amino acid sequence shown in any of sequence numbers 26-29, 32, 33, 37, 40, and 41, or an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences, A light chain CDR1 consisting of an amino acid sequence shown in any of sequence numbers 42-48 or an amino acid sequence in which 1-3 amino acids are mutated in these amino acid sequences, A light chain CDR2 consisting of an amino acid sequence shown in any of sequence numbers 51-54 and 57, or an amino acid sequence in which 1-3 amino acids are mutated in these amino acid sequences, or an amino acid sequence represented by KAN, A light chain CDR3 consisting of an amino acid sequence shown in any of sequence numbers 59-61, 64-68, and 71, or an amino acid sequence in which 1-3 amino acids are mutated in these amino acid sequences. The antibody or fragment thereof as described in item 2, including the antibody described in item 2. Section 6. The antibody or fragment thereof according to item 5, wherein the mutation is a conservative substitution. Section 6a. The antibody or fragment thereof according to item 5a, wherein the mutation is a conservative substitution. Section 7. An antibody or fragment thereof, as described in any of items 2 to 6, that specifically binds to the amino acid sequence shown in SEQ ID NO: 1. Section 7a. An antibody or fragment thereof, as described in item 5a or 6a, that specifically binds to the amino acid sequence shown in SEQ ID NO: 1. Section 8. The following formula (G-1) or (G-2): GX G21 X G31 X G41 X G51 X G61 X G71 X G81 (G-1) [In the formula, X G21 is F or L, X G31 is I, T, S, or F, X G41 is F or L, X G51 is S, N, T, R, or D, X G61 is N, T, or D, X G71 is Y, S, or H, X G81 [is F, T, Y, W, or G] KLSERY (G-2) A heavy chain CDR1 consisting of an amino acid sequence represented by, The following equations (H-1) to (H-3): IX H21 X H31 X H41 X H51 X H61 X H71 X H81 (H-1) [In the formula, X H21 is S, G, K, or N, X H31 is G, N, S, Y, T, or P, X H41 is D, S, T, or A, X H51 is S, G, or D, XH61 is T, S, A, G, or R, X H71 is Y, K, N, T, I, or S, X H81 [is I, T, P, or V] MWSDGDT (H-2) NDS (H-3) A heavy chain CDR2 consisting of an amino acid sequence represented by one of the following, The following equations (I-1) to (I-11): X I11 X I21 X I31 X I41 X I51 X I61 X I71 (I-1) [In the formula, X I11 is S or T, X I21 is I or T, X I31 is N, L, D, or T, X I41 is W or G, X I51 is G, V, F, or N, X I61 is D, G, F, or N, X I71 [is V or L] ARTSRIVADLVTMGYALDF (I-2) ARDVVKTGTTGLPFDL (I-3) AREGVITVGTWGDV (I-4) ATSEY (I-5) TTATDV (I-6) TPATDV (I-7) SPMTIHGLDT (I-8) GSGWV (I-9) GSGWY (I-10) LSSESDDARV (I-11) A heavy chain CDR3 consisting of an amino acid sequence represented by one of the following, The following equations (J-1) to (J-3): X J11 LX J31 X J41 X J51 Y (J-1) [In the formula, X J11 is K or A, X J31 is S or P, X J41 is N, E, K, T, or D, X J51 [is Q, R, K, or E] SX J23 SLX J53 X J63 X J73 DGX J103 TX J123 (J-2) [In the formula, X J23 is E or Q, X J53 is L or V, X J63 is H, D, or Y, X J73 is S or G, X J103 is N, K, or Y, X J123 [is Y or F] NIGGKA (J-3) A light chain CDR1 consisting of an amino acid sequence represented by one of the following, The following formula (K-1): X K11 X K21 X K31 (K-1) [In the formula, X K11 is K, N, L, or E, X K21 is D, G, V, or I, X K31 [is S, N, or D] A light chain CDR2 consisting of an amino acid sequence represented by, The following equations (L-1) to (L-6): LSX L31 X L41 X L51 XL61 X L71 X L81 X L91 X L101 X L111 (L-1) [wherein, X L31 is R, S, A, or G, X L41 is Q, E, or D, X L51 is S or G, X L61 is D, S, or N, X L71 is D, G, or T, X L81 is A, T, or Y, X L91 is A, T, or Y, X L101 is S, R, or W, X L111 [is V, L, or Y] LSX L33 X L43 X L53 X L63 X L73 X L83 X L93 X L103 (L-2) [In the formula, X L33 is R, S, A, or G, X L43 is Q, E, or D, X L53 is S or G, X L63 is D, S, or N, X L73 is D, G, or T, X L83 is A, T, or Y, X L93 is S, R, or W, X L103 [is V, L, or Y] X L14 QX L34 X L44 X L54 X L64 PX L84 XL94 (L-3) [In the formula, X L14 is L or V, X L34 is A or T, X L44 is T or S, X L54 is H or N, X L64 is D, N, or V, X L84 is Y, V, I, or L, X L94 [is T or S] QX L27 X L37 X L47 X L57 PX L77 X L87 (L-4) [In the formula, X L27 is A or T, X L37 is T or S, X L47 is H or N, X L57 is D, N, or V, X L77 is Y, V, I, or L, X L87 [is T or S] QVYDSSSFV (L-5) WQGTDFPR (L-6) A light chain CDR3 consisting of an amino acid sequence represented by one of the following: An antibody or a fragment thereof (antigen-binding fragment) containing [the specified substance]. Section 9. A heavy chain CDR1 consisting of an amino acid sequence represented by formula (G-1), A heavy chain CDR2 consisting of an amino acid sequence represented by formula (H-1) or (H-2), A heavy chain CDR3 consisting of an amino acid sequence represented by any of formulas (I-1) to (I-3) and (I-5) to (I-9), A light chain CDR1 consisting of an amino acid sequence represented by any of formulas (J-1) to (J-3), A light chain CDR2 consisting of an amino acid sequence represented by formula (K-1), A light chain CDR3 consisting of an amino acid sequence represented by any of the formulas (L-1) to (L-3), (L-5), and (L-6) The antibody or fragment thereof as described in item 8, including the antibody described in item 8. Section 10. The following formula (G-1-1): GX G22 X G32 X G42 X G52 X G62 X G72 X G82 (G-1-1) [In the formula, X G22 is F or L, X G32 is I, T, or S, X G42 is F or L, X G52 is S, N, T, or R, X G62 is N or T, X G72 is Y, S, or H, X G82 [is F, T, Y, or W] A heavy chain CDR1 consisting of an amino acid sequence represented by, The following formula (H-1-1) or (H-2): IX H22 X H32 X H42 X H52 X H62 X H72 X H82 (H-1-1) [In the formula, X H22 is S, G, K, or N, X H32 is G, N, S, T, or P, X H42 is D, S, or A, X H52 is S, G, or D, X H62 is T, S, G, or R, X H72 is Y, K, N, T, I, or S, X H82 [is I, T, P, or V] MWSDGDT (H-2) A heavy chain CDR2 consisting of the amino acid sequence represented by, The following equations (I-1-1), (I-2), (I-3), and (I-5)~(I-9): X I12 X I22 X I32 X I42 X I52 X I62 X I72 (I-1-1) [In the formula, X I12 is S or T, X I22 is I or T, X I32 is N, L, or T, X I42 is W or G, X I52 is G, V, F, or N, X I62 is D, F, or G, X I72 [is V or L] ARTSRIVADLVTMGYALDF (I-2) ARDVVKTGTTGLPFDL (I-3) ATSEY (I-5) TTATDV (I-6) TPATDV (I-7) SPMTIHGLDT (I-8) GSGWV (I-9) A heavy chain CDR3 consisting of an amino acid sequence represented by one of the following, The following formulas (J-1-1), (J-2-1), and (J-3): KLSX J42 X J52 Y (J-1-1) [In the formula, X J42 is N, E, or K, X J52[is Q, R, or E] SX J24 SLX J54 X J64 SDGX J104 TX J124 (J-2-1) [In the formula, X J24 is E or Q, X J54 is L or V, X J64 is H, D, or Y, X J104 is N, K, or Y, X J124 [is Y or F] NIGGKA (J-3) A light chain CDR1 consisting of an amino acid sequence represented by one of the following, The following equations (K-1-1) to (K-1-7): NDD (K-1-1) KDS (K-1-2) NGN (K-1-3) NDS (K-1-4) KVS (K-1-5) LIS (K-1-6) EVS (K-1-7) A light chain CDR2 consisting of an amino acid sequence represented by one of the following, The following equations (L-1-1), (L-2-1), (L-3-1), (L-5), and (L-6): LSX L32 X L42 SX L62 DAAX L102 V (L-1-1) [In the formula, X L32 is R or S, X L42 is Q or E, X L62 is D or N, X L102 [is S, R, or W] LSGESDDAWV (L-2-1) X L15 QX L35THDPYT (L-3-1) [In the formula, X L15 is L or V, X L35 [is A or T] QVYDSSSFV (L-5) WQGTDFPR (L-6) A light chain CDR3 consisting of an amino acid sequence represented by one of the following: An antibody or a fragment thereof (antigen-binding fragment) containing [the specified substance]. Section 11. A heavy chain CDR1 consisting of an amino acid sequence shown in any of sequence numbers 74-84 or an amino acid sequence in which 1-3 amino acids are mutated in these amino acid sequences, A heavy chain CDR2 consisting of an amino acid sequence shown in any of sequence numbers 85-101, an amino acid sequence in which 1-3 amino acids are mutated in these amino acid sequences, or an amino acid sequence represented by NDS, A heavy chain CDR3 consisting of an amino acid sequence shown in any of sequence numbers 102-118 or an amino acid sequence in which 1-3 amino acids are mutated in these amino acid sequences, A light chain CDR1 consisting of an amino acid sequence shown in any of sequence numbers 119-133 or an amino acid sequence in which 1-3 amino acids are mutated in these amino acid sequences, A light chain CDR2 consisting of an amino acid sequence represented by NDD, KDS, NGN, NDS, KVS, LIS, or EVS, A light chain CDR3 consisting of the amino acid sequence shown in any of sequence numbers 134-149 or an amino acid sequence in which 1-3 amino acids are mutated in these sequences. The antibody or fragment thereof as described in item 8, including the antibody described in item 8. Section 11a. A heavy chain CDR1 consisting of an amino acid sequence shown in any of sequence numbers 74-81 or an amino acid sequence in which 1-3 amino acids are mutated in these amino acid sequences, A heavy chain CDR2 consisting of an amino acid sequence shown in any of sequence numbers 85-90, 92-95, and 101, or an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences, A heavy chain CDR3 consisting of an amino acid sequence shown in any of sequence numbers 102-106, 109, 110, and 112-116, or an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences, A light chain CDR1 consisting of an amino acid sequence shown in any of sequence numbers 119-121, 125-129, and 133, or an amino acid sequence in which 1 to 3 amino acids are mutated in these amino acid sequences, A light chain CDR2 consisting of an amino acid sequence represented by NDD, KDS, NGN, NDS, KVS, LIS, or EVS, A light chain CDR3 consisting of an amino acid sequence shown in any of sequence numbers 134-136, 140-142, 148, and 149, or an amino acid sequence in which 1 to 3 amino acids are mutated in these sequences. The antibody or fragment thereof as described in item 8, including the antibody described in item 8. Section 12. The antibody or fragment thereof according to item 11, wherein the mutation is a conservative substitution. Section 12a. The antibody or fragment thereof as described in item 11a, wherein the mutation is a conservative substitution. Section 13. An antibody or fragment thereof, as described in any of items 8 to 12, that specifically binds to the amino acid sequence shown in SEQ ID NO: 2. Section 13a. An antibody or fragment thereof according to item 11a or 12a, which specifically binds to the amino acid sequence shown in SEQ ID NO: 2. Section 14. An antibody or fragment thereof against the nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), wherein the epitope is located in a region of the nucleocapsid protein consisting of the amino acid sequence shown in any of SEQ ID NOs. 150 to 152 (antigen-binding fragment). Section 15. The antibody or fragment thereof according to any one of items 1 to 14, wherein the antibody is a monoclonal antibody. Section 15a. The antibody or fragment thereof according to any one of items 5a, 6a, 11a, and 12a, wherein the antibody is a monoclonal antibody. Section 16. A polynucleotide containing the coding sequence of an antibody or fragment thereof as described in any of items 1 to 15. Section 16a. A polynucleotide comprising the coding sequence of an antibody or fragment thereof as described in any of items 5a, 6a, 11a, 12a, and 15a. Section 17. Cells containing the polynucleotides described in item 16. Section 17a. Cells containing the polynucleotides described in item 16a. Section 18. A reagent or pharmaceutical comprising an antibody or fragment thereof as described in any of items 1 to 15, a polynucleotide as described in item 16, or a cell as described in item 17. Section 18a. A reagent or pharmaceutical comprising an antibody or fragment thereof as described in any of items 5a, 6a, 11a, 12a, and 15a, a polynucleotide as described in item 16a, or a cell as described in item 17a. Section 19. A kit for detecting the nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprising an antibody or fragment thereof as described in any of items 1 to 15. Section 19a. A kit for detecting the nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprising an antibody or fragment thereof as described in any of items 5a, 6a, 11a, 12a, and 15a. Section 20. Two or more antibodies or fragments thereof that specifically bind to the amino acid sequence shown in SEQ ID NO: 1, and each of the two or more antibodies or fragments thereof recognizes a different epitope in the amino acid sequence shown in SEQ ID NO: 1, and / or Two or more antibodies or fragments thereof that specifically bind to the amino acid sequence shown in SEQ ID NO: 2, and each of the two or more antibodies or fragments thereof recognizes a different epitope in the amino acid sequence shown in SEQ ID NO: 2. A kit for detecting the nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including [specific protein name]. Section 21. Two or more antibodies or fragments thereof that specifically bind to the amino acid sequence shown in Sequence ID No. 1 are antibodies or fragments thereof described in any of items 2 to 7, or antibodies or fragments thereof consisting of an amino acid sequence having 90% or more sequence identity with respect to the amino acid sequence of said antibody or fragment. Two or more antibodies or fragments thereof that specifically bind to the amino acid sequence shown in Sequence ID No. 2 are antibodies or fragments thereof described in any of items 8 to 14, or antibodies or fragments thereof consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of said antibody or fragment. The kit described in item 20. Section 21a. Two or more antibodies or fragments thereof that specifically bind to the amino acid sequence shown in Sequence ID No. 1 are the antibodies or fragments thereof described in item 5a or 6a. Two or more antibodies or fragments thereof that specifically bind to the amino acid sequence shown in Sequence ID No. 2 are the antibodies or fragments thereof described in item 11a or 12a. The kit described in item 20. Section 22. A kit for detecting the nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by immunochromatography, enzyme immunoassay, chemiluminescence immunoassay, chemiluminescence enzyme immunoassay, radioimmunoassay, electrochemiluminescence immunoassay, immunoturbidimetry, or latex agglutination, as described in any of sections 19-21. Section 22a. A kit according to item 19a or 21a for detecting the nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by immunochromatography, enzyme immunoassay, chemiluminescence immunoassay, chemiluminescence enzyme immunoassay, radioimmunoassay, electrochemiluminescence immunoassay, immunoturbidimetry, or latex agglutination. Section 23. A kit according to any one of claims 19 to 22, wherein two or more antibodies or fragments thereof that specifically bind to the amino acid sequence indicated by Sequence ID No. 1 or 2 are immobilized, and the remainder is labeled. Section 23a. The kit according to any one of claims 19a, 21a, and 22a, wherein two or more antibodies or fragments thereof that specifically bind to the amino acid sequence indicated by SEQ ID NO: 1 or 2 are immobilized, and the remainder is labeled. Section 24. A method for detecting the nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprising the following steps (1) to (3): (1) A step of immobilizing a first antibody or a fragment thereof that specifically binds to the amino acid sequence indicated by SEQ ID NO: 1 or 2 onto a solid phase. (2) A step of applying to the solid phase a second antibody or fragment thereof that specifically binds to the nucleocapsid protein of SARS-CoV-2 and the amino acid sequence indicated by SEQ ID NO: 1 or 2 (wherein the second antibody or fragment thereof recognizes a different epitope in the amino acid sequence to which the first antibody or fragment thereof specifically binds and is labeled with a labeling substance), and (3) A step of detecting a label on the solid phase that originates from a labeling substance of the second antibody or a fragment thereof bound to the nucleocapsid protein via the nucleocapsid protein. Methods that include... Section 25. The method according to claim 24, wherein the first antibody or fragment thereof and the second antibody or fragment thereof are selected from the group consisting of antibodies or fragment thereof described in any of claims 2 to 15, and antibodies or fragment thereof having an amino acid sequence having 90% or more sequence identity with respect to the amino acid sequence of said antibody or fragment thereof. Section 25a. The method according to claim 24, wherein the first antibody or fragment thereof and the second antibody or fragment thereof are selected from the group consisting of antibodies or fragment thereof described in any of claims 5a, 6a, 11a, 12a, and 15a, and antibodies or fragment thereof consisting of an amino acid sequence having 90% or more sequence identity with respect to the amino acid sequence of said antibody or fragment thereof. [Effects of the Invention]

