WO2020009165A1 - Modified antibody and method for producing same - Google Patents

Modified antibody and method for producing same Download PDF

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Publication number
WO2020009165A1
WO2020009165A1 PCT/JP2019/026521 JP2019026521W WO2020009165A1 WO 2020009165 A1 WO2020009165 A1 WO 2020009165A1 JP 2019026521 W JP2019026521 W JP 2019026521W WO 2020009165 A1 WO2020009165 A1 WO 2020009165A1
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residue
antibody
modified
lysine residue
side chain
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PCT/JP2019/026521
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French (fr)
Japanese (ja)
Inventor
山崎　俊介
奈都紀 敷田
和高 新保
松田　豊
公太 井上
康博 三原
セルゲイ ヴァシリエヴィッチ スミルノフ
イリーナ リヴォヴナ トクマコーヴァ
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味の素株式会社
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes

Definitions

  • the present invention relates to a modified antibody and a method for producing the same.
  • ADC antibody-drug conjugates
  • ADC is a drug obtained by conjugating a drug (eg, an anticancer agent) to an antibody, and has a direct cell killing activity against cancer cells and the like.
  • a typical ADC is T-DM1 (trade name: Kadcyla (registered trademark)) jointly developed by Immunogene and Roche. Therefore, there is a need to develop a technique that is useful for modifying antibodies with drugs.
  • antibodies are a type of protein. Modification of a protein is useful for modulating the function of the desired protein. As a protein modification, modification using functions of various naturally occurring enzymes has been attempted. For example, phosphopantetheinyl transferase (PPTase), biotin ligase, and lipoic acid protein ligase (Lpl) are known as enzymes capable of modifying proteins.
  • PPTase phosphopantetheinyl transferase
  • biotin ligase biotin ligase
  • Lpl lipoic acid protein ligase
  • PPTase is an enzyme that forms a phosphate bond between a hydroxyl group of a serine residue in a protein and coenzyme A (CoA).
  • H / D S
  • L / I has been reported as a PPTase recognition amino acid sequence, and PPTase is considered to recognize an ⁇ -helix structure around a serine residue.
  • Non-patent Document 1 Since PPTase is an enzyme widely conserved in prokaryotes, it is considered to be suitable for searching for a target protein modifying enzyme.
  • PPTase has a short recognition amino acid sequence consisting of three amino acid residues, and is not strict in recognition of both N-terminal and C-terminal amino acid residues in the recognition amino acid sequence. It is thought that it is not high.
  • Biotin ligase is an enzyme that covalently binds biotin to lysine residues in proteins. Biotin has a very high affinity for avidin (Kd ⁇ 10 ⁇ 15 M), so that biotin ligase can add various drugs to lysine residues of proteins via avidin.
  • E. coli a kind of biotin ligase. It has been reported that E. coli BirA can recognize an amino acid sequence consisting of 15 amino acid residues represented by GLNDIFEAQ [K] IEWHE ([K] indicates a lysine residue to which biotin is added).
  • FIG. A fusion protein of an acceptor peptide having an E. coli BirA-recognizing amino acid sequence with a target protein is prepared. A technique for preparing an avidin-modified fusion protein by treating with E. coli BirA has been reported (Non-Patent Document 2).
  • Lpl is an enzyme that binds lipoic acid to a lysine residue in a protein for acylation.
  • Escherichia coli Escherichia coli LplA exists.
  • Escherichia coli (Escherichia coli) LplA is known to function as a coenzyme for enzymes such as pyruvate dehydrogenase, ⁇ -ketoglutarate dehydrogenase, and glycine cleavage system enzymes (Non-patent Documents 3 and 4).
  • LplA recognizes the amino acid sequence of E2 protein (E2p), which is one of the subunits of these enzymes, and activates E2p by adding lipoic acid (lipoylation) to lysine residues in the recognition sequence. As shown in Table 1, LplA is reported to have a relatively long recognition amino acid sequence consisting of 12 amino acid residues (Non-Patent Document 5).
  • N-terminal amino acid residue and the C-terminal amino acid residue of lysylated lysine residues are aspartic acid residue (D) and It is restricted to amino acid residues having a hydrocarbon group as a side chain [alanine (A), valine (V), or leucine (L)] (Table 1).
  • Patent Document 1 As a modification of a protein with LplA, a method of modifying a target protein with lipoic acid and a lipoic acid analog via the LplA recognition peptide tag in a target protein to which an LplA recognition peptide tag has been added is known (Patent Document 1). . Further, as a modification of a protein (antibody) by LplA, a method of modifying a Fab fragment with lipoic acid via an LplA recognition polypeptide in a Fab fragment to which an LplA recognition polypeptide has been added has been reported (Patent Document 2). .
  • the purpose of the present invention is to develop a technology that allows modification of an antibody.
  • the present inventors have conducted intensive studies on antibody modification by an enzymatic method. As a result, among the tested protein-modifying enzymes, phosphopantetheinyltransferase (PPTase) which has the shortest recognized amino acid sequence and is expected to have low substrate specificity ) And biotin ligase did not have the ability to modify an antibody, but lipoic acid protein ligase (Lpl) could modify an antibody despite having no known Lpl-recognizing amino acid sequence. Was found. Next, the present inventors analyzed the modification site of the antibody with Lpl.
  • PPTase phosphopantetheinyltransferase
  • Lpl lipoic acid protein ligase
  • the present invention is as follows. [1] including reacting the antibody with a lipoic acid analog having a modifying moiety in the presence of lipoic acid protein ligase to produce a modified antibody having a modifying moiety on the side chain of a lysine residue in the constant region. , A method for producing a modified antibody having a modified moiety. [2] The method of [1], wherein the antibody has a natural polypeptide chain structure. [3] The method of [1] or [2], wherein the antibody is a monoclonal antibody.
  • the lipoic acid analog having a modified moiety is a C 4 -C 10 alkyl-carboxylic acid having a modified moiety;
  • a modified antibody having a modification portion in the side chain of a lysine residue in the constant region is an antibody having a C 4 to C 10 alkyl-carbonyl having a modification portion in the side chain of a lysine residue in the constant region, [ Any one of 1) to [7].
  • the lipoic acid analog having a modified moiety is represented by the following formula (I): R—C n H 2n —COOH (I) (In the formula, R is a modifying moiety; n is an integer of 4 to 10.
  • a modified antibody having a modified portion on the side chain of a lysine residue in the constant region is represented by the following formula (II): R—C n H 2n —CO—NH— (II) (In the formula, R and n are the same as in formula (I); NH- is a group present on the side chain of a lysine residue in the constant region.
  • Is a modified antibody having a portion represented by the side chain of a lysine residue in the constant region The method according to any one of [1] to [8]. [10] The method of [9], wherein n is 7.
  • Lysine residues present in both the CH1 region of the heavy chain and the CL region of the light chain are a lysine residue at position 133 in a human IgG heavy chain and a lysine residue at position 169 in a human IgG light chain. , [13].
  • the lipoic acid protein ligase is derived from a microorganism; [16] the lipoic acid protein ligase is derived from a bacterium belonging to the genus Escherichia, a bacterium belonging to the genus Bacillus, a bacterium belonging to the genus Corynebacterium, or a bacterium belonging to the genus Staphylococcus; 15].
  • the lipoic acid protein ligase is obtained from Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum (Corynebacterium glutamicum, or Staphylococcus epsimid sp.). Any one of 1) to [16].
  • the lipoic acid protein ligase is a protein selected from the group consisting of the following (A) to (C): (A) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8; (B) an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8, including an amino acid sequence containing substitution, deletion, insertion, or addition of one or several amino acids, and lipoic acid A protein having protein ligase activity; and (C) a lipoic acid protein comprising an amino acid sequence having 90% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8.
  • a protein having ligase activity [19] The method of any one of [1] to [17], wherein the lipoic acid protein ligase is a protein selected from the group consisting of the following (A ′) to (C ′): (A ′) a protein having an amino acid sequence having one or more mutations selected from the group consisting of the following (i) to (vii) in the amino acid sequence of SEQ ID NO: 2, and having lipoic acid protein ligase activity: (I) substitution with an arginine residue at position 121, an alanine residue or a threonine residue; (Ii) substitution of a serine residue at position 136 with a leucine residue; (Iii) substitution with a tyrosine residue at position 140, an alanine residue or a valine residue; (Iv) substitution at position 142 of a glutamic acid serine residue, threonine residue, or valine residue; (V) deletion of histidine residue at position 149;
  • a bioorthogonal functional group is an azide residue, aldehyde residue, thiol residue, alkyne residue, alkene residue, halogen residue, tetrazine residue, nitrone residue, hydroxylamine residue, nitrile residue , Hydrazine residue, ketone residue, boronic acid residue, cyanobenzothiazole residue, allyl residue, phosphine residue, maleimide residue, disulfide residue, thioester residue, ⁇ -halocarbonyl residue, isonitrile residue [20] the method of [20], wherein the method is selected from the group consisting of: [22] A method for producing a modified antibody having a functional substance, comprising the following (1) and (2): (1) An antibody is reacted with a lipoic acid analog having a modified portion containing a bioorthogonal functional group.
  • the lipoic acid analog having a modified moiety containing a bioorthogonal functional group is a C 4 -C 10 alkyl-carboxylic acid having a modified moiety containing a bioorthogonal functional group;
  • a modified antibody having a modified moiety containing a bioorthogonal functional group on the side chain of a lysine residue in the constant region is used to convert a C 4 -C 10 alkyl-carbonyl having a modified moiety containing a bioorthogonal functional group into the constant region.
  • An antibody having a side chain of a lysine residue of A modified antibody having a functional substance on the side chain of a lysine residue in the constant region is characterized by a C 4 -C 10 alkyl-carbonyl having a functional substance and a modified moiety containing a bioorthogonal functional group reacted therewith, [20]
  • a lipoic acid analog having a modified moiety containing a bioorthogonal functional group is represented by the following formula (I): R—C n H 2n —COOH (I) (In the formula, R is a modifying moiety containing a bioorthogonal functional group, n is an integer of 4 to 10.
  • a modified antibody having a modified portion containing a bioorthogonal functional group on the side chain of a lysine residue in the constant region is represented by the following formula (II): R—C n H 2n —CO—NH— (II) (In the formula, R and n are the same as in formula (I); NH- is a group present on the side chain of a lysine residue in the constant region.
  • a modified antibody having a portion represented by a side chain of a lysine residue in the constant region is represented by the following formula (III): F-R'-C n H 2n -CO-NH- (III) (In the formula, n is the same as that of formula (I); NH- is the same as that of formula (II), F is a functional substance, R ′ is a divalent group including a moiety generated by a reaction between a functional substance and a bioorthogonal functional group.
  • a modified antibody having a modified portion on the side chain of a lysine residue in the constant region is represented by the following formula (II): R—C n H 2n —CO—NH— (II) (In the formula, R is a modifying moiety; n is an integer of 4 to 10, NH- is a group present on the side chain of a lysine residue in the constant region.
  • [28] The modified antibody according to [28], which has a portion represented by the formula (1) only in the side chain of a lysine residue specific to the antibody.
  • [30] the modified antibody of [28] or [29], wherein n is 7;
  • [32] the modified antibody of any of [28] to [31], wherein the lysine residue in the constant region is a lysine residue in the CH1 region;
  • [33] The modified antibody of any of [28] to [32], wherein the lysine residue in the constant region is a lysine residue present in both the CH1 region of the heavy chain and the CL region of the light chain.
  • Lysine residues present in both the CH1 region of the heavy chain and the CL region of the light chain are a lysine residue at position 133 in a human IgG heavy chain and a lysine residue at position 169 in a human IgG light chain.
  • the modified antibody of [33] a modified antibody having a functional substance on the side chain of a lysine residue in the constant region, A modified antibody comprising a functional substance and a C 4 -C 10 alkyl-carbonyl having a modifying moiety containing a bioorthogonal functional group reacted therewith only in the side chain of a lysine residue unique to the antibody.
  • a modified antibody having a functional substance on a side chain of a lysine residue in the constant region is represented by the following formula (III): F-R'-C n H 2n -CO-NH- (III) (In the formula, F is a functional substance, R ′ is a divalent group including a moiety generated by a reaction between a functional substance and a bioorthogonal functional group, n is an integer of 4 to 10, NH- is a group present on the side chain of a lysine residue in the constant region.
  • the modified antibody according to [35] which has a portion represented by [1] only in the side chain of a lysine residue specific to the antibody.
  • the method of the present invention relates to a method for preparing a constant region (eg, a CH1 region such as a lysine residue at position 133 in a human IgG heavy chain, a lysine residue at position 169 in a human IgG light chain, etc.) In the CL region).
  • a constant region eg, a CH1 region such as a lysine residue at position 133 in a human IgG heavy chain, a lysine residue at position 169 in a human IgG light chain, etc.
  • the modified antibodies of the present invention are useful, for example, as pharmaceuticals and reagents (eg, diagnostics, research reagents), and intermediates for their preparation.
  • FIG. 1-1 shows the amino acid sequence of the heavy chain of trastuzumab (SEQ ID NO: 14) in which the sugar chain has been cleaved with PNGase. Boxes and double underlined letters indicate peptide fragments (SEQ ID NOs: 16-22) by trypsin digestion. The shaded letters and the numbers subscripted indicate the modified lysine residue in the modified trastuzumab and its amino acid number based on EU numbering.
  • FIG. 1-2 shows the amino acid sequence of the light chain of trastuzumab (SEQ ID NO: 15). Boxes, single underscores, and double underscores indicate peptide fragments (SEQ ID NOs: 23-27) by trypsin digestion.
  • FIG. 2 shows the peptide fragment of LSCAASGFNIKDTYIHWVR (SEQ ID NO: 16) containing a site for modification of lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows an MS spectrum (m / z 1182.61437, bivalence).
  • FIG. 3 shows a peptide fragment of LSCAASGFNIKDTYIHWVR (SEQ ID NO: 16) containing a site for modification of lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows a CID spectrum.
  • FIG. 4 shows a peptide fragment of FTISADTSKNTAYLQMNSLR (SEQ ID NO: 17) containing a site for modification to lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2.
  • FIG. 4 is a view showing an MS spectrum (m / z.796.5363, trivalent).
  • FIG. 5 shows a peptide fragment of FTISADTSKNTAYLQMNSLR (SEQ ID NO: 17) containing a site for modification to lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows a CID spectrum.
  • FIG. 17 shows a peptide fragment of FTISADTSKNTAYLQMNSLR (SEQ ID NO: 17) containing a site for modification to lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows a CID spectrum.
  • FIG. 6 shows a peptide fragment of GPSVFPLAPSSSKSTSGTAGLGCLVK (SEQ ID NO: 18) containing a site for modification of a lysine residue (octanoic acid-introduced substance (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows MS spectrum (m / z 654.60949, 4 valence).
  • FIG. 7 shows a peptide fragment of GPSVFPLAPSSSKSTSGTAGLGCLVK (SEQ ID NO: 18) containing a site for modification of lysine residues by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2 (octanoic acid-introduced substance (+126.105 Da)). It is a figure which shows a CID spectrum.
  • FIG. 18 shows a peptide fragment of GPSVFPLAPSSSKSTSGTAGLGCLVK
  • FIG. 8 shows a peptide fragment of SCDKTHTCPPCPAPELLGGGPSVFFLPPKPK (SEQ ID NO: 19) containing a site for modification to lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2.
  • FIG. 4 is a view showing an MS spectrum (m / z 1154.25572, trivalent).
  • FIG. 9 shows a peptide fragment of SCDKTHTCPPCPAPELLGGGPSVFFLPPKPK (SEQ ID NO: 19) containing a site for modification to lysine residues by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2 (octanoic acid-introduced substance (+126.105 Da)). It is a figure which shows a CID spectrum.
  • SCDKTHTCPPCPAPELLGGGPSVFFLPPKPKPK SEQ ID NO: 19
  • FIG. 10 shows a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPK (SEQ ID NO: 20) containing a site for modification of lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows MS spectrum (m / z 1485.78552, bivalence).
  • FIG. 11 shows a peptide fragment of THTCPPCPAPELLGGPSVFLFPPPKPK (SEQ ID NO: 20) containing a site for modification of lysine residues by lysine residue by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2 (octaneic acid-introduced substance (+126.105 Da)). It is a figure which shows a CID spectrum.
  • FIG. 12 shows the peptide fragment of VSNKALPAPIEK (SEQ ID NO: 21) containing a site for modification to lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2.
  • FIG. 4 is a view showing an MS spectrum (m / z.696.8122, divalent).
  • FIG. 13 shows the peptide fragment of VSNKALPAPIEK (SEQ ID NO: 21) containing a site for modification to lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows a CID spectrum.
  • FIG. 21 shows the peptide fragment of VSNKALPAPIEK (SEQ ID NO: 21) containing a site for modification to lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows a CID spectrum.
  • FIG. 14 shows a peptide fragment of EPQVYTLPSPSREEMTKNQVSLTCLVK (SEQ ID NO: 22) containing a site for modification of a lysine residue by lysine digestion of octanoic acid-modified trastuzumab obtained in Example 2 (octanoic acid-introduced substance (+126.105 Da)). It is a figure which shows MS spectrum (m / z 794.17197, tetravalent).
  • FIG. 15 shows a peptide fragment of EPQVYTLPSPSREEMTKNQVSLTCLVK (SEQ ID NO: 22) containing a site for modification to lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows a CID spectrum.
  • SEQ ID NO: 22 a peptide fragment of EPQVYTLPSPSREEMTKNQVSLTCLVK
  • FIG. 16 shows the peptide fragment of ASQDVNTAVAWYQQKPGKAPK (SEQ ID NO: 23) containing a site for modification of lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows MS spectrum (m / z 1207.14994, bivalence).
  • FIG. 17 shows a peptide fragment of ASQDVNTAVAWYQQKPGKAPK (SEQ ID NO: 23) containing a site for modification of lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows a CID spectrum.
  • SEQ ID NO: 23 SEQ ID NO: 23
  • FIG. 18 shows a peptide fragment of VDNALQSGNSQESVTEQDSKDSTYSLSLSTLTLSK (SEQ ID NO: 24) containing a site for modification of lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows MS spectrum (m / z 1249.27813, trivalence).
  • FIG. 19 shows a peptide fragment of VDNALQSGNSQESVTEQDSKDSTYSLSSSTTLSK (SEQ ID NO: 24) containing a site for modification to lysine residues by lysine residue obtained by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2 (SEQ ID NO: 24). It is a figure which shows a CID spectrum.
  • FIG. 20 shows a peptide fragment of ADYEKHK (SEQ ID NO: 25) containing a site for modification to lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2.
  • FIG. 3 is a view showing an MS spectrum (m / z 508.77751, divalent).
  • FIG. 21 shows the peptide fragment of ADYEKHK (SEQ ID NO: 25) containing a site for modification to lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows a CID spectrum.
  • FIG. 21 shows the peptide fragment of ADYEKHK (SEQ ID NO: 25) containing a site for modification to lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows a CID spectrum.
  • FIG. 21 shows the peptide fragment of ADYEKHK (SEQ ID NO: 25) containing a
  • FIG. 22 shows a peptide fragment of HKVYACEVTHQGLSSPVTK (SEQ ID NO: 26) containing a site for modification to lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows MS spectrum (m / z 1134.08444, bivalence).
  • FIG. 23 shows a peptide fragment of HKVYACEVTHQGLSSPVTK (SEQ ID NO: 26) containing a site for modification to lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows a CID spectrum.
  • FIG. 24 shows a peptide fragment of HKVYACEVTHQGLSSPVTKSFNR (SEQ ID NO: 27) containing a site for modification of lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows MS spectrum (m / z 924.48353, trivalence).
  • FIG. 25 shows a peptide fragment of HKVYACEVTHQGLSSPVTKSFNR (SEQ ID NO: 27) containing a site for modification to lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows a CID spectrum.
  • FIG. 26 shows a CLUSTAL O (1.2.4) multiple sequence alignment of modified variants of LplA. Amino acid substitutions are underlined in bold.
  • FIG. 27 shows (A) SDS-PAGE of the reaction mixture and (B) a fluorogram of the gel.
  • the present invention provides a method for producing a modified antibody having a modifying moiety.
  • the method of the invention comprises reacting an antibody with a lipoic acid analog having a modifying moiety in the presence of lipoic acid protein ligase to produce a modified antibody having a modifying moiety on the side chain of a lysine residue in the constant region. Including.
  • the antibody used in the method of the present invention is a polyclonal antibody or a monoclonal antibody.
  • the antibody used in the method of the present invention may be an antibody (eg, glycoprotein) modified with a biomolecule (eg, sugar) or an antibody not modified with a biomolecule.
  • a biomolecule eg, sugar
  • an antibody against a biological-derived component or a virus-derived component is preferable.
  • biological components include components (eg, proteins) derived from animals such as mammals and birds (eg, chickens), insects, microorganisms, plants, fungi, and fish.
  • the biological component is a component derived from a mammal.
  • mammals include primates (eg, humans, monkeys, chimpanzees), rodents (eg, mice, rats, guinea pigs, hamsters, rabbits), pets (eg, dogs, cats), livestock (eg, Cattle, pigs, goats) and working animals (eg, horses, sheep).
  • the biological component is more preferably a primate or rodent-derived component (eg, a protein), and even more preferably a human-derived component (eg, a protein) from the viewpoint of clinical application of the present invention. is there.
  • virus-derived components include components (eg, proteins) derived from influenza virus (eg, avian influenza virus, swine influenza virus), AIDS virus, Ebola virus, and phage virus.
  • Antibody is an antibody against any antigen.
  • an antigen may be a component found in an organism or virus as described above.
  • antigens also include, for example, proteins [including oligopeptides and polypeptides. It may be a protein modified with a biomolecule such as sugar (eg, glycoprotein)], a sugar chain, a nucleic acid, and a low molecular compound.
  • the antibody may be an antibody having a protein as an antigen.
  • proteins include cell membrane receptors, cell membrane proteins other than cell membrane receptors (eg, extracellular matrix proteins), ligands, and soluble receptors.
  • the protein that is the antigen of the antibody may be a disease target protein.
  • diseases target protein include the following.
  • Ocular diseases Factor D IGF-1R, PGDFR, Ang2, VEGF-A, CD-105 (Endoglin), IGF-1R, ⁇ -amyloid
  • the antibody has a native polypeptide chain structure.
  • the “natural polypeptide chain structure” of an antibody refers to the first polypeptide chain consisting of the variable region (VH) and the constant regions (CH1, CH2, CH3) in the naturally occurring heavy chain of the antibody, and the naturally occurring antibody. It refers to the structure consisting of the second polypeptide chain consisting of the variable region (VL) and the constant region (CL) in the light chain. Therefore, antibodies (ie, VH, CH1, CH2, CH3, VL, and CL) which are fusion proteins having a structure in which a predetermined amino acid sequence (eg, a tag sequence, a spacer sequence) has been added or inserted into a predetermined region of the antibody. Antibodies having other portions) are excluded from antibodies having a natural polypeptide chain structure.
  • the antibodies used in the method of the invention are monoclonal antibodies.
  • the monoclonal antibody for example, a chimeric antibody, a humanized antibody, a human antibody, an antibody to which a predetermined sugar chain is added (for example, modified to have a sugar chain binding consensus sequence such as an N-type sugar chain binding consensus sequence) Antibodies), bispecific antibodies, scFv antibodies, Fab antibodies, F (ab ') 2 antibodies, VHH antibodies, Fc region proteins, and Fc fusion proteins.
  • Isotypes of antibodies such as monoclonal antibodies include, for example, IgG, IgM, IgA, IgD, IgE, and IgY.
  • the antibody may also be a bivalent antibody (eg, IgG, IgD, IgE) or a tetravalent or higher-valent antibody (eg, IgA antibody, IgM antibody).
  • the antibody used in the method of the present invention may be a full-length antibody, or an antibody fragment (eg, Fab, F (ab ') 2 , Fab', Fv, single chain antibody).
  • the antibody fragment preferably has either the CH1 region of the heavy chain or the CL region of the light chain, and more preferably has both the CH1 region of the heavy chain and the CL region of the light chain.
  • the antibody used in the method of the present invention is a human antibody or a humanized antibody.
  • a high degree of modification has been confirmed in a specific constant region of a humanized antibody (trastuzumab). Therefore, according to the method of the present invention, not only a humanized antibody but also a human antibody having the same constant region as a humanized antibody can highly modify a specific constant region.
  • adalimumab panitumumab, golimumab, ustekinumab, canakinumab, ofatumumab, denosumab (IgG2), ipilimumab, belimumab, raxivacumab, ramcilumab, nivolumabumalb (IgG4), Secukinumabumabu, g IgG2) and oraratumab (if no IgG subtype is mentioned, it indicates that it is an IgG1).
  • humanized antibodies examples include daclizumab, palivizumab, trastuzumab, alentuzumab, omalizumab, efalizumab, bevacizumab, natalizumab (IgG4), tocilizumab, eclidumab (IgG2), mogamulizumab, pertuzumab, pertuzumab, pertuzumab, pertuzumab, pertuzumab, pertuzumab, pertuzumab, penuzunumab , Dartatumumab, ikesekidumab (IgG4), reslizumab (IgG4), and atezolizumab (if no IgG subtype is mentioned, it indicates that it is IgG1).
  • the antibody used in the method of the present invention is an IgG.
  • IgG examples include IgG1, IgG2, IgG3, and IgG4.
  • IgG human IgG is preferable.
  • the antibody used in the method of the present invention is Fab, or F (ab ') 2 .
  • Fab or F (ab ') 2 .
  • a high degree of modification has been confirmed in specific constant regions (CH1, CL) of Fab. Therefore, according to the method of the present invention, not only Fabs but also F (ab ') 2 having the same constant regions as Fabs can be highly modified in specific constant regions.
  • Lipoic acid protein ligase (Lpl) used in the method of the present invention is an enzyme that catalyzes a reaction in which lipoic acid is bound to a lysine residue in a protein and acylated.
  • Lpl is widely found in various organisms such as microorganisms, and is known to be an enzyme capable of utilizing lipoic acid analogs having various structures as substrates (Fernandez-Suarez M et al. Nat Biotechnol). 2007 25: 1483-1487; U.S. Patent No. 8,137,925; U.S. Patent No. 9,284,541; WO 2017/095806).
  • the Lpl used in the method of the present invention is preferably Lpl derived from a microorganism from the viewpoint of easy availability.
  • Lpl used in the method of the present invention include bacteria belonging to the genus Escherichia (eg, Escherichia coli), bacteria belonging to the genus Bacillus (eg, Bacillus subtilis), and coli.
  • Bacteria belonging to the genus Corynebacterium eg, Corynebacterium glutamicum
  • bacteria belonging to the genus Staphylococcus eg, Staphylococcus epidermis from Staphylococcus epidermis
  • Staphylococcus epidermis Staphylococcus epidermis
  • Lpl used in the method of the present invention includes, for example, a protein selected from the group consisting of the following (A) to (C): (A) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8; (B) an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8, including an amino acid sequence containing substitution, deletion, insertion, or addition of one or several amino acids, and lipoic acid A protein having protein ligase activity; and (C) a lipoic acid protein comprising an amino acid sequence having 90% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8. A protein having ligase activity.
