WO2023277122A1 - ポリシアル酸又はポリシアル酸保持体の検出又は分離方法 - Google Patents

ポリシアル酸又はポリシアル酸保持体の検出又は分離方法 Download PDF

Info

Publication number
WO2023277122A1
WO2023277122A1 PCT/JP2022/026183 JP2022026183W WO2023277122A1 WO 2023277122 A1 WO2023277122 A1 WO 2023277122A1 JP 2022026183 W JP2022026183 W JP 2022026183W WO 2023277122 A1 WO2023277122 A1 WO 2023277122A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
seq
acid sequence
antibody
heavy chain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2022/026183
Other languages
English (en)
French (fr)
Japanese (ja)
Inventor
ちひろ 佐藤
正弥 羽根
開都 早川
健 北島
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tokai National Higher Education and Research System NUC
Original Assignee
Tokai National Higher Education and Research System NUC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tokai National Higher Education and Research System NUC filed Critical Tokai National Higher Education and Research System NUC
Priority to JP2023532047A priority Critical patent/JPWO2023277122A1/ja
Publication of WO2023277122A1 publication Critical patent/WO2023277122A1/ja
Anticipated expiration legal-status Critical
Priority to JP2025065498A priority patent/JP2025108564A/ja
Ceased legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to methods for detecting and separating polysialic acid or polysialic acid-supporting materials.
  • Polysialic acid exists mainly as a modified sugar chain of NCAM, which is an intercellular adhesion molecule, and it is known that it is responsible for NCAM function regulation and has a unique function of polysialic acid. ing. Polysialic acid has been previously reported to present repulsive fields between cells and modulate intercellular spaces to control intracellular signal strength. However, in recent years, it has been reported that polysialic acid presents an attractive field and specifically binds to various molecular groups. In addition, polysialic acid is expressed in cancer cells, is abnormally expressed in patients with psychiatric disorders, is abnormally expressed in patients with neurodegenerative diseases, and is found on the surface of immune cells (NK cells, etc.). It is known to exist (Non-Patent Document 1).
  • An object of the present invention is to provide a technique for detecting or separating polysialic acid or a polysialic acid-bearing material.
  • antibody A an anti-polysialic acid negative antibody in which the tyrosine residue on the C-terminal side in the light chain CDR1 is substituted with another amino acid
  • AX light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:33 or SEQ ID NO:34, light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:5, and light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:6 a light chain variable region and/or 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 the amino acid sequence represented by SEQ ID NO: 9
  • antibody B an amino acid sequence BH2 whose heavy chain variable region has 95% or more identity to the amino acid sequence BH1 shown in SEQ ID NO: 16 (antibody C)
  • the antibody A is (Antibody A1) Corresponding to the 39th tyrosine residue from the N-terminus of the amino acid sequence A1L1 shown in SEQ ID NO: 2, or the tyrosine residue in the amino acid sequence A1L2 having 95% or more identity to the amino acid sequence A1L1 an anti-polysialic acid negative antibody, or (Antibody A2) the N-terminus of the amino acid sequence A2L1 represented by SEQ ID NO: 20, comprising a light chain variable region comprising an amino acid sequence in which the tyrosine residue is mutated to another amino acid residue 37th tyrosine residue from 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 Item 2.
  • the kit according to Item 1 which is an anti-polysialic acid negative antibody, comprising a light chain variable region comprising an amino acid sequence.
  • Section 3. Item 1 or 2, wherein the antibody B is an anti-polysialic acid humanized antibody, the heavy chain variable region of which comprises the amino acid sequence shown in SEQ ID NO: 16.
  • Section 4 The kit according to any one of items 1 to 3, wherein said antibody C and/or said antibody D is an IgG antibody.
  • Item 5 Items 1 to 4, 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.
  • 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-bearing material.
  • Item 7 The kit according to any one of Items 1 to 6, which is for testing polysialic acid-related diseases or for isolating polysialic acid-expressing cells.
  • Antibody A an anti-polysialic acid negative antibody in which the C-terminal tyrosine residue in the light chain CDR1 is substituted with another amino acid
  • Antibody AX Light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:33 or SEQ ID NO:34, light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:5, and light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:6 and/or 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 the amino acid represented by SEQ ID NO:9 an anti-polysialic acid antibody comprising a heavy chain variable region comprising a heavy chain CDR3 comprising sequence;
  • Antibody B an anti-polysialic acid humanized antibody whose heavy chain variable region comprises an amino acid sequence BH2 having 95% or more identity
  • Item 10 The detection method according to Item 9, comprising visualizing a complex containing the antibody and polysialic acid or a polysialic acid-supporting material.
  • Item 11 The detection method according to Item 9 or 10, which is an ELISA method.
  • Item 12. The detection method according to Item 11, wherein the ELISA method is a sandwich ELISA method.
  • Antibody A an anti-polysialic acid negative antibody in which the C-terminal tyrosine residue in the light chain CDR1 is substituted with another amino acid
  • Antibody AX Light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO:33 or SEQ ID NO:34, light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO:5, and light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:6 and/or 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 the amino acid represented by SEQ ID NO:9 an anti-polysialic acid antibody comprising a heavy chain variable region comprising a heavy chain CDR3 comprising sequence;
  • Antibody B an anti-polysialic acid antibody comprising a heavy chain variable region comprising a heavy chain CDR3 comprising sequence
  • Antibody B an anti
  • a technique for detecting or separating polysialic acid or a polysialic acid-bearing material can be provided.
  • FIG. 1 shows the results of Western blotting using pig fetal brain and ELISA using mouse fetal brain (MEB) in Example 1.