[0011] The present invention provides an antibody or fragment thereof that specifically binds to the NTD or CTD of the SARS-CoV-2 N protein. The present invention also provides a kit and method for detecting the SARS-CoV-2 N protein with high sensitivity. [Modes for carrying out the invention]

[0012] 1. Definitions etc. In this specification, amino acids may be natural or unnatural amino acids. Examples of unnatural amino acids include, but are not limited to, citrulline, ornithine, ε-acetyl-lysine, β-alanine, aminobenzoic acid, 6-aminocaproic acid, aminobutyric acid, hydroxyproline, mercaptopropionic acid, 3-nitrotyrosine, norleucine, and pyroglutamic acid. Furthermore, amino acids may be, for example, L-amino acids, D-amino acids, or DL-amino acids.

[0013] In this specification, amino acid sequence identity refers to the degree of amino acid agreement when two or more amino acid sequences being compared are optimally aligned. Amino acid sequence identity can be calculated using commercially available analysis tools or tools available via telecommunication lines (the Internet). For example, it can be calculated using the commercially available software GENETYX (Genetics Co., Ltd.) or using the default parameters of the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignment search tool) (http: / / www.ncbi.nlm.nih.gov / BLAST / ).

[0014] The amino acid sequences disclosed herein may have one or more amino acids (e.g., 1, 2, or 3) deleted, substituted, or modified, or one or more amino acids (e.g., 1, 2, or 3) inserted or added to the sequence, as long as they do not inhibit the binding of SARS-CoV-2 to the N protein.

[0015] Amino acid substitutions are preferably substitutions with other amino acids that have similar structure and / or properties (conservative substitutions). Examples of conservative substitutions include substitutions within the groups shown in Table 1.

[0016] [Table 1]

[0017] Examples of amino acid modifications include the modification of functional groups such as amino groups, carboxyl groups, hydroxyl groups, and sulfhydryl (SH) groups. These functional group modifications may include, for example, glycosylation, methylation, esterification, amidation, PEGylation, phosphorylation, hydroxylation, binding with protecting groups such as t-butoxycarbonyl (Boc) and 9-fluorenylmethyloxycarbonyl (Fmoc) groups, biotinylation, binding with fluorescent dyes such as fluorescein isothiocyanate (FITC), and binding with enzymes such as peroxidase (HRP) and alkaline phosphatase (ALP).

[0018] In this specification, the antibody may be a monoclonal antibody or a polyclonal antibody, but it is preferably a monoclonal antibody. The antibody can be any isotype, such as IgG, IgA, IgD, IgE, IgM, etc. The antibody may be a human antibody or a non-human antibody. Examples of non-human antibodies include, but are not limited to, mouse antibodies, rat antibodies, guinea pig antibodies, etc. The antibody may be a chimeric antibody, such as a mouse-human chimeric antibody. The antibody may be a partially or fully humanized antibody.

[0019] In this specification, the antibody fragment is not particularly limited as long as it includes heavy chain CDR1-3 and light chain CDR1-3, and examples include Fv, Fab, Fab', (Fab')2, scFv, scFv-Fc, diabody, triabody, tetrabody, minibody, etc. The antibody fragment is preferably an antigen-binding fragment (antigen-binding fragment).

[0020] In this specification, heavy chain CDR1-3 and light chain CDR1-3 are defined based on the IMGT method.