  • one or several amino acid residues are changed by one, two, three or four kinds of mutations selected from the group consisting of deletion, substitution, addition and insertion of amino acid residues.
  • the amino acid residue mutation may be introduced into one region in the amino acid sequence, or may be introduced into a plurality of different regions.
  • the term "one or several” refers to numbers that do not significantly impair the activity of the protein.
  • the number indicated by the term “one or several” is, for example, 1 to 100, preferably 1 to 80, more preferably 1 to 50, 1 to 30, 1 to 20, 1 to 10 or 1 to 5 (eg, 1, 2, 3, 4, or 5).
  • the above may be 97% or more, 98% or more, or 99% or more.
  • NCBI National Center for Biotechnology Information
  • Lpl used in the method of the present invention may be a protein selected from the group consisting of (A ′) to (C ′): (A ′) In the amino acid sequence of SEQ ID NO: 2, an amino acid sequence having one or more (eg, 1 to 6, preferably 1 to 5) mutations selected from the group consisting of the following (i) to (vii): A protein comprising and having lipoic acid protein ligase activity: (I) substitution with an arginine residue at position 121, an alanine residue or a threonine residue; (Ii) substitution of a serine residue at position 136 with a leucine residue; (Iii) substitution with a tyrosine residue at position 140, an alanine residue or a valine residue; (Iv) substitution at position 142 of a glutamic acid serine residue, threonine residue, or valine residue; (V) deletion of histidine residue at position 149; (Vi) substitution with
  • the protein selected from the group consisting of (A ') to (C') preferably has a higher lipoic acid protein ligase activity than the protein consisting of the amino acid sequence of SEQ ID NO: 2.
  • Lipoic acid protein ligase activity can be measured by an LplA activity (labeling of IgG with octanoic azide) assay using IgG (IgG H chain or L chain or a combination thereof) as a substrate (see Example 7).
  • Lipoic acid protein ligase activity refers to the activity of binding lipoic acid to a lysine residue in a protein for acylation.
  • Lpl may have a mutation introduced at a specific site as long as it retains lipoic acid protein ligase activity.
  • the positions of amino acid residues at which mutations can be introduced, which can retain desired properties, will be apparent to those skilled in the art. Specifically, one of skill in the art would: 1) compare the amino acid sequences of multiple proteins with similar properties, and 2) identify relatively conserved and relatively non-conserved regions, 3) From the relatively conserved region and the relatively unconserved region, a region that can play a significant role in the function and a region that cannot play a significant role in the function can be predicted, respectively. We can recognize the correlation between structure and function. Therefore, those skilled in the art can specify the position of an amino acid residue at which a mutation may be introduced in the amino acid sequence of Lpl.
  • substitution of the amino acid residue may be a conservative substitution.
  • conservative substitution refers to the replacement of a given amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are well known in the art.
  • such families include amino acids having a basic side chain (eg, lysine, arginine, histidine), amino acids having an acidic side chain (eg, aspartic acid, glutamic acid), and amino acids having an uncharged polar side chain (Eg, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having a nonpolar side chain (eg, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), ⁇ -branched side chain (Eg, threonine, valine, isoleucine), an amino acid having an aromatic side chain (eg, tyrosine, phenylalanine, tryptophan, histidine), an amino acid having a hydroxyl group (eg, alcoholic, phenolic) -containing side chain ( Example, serine, thread Nin, tyrosine), and
  • conservative substitutions of amino acids include substitution between aspartic acid and glutamic acid, substitution between arginine and lysine and histidine, substitution between tryptophan and phenylalanine, and substitution between phenylalanine and valine.
  • Lpl may also be a fusion protein linked via a peptide bond to a heterologous moiety.
  • a heterologous portion include a peptide component that facilitates purification of a target protein (eg, a histidine tag, a tag portion such as Strep-tag II; glutathione-S-transferase, maltose binding protein, and mutants thereof).
  • a peptide component that improves the solubility of the target protein eg, Nus-tag
  • a peptide component that functions as a chaperone eg, a trigger factor
  • a peptide component having other functions eg, For example, a full-length protein or a portion thereof
  • the lipoic acid analog having a modifying moiety used in the method of the present invention is not particularly limited as long as it acts as a substrate for Lpl.
  • the lipoic acid analog in the lipoic acid analog having a modified portion is a carboxylic acid having a linear or branched hydrocarbon group and optionally having a cyclic portion such as a cyclic hydrocarbon group. Lipoic acid analogs with such modified moieties are known to act as substrates for Lpl (see references above, especially US Pat. No. 8,137,925).
  • the lipoic acid analog having a modified moiety is a lipoic acid having a modified moiety.
  • the lipoic acid in the lipoic acid having a modifying moiety is a carboxylic acid having a linear or branched hydrocarbon group.
  • the lipoic acid analog having a modified moiety has a structure in which a modified moiety is added to an alkyl-carboxylic acid having the following structure (in other words, a structure in which a hydrogen atom in lipoic acid is substituted by a modified moiety).
  • the lipoic acid analog having a modified moiety is preferably a C 4 -C 10 alkyl-carboxylic acid having a modified moiety, more preferably a C 5 -C 9 alkyl-carboxylic acid having a modified moiety, and more preferably a C 4 -C 9 alkyl-carboxylic acid having a modified moiety.
  • 6 ⁇ C 8 alkyl - are more preferred carboxylic acids
  • C 7 alkyl having a modifying moiety - carboxylic acid is particularly preferred.
  • the lipoic acid analog having a modified moiety is a compound represented by the following formula (I) having a modified moiety at the alkyl terminal of “alkyl-carboxylic acid” from the viewpoint of improving reaction efficiency and the like. is there.
  • R—C n H 2n —COOH (I) (In the formula, R is a modifying moiety; n is an integer of 4 to 10. ]
  • a hyphen (-) has two moieties on both sides thereof (eg, two moieties of R and C n H 2n and two moieties of C n H 2n and COOH) covalently bonded to each other. (The same applies to other expressions).
  • n is preferably an integer of 5 to 9, more preferably an integer of 6 to 8, and particularly preferably 7.
  • an antibody having a modified moiety on the side chain of a lysine residue in the constant region is represented by the following formula (II)
  • Modified antibodies having a moiety on the side chain of a lysine residue in the constant region are generated.
  • R—C n H 2n —CO—NH— (II) (In the formula, R and n are the same as in formula (I); NH- is a group present on the side chain of a lysine residue in the constant region. ]
  • the modified moiety is not particularly limited as long as the moiety imparts an arbitrary function to such an antibody.
  • a bioorthogonal functional group or a functional substance may be used.
  • one or more (eg, two, three, four) modified moieties may be added to lipoic acid, but preferably, one modified moiety is added to lipoic acid. It may be.
  • a bio-orthogonal functional group does not react with biological constituents (eg, amino acids, nucleic acids, lipids, sugars, and phosphates) or has a slow reaction rate with biological constituents, but does not react with components other than biological constituents. Refers to a group that reacts selectively.
  • Bioorthogonal functional groups are well known in the art (eg, Sharpless ⁇ KB et al., Angew. Chem. Int. Ed. 40, 2004 (2015); Bertozzi ⁇ CR et al., Science 291, 257 (2001); Bertozzi CR et al., Nature Chemical Biology 1, 13 (2005)).
  • a bioorthogonal functional group is retained in an antibody after the reaction according to the method of the present invention, it can impart reactivity to a functional substance to such an antibody.
  • the bio-orthogonal functional group is a bio-orthogonal functional group for an antibody (protein).
  • the bioorthogonal functional group for an antibody is a group that reacts with a predetermined functional group without reacting with the side chains of the 20 natural amino acid residues constituting the antibody.
  • A alanine
  • N asparagine
  • C cysteine
  • C glutamine
  • Q glycine
  • G isoleucine
  • I isoleucine
  • L isoleucine
  • M isoleucine
  • M isoleucine
  • M isoleucine
  • M isoleucine
  • P proline
  • S serine
  • T threonine
  • W tryptophan
  • Y valine
  • V valine
  • D glutamic acid
  • E Arginine
  • H histidine
  • L lysine
  • glycine without side chains ie, being a hydrogen atom
  • glycine whose side chains are hydrocarbon groups (ie, from the group consisting of sulfur, nitrogen, and oxygen atoms)
  • Alanine, isoleucine, leucine, phenylalanine, and valine are inert to normal reactions. Therefore, the bioorthogonal functional group for the antibody (protein) has asparagine, glutamine, methionine, proline, serine, threonine, as well as the side chains of those amino acids having side chains that are inactive to normal reactions.
  • bioorthogonal functional groups that cannot react with proteins include azide residues, aldehyde residues, thiol residues, and alkene residues (in other words, the minimum unit having a carbon-carbon double bond is It is only necessary to have a certain vinylene (ethenylene) moiety. The same applies to the following.)
  • An alkyne residue in other words, it suffices to have an ethinylene moiety which is a minimum unit having a triple bond between carbon atoms. The same applies to the following).
  • Halogen residue tetrazine residue, nitrone residue, hydroxylamine residue, nitrile residue, hydrazine residue, ketone residue, boronic acid residue, cyanobenzothiazole residue, allyl residue, phosphine residue, maleimide Residue, disulfide residue, thioester residue, ⁇ -halocarbonyl residue (eg, a fluorine atom, a chlorine atom, a bromine atom or an iodine atom at the ⁇ -position Carbonyl residue, the same applies hereinafter), an isonitrile residue, a sydnone residue, and a selenium residue.
  • ⁇ -halocarbonyl residue eg, a fluorine atom, a chlorine atom, a bromine atom or an iodine atom at the ⁇ -position Carbonyl residue, the same applies hereinafter
  • an isonitrile residue a sydnone residue,
  • Antibodies can be proteins that cannot contain free thiols. In proteins that cannot contain free thiols, thiols function as bioorthogonal functional groups. Therefore, in the case of the present invention, the bioorthogonal functional group includes a thiol.
  • the bio-orthogonal functional group is an azide residue, a thiol residue, an alkyne residue, a maleimide residue, and a disulfide residue among the above-described bio-orthogonal functional groups from the viewpoint of improving reaction efficiency and the like.
  • the functional substance is not particularly limited as long as it is a substance that imparts an arbitrary function to such an antibody when retained in the antibody after the reaction according to the method of the present invention. Details of the functional substance will be described later.
  • reaction of Antibody with Lipoic Acid Analog Having Modified Portion in the Presence of Lipoic Acid Protein Ligase The reaction can be appropriately performed under conditions (mild conditions) that cannot cause denaturation / decomposition (eg, cleavage of amide bond) of the antibody.
  • a suitable reaction system for example, a buffer (eg, a phosphate buffer) at ordinary temperature (for example, about 15 to 35 ° C., preferably about 20 to 30 ° C.).
  • the pH of the buffer is, for example, 5 to 9, preferably 5.5 to 8.5, and more preferably 6.0 to 8.0.
  • the buffer may contain a suitable cofactor such as ATP.
  • the reaction time is, for example, 10 minutes to 150 hours, preferably 20 minutes to 120 hours, more preferably 30 minutes to 100 hours, and even more preferably 60 minutes to 80 hours.
  • concentration of Lpl can be appropriately adjusted.
  • the molar ratio (Y / X) of the lipoic acid analog having the modified moiety to the antibody (X) is determined by the type of the antibody and the lipoic acid analog having the modified moiety, and the lipoic acid analog having the modified moiety. It is not particularly limited since it varies depending on the number of sites in the antibody to be modified by (e.g., DAR) and the like, but is, for example, 0.1 to 100, preferably 0.5 to 80, and more preferably Is from 1 to 70, still more preferably from 2 to 50, and particularly preferably from 3 to 30.
  • Confirmation of generation of a modified antibody having a modified portion on the side chain of a lysine residue in the constant region depends on the type of the specific raw material (antibody and lipoic acid analog having the modified portion) and the molecular weight of the product
  • Confirmation of the position of the modified lysine residue in the antibody can be performed, for example, by peptide mapping.
  • Peptide mapping can be performed, for example, by protease (eg, trypsin, chymotrypsin) treatment and mass spectrometry.
  • protease eg, trypsin, chymotrypsin
  • an endoprotease is preferable.
  • Such endoproteases include, for example, trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, Asp-N.
  • Confirmation of the number of modified moieties possessed by the modified antibody having a modified moiety on the side chain of a lysine residue in the constant region is performed, for example, by electrophoresis, chromatography, or mass spectrometry, preferably by mass spectrometry. be able to.
  • the modified antibody having a modified portion on the side chain of a lysine residue in the constant region can be appropriately purified by any method such as chromatography (eg, the above-described chromatography and affinity
  • the modifying moiety is a modifying moiety that includes a bioorthogonal functional group.
  • the method of the present invention may be performed by the following method, which includes an additional step for producing a modified antibody having a functional substance. (1) An antibody is reacted with a lipoic acid analog having a modified portion containing a bioorthogonal functional group in the presence of lipoic acid protein ligase A, so that the side chain of a lysine residue in the constant region has a bioorthogonal function.
  • the above step (1) comprises the step of reacting an antibody with a lipoic acid analog having a modifying portion in the presence of a lipoic acid protein ligase to form a modified portion on the side chain of a lysine residue in the constant region. Generating a modified antibody).
  • the above step (2) is an antibody for reacting a bioorthogonal functional group in the modified antibody with a functional substance.
  • the functional substance is not particularly limited as long as it is a substance that imparts an arbitrary function to such an antibody when retained in the antibody after the reaction according to the method of the present invention, and includes, for example, a drug, a labeling substance, and a stabilizing agent. And preferably a drug or a labeling substance.
  • the functional substance may also be a single functional substance or a substance in which two or more functional substances are linked.
  • the drug may be a drug for any disease.
  • diseases include, for example, cancer (eg, lung cancer, gastric cancer, colon cancer, pancreatic cancer, kidney cancer, liver cancer, thyroid cancer, prostate cancer, bladder cancer, ovarian cancer, uterine cancer, bone cancer, skin cancer, Brain tumor, melanoma), autoimmune disease / inflammatory disease (eg, allergic disease, rheumatoid arthritis, systemic lupus erythematosus), cranial nerve disease (eg, cerebral infarction, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis), Infections (eg, bacterial infections, viral infections), hereditary or rare diseases (eg, hereditary spherocytosis, non-dystrophic myotonia), eye diseases (eg, age-related macular degeneration, diabetic retinopathy, Retinitis pigmentosa), bone and orthopedic diseases (eg, osteoarthritis), blood diseases (eg, leukemia
  • the drug is an anti-cancer agent.
  • the anticancer agent include a chemotherapeutic agent, a toxin, a radioisotope, and a substance containing the same.
  • Chemotherapeutic agents include, for example, DNA damaging agents, antimetabolites, enzyme inhibitors, DNA intercalating agents, DNA cleaving agents, topoisomerase inhibitors, DNA binding inhibitors, tubulin binding inhibitors, cytotoxic nucleosides, Platinum compounds may be mentioned.
  • toxins include bacterial toxins (eg, diphtheria toxin) and plant toxins (eg, ricin).
  • radioactive isotope examples include a radioactive isotope of a hydrogen atom (eg, 3 H), a radioactive isotope of a carbon atom (eg, 14 C), a radioactive isotope of a phosphorus atom (eg, 32 P), and a radioactive isotope of a sulfur atom.
  • a radioactive isotope of a hydrogen atom eg, 3 H
  • a radioactive isotope of a carbon atom eg, 14 C
  • a radioactive isotope of a phosphorus atom eg, 32 P
  • a radioactive isotope of a sulfur atom examples include a radioactive isotope of a sulfur atom.
  • Radioactive isotope (eg, 35 S ), radioactive isotope of yttrium (eg, 90 Y), radioactive isotope of technetium (eg, 99m Tc), radioactive isotope of indium (eg, 111 In), radioactivity of iodine atom Isotopes (eg, 123 I, 125 I, 129 I, 131 I), radioisotopes of samarium (eg, 153 Sm), radioisotopes of rhenium (eg, 186 Re), radioisotopes of astatine (eg, 211 At) and a radioisotope of bismuth (eg, 212 Bi).
  • iodine atom Isotopes eg, 123 I, 125 I, 129 I, 131 I
  • radioisotopes of samarium eg, 153 Sm
  • radioisotopes of rhenium e
  • the labeling substance examples include enzymes (eg, peroxidase, alkaline phosphatase, luciferase, ⁇ -galactosidase), affinity substances (eg, streptavidin, biotin, digoxigenin, aptamer), fluorescent substances (eg, fluorescein, fluorescein isothiocyanate, rhodamine) , Green fluorescent protein, red fluorescent protein), luminescent substance (eg, luciferin, aequorin, acridinium ester, tris (2,2′-bipyridyl) ruthenium, luminol), radioisotope (eg, those described above) or And the like.
  • enzymes eg, peroxidase, alkaline phosphatase, luciferase, ⁇ -galactosidase
  • affinity substances eg, streptavidin, biotin, digoxigenin, aptamer
  • fluorescent substances eg,
  • the functional substance is also a high molecular compound, a medium molecular compound, or a low molecular compound, and is preferably a low molecular compound.
  • a low molecular compound is a compound having a molecular weight of 1500 or less.
  • a low molecular compound is a natural compound or a synthetic compound.
  • the molecular weight of the low molecular weight compound may be 1200 or less, 1000 or less, 900 or less, 800 or less, 700 or less, 600 or less, 500 or less, 400 or less, or 300 or less.
  • the molecular weight of the low molecular weight compound may also be 30 or higher, 40 or higher, or 50 or higher.
  • the low molecular weight compound may be a drug or a labeling substance as described above.
  • Examples of the low-molecular compound include amino acids, oligopeptides, vitamins, nucleosides, nucleotides, oligonucleotides, monosaccharides, oligosaccharides, lipids, fatty acids, and salts thereof.
  • the functional substance may be a peptide compound.
  • the peptide compound may be a drug such as an anticancer agent as described above, a labeling substance, or a low molecular compound.
  • the functional substance has various functional groups according to its structure.
  • the functional group of the functional substance and the bio-orthogonal functional group can be appropriately reacted.
  • the functional group that easily reacts with the bio-orthogonal functional group may vary depending on the specific type of the bio-orthogonal functional group. Those skilled in the art can appropriately select an appropriate functional group as a functional group that easily reacts with a bioorthogonal functional group (eg, Boutureira et al., Chem. Rev., 2015, 115, 2174-2195). ).
  • Examples of the functional group that easily reacts with the bioorthogonal functional group include, for example, an azide residue when the bioorthogonal functional group is an alkyne residue, and an aldehyde or ketone residue when the bioorthogonal functional group is an aldehyde residue or a ketone residue.
  • the functional substance and the bioorthogonal functional group may be a divalent group containing a triazole residue (which may or may not be condensed with another ring).
  • the divalent group containing the moiety generated by the reaction between the orthogonal functional group and the orthogonal functional group may be a divalent group containing a hydrazone residue;
  • the biological orthogonal functional group is a thiol residue, and Functional groups that readily react with the orthogonal functional groups Is a disulfide residue (or vice versa),
  • a divalent group containing a moiety formed by a reaction between a functional substance and a bioorthogonal functional group is a divalent group containing a thiosuccinimide residue Or a divalent group containing a disulfide residue (eg, Boutureira et al., Chem.
  • a divalent group containing a triazole residue (which may or may not be condensed with another ring), a divalent group containing a hydrazone residue, a divalent group containing a thiosuccinimide residue, or A divalent group containing a disulfide residue is a preferred example of a divalent group containing a moiety generated by a reaction between a functional substance and a bioorthogonal functional group.
  • a functional substance derivatized so as to have a desired functional group can be used.
  • the functional substance is a soluble protein
  • a substance derivatized so that the soluble protein has a functional group which does not naturally exist can be used.
  • Derivatization is a common technical knowledge in the art (eg, WO 2004/010957, US Patent Application Publication No. 2006/0074008, US Patent Application Publication No. 2005/0238649).
  • derivatization may be performed using a crosslinking agent as described above.
  • derivatization may be performed using a specific linker having a desired functional group.
  • such a linker may be capable of separating a functional substance and an antibody by cleavage of the linker in an appropriate environment (eg, inside or outside a cell).
  • a linker for example, a peptidyl linker (eg, a protease that is present in an intracellular protease (eg, a protease present in a lysosome or an endosome) or an extracellular protease (eg, a secretory protease)) (eg, a protease)
  • a linker that can be cleaved at a local acidic site present in vivo (eg, US Patent No. 5).
  • the linker may be self-immortal (eg, WO 02/083180, WO 04/044933, WO 05/112919).
  • the derivatized functional substance can also be simply referred to as “functional substance”.
  • the modified antibody having a functional substance on the side chain of a lysine residue in the constant region, obtained by the above steps (1) and (2), is produced by a reaction between the functional substance and a bio-orthogonal functional group.
  • divalent groups containing moieties includes, for example, a triazole residue, a hydrazone residue, a thiosuccinimide residue, a disulfide residue, an acetal residue, a ketal residue, an ester residue, a carbamoyl residue, an alkoxyalkyl residue, an imine residue.
  • Residue tertiary alkyloxycarbamate residue, silane residue, hydrazone-containing residue, phosphoramidate residue, aconityl residue, trityl residue, azo residue, vicinal diol residue, selenium residue, electron Aromatic ring-containing residue having a suction group (eg, halogen atom, boronic acid residue, mesyl, tosyl, triflate), coumarin-containing residue, sulfone-containing residue, unsaturated bond-containing chain residue, glycosyl residue Or a divalent group containing a residue selected from the group consisting of: Preferably, such a divalent group is a triazole residue, a hydrazone residue, a thiosuccinimide residue, or a disulfide residue.
  • a suction group eg, halogen atom, boronic acid residue, mesyl, tosyl, triflate
  • coumarin-containing residue s
  • the reaction in step (2) can be appropriately performed under conditions (mild conditions) that do not cause denaturation / decomposition of the antibody (eg, cleavage of an amide bond).
  • a reaction can be carried out in a suitable reaction system, for example, a buffer (eg, a phosphate buffer) at normal temperature (for example, about 15 to 35 ° C., preferably about 20 to 30 ° C.).
  • the pH of the buffer is, for example, 5 to 9, preferably 5.5 to 8.5, and more preferably 6.0 to 8.0.
  • the buffer may contain a suitable cofactor such as ATP.
  • the reaction time is, for example, 10 minutes to 150 hours, preferably 20 minutes to 120 hours, more preferably 30 minutes to 100 hours, and even more preferably 60 minutes to 80 hours.
  • concentration of Lpl can be appropriately adjusted.
  • Reactions of bioorthogonal functional groups are well known (eg, Sharless @ KB et al., Angew. Chem. Int. Ed. 40, 2004 (2015); Bertozzi ⁇ CR et al., Science # 291). , 2357 (2001); Bertozzi CR et al., Nature Chemical Biology 1, 13 (2005)).
  • the molar ratio (Z / Y) of the functional substance (Z) to the modified antibody (Y) having a modified portion containing a bioorthogonal functional group in the side chain of a lysine residue in the constant region is determined by It is not particularly limited since it varies depending on the type of the orthogonal functional group, the functional substance, and the antibody, and the number of sites in the antibody to be modified (eg, DAR). Yes, preferably from 0.5 to 80, more preferably from 1 to 70, even more preferably from 2 to 50, particularly preferably from 3 to 30.
  • Confirmation of the generation of a modified antibody having a functional substance on the side chain of a lysine residue in the constant region depends on the specific raw material and the molecular weight of the product. For example, electrophoresis, chromatography (eg, Gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC) or mass spectrometry, preferably mass spectrometry. Confirmation of the position of the modified lysine residue in the antibody can be performed, for example, by peptide mapping. Peptide mapping can be performed, for example, by protease (eg, trypsin, chymotrypsin) treatment and mass spectrometry. As the protease, an endoprotease is preferable.
  • protease eg, trypsin, chymotrypsin
  • Such endoproteases include, for example, trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, Asp-N.
  • Confirmation of the number of functional substances carried by a modified antibody having a functional substance on the side chain of a lysine residue in the constant region is performed, for example, by electrophoresis, chromatography, or mass spectrometry, preferably by mass spectrometry. Can be performed.
  • the modified antibody having a functional substance on the side chain of a lysine residue in the constant region can be appropriately purified by any method such as chromatography (eg, the above-described chromatography and affinity chromatography).
  • the present invention also provides a modified antibody having a target portion (modified portion or functional substance) on the side chain of a lysine residue in the constant region.
  • the modified antibody of the present invention has a C 4 -C 10 alkyl-carbonyl having a modified portion only on the side chain of a lysine residue unique to the antibody.
  • "An antibody-specific lysine residue” refers to the variable region (VH) and constant region (CH1, CH2, CH3) in the heavy chain of an antibody, and the variable region (VL) and constant region (CL) in the light chain of an antibody. ) refers to one or more lysine residues present in a full length antibody or antibody fragment thereof.
  • an antibody that is a fusion protein having a structure in which a predetermined amino acid sequence (eg, a tag sequence, a spacer sequence) is added or inserted into a predetermined region of the antibody a C 4 -C 10 alkyl-carbonyl having a modified portion is substituted with A modified antibody having a side chain of a lysine residue corresponding to a predetermined amino acid sequence added or inserted is excluded from the modified antibody of the present invention.
  • a predetermined amino acid sequence eg, a tag sequence, a spacer sequence
  • the modified antibody of the present invention is produced as an antibody having a different structure depending on the type of the lipoic acid analog having a modified moiety used in the above-described method of the present invention.
  • the lipoic acid analog having a modified moiety is a C 4 -C 10 alkyl-carboxylic acid having a modified moiety
  • the antibody having the modified moiety on the side chain of a lysine residue in the constant region has the modified moiety.
  • Antibodies are generated that have a C 4 -C 10 alkyl-carbonyl in the side chain of a lysine residue in the constant region.
  • the lipoic acid analog having a modified moiety is a C 6 -C 8 alkyl-carboxylic acid having a modified moiety
  • an antibody having a modified moiety on the side chain of a lysine residue in the constant region may be a C 6 having a modified moiety.
  • Antibodies are generated that have ⁇ C 8 alkyl-carbonyl in the side chain of a lysine residue in the constant region.
  • the lipoic acid analog having a modified moiety is a C 7 alkyl-carboxylic acid having a modified moiety
  • an antibody having a modified moiety on the side chain of a lysine residue in the constant region may be used as a C 7 alkyl-carbonyl having a modified moiety.
  • a modified antibody having a modified portion in the side chain of a lysine residue in a specific constant region (CH1 region of a heavy chain, CL region of a light chain) can be produced.
  • CH1 region of a heavy chain, CL region of a light chain CH1 region of a heavy chain, CL region of a light chain
  • the modified antibody of the present invention may also be a modified antibody having a modified portion on the side chain of a lysine residue present in both the CH1 region of the heavy chain and the CL region of the light chain.
  • the modified antibody of the present invention is a modified antibody having a modified portion in the side chain of both the lysine residue at position 133 in the human IgG heavy chain and the lysine residue at position 169 in the human IgG light chain.
  • the position of the amino acid residue in the light chain is indicated by the position in amino acid sequence number 15, and the position of the amino acid residue before the 120th position in the amino acid sequence number 14 in the heavy chain is defined by the position in amino acid sequence number 14.