  • FIG. 2 shows the results of Western blotting using pig fetal brain and ELISA using mouse fetal brain (MEB) in Example 2.
  • FIG. 2 shows the results of tissue staining in Example 2.
  • FIG. 3 shows the results of Western blotting using porcine fetal brain in Example 3.
  • FIG. 2 shows the results of sandwich ELISA using two types of anti-polysialic acid antibodies with different recognition abilities in Example 4.
  • FIG. 2 shows the results of Western blotting using pig fetal brain and ELISA using mouse fetal brain (MEB) in Example 5.
  • FIG. 6 shows the results of Western blotting using pig fetal brain and ELISA using mouse fetal brain (MEB) in Example 6.
  • FIG. 2 shows the results of ELISA using mouse fetal brain (MEB) in Example 7.
  • FIG. The amino acid sequences of the materials used or produced in Example 1 are shown.
  • the amino acid sequences of the materials used or produced in Example 5 are shown.
  • 2 shows the results of ELISA using mouse fetal brain (MEB) in Example 9.
  • FIG. 2 shows the results of ELISA using ganglioside GD3 in Example 10.
  • amino acids/amino acid residues are sometimes expressed in single letter notation.
  • Identity of amino acid sequences refers to the degree of matching of two or more comparable amino acid sequences to each other. Therefore, the higher the identity or similarity between two amino acid sequences, the higher the identity or similarity between those sequences.
  • the level of amino acid sequence identity is determined, for example, using the sequence analysis tool FASTA, using default parameters. or Algorithm BLAST by Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc Natl Acad Sci USA. "Applications and statistics for multiple high-scoring segments in molecular sequences.” Proc Natl Acad Sci USA. 90:5873-7 (1993)).
  • BLASTX based on such a BLAST algorithm has been developed. Specific methods of these analysis methods are known, and the website of the National Center of Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/) can be referred to. "Identity" of base sequences is also defined according to the above.
  • conservative substitution means that an amino acid residue is replaced with an amino acid residue having a similar side chain.
  • substitutions between amino acid residues having basic side chains such as lysine, arginine, and histidine correspond to conservative substitutions.
  • 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
  • alanine, valine, leucine, isoleucine amino acid residues with nonpolar side chains such as proline, phenylalanine, methionine, tryptophan
  • amino acid residues with ⁇ -branched side chains such as threonine, valine, isoleucine
  • aromatic side chains such as tyrosine, phenylalanine, tryptophan, histidine. Substitutions between amino acid residues are also conservative substitutions.
  • CDR is an abbreviation for Complementarity Determining Region, and is also called complementarity determining region.
  • a CDR is a region present in the variable region of immunoglobulin, and is a region deeply involved in the specific binding of an antibody to an antigen.
  • Light chain CDR means a CDR present in the immunoglobulin light chain variable region
  • heavy chain CDR means a CDR present in the immunoglobulin heavy chain variable region.
  • variable region means a region containing the aforementioned CDR1 to CDR3 (hereinafter simply referred to as "CDRs1-3").
  • CDRs1-3 CDRs1-3
  • the arrangement order of these CDRs1-3 is not particularly limited, but preferably, from the N-terminal side to the C-terminal side, in the order of CDR1, CDR2, and CDR3, or in the reverse order, continuous or the frame described later It refers to regions interspersed with other amino acid sequences called work regions (FR).
  • the "heavy chain variable region” is the region in which the above-mentioned heavy chain CDRs1-3 are arranged, and the “light chain variable region” is the region in which the above-mentioned light chain CDRs1-3 are arranged.
  • FR1 is the region between the N-terminus of the variable region and the CDR1
  • FR2 is the region between CDR1 and CDR2
  • FR3 is the region between CDR2 and CDR3
  • FR3 is the region between CDR3 and the C-terminus of the variable region. defined respectively as FR4.
  • the FR also functions as a linker sequence connecting the particularly important CDRs1-3 as the antigen recognition sequence mentioned above, and is a region that contributes to the formation of the three-dimensional structure of the entire variable region.
  • antibody A, antibody AX, antibody B, antibody C, antibody D, and antibody E in the present specification, these may be collectively referred to as "the antibody of the present invention”). Regarding. These are described below.
  • Antibody A is an anti-polysialic acid negative antibody in which the C-terminal tyrosine residue in the light chain CDR1 is substituted with another amino acid.
  • the C-terminal tyrosine residue in the light chain CDR1 is a tyrosine residue in the light chain CDR1, a tyrosine residue present on the C-terminal side in the light chain CDR1, and is not particularly limited in this respect.
  • C-terminal side means, for example, 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 indicates a region consisting of
  • the above tyrosine residue is preferably the most C-terminal tyrosine residue when there are multiple tyrosine residues on the C-terminal side in the light chain CDR1.
  • Antibody A is an anti-polysialic acid antibody into which the above tyrosine residue substitution has been introduced, and the tyrosine residue substitution greatly reduces the reactivity/binding to polysialic acid (see Example 2 below).
  • the reactivity/binding to polysialic acid measured by ELISA is, for example, 50% or less, preferably 40% or less, more preferably 30%, relative to 100% reactivity/binding before the introduction of tyrosine residue substitution. (more preferably 20% or less, still more preferably 10% or less, particularly preferably 5% or less), so it can be used as a polysialic acid negative antibody.
  • “Other amino acid residues” are not particularly limited as long as they are amino acid residues other than tyrosine residues.
