Method for detecting or separating polysialic acid or polysialic acid-carrying body

Abstract

To provide a technique for detecting or separating polysialic acid or a polysialic acid-carrying body.SOLUTION: The present invention provides a kit comprising an antibody to which a multimerization domain and / or a streptavidin-binding protein is added, and / or a polynucleotide encoding the antibody. Also provided is a kit comprising at least one antibody selected from the group consisting of an anti-polysialic acid antibody comprising a specific amino acid sequence and an anti-polysialic acid-negative antibody, and / or a polynucleotide encoding the antibody.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for detecting and separating polysialic acid or a polysialic acid carrier, etc.

Background Art

[0002] Polysialic acid (sialic acid polymer) mainly exists as a modified sugar chain of NCAM, which is an intercellular adhesion molecule, and is known to be responsible for controlling the function of NCAM and also for the unique functions of polysialic acid. Conventionally, polysialic acid has been reported to present a repulsive field between cells, regulate the intercellular space, and control the intracellular signal intensity. However, in recent years, it has been reported that polysialic acid presents an inductive field and specifically binds to various molecular groups. In addition, it is known that polysialic acid is expressed in cancer cells, shows abnormal expression in patients with mental diseases, shows abnormal expression in patients with neurodegenerative diseases, and exists on the surface of immune cells (such as NK cells) (Non-Patent Document 1).

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] An object of the present invention is to provide a technique for detecting or separating polysialic acid or a polysialic acid carrier.

Means for Solving the Problems

[0005] As a result of intensive research in view of the above problems, the present inventors have found that the following antibodies can solve the above problems: an anti-polysialic acid negative antibody in which the tyrosine residue on the C-terminal side within the light chain CDR1 of (antibody A) is substituted with another amino acid; an anti-polysialic acid antibody (antibody AX) comprising a light chain variable region including a light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 33 or SEQ ID NO: 34, a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 6, and / or a heavy chain variable region including a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 35 or SEQ ID NO: 36, a heavy chain CDR2 having the amino acid sequence represented by SEQ ID NO: 8, and a heavy chain CDR3 having the amino acid sequence represented by SEQ ID NO: 9; an anti-polysialic acid humanized antibody (antibody B) in which the heavy chain variable region includes an amino acid sequence BH2 having 95% or more identity to the amino acid sequence BH1 represented by SEQ ID NO: 16; an anti-polysialic acid antibody (antibody C) comprising a light chain variable region including a light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 17, a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 18, and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 19, and / or a heavy chain variable region including a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 21, a heavy chain CDR2 having the amino acid sequence represented by SEQ ID NO: 22, and a heavy chain CDR3 having the amino acid sequence represented by SEQ ID NO: 23; an anti-polysialic acid antibody (antibody D) comprising a light chain variable region including a light chain CDR1 having the amino acid sequence represented by SEQ ID NO: 25 or SEQ ID NO: 38, a light chain CDR2 having the amino acid sequence represented by SEQ ID NO: 26, and a light chain CDR3 having the amino acid sequence represented by SEQ ID NO: 27, and / or a heavy chain variable region including a heavy chain CDR1 having the amino acid sequence represented by SEQ ID NO: 29, a heavy chain CDR2 having the amino acid sequence represented by SEQ ID NO: 30, and a heavy chain CDR3 having the amino acid sequence represented by SEQ ID NO: 31; and at least one antibody selected from the group consisting of antibodies (antibody E) to which a streptavidin-binding protein and / or a multimerization domain is added. That is, the present invention includes the following aspects.

[0006] Item 1. An anti-polysialic acid negative antibody in which the tyrosine residue on the C-terminal side within the light chain CDR1 of (antibody A) is substituted with another amino acid; (Antibody AX) A light chain variable region comprising a light chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 33 or SEQ ID NO: 34, a light chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 6, and / or a heavy chain variable region comprising a heavy chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 35 or SEQ ID NO: 36, a heavy chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 8, and a heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 9, an anti-polysialic acid antibody; (Antibody B) An anti-polysialic acid humanized antibody in which the heavy chain variable region contains an amino acid sequence BH2 having 95% or more identity to the amino acid sequence BH1 represented by SEQ ID NO: 16; (Antibody C) A light chain variable region comprising a light chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 17, a light chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 18, and a light chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 19, and / or a heavy chain variable region comprising a heavy chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 21, a heavy chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 22, and a heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 23, an anti-polysialic acid antibody; (Antibody D) A light chain variable region comprising a light chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 25 or SEQ ID NO: 38, a light chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 26, and a light chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 27, and / or a heavy chain variable region comprising a heavy chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 29, a heavy chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 30, and a heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 31, an anti-polysialic acid antibody; and (Antibody E) An antibody to which a streptavidin-binding protein and / or a multimerization domain is added A kit comprising at least one antibody selected from the group consisting of, and / or a polynucleotide encoding said antibody.

[0007] Item 2. The antibody A is (Antibody A1) The 39th tyrosine residue from the N-terminus of the amino acid sequence A1L1 represented by SEQ ID NO: 2, or the tyrosine residue corresponding to the tyrosine residue in the amino acid sequence A1L2 having 95% or more identity to the amino acid sequence A1L1 is mutated to another amino acid residue, and includes a light chain variable region containing an amino acid sequence, an anti-polysialic acid negative antibody, or (Antibody A2) The 37th tyrosine residue from the N-terminus of the amino acid sequence A2L1 represented by SEQ ID NO: 20, or the tyrosine residue corresponding to the tyrosine residue in the amino acid sequence A2L2 having 95% or more identity to the amino acid sequence A2L1 is mutated to another amino acid residue, and includes a light chain variable region containing an amino acid sequence, an anti-polysialic acid negative antibody The kit according to item 1, which is

[0008] Item 3. The antibody B is a humanized anti-polysialic acid antibody, and the heavy chain variable region includes the amino acid sequence represented by SEQ ID NO: 16. The kit according to item 1 or 2.

[0009] Item 4. The antibody C and / or the antibody D is an IgG antibody. The kit according to any one of items 1 to 3.

[0010] Item 5. The antibody E is an anti-polysialic acid antibody obtained by adding a streptavidin-binding protein and / or a multimerization domain to the antibody AX, the antibody B, the antibody C, or the antibody D. The kit according to any one of items 1 to 4.

[0011] Item 6. The kit according to any one of items 1 to 5, which is for detecting or separating polysialic acid or a polysialic acid carrier.

[0012] Item 7. The kit according to any one of items 1 to 6, which is for examining polysialic acid-related diseases or separating polysialic acid-expressing cells.

[0013] Item 8. An anti-polysialic acid negative antibody, wherein the tyrosine residue on the C-terminal side within the light chain CDR1 of (antibody A) is substituted with another amino acid; (Antibody AX) A light chain variable region comprising a light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 33 or SEQ ID NO: 34, a light chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 6, and / or a heavy chain variable region comprising a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 35 or SEQ ID NO: 36, a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 8, and a heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 9, an anti-polysialic acid antibody; (Antibody B) An anti-polysialic acid humanized antibody, wherein the heavy chain variable region contains an amino acid sequence BH2 having 95% or more identity to the amino acid sequence BH1 shown in SEQ ID NO: 16; (Antibody C) A light chain variable region comprising a light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 17, a light chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 18, and a light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 19, and / or a heavy chain variable region comprising a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 21, a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 22, and a heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 23, an anti-polysialic acid antibody; (Antibody D) A light chain variable region comprising a light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 25 or SEQ ID NO: 38, a light chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 26, and a light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 27, and / or a heavy chain variable region comprising a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 29, a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 30, and a heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 31, an anti-polysialic acid antibody; and (Antibody E) An antibody to which a streptavidin-binding protein and / or a multimerization domain is added A reagent comprising at least one antibody selected from the group consisting of, and / or a polynucleotide encoding said antibody.