[0021] In this specification, nucleotides such as DNA and RNA may be subjected to known chemical modifications as exemplified below. To prevent degradation by hydrolytic enzymes such as nucleases, the phosphate residue of each nucleotide may be replaced with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithionate. The hydroxyl group at position 2 of the sugar (ribose) of each ribonucleotide may also be replaced with -OR (where R represents, for example, -CH3, -CH2CH2OCH3, -CH2CH2NHC(NH)NH2, -CH2CONHCH3, -CH2CH2CN, etc.). Furthermore, the base portion (pyrimidine, purine) may be chemically modified, for example, by introducing a methyl group or cationic functional group at position 5 of the pyrimidine base, or by substituting the carbonyl group at position 2 with a thiocarbonyl group. Furthermore, the phosphate portion or hydroxyl portion may be modified with, for example, biotin, an amino group, a lower alkylamine group, or an acetyl group, but is not limited to these.

[0022] In this specification, the SARS-CoV-2 N protein (antigen) has the amino acid sequence disclosed in GenBank accession number (MN908947). Furthermore, the SARS-CoV N protein has the amino acid sequence disclosed in GenBank accession number (AY278741).

[0023] 2. An antibody or fragment thereof that specifically binds to the NTD of the SARS-CoV-2 N protein. The NTD (sometimes simply referred to as NTD) of the SARS-CoV-2 N protein refers to the amino acid region from the 1st to the 172nd N-terminus of the N protein, and has the following amino acid sequence: MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFY(Sequence ID 1)

[0024] It is preferable that an antibody or fragment thereof that specifically binds to the NTD of SARS-CoV-2 does not bind to the NTD of SARS-CoV, or has low binding affinity to the NTD of SARS-CoV. In the amino acid sequence shown in SEQ ID NO: 1, for example, the 63rd D, 65th K, 79th S, 94th I, 103rd D, 120th G, 128th D, 131st I, 152nd A, and 157th I are different from the corresponding amino acids of the NTD of SARS-CoV. It is preferable that an antibody or fragment thereof that specifically binds to the NTD of SARS-CoV-2 binds to a region (or epitope) in SEQ ID NO: 1 that contains one or more amino acids selected from the group consisting of the 63rd D, 65th K, 79th S, 94th I, 103rd D, 120th G, 128th D, 131st I, 152nd A, and 157th I.

[0025] In one embodiment, an antibody or fragment thereof that specifically binds to the NTD of SARS-CoV-2 may bind to the amino acid region from the 1st to 180th position from the N-terminus of the N protein, preferably to the RNA-binding domain of the NTD, and preferably to the amino acid region from the 44th to 180th or 44th to 172nd position from the N-terminus of the N protein.

[0026] Antibodies or fragments thereof that specifically bind to SARS-CoV-2 NTDs can be produced by combining known methods, for example, by a method including the following steps (P1) to (P6): (P1) A step to obtain cells that produce antibodies that specifically bind to SARS CoV2 N protein from antibody-producing cells of animals immunized with SARS CoV2 N protein, using SARS CoV2 N protein labeled with a fluorescent label that has higher staining selectivity for the endoplasmic reticulum compared to staining selectivity for organelles other than the endoplasmic reticulum. (P2) A step to obtain an antibody variable region gene fragment from the cells obtained in step (P1), (P3) A step to obtain a gene expression unit from the antibody variable region gene fragment obtained in step (P2), (P4) A step of introducing the gene expression unit obtained in step (P3) into cells to express an antibody that binds to the N protein of SARS CoV2. (P5) A step of recovering the antibodies expressed in step (P4), and (P6) A step to obtain an antibody that specifically binds to NTDs from the antibody recovered in step (P5).

[0027] Step (P1) can be carried out in accordance with the method described in, for example, U.S. Patent Application Publication 2013 / 0029325 (which is incorporated into the specification in its entirety by reference) (ERIAA method).

[0028] Step (P2) can be carried out according to the method described in the literature (Rapid production of antigen-specific monoclonal antibodies from a variety of animals, BMC Biology 2012, 10:80) (MAGrahd method).

[0029] Step (P3) can be carried out in accordance with the method described in, for example, U.S. Patent Application Publication No. 2013 / 0023009 (which is incorporated into the specification in its entirety by reference) (TS-jPCT method).

[0030] Step (P4) can be carried out by known gene transfer methods, such as the calcium phosphate method, lipofection method, DEAE dextran method, electroporation method, or microinjection method. The cells into which the gene expression unit is introduced are not particularly limited as long as they can transiently express the antibody, and examples include mammalian cells such as CHO cells, HEK293 cells, Expi293 cells, 293F cells, 293T cells, and 293FT cells.

[0031] Step (P6) can be carried out by selecting an antibody that specifically binds to NTDs using a known screening method, such as enzyme immunosorbent assay (ELISA), Western blotting, or immunohistochemical staining.

[0032] Furthermore, the antibodies may be purified as needed by known methods, such as salting-out methods using ammonium sulfate, sodium sulfate, etc.; affinity chromatography using protein A, etc.; ion exchange chromatography using DEAE cellulose, etc.; gel filtration; or purification methods using His-tag, FLAG-tag, etc.

[0033] A method comprising steps (P1) to (P6) can be used to obtain multiple types of antibodies or fragments thereof that specifically bind to the NTDs of SARS-CoV-2 (where each antibody or fragment thereof differs from the others in the amino acid sequence of at least one of the heavy chain CDR1-3 and light chain CDR1-3).

[0034] In one embodiment, the heavy chain CDR1 of an antibody or fragment thereof that specifically binds to the NTD of SARS-CoV-2 is preferably composed of the following amino acid sequence: • An amino acid sequence represented by either formula (A-1) or (A-2) shown in Table 2, • An amino acid sequence having 90% or more (preferably 95% or more) identity with the said amino acid sequence, An amino acid sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) amino acids are mutated (preferably conserved substitutions).

[0035] [Table 2]

[0036] In equation (A-1), X A41 is preferably F or L, X A51 is preferably D, S, N, or T, and X A61 is preferably D, N, S, or I, and more preferably D, N, or S, X A71 is preferably Y or F, and X A81 Preferably, it is G, W, S, or Y.

[0037] Formula (A-1) is preferably one selected from the group consisting of formula (A-1-1) or formulas (A-1-2) to (A-1-9), and more preferably one selected from the group consisting of formulas (A-1-2) to (A-1-8).

[0038] In equation (A-2), X A33 is preferably S, X A43 is preferably I, X A53 is preferably T, X A63 is preferably T, X A73 is preferably N or S, X A83 is preferably G or Y, and X A93 is preferably Y or F, more preferably Y and X A103 is preferably T, G, or W, and more preferably T or G.

[0039] Formula (A-2) is preferably formula (A-2-1), and more preferably formula (A-2-2).

[0040] The heavy chain CDR1 of an antibody or fragment thereof that specifically binds to the NTD of SARS-CoV-2 is preferably an amino acid sequence selected from formula (A-1), more preferably formula (A-1-1) or the group consisting of formulas (A-1-2) to (A-1-9), and even more preferably the group consisting of formulas (A-1-2) to (A-1-8), or an amino acid sequence having 90% or more identity with said amino acid sequence. The identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.

[0041] In one embodiment, the heavy chain CDR2 of an antibody or fragment thereof that specifically binds to the NTD of SARS-CoV-2 is preferably composed of the following amino acid sequence: • An amino acid sequence represented by either formula (B-1) or (B-2) shown in Table 3, • An amino acid sequence having 90% or more (preferably 95% or more) identity with the said amino acid sequence, An amino acid sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) amino acids are mutated (preferably conserved substitutions).

[0042] [Table 3]

[0043] In equation (B-1), X B21 is preferably S, N, D, or H, and X B31 is preferably Y, G, or P, and X B41 is preferably S or D, X B51 is preferably G or S, X B61 is preferably G, D, T, or N, and X B71 is preferably R, K, T, S, G, or Y, and X B81 It is preferably T or I.

[0044] Formula (B-1) is preferably one selected from the group consisting of formula (B-1-1) or formulas (B-1-2) to (B-1-13), more preferably one selected from the group consisting of formulas (B-1-2) to (B-1-10), and even more preferably one selected from the group consisting of formulas (B-1-2) to (B-1-9).

[0045] In equation (B-2), X B23 is preferably S, N, D, or W, and more preferably W, X B33 is preferably Y, G, or S, and more preferably Y or S, X B43 is preferably D or G, X B53 is preferably G, S, or A, and more preferably G or A, X B63 is preferably R, K, T, or D, and more preferably R or D, X B73 It is preferably T or I.

[0046] Formula (B-2) is preferably formula (B-2-1), more preferably formula (B-2-2) or (B-2-3), and even more preferably formula (B-2-3).

[0047] The heavy chain CDR2 of an antibody or fragment thereof that specifically binds to the NTD of SARS-CoV-2 is preferably an amino acid sequence selected from the group consisting of formulas (B-1-1) and (B-2-1), or the group consisting of formulas (B-1-2) to (B-1-13), (B-2-2), and (B-2-3), more preferably the group consisting of formulas (B-1-2) to (B-1-10), (B-2-2), and (B-2-3), and particularly preferably the group consisting of formulas (B-1-2) to (B-1-9) and (B-2-3), or an amino acid sequence having 90% or more identity with said amino acid sequence. The identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.

[0048] In one embodiment, the heavy chain CDR3 of an antibody or fragment thereof that specifically binds to the NTD of SARS-CoV-2 is preferably composed of the following amino acid sequence: • An amino acid sequence represented by any of the formulas (C-1) to (C-8) shown in Table 4, • An amino acid sequence having 90% or more (preferably 95% or more) identity with the said amino acid sequence, An amino acid sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) amino acids are mutated (preferably conserved substitutions).

[0049] [Table 4] TIFF2026071287000005.tif245170

[0050] In equation (C-1), X C21 is preferably R, and X C31 It is preferably D, X C41 is preferably G, X C51 is preferably G, X C61is preferably S, X C71 is preferably Y or V, more preferably Y and X C81 is preferably Y, H, S, T, or K, and more preferably Y, H, or S, X C91 is preferably S, X C101 is preferably P, X C111 is preferably I, X C121 is preferably N, S, or Y, and more preferably N or S, X C131 is preferably F or C, X C141 Q is preferable, X C151 is preferably F, Y, or V, and more preferably F or Y.