  • Other amino acid residues include, for example, aliphatic amino acids such as G, A, V, I and L; sulfur-containing amino acids such as C and M; basic amino acids such as K, H and R; amides such as N and Q group-containing amino acids; hydroxy group-containing amino acids such as S and T; aromatic amino acids such as F and W; acidic amino acids such as D and E; Among these, preferably aliphatic amino acids such as G, A, V, I and L; basic amino acids such as K, H and R; hydroxy group-containing amino acids such as S and T; aromatic amino acids such as W; Examples include acidic amino acids such as D and E, and more preferably A, S, I, D, K, W, and the like.
  • Antibody A usually contains a light chain variable region and a heavy chain variable region.
  • Antibody A may be an anti-polysialic acid antibody into which mutations other than the above tyrosine residue substitutions have been introduced. Mutations include amino acid substitutions, deletions, insertions, and the like.
  • the number of amino acid residues for the mutation is not particularly limited as long as the antibody structure is maintained, but is, for example, 0-20, 0-10, or 0-5, For example, it is 10% or less, 5% or less, 2% or less, or 1% or less relative to 100% of the total number of amino acid residues in the heavy chain/light chain constant region).
  • antibody A1 the 39th tyrosine residue from the N-terminus of the amino acid sequence A1L1 shown in SEQ ID NO: 2, or an amino acid having 95% or more identity to the amino acid sequence A1L1
  • It is preferably an anti-polysialic acid negative antibody comprising a light chain variable region comprising an amino acid sequence in which the tyrosine residue corresponding to the tyrosine residue in sequence A1L2 is mutated to another amino acid residue.
  • Antibody A1 is an anti-polysialic acid antibody containing the amino acid sequence A1L1 as a light chain variable region, into which the above tyrosine residue substitution has been introduced.
  • 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%.
  • the number of amino acid residues mutated from the amino acid sequence A1L1 is, for example, 1-5, preferably 1-2, more preferably 1.
  • corresponding tyrosine residues indicate tyrosine residues at the same position on the aligned sequences when two sequences are compared by BLAST (default setting).
  • the tyrosine residue corresponding to the tyrosine residue in the amino acid sequence A1L2 is an alignment sequence obtained by comparing the amino acid sequence A1L1 and the amino acid sequence A1L2, in the amino acid sequence A1L2, The tyrosine residue at the same position as the 39th tyrosine residue from the N-terminus of the amino acid sequence A1L1 is shown.
  • Antibody A1 preferably comprises a heavy chain variable region comprising amino acid sequence A1H2 having 95% or more identity to amino acid sequence A1H1 shown in SEQ ID NO:3.
  • 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.
  • the number of amino acid residues mutated from the amino acid sequence A1H1 is, for example, 0-5, preferably 0-2, more preferably 0-1.
  • Antibody A2 is an anti-polysialic acid antibody containing the amino acid sequence A2L1 as a light chain variable region, into which the above tyrosine residue substitution has been introduced.
  • 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%.
  • the number of amino acid residues mutated from the amino acid sequence A2L1 is, for example, 1-5, preferably 1-2, more preferably 1.
  • Antibody A2 preferably comprises a heavy chain variable region comprising amino acid sequence A2H2 having 95% or more identity to amino acid sequence A2H1 shown in SEQ ID NO:24.
  • 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.
  • the number of amino acid residues mutated from the amino acid sequence A2H1 is, for example, 0-5, preferably 0-2, more preferably 0-1.
  • Antibody A has low reactivity to polysialic acid and can be used as a negative control in the detection and separation of polysialic acid.
  • Antibody AX has a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO:33 or SEQ ID NO:34 (preferably SEQ ID NO:33), a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO:5, and SEQ ID NO:6.
  • Antibody AX preferably contains an amino acid sequence AXL2 whose light chain variable region has 95% or more identity to the amino acid sequence AXL1 shown in SEQ ID NO:2.
  • 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.
  • the number of amino acid residues mutated from the amino acid sequence AXL1 is, for example, 0-5, preferably 0-2, more preferably 0-1.
  • Antibody AX preferably contains an amino acid sequence AXH2 whose heavy chain variable region has 95% or more identity to the amino acid sequence AXH1 shown in SEQ ID NO:3.
  • 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.
  • the number of amino acid residues mutated from the amino acid sequence AXH1 is, for example, 0-5, preferably 0-2, more preferably 0-1.
  • Antibody AX is preferably an IgG antibody.
  • Antibody AX can be used to detect/separate polysialic acid, and can be used for assays that require multiple types of anti-polysialic acid antibodies, such as sandwich ELISA.
  • Antibody B is an anti-polysialic acid humanized antibody whose heavy chain variable region comprises an amino acid sequence BH2 having 95% or more identity to the amino acid sequence BH1 shown in SEQ ID NO:16.
  • Antibody B is a humanized antibody with mouse-derived CDR sequences.
  • 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.
  • the number of amino acid residues mutated from the amino acid sequence BH1 is, for example, 0-5, preferably 0-2, more preferably 0-1.
  • Antibody B preferably contains a light chain variable region comprising an amino acid sequence BL2 having 95% or more identity to the amino acid sequence BL1 shown in SEQ ID NO:14.
  • 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.
  • the number of amino acid residues mutated from the amino acid sequence BL1 is, for example, 0-5, preferably 0-2, more preferably 0-1.
  • the mutation site and mutation content do not significantly impair the reactivity to polysialic acid, and the humanized sequence portion is not significantly mutated.
  • the site of mutation and the content of the mutation can be appropriately determined based on the antibody sequence information of each biological species, the information of Example 5 (FIG. 10, etc.), and the like.
  • antibody B has 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 an amino acid shown in SEQ ID NO: 6.
  • a light chain variable region comprising a light chain CDR3 comprising the sequence, and/or a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO:7, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO:8, and a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO:9