[0014] Item 9. An anti-polysialic acid negative antibody, wherein the tyrosine residue on the C-terminal side within the light chain CDR1 of (antibody A) is substituted with another amino acid; (Antibody AX) A light chain variable region comprising a light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 33 or SEQ ID NO: 34, a light chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 6, and / or a heavy chain variable region comprising a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 35 or SEQ ID NO: 36, a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 8, and a heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 9, an anti-polysialic acid antibody; (Antibody B) An anti-polysialic acid humanized antibody, wherein the heavy chain variable region contains an amino acid sequence BH2 having 95% or more identity to the amino acid sequence BH1 shown in SEQ ID NO: 16; (Antibody C) A light chain variable region comprising a light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 17, a light chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 18, and a light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 19, and / or a heavy chain variable region comprising a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 21, a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 22, and a heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 23, an anti-polysialic acid antibody; (Antibody D) A light chain variable region comprising a light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 25 or SEQ ID NO: 38, a light chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 26, and a light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 27, and / or a heavy chain variable region comprising a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 29, a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 30, and a heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 31, an anti-polysialic acid antibody; and (Antibody E) An antibody to which a streptavidin-binding protein and / or a multimerization domain is added A method for detecting polysialic acid or a polysialic acid carrier, comprising a step of contacting a sample with at least one antibody selected from the group consisting of

[0015] Item 10. The detection method according to Item 9, including visualizing a complex comprising the antibody and poly sialic acid or a poly sialic acid carrier.

[0016] Item 11. The detection method according to Item 9 or 10, which is an ELISA method.

[0017] Item 12. The detection method according to Item 11, wherein the ELISA method is a sandwich ELISA method.

[0018] Item 13. An anti-poly sialic acid negative antibody in which the tyrosine residue on the C-terminal side within the light chain CDR1 of (antibody A) is substituted with another amino acid; (Antibody AX) A light chain variable region comprising a light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 33 or SEQ ID NO: 34, a light chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 6, and / or a heavy chain variable region comprising a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 35 or SEQ ID NO: 36, a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 8, and a heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 9, an anti-poly sialic acid antibody; (Antibody B) An anti-poly sialic acid humanized antibody, the heavy chain variable region of which contains an amino acid sequence BH2 having 95% or more identity to the amino acid sequence BH1 shown in SEQ ID NO: 16; (Antibody C) A light chain variable region comprising a light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 17, a light chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 18, and a light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 19, and / or a heavy chain variable region comprising a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 21, a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 22, and a heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 23, an anti-poly sialic acid antibody; An anti-polysialic acid antibody comprising a light chain variable region containing a light chain CDR1 comprising the amino acid sequence shown by SEQ ID NO: 25 or SEQ ID NO: 38, a light chain CDR2 comprising the amino acid sequence shown by SEQ ID NO: 26, and a light chain CDR3 comprising the amino acid sequence shown by SEQ ID NO: 27, and / or a heavy chain variable region containing a heavy chain CDR1 comprising the amino acid sequence shown by SEQ ID NO: 29, a heavy chain CDR2 comprising the amino acid sequence shown by SEQ ID NO: 30, and a heavy chain CDR3 comprising the amino acid sequence shown by SEQ ID NO: 31; and (Antibody E) An antibody obtained by adding a streptavidin-binding protein and / or a multimerization domain A method for separating polysialic acid or a polysialic acid carrier, comprising a step of contacting a sample with at least one antibody selected from the group consisting of

[0019] Item 14. Any one of the following antibodies A to E, and / or a polynucleotide encoding said antibody: (Antibody A) An anti-polysialic acid negative antibody in which the tyrosine residue on the C-terminal side within the light chain CDR1 is substituted with another amino acid; (Antibody AX) An anti-polysialic acid antibody comprising a light chain variable region containing a light chain CDR1 comprising the amino acid sequence shown by SEQ ID NO: 33 or SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence shown by SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence shown by SEQ ID NO: 6, and / or a heavy chain variable region containing a heavy chain CDR1 comprising the amino acid sequence shown by SEQ ID NO: 35 or SEQ ID NO: 36, a heavy chain CDR2 comprising the amino acid sequence shown by SEQ ID NO: 8, and a heavy chain CDR3 comprising the amino acid sequence shown by SEQ ID NO: 9; (Antibody B) An anti-polysialic acid humanized antibody comprising an amino acid sequence BH2 having 95% or more identity to the amino acid sequence BH1 shown by SEQ ID NO: 16 in the heavy chain variable region; An anti-polysialic acid antibody comprising a light chain variable region containing a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 17, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 18, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 19, and / or a heavy chain variable region containing a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 21, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 22, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 23; An anti-polysialic acid antibody comprising a light chain variable region containing a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 25 or SEQ ID NO: 38, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 26, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 27, and / or a heavy chain variable region containing a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 29, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 30, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 31; and An antibody (antibody E) to which a streptavidin-binding protein and / or a multimerization domain is added. [[Effect of the Invention]]

[0020] According to the present invention, a technique for detecting or separating polysialic acid or a polysialic acid carrier can be provided. [[Brief Description of the Drawings]]

[0021]

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Mode for Carrying Out the Invention

[0022] 1. Definition In this specification, the expressions "containing" and "comprising" include the concepts of "containing", "comprising", "consisting essentially of", and "consisting only of".

[0023] In this specification, amino acids / amino acid residues may also be represented by one-letter notation.

[0024] The "identity" of amino acid sequences refers to the degree of coincidence of the amino acid sequences of two or more comparable amino acid sequences with respect to each other. Therefore, the higher the identity of two amino acid sequences, the higher their identity or similarity. The level of identity of amino acid sequences is determined, for example, using FASTA, a sequence analysis tool, with default parameters. Alternatively, it can be determined using the algorithm BLAST by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc Natl Acad Sci USA. 87:2264-2268 (1990), Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences." Proc Natl Acad Sci USA. 90:5873-7 (1993)). A program called BLASTX, based on such BLAST algorithms, has been developed. The specific methods of these analysis methods are known, and reference may be made to the website of the National Center of Biotechnology Information (NCBI) (http: / / www.ncbi.nlm.nih.gov / ). Also, the "identity" of nucleotide sequences is defined in accordance with the above.

[0025] As used herein, "conservative substitution" means that an amino acid residue is substituted with an amino acid residue having a similar side chain. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine is a conservative substitution. In addition, amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; substitution between amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine is also a conservative substitution.

[0026] As used herein, "CDR" means C omplementarity D etermining R the abbreviation of region, also referred to as the complementarity determining region. CDR is a region present in the variable region of an immunoglobulin and is deeply involved in the specific binding of an antibody to an antigen. And "light chain CDR" means a CDR present in the variable region of the light chain of an immunoglobulin, and "heavy chain CDR" means a CDR present in the variable region of the heavy chain of an immunoglobulin.

[0027] As used herein, "variable region" means a region containing the above-described CDR1 to CDR3 (hereinafter simply referred to as "CDRs1-3"). The arrangement order of these CDRs1-3 is not particularly limited, but preferably, in the direction from the N-terminal side to the C-terminal side, in the order of CDR1, CDR2, and CDR3, or in the reverse order, through a continuous or other amino acid sequence referred to as the framework region (FR) described below. And "heavy chain variable region" means a region in which the above-described heavy chain CDRs1-3 are arranged, and "light chain variable region" means a region in which the above-described light chain CDRs1-3 are arranged.

[0028] Regions of each variable region other than the above-mentioned CDR1-3 are referred to as framework regions (FR) as described above. In particular, the region between the N-terminus of the variable region and CDR1 is defined as FR1, the region between CDR1 and CDR2 is defined as FR2, the region between CDR2 and CDR3 is defined as FR3, and the region between CDR3 and the C-terminus of the variable region is defined as FR4, respectively.

[0029] FR also functions as a linker sequence connecting CDRs1-3, which are particularly important as the antigen recognition sequences described above, and is a region contributing to the three-dimensional structure formation of the entire variable region.

[0030] 2. Antibody In one aspect, the present invention relates to antibody A, antibody AX, antibody B, antibody C, antibody D, and antibody E (these may also be collectively referred to as "the antibodies of the present invention" in this specification). These will be described below.

[0031] 2-1. Antibody A Antibody A is an anti-polysialic acid negative antibody in which the tyrosine residue on the C-terminal side within the light chain CDR1 is substituted with another amino acid.

[0032] The tyrosine residue on the C-terminal side within the light chain CDR1 is a tyrosine residue within the light chain CDR1 and is a tyrosine residue present on the C-terminal side within the light chain CDR1, and is not particularly limited as long as this is the case. "C-terminal side" refers to a region consisting of 1 to 5 (preferably 1 to 3, more preferably 1 to 2, still more preferably 1) amino acid residues including the C-terminal amino acid residue of the light chain CDR1. When there are a plurality of tyrosine residues on the C-terminal side within the light chain CDR1, the tyrosine residue is preferably the most C-terminal tyrosine residue.