  • amino acid residues from position 121 onwards in the amino acid sequence number 14 of the heavy chain are shown at the position of EU numbering (therefore, the lysine residue at position 133 in the human IgG heavy chain and 169 in the human IgG light chain).
  • the position of the lysine residue at position follows Eu @ numbering in human IgG, as in the present specification.).
  • the CH1 region of the heavy chain and the CL region of the light chain are amongst the constant regions near the antigen binding site, and the desired modifications present in these regions also depend on the type of modification and the target containing the antigen. However, it may be able to interact strongly with targets, including antigens.
  • modified antibodies of the present invention having desired modifications in the CH1 region and CL region may be useful as medicines or reagents.
  • the modified antibody of the present invention can have a modified portion in the side chain of two lysine residues (eg, two lysine residues at position 133) in the CH1 region of two heavy chains.
  • the modified antibodies of the present invention can also have a modified moiety in the side chain of two lysine residues (eg, two lysine residues at position 169) in the CL regions of the two light chains.
  • the modified antibody of the present invention may be one in which such a lysine residue is highly modified.
  • the modification ratio of the lysine residue at position 133 in the total modification of human IgG CH1 is 30% or more, preferably 40% or more, more preferably 50% or more, and even more preferably 60% or more. %, Particularly preferably 70% or more.
  • the modification ratio of lysine residues at positions other than position 133 in human IgG @ CH1 in the modification of human IgG @ CH1 is low (eg, less than 30%, less than 20%, less than 10%, or 5%). Less than).
  • the modification ratio of the lysine residue at position 169 in the modification of the whole human IgG @ CL may be 30% or more.
  • the modification ratio of lysine residues at positions other than position 169 in human IgG @ CL in the total modification of human IgG @ CL is low (eg, less than 30%, less than 20%, less than 10%, or 5%). Less than).
  • the modified antibodies of the present invention also include lysine residues at other positions in the heavy chain of the antibody (eg, lysine residues in the constant region (eg, lysine residue at position 222) or lysine residues in the variable region). May be modified.
  • the modification ratio of the lysine residue at position 222 in the modification of human IgGICH2 may be 30% or more.
  • the modification ratio of lysine residues at positions other than position 222 in human IgG @ CH2 in the modification of human IgG @ CH2 is low (eg, less than 30%, less than 20%, (Less than 10%, or less than 5%).
  • the modified antibody of the present invention is also a polyclonal antibody or a monoclonal antibody.
  • the modified antibody of the present invention may be an antibody (eg, glycoprotein) modified with a biomolecule (eg, sugar) or an antibody not modified with a biomolecule.
  • the modified antibody of the present invention may be any antibody against any component such as a biological component, a virus-derived component, and a component found in the environment, but is preferably an antibody against a biological-derived component or a virus-derived component.
  • the biological component include components (eg, proteins) derived from animals such as mammals and birds (eg, chickens), insects, microorganisms, plants, fungi, and fish.
  • the biological component is a component derived from a mammal.
  • mammals examples include primates (eg, humans, monkeys, chimpanzees), rodents (eg, mice, rats, guinea pigs, hamsters, rabbits), companion animals (eg, dogs, cats), livestock (eg, Cattle, pigs, goats) and working animals (eg, horses, sheep).
  • the biological component is more preferably a primate or rodent-derived component (eg, a protein), and even more preferably, from the viewpoint of clinical application of the present invention, a human-derived component (eg, a protein).
  • virus-derived components include components (eg, proteins) derived from influenza virus (eg, avian influenza virus, swine influenza virus), AIDS virus, Ebola virus, and phage virus.
  • the modified antibody of the present invention is also an antibody against any antigen.
  • an antigen may be a component found in an organism or virus as described above.
  • antigens also include, for example, proteins [including oligopeptides and polypeptides. It may be a protein modified with a biomolecule such as sugar (eg, glycoprotein)], a sugar chain, a nucleic acid, and a low molecular compound.
  • the modified antibody of the present invention may be an antibody having a protein as an antigen.
  • proteins include cell membrane receptors, cell membrane proteins other than cell membrane receptors (eg, extracellular matrix proteins), ligands, and soluble receptors.
  • the protein that is the antigen of the modified antibody of the present invention may be a disease target protein.
  • diseases target proteins include (1) cancer regions, (2) autoimmune diseases / inflammatory diseases, (3) cranial nerve diseases, (4) infectious diseases, (5) hereditary / rare diseases, and (6) Examples include the above-mentioned proteins in eye diseases, (7) bone and orthopedic fields, (8) blood diseases, and (9) other diseases.
  • the modified antibodies of the present invention have a native polypeptide chain structure, as described above.
  • the modified antibodies of the present invention are monoclonal antibodies.
  • the monoclonal antibody for example, a chimeric antibody, a humanized antibody, a human antibody, an antibody to which a predetermined sugar chain is added (for example, modified to have a sugar chain binding consensus sequence such as an N-type sugar chain binding consensus sequence) Antibodies), bispecific antibodies, scFv antibodies, Fab antibodies, F (ab ') 2 antibodies, VHH antibodies, Fc region proteins, and Fc fusion proteins.
  • Isotypes of antibodies such as monoclonal antibodies include, for example, IgG, IgM, IgA, IgD, IgE, and IgY.
  • the antibody may also be a bivalent antibody (eg, IgG, IgD, IgE) or a tetravalent or higher-valent antibody (eg, IgA antibody, IgM antibody).
  • the modified antibodies of the present invention may be full length antibodies, or antibody fragments (eg, Fab, F (ab ') 2 , Fab', Fv, single chain antibodies).
  • the antibody fragment preferably has either the CH1 region of the heavy chain or the CL region of the light chain, and more preferably has both the CH1 region of the heavy chain and the CL region of the light chain.
  • the modified antibodies of the present invention are human or humanized antibodies.
  • specific examples of the human antibody and the humanized antibody are the same as those described above.
  • the modified antibodies of the invention are IgG.
  • IgG examples include IgG1, IgG2, IgG3, and IgG4.
  • IgG human IgG is preferable.
  • the modified antibodies of the invention are Fab, or F (ab ') 2 .
  • the modified antibody of the present invention is a modified antibody having a modification portion on the side chain of a lysine residue in the constant region.
  • the modified antibody of the present invention having a modified portion on the side chain of a lysine residue in the constant region has a portion represented by the following formula (II) only on the side chain of the antibody-specific lysine residue: R—C n H 2n —CO—NH— (II) (In the formula, R is a modifying moiety; n is an integer of 4 to 10, NH- is a group present on the side chain of a lysine residue in the constant region. ]
  • n is preferably an integer of 5 to 9, more preferably an integer of 6 to 8, and particularly preferably 7.
  • the modifying moiety in the modified antibodies of the present invention comprises a bioorthogonal functional group or a functional substance.
  • a modified antibody of the present invention can be prepared by reacting the antibody with a lipoic acid analog having a modified portion containing a bioorthogonal functional group in the presence of lipoic acid protein ligase A as described above. Producing a modified antibody having a modified moiety containing a bioorthogonal functional group on the side chain of a lysine residue in the region.
  • the modified antibody of the present invention (containing a bioorthogonal functional group as a modifying moiety) thus obtained is useful as an intermediate for the synthesis of an antibody having a functional substance.
  • the thus obtained modified antibody of the present invention (containing a functional substance as a modifying moiety) is useful as a medicine or a reagent (eg, a diagnostic agent, a research reagent).
  • the modifying moiety in the modified antibodies of the present invention comprises a bioorthogonal functional group.
  • the modified antibody of the present invention (containing a bioorthogonal functional group as a modified moiety) thus obtained is useful as an intermediate for the synthesis of antibodies having various functional substances (in particular, the modified moiety).
  • Lipoic acid analogs having a functional substance cannot function as an Lpl substrate due to the size of the functional substance, or can function as an Lpl substrate but have a low reaction rate as an enzyme).
  • the modified antibody of the present invention is a modified antibody having a functional substance on the side chain of a lysine residue in the constant region.
  • the modified antibody of the present invention having a functional substance on the side chain of a lysine residue in the constant region has a portion represented by the following formula (III) only in the side chain of the antibody-specific lysine residue: F-R'-C n H 2n -CO-NH- (III) (In the formula, F is a functional substance, R ′ is a divalent group including a moiety generated by a reaction between a functional substance and a bioorthogonal functional group, n is an integer of 4 to 10, NH- is a group present on the side chain of a lysine residue in the constant region. ]
  • the functional substance, the bio-orthogonal functional group, and the divalent group containing the moiety generated by the reaction between the functional substance and the bio-orthogonal functional group are the same as those described above. is there.
  • n is preferably an integer of 5 to 9, more preferably an integer of 6 to 8, and particularly preferably 7.
  • the modified antibody of the present invention having a functional substance on the side chain of a lysine residue in the constant region can be obtained by the method of the present invention including the above-described steps (1) and (2).
  • the modified antibody of the present invention thus obtained is useful as a medicine or a reagent (eg, a diagnostic agent, a research reagent).
  • Example 1 Search for Modification Activity of IgG Antibody (1-1) Expression and purification of E. coli LplA As lipoic acid protein ligase (Lpl), a fusion protein of LplA (EcLplA) derived from Escherichia coli and glutathione-S-transferase (GST) was used.
  • Lpl lipoic acid protein ligase
  • EcLplA fusion protein of LplA derived from Escherichia coli and glutathione-S-transferase
  • EcLplA expression plasmid In order to obtain a plasmid that expresses a fusion protein of EcLplA and GST (hereinafter simply referred to as “EcLplA expression plasmid”), the base sequence encoding the full-length LplA sequence is cloned into a pCold GST DNA vector (TaKaRa), and pCold -LplA (Ec) was constructed. A histidine tag derived from a vector is added to pCold-IplA (Ec).
  • PCold-LplA (Ec) was constructed by the following method.
  • E. FIG. A DNA fragment containing a base sequence encoding EcLplA by PCR using genomic DNA of E. coli strain MG1655 as a template chain and lplA (Ec) fw (SEQ ID NO: 9) and lplA (Ec) rv (SEQ ID NO: 10) as primers I got PCR was performed using PrimeStar polymerase (TaKaRa) with the reaction composition described in the attached protocol. The PCR cycle was performed at 94 ° C for 5 minutes, followed by 30 cycles of 98 ° C for 5 seconds, 55 ° C for 10 seconds, and 72 ° C for 2 minutes, and finally, at 4 ° C.
  • TaKaRa PrimeStar polymerase
  • a DNA fragment of pCold ⁇ GST ⁇ DNA was obtained by PCR using pCold ⁇ GST ⁇ DNA (SEQ ID NO: 11) as a template DNA and oligonucleotides of SEQ ID NOS: 12 and 13 as primers.
  • the PCR was performed using PrimeStar polymerase with the reaction composition described in the attached protocol.
  • the PCR cycle was 94 ° C for 5 minutes, followed by 30 cycles of 98 ° C for 5 seconds, 55 ° C for 10 seconds and 72 ° C for 5 minutes, and finally 4 cycles.
  • the test was performed under the condition of keeping the temperature at °C.
  • the obtained DNA fragments were ligated using an In-Fusion @ HD cloning kit (Clontech) to construct an EcLplA expression plasmid pCold-lplA (Ec).
  • the nucleotide sequence of lplA (Ec) and the amino acid sequence encoded thereby are shown in SEQ ID NOs: 1 and 2, respectively.
  • the EcLplA expression plasmid pCold-lplA (Ec) was coli JM109 strain (TaKaRa) was introduced by a transformation method using heat shock to obtain a JM109 / pCold-lplA (Ec) strain. Transformation using heat shock was performed according to the protocol described in the attached protocol. This strain was transformed into an LB medium (1.0% (w / v) peptone, 0.50% (w / v) yeast extract, 1.0% (w / v) NaCl) supplemented with 100 ⁇ g / mL ampicillin. The cells were inoculated and cultured with shaking at 37 ° C. overnight.
  • the culture solution was inoculated to a final concentration of 1% in an LB medium in which 100 mL of the medium was inserted into a Sakaguchi flask. After culturing with shaking at 37 ° C. for 3 hours, isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) was added to a final concentration of 1.0 mM, and the culture temperature was further reduced to 15 ° C. Culture was performed for 16 hours.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • an equilibration buffer (elution buffer) to which imidazole having a final concentration of 300 mM was added was passed through the column to collect a fraction containing EcLplA, and 1.0 mL of a 0.63 mg / mL fraction was used as purified EcLplA.
  • IgG antibody modifying activities of various enzymes IgG antibody modifying activities of various enzymes were evaluated.
  • the enzymes Lpl, 4'-phosphopantetheinyl transferase (4'-phosphopantheinyl transferase, PPTase), and biotin ligase were used.
  • an IgG antibody an anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical) was used.
  • the antibody modification reaction with ⁇ ⁇ ⁇ Lpl was performed as follows. EcLplA prepared in (1-1) was used as Lpl. 8-Azidooctanoic acid was used as a modification.
  • the reaction composition is as follows. 500 ⁇ g of trastuzumab was dissolved in 250 ⁇ L of 1.0 mM sodium phosphate buffer (pH 6.0) and used. A mixed solution having a final concentration of 1.0 mg / mL trastuzumab, ATP 2.5 mM, 8-azidooctanoic acid (Sundia) 0.50 mM, magnesium sulfate 3.2 mM, and sodium phosphate buffer 25 mM (pH 7.0).
  • EcLplA having a final concentration of 0.050 mg / mL was added thereto to prepare a reaction solution having a total volume of 100 ⁇ L, and reacted at 30 ° C. for 15 hours to produce 8-azidooctanoic acid-modified trastuzumab as a modified antibody.
  • 2 ⁇ L of an EDTA-2Na solution was added to the reaction solution (final concentration: 10 mM) to stop the reaction.
  • ⁇ Antibody modification reaction with PPTase was performed as follows. SFP @ synthase (New @ England @ Biolabs) was used as the PPTase. CoA lithium salt (Sigma-Aldrich) was used as a modifier.
  • the reaction composition is as follows. A final concentration of a mixture of trastuzumab 1.0 mg / mL, CoA lithium salt (Sigma-Aldrich) 0.50 mM, magnesium sulfate 2.0 mM, Hepes-KOH buffer 50 mM (pH 7.5) as a reaction solution was added to the final concentration. 1.0 ⁇ M SFP @ synthase was added to make a total volume of 100 ⁇ L, and reacted at 30 ° C. for 15 hours to generate CoA-modified trastuzumab as a modified antibody. 2 ⁇ L of an EDTA-2Na solution was added to the reaction solution (final concentration: 10 mM) to stop the reaction.
  • Antibody modification with biotin ligase was performed as follows. BirA (Abcam) was used as biotin ligase. Biotin was used as a modification.
  • the reaction composition is as follows. A final concentration of 1.050 mg / mL of trastuzumab, 1.0 mM of ATP, 0.50 mM of biotin, 1.0 mM of magnesium sulfate, and 50 mM of Tris-HCl buffer (pH 8.3) was added to a mixed solution having a final concentration of 0.050 mg / mL. mL of BirA was added to make a total volume of 100 ⁇ L, and reacted at 30 ° C. for 15 hours to generate biotin-modified trastuzumab as a modified antibody. 2 ⁇ L of an EDTA-2Na solution was added to the reaction solution (final concentration: 10 mM) to stop the reaction.
  • the modified antibody was purified using a Protein A column (Protein A HP MultiTrap (96 well), GE Healthcare). The Protein A column used was equilibrated with 20 mM Tris (pH 7.6) and 20% (v / v) ethanol. A centrifugal separator was used to pass the solution, and centrifugation was performed at 900 g for 1 minute at each stage.
  • 500 ⁇ L of the enzyme reaction solution containing 500 ⁇ g of trastuzumab was divided into two wells of 250 ⁇ L each, applied, and washed twice with 300 ⁇ L of equilibration buffer. Further, 200 ⁇ L of 100 mM glycine buffer (pH 2.7) was added and eluted. Immediately, 15 ⁇ L of 1.0 M Tris-HCl was added to adjust the pH to around 8.5 to obtain a modified antibody sample.
  • the Lpl reaction product and the PPTase reaction product were evaluated for antibody modification by the following method.
  • the modified antibody sample fluorescently labeled in (1-4) was purified in the same manner as in (1-3).
  • the modification to the antibody was evaluated by quantifying the amount of the fluorescent label in the purified sample after the fluorescent labeling. All analyzes were performed on a 96-well plate, and fluorescence was detected at an excitation wavelength of 520 nm and a fluorescence wavelength of 580-640 nm.
  • the biotin ligase reaction product was evaluated for antibody modification by the following method.
  • the modification to the antibody was evaluated by quantifying the concentration of side chain biotin in the modified antibody sample purified in (1-3) using a Fluorescence Biotin Quantitation Kit (manufactured by Thermo Fisher Scientific).
  • the Fluorescence Biotin Quantitation Kit is capable of quantifying the concentration of side-chain biotin in a solution by fluorescent labeling, and the quantification has been confirmed from 0 to 6.0 ⁇ M.
  • detection was performed at an excitation wavelength of 475 nm and a fluorescence wavelength of 500 to 550 nm. GroMAX (Promega) was used as a plate reader.
  • modification to the antibody was observed only in the Lpl reaction product (Table 2).
  • Example 2 Modification of IgG antibody by LplA and analysis by ESI-MS (2-1) Enzyme reaction and analysis by MS As IgG antibody and LplA, anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical) and (1-1) The prepared EcLplA was used.
  • the reaction composition is as follows. 500 ⁇ g of trastuzumab was dissolved in 250 ⁇ L of 1 mM sodium phosphate buffer (pH 6.0) and used.
  • a final concentration of 1.0 mg / mL of trastuzumab, 2.5 mM of ATP, 0.5 mM of octanoic acid, 3.2 mM of magnesium sulfate, and 25 mM of sodium phosphate buffer (pH 7.0) in the final concentration was added to the final mixture.
  • a total of 500 ⁇ L of a reaction solution was prepared by adding 0.05 mg / mL EcLplA, and reacted at 30 ° C. for 64 hours to produce octanoic acid-modified trastuzumab as a modified antibody.
  • the reaction was terminated by adding 10 ⁇ L of an EDTA-2Na solution (final concentration: 10 mM) to the reaction solution.
  • LplA has the ability to modify antibodies, and modifies antibodies with 1 to 10 modifications per antibody.
  • Example 3 Modification of IgG with various LplAs and analysis by ESI-MS (3-1) Expression and purification of various LplAs As other LplAs described in Example 1, LplA derived from Bacillus subtilis (BsLplA) and Corynebacterium glutamicum. LplA (CgLplA) and LplA derived from Staphylococcus epidermides (SeLplA) were used.
  • nucleotide sequences encoding various LplA full-length sequences were cloned into a pCold GST DNA vector, and lplA expression plasmids pCold-lplA (Bs), pCold-lplA (Cg), and pCold-lplA (Se) were constructed.
  • lplA (Bs), lplA (Cg), and lplA (Se) base sequences (6 bases each having a BamHI and HindIII recognition sequence added to the 5′- and 3′-ends), and lplA (Bs), lplA
  • the amino acid sequence of (Cg), lplA (Se) is shown in SEQ ID NOs: 3, 5, and 7, and SEQ ID NOs: 4, 6, and 8.
  • ⁇ pCold-lplA (Bs), pCold-lplA (Cg) and pCold-lplA (Se) were purchased from E. coli. coli JM109 strain.
  • a purified enzyme solution was prepared from a cell extract prepared by culturing the transformant by the method described in (1-1).
  • the modified antibody was purified by a Protein A column in the same manner as in (1-3) to prepare a purified and modified antibody sample.
  • DIBAC-HHHHHHG-OH peptide (SEQ ID NO: 28) DIBAC-HHHHHHG-OH peptide (SEQ ID NO: 28) as a functional substance capable of binding to an azide group by a click reaction was prepared by the following reaction It was synthesized according to the scheme.
  • the peptide synthesizer used was Initiator + Alstra manufactured by Biotage. All reagents used were made by Watanabe Chemical Industry.
  • As the resin H-Gly-Trt (2-Cl) -Resin (0.89 mmol / g) was used.
  • the antibody was purified from the reaction solution after the click reaction using a Protein A column in the same manner as in (1-3).
  • the fraction containing the eluted antibody was solvent-exchanged with a 1,000-fold volume of a 20 mM ammonium acetate solution (pH 6.4) using an ultrafiltration unit (Amicon Ultra 0.5 mL 10 kDa, Merck Millipore), and then concentrated to concentrate IgG. After confirming that the concentration was 0.50 mg / mL or more, analysis by MS was performed.
  • Example 4 Identification of Modification Position by Peptide Mapping (4-1) Identification of Modification Position by Peptide Mapping 10 ⁇ L of the sample solution obtained in Example 2 in 50 mL low-adsorption microtest tube, 50 mM ammonium bicarbonate 10 ⁇ L of a 20 mM dithiothreitol aqueous solution dissolved in a buffer solution and 40% trifluoroethanol was added, and the mixture was heated at 65 ° C. for 1 hour. Reacted. After the reaction, 40 ⁇ L of a 50 mM ammonium bicarbonate buffer was added and stirred, and 10 ⁇ L of a 20 ng / ⁇ L aqueous trypsin solution was added, followed by enzyme digestion at 37 ° C. for 18 hours. After the digestion, 2 ⁇ L of a 20% aqueous trifluoroacetic acid solution was added to stop the reaction, and LC / MS / MS measurement was performed.
  • Sequence ⁇ HT was used as a search engine, and the range of the precursor ion was set to 350 to 5000 Da.
  • trypsin was set as a digestive enzyme, and Maximum ⁇ Missed ⁇ Clearage ⁇ Sites was set to 3.
  • Mass @ Tolerance of the precursor and the fragment ion were set to 5 ppm and 0.5 Da, respectively.
  • Static @ Modification Carbamidomethyl (+57.021 Da) was set as a modification of cysteine residue with iodoacetamide.
  • the divalent y17 also shows the modification of the lysine residue at position 133 in EU numbering of the heavy chain. A corresponding product ion of m / z 875.67 (theoretical value: 875.47) was confirmed (FIG. 7).
  • MS spectrum of peptide fragment of SCDKTHTCPPCPAPELLGGPSVFLFPPPKPK (SEQ ID NO: 19), which is a peptide consisting of 30 amino acids including a site for modification to lysine residue (octanoic acid-introduced substance (+126.105 Da)) by trypsin digestion (actual value: m / Z 1154.252572, theoretical value: 1154.25381; trivalent) (FIG.
  • the monovalent y5 also shows the modification of the lysine residue at position 246 or 248 of the heavy chain.
  • a corresponding product ion of m / z 692.46 (theoretical value: 692.47) was confirmed (FIG. 11).
  • an MS spectrum (actual measurement: m / Z 696.92812, theoretical value: 696.992724, divalent) was observed (FIG. 12), and the CID spectrum also shows modification of the lysine residue at position 326 of the heavy chain, m corresponding to monovalent b4.
  • a product ion of / z 555.38 (theoretical value: 555.35) was confirmed (FIG. 13).
  • MS spectrum of peptide fragment of EPQVYTLPPPSREEMTKNQVSLTCLVK (SEQ ID NO: 22) consisting of a peptide consisting of 26 amino acids including a modification site to lysine residue by lysine digestion (octanoic acid-introduced substance (+126.105 Da)) (actual value: m / Z 794.17197, theoretical value: 794.17052, 4 valences) (FIG. 14), and the CID spectrum shows a modification of the lysine residue at the 360-position of the heavy chain, which corresponds to divalent b18. A product ion of / z 1128.23 (theoretical value: 1128.07) was confirmed (FIG. 15).
  • MS spectrum of a peptide fragment of ASQDVNTAVAWYQQKPGKAPK (SEQ ID NO: 23) comprising a peptide consisting of 21 amino acids including a modification site to lysine residue by lysine digestion (octanoic acid-introduced substance (+126.105 Da)) (actual value: m / Z 1207.14994, theoretical value: 1207.14792, divalent) (FIG. 16), and the CID spectrum shows the modification of the lysine residue at position 42 in amino acid SEQ ID NO: 15 of the light chain.
  • the product ion of m / z 626.41 (theoretical value: 626.42) corresponding to was obtained (FIG. 17).
  • MS spectrum (actual value: m) of a peptide fragment of VDNALQSGNSQESVTEQDSKDSTYSLSLSTLTLSK (SEQ ID NO: 24), which is a peptide consisting of 34 amino acids including a site for modification to a lysine residue (octanoic acid-introduced substance (+126.105 Da)) by trypsin digestion. / Z 1249.227813, theoretical value: 1249.276768, trivalent) (FIG. 18), and the CID spectrum shows a modification of the lysine residue at position 169 of the light chain, which corresponds to divalent b22.
  • Example 5 Using the peptide-spectrum match (Psm) of the tryptic digest of the modified trastuzumab obtained in Example 1 as an index, the ratio of highly modified positions in trastuzumab was calculated to be about 79% in the heavy chain CH1 region. % was a modification to the lysine residue at position 133, and about 33% of the light chain CL region was confirmed to be a modification to the lysine residue at position 169 (Table 3). In the heavy chain CH2 region, modification to the lysine residue at position 222 was about 32% (Table 3).
  • Fab was purified by a Protein G column (HiTrap Protein G HP Columns, 1 mL, GE Healthcare).
  • a Protein G column a column equilibrated with 20 mM Tris (pH 7.6) and 20% (v / v) ethanol was used.
  • 500 ⁇ L of the enzyme reaction solution containing 1 mg of Fab the plate was washed with 4 mL of equilibration buffer. Further, 2 mL of a 100 mM glycine buffer (pH 2.7) was added and eluted. Immediately, 150 ⁇ L of 1 M Tris-HCl was added to adjust the pH to around 8.5 to obtain a purified Fab sample.
  • the purified Fab was subjected to solvent exchange and concentration in the same manner as in (3-4), and the mass was measured by ESI-MS. As a result, a peak was observed at 47638 for the raw Fab, and one octanoic acid azide was introduced into the purified product. Peaks were observed at 47805 introduced and 47971 introduced at two sites.
  • PCR1 primers P4, P5; DNA template pET15-wt-lplA. Protocol: 95 ° C, 3 minutes; 98 ° C, 20 seconds; 60 ° C, 15 seconds; 72 ° C, 15 seconds; 25 cycles. As a result, a DNA fragment F1 was obtained.
  • PCR2 primers P3, P6; DNA template pET15-wt-lplA.
  • Protocol 95 ° C, 3 minutes; 98 ° C, 20 seconds; 60 ° C, 15 seconds; 72 ° C, 15 seconds; 25 cycles.
  • a DNA fragment F2 was obtained.
  • the pET15b vector was digested with XbaI and BamHI, and ligated to a DNA fragment F1 digested with XbaI-SacI and a DNA fragment F2 digested with SacI-BamHI.
  • a pET15b-wt-lplA (SacI) plasmid was constructed.
  • PCR2 primers P9 and P11; DNA template 1 ⁇ L of the obtained PCR1 mixture. Protocol: 95 ° C, 3 minutes; 98 ° C, 20 seconds; 64 ° C, 15 seconds; 72 ° C, 30 seconds; 25 cycles. As a result, a DNA fragment F2 was obtained.