  • antibody B When antibody B has a constant region, its amino acid sequence is the amino acid sequence of the constant region of a human antibody.
  • Antibody B has a reactivity/binding to polysialic acid measured by ELISA in Example 5 described later, which is 100% of the reactivity/binding of the humanized antibody obtained in Example 5, for example, 50 % or more, preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, and even more preferably 90% or more.
  • the upper limit of reactivity/binding is not particularly limited, and is, for example, 500%, 300%, 200%, 150%, or 120%.
  • Antibody B can be suitably used as a humanized antibody for in vivo diagnosis.
  • Antibody C comprises a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 17, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 18, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 19.
  • a heavy chain CDR1 comprising a variable region and/or an amino acid sequence represented by SEQ ID NO: 21, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 22, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 23
  • An anti-polysialic acid antibody comprising a heavy chain variable region.
  • Antibody C has an amino acid sequence CL2 whose light chain variable region has 95% or more (preferably 100%) identity to the amino acid sequence CL1 represented by SEQ ID NO: 20 or SEQ ID NO: 37 (preferably SEQ ID NO: 37). preferably included.
  • 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.
  • the number of amino acid residues mutated from the amino acid sequence CL1 is, for example, 0-5, preferably 0-2, more preferably 0-1.
  • Antibody C preferably contains an amino acid sequence CH2 whose heavy chain variable region has 95% or more identity to the amino acid sequence CH1 shown in SEQ ID NO:24.
  • 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.
  • the number of amino acid residues mutated from the amino acid sequence CH1 is, for example, 0-5, preferably 0-2, more preferably 0-1.
  • Antibody C is preferably an IgG antibody.
  • Antibody C can be used to detect/separate polysialic acid, and can be used in assays that require multiple types of anti-polysialic acid antibodies, such as sandwich ELISA.
  • Antibody D has 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 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 comprising the amino acid sequence represented by SEQ ID NO: 31
  • Antibody D preferably contains an amino acid sequence DL2 whose light chain variable region has 95% or more identity to the amino acid sequence DL1 shown in SEQ ID NO:28.
  • 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.
  • the number of amino acid residues mutated from the amino acid sequence DL1 is, for example, 0-5, preferably 0-2, more preferably 0-1.
  • Antibody D preferably contains an amino acid sequence DH2 whose heavy chain variable region has 95% or more identity to the amino acid sequence DH1 shown in SEQ ID NO:32.
  • 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.
  • the number of amino acid residues mutated from the amino acid sequence DH1 is, for example, 0-5, preferably 0-2, more preferably 0-1.
  • Antibody D is preferably an IgG antibody.
  • Antibody D can be used to detect/separate polysialic acid, and can be used in assays that require multiple types of anti-polysialic acid antibodies, such as sandwich ELISA.
  • Antibody E is an antibody to which a streptavidin-binding protein and/or a multimerization domain has been added. Addition of streptavidin-binding proteins and/or multimerization domains can improve detection sensitivity.
  • the target of antibody E is not particularly limited, and examples include sugar chains such as polysialic acid, proteins, and peptides.
  • the streptavidin-binding protein is not particularly limited as long as it is a protein that has binding properties to streptavidin.
  • streptavidin-binding proteins for example, those derived from various organisms (animals, plants, and microorganisms) can be employed.
  • Specific examples of streptavidin-binding proteins include proteins consisting of an amino acid sequence E2 having 70% or more identity with the amino acid sequence E1 shown in 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%. It is below.
  • the number of amino acid residues mutated from the amino acid sequence E1 is, for example, 0-30, preferably 0-15, more preferably 0-10, more preferably 0-5, still more preferably 0. ⁇ 2.
  • a multimerization domain is a domain that can bind to each other to form a multimer, and is not particularly limited as long as it is.
  • Multimerization domains include, for example, cartilage oligomeric matrix protein domains, leucine zipper domains, collagen-like domains, cholera toxin B subunit domains, tetrabrachion coiled core domains, reovirus ⁇ 1 protein domains, hepatitis delta antigen domains, and the like.
  • a specific example of the multimerization domain is a protein consisting of an amino acid sequence E4 having 70% or more identity with the amino acid sequence E3 shown in 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%. It is below.
  • the number of amino acid residues mutated from the amino acid sequence E3 is, for example, 0-30, preferably 0-15, more preferably 0-10, more preferably 0-5, even more preferably 0. ⁇ 2.
  • the mode of addition of the streptavidin-binding protein and/or the multimerization domain is not particularly limited, and may be added in the form of a fusion protein with the antibody, or may be added with the antibody by chemical cross-linking.
  • the streptavidin-binding protein and/or the multimerization domain may be directly attached to the antibody, or may be a linker (peptide linker (for example, a linker composed of G and S), a chain-like bivalent group (e.g., optionally substituted alkylene group, optionally substituted heteroalkylene group, etc.), peptide tag (e.g., purification tag such as His tag, etc.), etc.
  • the position where the streptavidin-binding protein and/or the multimerization domain are added is preferably the end opposite to the antibody variable region.
  • Antibody E is preferably an IgG antibody.
  • Antibody E also typically comprises a light chain variable region and a heavy chain variable region. These variable regions or CDR sequences in these variable regions are preferably variable regions or CDR antibodies derived from IgM antibodies.
  • the target binding of the obtained IgG antibody is can drop significantly. In this case, binding can be improved by adding a streptavidin-binding protein and/or a multimerization domain.