[0033] Antibody A is an antibody in which the above tyrosine residue substitution has been introduced in an anti-polysialic acid antibody, and the reactivity / binding ability to polysialic acid is greatly reduced by this tyrosine residue substitution (measured by ELISA in Example 2 described later, the reactivity / binding ability to polysialic acid is, for example, 50% or less, preferably 40% or less, more preferably 30% or less, still more preferably 20% or less, even more preferably 10% or less, particularly preferably 5% or less with respect to 100% of the reactivity / binding ability before the introduction of the tyrosine residue substitution), so it can be used as a polysialic acid-negative antibody.

[0034] "Other amino acid residues" are not particularly limited as long as they are amino acid residues other than tyrosine residues. Examples of other amino acid residues include aliphatic amino acids such as G, A, V, I, L; sulfur-containing amino acids such as C, M; basic amino acids such as K, H, R; amide group-containing amino acids such as N, Q; hydroxy group-containing amino acids such as S, T; aromatic amino acids such as F, W; acidic amino acids such as D, E; imino acids such as P, etc. Among these, preferably, aliphatic amino acids such as G, A, V, I, L; basic amino acids such as K, H, R; hydroxy group-containing amino acids such as S, T; aromatic amino acids such as W; acidic amino acids such as D, E, etc. are mentioned, and more preferably, A, S, I, D, K, W, etc. are mentioned.

[0035] Antibody A usually contains a light chain variable region and a heavy chain variable region.

[0036] Antibody A may be one in which mutations other than the above tyrosine residue substitution have been introduced into the anti-polysialic acid antibody. Examples of mutations include amino acid substitution, deletion, insertion, etc. The number of amino acid residues of the mutation is not particularly limited as long as the antibody structure is maintained, but is, for example, 0 to 20, 0 to 10, or 0 to 5, or, for example, 10% or less, 5% or less, 2% or less, or 1% or less with respect to 100% of the total number of amino acid residues of the antibody (or the heavy chain / light chain variable region, heavy chain / light chain constant region).

[0037] In one aspect, antibody A is preferably an anti-polysialic acid negative antibody comprising a light chain variable region containing an amino acid sequence in which the 39th tyrosine residue from the N-terminus of the amino acid sequence A1L1 shown in SEQ ID NO: 2 (antibody A1), or the tyrosine residue corresponding to the tyrosine residue in the amino acid sequence A1L2 having 95% or more identity to the amino acid sequence A1L1, is mutated to another amino acid residue.

[0038] Antibody A1 is an antibody in which the above tyrosine residue substitution is introduced in an anti-polysialic acid antibody containing the amino acid sequence A1L1 as the light chain variable region.

[0039] The identity of the amino acid sequence A1L2 to the amino acid sequence A1L1 is preferably 97% or more, more preferably 98% or more, still more preferably 99% or more, and in any case less than 100%. In the amino acid sequence A1L2, the number of amino acid residues mutated from the amino acid sequence A1L1 is, for example, 1 to 5, preferably 1 to 2, more preferably 1.

[0040] In the present specification, the "corresponding tyrosine residue" indicates a tyrosine residue at the same position on the alignment sequence when two sequences are compared by BLAST (default settings). For example, in the above case, the "tyrosine residue corresponding to the tyrosine residue in the amino acid sequence A1L2" refers to the tyrosine residue at the same position as the 39th tyrosine residue from the N-terminus of the amino acid sequence A1L1 in the amino acid sequence A1L2 in the alignment sequence obtained by comparing the amino acid sequence A1L1 and the amino acid sequence A1L2.

[0041] Antibody A1 preferably comprises a heavy chain variable region containing an amino acid sequence A1H2 having 95% or more identity to the amino acid sequence A1H1 shown in SEQ ID NO: 3.

[0042] The identity of the amino acid sequence A1H2 to the amino acid sequence A1H1 is preferably 97% or more, more preferably 98% or more, still more preferably 99% or more, and in any case, 100% or less. In the amino acid sequence A1H2, the number of amino acid residues mutated from the amino acid sequence A1H1 is, for example, 0 to 5, preferably 0 to 2, more preferably 0 to 1.

[0043] In one aspect, antibody A is preferably an anti-polysialic acid negative antibody that includes a light chain variable region including an amino acid sequence in which the 37th tyrosine residue from the N-terminus of the amino acid sequence A2L1 represented by SEQ ID NO: 20, or the tyrosine residue corresponding to the tyrosine residue in the amino acid sequence A2L2 having 95% or more identity to the amino acid sequence A2L1 is mutated to another amino acid residue.

[0044] Antibody A2 is an antibody in which the above tyrosine residue substitution is introduced in an anti-polysialic acid antibody including the amino acid sequence A2L1 as the light chain variable region.

[0045] The identity of the amino acid sequence A2L2 to the amino acid sequence A2L1 is preferably 97% or more, more preferably 98% or more, still more preferably 99% or more, and in any case, less than 100%. In the amino acid sequence A2L2, the number of amino acid residues mutated from the amino acid sequence A2L1 is, for example, 1 to 5, preferably 1 to 2, more preferably 1.

[0046] Antibody A2 preferably includes a heavy chain variable region including an amino acid sequence A2H2 having 95% or more identity to the amino acid sequence A2H1 represented by SEQ ID NO: 24.

[0047] The identity of the amino acid sequence A2H2 to the amino acid sequence A2H1 is preferably 97% or more, more preferably 98% or more, still more preferably 99% or more, and in any case 100% or less. In the amino acid sequence A2H2, the number of amino acid residues mutated from the amino acid sequence A2H1 is, for example, 0 to 5, preferably 0 to 2, more preferably 0 to 1.

[0048] Antibody A has low reactivity to polysialic acid and can be used as a negative control in polysialic acid detection and separation.

[0049] 2-1X. Antibody AX Antibody AX is an anti-polysialic acid antibody comprising a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 33 or SEQ ID NO: 34 (preferably SEQ ID NO: 33), a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6, and / or a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 35 or SEQ ID NO: 36 (preferably SEQ ID NO: 36), a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 8, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 9.

[0050] Antibody AX preferably comprises an amino acid sequence AXL2 having 95% or more identity to the amino acid sequence AXL1 represented by SEQ ID NO: 2 in the light chain variable region.

[0051] The identity of the amino acid sequence AXL2 to the amino acid sequence AXL1 is preferably 97% or more, more preferably 98% or more, still more preferably 99% or more, and in any case 100% or less. In the amino acid sequence AXL2, the number of amino acid residues mutated from the amino acid sequence AXL1 is, for example, 0 to 5, preferably 0 to 2, more preferably 0 to 1.

[0052] Antibody AX preferably comprises an amino acid sequence AXH2 having 95% or more identity to the amino acid sequence AXH1 represented by SEQ ID NO: 3 in the heavy chain variable region.

[0053] The identity of the amino acid sequence AXH2 to the amino acid sequence AXH1 is preferably 97% or more, more preferably 98% or more, still more preferably 99% or more, and in any case 100% or less. In the amino acid sequence AXH2, the number of amino acid residues mutated from the amino acid sequence AXH1 is, for example, 0 to 5, preferably 0 to 2, more preferably 0 to 1.

[0054] Antibody AX is preferably an IgG antibody.

[0055] Antibody AX can be used for the detection / separation of polysialic acid, and can be used, for example, in assays that require multiple anti-polysialic acid antibodies such as sandwich ELISA.

[0056] 2-2. Antibody B Antibody B is a humanized anti-polysialic acid antibody comprising an amino acid sequence BH2 having 95% or more identity to the amino acid sequence BH1 represented by SEQ ID NO: 16.

[0057] Antibody B is a humanized antibody of an antibody having a CDR sequence derived from a mouse.

[0058] The identity of the amino acid sequence BH2 to the amino acid sequence BH1 is preferably 97% or more, more preferably 98% or more, still more preferably 99% or more, and in any case 100% or less. In the amino acid sequence BH2, the number of amino acid residues mutated from the amino acid sequence BH1 is, for example, 0 to 5, preferably 0 to 2, more preferably 0 to 1.