[0051] Formula (C-1) is preferably one selected from the group consisting of formula (C-1-1) or formulas (C-1-2) to (C-1-7), and more preferably one selected from the group consisting of formulas (C-1-2) to (C-1-4).

[0052] In equation (C-2), X C13 It is preferably A, and X C23 is preferably T, X C33 is preferably N or S, X C43 is preferably Y or G, and X C53 is preferably N, S, or P, and more preferably N or P, X C63 is preferably F, C, or M, more preferably F or M, and X C73 is preferably Q or D, and X C83 is preferably F, Y, or V, and more preferably Y or V.

[0053] Formula (C-2) is preferably formula (C-2-1), and more preferably formula (C-2-2) or (C-2-3).

[0054] In equation (C-3), X C15 It is preferably A, and X C25 is preferably R, and X C35is preferably S, X C45 is preferably I, X C55 is preferably I, X C65 It is preferably A, and X C75 is preferably E, X C85 is preferably I, X C95 is preferably P, X C155 is preferably P, X C165 is preferably N, S, or P, and more preferably P and X C175 is preferably F, C, or M, and more preferably F and X C185 is preferably Q or D, more preferably D and X C195 is preferably F, Y, or V, and more preferably V.

[0055] Formula (C-3) is preferably formula (C-3-1).

[0056] In equation (C-5), X C16 It is preferably A, and X C26 is preferably K, X C36 Preferably Y, X C46 Preferably Y, X C56 It is preferably D, X C66 Preferably Y, X C76 It is preferably D, X C86 Preferably Y, X C96 is preferably T, X C106 is preferably G, X C116 is preferably F, C, or M, and more preferably F and X C126 is preferably Q or D, more preferably D and X C136 is preferably F, Y, or V, and more preferably V.

[0057] Formula (C-5) is preferably formula (C-5-1).

[0058] In equation (C-6), X C107 is preferably P, XC117 is preferably F, X C127 is preferably F, C, or M, and more preferably F and X C137 is preferably Q or D, more preferably Q and X C147 It is preferably Y or S.

[0059] Formula (C-6) is preferably one selected from the group consisting of formulas (C-6-1) to (C-6-3).

[0060] In equation (C-7), X C58 is preferably G, X C68 It is preferably A, and X C78 is preferably G, X C88 is preferably L, X C98 is preferably Q or D, more preferably D and X C108 is preferably F, Y, or V, and more preferably V.

[0061] Formula (C-7) is preferably formula (C-7-1).

[0062] In equation (C-8), X C49 It is preferably A, and X C59 is preferably V, X C69 is preferably G, X C79 is preferably F, C, or M, and more preferably F and X C89 is preferably Q or D, more preferably D and X C99 is preferably F, Y, or V, and more preferably V.

[0063] Formula (C-8) is preferably formula (C-8-1).

[0064] The heavy chain CDR3 of an antibody or fragment thereof that specifically binds to the NTD of SARS-CoV-2 is preferably from the group consisting of formulas (C-1), (C-2), (C-4), and (C-6) to (C-8), more preferably from the group consisting of formulas (C-1-1), (C-2-1), and (C-6) to (C-8), or formulas (C-1-2) to (C-1-7), (C-2-2), (C-2-3), (C-4), (C- The amino acid sequence is selected from the group consisting of formulas (C-1-2) to (C-1-5), (C-2-2), (C-2-3), (C-4), (C-6-1), (C-7-1), and (C-8-1), or from the group consisting of formulas (C-1-2) to (C-1-5), (C-2-2), (C-2-3), (C-4), (C-6-1), (C-7-1), and (C-8-1), or from an amino acid sequence having 90% or more identity with the said amino acid sequence. The identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.

[0065] In one embodiment, the light chain CDR1 of an antibody or fragment thereof that specifically binds to the NTD of SARS-CoV-2 is preferably composed of the following amino acid sequence: • An amino acid sequence represented by any of the formulas (D-1) to (D-3) shown in Table 5, • An amino acid sequence having 90% or more (preferably 95% or more) identity with the said amino acid sequence, An amino acid sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) amino acids are mutated (preferably conserved substitutions).

[0066] [Table 5]

[0067] Formula (D-1) is preferably one selected from the group consisting of formulas (D-1-1) to (D-1-7).

[0068] In equation (D-2), X D23 Q is preferable, X D33 is preferably S, X D43 is preferably L, X D53is preferably F, X D63 is preferably Y or K, more preferably Y and X D73 is preferably Y, S, or W, and more preferably S.

[0069] Formula (D-2) is preferably formula (D-2-1).

[0070] The light chain CDR1 of an antibody or fragment thereof that specifically binds to the NTD of SARS-CoV-2 preferably consists of an amino acid sequence selected from the group consisting of formula (D-1), more preferably formulas (D-1-1) to (D-1-7), or an amino acid sequence having 90% or more identity with said amino acid sequence. The identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.

[0071] In one embodiment, the light chain CDR2 of an antibody or fragment thereof that specifically binds to the NTD of SARS-CoV-2 is preferably composed of the following amino acid sequence: • An amino acid sequence represented by any of the formulas (E-1) to (E-3) shown in Table 6, • An amino acid sequence having 90% or more (preferably 95% or more) identity with the said amino acid sequence, An amino acid sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) amino acids are mutated (preferably conserved substitutions).

[0072] [Table 6]

[0073] In equation (E-1), X E21 is preferably K or E, and X E41 It is preferably D.

[0074] Formula (E-1) is preferably one selected from the group consisting of formula (E-1-1) or formulas (E-1-2) to (E-1-8), and more preferably one selected from the group consisting of formulas (E-1-2) to (E-1-5).

[0075] In equation (E-3), X E13 is preferably K, X E23 It is preferably A, and X E33 It is preferably N.

[0076] Formula (E-3) is preferably one selected from the group consisting of formulas (E-3-1) to (E-3-3), and more preferably formula (E-3-1).

[0077] The light chain CDR2 of an antibody or fragment thereof that specifically binds to the NTD of SARS-CoV-2 is preferably an amino acid sequence selected from the group consisting of formulas (E-1-1), (E-2), and (E-3), or the group consisting of formulas (E-1-2) to (E-1-8), (E-2), and (E-3-1) to (E-3-3), and more preferably from the group consisting of formulas (E-1-2) to (E-1-5), (E-2), and (E-3-1), or an amino acid sequence having 90% or more identity with said amino acid sequence. The identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.

[0078] In one embodiment, the light chain CDR3 of an antibody or fragment thereof that specifically binds to the NTD of SARS-CoV-2 is preferably composed of the following amino acid sequence: • An amino acid sequence represented by any of the formulas (F-1) to (F-7) shown in Table 7, • An amino acid sequence having 90% or more (preferably 95% or more) identity with the said amino acid sequence, An amino acid sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) amino acids are mutated (preferably conserved substitutions).

[0079] [Table 7]

[0080] In equation (F-1), X F21 is preferably V, X F31 is preferably S, X F41 is preferably Y, F, V, or L, and more preferably Y or F, X F51 is preferably S or K, more preferably S and X F61 is preferably G, X F71 is preferably G, X F81 is preferably H, X F91 is preferably N or G, X F101 is preferably I, V, or L, and more preferably I or V.

[0081] Formula (F-1) is preferably one selected from the group consisting of formula (F-1-1) or formulas (F-1-2) to (F-1-6), and more preferably formulas (F-1-2) to (F-1-4).

[0082] In equation (F-2), X F23 It is preferably A, and X F33 is preferably S or D, more preferably D and X F43 is preferably Y, F, V, or L, more preferably Y or F, and even more preferably Y and X F53 is preferably S or K, more preferably S and X F63 is preferably G, X F73 is preferably G or A, more preferably A and X F83 is preferably H or S, preferably S and X F93 is preferably S, X F113 is preferably I, V, or L, more preferably I or V, and even more preferably V.

[0083] Formula (F-2) is preferably formula (F-2-1).

[0084] In equation (F-3), X F24 It is preferably A, and X F34 is preferably S, D, or N, and more preferably N and X F44 is preferably Y, F, or H, and more preferably H and X F54 is preferably S or N, more preferably N and X F74 is preferably G, X F84 is preferably G or E, more preferably E and X F94 is preferably H or S, preferably S and X F114 is preferably G, X F134 is preferably I, V, or L, more preferably I or V, and even more preferably V.

[0085] Formula (F-3) is preferably formula (F-3-1).

[0086] In equation (F-4), X F25 is preferably V, X F35 is preferably S, G, or K, and X F45 Preferably Y, X F55 is preferably S or N, X F65 is preferably G or N, X F75 is preferably G or K, and X F85 is preferably Y or V, and X F95 It is preferably V or Y.

[0087] Formula (F-4) is preferably formula (F-4-1), more preferably one selected from the group consisting of formulas (F-4-2) to (F-4-6), and even more preferably one selected from the group consisting of formulas (F-4-2) to (F-4-4).

[0088] The light chain CDR3 of an antibody or fragment thereof that specifically binds to the NTD of SARS-CoV-2 is preferably an amino acid sequence selected from the group consisting of formulas (F-1) to (F-5), more preferably the group consisting of formulas (F-1-1), (F-2-1), (F-3-1), (F-4-1), and (F-5), even more preferably the group consisting of formulas (F-1-2) to (F-1-6), (F-2-1), (F-3-1), (F-4-2) to (F-4-6), and (F-5), and particularly preferably the group consisting of formulas (F-1-2) to (F-1-4), (F-2-1), (F-3-1), (F-4-2) to (F-4-4), and (F-5), or an amino acid sequence having 90% or more identity with said amino acid sequence. The aforementioned identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.

[0089] Table 8 shows preferred examples of combinations of heavy chain CDR1-3 and light chain CDR1-3 of antibodies or fragments thereof that specifically bind to SARS-CoV-2 NTDs.

[0090] [Table 8]

[0091] In combinations N-3 to N-14, it is also preferable that at least one CDR among the heavy chain CDR1 to 3 and light chain CDR1 to 3 has 1 to 3 mutated amino acids (preferably conservative substitutions).