  • Antibody E is preferably an anti-polysialic acid antibody obtained by adding a streptavidin-binding protein and/or a multimerization domain to antibody AX, antibody B, antibody C, or antibody D.
  • the antibodies of the present invention are preferably monoclonal antibodies.
  • 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. .
  • the structure of the antibody of the present invention is not particularly limited.
  • the antibodies of the present invention may or may not contain a constant region. When it contains a constant region, it may contain all of the heavy chain constant region (CH1, CH2, and CH3) and the light chain constant region (CL), or any one or more of these. may include a combination of
  • antibody structures of the invention include immunoglobulin, Fab, F(ab') 2 , minibody, scFv-Fc, Fv, scFv, diabody, triabody, A tetrabody and the like can be mentioned. Among these, immunoglobulins are preferred from the viewpoint of the effects of the present invention.
  • An immunoglobulin has a structure in which two structures are combined, one heavy chain having a heavy chain variable region and heavy chain constant region and one light chain having a light chain variable region and light chain constant region.
  • Fab includes a heavy chain fragment containing 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). It has a structure in which it associates with the chain variable region through the above-described non-covalent intermolecular interaction or binds through a disulfide bond.
  • CH1 and CL may form a disulfide bond between the thiol groups of the cysteine residues present in each.
  • F(ab') 2 has two pairs of the above Fab, and has a structure in which CH1s are disulfide-bonded between thiol groups of cysteine residues contained therein.
  • a minibody is a structure in which two fragments in which CH3 is bound to the heavy chain variable region that constitutes the scFv below are associated through non-covalent intermolecular interactions between CH3.
  • scFv-Fc means that two antibody fragments containing the following scFv, CH2, and CH3 are associated by non-covalent intermolecular interactions between CH3, similar to the above minibody, and the cysteine residue contained in each CH3 It is a structure in which the thiol groups of the groups are disulfide-bonded.
  • Fv is also called the minimum structural unit of an antibody, and is a structure in which heavy chain variable regions and light chain variable regions are associated through non-covalent intermolecular interactions.
  • the thiol groups of cysteine residues present in the heavy chain variable region and the light chain variable region may be disulfide bonded.
  • 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 the N-terminus of the heavy chain variable region and the C-terminus of the light chain variable region are connected by a linker.
  • structure also called a single-chain antibody.
  • Diabodies, triabodies, and tetrabodies are the above-described scFvs that form dimers, trimers, and tetramers, respectively, and have non-covalent intermolecular interactions between variable regions, etc., similar to Fv. It is a structure that associates in a structurally stable state.
  • the antibody of the present invention is an immunoglobulin
  • its class is not particularly limited.
  • the classes include, for example, IgA, IgD, IgE, IgG, IgM, and subclasses thereof.
  • Preferred classes include, for example, IgG.
  • Antibodies of the present invention can be, for example, human-derived antibodies, mouse-derived antibodies, rat-derived antibodies, rabbit-derived antibodies, monkey-derived antibodies, chimpanzee-derived antibodies, and the like. Further, the antibodies of the present invention include chimeric antibodies (for example, antibodies obtained by replacing the amino acid sequence of the constant region of an antibody derived from a non-human organism (such as a mouse) with the amino acid sequence of the constant region of a human-derived antibody), humanized antibodies, It may be a fully humanized antibody or the like.
  • the antibody of the present invention can be produced, for example, by culturing a host transformed with a polynucleotide encoding the antibody of the present invention and collecting fractions containing the antibody of the present invention.
  • a 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 besides the coding sequence of the antibody of the present invention. Other sequences include secretory signal peptide coding sequences, promoter sequences, enhancer sequences, repressor sequences, insulator sequences, origins of replication, drug resistance gene coding sequences, etc., which are located adjacent to the antibody coding sequences of the invention. be done.
  • a polynucleotide encoding the antibody of the present invention may be a linear polynucleotide or a circular polynucleotide (vector etc.).
  • polynucleotides include (I) a polynucleotide comprising a nucleotide sequence encoding at least one selected from the group consisting of the heavy chain, heavy chain variable region, and heavy chain CDRs 1-3 of the antibody of the present invention; (II) a polynucleotide comprising a base sequence encoding at least one selected from the group consisting of the light chain, light chain variable region, and light chain CDRs 1-3 of the antibody of the present invention; A polynucleotide comprising a nucleotide sequence encoding at least one selected from the group consisting of a heavy chain, a heavy chain variable region, and heavy chain CDRs 1-3, and a light chain, a light chain variable region, and a light chain of the antibody of the present invention A polynucleotide containing a base sequence encoding at least one selected from the group consisting of chain CDRs 1-3 and the like.
  • the host is not particularly limited, and examples include insect cells, eukaryotic cells, mammalian cells, and the like. Among them, mammalian cells such as HEK cells, CHO cells, NS0 cells, SP2/O cells, and P3U1 cells are preferred from the viewpoint of more efficient antibody expression.
  • Methods for transformation, culture and collection are not particularly limited, and known methods for antibody production can be employed. After recovery, the antibody of the present invention may be purified as necessary. Purification can be performed by known methods for antibody production, such as chromatography, dialysis, and the like.
  • the present invention relates to reagents or kits containing the antibody of the present invention and/or a polynucleotide encoding the antibody of the present invention.