[0059] Antibody B preferably includes a light chain variable region comprising an amino acid sequence BL2 having 95% or more identity to the amino acid sequence BL1 represented by SEQ ID NO: 14.

[0060] The identity of the amino acid sequence BL2 to the amino acid sequence BL1 is preferably 97% or more, more preferably 98% or more, still more preferably 99% or more, and in any case 100% or less. In the amino acid sequence BL2, the number of amino acid residues mutated from the amino acid sequence BL1 is, for example, 0 to 5, preferably 0 to 2, more preferably 0 to 1.

[0061] In the above, when it is a sequence mutated with respect to the amino acid sequence BH1 or the amino acid sequence BL1, the mutation site and the mutation content are not particularly limited as long as the reactivity to polysialic acid is not significantly impaired and the humanized sequence portion is not significantly mutated. The mutation site and the mutation content can be appropriately determined based on the antibody sequence information of each species, the information of the content of Example 5 (Figure 10, etc.), and the like.

[0062] From the viewpoint of reactivity to polysialic acid, antibody B preferably includes a light chain variable region including a light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 6, and / or a heavy chain variable region including a heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 7, a heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NO: 8, and a heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 9.

[0063] When antibody B has a constant region, its amino acid sequence is the amino acid sequence of the constant region of a human antibody.

[0064] The reactivity / binding property of antibody B to polysialic acid, measured by the ELISA of Example 5 described below, is, for example, 50% or more, preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, even more preferably 90% or more, based on the reactivity / binding property of the humanized antibody obtained in Example 5, which is 100%. The upper limit of the reactivity / binding property is not particularly limited, and is, for example, 500%, 300%, 200%, 150%, or 120%.

[0065] Antibody B, as a humanized antibody, can be suitably used in in vivo diagnosis and the like.

[0066] 2-3. Antibody C Antibody C is an anti-polysialic acid antibody comprising a light chain variable region containing a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 17, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 18, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 19, and / or a heavy chain variable region containing a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 21, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 22, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 23.

[0067] Antibody C preferably contains an amino acid sequence CL2 having 95% or more (preferably 100%) identity to the amino acid sequence CL1 shown in SEQ ID NO: 20 or SEQ ID NO: 37 (preferably SEQ ID NO: 37).

[0068] The identity of the amino acid sequence CL2 to the amino acid sequence CL1 is preferably 97% or more, more preferably 98% or more, still more preferably 99% or more, and in any case 100% or less. In the amino acid sequence CL2, the number of amino acid residues mutated from the amino acid sequence CL1 is, for example, 0 to 5, preferably 0 to 2, more preferably 0 to 1.

[0069] Antibody C preferably contains an amino acid sequence CH2 having 95% or more identity to the amino acid sequence CH1 shown in SEQ ID NO: 24.

[0070] The identity of the amino acid sequence CH2 to the amino acid sequence CH1 is preferably 97% or more, more preferably 98% or more, still more preferably 99% or more, and in any case 100% or less. In the amino acid sequence CH2, the number of amino acid residues mutated from the amino acid sequence CH1 is, for example, 0 to 5, preferably 0 to 2, more preferably 0 to 1.

[0071] Antibody C is preferably an IgG antibody.

[0072] Antibody C can be used for the detection / separation of polysialic acid, and can be utilized in assays that require multiple anti-polysialic acid antibodies, such as sandwich ELISA.

[0073] 2-4. Antibody D Antibody D is an anti-polysialic acid antibody that includes a light chain variable region containing a light chain CDR1 with the amino acid sequence shown in SEQ ID NO: 25, a light chain CDR2 with the amino acid sequence shown in SEQ ID NO: 26, and a light chain CDR3 with the amino acid sequence shown in SEQ ID NO: 27, and / or a heavy chain variable region containing a heavy chain CDR1 with the amino acid sequence shown in SEQ ID NO: 29, a heavy chain CDR2 with the amino acid sequence shown in SEQ ID NO: 30, and a heavy chain CDR3 with the amino acid sequence shown in SEQ ID NO: 31.

[0074] Antibody D preferably includes an amino acid sequence DL2 that has 95% or more identity to the amino acid sequence DL1 shown in SEQ ID NO: 28 in the light chain variable region.

[0075] The identity of the amino acid sequence DL2 to the amino acid sequence DL1 is preferably 97% or more, more preferably 98% or more, still more preferably 99% or more, and in any case 100% or less. In the amino acid sequence DL2, the number of amino acid residues mutated from the amino acid sequence DL1 is, for example, 0 to 5, preferably 0 to 2, more preferably 0 to 1.

[0076] Antibody D preferably includes an amino acid sequence DH2 that has 95% or more identity to the amino acid sequence DH1 shown in SEQ ID NO: 32 in the heavy chain variable region.

[0077] The identity of the amino acid sequence DH2 to the amino acid sequence DH1 is preferably 97% or more, more preferably 98% or more, still more preferably 99% or more, and in any case 100% or less. In the amino acid sequence DH2, the number of amino acid residues mutated from the amino acid sequence DH1 is, for example, 0 to 5, preferably 0 to 2, more preferably 0 to 1.

[0078] Antibody D is preferably an IgG antibody.

[0079] Antibody D can be used for the detection / separation of polysialic acid, and can be used, for example, in assays that require multiple anti-polysialic acid antibodies such as sandwich ELISA.

[0080] 2-5. Antibody E Antibody E is an antibody to which a streptavidin-binding protein and / or a multimerization domain is added. The addition of the streptavidin-binding protein and / or the multimerization domain can improve the detection sensitivity.

[0081] The target of antibody E is not particularly limited, and examples include sugar chains such as polysialic acid, proteins, peptides, and the like.

[0082] The streptavidin-binding protein is not particularly limited as long as it is a protein having binding affinity for streptavidin. As the streptavidin-binding protein, for example, those derived from various organisms (animals, plants, microorganisms) can be adopted. Specific examples of the streptavidin-binding protein include a protein consisting of an amino acid sequence E2 having 70% or more identity to the amino acid sequence E1 represented by SEQ ID NO: 11. The identity of the amino acid sequence E2 to the amino acid sequence E1 is preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, even more preferably 98% or more, and in any case 100% or less. In the amino acid sequence E2, the number of amino acid residues mutated from the amino acid sequence E1 is, for example, 0 to 30, preferably 0 to 15, more preferably 0 to 10, still more preferably 0 to 5, and even more preferably 0 to 2.

[0083] The multimerization domain is a domain capable of binding to each other to form a multimer, and is not particularly limited as long as it is so. Examples of the multimerization domain include a cartilage oligomeric matrix protein domain, a leucine zipper domain, a collagen-like domain, a cholera toxin B subunit domain, a tetrabrachion coiled coil core domain, a reovirus σ1 protein domain, a hepatitis delta antigen domain, and the like. Specific examples of the multimerization domain include a protein consisting of an amino acid sequence E4 having 70% or more identity to the amino acid sequence E3 represented by SEQ ID NO: 10. The identity of the amino acid sequence E4 to the amino acid sequence E3 is preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, even more preferably 98% or more, and in any case 100% or less. In the amino acid sequence E4, the number of amino acid residues mutated from the amino acid sequence E3 is, for example, 0 to 30, preferably 0 to 15, more preferably 0 to 10, still more preferably 0 to 5, and even more preferably 0 to 2.

[0084] The mode of addition of streptavidin-binding protein and / or multimerization domain is not particularly limited, and it may be added in the form of a fusion protein with an antibody, or may be added by chemical cross-linking with an antibody. Further, the streptavidin-binding protein and / or multimerization domain may be directly added to the antibody, or may be added to the antibody via other structures such as a linker (peptide linker (e.g., a linker composed of G and S, etc.), a divalent group having a chain structure (e.g., an optionally substituted alkylene group, an optionally substituted heteroalkylene group, etc.)), a peptide tag (e.g., a purification tag such as His tag, etc.). In order to suppress the influence on the binding property of the antibody, the position where the streptavidin-binding protein and / or multimerization domain is added is preferably the terminal on the opposite side of the antibody variable region.