[0092] 3. An antibody or fragment thereof that specifically binds to the CTD of the SARS-CoV-2 N protein. The CTD (sometimes simply referred to as CTD) of the SARS-CoV-2 N protein refers to the amino acid region from the N-terminus of the N protein, specifically from position 247 to 419, and has the following amino acid sequence: TKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTAAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA (Sequence ID 2)

[0093] It is preferable that an antibody or fragment thereof that specifically binds to the CTD of SARS-CoV-2 does not bind to the CTD of SARS-CoV, or has low binding affinity to the CTD of SARS-CoV. In the amino acid sequence shown in SEQ ID NO: 2, for example, the 267th A, 290th E, 334th T, 345th N, and 349th Q from the N-terminus of the N protein are different from the corresponding amino acids of the CTD of SARS-CoV. It is preferable that an antibody or fragment thereof against the N protein of SARS-CoV-2 (particularly an antibody or fragment thereof that specifically binds to the CTD of SARS-CoV-2) binds to a region (or epitope) in SEQ ID NO: 2 that contains one or more amino acids selected from the group consisting of the 267th A, 290th E, 334th T, 345th N, and 349th Q from the N-terminus of the N protein.

[0094] In one embodiment, an antibody or fragment thereof against the SARS-CoV-2 N protein (particularly an antibody or fragment thereof that specifically binds to the SARS-CoV-2 CTD) may bind to the amino acid region from the N-terminus of the N protein between positions 247 and 419, preferably to the dimerization domain of the CTD, and preferably to the amino acid region from the N-terminus of the N protein between positions 247 and 364 or between positions 256 and 364.

[0095] In one embodiment, it is preferable that an antibody or fragment thereof against the SARS-CoV-2 N protein (particularly an antibody or fragment thereof that specifically binds to the SARS-CoV-2 CTD) binds to the amino acid region at positions 250-257, 374-382, or 394-405 from the N-terminus of the N protein, i.e., an epitope exists in the region of the N protein consisting of the amino acid sequence shown in any of SEQ ID NOs. 150-152.

[0096] Antibodies or fragments thereof that specifically bind to the CTD of SARS-CoV-2 can be produced by combining known methods, for example, by a method comprising the above steps (P1) to (P5) and the following step (P6'). (P6') A step to obtain an antibody that specifically binds to CTD from the antibody recovered in step (P5).

[0097] Step (P6') can be carried out in the same way as step (P6) by selecting an antibody that specifically binds to CTD using a known screening method, such as enzyme immunosorbent assay (ELISA), Western blotting, or immunohistochemical staining.

[0098] A method comprising steps (P1) to (P5) and (P6') can be used to obtain multiple antibodies or fragments thereof that specifically bind to the CTD of SARS-CoV-2 (where each antibody or fragment thereof differs from the others in the amino acid sequence of at least one of the heavy chain CDR1-3 and light chain CDR1-3).

[0099] In one embodiment, the heavy chain CDR1 of an antibody or fragment thereof that specifically binds to the CTD of SARS-CoV-2 preferably consists of the following amino acid sequence: • An amino acid sequence represented by either formula (G-1) or (G-2) shown in Table 9, • An amino acid sequence having 90% or more (preferably 95% or more) identity with the said amino acid sequence, An amino acid sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) amino acids are mutated (preferably conserved substitutions).

[0100] [Table 9]

[0101] In equation (G-1), X G31 is preferably I, T, or S, and X G51 is preferably S, N, T, or R, and X G61 is preferably N or T, X G71 is preferably Y, S, or H, and X G81 Preferably, it is F, T, Y, or W.

[0102] Formula (G-1) is preferably formula (G-1-1), more preferably one selected from the group consisting of formulas (G-1-2) to (G-1-11), and even more preferably one selected from the group consisting of formulas (G-1-2) to (G-1-9).

[0103] The heavy chain CDR1 of an antibody or fragment thereof that specifically binds to the CTD of SARS-CoV-2 preferably consists of an amino acid sequence selected from the group consisting of formula (G-1-1), more preferably from the group consisting of formulas (G-1-2) to (G-1-11), and even more preferably from the group consisting of formulas (G-1-2) to (G-1-9), or an amino acid sequence having 90% or more identity with said amino acid sequence. The identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.

[0104] In one embodiment, the heavy chain CDR2 of an antibody or fragment thereof that specifically binds to the CTD of SARS-CoV-2 preferably consists of the following amino acid sequence: • An amino acid sequence represented by any of the formulas (H-1) to (H-3) shown in Table 10. • An amino acid sequence having 90% or more (preferably 95% or more) identity with the said amino acid sequence, An amino acid sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) amino acids are mutated (preferably conserved substitutions).

[0105] [Table 10]

[0106] In equation (H-1), X H31 is preferably G, N, S, T, or P, X H41 is preferably D, S, or A, and X H61 The element is preferably T, S, G, or R.

[0107] Formula (H-1) is preferably one selected from the group consisting of formula (H-1-1) or formulas (H-1-2) to (H-1-17), more preferably one selected from the group consisting of formulas (H-1-2) to (H-1-15), and even more preferably one selected from the group consisting of formulas (H-1-2) to (H-1-7) and (H-1-9) to (H-1-12).

[0108] The heavy chain CDR2 of an antibody or fragment thereof that specifically binds to the CTD of SARS-CoV-2 is preferably an amino acid sequence selected from the group consisting of formulas (H-1) and (H-2), more preferably the group consisting of formulas (H-1-1) and (H-2), or the group consisting of formulas (H-1-2) to (H-1-17) and (H-2), even more preferably the group consisting of formulas (H-1-2) to (H-1-15) and (H-2), particularly preferably the group consisting of formulas (H-1-2) to (H-1-7), (H-1-9) to (H-1-12), and (H-2), or an amino acid sequence having 90% or more identity with said amino acid sequence. The identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.

[0109] In one embodiment, the heavy chain CDR3 of an antibody or fragment thereof that specifically binds to the CTD of SARS-CoV-2 preferably consists of the following amino acid sequence: • An amino acid sequence represented by any of the formulas (I-1) to (I-11) shown in Table 11, • An amino acid sequence having 90% or more (preferably 95% or more) identity with the said amino acid sequence, An amino acid sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) amino acids are mutated (preferably conserved substitutions).

[0110] [Table 11]

[0111] In equation (I-1), X I31 is preferably N, L, or T, and X I61 It is preferably D, F, or G.

[0112] Formula (I-1) is preferably one selected from the group consisting of formula (I-1-1) or formulas (I-1-2) to (I-1-8), more preferably one selected from the group consisting of formulas (I-1-2) to (I-1-7), and even more preferably one selected from the group consisting of formulas (I-1-2) to (I-1-6).

[0113] The heavy chain CDR3 of an antibody or fragment thereof that specifically binds to the CTD of SARS-CoV-2 is preferably an amino acid sequence selected from the group consisting of formulas (I-1) to (I-3) and (I-5) to (I-9), more preferably the group consisting of formulas (I-1-1), (I-2), (I-3), and (I-5) to (I-9), or the group consisting of formulas (I-1-2) to (I-1-8), (I-2), (I-3), and (I-5) to (I-9), even more preferably the group consisting of formulas (I-1-2) to (I-1-7), (I-2), (I-3), and (I-5) to (I-9), and particularly preferably the group consisting of formulas (I-1-2) to (I-1-6), (I-2), (I-3), and (I-5) to (I-9), or an amino acid sequence having 90% or more identity with said amino acid sequence. The aforementioned identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.

[0114] In one embodiment, the light chain CDR1 of an antibody or fragment thereof that specifically binds to the CTD of SARS-CoV-2 preferably consists of the following amino acid sequence: • An amino acid sequence represented by any of the formulas (J-1) to (J-3) shown in Table 12. • An amino acid sequence having 90% or more (preferably 95% or more) identity with the said amino acid sequence, An amino acid sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) amino acids are mutated (preferably conserved substitutions).

[0115] [Table 12]

[0116] In equation (J-1), X J11 is preferably K, X J31 is preferably S, X J41 is preferably N, E, or K, and X J51 It is preferably Q, R, or E.

[0117] Formula (J-1) is preferably one selected from the group consisting of formula (J-1-1) or formulas (J-1-2) to (J-1-7), and more preferably one selected from the group consisting of formulas (J-1-2) to (J-1-4).

[0118] In equation (J-2), X J73 It is preferably S.

[0119] Formula (J-2) is preferably one selected from the group consisting of formula (J-2-1) or formulas (J-2-2) to (J-2-9), more preferably one selected from the group consisting of formulas (J-2-2) to (J-2-8), and even more preferably one selected from the group consisting of formulas (J-2-2) to (J-2-6).

[0120] The heavy chain CDR3 of an antibody or fragment thereof that specifically binds to the CTD of SARS-CoV-2 is preferably an amino acid sequence selected from the group consisting of formulas (J-1-1), (J-2-1), and (J-3), or the group consisting of formulas (J-1-2) to (J-1-7), (J-2-2) to (J-2-9), and (J-3), and more preferably from the group consisting of formulas (J-1-2) to (J-1-4), (J-2-2) to (J-2-6), and (J-3), or an amino acid sequence having 90% or more identity with said amino acid sequence. The identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.

[0121] In one embodiment, the light chain CDR2 of an antibody or fragment thereof that specifically binds to the CTD of SARS-CoV-2 preferably consists of the following amino acid sequence: • The amino acid sequence represented by formula (K-1) shown in Table 13, • An amino acid sequence having 90% or more (preferably 95% or more) identity with the said amino acid sequence, An amino acid sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) amino acids are mutated (preferably conserved substitutions).

[0122] [Table 13]

[0123] Formula (K-1) is preferably one selected from the group consisting of formulas (K-1-1) to (K-1-7).

[0124] The light chain CDR2 of an antibody or fragment thereof that specifically binds to the CTD of SARS-CoV-2 preferably consists of an amino acid sequence selected from the group consisting of formulas (K-1-1) to (K-1-7), or an amino acid sequence having 90% or more identity with said amino acid sequence. The identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.

[0125] In one embodiment, the light chain CDR3 of an antibody or fragment thereof that specifically binds to the CTD of SARS-CoV-2 preferably consists of the following amino acid sequence: • An amino acid sequence represented by any of the formulas (L-1) to (L-6) shown in Table 14, • An amino acid sequence having 90% or more (preferably 95% or more) identity with the said amino acid sequence, An amino acid sequence in which 1 to 3 (preferably 1 or 2, more preferably 1) amino acids are mutated (preferably conserved substitutions).