  • PCR3 primers P10, P12; DNA template pET15-lplA (SacI). Protocol: 95 ° C, 3 minutes; 98 ° C, 20 seconds; 64 ° C, 15 seconds; 72 ° C, 30 seconds; 25 cycles. As a result, a DNA fragment F3 was obtained.
  • PCR4 primers P13, P14; equimolar mixture of DNA templates F2 and F3 DNA fragments (0.3 ⁇ M each). Protocol: 95 ° C, 3 minutes; 98 ° C, 20 seconds; 64 ° C, 15 seconds; 72 ° C, 1 minute; 25 cycles.
  • a DNA fragment mixture (library 1) encoding four modified LplA variants (R121A, V, S, T) was obtained.
  • Modified LplA variant encodes a combinatorial library of R121 (A, V, S, T) * Y140 (A, V) * E142 (A, V, S, T) (total of 32) Construction of DNA fragment Primers P15 (SEQ ID NO: 52), P16 (SEQ ID NO: 53), P17 (SEQ ID NO: 54), P18 (SEQ ID NO: 55), P19 (SEQ ID NO: 56), P20 (SEQ ID NO: 57), P21 (SEQ ID NO: 57) No. 58) and P22 (SEQ ID NO: 59) were used.
  • PCR1 primers P15, P16; 1 ⁇ L of DNA template library 1 DNA mixture.
  • Protocol 95 ° C, 3 minutes; 98 ° C, 20 seconds; 65 ° C, 15 seconds; 72 ° C, 1 minute; 20 cycles.
  • PCR2 primers P17 and P19; DNA template 1 ⁇ L of the obtained PCR1 mixture.
  • Protocol 95 ° C, 3 minutes; 98 ° C, 20 seconds; 64 ° C, 15 seconds; 72 ° C, 30 seconds; 25 cycles.
  • PCR3 primers P18, P20; 1 ⁇ L of DNA template library 1 DNA mixture.
  • Protocol 95 ° C, 3 minutes; 98 ° C, 20 seconds; 64 ° C, 15 seconds; 72 ° C, 30 seconds; 25 cycles.
  • DNA fragment 3 was obtained.
  • PCR4 Primers P21 and P22; DNA templates F2 and F3 equimolar mixtures of DNA fragments (0.3 ⁇ M each). Protocol: 95 ° C, 3 minutes; 98 ° C, 20 seconds; 64 ° C, 15 seconds; 72 ° C, 1 minute; 25 cycles.
  • R121 (A, V, S, T) * Y140 (A, V) * E142 (A, V, S, T) was obtained.
  • PCR1 primers P23, P24, P25; DNA template library 2 1 ⁇ L of DNA mixture. Protocol: 95 ° C, 3 minutes; 98 ° C, 20 seconds; 65 ° C, 15 seconds; 72 ° C, 1 minute; 20 cycles.
  • PCR2 primers P26, P28; DNA-templates, 1 ⁇ L of the resulting PCR1 mixture. Protocol: 95 ° C, 3 seconds; 98 ° C, 20 seconds; 64 ° C, 15 seconds; 72 ° C, 30 seconds; 25 cycles. As a result, a DNA fragment F2 was obtained.
  • PCR3 primers P27, P28; 1 ⁇ L of DNA template library 2 DNA mixture.
  • Protocol 95 ° C, 3 minutes; 98 ° C, 20 seconds; 64 ° C, 15 seconds; 72 ° C, 30 seconds; 25 cycles.
  • PCR4 primers P30, P31; an equimolar mixture of DNA templates F2 and F3.
  • Protocol 95 ° C, 3 minutes; 98 ° C, 20 seconds; 64 ° C, 15 seconds; 72 ° C, 1 minute; 25 cycles.
  • the modified LplA variant R121 (A, V, S, T) * Y140 (A, V) * E142 (A, V, S, T) * K176 (A, V, S, T) * 178 (A , V) (mixture of DNA fragments encoding the combinatorial library) (library 3).
  • BL21 (DE3) / pE15b-wt-lplA strain capable of expressing wt-LplA and a BL21 (DE3) / pET15b strain capable of expressing one chromosome copy of the wt-lplA gene were constructed.
  • BL21 (DE3) / pET15b-wtLplA strain was inoculated into 10 wells to measure wtLplA activity expressed in the plasmid on all plates. Then, the microplate was cultured at 300 RPM and 37 ° C. for about 2 hours using a shaker (Lab-Therm LT-X, Kuhner (registered trademark)). When the OD 595 reaches about 1, IPTG (isopropyl ⁇ -D-1-thiogalactopyranoside) is added to each well to a concentration of 1 mM, and cultured for about 2 hours to obtain protein synthesis. Was induced.
  • the cells were centrifuged at 4 ° C., 4000 RPM for 10 minutes using an Eppendorf 5920R centrifuge equipped with an S-4x Universal-L rotor, and the cells were collected and the supernatant was removed.
  • Cells in each well were resuspended in 60 ⁇ L of cell lysis buffer containing: 50 mM Tris-HCl pH 7.5, 5% glycerol (v / v), 1 mM DTT (dithiothreitol), 1 mM PMSF (Phenylmethylsulfonyl fluoride). Thereafter, lysozyme was added to each well to a final concentration of 300 ⁇ g / mL, and a Kuhner shaker was used.
  • the cell suspension was incubated at 100 rpm for 4 hours at 10 ° C. Then, 5 mM MgCl 2 and 1 ⁇ g / mL of DNase I (Sigma) were added and the reaction mixture was incubated at 10 ° C. for 30 minutes at 100 rpm.
  • the obtained crude cell lysate was filtered using a 96-well filter plate (Corning; 0.2 ⁇ m, PDVF hydrophobic membrane), and the flow-through fraction was prepared as a cell-free extract containing the LplA protein.
  • LplA activity labeling of IgG with octanoic acid azide assay of crude cell lysate
  • the reaction was incubated at 30 ° C. for about 16 hours (overnight).
  • DBCO-Cy3 Sigma was added to each reaction solution until the final concentration became 30 ⁇ M, and the mixture was incubated at 30 ° C.
  • each mLplA was calculated by normalizing as follows: [F (i) -F (N)] / F (P), where F (i) is the fluorescence of the i fraction and F (i) (N) is the average fluorescence intensity of 10 reactions using the crude cell-free extract of BL21 (DE3) / pET15b strain, and F (P) is BL21 (DE3) / pET15b-wt-lplA (SacI).
  • Protocol 95 ° C, 3 minutes; 98 ° C, 20 seconds; 64 ° C, 15 seconds; 72 ° C, 1 minute; 25 cycles.
  • the obtained DNA fragment was digested with NcoI and BamHI restriction enzymes, and ligated with the pET15b plasmid digested with the same restriction enzymes.
  • X wt, m231, m746, m766, m876) were constructed.
  • IPTG was added to a final concentration of 1 mM, and the cells were cultured at 37 ° C. for 2 hours.
  • the obtained cells were collected by centrifugation, washed twice with 50 mL of 0.9% NaCl solution, and frozen at ⁇ 20 ° C. until use.
  • the lysed cells were resuspended in 4 mL of buffer A (20 mM Tris-HCl, 20 mM imidazole, 500 mM NaCl, pH 7.4), and the suspension was divided into five 1.5 vials by 0.8 mL.
  • the cells were destroyed by sonication.
  • the insoluble fraction of the lysate was removed by centrifugation at 13000 RPM for about 20 minutes.
  • the resulting cell-free extract was applied to a 1 mL HiTrap Chelating column (GE healthcare) and purified according to standard protocols.
  • the buffer of the eluted fraction containing N-Tag6His-m-LplA was exchanged with a 50 mM NaPi buffer (pH 7) using a 5 mL HiTrap desalting column (GE Healthcare).
  • the resulting solution was concentrated on an Amicon 10K membrane until the final protein concentration was 0.5-1 mg / L.
  • the reaction was incubated at 30 ° C. for 48 hours, and 20 ⁇ L of the reaction mixture was sampled over time. The reaction was stopped by the addition of EDTA (10 mM) and stored frozen ( ⁇ 20 ° C.) until analysis. To this reaction solution, DBCO-Cy3 was added at 100 ⁇ M, and incubated at 30 ° C. for 5 hours. The reaction solution after labeling was applied to an Amicon Ultra 0.5 ml 10 kDa filter, and washed three times with 0.35 mL of PBS to which ethanol (20%) was added.
  • the present invention is useful, for example, for producing regioselectively modified antibodies.
  • SEQ ID NOs: 1 and 2 show the nucleotide sequence of LplA (EcLplA) derived from Escherichia coli and the amino acid sequence encoded thereby, respectively.
  • SEQ ID NOS: 3 and 4 show the amino acid sequences of Bacillus subtilis-derived LplA (BsLplA) (having 6 bases of BamHI and HindIII recognition sequences added at the 5′- and 3′-ends) and BsLplA, respectively. .
  • SEQ ID NOs: 5 and 6 show the amino acid sequences of Corynebacterium glutamicum-derived LplA (CgLplA) (having 6 bases of BamHI and HindIII recognition sequences at the 5′- and 3′-ends) and CgLplA, respectively.
  • SEQ ID NOs: 7 and 8 show the amino acid sequences of Staphylococcus epidermides-derived LplA (SeLplA) (having 6 bases of BamHI and HindIII recognition sequences added at the 5′- and 3′-ends) and BsLplA, respectively.
  • SEQ ID NOS: 9 and 10 show the nucleotide sequences of PCR primers for amplification of EcLplA (lplA (Ec) fw and lplA (Ec) rv), respectively.
  • SEQ ID NO: 11 shows the nucleotide sequence of plasmid vector pCold GST DNA.
  • SEQ ID NOs: 12 and 13 show the nucleotide sequences of PCR primers for pCold GST DNA amplification (pCold GST fw and pCold GST rv), respectively.
  • SEQ ID NOS: 14 and 15 show the amino acid sequence of the heavy chain of trastuzumab and the amino acid sequence of the light chain of trastuzumab, respectively, in which the sugar chain is cleaved with PNGase.
  • SEQ ID NOs: 16 to 22 show the amino acid sequences of peptide fragments of the heavy chain region obtained by trypsin digestion of trastuzumab.
  • SEQ ID NOs: 23 to 27 show the amino acid sequences of peptide fragments of the light chain region obtained by trypsin digestion of trastuzumab.
  • SEQ ID NO: 28 shows the amino acid sequence of a model peptide of a functional substance that modifies trastuzumab in Example 3.
  • SEQ ID NOs: 29 to 37 show known LplA recognition amino acid sequences.
  • SEQ ID NOs: 38 to 68 show the nucleotide sequences of primers P1 to P31, respectively.
  • SEQ ID NOS: 69 and 70 show the base sequence of the modified LplA variant m231 and the amino acid sequence encoded thereby, respectively.
  • SEQ ID NOs: 71 and 72 show the nucleotide sequence of the modified LplA variant m746 and the amino acid sequence encoded thereby, respectively.
  • SEQ ID NOS: 73 and 74 show the nucleotide sequence of the modified LplA variant m766 and the amino acid sequence encoded thereby, respectively.
  • SEQ ID NOs: 75 and 76 show the base sequence of the modified LplA variant m876 and the amino acid sequence encoded thereby, respectively.
  • SEQ ID NOs: 77 and 78 show the nucleotide sequences of primers P32 and P33, respectively.

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Abstract

The present invention provides a technique that enables the modification of an antibody. More specifically, the present invention provides the following inventions: (1) a method for producing a modified antibody having a modified moiety, the method including reacting an antibody with a lipoic acid analog having a modified moiety in the presence of lipoic acid protein ligase A to produce a modified antibody having a modified moiety in a side chain of a lysine residue in a constant region; (2) a modified antibody having a modified moiety in a side chain of a lysine residue in a constant region, wherein the modified antibody has a C4-C10 alkyl-carbonyl having a modified moiety in only a side chain of an antibody-specific lysine residue; etc.

Description

修飾抗体およびその製造方法Modified antibody and method for producing the same
　本発明は、修飾抗体およびその製造方法に関する。 << The present invention relates to a modified antibody and a method for producing the same.
　近年、抗体薬物複合体（Ａｎｔｉｂｏｄｙ　Ｄｒｕｇ　Ｃｏｎｊｕｇａｔｉｏｎ：ＡＤＣ）の研究開発が盛んに行われている。ＡＤＣはその名の通り、抗体に薬物（例、抗がん剤）をコンジュゲーションした薬剤であり、がん細胞などに対して直接的な殺細胞活性を有する。代表的なＡＤＣとしては、Ｉｍｍｕｎｏｇｅｎｅ社およびＲｏｃｈｅ社が共同開発したＴ－ＤＭ１（商品名：カドサイラ（登録商標））がある。したがって、薬物による抗体の修飾に役立つ技術の開発が求められている。 In recent years, research and development of antibody-drug conjugates (Antibody Drug Conjugation: ADC) have been actively conducted. As the name implies, ADC is a drug obtained by conjugating a drug (eg, an anticancer agent) to an antibody, and has a direct cell killing activity against cancer cells and the like. A typical ADC is T-DM1 (trade name: Kadcyla (registered trademark)) jointly developed by Immunogene and Roche. Therefore, there is a need to develop a technique that is useful for modifying antibodies with drugs.
　ところで、抗体は、タンパク質の一種である。タンパク質の修飾は、所望のタンパク質の機能の調節に有用である。タンパク質の修飾としては、天然に存在する種々の酵素の機能を利用した修飾が試みられている。例えば、タンパク質を修飾できる酵素として、ホスホパンテテイニルトランスフェラーゼ（ＰＰＴａｓｅ）、ビオチンリガーゼ、およびリポ酸タンパク質リガーゼ（Ｌｐｌ）が知られている。 By the way, antibodies are a type of protein. Modification of a protein is useful for modulating the function of the desired protein. As a protein modification, modification using functions of various naturally occurring enzymes has been attempted. For example, phosphopantetheinyl transferase (PPTase), biotin ligase, and lipoic acid protein ligase (Lpl) are known as enzymes capable of modifying proteins.
　ＰＰＴａｓｅは、タンパク質中のセリン残基の水酸基と補酵素Ａ（ＣｏＡ）間でリン酸エステル結合を形成する酵素である。ＰＰＴａｓｅ認識アミノ酸配列として（Ｈ／Ｄ）Ｓ（Ｌ／Ｉ）が報告されており、ＰＰＴａｓｅは、セリン残基周辺のα－ヘリックス構造を認識していると考察されている（非特許文献１）。ＰＰＴａｓｅは原核生物において広く保存される酵素であることから、標的タンパク質の修飾酵素の探索に適すると考えられている。ＰＰＴａｓｅは、その認識アミノ酸配列が３個のアミノ酸残基からなる短いものであり、また、その認識アミノ酸配列におけるＮ末端およびＣ末端の双方のアミノ酸残基の認識も厳密ではないことから、基質特異性が高くないと考えられる。 PPTase is an enzyme that forms a phosphate bond between a hydroxyl group of a serine residue in a protein and coenzyme A (CoA). (H / D) S (L / I) has been reported as a PPTase recognition amino acid sequence, and PPTase is considered to recognize an α-helix structure around a serine residue (Non-patent Document 1). . Since PPTase is an enzyme widely conserved in prokaryotes, it is considered to be suitable for searching for a target protein modifying enzyme. PPTase has a short recognition amino acid sequence consisting of three amino acid residues, and is not strict in recognition of both N-terminal and C-terminal amino acid residues in the recognition amino acid sequence. It is thought that it is not high.
　ビオチンリガーゼは、ビオチンをタンパク質のリジン残基に共有結合させる酵素である。ビオチンはアビジンと極めて高い親和性（Ｋｄ≧１０－１５Ｍ）を有するため、ビオチンリガーゼは、アビジンを介して様々な薬剤をタンパク質のリジン残基に付加することができる。ビオチンリガーゼの１種であるＥ．ｃｏｌｉ　ＢｉｒＡは、ＧＬＮＤＩＦＥＡＱ［Ｋ］ＩＥＷＨＥで表される１５個のアミノ酸残基からなるアミノ酸配列（［Ｋ］は、ビオチンが付加されるリジン残基を示す。）を認識できることが報告されている。Ｅ．ｃｏｌｉ　ＢｉｒＡ認識アミノ酸配列を有するアクセプターペプチドと目的タンパク質との融合タンパク質を調製し、次いで、この融合タンパク質をＥ．ｃｏｌｉ　ＢｉｒＡで処理することにより、アビジンで修飾された融合タンパク質を調製する技術が報告されている（非特許文献２）。 Biotin ligase is an enzyme that covalently binds biotin to lysine residues in proteins. Biotin has a very high affinity for avidin (Kd ≧ 10 −15 M), so that biotin ligase can add various drugs to lysine residues of proteins via avidin. E. coli, a kind of biotin ligase. It has been reported that E. coli BirA can recognize an amino acid sequence consisting of 15 amino acid residues represented by GLNDIFEAQ [K] IEWHE ([K] indicates a lysine residue to which biotin is added). E. FIG. A fusion protein of an acceptor peptide having an E. coli BirA-recognizing amino acid sequence with a target protein is prepared. A technique for preparing an avidin-modified fusion protein by treating with E. coli BirA has been reported (Non-Patent Document 2).
　Ｌｐｌは、タンパク質中のリジン残基にリポ酸を結合させてアシル化する酵素である。Ｌｐｌとしては、例えば、エシェリヒア・コリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）ＬｐｌＡが存在する。エシェリヒア・コリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）ＬｐｌＡは、ピルビン酸デヒドロゲナーゼ、α－ケトグルタル酸デヒドロゲナーゼ、およびグリシン開裂系酵素等の酵素の補酵素として機能することが知られている（非特許文献３および４）。ＬｐｌＡは、これらの酵素のサブユニットの１つであるＥ２タンパク質（Ｅ２ｐ）のアミノ酸配列を認識し、認識配列中のリジン残基にリポ酸を付加（リポイル化）してＥ２ｐを活性化させる。ＬｐｌＡは、表１に示されるように、その認識アミノ酸配列が１２個のアミノ酸残基からなる比較的長いものであることが報告されている（非特許文献５）。ＬｐｌＡの認識アミノ酸配列には、アミノ酸残基のバリエーションが認められるが、リポイル化されるリジン残基のＮ末端側アミノ酸残基およびＣ末端側アミノ酸残基はそれぞれ、アスパラギン酸残基（Ｄ）および炭化水素基を側鎖として有するアミノ酸残基〔アラニン（Ａ）、バリン（Ｖ）、またはロイシン（Ｌ）〕に制限されている（表１）。 Lpl is an enzyme that binds lipoic acid to a lysine residue in a protein for acylation. As Lpl, for example, Escherichia coli (Escherichia coli) LplA exists. Escherichia coli (Escherichia coli) LplA is known to function as a coenzyme for enzymes such as pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and glycine cleavage system enzymes (Non-patent Documents 3 and 4). LplA recognizes the amino acid sequence of E2 protein (E2p), which is one of the subunits of these enzymes, and activates E2p by adding lipoic acid (lipoylation) to lysine residues in the recognition sequence. As shown in Table 1, LplA is reported to have a relatively long recognition amino acid sequence consisting of 12 amino acid residues (Non-Patent Document 5). Although variations in amino acid residues are recognized in the recognition amino acid sequence of LplA, the N-terminal amino acid residue and the C-terminal amino acid residue of lysylated lysine residues are aspartic acid residue (D) and It is restricted to amino acid residues having a hydrocarbon group as a side chain [alanine (A), valine (V), or leucine (L)] (Table 1).
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　ＬｐｌＡによるタンパク質の修飾としては、ＬｐｌＡ認識ペプチドタグを付加した目的タンパク質において、ＬｐｌＡ認識ペプチドタグを介して、目的タンパク質をリポ酸およびリポ酸アナログで修飾する方法が知られている（特許文献１）。また、ＬｐｌＡによるタンパク質（抗体）の修飾として、ＬｐｌＡ認識ポリペプチドを付加したＦａｂ断片において、ＬｐｌＡ認識ポリペプチドを介して、Ｆａｂ断片をリポ酸で修飾する方法（特許文献２）が報告されている。 As a modification of a protein with LplA, a method of modifying a target protein with lipoic acid and a lipoic acid analog via the LplA recognition peptide tag in a target protein to which an LplA recognition peptide tag has been added is known (Patent Document 1). . Further, as a modification of a protein (antibody) by LplA, a method of modifying a Fab fragment with lipoic acid via an LplA recognition polypeptide in a Fab fragment to which an LplA recognition polypeptide has been added has been reported (Patent Document 2). .
米国特許第８，１３７，９２５号明細書US Patent No. 8,137,925 国際公開第２０１７／０９５８０６号International Publication No. WO 2017/095806
　本発明は、抗体の修飾を可能にする技術を開発することを目的とする。 The purpose of the present invention is to develop a technology that allows modification of an antibody.
　本発明者らは、酵素的方法による抗体の修飾について鋭意検討した結果、試験したタンパク質修飾酵素のうち、認識アミノ酸配列が一番短く基質特異性が低いと予想されたホスホパンテテイニルトランスフェラーゼ（ＰＰＴａｓｅ）や、ビオチンリガーゼは抗体の修飾能を有していなかったものの、リポ酸タンパク質リガーゼ（Ｌｐｌ）は、従来知られていたＬｐｌ認識アミノ酸配列を有していないにもかかわらず、抗体を修飾できることを見出した。
　次いで、本発明者らは、Ｌｐｌによる抗体の修飾部位を解析したところ、予想外なことに、重鎖のＣＨ１領域、軽鎖のＣＬ領域といった抗体中の特定の定常領域を簡便な構造（アルキル－カルボニル構造）で高度に修飾できることなどを見出し、本発明を完成するに至った。本発明者らが把握する限り、これらの領域をこのような簡便な構造で修飾することに成功した事例は知られていない。 The present inventors have conducted intensive studies on antibody modification by an enzymatic method. As a result, among the tested protein-modifying enzymes, phosphopantetheinyltransferase (PPTase) which has the shortest recognized amino acid sequence and is expected to have low substrate specificity ) And biotin ligase did not have the ability to modify an antibody, but lipoic acid protein ligase (Lpl) could modify an antibody despite having no known Lpl-recognizing amino acid sequence. Was found.
Next, the present inventors analyzed the modification site of the antibody with Lpl. Unexpectedly, it was found that specific constant regions in the antibody, such as the heavy chain CH1 region and the light chain CL region, had a simple structure (alkyl -Carbonyl structure), and the present invention was completed. As far as the present inventors understand, there has been no known case in which these regions have been successfully modified with such a simple structure.