[0085] Antibody E is preferably an IgG antibody. Further, antibody E usually includes a light chain variable region and a heavy chain variable region. These variable regions or the CDR sequences in these variable regions are preferably variable regions or CDR antibodies derived from IgM antibodies. When obtaining an IgG antibody using the variable region of an IgM antibody (for example, obtaining a chimeric antibody formed by fusing the variable region of an IgM antibody and the constant region of an IgG antibody), the binding property of the obtained IgG antibody to the target may be greatly reduced. In this case, the binding property can be improved by adding a streptavidin-binding protein and / or multimerization domain.

[0086] Antibody E is preferably an anti-polysialic acid antibody in which a streptavidin-binding protein and / or multimerization domain is added to antibody AX, antibody B, antibody C, or antibody D.

[0087] 2-6. Common Matters Regarding each antibody of the present invention, the constitution other than the constitution described above is as follows.

[0088] The antibody of the present invention is preferably a monoclonal antibody.

[0089] The molecular weight of the antibody of the present invention is not particularly limited, but the lower limit is, for example, 20,000, preferably 50,000, preferably 100,000, more preferably 120,000, and the upper limit is, for example, 1,000,000, preferably 500,000, more preferably 200,000.

[0090] The structure of the antibody of the present invention is not particularly limited. The antibody of the present invention may or may not include a constant region. When including a constant region, it may include all of the constant regions of the heavy chain (CH1, CH2, and CH3) and the constant region of the light chain (CL), or may include any one or a combination of two or more of these.

[0091] Specific examples of the structure of the antibody of the present invention include immunoglobulin, Fab, F(ab’)2, minibody, scFv-Fc, Fv, scFv, diabody, triabody, tetrabody, and the like. Among these, from the viewpoint of the effects of the present invention, immunoglobulin is preferably mentioned.

[0092] Immunoglobulin has a structure in which two structures each composed of one heavy chain having a heavy chain variable region and a heavy chain constant region and one light chain having a light chain variable region and a light chain constant region are combined.

[0093] Fab includes a fragment of the heavy chain containing the CH1 in the heavy chain variable region and the heavy chain constant region, and a light chain containing the light chain variable region and the light chain constant region (CL), and has a structure in which the heavy chain variable region and the light chain variable region are associated by the non-covalent intermolecular interaction described above or are bonded by a disulfide bond. In Fab, CH1 and CL may be disulfide-bonded by the thiol groups of the cysteine residues present therein.

[0094] F(ab’)2 has a structure in which two pairs of the above-mentioned Fab are present and CH1s are disulfide-bonded by the thiol groups of the cysteine residues contained therein.

[0095] A minibody is a structure in which two fragments, each having a CH3 bound to the heavy chain variable region constituting the following scFv, are associated with each other through non-covalent intermolecular interactions between the CH3s.

[0096] An scFv-Fc is a structure in which two antibody fragments containing the following scFv, CH2, and CH3 are associated with each other through non-covalent intermolecular interactions between the CH3s in the same manner as the above minibody, and the thiol groups of the cysteine residues contained in each CH3 are disulfide-bonded to each other.

[0097] An Fv, which is also said to be the minimum structural unit of an antibody, is a structure in which a heavy chain variable region and a light chain variable region are associated with each other through non-covalent intermolecular interactions. In the Fv, the thiol groups of the cysteine residues present in the heavy chain variable region and the light chain variable region may be disulfide-bonded to each other.

[0098] An scFv is a structure in which the C-terminus of the heavy chain variable region and the N-terminus of the light chain variable region are connected by a linker, or a structure in which the N-terminus of the heavy chain variable region and the C-terminus of the light chain variable region are connected by a linker, and is also called a single-chain antibody.

[0099] A diabody, a triabody, and a tetrabody are structures in which the above scFv forms a dimer, a trimer, and a tetramer, respectively, and is structurally stably associated through non-covalent intermolecular interactions between variable regions, etc., in the same manner as an Fv or the like.

[0100] When the antibody of the present invention is an immunoglobulin, its class is not particularly limited. Examples of such classes include IgA, IgD, IgE, IgG, IgM, etc., and further include subclasses thereof. Preferred classes include, for example, IgG.

[0101] The origin of the antibody of the present invention is not particularly limited. The antibody of the present invention can be, for example, a human-derived antibody, a mouse-derived antibody, a rat-derived antibody, a rabbit-derived antibody, a monkey-derived antibody, a chimpanzee-derived antibody, or the like. Further, the antibody of the present invention may be a chimeric antibody (for example, an antibody in which the amino acid sequence of the constant region of an antibody derived from an organism other than human (such as a mouse) is replaced with the amino acid sequence of the constant region of a human-derived antibody), a humanized antibody, a fully humanized antibody, or the like.

[0102] The antibody of the present invention can be produced, for example, by a method including a step of culturing a host transformed with a polynucleotide encoding the antibody of the present invention and recovering a fraction containing the antibody of the present invention.

[0103] The polynucleotide encoding the antibody of the present invention is not particularly limited as long as it contains the antibody of the present invention in an expressible state, and may contain other sequences in addition to the coding sequence of the antibody of the present invention. Examples of other sequences include a secretion signal peptide coding sequence arranged adjacent to the antibody coding sequence of the present invention, a promoter sequence, an enhancer sequence, a repressor sequence, an insulator sequence, a replication origin, a drug resistance gene coding sequence, and the like. Further, the polynucleotide encoding the antibody of the present invention may be a linear polynucleotide or a circular polynucleotide (such as a vector).

[0104] Specific examples of the polynucleotide include (I) a polynucleotide containing a base sequence encoding at least one selected from the group consisting of the heavy chain, the heavy chain variable region, and heavy chain CDRs1-3 of the antibody of the present invention, (II) a polynucleotide containing a base sequence encoding at least one selected from the group consisting of the light chain, the light chain variable region, and light chain CDRs1-3 of the antibody of the present invention, (III) a polynucleotide containing a base sequence encoding at least one selected from the group consisting of the heavy chain, the heavy chain variable region, and heavy chain CDRs1-3 of the antibody of the present invention, and a polynucleotide containing a base sequence encoding at least one selected from the group consisting of the light chain, the light chain variable region, and light chain CDRs1-3 of the antibody of the present invention.

[0105] The host is not particularly limited and examples include insect cells, eukaryotic cells, mammalian cells, etc. Among them, from the viewpoint of more efficiently expressing antibodies, mammalian cells such as HEK cells, CHO cells, NS0 cells, SP2 / O cells, P3U1 cells, etc. are preferred. The methods of transformation, culture, and recovery are not particularly limited, and known methods in antibody production can be adopted. After recovery, the antibody of the present invention may be purified as necessary. Purification can be performed by known methods in antibody production, such as chromatography, dialysis, etc.

[0106] 3. Reagent, Kit In one aspect, the present invention relates to a reagent or kit containing the antibody of the present invention and / or a polynucleotide encoding the antibody of the present invention.

[0107] The reagent of the present invention may further contain other components as necessary. The other components are not particularly limited, and examples include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, chelating agents, etc.

[0108] The kit of the present invention may appropriately contain other materials, reagents, instruments, etc. necessary for the implementation of the method of the present invention, such as gene transfection reagents, cells, buffers, etc., as necessary.

[0109] The reagent and kit of the present invention can be used for the detection or separation of polysialic acid or a polysialic acid carrier. More specifically, it can be used for the examination of polysialic acid-related diseases or the separation of polysialic acid-expressing cells. Specific embodiments of these uses will be described in the next section (4. Detection and separation methods).

[0110] Polysialic acid-related diseases include cancer (e.g., ovarian cancer, liver cancer, pancreatic cancer, bladder cancer, urethral cancer, colorectal cancer, skin cancer, malignant melanoma, osteosarcoma, squamous cell carcinoma of the head and neck, gastric cancer, prostate cancer, breast cancer, lung cancer, colon cancer, lymphoma, liver cancer, mesothelioma, melanoma, astrocytoma, oligodendroglioma, meningioma, neurofibroma, glioblastoma, epithelioma, schwannoma, neurofibrosarcoma, medulloblastoma, fibrosarcoma, squamous cell carcinoma, neuroectodermal cell carcinoma, thyroid tumor, pituitary tumor, epidermoid carcinoma, etc.), mental diseases (schizophrenia, bipolar disorder, depression), neurodegenerative diseases (Huntington's disease, Alzheimer's disease, ischemia), etc.