  • the reagent of the present invention may further contain other components as necessary.
  • other components include, but are not limited to, bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, and thickeners. agents, moisturizing agents, coloring agents, fragrances, chelating agents, and the like.
  • the kit of the present invention may optionally contain other materials, reagents, instruments, etc. necessary for carrying out the method of the present invention, such as gene introduction reagents, cells, buffer solutions, etc., as necessary.
  • the reagents and kits of the present invention can be used for detecting or separating polysialic acid or polysialic acid-bearing substances. More specifically, it can be used for examination of polysialic acid-related diseases or separation of polysialic acid-expressing cells. Specific aspects of these applications are described in the next section (4. Detection and Separation Methods).
  • Polysialic acid-related diseases include cancer (eg, ovarian cancer, liver cancer, pancreatic cancer, bladder cancer, urethral cancer, colon cancer, skin cancer, malignant melanoma, osteosarcoma, squamous epithelium of the head and neck).
  • cancer eg, ovarian cancer, liver cancer, pancreatic cancer, bladder cancer, urethral cancer, colon cancer, skin cancer, malignant melanoma, osteosarcoma, squamous epithelium of the head and neck).
  • stomach cancer prostate cancer, breast cancer, lung cancer, colon cancer, lymphoma, liver cancer, mesothelioma, melanoma, astrocytoma, oligodendroglioma, meningioma, neurofibroma, nerve glioblastoma, ependymoma, schwannoma, neurofibrosarcoma, neuromedulloblastoma, fibrosarcoma, squamous cell carcinoma, neuroectodermal cell carcinoma, thyroid tumor, pituitary tumor, epidermoid carcinoma cancer, etc.), psychiatric disorders (schizophrenia, bipolar disorder, depression), neurodegenerative diseases (Huntington's disease, Alzheimer's disease, ischemia), and the like.
  • the polysialic acid retainer is not particularly limited as long as it retains polysialic acid (for example, proteins, cells, etc.). Specific examples include polysialic acid-expressing cells (immune cells such as NK cells, etc.). Polysialic acid-expressing cells are cells in which polysialic acid is retained on the cell surface.
  • the present invention relates to a method for detecting polysialic acid or a polysialic acid-bearing material (detection method of the present invention), comprising a step of contacting the antibody of the present invention with a sample (contacting step).
  • detection method of the present invention polysialic acid or a polysialic acid-supported polysialic acid or polysialic acid-supported material comprising a contacting step and a step of separating the antibody-bound fraction and the antibody-unbound fraction (separation step) Separation method (separation method of the present invention).
  • the sample is not particularly limited as long as it can contain polysialic acid or a polysialic acid support, and can be, for example, a biological sample.
  • the species from which the biological sample is derived is not particularly limited, and examples thereof include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits, other deuterostomes such as sea urchins, and capsular polysaccharides of bacteria. and preferably humans.
  • the biological sample is not particularly limited, and includes tissue (e.g., brain tissue, tissue in which various cancers may exist, etc.), body fluid (e.g., whole blood, serum, plasma, cerebrospinal fluid, saliva, synovial fluid, urine, tissue fluid (bronchial (including alveolar lavage fluid), sweat, tears, sputum, nasal discharge, etc.), and samples derived therefrom (for example, obtained through various treatments such as enzyme treatment and purification treatment).
  • tissue e.g., brain tissue, tissue in which various cancers may exist, etc.
  • body fluid e.g., whole blood, serum, plasma, cerebrospinal fluid, saliva, synovial fluid, urine, tissue fluid (bronchial (including alveolar lavage fluid), sweat, tears, sputum, nasal discharge, etc.
  • samples derived therefrom for example, obtained through various treatments such as enzyme treatment and purification treatment.
  • the contacting step can be performed in vitro or in vivo.
  • the antibody of the present invention can be brought into contact with the sample by administering the antibody of the present invention to a living body.
  • detection of polysialic acid or a polysialic acid-supporting substance can be performed by detecting (for example, visualizing) a complex containing the antibody of the present invention and polysialic acid or a polysialic acid-supporting substance.
  • detecting for example, visualizing
  • Specific examples include various immunological assays, more specifically immunohistochemical staining, ELISA, sandwich ELISA, EIA, RIA, Western blotting, and the like.
  • a polysialic acid-related disease can be tested based on the amount or concentration of polysialic acid or polysialic acid-supporting material detected by the detection method of the present invention.
  • the amount or concentration of polysialic acid or a polysialic acid carrier can aid in the determination of polysialic acid-related diseases.
  • the separation step is not particularly limited.
  • the antibody of the present invention is immobilized on a support (carrier particles, substrate, etc.) during the contacting step, or the antibody of the present invention is immobilized on the support after the contacting step, and the antibody of the present invention is immobilized on the support during the separation step. It can be carried out by separating the supernatant (for example, by washing the support). After separation, the complex of the antibody of the present invention and polysialic acid or polysialic acid-supported material, or polysialic acid or polysialic acid-supported material, can be liberated, if necessary.
  • Example 1 Preparation of Mouse Anti-polySia-hIgG1 Chimeric Antibody
  • the sequences (SEQ ID NOs: 1-3) in the description below are shown in FIG.
  • the sequence numbers of the L chain CDR sequences and the H chain CDR sequences are as follows.
  • H chain CDR1 SEQ ID NO: SEQ ID NO: 7
  • H chain CDR2 SEQ ID NO: 8
  • H chain CDR3 SEQ ID NO: 9.
  • pAb-mouse anti-polySia LC variant region, sequence 2-1: SEQ ID NO: 2
  • pAb - A mouse anti-polySia HC (variable region, sequence 2-2: SEQ ID NO: 3) was constructed. These were co-transfected into Expi293F cells, and the culture supernatant was obtained as a mouse anti-polySia-hIgG1 chimeric antibody fraction.