[0126] [Table 14]

[0127] In equation (L-1), X L31 is preferably R or S, X L41 is preferably Q or E, and X L51 is preferably S, X L61 is preferably D or N, X L71 It is preferably D, X L81 It is preferably A, and X L91 It is preferably A, and X L101 is preferably S, R, or W, and X L111 It is preferably V.

[0128] Formula (L-1) is preferably one selected from the group consisting of formula (L-1-1) or formulas (L-1-2) to (L-1-7), and more preferably one selected from the group consisting of formulas (L-1-2) to (L-1-4).

[0129] In equation (L-2), X L33 is preferably R, S, or G, and more preferably G and X L43 is preferably Q or E, more preferably E and X L53 is preferably S, X L63 It is preferably D, X L73 It is preferably D, XL83 It is preferably A, and X L93 is preferably W, X L103 It is preferably V.

[0130] Formula (L-2) is preferably formula (L-2-1).

[0131] In equation (L-3), X L54 is preferably H, X L64 is preferably D or N, or D or V, X L84 Preferably, it is Y, V, or I, or Y or L.

[0132] Formula (L-3) is preferably one selected from the group consisting of formula (L-3-1) or formulas (L-3-2) to (L-3-7), and more preferably formula (L-3-2) or (L-3-3).

[0133] In equation (L-4), X L27 is preferably T, X L37 is preferably T, X L47 is preferably N, and X L57 It is preferably D, X L77 Preferably Y, X L87 It is preferably T.

[0134] Formula (L-4) is preferably formula (L-4-1).

[0135] The light chain CDR3 of an antibody or fragment thereof that specifically binds to the CTD of SARS-CoV-2 is preferably an amino acid sequence selected from the group consisting of formulas (L-1) to (L-3), (L-5), and (L-6), more preferably the group consisting of formulas (L-1-1), (L-2-1), (L-3-1), (L-5), and (L-6), or the group consisting of (L-1-2) to (L-1-7), (L-2-1), (L-3-2), (L-3-3), (L-5), and (L-6), even more preferably the group consisting of formulas (L-1-2) to (L-1-4), (L-2-1), (L-3-2), (L-3-3), (L-5), and (L-6), or an amino acid sequence having 90% or more identity with said amino acid sequence. The aforementioned identity is preferably 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more.

[0136] Table 15 shows preferred examples of combinations of heavy chain CDR1-3 and light chain CDR1-3 of antibodies or fragments thereof that specifically bind to the CTD of SARS-CoV-2.

[0137] [Table 15]

[0138] In combinations C-3 to C-8, it is also preferable that at least one CDR among the heavy chain CDR1 to 3 and the light chain CDR1 to 3 has a mutation (preferably a conservative substitution) in the amino acid sequence of 1 to 3 amino acids.

[0139] Regions other than the heavy chain CDR1-3 and light chain CDR1-3 are not particularly limited and can consist of any amino acid sequence as long as they are suitable for use in an antibody. For example, the constant regions of IgG1, IgG2, IgG3, IgA1, IgA2, and IgM can be used, but are not limited to these.

[0140] The binding affinity of antibodies or fragments thereof to the SARS-CoV-2 N protein to the SARS-CoV-2 NTD or CTD is high, and the absorbance measured by the enzyme immunosorbent assay (ELISA) described in the examples below is, for example, 0.2 or higher, preferably 0.3 or higher, more preferably 0.4 or higher, and even more preferably 0.5 or higher.

[0141] The binding affinity (Kd) of an antibody or fragment thereof that specifically binds to the SARS-CoV-2 NTD or CTD for the SARS-CoV-2 N protein is, for example, 50 nM or less, preferably 10 nM or less, and for example, 1 pM or more. Kd can be measured using Biacore™ T200 (Cytiva). Specifically, an anti-guinea pig IgG antibody is covalently immobilized on carboxymethyl dextran of sensor chip CM5 by an amine coupling reaction using N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (NHS) and N-hydroxysuccinimide (EDC). An antibody or fragment thereof that specifically binds to the SARS-CoV-2 NTD or CTD is injected into the sensor chip for binding. Furthermore, the SARS-CoV-2 N protein is injected into the sensor chip and reacted with the antibody or fragment thereof that specifically binds to the SARS-CoV-2 NTD or CTD, and the reaction signal is analyzed to calculate Kd.

[0142] 4. Polynucleotides The polynucleotide of the present invention preferably contains a coding sequence for an antibody or fragment thereof that specifically binds to the NTD or CTD of SARS-CoV-2. Examples of such coding sequences include, but are not limited to, the base sequences encoding an antibody or fragment thereof that includes the heavy chain CDR1-3 and light chain CDR1-3 described in item 3 above.

[0143] In one embodiment, the polynucleotide of the present invention preferably comprises an expression cassette of an antibody or fragment thereof that specifically binds to an NTD or CTD of SARS-CoV-2. The expression cassette is not particularly limited as long as it enables expression in a host cell, and may include, for example, a promoter and a coding sequence positioned under the control of the promoter.

[0144] The promoter is not particularly limited and can be appropriately selected depending on the type of host cell. For example, various pol II system promoters can be used. While there are no particular limitations on pol II system promoters, examples include the CMV promoter, EF1 promoter, SV40 promoter, and MSCV promoter. Other examples of promoters include tryptophan promoters such as trc and tac; lac promoter; T7 promoter; T5 promoter; T3 promoter; SP6 promoter; arabinose-inducible promoter; cold shock promoter; and tetracycline-inducible promoter.

[0145] The expression cassette may contain other elements as needed. Examples of other elements include multiple cloning sites (MCS), drug resistance genes, origins of replication, enhancer sequences, repressor sequences, insulator sequences, reporter protein coding sequences, and drug resistance gene coding sequences. These may be present individually or in combination of two or more.

[0146] The polynucleotides of the present invention can be in the form of vectors, for example. An appropriate vector is selected depending on the intended use, the type of host cell, etc. Examples of vectors that use E. coli as a host include M13 phage or its variants, λ phage or its variants, pBR322 or its variants (e.g., pB325, pAT153, pUC8), etc. Examples of vectors that use yeast as a host include pYepSec1, pMFa, pYES2, pPIC3.5K, etc. Examples of vectors that use insect cells as a host include pAc, pVL, etc. Examples of vectors that use mammalian cells as a host include pcDNA, pCDM8, pMT2PC, etc.

[0147] 5. Cell The cells of the present invention preferably contain the polynucleotides described in section 4 above. Examples of cells include Escherichia coli K12 and other Escherichia coli, Bacillus bacteria such as Bacillus subtilis MI114, yeast such as Saccharomyces cerevisiae AH22, Sf cell lineage derived from Spodoptera frugiperda or HighFive cell lineage derived from Trichoplusia ni, insect cells such as olfactory nerve cells, and animal cells. Examples of animal cells include cultured cells derived from mammals, specifically COS7 cells, CHO cells, HEK293 cells, Expi293 cells, 293F cells, 293T cells, 293FT cells, Hela cells, PC12 cells, N1E-115 cells, SH-SY5Y cells, and the like.

[0148] In one embodiment, it is preferable that the cells of the present invention express an antibody or fragment thereof that specifically binds to the NTD or CTD of SARS-CoV-2.

[0149] In one embodiment, it is preferable that the cells of the present invention secrete or have on their cell surface an antibody or fragment thereof that specifically binds to the NTD or CTD of SARS-CoV-2.

[0150] 6. Reagents or pharmaceuticals The reagents or pharmaceuticals of the present invention preferably include antibodies or fragments thereof that specifically bind to the NTD or CTD of SARS-CoV-2, polynucleotides containing the coding sequence of said antibody or fragment, or cells containing said polynucleotide. The reagents or pharmaceuticals of the present invention preferably further include pharmaceutically acceptable excipients or carriers and / or additives.

[0151] Examples of the excipients or carriers include starch, lactose, crystalline cellulose, sorbitol, calcium hydrogen phosphate, water, ethanol, (poly)ethylene glycol, (poly)propylene glycol, glycerol, and vegetable oil. These can be used individually or in combination of two or more.

[0152] Examples of the aforementioned additives include buffering agents, isotonic agents, thickeners, chelating agents, emulsifiers, colorants, and preservatives. These can be used individually or in combination of two or more.

[0153] The reagent or pharmaceutical of the present invention may be an antibody or fragment thereof that specifically binds to the NTD or CTD of SARS-CoV-2 dissolved or dispersed in a solvent, or it may be lyophilized.

[0154] 7. Kit for detecting the N protein of SARS-CoV-2 The kit for detecting the SARS-CoV-2 N protein of the present invention preferably includes an antibody or fragment thereof that specifically binds to the NTD or CTD of SARS-CoV-2. The antibody or fragment thereof may be in the form of the reagent described in section 6 above, for example.

[0155] This kit is, • Two or more antibodies or fragments thereof that specifically bind to the NTD of SARS-CoV-2, each of which recognizes a different epitope of the NTD, and / or Two or more antibodies or fragments thereof that specifically bind to the CTD of SARS-CoV-2, and each of these antibodies or fragments recognizes a different epitope of the CTD. It is preferable to include it.

[0156] Preferably, two or more antibodies or fragments thereof that specifically bind to NTDs have different amino acid sequences in at least one of the heavy chain CDRs CDR1-3 and light chain CDRs CDR1-3. In one embodiment, it is preferable that a portion of the two or more antibodies or fragments thereof that specifically bind to NTDs (hereinafter referred to as "the first anti-NTD antibody or fragment thereof") is immobilized, and the remainder (hereinafter referred to as "the second anti-NTD antibody or fragment thereof") is labeled. Examples of the immobilized phase on which the first anti-NTD antibody or fragment thereof is immobilized include, but are not limited to, beads, wells, tips, strips, etc. There are no particular limitations on the method for immobilizing the first anti-NTD antibody or fragment thereof, but examples include contacting a biotinylated first anti-NTD antibody or fragment thereof with a solid phase surface having avidin. There are no particular limitations on the labeling substance for labeling the second anti-NTD antibody or fragment thereof, but examples include enzymes such as peroxidase (HRP) and alkaline phosphatase (ALP).