[0111] The polysialic acid carrier is not particularly limited as long as it can hold polysialic acid (e.g., proteins, cells, etc.). Specific examples include polysialic acid-expressing cells (immune cells such as NK cells, etc.). A polysialic acid-expressing cell is a cell in which polysialic acid is held on the cell surface.

[0112] 4. Detection, Separation Method In one aspect, the present invention relates to a method for detecting polysialic acid or a polysialic acid carrier (the detection method of the present invention), which includes a step of contacting the antibody of the present invention with a sample (contact step). Further, in one aspect, the present invention relates to a method for separating polysialic acid or a polysialic acid carrier (the separation method of the present invention), which includes a contact step and a step of separating the fraction bound to the antibody and the fraction unbound to the antibody (separation step). These will be described below.

[0113] The sample is not particularly limited as long as it can contain polysialic acid or a polysialic acid carrier, and can be, for example, a biological sample. The biological species from which the biological sample is derived is not particularly limited, and examples include various mammalian animals such as humans, monkeys, mice, rats, dogs, cats, rabbits, etc., other deuterostomes such as sea urchins, and capsular polysaccharides of bacteria. Preferably, humans are included.

[0114] The biological sample is not particularly limited, and examples include tissues (such as brain tissue, tissues where various cancers may be present, etc.), body fluids (such as whole blood, serum, plasma, cerebrospinal fluid, saliva, synovial fluid, urine, tissue fluid (including bronchoalveolar lavage fluid), sweat, tears, sputum, nasal discharge, etc.), and further samples derived therefrom (such as samples obtained through various treatments such as enzymatic treatment and purification treatment).

[0115] The contacting step can be carried out in vitro or in vivo. When the contacting is carried out in vivo, the antibody of the present invention can be administered to a living body to bring the antibody of the present invention into contact with the sample.

[0116] In the detection method of the present invention, the detection of poly sialic acid or a poly sialic acid carrier can be carried out by detecting (for example, visualizing) a complex containing the antibody of the present invention and poly sialic acid or a poly sialic acid carrier. Specifically, for example, various immunological measurement methods can be mentioned, and more specifically, immunohistochemical staining, ELISA, sandwich ELISA, EIA, RIA, Western blotting, etc. can be mentioned.

[0117] Based on the amount or concentration of poly sialic acid or a poly sialic acid carrier detected by the detection method of the present invention, a poly sialic acid-related disease can be examined. For example, based on the amount or concentration of poly sialic acid or a poly sialic acid carrier, the determination of a poly sialic acid-related disease can be assisted.

[0118] The specific embodiment of the separation step is not particularly limited. For example, during the contacting step, the antibody of the present invention can be immobilized on a support (carrier particles, substrate, etc.), or after the contacting step, the antibody of the present invention can be immobilized on a support, and in the separation step, the support and the supernatant can be separated (for example, by washing the support). After separation, if necessary, the complex of the antibody of the present invention and poly sialic acid or a poly sialic acid carrier, or poly sialic acid or a poly sialic acid carrier, can be released.

Example

[0119] The present invention will be described in detail below based on examples, but the present invention is not limited by these examples.

[0120] Example 1. Preparation of Mouse Anti-polySia-hIgG1 Chimeric Antibody The sequences (SEQ ID NOs: 1 to 3) in the following description are shown in FIG. 9. In FIG. 9, the SEQ ID NOs of the L-chain CDR sequences and the H-chain CDR sequences are as follows. L-chain CDR1: SEQ ID NO: 4, L-chain CDR2: SEQ ID NO: 5, L-chain CDR3: SEQ ID NO: 6, H-chain CDR1: SEQ ID NO: 7, H-chain CDR2: SEQ ID NO: 8, H-chain CDR3: SEQ ID NO: 9.

[0121] The mouse anti-polySia (=polysialic acid) antibody scFv variable light chain and the mouse anti-polySia variable heavy chain regions were amplified by PCR from the expression vector of mouse anti-polySia antibody scFv (pCold-mouse anti-polySia scFv (SEQ ID 1: SEQ ID NO: 1)), and subcloned into the pAb-LC and pAb-HC Expression Vectors having the sequences of the human IgG constant regions by the seamless ligation method, respectively, to construct pAb-mouse anti-polySia LC (variable region, SEQ ID 2-1: SEQ ID NO: 2) and pAb-mouse anti-polySia HC (variable region, SEQ ID 2-2: SEQ ID NO: 3). These were co-transfected into Expi293F cells, and the culture supernatant was obtained as a mouse anti-polySia-hIgG1 chimeric antibody fraction. Western blotting was performed using this mouse anti-polySia-hIgG1 chimeric antibody fraction with porcine fetal brain highly expressing polysialic acid, and ELISA was performed using mouse fetal brain (MEB) to confirm the recognition ability of the antibody (FIG. 1).

[0122] Example 2. Preparation of Mouse Anti-polySia -hIgG1 Chimeric (Negative) Antibody A negative control antibody was prepared by point-mutating the 39th tyrosine residue of the variable light chain (SEQ ID NO: 2) in the mouse anti-polySia-hIgG1 chimeric antibody prepared in Example 1 to an alanine residue. As a result, it was a negative antibody that could not be detected by Western blotting in the porcine fetal brain with high polysialic acid expression (Figure 2). Furthermore, in order to confirm the universality of the influence of introducing the point mutation into the 39th tyrosine residue, not only alanine but also serine, isoleucine, aspartic acid, lysine, phenylalanine, and tryptophan were mutated to prepare the Y39A mutant, Y39S mutant, Y39I mutant, Y39D mutant, Y39K mutant, Y39F mutant, and Y39W mutant, respectively. These amino acids were prepared as representatives of those with different properties such as charge and hydrophobicity. When the secreted antibody was confirmed by CBB staining, no significant effect was observed on the expression level and stability as a protein (Figure 2). And in Western blotting using porcine fetal brain, its activity remained about half in the Y39F mutant in which tyrosine was mutated to phenylalanine, an aromatic amino acid, but the other mutants, including tryptophan which is also aromatic, were negative antibodies below the detection limit (Figure 2). Furthermore, as a result of performing ELISA using MEB, the Y39I mutant was a completely negative antibody with no reactivity (Figure 2). Also, the usefulness of the mouse anti-polySia-hIgG1 chimeric antibody and the negative control antibody was shown by tissue staining (Figure 3).

[0123] Example 3. Preparation of High-performance Mouse Anti-polySia -hIgG1 Chimeric Antibody The mouse anti-polySia hIgG1 chimeric antibody prepared in Example 1 was multimerized and its functionality enhanced. An AVEXIS sequence (SEQ ID NO: 10) with a pentamerization domain, a 6xHisTag, and an SBP (streptavidin-binding protein) Tag (SEQ ID NO: 11) were fused to the constant region of pAb-mouse anti-polySiaHC to produce mouse anti-polySia-hIgG1-x5-His-SBP (FL-x5-His-SBP). Also, mouse anti-polySia-hIgG1(Hinge)-x5-His-SBP (Hinge-x5-His-SBP) and mouse anti-polySia-hIgG1(Hinge)-x5-His (Hinge-x5-His) with the hIgG1 constant region up to the hinge region were prepared. Furthermore, mouse anti-polySia-hIgG1-SBP with an SBP Tag fused although it is monomeric was prepared. Western blotting was performed using fetal pig brain for each to confirm the recognition ability of the antibody. The antibody fused with the SBP Tag could be detected by Streptavidin-POD (Figure 4).

[0124] Example 4. Establishment of Sandwich ELISA for Highly Sensitive Detection of Polysialic Acid To detect trace amounts of polysialic acid, a sandwich ELISA method using two types of anti-polysialic acid antibodies with different recognition abilities was developed. Specifically, mouse IgM anti-polysialic acid antibody (prepared in Example 6) was immobilized on a 96-well plate, a sample was added to capture the polysialic acid chain with the mouse IgM anti-polysialic acid antibody, and blocking was performed using BSA. The polysialic acid chain captured by mouse anti-polySia-hIgG1-SBP, one of the above-described highly functionalized mouse anti-polySia-hIgG1 chimeric antibodies, was recognized, and the SBP attached to the antibody was recognized by Streptavidin-POD. Then, a TMB solution was used as a substrate and color development was carried out using the activity of POD, and A450 was measured. As a result, quantification was possible up to 1 ng for MEB, 125 ng for the lysate of melanoma B16 cells transfected with the polysialyltransferase gene and expressing polysialic acid, 5 ng for colominic acid derived from the bacterial capsule, and 0.2 ng for purified polySia-NCAM-Fc (Figure 5).