　すなわち、本発明は、以下のとおりである。
〔１〕抗体を、リポ酸タンパク質リガーゼの存在下において、修飾部分を有するリポ酸アナログと反応させて、定常領域中のリジン残基の側鎖に修飾部分を有する修飾抗体を生成することを含む、修飾部分を有する修飾抗体の製造方法。
〔２〕抗体が天然ポリペプチド鎖構造を有する、〔１〕の方法。
〔３〕抗体がモノクローナル抗体である、〔１〕または〔２〕の方法。
〔４〕抗体が全長抗体または抗体断片である、〔１〕～〔３〕のいずれかの方法。
〔５〕抗体がヒト抗体またはヒト化抗体である、〔１〕～〔４〕のいずれかの方法。
〔６〕抗体がＩｇＧである、〔１〕～〔５〕のいずれかの方法。
〔７〕抗体がＦａｂまたはＦ（ａｂ’）２である、〔１〕～〔６〕のいずれかの方法。
〔８〕修飾部分を有するリポ酸アナログが、修飾部分を有するＣ４～Ｃ１０アルキル－カルボン酸であり、
　定常領域中のリジン残基の側鎖に修飾部分を有する修飾抗体が、修飾部分を有するＣ４～Ｃ１０アルキル－カルボニルを、定常領域中のリジン残基の側鎖に有する抗体である、〔１〕～〔７〕のいずれかの方法。
〔９〕修飾部分を有するリポ酸アナログが、下記式（Ｉ）：
Ｒ－ＣｎＨ２ｎ－ＣＯＯＨ　　　（Ｉ）
〔式中、
　Ｒは、修飾部分であり、
　ｎは、４～１０の整数である。〕で表されるものであり、かつ
　定常領域中のリジン残基の側鎖に修飾部分を有する修飾抗体が、下記式（ＩＩ）：
Ｒ－ＣｎＨ２ｎ－ＣＯ－ＮＨ－　　　（ＩＩ）
〔式中、
　Ｒおよびｎは、式（Ｉ）のものと同じであり、
　ＮＨ－は、定常領域中のリジン残基の側鎖に存在する基である。〕で表される部分を定常領域中のリジン残基の側鎖に有する修飾抗体である、
　〔１〕～〔８〕のいずれかの方法。
〔１０〕ｎが７である、〔９〕の方法。
〔１１〕定常領域中のリジン残基の側鎖に修飾部分を有する修飾抗体が、修飾部分を抗体特有のリジン残基の側鎖のみに有する、〔１〕～〔１０〕のいずれかの方法。
〔１２〕定常領域中のリジン残基がＣＨ１領域中のリジン残基である、〔１〕～〔１１〕のいずれかの方法。
〔１３〕定常領域中のリジン残基が、重鎖のＣＨ１領域および軽鎖のＣＬ領域の双方に存在するリジン残基である、〔１〕～〔１２〕のいずれかの方法。
〔１４〕重鎖のＣＨ１領域および軽鎖のＣＬ領域の双方に存在するリジン残基が、ヒトＩｇＧ重鎖における１３３位のリジン残基、およびヒトＩｇＧ軽鎖における１６９位のリジン残基である、〔１３〕の方法。
〔１５〕リポ酸タンパク質リガーゼが、微生物に由来する、〔１〕～〔１４〕のいずれかの方法。
〔１６〕リポ酸タンパク質リガーゼが、エシェリヒア（Ｅｓｃｈｅｒｉｃｈｉａ）属細菌、バチルス（Ｂａｃｉｌｌｕｓ）属細菌、コリネバクテリウム（Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ）属細菌、またはスタフィロコッカス（Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ）属細菌に由来する、〔１〕～〔１５〕のいずれかの方法。
〔１７〕リポ酸タンパク質リガーゼが、エシェリヒア・コリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）、バチルス・ズブチリス（Ｂａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓ）、コリネバクテリウム・グルタミカム（Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ　ｇｌｕｔａｍｉｃｕｍ）、またはスタフィロコッカス・エピダーミス（Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ　ｅｐｉｄｅｒｍｉｄｉｓ）に由来する、〔１〕～〔１６〕のいずれかの方法。
〔１８〕リポ酸タンパク質リガーゼが、下記（Ａ）～（Ｃ）からなる群より選ばれるタンパク質である、〔１〕～〔１７〕のいずれかの方法：
（Ａ）配列番号２、４、６、および８からなる群から選ばれるアミノ酸配列を含むタンパク質；
（Ｂ）配列番号２、４、６、および８からなる群から選ばれるアミノ酸配列において、１もしくは数個のアミノ酸の置換、欠失、挿入、もしくは付加を含むアミノ酸配列を含み、かつ、リポ酸タンパク質リガーゼ活性を有するタンパク質；ならびに
（Ｃ）配列番号２、４、６、および８からなる群から選ばれるアミノ酸配列に対して９０％以上の同一性を有するアミノ酸配列を含み、かつ、リポ酸タンパク質リガーゼ活性を有するタンパク質。
〔１９〕リポ酸タンパク質リガーゼが、下記（Ａ’）～（Ｃ’）からなる群より選ばれるタンパク質である、〔１〕～〔１７〕のいずれかの方法：
（Ａ’）配列番号２のアミノ酸配列において、下記（ｉ）～（ｖｉｉ）からなる群より選ばれる１個以上の変異を有するアミノ酸配列を含み、かつ、リポ酸タンパク質リガーゼ活性を有するタンパク質：
（ｉ）１２１位のアルギニン残基
　アラニン残基、またはスレオニン残基による置換；
（ｉｉ）１３６位のセリン残基
　ロイシン残基による置換；
（ｉｉｉ）１４０位のチロシン残基
　アラニン残基、またはバリン残基による置換；
（ｉｖ）１４２位のグルタミン酸
　セリン残基、スレオニン残基、またはバリン残基への置換；
（ｖ）１４９位のヒスチジン残基
　欠失；
（ｖｉ）１７６位のリジン残基
　アラニン残基、セリン残基、またはスレオニン残基による置換；
（ｖｉｉ）１７８位のイソロイシン残基
　アラニン残基、またはバリン残基による置換；
（Ｂ’）（Ａ’）のアミノ酸配列において、１もしくは数個のアミノ酸の置換、欠失、挿入、もしくは付加を含むアミノ酸配列を含み、かつ、リポ酸タンパク質リガーゼ活性を有するタンパク質；ならびに
（Ｃ’）（Ａ’）のアミノ酸配列に対して９０％以上の同一性を有するアミノ酸配列を含み、かつ、リポ酸タンパク質リガーゼ活性を有するタンパク質。
〔２０〕修飾部分が生体直交性官能基を含む、〔１〕～〔１９〕のいずれかの方法。
〔２１〕生体直交性官能基が、アジド残基、アルデヒド残基、チオール残基、アルキン残基、アルケン残基、ハロゲン残基、テトラジン残基、ニトロン残基、ヒドロキシルアミン残基、ニトリル残基、ヒドラジン残基、ケトン残基、ボロン酸残基、シアノベンゾチアゾール残基、アリル残基、ホスフィン残基、マレイミド残基、ジスルフィド残基、チオエステル残基、α―ハロカルボニル残基、イソニトリル残基、シドノン残基、およびセレン残基からなる群より選ばれる、〔２０〕の方法。
〔２２〕以下（１）および（２）を含む、機能性物質を有する修飾抗体の製造方法：
（１）抗体を、リポ酸タンパク質リガーゼＡの存在下において、生体直交性官能基を含む修飾部分を有するリポ酸アナログと反応させて、定常領域中のリジン残基の側鎖に生体直交性官能基を含む修飾部分を有する修飾抗体を生成すること；ならびに
（２）定常領域中のリジン残基の側鎖に生体直交性官能基を含む修飾部分を有する修飾抗体を、生体直交性官能基を介して機能性物質と反応させて、定常領域中のリジン残基の側鎖に機能性物質を有する修飾抗体を生成すること。
〔２３〕生体直交性官能基を含む修飾部分を有するリポ酸アナログが、生体直交性官能基を含む修飾部分を有するＣ４～Ｃ１０アルキル－カルボン酸であり、
　定常領域中のリジン残基の側鎖に生体直交性官能基を含む修飾部分を有する修飾抗体が、生体直交性官能基を含む修飾部分を有するＣ４～Ｃ１０アルキル－カルボニルを、定常領域中のリジン残基の側鎖に有する抗体であり、
　定常領域中のリジン残基の側鎖に機能性物質を有する修飾抗体が、機能性物質およびそれと反応した生体直交性官能基を含む修飾部分を有するＣ４～Ｃ１０アルキル－カルボニルを、定常領域中のリジン残基の側鎖に有する抗体である、〔２０〕の方法。
〔２４〕生体直交性官能基を含む修飾部分を有するリポ酸アナログが、下記式（Ｉ）：
Ｒ－ＣｎＨ２ｎ－ＣＯＯＨ　　　（Ｉ）
〔式中、
　Ｒは、生体直交性官能基を含む修飾部分であり、
　ｎは、４～１０の整数である。〕で表されるものであり、かつ
　定常領域中のリジン残基の側鎖に生体直交性官能基を含む修飾部分を有する修飾抗体が、下記式（ＩＩ）：
Ｒ－ＣｎＨ２ｎ－ＣＯ－ＮＨ－　　　（ＩＩ）
〔式中、
　Ｒおよびｎは、式（Ｉ）のものと同じであり、
　ＮＨ－は、定常領域中のリジン残基の側鎖に存在する基である。〕で表される部分を定常領域中のリジン残基の側鎖に有する修飾抗体であり、
　定常領域中のリジン残基の側鎖に機能性物質を有する修飾抗体が、下記式（ＩＩＩ）：
Ｆ－Ｒ’－ＣｎＨ２ｎ－ＣＯ－ＮＨ－　　　（ＩＩＩ）
〔式中、
　ｎは、式（Ｉ）のものと同じであり、
　ＮＨ－は、式（ＩＩ）のものと同じであり、
　Ｆは、機能性物質であり、
　Ｒ’は、機能性物質と生体直交性官能基との間の反応により生成する部分を含む２価の基である。〕で表される部分を定常領域中のリジン残基の側鎖に有する修飾抗体である、〔２２〕または〔２３〕の方法。
〔２５〕機能性物質が薬物または標識物質である、〔２０〕～〔２４〕のいずれかの方法。
〔２６〕機能性物質が低分子化合物である、〔２０〕～〔２５〕のいずれかの方法。
〔２７〕機能性物質がペプチド化合物である、〔２０〕～〔２６〕のいずれかの方法。
〔２８〕定常領域中のリジン残基の側鎖に修飾部分を有する修飾抗体であって、
　修飾部分を有するＣ４～Ｃ１０アルキル－カルボニルを、抗体特有のリジン残基の側鎖のみに有する、修飾抗体。
〔２９〕定常領域中のリジン残基の側鎖に修飾部分を有する修飾抗体が、下記式（ＩＩ）：
Ｒ－ＣｎＨ２ｎ－ＣＯ－ＮＨ－　　　（ＩＩ）
〔式中、
　Ｒは、修飾部分であり、
　ｎは、４～１０の整数であり、
　ＮＨ－は、定常領域中のリジン残基の側鎖に存在する基である。〕で表される部分を抗体特有のリジン残基の側鎖のみに有する、〔２８〕の修飾抗体。
〔３０〕ｎが７である、〔２８〕または〔２９〕の修飾抗体。
〔３１〕修飾部分が、生体直交性官能基を含む、〔２８〕～〔３０〕のいずれかの修飾抗体。
〔３２〕定常領域中のリジン残基がＣＨ１領域中のリジン残基である、〔２８〕～〔３１〕のいずれかの修飾抗体。
〔３３〕定常領域中のリジン残基が、重鎖のＣＨ１領域および軽鎖のＣＬ領域の双方に存在するリジン残基である、〔２８〕～〔３２〕のいずれかの修飾抗体。
〔３４〕重鎖のＣＨ１領域および軽鎖のＣＬ領域の双方に存在するリジン残基が、ヒトＩｇＧ重鎖における１３３位のリジン残基、およびヒトＩｇＧ軽鎖における１６９位のリジン残基である、〔３３〕の修飾抗体。
〔３５〕定常領域中のリジン残基の側鎖に機能性物質を有する修飾抗体であって、
　機能性物質およびそれと反応した生体直交性官能基を含む修飾部分を有するＣ４～Ｃ１０アルキル－カルボニルを、抗体特有のリジン残基の側鎖のみに有する、修飾抗体。
〔３６〕定常領域中のリジン残基の側鎖に機能性物質を有する修飾抗体が、下記式（ＩＩＩ）：
Ｆ－Ｒ’－ＣｎＨ２ｎ－ＣＯ－ＮＨ－　　　（ＩＩＩ）
〔式中、
　Ｆは、機能性物質であり、
　Ｒ’は、機能性物質と生体直交性官能基との間の反応により生成する部分を含む２価の基であり、
　ｎは、４～１０の整数であり、
　ＮＨ－は、定常領域中のリジン残基の側鎖に存在する基である。〕で表される部分を抗体特有のリジン残基の側鎖のみに有する、〔３５〕の修飾抗体。
〔３７〕ｎが７である、〔３５〕または〔３６〕の修飾抗体。
〔３８〕定常領域中のリジン残基がＣＨ１領域中のリジン残基である、〔３５〕～〔３７〕のいずれかの修飾抗体。
〔３９〕定常領域中のリジン残基が、重鎖のＣＨ１領域および軽鎖のＣＬ領域の双方に存在するリジン残基である、〔３５〕～〔３８〕のいずれかの修飾抗体。
〔４０〕重鎖のＣＨ１領域および軽鎖のＣＬ領域の双方に存在するリジン残基が、ヒトＩｇＧ重鎖における１３３位のリジン残基、およびヒトＩｇＧ軽鎖における１６９位のリジン残基である、〔３９〕の修飾抗体。 That is, the present invention is as follows.
[1] including reacting the antibody with a lipoic acid analog having a modifying moiety in the presence of lipoic acid protein ligase to produce a modified antibody having a modifying moiety on the side chain of a lysine residue in the constant region. , A method for producing a modified antibody having a modified moiety.
[2] The method of [1], wherein the antibody has a natural polypeptide chain structure.
[3] The method of [1] or [2], wherein the antibody is a monoclonal antibody.
[4] the method of any one of [1] to [3], wherein the antibody is a full-length antibody or an antibody fragment;
[5] the method of any one of [1] to [4], wherein the antibody is a human antibody or a humanized antibody;
[6] the method of any one of [1] to [5], wherein the antibody is an IgG;
[7] The method of any one of [1] to [6], wherein the antibody is Fab or F (ab ') 2 .
[8] the lipoic acid analog having a modified moiety is a C 4 -C 10 alkyl-carboxylic acid having a modified moiety;
A modified antibody having a modification portion in the side chain of a lysine residue in the constant region is an antibody having a C 4 to C 10 alkyl-carbonyl having a modification portion in the side chain of a lysine residue in the constant region, [ Any one of 1) to [7].
[9] The lipoic acid analog having a modified moiety is represented by the following formula (I):
R—C n H 2n —COOH (I)
(In the formula,
R is a modifying moiety;
n is an integer of 4 to 10. And a modified antibody having a modified portion on the side chain of a lysine residue in the constant region is represented by the following formula (II):
R—C n H 2n —CO—NH— (II)
(In the formula,
R and n are the same as in formula (I);
NH- is a group present on the side chain of a lysine residue in the constant region. Is a modified antibody having a portion represented by the side chain of a lysine residue in the constant region,
The method according to any one of [1] to [8].
[10] The method of [9], wherein n is 7.
[11] the method of any of [1] to [10], wherein the modified antibody having a modified portion on the side chain of a lysine residue in the constant region has the modified portion only on the side chain of a lysine residue specific to the antibody; .
[12] The method of any of [1] to [11], wherein the lysine residue in the constant region is a lysine residue in the CH1 region.
[13] The method of any of [1] to [12], wherein the lysine residue in the constant region is a lysine residue present in both the CH1 region of the heavy chain and the CL region of the light chain.
[14] Lysine residues present in both the CH1 region of the heavy chain and the CL region of the light chain are a lysine residue at position 133 in a human IgG heavy chain and a lysine residue at position 169 in a human IgG light chain. , [13].
[15] the method of any one of [1] to [14], wherein the lipoic acid protein ligase is derived from a microorganism;
[16] the lipoic acid protein ligase is derived from a bacterium belonging to the genus Escherichia, a bacterium belonging to the genus Bacillus, a bacterium belonging to the genus Corynebacterium, or a bacterium belonging to the genus Staphylococcus; 15].
[17] The lipoic acid protein ligase is obtained from Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum (Corynebacterium glutamicum, or Staphylococcus epsimid sp.). Any one of 1) to [16].
[18] The method of any one of [1] to [17], wherein the lipoic acid protein ligase is a protein selected from the group consisting of the following (A) to (C):
(A) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8;
(B) an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8, including an amino acid sequence containing substitution, deletion, insertion, or addition of one or several amino acids, and lipoic acid A protein having protein ligase activity; and (C) a lipoic acid protein comprising an amino acid sequence having 90% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8. A protein having ligase activity.
[19] The method of any one of [1] to [17], wherein the lipoic acid protein ligase is a protein selected from the group consisting of the following (A ′) to (C ′):
(A ′) a protein having an amino acid sequence having one or more mutations selected from the group consisting of the following (i) to (vii) in the amino acid sequence of SEQ ID NO: 2, and having lipoic acid protein ligase activity:
(I) substitution with an arginine residue at position 121, an alanine residue or a threonine residue;
(Ii) substitution of a serine residue at position 136 with a leucine residue;
(Iii) substitution with a tyrosine residue at position 140, an alanine residue or a valine residue;
(Iv) substitution at position 142 of a glutamic acid serine residue, threonine residue, or valine residue;
(V) deletion of histidine residue at position 149;
(Vi) substitution with a lysine residue at position 176 by an alanine residue, a serine residue or a threonine residue;
(Vii) substitution with an isoleucine residue at position 178, an alanine residue, or a valine residue;
(B ′) a protein comprising an amino acid sequence containing substitution, deletion, insertion or addition of one or several amino acids in the amino acid sequence of (A ′) and having lipoic acid protein ligase activity; ') A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence of (A'), and having lipoic acid protein ligase activity.
[20] The method of any one of [1] to [19], wherein the modifying moiety contains a bioorthogonal functional group.
[21] a bioorthogonal functional group is an azide residue, aldehyde residue, thiol residue, alkyne residue, alkene residue, halogen residue, tetrazine residue, nitrone residue, hydroxylamine residue, nitrile residue , Hydrazine residue, ketone residue, boronic acid residue, cyanobenzothiazole residue, allyl residue, phosphine residue, maleimide residue, disulfide residue, thioester residue, α-halocarbonyl residue, isonitrile residue [20] the method of [20], wherein the method is selected from the group consisting of:
[22] A method for producing a modified antibody having a functional substance, comprising the following (1) and (2):
(1) An antibody is reacted with a lipoic acid analog having a modified portion containing a bioorthogonal functional group in the presence of lipoic acid protein ligase A, so that the side chain of a lysine residue in the constant region has a bioorthogonal function. (2) generating a modified antibody having a modification moiety containing a bioorthogonal functional group on the side chain of a lysine residue in the constant region, To generate a modified antibody having a functional substance on the side chain of a lysine residue in the constant region.
[23] the lipoic acid analog having a modified moiety containing a bioorthogonal functional group is a C 4 -C 10 alkyl-carboxylic acid having a modified moiety containing a bioorthogonal functional group;
A modified antibody having a modified moiety containing a bioorthogonal functional group on the side chain of a lysine residue in the constant region is used to convert a C 4 -C 10 alkyl-carbonyl having a modified moiety containing a bioorthogonal functional group into the constant region. An antibody having a side chain of a lysine residue of
A modified antibody having a functional substance on the side chain of a lysine residue in the constant region is characterized by a C 4 -C 10 alkyl-carbonyl having a functional substance and a modified moiety containing a bioorthogonal functional group reacted therewith, [20] The method of [20], which is an antibody having a side chain of a lysine residue therein.
[24] A lipoic acid analog having a modified moiety containing a bioorthogonal functional group is represented by the following formula (I):
R—C n H 2n —COOH (I)
(In the formula,
R is a modifying moiety containing a bioorthogonal functional group,
n is an integer of 4 to 10. And a modified antibody having a modified portion containing a bioorthogonal functional group on the side chain of a lysine residue in the constant region is represented by the following formula (II):
R—C n H 2n —CO—NH— (II)
(In the formula,
R and n are the same as in formula (I);
NH- is a group present on the side chain of a lysine residue in the constant region. A modified antibody having a portion represented by a side chain of a lysine residue in the constant region,
A modified antibody having a functional substance on the side chain of a lysine residue in the constant region is represented by the following formula (III):
F-R'-C n H 2n -CO-NH- (III)
(In the formula,
n is the same as that of formula (I);
NH- is the same as that of formula (II),
F is a functional substance,
R ′ is a divalent group including a moiety generated by a reaction between a functional substance and a bioorthogonal functional group. ] The method according to [22] or [23], which is a modified antibody having a portion represented by the formula (1) on the side chain of a lysine residue in the constant region.
[25] The method of any one of [20] to [24], wherein the functional substance is a drug or a labeling substance.
[26] The method of any one of [20] to [25], wherein the functional substance is a low molecular compound.
[27] The method of any one of [20] to [26], wherein the functional substance is a peptide compound.
(28) a modified antibody having a modified portion on the side chain of a lysine residue in the constant region,
A modified antibody having a C 4 -C 10 alkyl-carbonyl having a modifying portion only on the side chain of a lysine residue unique to the antibody.
[29] A modified antibody having a modified portion on the side chain of a lysine residue in the constant region is represented by the following formula (II):
R—C n H 2n —CO—NH— (II)
(In the formula,
R is a modifying moiety;
n is an integer of 4 to 10,
NH- is a group present on the side chain of a lysine residue in the constant region. [28] The modified antibody according to [28], which has a portion represented by the formula (1) only in the side chain of a lysine residue specific to the antibody.
[30] the modified antibody of [28] or [29], wherein n is 7;
[31] The modified antibody of any one of [28] to [30], wherein the modified moiety contains a bioorthogonal functional group.
[32] the modified antibody of any of [28] to [31], wherein the lysine residue in the constant region is a lysine residue in the CH1 region;
[33] The modified antibody of any of [28] to [32], wherein the lysine residue in the constant region is a lysine residue present in both the CH1 region of the heavy chain and the CL region of the light chain.
[34] Lysine residues present in both the CH1 region of the heavy chain and the CL region of the light chain are a lysine residue at position 133 in a human IgG heavy chain and a lysine residue at position 169 in a human IgG light chain. And the modified antibody of [33].
(35) a modified antibody having a functional substance on the side chain of a lysine residue in the constant region,
A modified antibody comprising a functional substance and a C 4 -C 10 alkyl-carbonyl having a modifying moiety containing a bioorthogonal functional group reacted therewith only in the side chain of a lysine residue unique to the antibody.
[36] A modified antibody having a functional substance on a side chain of a lysine residue in the constant region is represented by the following formula (III):
F-R'-C n H 2n -CO-NH- (III)
(In the formula,
F is a functional substance,
R ′ is a divalent group including a moiety generated by a reaction between a functional substance and a bioorthogonal functional group,
n is an integer of 4 to 10,
NH- is a group present on the side chain of a lysine residue in the constant region. [35] The modified antibody according to [35], which has a portion represented by [1] only in the side chain of a lysine residue specific to the antibody.
[37] the modified antibody of [35] or [36], wherein n is 7;
[38] the modified antibody of any of [35] to [37], wherein the lysine residue in the constant region is a lysine residue in the CH1 region;
[39] The modified antibody of any of [35] to [38], wherein the lysine residue in the constant region is a lysine residue present in both the CH1 region of the heavy chain and the CL region of the light chain.
[40] Lysine residues present in both the CH1 region of the heavy chain and the CL region of the light chain are a lysine residue at position 133 in a human IgG heavy chain and a lysine residue at position 169 in a human IgG light chain. And the modified antibody of [39].
　本発明の方法は、抗体の抗原結合部位の近傍に存在する定常領域（例、ヒトＩｇＧ重鎖における１３３位のリジン残基等のＣＨ１領域、およびヒトＩｇＧ軽鎖における１６９位のリジン残基等のＣＬ領域）における抗体の高度な修飾に有用である。
　本発明の修飾抗体は、例えば、医薬、および試薬（例、診断薬、研究用試薬）、ならびにこれらの調製のための中間体として有用である。 The method of the present invention relates to a method for preparing a constant region (eg, a CH1 region such as a lysine residue at position 133 in a human IgG heavy chain, a lysine residue at position 169 in a human IgG light chain, etc.) In the CL region).
The modified antibodies of the present invention are useful, for example, as pharmaceuticals and reagents (eg, diagnostics, research reagents), and intermediates for their preparation.