  • Example 2 Preparation of mouse anti-polySia-hIgG1 chimeric (negative) antibody
  • the 39th tyrosine residue of the variable region light chain (SEQ ID NO: 2) was point-mutated to an alanine residue.
  • a negative control antibody was prepared.
  • the fetal porcine brain, in which polysialic acid is highly expressed was a negative antibody that could not be detected by Western blotting (Fig. 2).
  • Y39A and Y39S mutants were mutated not only to alanine but also to serine, isoleucine, aspartic acid, lysine, phenylalanine, and tryptophan, respectively.
  • Mutants, Y39I mutant, Y39D mutant, Y39K mutant, Y39F mutant and Y39W mutant were produced. These amino acids were prepared as representatives of different properties such as charge and hydrophobicity.
  • Example 3 Preparation of highly functionalized mouse anti -polySia-hIgG1 chimeric antibody
  • the mouse anti-polySia hIgG1 chimeric antibody prepared in Example 1 was multimerized and highly functionalized.
  • the pAb-mouse anti-polySiaHC constant region was fused with the AVEXIS sequence (SEQ ID NO: 10) having a pentamerization domain, 6xHisTag, and SBP (streptavidin-binding protein) Tag (SEQ ID NO: 11), resulting in mouse anti-polySia-hIgG1- x5-His-SBP (FL-x5-His-SBP) was generated.
  • mouse anti-polySia-hIgG1(Hinge)-x5-His-SBP Hinge-x5-His-SBP
  • mouse anti-polySia-hIgG1(Hinge)-x5-His Hinge- x5-His
  • SBP Tag mouse anti-polySia-hIgG1-SBP fused with SBP Tag. Western blotting was performed using fetal porcine brain to confirm the recognition ability of the antibodies. Antibody fused with SBP Tag became detectable with Streptavidin-POD (Fig. 4).
  • Example 4 Establishment of sandwich ELISA for highly sensitive detection of polysialic acid
  • a sandwich ELISA method using two types of anti-polysialic acid antibodies with different recognition abilities. Specifically, a mouse IgM anti-polysialic acid antibody (prepared in Example 6) is immobilized on a 96-well plate, a sample is added, polysialic acid chains are captured by the mouse IgM anti-polysialic acid antibody, and blocked using BSA.
  • mouse anti-polySia-hIgG1-SBP which recognizes the polysialic acid chain captured by mouse anti-polySia-hIgG1-SBP, which is one of the above-mentioned highly functional mouse anti-polySia-hIgG1 chimeric antibodies, and the SBP attached to the antibody is treated with Streptavidin-POD made me recognize it.
  • Streptavidin-POD made me recognize it.
  • TMB solution as a substrate, POD activity was used to develop color, and A450 was measured.
  • 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 mouse IgM anti-polysialic acid antibody, and the variable light chain and variable heavy chain regions were cloned by PCR.
  • pAb-LC and pAb-HC Expression Vectors having human IgG constant region sequences by the seamless ligation method, respectively, pAb-mIgMpolySiaLC and pAb-mIgMpolySiaHC, and multimerized mIgMpolySia-hIgG1-x5-His- SBP (FL-x5-His-SBP) was generated.
  • a negative control antibody was prepared by point-mutating the 37th tyrosine residue of the light chain in the CDR to an alanine residue in the same manner as the anti-polySia antibody.
  • 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: QVQLQQPGSELVRPGGSVKLSCRASGYTFTSYWIHWVKQRPGQGLEWIGNIYPGSGSTNYDEEFKR
  • 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 human IgG constant region sequences by the seamless ligation method, respectively, pAb-moligoSiaLC and pAb-moligoSiaHC, and moligoSia-hIgG1-x5-His- SBP (FL-x5-His-SBP) was generated. Using this moligoSia-hIgG1 chimeric antibody, ELISA using ganglioside GD3 was performed to confirm the recognition ability of the antibody (Fig. 8).
  • 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 QVQLKQSVAELVRPGASVKLSCTASGFNIKNTYIHWVNQRPEQGLEWIGRIDPANGNTKYAPKFQ
  • Example 8 Preparation of high-affinity mouse anti-polySia-hIgG1 chimeric antibody and evaluation of its activity Based on the mouse anti-polySia-hIgG1 chimeric antibody prepared in Example 1, the following five point mutants were prepared.
  • N35Q-mIgGpolySia, L chain CDR1 is SEQ ID NO: 33: RSSQSLVHSNGQTYLY) in which the 12th asparagine residue of L chain CDR1 (SEQ ID NO: 4: RSSQSLVHSNGNTYLY) is point-mutated to a glutamine residue
  • - Mutant antibody N35L-mIgGpolySia, L chain CDR1 is SEQ ID NO: 34: RSSQSLVHSNGLTYLY) in which the 12th asparagine residue of L chain CDR1 (SEQ ID NO: 4: RSSQSLVHSNGNTYLY) is point-mutated to a leucine residue
  • D158Q-mIgGpolySia, H chain CDR1 is SEQ ID NO: 35: GYTFTQY) in which the 6th aspartic acid residue of H chain CDR1 (SEQ ID NO: 7: GYTFTDY) is point-mutated to
  • mice anti-polySia-hIgG1 chimeric antibody (mIgGpolySia-WT) prepared in Example 1
  • the negative antibody (Y39I-mIgGpolySia) prepared in Example 2
  • biolayer interferometry (Octet) analyzed the affinity to polysialic acid. Table 1 shows the results. The introduction of point mutations improved the affinity to polysialic acid.
  • Example 9 Preparation of high-affinity mouse anti-polySia-hIgG1 chimeric antibody and evaluation of its activity Based on the mIgMpolySia-hIgG-x5-His-SBP antibody prepared in Example 6, the following point mutants were prepared.
  • Example 10 Preparation of low-affinity mouse IgM anti-oligosialic acid antibody and evaluation of its activity Based on the mIgMoligoSia-hIgG-x5-His-SBP antibody prepared in Example 7, the following point mutants were prepared.
  • - Mutant antibody N31A, light chain CDR1 is SEQ ID NO: 38: KSVDAYGISF
  • the fifth asparagine residue of light chain CDR1 SEQ ID NO: 25: KSVDNYGISF
  • the above mutant antibody or the mIgMoligoSia-hIgG-x5-His-SBP antibody prepared in Example 7 was subjected to ELISA using ganglioside GD3 to confirm the recognition ability of the antibody. The results are shown in FIG.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biomedical Technology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Psychiatry (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Psychology (AREA)
  • Hospice & Palliative Care (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Pain & Pain Management (AREA)
  • Pathology (AREA)
  • Vascular Medicine (AREA)
  • Cardiology (AREA)
PCT/JP2022/026183 2021-07-01 2022-06-30 ポリシアル酸又はポリシアル酸保持体の検出又は分離方法 Ceased WO2023277122A1 (ja)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP2023532047A JPWO2023277122A1 (https=) 2021-07-01 2022-06-30
JP2025065498A JP2025108564A (ja) 2021-07-01 2025-04-11 ポリシアル酸又はポリシアル酸保持体の検出又は分離方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2021110251 2021-07-01
JP2021-110251 2021-07-01