[0125] Example 5. Preparation of Fully Humanized Anti-polySia Antibody The sequences (SEQ ID NOs: 12 to 16) in the following description are shown in Figure 10.

[0126] The CDR regions of the mouse anti-polySia antibody were transplanted into a human IgG antibody to aim at producing a fully humanized anti-polySia antibody. As the human IgG antibody to be transplanted, the sequence of trastuzumab (variable region, Sequence 3: SEQ ID NOs: 12 and 13) was used (variable region, Sequence 4: SEQ ID NOs: 14 and 15). However, its activity was below the detection limit. Therefore, the sequences of other mouse IgG and human IgG were compared, and adjustment was made to the portions other than the CDR of the heavy chain sequence (variable region, Sequence 5: SEQ ID NO: 16). As a result, although its activity was inferior to that of the mouse anti-polySia-hIgG1 chimeric antibody, full humanization was achieved (Figure 6).

[0127] Example 6. Cloning of CDR Sequences of Mouse IgM Anti-polysialic Acid Antibody and Evaluation of Its Activity cDNA was prepared from a hybridoma producing a mouse IgM anti-polysialic acid antibody, and the variable light chain and variable heavy chain regions were cloned by the PCR method. Then, they were subcloned into pAb-LC and pAb-HC Expression Vectors having the sequence of the human IgG constant region by the seamless ligation method, respectively, to prepare pAb-mIgMpolySiaLC and pAb-mIgMpolySiaHC, and a multimeric mIgMpolySia-hIgG1-x5-His-SBP (FL-x5-His-SBP). In addition, similar to the anti-polysialic acid antibody, a negative control antibody was prepared by point-mutating the 37th tyrosine residue of the light chain in the CDR to an alanine residue. Using this mIgMpolySia-hIgG1 chimeric antibody, Western blotting was performed using porcine fetal brain highly expressing polysialic acid and ELISA was performed using mouse fetal brain (MEB) to confirm the recognition ability of the antibody (Figure 7).

[0128] The sequences of the obtained antibodies are as follows: Light chain CDR1: QSLLDSDGKTY (SEQ ID NO: 17) Light chain CDR2: LVS (SEQ ID NO: 18) Light chain CDR3: WQGTHFP (SEQ ID NO: 19) Light chain variable region: DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPFTFGSGTKLEIK (SEQ ID NO: 20) Heavy chain CDR1: GYTFTSYWI (SEQ ID NO: 21) Heavy chain CDR2: IYPGSGST (SEQ ID NO: 22) Heavy chain CDR3: IRSGVRRPHFDY (SEQ ID NO: 23) Heavy chain variable region: QVQLQQPGSELVRPGGSVKLSCRASGYTFTSYWIHWVKQRPGQGLEWIGNIYPGSGSTNYDEEFKRQATLTVDTSSTTAYMQLSSLTSEDSAVYYCIRSGVRRPHFDYWGQGTTLTVSS (SEQ ID NO: 24).

[0129] Example 7. Cloning of CDR Sequences of Mouse IgM Anti-oligosialic Acid Antibody and Evaluation of Its Activity cDNA was prepared from a hybridoma producing mouse IgMoligoSia, and the variable light chain and variable heavy chain regions were cloned by PCR. Then, they were subcloned into pAb-LC and pAb-HC Expression Vectors having the sequences of human IgG constant regions by seamless ligation method, respectively, to produce pAb-moligoSiaLC and pAb-moligoSiaHC, and multimeric moligoSia-hIgG1-x5-His-SBP (FL-x5-His-SBP). Using this moligoSia-hIgG1 chimeric antibody, ELISA using ganglioside GD3 was performed to confirm the recognition ability of the antibody (Figure 8).

[0130] The sequences of the obtained antibodies are as follows: Light chain CDR1: KSVDNYGISF (SEQ ID NO: 25) Light chain CDR2: AAS (SEQ ID NO: 26) Light chain CDR3: QQSKEVPYT (SEQ ID NO: 27) Light chain variable region: DIVLTQSPASLAVSLGQRATISCRASKSVDNYGISFMNWFQQKPGQPPKLLIYAASNQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVPYTFGGGTKLEIK (SEQ ID NO: 28) Heavy chain CDR1: GFNIKNTY (SEQ ID NO: 29) Heavy chain CDR2: IDPANGNTK (SEQ ID NO: 30) Heavy chain CDR3: ARRLRSSAGDYFDY (SEQ ID NO: 31) Heavy chain variable region: QVQLKQSVAELVRPGASVKLSCTASGFNIKNTYIHWVNQRPEQGLEWIGRIDPANGNTKYAPKFQGKATISAATSSNTAYLQLSSLTSEDTAMYYCARRLRSSAGDYFDYWGLGTTLTVSS (SEQ ID NO: 32).

[0131] Example 8. Preparation and Activity Evaluation of High-affinity Mouse Anti-polySia -hIgG1 Chimeric Antibody Based on the mouse anti-polySia-hIgG1 chimeric antibody prepared in Example 1, the following five point mutants were prepared. · A mutant antibody (N35Q-mIgGpolySia) in which the 12th asparagine residue of the L-chain CDR1 (SEQ ID NO: 4: RSSQSLVHSNGNTYLY) was point-mutated to a glutamine residue, and the L-chain CDR1 is SEQ ID NO: 33: RSSQSLVHSNGQTYLY), · A mutant antibody (N35L-mIgGpolySia) in which the 12th asparagine residue of the L-chain CDR1 (SEQ ID NO: 4: RSSQSLVHSNGNTYLY) was point-mutated to a leucine residue, and the L-chain CDR1 is SEQ ID NO: 34: RSSQSLVHSNGLTYLY), · A mutant antibody (D158Q-mIgGpolySia) in which the 6th aspartic acid residue of the H-chain CDR1 (SEQ ID NO: 7: GYTFTDY) was point-mutated to a glutamine residue, and the H-chain CDR1 is SEQ ID NO: 35: GYTFTQY), · A mutant antibody (D158E-mIgGpolySia) in which the 6th aspartic acid residue of the H-chain CDR1 (SEQ ID NO: 7: GYTFTDY) was point-mutated to a glutamic acid residue, and the H-chain CDR1 is SEQ ID NO: 36: GYTFTEY), and · A mutant antibody (N35Q / D158Q-mIgGpolySia) in which the 12th asparagine residue of the L-chain CDR1 (SEQ ID NO: 4: RSSQSLVHSNGNTYLY) was point-mutated to a glutamine residue, and the 6th aspartic acid residue of the H-chain CDR1 (SEQ ID NO: 7: GYTFTDY) was point-mutated to a glutamine residue, and the L-chain CDR1 is SEQ ID NO: 33: RSSQSLVHSNGQTYLY, and the H-chain CDR1 is SEQ ID NO: 35: GYTFTQY).

[0132] For each of these five mutant antibodies, the mouse anti-polySia-hIgG1 chimeric antibody (mIgGpolySia-WT) prepared in Example 1, and the negative antibody (Y39I-mIgGpolySia) prepared in Example 2, the affinity for polysialic acid was analyzed by biolayer interferometry (Octet). The results are shown in Table 1. The introduction of point mutations improved the affinity for polysialic acid.

[0133]

Table 1

[0134] Example 9. Preparation and Activity Evaluation of High-affinity Mouse Anti-polySia -hIgG1 Chimeric Antibody Based on the mIgMpolySia-hIgG-x5-His-SBP antibody prepared in Example 6, the following point mutants were prepared. · A mutant antibody (D60Q) in which the 60th aspartic acid residue of the light chain variable region (SEQ ID NO: 20: DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPFTFGSGTKLEIK) was point-mutated to a glutamine residue, and the light chain variable region is SEQ ID NO: 37: DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLQSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPFTFGSGTKLEIK).

[0135] Using the above mutant antibody or the mIgMpolySia-hIgG-x5-His-SBP antibody prepared in Example 6, ELISA was performed using mouse embryonic brain (MEB) to confirm the recognition ability of the antibody. The results are shown in Figure 11. The introduction of point mutations improved the affinity for polysialic acid. In addition, several point mutants other than the above were prepared, but all showed a significant decrease in affinity.