図１－１は、糖鎖をＰＮＧａｓｅで切断したトラスツズマブの重鎖のアミノ酸配列（配列番号１４）を示す図である。ボックス囲みおよび二重下線文字は、トリプシン消化によるペプチドフラグメント（配列番号１６～２２）を示す。影付き文字およびその下添えの数字は、修飾トラスツズマブにおける修飾リジン残基およびＥＵ　ｎｕｍｂｅｒｉｎｇに基づくそのアミノ酸番号を示す。FIG. 1-1 shows the amino acid sequence of the heavy chain of trastuzumab (SEQ ID NO: 14) in which the sugar chain has been cleaved with PNGase. Boxes and double underlined letters indicate peptide fragments (SEQ ID NOs: 16-22) by trypsin digestion. The shaded letters and the numbers subscripted indicate the modified lysine residue in the modified trastuzumab and its amino acid number based on EU numbering. 図１－２は、トラスツズマブの軽鎖のアミノ酸配列（配列番号１５）を示す図である。ボックス囲み、一重下線、および二重下線文字は、トリプシン消化によるペプチドフラグメント（配列番号２３～２７）を示す。影付き文字およびその下添えの数字は、修飾トラスツズマブにおける修飾リジン残基およびＥＵ　ｎｕｍｂｅｒｉｎｇに基づくそのアミノ酸番号を示す。FIG. 1-2 shows the amino acid sequence of the light chain of trastuzumab (SEQ ID NO: 15). Boxes, single underscores, and double underscores indicate peptide fragments (SEQ ID NOs: 23-27) by trypsin digestion. The shaded letters and the numbers subscripted indicate the modified lysine residue in the modified trastuzumab and its amino acid number based on EU numbering. 図２は、実施例２で得られたオクタン酸修飾トラスツズマブのトリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含む、ＬＳＣＡＡＳＧＦＮＩＫＤＴＹＩＨＷＶＲ（配列番号１６）のペプチドフラグメントのＭＳスペクトル（ｍ／ｚ　１１８２．６１４３７、２価）を示す図である。FIG. 2 shows the peptide fragment of LSCAASGFNIKDTYIHWVR (SEQ ID NO: 16) containing a site for modification of lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows an MS spectrum (m / z 1182.61437, bivalence). 図３は、実施例２で得られたオクタン酸修飾トラスツズマブのトリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含む、ＬＳＣＡＡＳＧＦＮＩＫＤＴＹＩＨＷＶＲ（配列番号１６）のペプチドフラグメントのＣＩＤスペクトルを示す図である。FIG. 3 shows a peptide fragment of LSCAASGFNIKDTYIHWVR (SEQ ID NO: 16) containing a site for modification of lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows a CID spectrum. 図４は、実施例２で得られたオクタン酸修飾トラスツズマブのトリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含む、ＦＴＩＳＡＤＴＳＫＮＴＡＹＬＱＭＮＳＬＲ（配列番号１７）のペプチドフラグメントのＭＳスペクトル（ｍ／ｚ　７９６．４１５３６、３価）を示す図である。FIG. 4 shows a peptide fragment of FTISADTSKNTAYLQMNSLR (SEQ ID NO: 17) containing a site for modification to lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. FIG. 4 is a view showing an MS spectrum (m / z．796.5363, trivalent). 図５は、実施例２で得られたオクタン酸修飾トラスツズマブのトリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含む、ＦＴＩＳＡＤＴＳＫＮＴＡＹＬＱＭＮＳＬＲ（配列番号１７）のペプチドフラグメントのＣＩＤスペクトルを示す図である。FIG. 5 shows a peptide fragment of FTISADTSKNTAYLQMNSLR (SEQ ID NO: 17) containing a site for modification to lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows a CID spectrum. 図６は、実施例２で得られたオクタン酸修飾トラスツズマブのトリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含む、ＧＰＳＶＦＰＬＡＰＳＳＫＳＴＳＧＧＴＡＡＬＧＣＬＶＫ（配列番号１８）のペプチドフラグメントのＭＳスペクトル（ｍ／ｚ　６５４．６０９４９、４価）を示す図である。FIG. 6 shows a peptide fragment of GPSVFPLAPSSSKSTSGTAGLGCLVK (SEQ ID NO: 18) containing a site for modification of a lysine residue (octanoic acid-introduced substance (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows MS spectrum (m / z 654.60949, 4 valence). 図７は、実施例２で得られたオクタン酸修飾トラスツズマブのトリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含む、ＧＰＳＶＦＰＬＡＰＳＳＫＳＴＳＧＧＴＡＡＬＧＣＬＶＫ（配列番号１８）のペプチドフラグメントのＣＩＤスペクトルを示す図である。FIG. 7 shows a peptide fragment of GPSVFPLAPSSSKSTSGTAGLGCLVK (SEQ ID NO: 18) containing a site for modification of lysine residues by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2 (octanoic acid-introduced substance (+126.105 Da)). It is a figure which shows a CID spectrum. 図８は、実施例２で得られたオクタン酸修飾トラスツズマブのトリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含む、ＳＣＤＫＴＨＴＣＰＰＣＰＡＰＥＬＬＧＧＰＳＶＦＬＦＰＰＫＰＫ（配列番号１９）のペプチドフラグメントのＭＳスペクトル（ｍ／ｚ　１１５４．２５５７２、３価）を示す図である。FIG. 8 shows a peptide fragment of SCDKTHTCPPCPAPELLGGGPSVFFLPPKPK (SEQ ID NO: 19) containing a site for modification to lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. FIG. 4 is a view showing an MS spectrum (m / z 1154.25572, trivalent). 図９は、実施例２で得られたオクタン酸修飾トラスツズマブのトリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含む、ＳＣＤＫＴＨＴＣＰＰＣＰＡＰＥＬＬＧＧＰＳＶＦＬＦＰＰＫＰＫ（配列番号１９）のペプチドフラグメントのＣＩＤスペクトルを示す図である。FIG. 9 shows a peptide fragment of SCDKTHTCPPCPAPELLGGGPSVFFLPPKPK (SEQ ID NO: 19) containing a site for modification to lysine residues by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2 (octanoic acid-introduced substance (+126.105 Da)). It is a figure which shows a CID spectrum. 図１０は、実施例２で得られたオクタン酸修飾トラスツズマブのトリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含む、ＴＨＴＣＰＰＣＰＡＰＥＬＬＧＧＰＳＶＦＬＦＰＰＫＰＫ（配列番号２０）のペプチドフラグメントのＭＳスペクトル（ｍ／ｚ　１４８５．７８５５２、２価）を示す図である。FIG. 10 shows a peptide fragment of THTCPPCPAPELLGGPSVFLFPPKPK (SEQ ID NO: 20) containing a site for modification of lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows MS spectrum (m / z 1485.78552, bivalence). 図１１は、実施例２で得られたオクタン酸修飾トラスツズマブのトリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含む、ＴＨＴＣＰＰＣＰＡＰＥＬＬＧＧＰＳＶＦＬＦＰＰＫＰＫ（配列番号２０）のペプチドフラグメントのＣＩＤスペクトルを示す図である。FIG. 11 shows a peptide fragment of THTCPPCPAPELLGGPSVFLFPPPKPK (SEQ ID NO: 20) containing a site for modification of lysine residues by lysine residue by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2 (octaneic acid-introduced substance (+126.105 Da)). It is a figure which shows a CID spectrum. 図１２は、実施例２で得られたオクタン酸修飾トラスツズマブのトリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含む、ＶＳＮＫＡＬＰＡＰＩＥＫ（配列番号２１）のペプチドフラグメントのＭＳスペクトル（ｍ／ｚ　６９６．９２８１２、２価）を示す図である。FIG. 12 shows the peptide fragment of VSNKALPAPIEK (SEQ ID NO: 21) containing a site for modification to lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. FIG. 4 is a view showing an MS spectrum (m / z．696.8122, divalent). 図１３は、実施例２で得られたオクタン酸修飾トラスツズマブのトリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含む、ＶＳＮＫＡＬＰＡＰＩＥＫ（配列番号２１）のペプチドフラグメントのＣＩＤスペクトルを示す図である。FIG. 13 shows the peptide fragment of VSNKALPAPIEK (SEQ ID NO: 21) containing a site for modification to lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows a CID spectrum. 図１４は、実施例２で得られたオクタン酸修飾トラスツズマブのトリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含む、ＥＰＱＶＹＴＬＰＰＳＲＥＥＭＴＫＮＱＶＳＬＴＣＬＶＫ（配列番号２２）のペプチドフラグメントのＭＳスペクトル（ｍ／ｚ　７９４．１７１９７、４価）を示す図である。FIG. 14 shows a peptide fragment of EPQVYTLPSPSREEMTKNQVSLTCLVK (SEQ ID NO: 22) containing a site for modification of a lysine residue by lysine digestion of octanoic acid-modified trastuzumab obtained in Example 2 (octanoic acid-introduced substance (+126.105 Da)). It is a figure which shows MS spectrum (m / z 794.17197, tetravalent). 図１５は、実施例２で得られたオクタン酸修飾トラスツズマブのトリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含む、ＥＰＱＶＹＴＬＰＰＳＲＥＥＭＴＫＮＱＶＳＬＴＣＬＶＫ（配列番号２２）のペプチドフラグメントのＣＩＤスペクトルを示す図である。FIG. 15 shows a peptide fragment of EPQVYTLPSPSREEMTKNQVSLTCLVK (SEQ ID NO: 22) containing a site for modification to lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows a CID spectrum. 図１６は、実施例２で得られたオクタン酸修飾トラスツズマブのトリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含む、ＡＳＱＤＶＮＴＡＶＡＷＹＱＱＫＰＧＫＡＰＫ（配列番号２３）のペプチドフラグメントのＭＳスペクトル（ｍ／ｚ　１２０７．１４９９４、２価）を示す図である。FIG. 16 shows the peptide fragment of ASQDVNTAVAWYQQKPGKAPK (SEQ ID NO: 23) containing a site for modification of lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows MS spectrum (m / z 1207.14994, bivalence). 図１７は、実施例２で得られたオクタン酸修飾トラスツズマブのトリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含む、ＡＳＱＤＶＮＴＡＶＡＷＹＱＱＫＰＧＫＡＰＫ（配列番号２３）のペプチドフラグメントのＣＩＤスペクトルを示す図である。FIG. 17 shows a peptide fragment of ASQDVNTAVAWYQQKPGKAPK (SEQ ID NO: 23) containing a site for modification of lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows a CID spectrum. 図１８は、実施例２で得られたオクタン酸修飾トラスツズマブのトリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含む、ＶＤＮＡＬＱＳＧＮＳＱＥＳＶＴＥＱＤＳＫＤＳＴＹＳＬＳＳＴＬＴＬＳＫ（配列番号２４）のペプチドフラグメントのＭＳスペクトル（ｍ／ｚ　１２４９．２７８１３、３価）を示す図である。FIG. 18 shows a peptide fragment of VDNALQSGNSQESVTEQDSKDSTYSLSLSTLTLSK (SEQ ID NO: 24) containing a site for modification of lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows MS spectrum (m / z 1249.27813, trivalence). 図１９は、実施例２で得られたオクタン酸修飾トラスツズマブのトリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含む、ＶＤＮＡＬＱＳＧＮＳＱＥＳＶＴＥＱＤＳＫＤＳＴＹＳＬＳＳＴＬＴＬＳＫ（配列番号２４）のペプチドフラグメントのＣＩＤスペクトルを示す図である。FIG. 19 shows a peptide fragment of VDNALQSGNSQESVTEQDSKDSTYSLSSSTTLSK (SEQ ID NO: 24) containing a site for modification to lysine residues by lysine residue obtained by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2 (SEQ ID NO: 24). It is a figure which shows a CID spectrum. 図２０は、実施例２で得られたオクタン酸修飾トラスツズマブのトリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含む、ＡＤＹＥＫＨＫ（配列番号２５）のペプチドフラグメントのＭＳスペクトル（ｍ／ｚ　５０８．７７５０１、２価）を示す図である。FIG. 20 shows a peptide fragment of ADYEKHK (SEQ ID NO: 25) containing a site for modification to lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. FIG. 3 is a view showing an MS spectrum (m / z 508.77751, divalent). 図２１は、実施例２で得られたオクタン酸修飾トラスツズマブのトリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含む、ＡＤＹＥＫＨＫ（配列番号２５）のペプチドフラグメントのＣＩＤスペクトルを示す図である。FIG. 21 shows the peptide fragment of ADYEKHK (SEQ ID NO: 25) containing a site for modification to lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows a CID spectrum. 図２２は、実施例２で得られたオクタン酸修飾トラスツズマブのトリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含む、ＨＫＶＹＡＣＥＶＴＨＱＧＬＳＳＰＶＴＫ（配列番号２６）のペプチドフラグメントのＭＳスペクトル（ｍ／ｚ　１１３４．０９８４４、２価）を示す図である。FIG. 22 shows a peptide fragment of HKVYACEVTHQGLSSPVTK (SEQ ID NO: 26) containing a site for modification to lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows MS spectrum (m / z 1134.08444, bivalence). 図２３は、実施例２で得られたオクタン酸修飾トラスツズマブのトリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含む、ＨＫＶＹＡＣＥＶＴＨＱＧＬＳＳＰＶＴＫ（配列番号２６）のペプチドフラグメントのＣＩＤスペクトルを示す図である。FIG. 23 shows a peptide fragment of HKVYACEVTHQGLSSPVTK (SEQ ID NO: 26) containing a site for modification to lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows a CID spectrum. 図２４は、実施例２で得られたオクタン酸修飾トラスツズマブのトリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含む、ＨＫＶＹＡＣＥＶＴＨＱＧＬＳＳＰＶＴＫＳＦＮＲ（配列番号２７）のペプチドフラグメントのＭＳスペクトル（ｍ／ｚ　９２４．４８３５３、３価）を示す図である。FIG. 24 shows a peptide fragment of HKVYACEVTHQGLSSPVTKSFNR (SEQ ID NO: 27) containing a site for modification of lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows MS spectrum (m / z 924.48353, trivalence). 図２５は、実施例２で得られたオクタン酸修飾トラスツズマブのトリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含む、ＨＫＶＹＡＣＥＶＴＨＱＧＬＳＳＰＶＴＫＳＦＮＲ（配列番号２７）のペプチドフラグメントのＣＩＤスペクトルを示す図である。FIG. 25 shows a peptide fragment of HKVYACEVTHQGLSSPVTKSFNR (SEQ ID NO: 27) containing a site for modification to lysine residues (octanoic acid-introduced (+126.105 Da)) by trypsin digestion of octanoic acid-modified trastuzumab obtained in Example 2. It is a figure which shows a CID spectrum. 図２６は、ＬｐｌＡの修飾バリアントのＣＬＵＳＴＡＬ　Ｏ（１．２．４）多重配列アライメントを示す図である。アミノ酸の置換は太字下線である。FIG. 26 shows a CLUSTAL O (1.2.4) multiple sequence alignment of modified variants of LplA. Amino acid substitutions are underlined in bold. 図２７は、（Ａ）反応混合液のＳＤＳ－ＰＡＧＥ、および（Ｂ）ゲルのフルオログラムを示す図である。レーン：１－コントロール反応１（Ｃ８－Ｎ３およびＬｐｌＡを除く全成分）、２－コントロール反応２（ＬｐｌＡを除く全成分）、３－野生型（ｗｔ）、４および５－ｍ２３１、６－ｍ７４６、７－ｍ７６６、８－ｍ８７６；ｍＬｐｌＡバンドは、自己標識ｍＬｐｌＡに対応する。FIG. 27 shows (A) SDS-PAGE of the reaction mixture and (B) a fluorogram of the gel. Lanes: 1-control reaction 1 (all components except C8-N3 and LplA), 2-control reaction 2 (all components except LplA), 3-wild type (wt), 4 and 5-m231, 6-m746, 7-m766, 8-m876; mLplA band corresponds to self-labeled mLplA.
（１．修飾部分を有する修飾抗体の製造方法）
　本発明は、修飾部分を有する修飾抗体の製造方法を提供する。本発明の方法は、抗体を、リポ酸タンパク質リガーゼの存在下において、修飾部分を有するリポ酸アナログと反応させて、定常領域中のリジン残基の側鎖に修飾部分を有する修飾抗体を生成することを含む。 (1. Method for producing modified antibody having modified moiety)
The present invention provides a method for producing a modified antibody having a modifying moiety. The method of the invention comprises reacting an antibody with a lipoic acid analog having a modifying moiety in the presence of lipoic acid protein ligase to produce a modified antibody having a modifying moiety on the side chain of a lysine residue in the constant region. Including.
（抗体）
　本発明の方法に用いられる抗体は、ポリクローナル抗体またはモノクローナル抗体である。本発明の方法に用いられる抗体はまた、生体分子（例、糖）で修飾された抗体（例、糖タンパク質）であっても、生体分子で未修飾の抗体であってもよい。抗体としては、生物由来成分、ウイルス由来成分、および環境中に見出される成分等の任意の成分に対する任意の抗体を用いることができるが、生物由来成分またはウイルス由来成分に対する抗体が好ましい。生物由来成分としては、例えば、哺乳動物、鳥類（例、ニワトリ）等の動物、昆虫、微生物、植物、菌類、および魚類由来の成分（例、タンパク質）が挙げられる。好ましくは、生物由来成分は、哺乳動物由来の成分である。哺乳動物としては、例えば、霊長類（例、ヒト、サル、チンパンジー）、齧歯類（例、マウス、ラット、モルモット、ハムスター、ウサギ）、愛玩動物（例、イヌ、ネコ）、家畜（例、ウシ、ブタ、ヤギ）、使役動物（例、ウマ、ヒツジ）が挙げられる。生物由来成分は、より好ましくは、霊長類または齧歯類由来の成分（例、タンパク質）であり、さらにより好ましくは、本発明の臨床応用の観点から、ヒト由来の成分（例、タンパク質）である。ウイルス由来成分としては、例えば、インフルエンザウイルス（例、トリインフルエンザウイルス、ブタインフルエンザウイルス）、エイズウイルス、エボラウイルス、ファージウイルスに由来する成分（例、タンパク質）が挙げられる。 (antibody)
The antibody used in the method of the present invention is a polyclonal antibody or a monoclonal antibody. The antibody used in the method of the present invention may be an antibody (eg, glycoprotein) modified with a biomolecule (eg, sugar) or an antibody not modified with a biomolecule. As the antibody, any antibody against any component such as a biological component, a virus-derived component, and a component found in the environment can be used, but an antibody against a biological-derived component or a virus-derived component is preferable. Examples of biological components include components (eg, proteins) derived from animals such as mammals and birds (eg, chickens), insects, microorganisms, plants, fungi, and fish. Preferably, the biological component is a component derived from a mammal. Examples of mammals include primates (eg, humans, monkeys, chimpanzees), rodents (eg, mice, rats, guinea pigs, hamsters, rabbits), pets (eg, dogs, cats), livestock (eg, Cattle, pigs, goats) and working animals (eg, horses, sheep). The biological component is more preferably a primate or rodent-derived component (eg, a protein), and even more preferably a human-derived component (eg, a protein) from the viewpoint of clinical application of the present invention. is there. Examples of virus-derived components include components (eg, proteins) derived from influenza virus (eg, avian influenza virus, swine influenza virus), AIDS virus, Ebola virus, and phage virus.
　抗体は、任意の抗原に対する抗体である。例えば、このような抗原は、上述したような生物またはウイルスにおいて見出される成分であってもよい。このような抗原としてはまた、例えば、タンパク質〔オリゴペプチド、ポリペプチドを含む。糖等の生体分子で修飾されたタンパク質（例、糖タンパク質）であってもよい〕、糖鎖、核酸、低分子化合物が挙げられる。 Antibody is an antibody against any antigen. For example, such an antigen may be a component found in an organism or virus as described above. Such antigens also include, for example, proteins [including oligopeptides and polypeptides. It may be a protein modified with a biomolecule such as sugar (eg, glycoprotein)], a sugar chain, a nucleic acid, and a low molecular compound.
　好ましくは、抗体は、タンパク質を抗原とする抗体であってもよい。タンパク質としては、例えば、細胞膜受容体、細胞膜受容体以外の細胞膜タンパク質（例、細胞外基質タンパク質）、リガンド、可溶性受容体が挙げられる。 Preferably, the antibody may be an antibody having a protein as an antigen. Examples of proteins include cell membrane receptors, cell membrane proteins other than cell membrane receptors (eg, extracellular matrix proteins), ligands, and soluble receptors.
　より具体的には、抗体の抗原であるタンパク質は、疾患標的タンパク質であってもよい。疾患標的タンパク質としては、例えば、以下が挙げられる。 More specifically, the protein that is the antigen of the antibody may be a disease target protein. Examples of the disease target protein include the following.
（１）がん領域
　ＰＤ－Ｌ１、ＧＤ２、ＰＤＧＦＲα（血小板由来成長因子受容体）、ＣＤ２２、ＨＥＲ２、ホスファチジルセリン（ＰＳ）、ＥｐＣＡＭ、フィブロネクチン、ＰＤ－１、ＶＥＧＦＲ－２、ＣＤ３３、ＨＧＦ、ｇｐＮＭＢ、ＣＤ２７、ＤＥＣ－２０５、葉酸受容体、ＣＤ３７、ＣＤ１９、Ｔｒｏｐ２、ＣＥＡＣＡＭ５、Ｓ１Ｐ、ＨＥＲ３、ＩＧＦ－１Ｒ、ＤＬＬ４、ＴＮＴ－１／Ｂ、ＣＰＡＡｓ、ＰＳＭＡ、ＣＤ２０、ＣＤ１０５（エンドグリン）、ＩＣＡＭ－１、ＣＤ３０、ＣＤ１６Ａ、ＣＤ３８、ＭＵＣ１、ＥＧＦＲ、ＫＩＲ２ＤＬ１，２，、ＮＫＧ２Ａ、ｔｅｎａｓｃｉｎ－Ｃ、ＩＧＦ（Ｉｎｓｕｌｉｎ－ｌｉｋｅ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ）、ＣＴＬＡ－４、ｍｅｓｏｔｈｅｌｉｎ、ＣＤ１３８、ｃ－Ｍｅｔ、Ａｎｇ２、ＶＥＧＦ－Ａ、ＣＤ７９ｂ、ＥＮＰＤ３、葉酸受容体α、ＴＥＭ－１、ＧＭ２、グリピカン３、ｍａｃｒｏｐｈａｇｅ　ｉｎｈｉｂｉｔｏｒｙ　ｆａｃｔｏｒ、ＣＤ７４、Ｎｏｔｃｈ１、Ｎｏｔｃｈ２、Ｎｏｔｃｈ３、ＣＤ３７、ＴＬＲ－２、ＣＤ３、ＣＳＦ－１Ｒ、ＦＧＦＲ２ｂ、ＨＬＡ－ＤＲ、ＧＭ－ＣＳＦ、ＥｐｈＡ３、Ｂ７－Ｈ３、ＣＤ１２３、ｇｐＡ３３、Ｆｒｉｚｚｌｅｄ７受容体、ＤＬＬ４、ＶＥＧＦ、ＲＳＰＯ、ＬＩＶ－１、ＳＬＩＴＲＫ６、Ｎｅｃｔｉｎ－４、ＣＤ７０、ＣＤ４０、ＣＤ１９、ＳＥＭＡ４Ｄ（ＣＤ１００）、ＣＤ２５、ＭＥＴ、Ｔｉｓｓｕｅ　Ｆａｃｔｏｒ、ＩＬ－８、ＥＧＦＲ、ｃＭｅｔ、ＫＩＲ３ＤＬ２、Ｂｓｔ１（ＣＤ１５７）、Ｐ－カドヘリン、ＣＥＡ、ＧＩＴＲ、ＴＡＭ（ｔｕｍｏｒ　ａｓｓｏｃｉａｔｅｄ　ｍａｃｒｏｐｈａｇｅ）、ＣＥＡ、ＤＬＬ４、Ａｎｇ２、ＣＤ７３、ＦＧＦＲ２、ＣＸＣＲ４、ＬＡＧ－３、ＧＩＴＲ、Ｆｕｃｏｓｙｌ　ＧＭ１、ＩＧＦ－１、Ａｎｇｉｏｐｏｉｅｔｉｎ　２、ＣＳＦ－１Ｒ、ＦＧＦＲ３、ＯＸ４０、ＢＣＭＡ、ＥｒｂＢ３、ＣＤ１３７（４－１ＢＢ）、ＰＴＫ７、ＥＦＮＡ４、ＦＡＰ、ＤＲ５、ＣＥＡ、Ｌｙ６Ｅ、ＣＡ６、ＣＥＡＣＡＭ５、ＬＡＭＰ１、ｔｉｓｓｕｅ　ｆａｃｔｏｒ、ＥＰＨＡ２、ＤＲ５、Ｂ７－Ｈ３、ＦＧＦＲ４、ＦＧＦＲ２、α２－ＰＩ、Ａ３３、ＧＤＦ１５、ＣＡＩＸ、ＣＤ１６６、ＲＯＲ１、ＧＩＴＲ、ＢＣＭＡ、ＴＢＡ、ＬＡＧ－３、ＥｐｈＡ２、ＴＩＭ－３、ＣＤ－２００、ＥＧＦＲｖＩＩＩ、ＣＤ１６Ａ、ＣＤ３２Ｂ、ＰＩＧＦ、Ａｘｌ、ＭＩＣＡ／Ｂ、Ｔｈｏｍｓｅｎ－Ｆｒｉｅｄｅｎｒｅｉｃｈ、ＣＤ３９、ＣＤ３７、ＣＤ７３、ＣＬＥＣ１２Ａ、Ｌｇｒ３、トランスフェリン受容体、ＴＧＦβ、ＩＬ－１７、５Ｔ４、ＲＴＫ、Ｉｍｍｕｎｅ　Ｓｕｐｐｒｅｓｓｏｒ　Ｐｒｏｔｅｉｎ、ＮａＰｉ２ｂ、ルイス血液型Ｂ抗原、Ａ３４、Ｌｙｓｉｌ－Ｏｘｉｄａｓｅ、ＤＬＫ－１、ＴＲＯＰ－２、α９インテグリン、ＴＡＧ－７２（ＣＡ７２－４）、ＣＤ７０ (1) Cancer area PD-L1, GD2, PDGFRα (platelet-derived growth factor receptor), CD22, HER2, phosphatidylserine (PS), EpCAM, fibronectin, PD-1, VEGFR-2, CD33, HGF, gpNMB, CD27, DEC-205, folate receptor, CD37, CD19, Trop2, CEACAM5, S1P, HER3, IGF-1R, DLL4, TNT-1 / B, CPAAs, PSMA, CD20, CD105 (endoglin), ICAM-1, CD30, CD16A, CD38, MUC1, EGFR, KIR2DL1, 2, NKG2A, tenascin-C, IGF (insulin-like growth factor), CTLA-4, mesothelin, CD138, c-Met, Ang , VEGF-A, CD79b, ENPD3, folate receptor α, TEM-1, GM2, glypican 3, macrophage inhibitory factor, CD74, Notch1, Notch2, Notch3, CD37, TLR-2, CD3, CSF-1R, FGFR2H, L -DR, GM-CSF, EphA3, B7-H3, CD123, gpA33, Frizzled7 receptor, DLL4, VEGF, RSPO, LIV-1, SLITRK6, Nectin-4, CD70, CD40, CD19, SEMA4D (CD100), CD25, MET, Tissue Factor, IL-8, EGFR, cMet, KIR3DL2, Bst1 (CD157), P-cadherin, CEA, GITR, TAM (tumor assoc) iatated macrophage), CEA, DLL4, Ang2, CD73, FGFR2, CXCR4, LAG-3, GITR, Fucosyl GM1, IGF-1, Angiopoietin 2, CSF-1R, FGFR3, OX40, BCMA, ErbB3, ErbB1 , PTK7, EFNA4, FAP, DR5, CEA, Ly6E, CA6, CEACAM5, LAMP1, tissue factor, EPHA2, DR5, B7-H3, FGFR4, FGFR2, α2-PI, A33, GDF15, CAIX, CD166, ROR1, GOR1 BCMA, TBA, LAG-3, EphA2, TIM-3, CD-200, EGFRvIII, CD16A, CD32B, PIGF, Axl, MICA / B, Thoms n-Friedenreich, CD39, CD37, CD73, CLEC12A, Lgr3, transferrin receptor, TGFβ, IL-17, 5T4, RTK, Immun Suppressor Protein, NaPi2b, Lewis blood group B antigen, A34, Lysil-Oxidase, DLK-1 TROP-2, α9 integrin, TAG-72 (CA72-4), CD70
（２）自己免疫疾患・炎症性疾患
　ＩＬ－１７、ＩＬ－６Ｒ、ＩＬ－１７Ｒ、ＩＮＦ－α、ＩＬ－５Ｒ、ＩＬ－１３、ＩＬ－２３、ＩＬ－６、ＡｃｔＲＩＩＢ、β７－Ｉｎｔｅｇｒｉｎ、ＩＬ－４αＲ、ＨＡＳ、Ｅｏｔａｘｉｎ－１、ＣＤ３、ＣＤ１９、ＴＮＦ－α、ＩＬ－１５、ＣＤ３ε、Ｆｉｂｒｏｎｅｃｔｉｎ、ＩＬ－１β、ＩＬ－１α、ＩＬ－１７、ＴＳＬＰ（Ｔｈｙｍｉｃ　Ｓｔｒｏｍａｌ　Ｌｙｍｐｈｏｐｏｉｅｔｉｎ）、ＬＡＭＰ（Ａｌｐｈａ４　Ｂｅｔａ　７　Ｉｎｔｅｇｒｉｎ）、ＩＬ－２３、ＧＭ－ＣＳＦＲ、ＴＳＬＰ、ＣＤ２８、ＣＤ４０、ＴＬＲ－３、ＢＡＦＦ－Ｒ、ＭＡｄＣＡＭ、ＩＬ－３１Ｒ、ＩＬ－３３、ＣＤ７４、ＣＤ３２Ｂ、ＣＤ７９Ｂ、ＩｇＥ（免疫グロブリンＥ）、ＩＬ－１７Ａ、ＩＬ－１７Ｆ、Ｃ５、ＦｃＲｎ、ＣＤ２８、ＴＬＲ４、ＭＣＡＭ、Ｂ７ＲＰ１、ＣＸＣＲ１，２　Ｌｉｇａｎｄｓ、ＩＬ－２１、Ｃａｄｈｅｒｉｎ－１１、ＣＸ３ＣＬ１、ＣＣＬ２０、ＩＬ－３６Ｒ、ＩＬ－１０Ｒ、ＣＤ８６、ＴＮＦ－α、ＩＬ－７Ｒ、Ｋｖ１．３、α９インテグリン、ＬＩＦＨＴ (2) Autoimmune disease / inflammatory disease IL-17, IL-6R, IL-17R, INF-α, IL-5R, IL-13, IL-23, IL-6, ActRIIB, β7-Integran, IL- 4αR, HAS, Eotaxin-1, CD3, CD19, TNF-α, IL-15, CD3ε, Fibronectin, IL-1β, IL-1α, IL-17, TSLP (Thymbolic Lymphopoietin), LAMP (Alpha4 Betain) , IL-23, GM-CSFR, TSLP, CD28, CD40, TLR-3, BAFF-R, MAdCAM, IL-31R, IL-33, CD74, CD32B, CD79B, IgE (immunoglobulin E), IL-17A, IL-17F, C5, FcRn CD28, TLR4, MCAM, B7RP1, CXCR1,2 Ligands, IL-21, Cadherin-11, CX3CL1, CCL20, IL-36R, IL-10R, CD86, TNF-α, IL-7R, Kv1.3, α9 integrin, LIFHT
（３）脳神経疾患
　ＣＧＲＰ、ＣＤ２０、βアミロイド、βアミロイドプロトフィブリン、Ｃａｌｃｉｔｏｎｉｎ　Ｇｅｎｅ－Ｒｅｌａｔｅｄ　Ｐｅｐｔｉｄｅ　Ｒｅｃｅｐｔｏｒ、ＬＩＮＧＯ（Ｉｇ　Ｄｏｍａｉｎ　Ｃｏｎｔａｉｎｉｎｇ１）、αシヌクレイン、細胞外ｔａｕ、ＣＤ５２、インスリン受容体、ｔａｕタンパク、ＴＤＰ－４３、ＳＯＤ１、ＴａｕＣ３、ＪＣウイルス (3) Cranial nerve disease CGRP, CD20, β-amyloid, β-amyloid protofibrin, Calcitonin Gene-Related Peptide Receptor, LINGO (Ig Domain Containing 1), α-synuclein, extracellular tau, CD52, Tp, t , SOD1, TauC3, JC virus
（４）感染症
　Ｃｌｏｓｔｒｉｄｉｕｍ　Ｄｉｆｆｉｃｉｌｅ　ｔｏｘｉｎ　Ｂ、サイトメガロウイルス、ＲＳウイルス、ＬＰＳ、Ｓ．Ａｕｒｅｕｓ　Ａｌｐｈａ－ｔｏｘｉｎ、Ｍ２ｅタンパク、Ｐｓｌ、ＰｃｒＶ、Ｓ．Ａｕｒｅｕｓ　ｔｏｘｉｎ、インフルエンザＡ、Ａｌｇｉｎａｔｅ、黄色ブドウ球菌、ＰＤ－Ｌ１、インフルエンザＢ、アシネトバクター、Ｆ－ｐｒｏｔｅｉｎ、Ｅｎｖ、ＣＤ３、病原性大腸菌、クレブシエラ、肺炎球菌 (4) Infectious disease Clostridium difficile toxin B, cytomegalovirus, RS virus, LPS, S. Aureus Alpha-toxin, M2e protein, Psl, PcrV, S.A. Aureus toxin, influenza A, Alginate, Staphylococcus aureus, PD-L1, influenza B, acinetobacter, F-protein, Env, CD3, pathogenic Escherichia coli, Klebsiella, pneumococcus
（５）遺伝性・希少疾患
　アミロイドＡＬ、ＳＥＭＡ４Ｄ（ＣＤ１００）、インスリン受容体、ＡＮＧＰＴＬ３、ＩＬ４、ＩＬ１３、ＦＧＦ２３、副腎皮質刺激ホルモン、トランスサイレチン、ハンチンチン (5) Hereditary and rare diseases amyloid AL, SEMA4D (CD100), insulin receptor, ANGPTL3, IL4, IL13, FGF23, adrenocorticotropic hormone, transthyretin, huntingtin
（６）眼疾患
　Ｆａｃｔｏｒ　Ｄ、ＩＧＦ－１Ｒ、ＰＧＤＦＲ、Ａｎｇ２、ＶＥＧＦ－Ａ、ＣＤ－１０５（Ｅｎｄｏｇｌｉｎ）、ＩＧＦ－１Ｒ、βアミロイド (6) Ocular diseases Factor D, IGF-1R, PGDFR, Ang2, VEGF-A, CD-105 (Endoglin), IGF-1R, β-amyloid
（７）骨・整形外科領域
　Ｓｃｌｅｒｏｓｔｉｎ、Ｍｙｏｓｔａｔｉｎ、Ｄｉｃｋｋｏｐｆ－１、ＧＤＦ８、ＲＮＡＫＬ、ＨＡＳ、Ｓｉｇｌｅｃ－１５ (7) Bone and Orthopedic Sclerostin, Myostatin, Dickkopf-1, GDF8, RNAKL, HAS, Siglec-15
（８）血液疾患
　ｖＷＦ、Ｆａｃｔｏｒ　ＩＸａ、Ｆａｃｔｏｒ　Ｘ、ＩＦＮγ、Ｃ５、ＢＭＰ－６、Ｆｅｒｒｏｐｏｒｔｉｎ、ＴＦＰＩ (8) Blood diseases vWF, Factor IXa, Factor X, IFNγ, C5, BMP-6, Ferroportin, TFPI
（９）その他の疾患
　ＢＡＦＦ（Ｂ　ｃｅｌｌ　ａｃｔｉｖａｔｉｎｇ　ｆａｃｔｏｒ）、ＩＬ－１β、ＰＣＳＫ９、ＮＧＦ、ＣＤ４５、ＴＬＲ－２、ＧＬＰ－１、ＴＮＦＲ１、Ｃ５、ＣＤ４０、ＬＰＡ、プロラクチン受容体、ＶＥＧＦＲ－１、ＣＢ１、Ｅｎｄｏｇｌｉｎ、ＰＴＨ１Ｒ、ＣＸＣＬ１、ＣＸＣＬ８、ＩＬ－１β、ＡＴ２－Ｒ、ＩＡＰＰ (9) Other diseases BAFF (B cell activating factor), IL-1β, PCSK9, NGF, CD45, TLR-2, GLP-1, TNFR1, C5, CD40, LPA, prolactin receptor, VEGFR-1, CB1, Endoglin, PTH1R, CXCL1, CXCL8, IL-1β, AT2-R, IAPP
　好ましい実施形態では、抗体は、天然ポリペプチド鎖構造を有する。抗体の「天然ポリペプチド鎖構造」とは、天然に生じる抗体の重鎖における可変領域（ＶＨ）、および定常領域（ＣＨ１、ＣＨ２、ＣＨ３）からなる第１ポリペプチド鎖、ならびに天然に生じる抗体の軽鎖における可変領域（ＶＬ）、および定常領域（ＣＬ）からなる第２ポリペプチド鎖からなる構造をいう。したがって、抗体の所定の領域に所定のアミノ酸配列（例、タグ配列、スペーサー配列）が付加または挿入された構造を有する融合タンパク質である抗体（すなわち、ＶＨ、ＣＨ１、ＣＨ２、ＣＨ３、ＶＬ、およびＣＬ以外の部分を有する抗体）は、天然ポリペプチド鎖構造を有する抗体から除かれる。 In a preferred embodiment, the antibody has a native polypeptide chain structure. The “natural polypeptide chain structure” of an antibody refers to the first polypeptide chain consisting of the variable region (VH) and the constant regions (CH1, CH2, CH3) in the naturally occurring heavy chain of the antibody, and the naturally occurring antibody. It refers to the structure consisting of the second polypeptide chain consisting of the variable region (VL) and the constant region (CL) in the light chain. Therefore, antibodies (ie, VH, CH1, CH2, CH3, VL, and CL) which are fusion proteins having a structure in which a predetermined amino acid sequence (eg, a tag sequence, a spacer sequence) has been added or inserted into a predetermined region of the antibody. Antibodies having other portions) are excluded from antibodies having a natural polypeptide chain structure.