Publications (1)

Publication Number Publication Date
WO2023277122A1 true WO2023277122A1 (ja) 2023-01-05

Family

ID=84691903

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2022/026183 Ceased WO2023277122A1 (ja) 2021-07-01 2022-06-30 ポリシアル酸又はポリシアル酸保持体の検出又は分離方法

Country Status (2)

Country Link
JP (2) JPWO2023277122A1 (https=)
WO (1) WO2023277122A1 (https=)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019178218A1 (en) * 2018-03-14 2019-09-19 Memorial Sloan Kettering Cancer Center Anti-polysialic acid antibodies and uses thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3478717B1 (en) * 2016-07-04 2022-01-05 F. Hoffmann-La Roche AG Novel antibody format

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019178218A1 (en) * 2018-03-14 2019-09-19 Memorial Sloan Kettering Cancer Center Anti-polysialic acid antibodies and uses thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NAGAE MASAMICHI, IKEDA AKEMI, HANE MASAYA, HANASHIMA SHINYA, KITAJIMA KEN, SATO CHIHIRO, YAMAGUCHI YOSHIKI: "Crystal Structure of Anti-polysialic Acid Antibody Single Chain Fv Fragment Complexed with Octasialic Acid", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 288, no. 47, 7 October 2013 (2013-10-07), US , pages 33784 - 33796, XP093018087, ISSN: 0021-9258, DOI: 10.1074/jbc.M113.496224 *
TSUMOTO, KOUHEI: "Dissection of antibody-antigen interactions for development of antibodies", DRUG DELIVERY SYSTEM, vol. 28, no. 5, 31 January 2013 (2013-01-31), pages 412 - 423, XP093018086, DOI: 10.2745/dds.28.412 *

Also Published As

Publication number Publication date
JP2025108564A (ja) 2025-07-23
JPWO2023277122A1 (https=) 2023-01-05

Similar Documents

Publication Publication Date Title
US10604586B2 (en) Humanized monoclonal antibody and uses thereof
US12007396B2 (en) Methods for characterizing low-abundance host cell proteins
KR102785081B1 (ko) 항 인간 심근 트로포닌 i의 항체 및 그 사용
WO2022247804A1 (zh) 抗gprc5d抗体、其制备方法与用途
CA3242041A1 (en) Antibody or antigen-binding fragment thereof
CN118373910B (zh) 抗lgi1抗体及其应用
CN115028731A (zh) 一种抗Flag标签的抗体及其应用
US10995133B2 (en) Method for producing antibody
CN113880948B (zh) 抗ca724抗体或其抗原结合片段及其制备方法和应用
CN118852441B (zh) 一种抗ceacam5的纳米抗体及其相关应用
WO2023277122A1 (ja) ポリシアル酸又はポリシアル酸保持体の検出又は分離方法
CN112979798B (zh) 一种包含c反应蛋白抗原结合结构域的分离的结合蛋白
WO2022218277A1 (zh) 一种抗fgf21羧基末端的抗体及其应用
CN115850458B (zh) 一种抗冠状病毒rbd蛋白的抗体、制备方法和应用
JP7366411B2 (ja) ヒトαディフェンシンＨＤ５を検出する方法及びキット、並びにこれらにおいて用いられる抗体
CN112724251B (zh) 一种包含c反应蛋白抗原结合结构域的结合蛋白
EP4032914A1 (en) Antibody for detecting acetylation of cox2 protein, and uses thereof
CN121135881A (zh) 一种鼠抗人CD8a重组单克隆抗体及其应用
CN115433279A (zh) 靶向cd123兔重组单克隆抗体的制备及应用
WO2023035226A1 (zh) 抗ang2抗体及其制备方法和应用
KR101630228B1 (ko) 단일클론항체 ｃａ２７ 및 이를 이용한 검출방법
CN116635718A (zh) 用于在免疫组织化学（ihc）方案中使用以诊断癌症的抗人cd10抗体
CN121319202A (zh) Atp7b抗体及其应用
CN114478779A (zh) 人源化的磷脂酰肌醇蛋白聚糖3的单克隆抗体及其应用
JPWO2009116491A1 (ja) ヒトインターフェロンαサブタイプα８及びその変異蛋白質を特異的に認識するモノクローナル抗体

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22833268

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2023532047

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22833268

Country of ref document: EP

Kind code of ref document: A1