[0157] The first and second anti-NTD antibodies or fragments thereof can be selected, for example, from the antibodies or fragments thereof described in 2 above, preferably from N-1 in Table 8, more preferably from N-2 in Table 8, even more preferably from N-3 in Table 8, and particularly preferably from combinations of N-4 and N-5, N-4 and N-6, N-4 and N-7, N-5 and N-6, N-5 and N-7, N-6 and The combinations of N-7, N-6 and N-13, N-10 and N-12, N-5 and N-8, and N-9 and N-10 can be selected (wherein the amino acid sequences of the heavy chain CDR1-3 and light chain CDR1-3 of N-4-N-10, N-12, and N-13 may be amino acid sequences that have 90% or more identity with the original amino acid sequence, or may be sequences in which 1-3 amino acids have been mutated).

[0158] Preferably, two or more antibodies or fragments thereof that specifically bind to CTD have different amino acid sequences in at least one of the heavy chain CDR1-3 and light chain CDR1-3. In one embodiment, it is preferable that a portion of the two or more antibodies or fragments thereof that specifically bind to CTD (hereinafter referred to as "the first anti-CTD antibody or fragment thereof") is immobilized, and the remainder (hereinafter referred to as "the second anti-CTD antibody or fragment thereof") is labeled. Examples of the immobilized phase on which the first anti-CTD antibody or fragment thereof is immobilized include, but are not limited to, beads, wells, tips, strips, etc. There are no particular limitations on the method for immobilizing the first anti-CTD antibody or fragment thereof, but examples include contacting the biotinylated first anti-CTD antibody or fragment thereof with a solid phase surface having avidin. There are no particular limitations on the labeling substance for labeling the second anti-CTD antibody or fragment thereof, but examples include enzymes such as peroxidase (HRP) and alkaline phosphatase (ALP).

[0159] The first and second anti-CTD antibodies or fragments thereof can be selected, for example, from the antibodies or fragments thereof described in 3 above, preferably from C-1 in Table 15, more preferably from C-2 in Table 15, even more preferably from C-3 in Table 15, and particularly preferably from the combinations of C-4 and C-5, C-4 and C-6, C-4 and C-7, C-5 and C-6, and C-5 and C-7 in Table 15. The following combinations can be selected: C-6 and C-7, C-4 and C-12, C-9 and C-11, and C-7 and C-16 (wherein the amino acid sequences of the heavy chain CDR1-3 and light chain CDR1-3 of C-4-C-7, C-9, C-11, C-12, and C-16 may be amino acid sequences that have 90% or more identity with the original amino acid sequence, or they may be sequences in which 1-3 amino acids have been mutated).

[0160] The kit is preferably for detecting the N protein of SARS-CoV-2 in a test sample. The test sample may be a biological sample or a non-biological sample. The test sample may include not only the sample as it was collected, but also samples that have undergone pretreatment, such as the removal of impurities.

[0161] Examples of biological samples include, but are not limited to, blood, serum, plasma, bone marrow fluid, lymph fluid, tears, nasal secretions, nasal lavage fluid, nasal swabs, saliva, gargle fluid, sputum, pharyngeal swabs, sweat, tracheal aspirate, bronchial lavage fluid, pleural fluid, ascites, amniotic fluid, bowel lavage fluid, urine, feces, cell extracts, tissue extracts, and organ extracts.

[0162] Examples of non-biological samples include, but are not limited to, samples taken from environmental surfaces such as faucets, handles, handrails, straps, switches, walls, floors, desks, chairs, and toilet seats.

[0163] There are no particular limitations on the methods used to detect the SARS-CoV-2 N protein using this kit, but examples include immunochromatography, enzyme immunoassay, chemiluminescence immunoassay, chemiluminescence enzyme immunoassay, radioimmunoassay, electrochemiluminescence immunoassay, immunoturbidimetry, and latex agglutination.

[0164] Depending on the method for preparing the test sample, the method for detecting the SARS-CoV-2 N protein, etc., the kit may also include other substances (e.g., buffer solution, enzyme solution, diluent, secondary antibody, etc.), equipment for use, instructions for use, etc.

[0165] 8. Method for detecting the N protein of SARS-CoV-2 The method for detecting the N protein of SARS-CoV-2 involves the following steps (1) to (3): (1) A step of immobilizing a first antibody or fragment thereof that specifically binds to an NTD or CTD onto a solid phase. (2) A step of applying to the solid phase a second antibody or fragment thereof that specifically binds to the N protein of SARS-CoV-2 and the amino acid sequence indicated by SEQ ID NO: 1 or 2 (wherein the second antibody or fragment thereof recognizes a different epitope in the amino acid sequence to which the first antibody or fragment thereof specifically binds and is labeled with a labeling substance), and (3) A step of detecting a label on the solid phase that originates from a labeling substance of the second antibody or a fragment thereof bound to the nucleocapsid protein via the nucleocapsid protein. It is preferable to include it.

[0166] The first and second antibodies or fragments thereof are preferably the first and second anti-NTD antibodies or fragments thereof described in item 7 above, and / or the first and second anti-CTD antibodies or fragments thereof.

[0167] For example, those described in item 7 above can be selected as the solid phase and labeling substance. [Examples]

[0168] (Acquisition of antibody-producing cells through animal immunization) As a primary immunization, 195 μg of SARS-CoV-2 N protein suspended in PBS was mixed with the adjuvant TiterMAX Gold (Funakoshi Co., Ltd.) and administered intramuscularly to the left and right hind limbs of guinea pigs. Fourteen days after the primary immunization, 100 μg of SARS-CoV-2 N protein suspended in PBS was mixed with TiterMAX Gold and administered as a booster immunization intramuscularly to the left and right hind limbs of the guinea pigs. Furthermore, 31 days after the booster immunization, 100 μg of SARS-CoV-2 N protein suspended in PBS was administered again intramuscularly to the left and right hind limbs of the guinea pigs. Eight days after the second booster immunization, the two guinea pigs were anesthetized, had their entire blood drawn, euthanized, and their lumbar lymph nodes removed.

[0169] (Sorting of plasma cells producing anti-SARS-CoV2 N protein antibodies) The procedure was carried out in accordance with the method (ERIAA method) described in "Fluorescent probe for plasma cell identification and isolation, and method for identifying or isolating plasma cells using the probe" (U.S. Patent Application Publication 2013 / 0029325).

[0170] Cells obtained from excised lumbar lymph nodes were fixed in formaldehyde PBS solution on ice for 10 minutes. The fixed cells were stained with Dylight488-labeled SARS-CoV2 N protein, Dylight550-labeled SARS-CoV N protein, Dylight650-labeled anti-guinea pig IgG antibody, and DAPI (4',6-diamidino-2-phenylindole). Plasma cells that were strongly positive for SARS-CoV2 N protein, negative for SARS-CoV N protein, and strongly positive for anti-guinea pig IgG were sorted by flow cytometry.

[0171] (Synthesis of antibody variable region gene fragments) For each cell obtained by sorting, mRNA extraction, cDNA synthesis, and amplification of antibody variable region gene fragments were performed according to the method of Kurosawa et al. (MAGrahd method) (Rapid production of antigen-specific monoclonal antibodies from a variety of animals, BMC Biology 2012, 10:80).

[0172] (Production of antibody gene expression units) Antibody gene expression units were constructed from amplified antibody variable region gene fragments according to the method (TS-jPCR method) described in "Specific method for producing linked DNA fragments containing target gene-derived sequences" (U.S. Patent Application Publication No. 2013 / 0023009).

[0173] (Transient antibody expression in 293FT cells) The prepared antibody gene expression unit, FuGene HD Transfection Reagent (trademark), and Opti-MEM (trademark) were mixed in a 1:1:50 liquid-volume ratio and left at room temperature for 20 minutes. The antibody gene expression unit was then introduced into 293FT cells suspended in 10% (w / v) FBS / IMDM medium. The 293FT cells introduced with the antibody gene expression unit were seeded into a 24-well plate and incubated at 37°C in an 8% (v / v) CO2 environment for 2 days. The culture supernatant was then collected. The antibodies released into the culture supernatant were used for subsequent screening.

[0174] (Antibody screening by ELISA) Screening was performed by evaluating the reaction strength of each antibody with the SARS-CoV-2 N protein using ELISA. The ELISA was prepared by immobilizing 15 ng / well of SARS-CoV-2 N protein on the bottom of a 96-well plate, adding 100 μL of the culture supernatant from 293FT cells transiently expressing the antibody, and performing the antigen-antibody reaction at room temperature for 2 hours. Guinea pig antibodies bound to the antigen were detected using goat anti-guinea pig antibodies conjugated with horseradish peroxidase, and the absorbance at 450 nm was measured using a microplate reader.

[0175] (Antibody reactivity screening by immunohistochemical staining) To determine whether the acquired antibody recognizes the N-terminal region (NTD) or the C-terminal region (CTD) of the SARS-CoV-2 N protein, DNA fragments encoding the NTD and CTD, respectively, were transiently expressed in 293FT cells. The reactivity of the antibody with the NTD and CTD-encoding DNA fragments was then evaluated by immunostaining after adding the acquired antibody.

[0176] The amino acid sequences of NTD and CTD are shown in Table 16. [Table 16]

[0177] Using a plasmid containing the ORF region of the SARS-CoV2 N protein as a template, DNA fragments encoding NTD and CTD, respectively, were prepared by PCR. The prepared DNA fragments (50 ng / μL) were mixed with FuGene HD Transfection Reagent (Promega) and Opti-MEM (Thermo Fisher) in a 1:1:50 liquid-volume ratio and left at room temperature for 20 minutes. After that, the mixture was added to 293FT cells suspended in 10% (w / v) FBS / IMDM medium.

[0178] 293FT cells, into which DNA fragments were introduced, were seeded in collagen-coated 96-well plates and incubated at 37°C in an 8% (v / v) CO2 environment for 2 days. Afterward, the culture medium was removed and the plates were washed with PBS. 100 μL / well of 3% (w / v) formaldehyde PBS solution was added, and the plates were incubated at room temperature for 20 minutes to fix the cells. Then, 200 μL / well of 0.1% (w / v) Triton PBS solution was added, and the plates were incubated at room temperature for 5 minutes to permeabilize the cell membrane. After removing the solution from the wells, 100 μL of the culture supernatant from 293FT cells transiently expressing the antibody was added, and the plates were incubated at room temperature for 3 hours to allow the reaction to occur. After removing the culture supernatant and washing with PBS, Dylight594-labeled anti-guinea pig IgG antibody was added, and the plates were incubated at room temperature under light-shielding conditions for 1 hour to allow the reaction to occur. After further washing with PBS, the nuclei were stained using PBS containing DAPI.