　別の好ましい実施形態では、本発明の方法に用いられる抗体は、モノクローナル抗体である。モノクローナル抗体としては、例えば、キメラ抗体、ヒト化抗体、ヒト抗体、所定の糖鎖が付加された抗体（例、Ｎ型糖鎖結合コンセンサス配列等の糖鎖結合コンセンサス配列を有するように改変された抗体）、二重特異性抗体、ｓｃＦｖ抗体、Ｆａｂ抗体、Ｆ（ａｂ‘）２抗体、ＶＨＨ抗体、Ｆｃ領域タンパク質、Ｆｃ融合タンパク質が挙げられる。モノクローナル抗体等の抗体のアイソタイプとしては、例えば、ＩｇＧ、ＩｇＭ、ＩｇＡ、ＩｇＤ、ＩｇＥ、およびＩｇＹが挙げられる。抗体はまた、２価の抗体（例、ＩｇＧ、ＩｇＤ、ＩｇＥ）、または４価以上の抗体（例、ＩｇＡ抗体、ＩｇＭ抗体）であってもよい。 In another preferred embodiment, the antibodies used in the method of the invention are monoclonal antibodies. As the monoclonal antibody, for example, a chimeric antibody, a humanized antibody, a human antibody, an antibody to which a predetermined sugar chain is added (for example, modified to have a sugar chain binding consensus sequence such as an N-type sugar chain binding consensus sequence) Antibodies), bispecific antibodies, scFv antibodies, Fab antibodies, F (ab ') 2 antibodies, VHH antibodies, Fc region proteins, and Fc fusion proteins. Isotypes of antibodies such as monoclonal antibodies include, for example, IgG, IgM, IgA, IgD, IgE, and IgY. The antibody may also be a bivalent antibody (eg, IgG, IgD, IgE) or a tetravalent or higher-valent antibody (eg, IgA antibody, IgM antibody).
　さらに別の好ましい実施形態では、本発明の方法に用いられる抗体は、全長抗体、または抗体断片（例、Ｆａｂ、Ｆ（ａｂ’）２、Ｆａｂ’、Ｆｖ、単鎖抗体）であってもよい。抗体断片は、重鎖のＣＨ１領域または軽鎖のＣＬ領域のいずれかを有することが好ましく、重鎖のＣＨ１領域および軽鎖のＣＬ領域の双方を有することがより好ましい。 In yet another preferred embodiment, the antibody used in the method of the present invention may be a full-length antibody, or an antibody fragment (eg, Fab, F (ab ') 2 , Fab', Fv, single chain antibody). . The antibody fragment preferably has either the CH1 region of the heavy chain or the CL region of the light chain, and more preferably has both the CH1 region of the heavy chain and the CL region of the light chain.
　さらに別の好ましい実施形態では、本発明の方法に用いられる抗体は、ヒト抗体またはヒト化抗体である。本発明の方法では、ヒト化抗体（トラスツズマブ）の特定の定常領域において高度な修飾が確認されている。したがって、本発明の方法によれば、ヒト化抗体のみならず、ヒト化抗体と同じ定常領域を有するヒト抗体においても特定の定常領域を高度に修飾することができる。ヒト抗体としては、例えば、アダリムマブ、パニツムマブ、ゴリムマブ、ウステキヌマブ、カナキヌマブ、オファツムマブ、デノスマブ（ＩｇＧ２）、イピリムマブ、ベリムマブ、ラキシバクマブ、ラムシルマブ、ニボルマブ（ＩｇＧ４）、セクキヌマブ、エボロクマブ（ＩｇＧ２）、アリロクマブ、ネシツムマブ、ブロダルマブ（ＩｇＧ２）、オララツマブが挙げられる（ＩｇＧサブタイプに言及していない場合、ＩｇＧ１であることを示す）。ヒト化抗体としては、例えば、ダクリヅマブ、パリビズマブ、トラスツズマブ、アレンツズマブ、オマリヅマブ、エファリヅマブ、ベバシヅマブ、ナタリヅマブ（ＩｇＧ４）、トシリヅマブ、エクリヅマブ（ＩｇＧ２）、モガムリヅマブ、ペルツヅマブ、オビヌツヅマブ、ベドリヅマブ、ペンプロリヅマブ（ＩｇＧ４）、メポリヅマブ、エロツヅマブ、ダラツムマブ、イケセキヅマブ（ＩｇＧ４）、レスリヅマブ（ＩｇＧ４）、アテゾリヅマブが挙げられる（ＩｇＧサブタイプに言及していない場合、ＩｇＧ１であることを示す）。 で は In yet another preferred embodiment, the antibody used in the method of the present invention is a human antibody or a humanized antibody. In the method of the present invention, a high degree of modification has been confirmed in a specific constant region of a humanized antibody (trastuzumab). Therefore, according to the method of the present invention, not only a humanized antibody but also a human antibody having the same constant region as a humanized antibody can highly modify a specific constant region. As human antibodies, for example, adalimumab, panitumumab, golimumab, ustekinumab, canakinumab, ofatumumab, denosumab (IgG2), ipilimumab, belimumab, raxivacumab, ramcilumab, nivolumabumalb (IgG4), Secukinumabumabu, g IgG2) and oraratumab (if no IgG subtype is mentioned, it indicates that it is an IgG1). Examples of the humanized antibodies include daclizumab, palivizumab, trastuzumab, alentuzumab, omalizumab, efalizumab, bevacizumab, natalizumab (IgG4), tocilizumab, eclidumab (IgG2), mogamulizumab, pertuzumab, pertuzumab, pertuzumab, pertuzumab, pertuzumab, pertuzumab, pertuzumab, penuzunumab , Dartatumumab, ikesekidumab (IgG4), reslizumab (IgG4), and atezolizumab (if no IgG subtype is mentioned, it indicates that it is IgG1).
　さらに別の好ましい実施形態では、本発明の方法に用いられる抗体は、ＩｇＧである。ＩｇＧとしては、例えば、ＩｇＧ１、ＩｇＧ２、ＩｇＧ３、ＩｇＧ４が挙げられる。ＩｇＧとしては、ヒトＩｇＧが好ましい。 で は In yet another preferred embodiment, the antibody used in the method of the present invention is an IgG. Examples of IgG include IgG1, IgG2, IgG3, and IgG4. As IgG, human IgG is preferable.
　さらに別の好ましい実施形態では、本発明の方法に用いられる抗体は、Ｆａｂ、またはＦ（ａｂ’）２である。本発明の方法では、Ｆａｂの特定の定常領域（ＣＨ１、ＣＬ）において高度な修飾が確認されている。したがって、本発明の方法によれば、Ｆａｂのみならず、Ｆａｂと同じ定常領域を有するＦ（ａｂ’）２においても特定の定常領域を高度に修飾することができる。 In yet another preferred embodiment, the antibody used in the method of the present invention is Fab, or F (ab ') 2 . In the method of the present invention, a high degree of modification has been confirmed in specific constant regions (CH1, CL) of Fab. Therefore, according to the method of the present invention, not only Fabs but also F (ab ') 2 having the same constant regions as Fabs can be highly modified in specific constant regions.
（リポ酸タンパク質リガーゼ）
　本発明の方法で用いられるリポ酸タンパク質リガーゼ（Ｌｐｌ）は、タンパク質中のリジン残基にリポ酸を結合させてアシル化する反応を触媒する酵素である。このようなＬｐｌは、微生物等の種々の生物において広範に存在し、多様な構造を有するリポ酸アナログを基質として利用できる酵素であることが知られている（Ｆｅｒｎａｎｄｅｚ－Ｓｕａｒｅｚ　Ｍ　ｅｔ　ａｌ．　Ｎａｔ　Ｂｉｏｔｅｃｈｎｏｌ　２００７　２５：１４８３－１４８７；米国特許第８，１３７，９２５号；米国特許第９，２８４，５４１号；国際公開第２０１７／０９５８０６号）。本発明の方法で用いられるＬｐｌは、入手の容易性等の観点から、微生物に由来するＬｐｌが好ましい。本発明の方法で用いられるＬｐｌとしては、例えば、エシェリヒア（Ｅｓｃｈｅｒｉｃｈｉａ）属細菌〔例、エシェリヒア・コリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）〕、バチルス（Ｂａｃｉｌｌｕｓ）属細菌〔例、バチルス・ズブチリス（Ｂａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓ）〕、コリネバクテリウム（Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ）属細菌〔例、コリネバクテリウム・グルタミカム（Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ　ｇｌｕｔａｍｉｃｕｍ）〕、またはスタフィロコッカス（Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ）属細菌〔例、スタフィロコッカス・エピダーミス（Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ　ｅｐｉｄｅｒｍｉｄｉｓ）〕に由来するＬｐｌがより好ましい。 (Lipoic acid protein ligase)
Lipoic acid protein ligase (Lpl) used in the method of the present invention is an enzyme that catalyzes a reaction in which lipoic acid is bound to a lysine residue in a protein and acylated. Such Lpl is widely found in various organisms such as microorganisms, and is known to be an enzyme capable of utilizing lipoic acid analogs having various structures as substrates (Fernandez-Suarez M et al. Nat Biotechnol). 2007 25: 1483-1487; U.S. Patent No. 8,137,925; U.S. Patent No. 9,284,541; WO 2017/095806). The Lpl used in the method of the present invention is preferably Lpl derived from a microorganism from the viewpoint of easy availability. Examples of Lpl used in the method of the present invention include bacteria belonging to the genus Escherichia (eg, Escherichia coli), bacteria belonging to the genus Bacillus (eg, Bacillus subtilis), and coli. Bacteria belonging to the genus Corynebacterium (eg, Corynebacterium glutamicum) or bacteria belonging to the genus Staphylococcus (eg, Staphylococcus epidermis from Staphylococcus epidermis) are preferred. .
　本発明の方法で用いられるＬｐｌとしては、例えば、下記（Ａ）～（Ｃ）からなる群より選ばれるタンパク質が挙げられる：
（Ａ）配列番号２、４、６、および８からなる群から選ばれるアミノ酸配列を含むタンパク質；
（Ｂ）配列番号２、４、６、および８からなる群から選ばれるアミノ酸配列において、１もしくは数個のアミノ酸の置換、欠失、挿入、もしくは付加を含むアミノ酸配列を含み、かつ、リポ酸タンパク質リガーゼ活性を有するタンパク質；ならびに
（Ｃ）配列番号２、４、６、および８からなる群から選ばれるアミノ酸配列に対して９０％以上の同一性を有するアミノ酸配列を含み、かつ、リポ酸タンパク質リガーゼ活性を有するタンパク質。 Lpl used in the method of the present invention includes, for example, a protein selected from the group consisting of the following (A) to (C):
(A) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8;
(B) an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8, including an amino acid sequence containing substitution, deletion, insertion, or addition of one or several amino acids, and lipoic acid A protein having protein ligase activity; and (C) a lipoic acid protein comprising an amino acid sequence having 90% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8. A protein having ligase activity.
　本発明の方法で用いられるＬｐｌでは、アミノ酸残基の欠失、置換、付加および挿入からなる群より選ばれる１、２、３または４種の変異により、１個または数個のアミノ酸残基が改変され得る。アミノ酸残基の変異は、アミノ酸配列中の１つの領域に導入されてもよいが、複数の異なる領域に導入されてもよい。用語「１個または数個」は、タンパク質の活性を大きく損なわない個数を示す。用語「１個または数個」が示す数は、例えば、１～１００個、好ましくは１～８０個、より好ましくは１～５０個、１～３０個、１～２０個、１～１０個または１～５個（例、１個、２個、３個、４個、または５個）である。 In the Lpl used in the method of the present invention, one or several amino acid residues are changed by one, two, three or four kinds of mutations selected from the group consisting of deletion, substitution, addition and insertion of amino acid residues. Can be modified. The amino acid residue mutation may be introduced into one region in the amino acid sequence, or may be introduced into a plurality of different regions. The term "one or several" refers to numbers that do not significantly impair the activity of the protein. The number indicated by the term “one or several” is, for example, 1 to 100, preferably 1 to 80, more preferably 1 to 50, 1 to 30, 1 to 20, 1 to 10 or 1 to 5 (eg, 1, 2, 3, 4, or 5).
　本発明の方法で用いられるＬｐｌでは、所定の配列番号のアミノ酸配列との同一性％は、９０％以上、９１％以上、９２％以上、９３％以上、９４％以上、９５％以上、９６％以上、９７％以上、９８％以上または９９％以上であってもよい。本発明では、ペプチドまたはポリペプチド（タンパク質）の同一性％の算出は、アルゴリズムｂｌａｓｔｐにより行うことができる。より具体的には、ポリペプチドの同一性の算定％は、Ｎａｔｉｏｎａｌ　Ｃｅｎｔｅｒ　ｆｏｒ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ　Ｉｎｆｏｒｍａｔｉｏｎ（ＮＣＢＩ）において提供されているアルゴリズムｂｌａｓｔｐにおいて、デフォルト設定のＳｃｏｒｉｎｇ　Ｐａｒａｍｅｔｅｒｓ（Ｍａｔｒｉｘ：ＢＬＯＳＵＭ６２；Ｇａｐ　Ｃｏｓｔｓ：Ｅｘｉｓｔｅｎｃｅ＝１１　Ｅｘｔｅｎｓｉｏｎ＝１；Ｃｏｍｐｏｓｉｔｉｏｎａｌ　Ａｄｊｕｓｔｍｅｎｔｓ：Ｃｏｎｄｉｔｉｏｎａｌ　ｃｏｍｐｏｓｉｔｉｏｎａｌ　ｓｃｏｒｅ　ｍａｔｒｉｘ　ａｄｊｕｓｔｍｅｎｔ）を用いて行うことができる。また、ポリヌクレオチド（遺伝子）の同一性％の算出は、アルゴリズムｂｌａｓｔｎにより行うことができる。より具体的には、ポリヌクレオチドの同一性％の算定は、ＮＣＢＩにおいて提供されているアルゴリズムｂｌａｓｔｎにおいて、デフォルト設定のＳｃｏｒｉｎｇ　Ｐａｒａｍｅｔｅｒｓ（Ｍａｔｃｈ／Ｍｉｓｍａｔｃｈ　Ｓｃｏｒｅｓ＝１，－２；Ｇａｐ　Ｃｏｓｔｓ＝Ｌｉｎｅａｒ）を用いて行うことができる。 In the Lpl used in the method of the present invention, the percent identity with the amino acid sequence of the given SEQ ID NO: 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% The above may be 97% or more, 98% or more, or 99% or more. In the present invention, calculation of the percent identity of a peptide or polypeptide (protein) can be performed by the algorithm blastp. More specifically, the calculation% of the polypeptide identity is determined by the algorithm blastp provided in National Center for Biotechnology Information (NCBI) by default Scoring Parameters (Matrix: BLOSUM62; Gap Costs: Existence = 11). = 1; Compositional Adjustments: Conditional compositional score matrix adjustment. The calculation of the percent identity of the polynucleotide (gene) can be performed by the algorithm blastn. More specifically, the calculation of the percent identity of the polynucleotide is performed by using the default setting Scoring \ Parameters (Match / Mismatch \ Scores = 1, -2; Gap \ Costs = Linear) in the algorithm blastn provided in NCBI. It can be carried out.
　特定の実施形態では、本発明の方法で用いられるＬｐｌは、下記（Ａ’）～（Ｃ’）からなる群より選ばれるタンパク質であってもよい：
（Ａ’）配列番号２のアミノ酸配列において、下記（ｉ）～（ｖｉｉ）からなる群より選ばれる１個以上（例えば１～６個、好ましくは１～５個）の変異を有するアミノ酸配列を含み、かつ、リポ酸タンパク質リガーゼ活性を有するタンパク質：
（ｉ）１２１位のアルギニン残基
　アラニン残基、またはスレオニン残基による置換；
（ｉｉ）１３６位のセリン残基
　ロイシン残基による置換；
（ｉｉｉ）１４０位のチロシン残基
　アラニン残基、またはバリン残基による置換；
（ｉｖ）１４２位のグルタミン酸
　セリン残基、スレオニン残基、またはバリン残基への置換；
（ｖ）１４９位のヒスチジン残基
　欠失；
（ｖｉ）１７６位のリジン残基
　アラニン残基、セリン残基、またはスレオニン残基による置換；
（ｖｉｉ）１７８位のイソロイシン残基
　アラニン残基、またはバリン残基による置換；
（Ｂ’）（Ａ’）のアミノ酸配列において、１もしくは数個のアミノ酸の置換、欠失、挿入、もしくは付加を含むアミノ酸配列を含み、かつ、リポ酸タンパク質リガーゼ活性を有するタンパク質；ならびに
（Ｃ’）（Ａ’）のアミノ酸配列に対して９０％以上の同一性を有するアミノ酸配列を含み、かつ、リポ酸タンパク質リガーゼ活性を有するタンパク質。
　（Ａ’）～（Ｃ’）からなる群より選ばれるタンパク質は、配列番号２のアミノ酸配列からなるタンパク質よりも高いリポ酸タンパク質リガーゼ活性を有することが好ましい。リポ酸タンパク質リガーゼ活性は、ＩｇＧ（ＩｇＧ　Ｈ鎖もしくはＬ鎖またはそれらの組合せ）を基質とするＬｐｌＡ活性（オクタン酸アジドによるＩｇＧの標識）アッセイ（実施例７を参照）により測定することができる。（Ａ’）～（Ｃ’）からなる群より選ばれるタンパク質のリポ酸タンパク質リガーゼ活性は、配列番号２のアミノ酸配列からなるタンパク質のリポ酸タンパク質リガーゼ活性よりも１．１倍以上、１．２倍以上、１．３倍以上、１．４倍以上または１．５倍以上であることが好ましい。 In a specific embodiment, Lpl used in the method of the present invention may be a protein selected from the group consisting of (A ′) to (C ′):
(A ′) In the amino acid sequence of SEQ ID NO: 2, an amino acid sequence having one or more (eg, 1 to 6, preferably 1 to 5) mutations selected from the group consisting of the following (i) to (vii): A protein comprising and having lipoic acid protein ligase activity:
(I) substitution with an arginine residue at position 121, an alanine residue or a threonine residue;
(Ii) substitution of a serine residue at position 136 with a leucine residue;
(Iii) substitution with a tyrosine residue at position 140, an alanine residue or a valine residue;
(Iv) substitution at position 142 of a glutamic acid serine residue, threonine residue, or valine residue;
(V) deletion of histidine residue at position 149;
(Vi) substitution with a lysine residue at position 176 by an alanine residue, a serine residue or a threonine residue;
(Vii) substitution with an isoleucine residue at position 178, an alanine residue, or a valine residue;
(B ′) a protein comprising an amino acid sequence containing substitution, deletion, insertion or addition of one or several amino acids in the amino acid sequence of (A ′) and having lipoic acid protein ligase activity; ') A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence of (A'), and having lipoic acid protein ligase activity.
The protein selected from the group consisting of (A ') to (C') preferably has a higher lipoic acid protein ligase activity than the protein consisting of the amino acid sequence of SEQ ID NO: 2. Lipoic acid protein ligase activity can be measured by an LplA activity (labeling of IgG with octanoic azide) assay using IgG (IgG H chain or L chain or a combination thereof) as a substrate (see Example 7). The lipoic acid protein ligase activity of the protein selected from the group consisting of (A ′) to (C ′) is 1.1 times or more, 1.2 times or more, of the lipoic acid protein ligase activity of the protein consisting of the amino acid sequence of SEQ ID NO: 2. It is preferred that the ratio be at least 1.3 times, at least 1.4 times, or at least 1.5 times.
　リポ酸タンパク質リガーゼ活性とは、タンパク質中のリジン残基にリポ酸を結合させてアシル化する活性をいう。 Lipoic acid protein ligase activity refers to the activity of binding lipoic acid to a lysine residue in a protein for acylation.
　Ｌｐｌは、リポ酸タンパク質リガーゼ活性を保持する限り、特定の部位に、変異が導入されていてもよい。目的の特性を保持し得る、変異が導入されてもよいアミノ酸残基の位置は、当業者に明らかである。具体的には、当業者は、１）同種の特性を有する複数のタンパク質のアミノ酸配列を比較し、２）相対的に保存されている領域、および相対的に保存されていない領域を明らかにし、次いで、３）相対的に保存されている領域および相対的に保存されていない領域から、それぞれ、機能に重要な役割を果たし得る領域および機能に重要な役割を果たし得ない領域を予測できるので、構造・機能の相関性を認識することができる。したがって、当業者は、Ｌｐｌのアミノ酸配列において変異が導入されてもよいアミノ酸残基の位置を特定できる。 Lpl may have a mutation introduced at a specific site as long as it retains lipoic acid protein ligase activity. The positions of amino acid residues at which mutations can be introduced, which can retain desired properties, will be apparent to those skilled in the art. Specifically, one of skill in the art would: 1) compare the amino acid sequences of multiple proteins with similar properties, and 2) identify relatively conserved and relatively non-conserved regions, 3) From the relatively conserved region and the relatively unconserved region, a region that can play a significant role in the function and a region that cannot play a significant role in the function can be predicted, respectively. We can recognize the correlation between structure and function. Therefore, those skilled in the art can specify the position of an amino acid residue at which a mutation may be introduced in the amino acid sequence of Lpl.
　アミノ酸残基が置換により変異される場合、アミノ酸残基の置換は、保存的置換であってもよい。本明細書中で用いられる場合、用語「保存的置換」とは、所定のアミノ酸残基を、類似の側鎖を有するアミノ酸残基で置換することをいう。類似の側鎖を有するアミノ酸残基のファミリーは、当該分野で周知である。例えば、このようなファミリーとしては、塩基性側鎖を有するアミノ酸（例、リジン、アルギニン、ヒスチジン）、酸性側鎖を有するアミノ酸（例、アスパラギン酸、グルタミン酸）、非荷電性極性側鎖を有するアミノ酸（例、アスパラギン、グルタミン、セリン、スレオニン、チロシン、システイン）、非極性側鎖を有するアミノ酸（例、グリシン、アラニン、バリン、ロイシン、イソロイシン、プロリン、フェニルアラニン、メチオニン、トリプトファン）、β位分岐側鎖を有するアミノ酸（例、スレオニン、バリン、イソロイシン）、芳香族側鎖を有するアミノ酸（例、チロシン、フェニルアラニン、トリプトファン、ヒスチジン）、ヒドロキシル基（例、アルコール性、フェノール性）含有側鎖を有するアミノ酸（例、セリン、スレオニン、チロシン）、および硫黄含有側鎖を有するアミノ酸（例、システイン、メチオニン）が挙げられる。好ましくは、アミノ酸の保存的置換は、アスパラギン酸とグルタミン酸との間での置換、アルギニンとリジンとヒスチジンとの間での置換、トリプトファンとフェニルアラニンとの間での置換、フェニルアラニンとバリンとの間での置換、ロイシンとイソロイシンとアラニンとの間での置換、およびグリシンとアラニンとの間での置換であってもよい。 場合 When an amino acid residue is mutated by substitution, the substitution of the amino acid residue may be a conservative substitution. As used herein, the term "conservative substitution" refers to the replacement of a given amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are well known in the art. For example, such families include amino acids having a basic side chain (eg, lysine, arginine, histidine), amino acids having an acidic side chain (eg, aspartic acid, glutamic acid), and amino acids having an uncharged polar side chain (Eg, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having a nonpolar side chain (eg, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chain (Eg, threonine, valine, isoleucine), an amino acid having an aromatic side chain (eg, tyrosine, phenylalanine, tryptophan, histidine), an amino acid having a hydroxyl group (eg, alcoholic, phenolic) -containing side chain ( Example, serine, thread Nin, tyrosine), and amino acids (e.g. having sulfur-containing side chains, cysteine, methionine) and the like. Preferably, conservative substitutions of amino acids include substitution between aspartic acid and glutamic acid, substitution between arginine and lysine and histidine, substitution between tryptophan and phenylalanine, and substitution between phenylalanine and valine. , A substitution between leucine and isoleucine and alanine, and a substitution between glycine and alanine.