[0136] Example 10. Preparation and Activity Evaluation of Low-affinity Mouse IgM Anti-oligosialic Acid Antibody Based on the mIgMoligoSia-hIgG-x5-His-SBP antibody prepared in Example 7, the following point mutants were prepared. · A mutant antibody (N31A, light chain CDR1 is SEQ ID NO: 38: KSVDAYGISF) in which the 5th asparagine residue of the light chain CDR1 (SEQ ID NO: 25: KSVDNYGISF) was point-mutated to an alanine residue.

[0137] For the above mutant antibody or the mIgMoligoSia-hIgG-x5-His-SBP antibody prepared in Example 7, ELISA using ganglioside GD3 was performed to confirm the recognition ability of the antibody. The results are shown in Fig. 12.

Claims

1. An anti-polysialic acid negative antibody in which the tyrosine residue on the C-terminal side within the light chain CDR1 of (antibody A) is substituted with another amino acid; An anti-polysialic acid antibody comprising a light chain variable region containing a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 33 or SEQ ID NO: 34, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6, and / or a heavy chain variable region containing a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 35 or SEQ ID NO: 36, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 8, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 9; An anti-polysialic acid humanized antibody (antibody B) in which the heavy chain variable region contains an amino acid sequence BH2 having 95% or more identity to the amino acid sequence BH1 shown in SEQ ID NO: 16; An anti-polysialic acid antibody comprising a light chain variable region containing a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 17, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 18, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 19, and / or a heavy chain variable region containing a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 21, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 22, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 23; An anti-polysialic acid antibody comprising a light chain variable region containing a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 25 or SEQ ID NO: 38, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 26, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 27, and / or a heavy chain variable region containing a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 29, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 30, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 31; and An antibody (antibody E) to which a streptavidin-binding protein and / or a multimerization domain is added A kit comprising at least one antibody selected from the group consisting of the above antibodies, and / or a polynucleotide encoding the above antibody.

2. Said antibody A is An anti-polysialic acid negative antibody, or An anti-polysialic acid negative antibody, comprising a light chain variable region containing an amino acid sequence in which the 39th tyrosine residue from the N-terminus of the amino acid sequence A1L1 represented by SEQ ID NO: 2, or the tyrosine residue corresponding to the tyrosine residue in the amino acid sequence A1L2 having 95% or more identity to the amino acid sequence A1L1, is mutated to another amino acid residue; The kit according to claim 1, which is

3. The kit according to claim 1, wherein the antibody B is an anti-polysialic acid humanized antibody, the heavy chain variable region of which contains the amino acid sequence represented by SEQ ID NO:

16.

4. The kit according to claim 1, wherein the antibody C and / or the antibody D is an IgG antibody.

5. The kit according to claim 1, wherein the antibody E is an anti-polysialic acid antibody obtained by adding a streptavidin-binding protein and / or a multimerization domain to the antibody AX, the antibody B, the antibody C, or the antibody D.

6. The kit according to any one of claims 1 to 5, which is for detecting or separating polysialic acid or a polysialic acid carrier.

7. The kit according to any one of claims 1 to 5, which is for examining polysialic acid-related diseases or separating polysialic acid-expressing cells.

8. An anti-polysialic acid negative antibody in which the tyrosine residue on the C-terminal side within the light chain CDR1 is substituted with another amino acid; An anti-polysialic acid antibody, comprising a light chain variable region containing a light chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 33 or SEQ ID NO: 34, a light chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 6, and / or a heavy chain variable region containing a heavy chain CDR1 containing the amino acid sequence represented by SEQ ID NO: 35 or SEQ ID NO: 36, a heavy chain CDR2 containing the amino acid sequence represented by SEQ ID NO: 8, and a heavy chain CDR3 containing the amino acid sequence represented by SEQ ID NO: 9; An anti-polysialic acid humanized antibody comprising an amino acid sequence BH2 in the heavy chain variable region having 95% or more identity to the amino acid sequence BH1 represented by SEQ ID NO: 16; (Antibody C) A light chain variable region comprising a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 17, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 18, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 19, and / or a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 21, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 22, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 23, an anti-polysialic acid antibody; (Antibody D) A light chain variable region comprising a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 25 or SEQ ID NO: 38, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 26, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 27, and / or a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 29, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 30, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 31, an anti-polysialic acid antibody; and (Antibody E) An antibody obtained by adding a streptavidin-binding protein and / or a multimerization domain A reagent comprising at least one antibody selected from the group consisting of, and / or a polynucleotide encoding said antibody.

9. (Antibody A) An anti-polysialic acid negative antibody in which the tyrosine residue on the C-terminal side within the light chain CDR1 is substituted with another amino acid; (Antibody AX) A light chain variable region comprising a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 33 or SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6, and / or a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 35 or SEQ ID NO: 36, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 8, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 9, an anti-polysialic acid antibody; (Antibody B) An anti-polysialic acid humanized antibody comprising an amino acid sequence BH2 in the heavy chain variable region having 95% or more identity to the amino acid sequence BH1 represented by SEQ ID NO: 16; An anti-polysialic acid antibody comprising a light chain variable region containing a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 17, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 18, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 19, and / or a heavy chain variable region containing a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 21, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 22, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 23; An anti-polysialic acid antibody comprising a light chain variable region containing a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 25 or SEQ ID NO: 38, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 26, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 27, and / or a heavy chain variable region containing a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 29, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 30, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 31; and (Antibody E) An antibody having a streptavidin-binding protein and / or a multimerization domain added thereto A method for detecting polysialic acid or a polysialic acid carrier, comprising the step of contacting a sample with at least one antibody selected from the group consisting of the antibodies.

10. The detection method according to claim 9, comprising visualizing a complex comprising the antibody and polysialic acid or a polysialic acid carrier.

11. The detection method according to claim 9 or 10, which is an ELISA method.

12. The detection method according to claim 11, wherein the ELISA method is a sandwich ELISA method.

13. (Antibody A) An anti-polysialic acid negative antibody in which the tyrosine residue on the C-terminal side within the light chain CDR1 is substituted with another amino acid; (Antibody AX) A light chain variable region containing a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 33 or SEQ ID NO: 34, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6, and / or a heavy chain variable region containing a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 35 or SEQ ID NO: 36, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 8, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 9, an anti-polysialic acid antibody; An anti-polysialic acid humanized antibody comprising an amino acid sequence BH2 having 95% or more identity to the amino acid sequence BH1 represented by SEQ ID NO: 16 in the heavy chain variable region of (antibody B); (Antibody C) A light chain variable region comprising a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 17, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 18, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 19, and / or a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 21, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 22, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 23, an anti-polysialic acid antibody; (Antibody D) A light chain variable region comprising a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 25 or SEQ ID NO: 38, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 26, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 27, and / or a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 29, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 30, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 31, an anti-polysialic acid antibody; and (Antibody E) An antibody obtained by adding a streptavidin-binding protein and / or a multimerization domain A method for separating polysialic acid or a polysialic acid carrier, comprising the step of contacting a sample with at least one antibody selected from the group consisting of the antibodies, and the step of separating the bound fraction to the antibody and the unbound fraction to the antibody.

14. Any one of the following antibodies A to E, and / or a polynucleotide encoding said antibody: (Antibody A) An anti-polysialic acid negative antibody in which the tyrosine residue on the C-terminal side within the light chain CDR1 is substituted with another amino acid; (Antibody AX) A light chain variable region comprising a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 33 or SEQ ID NO: 34, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 6, and / or a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 35 or SEQ ID NO: 36, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 8, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 9, an anti-polysialic acid antibody; A humanized anti-polysialic acid antibody comprising an amino acid sequence BH2 of the heavy chain variable region having 95% or more identity to the amino acid sequence BH1 represented by SEQ ID NO: 16; An anti-polysialic acid antibody comprising a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 17, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 18, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 19, and / or a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 21, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 22, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 23; An anti-polysialic acid antibody comprising a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 25 or SEQ ID NO: 38, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 26, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 27, and / or a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 29, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 30, and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 31; and An antibody comprising a streptavidin-binding protein and / or a multimerization domain added thereto.

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