[0179] By observing the reactivity between the immobilized SARS-CoV-2 N protein (full length) and the antibody, as well as the reactivity between the antibody and cells expressing DNA fragments encoding NTD and CTD, respectively, we determined whether each antibody was NTD-reactive or CTD-reactive.

[0180] (Transient expression and purification of antibodies in Expi293 cells) Based on ELISA evaluation, antibodies showing good reactivity were synthesized by transfecting Expi293 cells with the prepared gene expression units. Expi293 Expression Medium (GIBCO) and Expi Fectamine 293 Transfection Kit (GIBCO) were used for transfection. For each E250 culture flask, 50 μg of the gene expression unit for each antibody, 3 mL of OptiMEM I, and 0.16 mL of Expi Fectamine 293 were mixed and allowed to stand for 10 minutes before being added to Expi293 cells for gene transfection. After 1 day of culture, Expi Fectamine 293 Transfection Enhancer was added as feed, and the cells were cultured for a further 4 days. The supernatant was then collected and purified using a Protein A column to obtain the antibodies.

[0181] Tables 17 and 18 show the amino acid sequences (based on IMGT method) of the heavy chain CDR1-3 and light chain CDR1-3 of the 16 antibody clones (N1-N16) that specifically bound to NTDs and the 27 antibody clones (C1-C27) that specifically bound to CTDs, respectively, obtained by screening the 260 antibody clones acquired.

[0182] [Table 17]

[0183] [Table 18]

[0184] The effects of the present invention will be explained below using several clones that are responsive to NTDs or CTDs, but the present invention is not limited to these clones. Table 19 shows the results of immunohistochemical staining and ELISA for representative clones. Similar effects may be obtained using other clones.

[0185] [Table 19]

[0186] (Sandwich ELISA of NP antigen after degradation) Each biotin-labeled clone (immobilized antibody) was diluted to 5 μg / mL with TBS, and 50 μL / well was added to a 96-well microplate. The plates were coated at room temperature for 2 hours. After blocking with 1% (w / v) BSA-TBS-T and washing with TBS-T, each clone (detection antibody) labeled with 0.25 μg / mL alkaline phosphatase and 50 μL / well of 10 ng / mL antigen solution (diluted with 1% (w / v) TBS-T) were added, and the plates were reacted at room temperature for 2 hours. As antigen solutions, in addition to sample 1 containing the full-length SARS-CoV2 N protein, sample 2 was used, which simulated a sample in which the entire amount of SARS-CoV2 N protein was replaced with equimolar amounts of NTD and CTD to mimic a sample in which degradation of the SARS-CoV2 N protein had occurred. After washing with TBS-T, 100 μL / well of pNPP was added as a chromogenic substrate, and the mixture was reacted at room temperature for 15 minutes. The absorbance at 405 nm was then measured using a microplate reader. The results are shown in Table 20. The percentages in Table 20 represent the relative absorbance values ​​to the absorbance measurements of Sample 1.

[0187] [Table 20]

[0188] When two types of antibodies recognizing NTDs were combined, and when two types of antibodies recognizing CTDs were combined, the antigen fragments in sample 2, which mimicked a sample in which SARS-CoV-2 N protein degradation had occurred, were presumed to have undergone a change in three-dimensional structure and a decrease in reactivity with the antigen fragments, resulting in a decrease in absorbance measurements, although the level of decrease was small. On the other hand, when an NTD-recognizing antibody and a CTD-recognizing antibody were combined, a significant decrease in absorbance measurements was observed in samples 2-4.

[0189] (Sandwich ELISA using oligoclonal antibodies) A solid-phase antibody containing a mixture of biotin-labeled clones was diluted to 5 μg / mL with TBS, and 50 μL / well was added to a 96-well microplate. The plate was coated at room temperature for 2 hours. After blocking with 1% (w / v) BSA-TBS-T and washing with TBS-T, 50 μL / well each of a 0.25 μg / mL detection antibody containing a mixture of alkaline phosphatase-labeled clones and a 10 ng / mL antigen solution (diluted with 1% (w / v) TBS-T) were added, and the mixture was reacted at room temperature for 2 hours. When using two types of solid-phase antibodies, the amount of each antibody added was halved to achieve a total volume of 50 μL / well. The same procedure was followed when using two types of detection antibodies. As antigen solutions, sample 1, containing the full-length SARS-CoV-2 N protein, was used, along with sample 2, which simulated a sample in which the entire amount of SARS-CoV-2 N protein was replaced with equimolar amounts of NTD and CTD. After washing with TBS-T, 100 uL / well of pNPP was added as a chromogenic substrate, and the mixture was reacted at room temperature for 15 minutes. The absorbance at 405 nm was then measured using a microplate reader. The results are shown in Table 21.

[0190] [Table 21]

[0191] It was confirmed that the detection sensitivity was improved when multiple antibodies were combined compared to when a single antibody was used as the immobilized antibody or detection antibody.

[0192] (Antibody epitope mapping) Epitope mapping was determined using PEPperPRINT's Conformational Epitope Mappings service. Peptides consisting of 7, 10, and 13 amino acids from the 200-419 amino acid region from the N-terminus of the SARS-CoV-2 N protein were synthesized on a peptide array, with overlaps of 6, 9, and 12 amino acids respectively, shifted by one amino acid. The reactivity of the antibody, i.e., the detection signal, was used to identify which peptides were antibody-bound epitopes.

[0193] (result) By epitope mapping of monoclonal antibodies, Of the clones shown in Table 18, the epitopes of three clones were confirmed to be located in the following regions, respectively: SEQ ID NO: 150: LLPAADLDDFSK (positions 394-405 from the N-terminus of the SARS-CoV-2 N protein), SEQ ID NO: 151: KKADETQAL (positions 374-382 from the N-terminus of the SARS-CoV-2 N protein), and SEQ ID NO: 152: SAAEASKK (positions 250-257 from the N-terminus of the SARS-CoV-2 N protein).

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to the amino acid sequence shown in Sequence ID No. 2, and is selected from the group consisting of (C1), (C2), (C3), (C4), (C6), (C9), (C13), (C14), (C16), (C18), (C20), (C23), and (C27): (C1) An antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 74, a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 85, a heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 102, a light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 119, a light chain CDR2 containing the amino acid sequence shown in KDS, and a light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 134; (C2) An antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 75, a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 86, a heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 109, a light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 133, a light chain CDR2 containing the amino acid sequence shown in NGN, and a light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 148; (C3) An antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 76, a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 87, a heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 103, a light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 120, a light chain CDR2 containing the amino acid sequence shown in NDS, and a light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 135; (C4) An antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 77, a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 101, a heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 112, a light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 125, a light chain CDR2 containing the amino acid sequence shown in KVS, and a light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 149; (C6) An antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 78, a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 89, a heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 116, a light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 126, a light chain CDR2 containing the amino acid sequence shown in LIS, and a light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 141; (C9) An antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 78, a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 89, a heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 116, a light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 127, a light chain CDR2 containing the amino acid sequence shown in LIS, and a light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 142; (C13) An antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 80, a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 92, a heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 113, a light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 128, a light chain CDR2 containing the amino acid sequence shown in EVS, and a light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 142; (C14) An antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 81, a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 93, a heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 114, a light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 129, a light chain CDR2 containing the amino acid sequence shown in EVS, and a light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 142; (C16) An antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 79, a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 90, a heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 110, a light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 120, a light chain CDR2 containing the amino acid sequence shown in NDS, and a light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 140; (C18) An antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 79, a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 94, a heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 105, a light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 130, a light chain CDR2 containing the amino acid sequence shown in EVS, and a light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 145; (C20) An antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 76, a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 88, a heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 115, a light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 121, a light chain CDR2 containing the amino acid sequence shown in KDS, and a light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 136; (C23) An antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 76, a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 95, a heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 104, a light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 120, a light chain CDR2 containing the amino acid sequence shown in NDS, and a light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 135; (C27) An antibody or antigen-binding fragment thereof comprising a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 76, a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 96, a heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 106, a light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 120, a light chain CDR2 containing the amino acid sequence shown in NDD, and a light chain CDR3 containing the amino acid sequence shown in SEQ ID NO:

135.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a monoclonal antibody.

3. A polynucleotide comprising the coding sequence of an antibody or antigen-binding fragment thereof according to claim 1 or 2.

4. A cell containing the polynucleotide described in claim 3.

5. A reagent or pharmaceutical comprising an antibody or antigen-binding fragment thereof according to claim 1 or 2, a polynucleotide according to claim 3, or a cell according to claim 4.

6. A kit for detecting the nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprising the antibody or antigen-binding fragment thereof according to claim 1 or 2.

7. The kit according to claim 6, for detecting the nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by immunochromatography, enzyme immunoassay, chemiluminescence immunoassay, chemiluminescence enzyme immunoassay, radioimmunoassay, electrochemiluminescence immunoassay, immunoturbidimetry, or latex agglutination.

8. A method for detecting the nucleocapsid protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprising the following steps (1) to (3): (1) A step of immobilizing a first antibody or its antigen-binding fragment that specifically binds to the amino acid sequence indicated by SEQ ID NO: 1 or 2 onto a solid phase. (2) A step of applying to the solid phase a second antibody or its antigen-binding fragment that specifically binds to the nucleocapsid protein of SARS-CoV-2 and the amino acid sequence indicated by SEQ ID NO: 1 or 2 (wherein the second antibody or its antigen-binding fragment recognizes a different epitope in the amino acid sequence to which the first antibody or its antigen-binding fragment specifically binds, and is labeled with a labeling substance), and (3) A step of detecting a label on the solid phase that originates from a labeling substance of the second antibody or its antigen-binding fragment, which is bound to the nucleocapsid protein via the nucleocapsid protein. A method comprising, wherein at least one of the first antibody or its antigen-binding fragment and the second antibody or its antigen-binding fragment comprises the antibody or its antigen-binding fragment according to claim 1 or 2.

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