　Ｌｐｌはまた、異種部分とペプチド結合を介して連結された融合タンパク質であってもよい。このような異種部分としては、例えば、目的タンパク質の精製を容易にするペプチド成分（例、ヒスチジンタグ、Ｓｔｒｅｐ－ｔａｇ　ＩＩ等のタグ部分；グルタチオン－Ｓ－トランスフェラーゼ、マルトース結合タンパク質、およびこれらの変異型等の目的タンパク質の精製に利用されるタンパク質）、目的タンパク質の可溶性を向上させるペプチド成分（例、Ｎｕｓ－ｔａｇ）、シャペロンとして働くペプチド成分（例、トリガーファクター）、他の機能を有するペプチド成分（例、全長タンパク質またはその一部）、ならびにリンカーが挙げられる。 Lpl may also be a fusion protein linked via a peptide bond to a heterologous moiety. Examples of such a heterologous portion include a peptide component that facilitates purification of a target protein (eg, a histidine tag, a tag portion such as Strep-tag II; glutathione-S-transferase, maltose binding protein, and mutants thereof). , A peptide component that improves the solubility of the target protein (eg, Nus-tag), a peptide component that functions as a chaperone (eg, a trigger factor), a peptide component having other functions (eg, For example, a full-length protein or a portion thereof), as well as a linker.
（修飾部分を有するリポ酸アナログ）
　本発明の方法で用いられる、修飾部分を有するリポ酸アナログは、Ｌｐｌの基質として作用する限り特に限定されない。修飾部分を有するリポ酸アナログにおけるリポ酸アナログは、直鎖または分岐鎖の炭化水素基を有し、さらに環状の炭化水素基等の環状部分を有していてもよいカルボン酸である。このような修飾部分を有するリポ酸アナログは、Ｌｐｌの基質として作用することが知られている（上述の参考文献、特に米国特許第８，１３７，９２５号明細書を参照）。 (Lipoic acid analog having a modified moiety)
The lipoic acid analog having a modifying moiety used in the method of the present invention is not particularly limited as long as it acts as a substrate for Lpl. The lipoic acid analog in the lipoic acid analog having a modified portion is a carboxylic acid having a linear or branched hydrocarbon group and optionally having a cyclic portion such as a cyclic hydrocarbon group. Lipoic acid analogs with such modified moieties are known to act as substrates for Lpl (see references above, especially US Pat. No. 8,137,925).
　好ましくは、修飾部分を有するリポ酸アナログは、修飾部分を有するリポ酸である。修飾部分を有するリポ酸におけるリポ酸は、直鎖または分岐鎖の炭化水素基を有するカルボン酸である。 Preferably, the lipoic acid analog having a modified moiety is a lipoic acid having a modified moiety. The lipoic acid in the lipoic acid having a modifying moiety is a carboxylic acid having a linear or branched hydrocarbon group.
　より好ましくは、修飾部分を有するリポ酸アナログは、下記構造を含むアルキル－カルボン酸において修飾部分が付加された構造（換言すれば、リポ酸中の水素原子が修飾部分に置換された構造）を有する化合物である：
Figure JPOXMLDOC01-appb-C000002
 （式中、ｎは、１以上の任意の整数である。）
　反応効率の向上等の観点からは、ｎは、好ましくは３～９の整数、より好ましくは４～８の整数、さらにより好ましくは５～７の整数、特に好ましくは６である。 More preferably, the lipoic acid analog having a modified moiety has a structure in which a modified moiety is added to an alkyl-carboxylic acid having the following structure (in other words, a structure in which a hydrogen atom in lipoic acid is substituted by a modified moiety). Is a compound having:
Figure JPOXMLDOC01-appb-C000002
 (In the formula, n is any integer of 1 or more.)
From the viewpoint of improving the reaction efficiency and the like, n is preferably an integer of 3 to 9, more preferably an integer of 4 to 8, still more preferably an integer of 5 to 7, and particularly preferably 6.
　したがって、修飾部分を有するリポ酸アナログとしては、修飾部分を有するＣ４～Ｃ１０アルキル－カルボン酸が好ましく、修飾部分を有するＣ５～Ｃ９アルキル－カルボン酸がより好ましく、修飾部分を有するＣ６～Ｃ８アルキル－カルボン酸がより好ましく、修飾部分を有するＣ７アルキル－カルボン酸が特に好ましい。 Accordingly, the lipoic acid analog having a modified moiety is preferably a C 4 -C 10 alkyl-carboxylic acid having a modified moiety, more preferably a C 5 -C 9 alkyl-carboxylic acid having a modified moiety, and more preferably a C 4 -C 9 alkyl-carboxylic acid having a modified moiety. 6 ~ C 8 alkyl - are more preferred carboxylic acids, C 7 alkyl having a modifying moiety - carboxylic acid is particularly preferred.
　好ましい実施形態では、修飾部分を有するリポ酸アナログは、反応効率の向上等の観点から、「アルキル－カルボン酸」のアルキル末端部分に修飾部分を有する、下記式（Ｉ）で表される化合物である。
Ｒ－ＣｎＨ２ｎ－ＣＯＯＨ　　　（Ｉ）
〔式中、
　Ｒは、修飾部分であり、
　ｎは、４～１０の整数である。〕
　式中のハイフン（－）は、その両側に存在する２つの部分（例、ＲおよびＣｎＨ２ｎの２つの部分、ならびにＣｎＨ２ｎおよびＣＯＯＨの２つの部分）が互いに共有結合していることを示す（他の式も同様）。 In a preferred embodiment, the lipoic acid analog having a modified moiety is a compound represented by the following formula (I) having a modified moiety at the alkyl terminal of “alkyl-carboxylic acid” from the viewpoint of improving reaction efficiency and the like. is there.
R—C n H 2n —COOH (I)
(In the formula,
R is a modifying moiety;
n is an integer of 4 to 10. ]
In the formula, a hyphen (-) has two moieties on both sides thereof (eg, two moieties of R and C n H 2n and two moieties of C n H 2n and COOH) covalently bonded to each other. (The same applies to other expressions).
　上記式（Ｉ）において、ｎは、好ましくは５～９の整数、より好ましくは６～８の整数、特に好ましくは７である。 に お い て In the above formula (I), n is preferably an integer of 5 to 9, more preferably an integer of 6 to 8, and particularly preferably 7.
　修飾部分を有するリポ酸アナログが、上記式（Ｉ）で表される化合物である場合、定常領域中のリジン残基の側鎖に修飾部分を有する抗体として、下記式（ＩＩ）で表される部分を定常領域中のリジン残基の側鎖に有する修飾抗体が生成される。
Ｒ－ＣｎＨ２ｎ－ＣＯ－ＮＨ－　　　（ＩＩ）
〔式中、
　Ｒおよびｎは、式（Ｉ）のものと同じであり、
　ＮＨ－は、定常領域中のリジン残基の側鎖に存在する基である。〕 When the lipoic acid analog having a modified moiety is the compound represented by the above formula (I), an antibody having a modified moiety on the side chain of a lysine residue in the constant region is represented by the following formula (II) Modified antibodies having a moiety on the side chain of a lysine residue in the constant region are generated.
R—C n H 2n —CO—NH— (II)
(In the formula,
R and n are the same as in formula (I);
NH- is a group present on the side chain of a lysine residue in the constant region. ]
　修飾部分は、本発明の方法による反応後に抗体に保有された場合、そのような抗体に任意の機能を付与する部分である限り特に限定されず、例えば、生体直交性官能基または機能性物質を含むものが挙げられる。本発明では、１種または２種以上（例、２種、３種、４種）の修飾部分がリポ酸に付加されていてもよいが、好ましくは、１種の修飾部分がリポ酸に付加されていてもよい。 When the modified moiety is retained in the antibody after the reaction according to the method of the present invention, the modified moiety is not particularly limited as long as the moiety imparts an arbitrary function to such an antibody.For example, a bioorthogonal functional group or a functional substance may be used. Including. In the present invention, one or more (eg, two, three, four) modified moieties may be added to lipoic acid, but preferably, one modified moiety is added to lipoic acid. It may be.
　生体直交性官能基とは、生体構成成分（例、アミノ酸、核酸、脂質、糖、リン酸）とは反応しない、もしくは生体構成成分に対する反応の速度が遅いが、生体構成成分以外の成分に対して選択的に反応する基をいう。生体直交性官能基は、当該技術分野において周知である（例、Ｓｈａｒｐｌｅｓｓ　Ｋ．Ｂ．ｅｔ　ａｌ．，Ａｎｇｅｗ．Ｃｈｅｍ．Ｉｎｔ．Ｅｄ．４０，２００４（２０１５）；Ｂｅｒｔｏｚｚｉ　Ｃ．Ｒ．ｅｔ　ａｌ．，Ｓｃｉｅｎｃｅ　２９１，２３５７（２００１）；Ｂｅｒｔｏｚｚｉ　Ｃ．Ｒ．ｅｔ　ａｌ．，Ｎａｔｕｒｅ　Ｃｈｅｍｉｃａｌ　Ｂｉｏｌｏｇｙ　１，１３（２００５）を参照）。生体直交性官能基は、本発明の方法による反応後に抗体に保有された場合、そのような抗体に対して機能性物質に対する反応性を付与することができる。 A bio-orthogonal functional group does not react with biological constituents (eg, amino acids, nucleic acids, lipids, sugars, and phosphates) or has a slow reaction rate with biological constituents, but does not react with components other than biological constituents. Refers to a group that reacts selectively. Bioorthogonal functional groups are well known in the art (eg, Sharpless {KB et al., Angew. Chem. Int. Ed. 40, 2004 (2015); Bertozzi {CR et al., Science 291, 257 (2001); Bertozzi CR et al., Nature Chemical Biology 1, 13 (2005)). When a bioorthogonal functional group is retained in an antibody after the reaction according to the method of the present invention, it can impart reactivity to a functional substance to such an antibody.
　本発明では、生体直交性官能基は、抗体（タンパク質）に対する生体直交性官能基である。抗体に対する生体直交性官能基とは、抗体を構成する天然の２０種のアミノ酸残基の側鎖と反応せずに、所定の官能基と反応する基である。抗体（タンパク質）を構成する天然の２０種のアミノ酸は、アラニン（Ａ）、アスパラギン（Ｎ）、システイン（Ｃ）、グルタミン（Ｑ）、グリシン（Ｇ）、イソロイシン（Ｉ）、ロイシン（Ｌ）、メチオニン（Ｍ）、フェニルアラニン（Ｆ）、プロリン（Ｐ）、セリン（Ｓ）、スレオニン（Ｔ）、トリプトファン（Ｗ）、チロシン（Ｙ）、バリン（Ｖ）、アスパラギン酸（Ｄ）、グルタミン酸（Ｅ）、アルギニン（Ｒ）、ヒスチジン（Ｈ）、およびリジン（Ｌ）である。これらの天然の２０種のアミノ酸のうち、側鎖がない（すなわち、水素原子である）グリシン、ならびに側鎖が炭化水素基である（すなわち、硫黄原子、窒素原子、および酸素原子からなる群より選ばれるヘテロ原子を側鎖に含まない）アラニン、イソロイシン、ロイシン、フェニルアラニン、およびバリンは、通常の反応に対して不活性である。したがって、抗体（タンパク質）に対する生体直交性官能基は、通常の反応に対して不活性である側鎖を有するこれらのアミノ酸の側鎖に加えて、アスパラギン、グルタミン、メチオニン、プロリン、セリン、スレオニン、トリプトファン、チロシン、アスパラギン酸、グルタミン酸、アルギニン、ヒスチジン、およびリジンの側鎖に対しても反応できない官能基である。 で は In the present invention, the bio-orthogonal functional group is a bio-orthogonal functional group for an antibody (protein). The bioorthogonal functional group for an antibody is a group that reacts with a predetermined functional group without reacting with the side chains of the 20 natural amino acid residues constituting the antibody. Twenty natural amino acids constituting the antibody (protein) are alanine (A), asparagine (N), cysteine (C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), Methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), valine (V), aspartic acid (D), glutamic acid (E) , Arginine (R), histidine (H), and lysine (L). Of these naturally occurring 20 amino acids, glycine without side chains (ie, being a hydrogen atom), and glycine whose side chains are hydrocarbon groups (ie, from the group consisting of sulfur, nitrogen, and oxygen atoms) Alanine, isoleucine, leucine, phenylalanine, and valine (without the selected heteroatom in the side chain) are inert to normal reactions. Therefore, the bioorthogonal functional group for the antibody (protein) has asparagine, glutamine, methionine, proline, serine, threonine, as well as the side chains of those amino acids having side chains that are inactive to normal reactions. A functional group that cannot react with the side chains of tryptophan, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine.
　タンパク質に対して反応できないこのような生体直交性官能基としては、例えば、アジド残基、アルデヒド残基、チオール残基、アルケン残基（換言すれば、炭素原子間二重結合を有する最小単位であるビニレン（エテニレン）部分を有していればよい。以下同様）、アルキン残基（換言すれば、炭素原子間三重結合を有する最小単位であるエチニレン部分を有していればよい。以下同様）、ハロゲン残基、テトラジン残基、ニトロン残基、ヒドロキシルアミン残基、ニトリル残基、ヒドラジン残基、ケトン残基、ボロン酸残基、シアノベンゾチアゾール残基、アリル残基、ホスフィン残基、マレイミド残基、ジスルフィド残基、チオエステル残基、α―ハロカルボニル残基（例、α位にフッ素原子、塩素原子、臭素原子またはヨウ素原子を有するカルボニル残基。以下同様）、イソニトリル残基、シドノン残基、セレン残基が挙げられる。抗体は、遊離のチオールを含み得ないタンパク質であり得る。遊離のチオールを含み得ないタンパク質においては、チオールが生体直交性官能基として機能する。したがって、本発明の場合、生体直交性官能基にはチオールが含まれる。 Examples of such bioorthogonal functional groups that cannot react with proteins include azide residues, aldehyde residues, thiol residues, and alkene residues (in other words, the minimum unit having a carbon-carbon double bond is It is only necessary to have a certain vinylene (ethenylene) moiety. The same applies to the following.) An alkyne residue (in other words, it suffices to have an ethinylene moiety which is a minimum unit having a triple bond between carbon atoms. The same applies to the following). , Halogen residue, tetrazine residue, nitrone residue, hydroxylamine residue, nitrile residue, hydrazine residue, ketone residue, boronic acid residue, cyanobenzothiazole residue, allyl residue, phosphine residue, maleimide Residue, disulfide residue, thioester residue, α-halocarbonyl residue (eg, a fluorine atom, a chlorine atom, a bromine atom or an iodine atom at the α-position Carbonyl residue, the same applies hereinafter), an isonitrile residue, a sydnone residue, and a selenium residue. Antibodies can be proteins that cannot contain free thiols. In proteins that cannot contain free thiols, thiols function as bioorthogonal functional groups. Therefore, in the case of the present invention, the bioorthogonal functional group includes a thiol.
　好ましくは、生体直交性官能基は、反応効率の向上等の観点より、上述の生体直交性官能基のなかでも、アジド残基、チオール残基、アルキン残基、マレイミド残基、およびジスルフィド残基からなる群より選ばれる基であってもよい。 Preferably, the bio-orthogonal functional group is an azide residue, a thiol residue, an alkyne residue, a maleimide residue, and a disulfide residue among the above-described bio-orthogonal functional groups from the viewpoint of improving reaction efficiency and the like. Or a group selected from the group consisting of
　機能性物質は、本発明の方法による反応後に抗体に保有された場合、そのような抗体に任意の機能を付与する物質である限り特に限定されない。機能性物質の詳細については、後述する。 The functional substance is not particularly limited as long as it is a substance that imparts an arbitrary function to such an antibody when retained in the antibody after the reaction according to the method of the present invention. Details of the functional substance will be described later.
（リポ酸タンパク質リガーゼの存在下における、修飾部分を有するリポ酸アナログとの抗体の反応）
　反応は、抗体の変性・分解（例、アミド結合の切断）を引き起こし得ない条件（温和な条件）下で適宜行うことができる。例えば、このような反応は、適切な反応系、例えば緩衝液（例、リン酸緩衝液）中において、常温（例えば約１５～３５℃、好ましくは約２０～３０℃）で行うことができる。緩衝液のｐＨは、例えば５～９であり、好ましくは５．５～８．５であり、より好ましくは６．０～８．０である。緩衝液は、ＡＴＰ等の適切な補因子を含んでいてもよい。反応時間は、例えば１０分～１５０時間、好ましくは２０分～１２０時間、より好ましくは３０分～１００時間、さらにより好ましくは６０分～８０時間である。Ｌｐｌの濃度は、適宜調製することができる。このような反応の詳細については、例えば、Ｇｒｅｅｎ　ＤＥ　ｅｔ　ａｌ．　Ｂｉｏｃｈｅｍ　Ｊ　３０９　１９９５　：８５３－８６２；米国特許第８，１３７，９２５号；米国特許第９，２８４，５４１号；国際公開第２０１７／０９５８０６号を参照のこと。 (Reaction of Antibody with Lipoic Acid Analog Having Modified Portion in the Presence of Lipoic Acid Protein Ligase)
The reaction can be appropriately performed under conditions (mild conditions) that cannot cause denaturation / decomposition (eg, cleavage of amide bond) of the antibody. For example, such a reaction can be carried out in a suitable reaction system, for example, a buffer (eg, a phosphate buffer) at ordinary temperature (for example, about 15 to 35 ° C., preferably about 20 to 30 ° C.). The pH of the buffer is, for example, 5 to 9, preferably 5.5 to 8.5, and more preferably 6.0 to 8.0. The buffer may contain a suitable cofactor such as ATP. The reaction time is, for example, 10 minutes to 150 hours, preferably 20 minutes to 120 hours, more preferably 30 minutes to 100 hours, and even more preferably 60 minutes to 80 hours. The concentration of Lpl can be appropriately adjusted. For details of such reactions, see, for example, Green DE et al. Biochem J 309 1995: 853-862; U.S. Patent No. 8,137,925; U.S. Patent No. 9,284,541; WO 2017/095806.
　反応系において、抗体（Ｘ）に対する、修飾部分を有するリポ酸アナログ（Ｙ）のモル比率（Ｙ／Ｘ）は、抗体、および修飾部分を有するリポ酸アナログの種類、修飾部分を有するリポ酸アナログによって修飾されるべき抗体中の部位の数（例、ＤＡＲ）等に応じて変動することから特に限定されないが、例えば０．１～１００であり、好ましくは０．５～８０であり、より好ましくは１～７０であり、さらにより好ましくは２～５０であり、特に好ましくは３～３０である。 In the reaction system, the molar ratio (Y / X) of the lipoic acid analog having the modified moiety to the antibody (X) is determined by the type of the antibody and the lipoic acid analog having the modified moiety, and the lipoic acid analog having the modified moiety. It is not particularly limited since it varies depending on the number of sites in the antibody to be modified by (e.g., DAR) and the like, but is, for example, 0.1 to 100, preferably 0.5 to 80, and more preferably Is from 1 to 70, still more preferably from 2 to 50, and particularly preferably from 3 to 30.
　定常領域中のリジン残基の側鎖に修飾部分を有する修飾抗体の生成の確認は、その具体的な原料（抗体、および修飾部分を有するリポ酸アナログ）の種類および生成物の分子量にもよるが、例えば、電気泳動法、クロマトグラフィー（例、ゲル濾過クロマトグラフィー、イオン交換クロマトグラフィー、逆相カラムクロマトグラフィー、ＨＰＬＣ）、または質量分析により、好ましくは、質量分析により行うことができる。抗体において修飾されたリジン残基の位置の確認は、例えば、ペプチドマッピングにより行うことができる。ペプチドマッピングは、例えば、プロテアーゼ（例、トリプシン、キモトリプシン、）処理および質量分析により行うことができる。プロテアーゼとしては、エンドプロテアーゼが好ましい。このようなエンドプロテアーゼとしては、例えば、トリプシン、キモトリプシン、Ｇｌｕ－Ｃ、Ｌｙｓ－Ｎ、Ｌｙｓ－Ｃ、Ａｓｐ－Ｎが挙げられる。定常領域中のリジン残基の側鎖に修飾部分を有する修飾抗体が保有する、修飾部分の個数の確認は、例えば、電気泳動法、クロマトグラフィー、または質量分析により、好ましくは、質量分析により行うことができる。定常領域中のリジン残基の側鎖に修飾部分を有する修飾抗体は、クロマトグラフィー（例、上述したクロマトグラフィー、およびアフィニティークロマトグラフィー）等の任意の方法により適宜精製することができる。 Confirmation of generation of a modified antibody having a modified portion on the side chain of a lysine residue in the constant region depends on the type of the specific raw material (antibody and lipoic acid analog having the modified portion) and the molecular weight of the product Can be performed by, for example, electrophoresis, chromatography (eg, gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC), or mass spectrometry, preferably mass spectrometry. Confirmation of the position of the modified lysine residue in the antibody can be performed, for example, by peptide mapping. Peptide mapping can be performed, for example, by protease (eg, trypsin, chymotrypsin) treatment and mass spectrometry. As the protease, an endoprotease is preferable. Such endoproteases include, for example, trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, Asp-N. Confirmation of the number of modified moieties possessed by the modified antibody having a modified moiety on the side chain of a lysine residue in the constant region is performed, for example, by electrophoresis, chromatography, or mass spectrometry, preferably by mass spectrometry. be able to. The modified antibody having a modified portion on the side chain of a lysine residue in the constant region can be appropriately purified by any method such as chromatography (eg, the above-described chromatography and affinity chromatography).
　好ましい特定の実施形態では、修飾部分は、生体直交性官能基を含む修飾部分である。この場合、本発明の方法は、機能性物質を有する修飾抗体の製造のためのさらなる工程を含む、以下の方法により行われてもよい。
（１）抗体を、リポ酸タンパク質リガーゼＡの存在下において、生体直交性官能基を含む修飾部分を有するリポ酸アナログと反応させて、定常領域中のリジン残基の側鎖に生体直交性官能基を含む修飾部分を有する修飾抗体を生成すること；ならびに
（２）定常領域中のリジン残基の側鎖に生体直交性官能基を含む修飾部分を有する修飾抗体を、生体直交性官能基を介して機能性物質と反応させて、定常領域中のリジン残基の側鎖に機能性物質を有する修飾抗体を生成すること。 In certain preferred embodiments, the modifying moiety is a modifying moiety that includes a bioorthogonal functional group. In this case, the method of the present invention may be performed by the following method, which includes an additional step for producing a modified antibody having a functional substance.
(1) An antibody is reacted with a lipoic acid analog having a modified portion containing a bioorthogonal functional group in the presence of lipoic acid protein ligase A, so that the side chain of a lysine residue in the constant region has a bioorthogonal function. (2) generating a modified antibody having a modification moiety containing a bioorthogonal functional group on the side chain of a lysine residue in the constant region, To generate a modified antibody having a functional substance on the side chain of a lysine residue in the constant region.
　上記工程（１）は、上述した工程（抗体を、リポ酸タンパク質リガーゼの存在下において、修飾部分を有するリポ酸アナログと反応させて、定常領域中のリジン残基の側鎖に修飾部分を有する修飾抗体を生成すること）と同様にして行うことができる。 The above step (1) comprises the step of reacting an antibody with a lipoic acid analog having a modifying portion in the presence of a lipoic acid protein ligase to form a modified portion on the side chain of a lysine residue in the constant region. Generating a modified antibody).
　上記工程（２）は、修飾抗体中の生体直交性官能基を機能性物質と反応させるための抗体である。 The above step (2) is an antibody for reacting a bioorthogonal functional group in the modified antibody with a functional substance.
　機能性物質としては、本発明の方法による反応後に抗体に保有された場合、そのような抗体に任意の機能を付与する物質である限り特に限定されず、例えば、薬物、標識物質、安定化剤が挙げられるが、好ましくは薬物または標識物質である。機能性物質はまた、単一の機能性物質であってもよく、または２以上の機能性物質が連結された物質であってもよい。 The functional substance is not particularly limited as long as it is a substance that imparts an arbitrary function to such an antibody when retained in the antibody after the reaction according to the method of the present invention, and includes, for example, a drug, a labeling substance, and a stabilizing agent. And preferably a drug or a labeling substance. The functional substance may also be a single functional substance or a substance in which two or more functional substances are linked.
　薬物としては、任意の疾患に対する薬物であってもよい。このような疾患としては、例えば、癌（例、肺癌、胃癌、大腸癌、膵臓癌、腎臓癌、肝臓癌、甲状腺癌、前立腺癌、膀胱癌、卵巣癌、子宮癌、骨癌、皮膚癌、脳腫瘍、黒色腫）、自己免疫疾患・炎症性疾患（例、アレルギー疾患、関節リウマチ、全身性エリテマトーデス）、脳神経疾患（例、脳梗塞、アルツハイマー病、パーキンソン病、筋萎縮性側索硬化症）、感染症（例、細菌感染症、ウイルス感染症）、遺伝性・希少疾患（例、遺伝性球状赤血球症、非ジストロフィー筋緊張症）、眼疾患（例、加齢黄斑変性症、糖尿病網膜症、網膜色素変性症）、骨・整形外科領域の疾患（例、変形性関節症）、血液疾患（例、白血病、紫斑病）、その他の疾患（例、糖尿病、高脂血症等の代謝異常症、肝臓疾患、腎疾患、肺疾患、循環器系疾患、消化器官系疾患）が挙げられる。薬物は、所定の疾患を治療または予防する薬物、または所定の疾患の標的タンパク質に対する抗体の使用に伴う副作用を緩和する薬物であってもよい。 The drug may be a drug for any disease. Such diseases include, for example, cancer (eg, lung cancer, gastric cancer, colon cancer, pancreatic cancer, kidney cancer, liver cancer, thyroid cancer, prostate cancer, bladder cancer, ovarian cancer, uterine cancer, bone cancer, skin cancer, Brain tumor, melanoma), autoimmune disease / inflammatory disease (eg, allergic disease, rheumatoid arthritis, systemic lupus erythematosus), cranial nerve disease (eg, cerebral infarction, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis), Infections (eg, bacterial infections, viral infections), hereditary or rare diseases (eg, hereditary spherocytosis, non-dystrophic myotonia), eye diseases (eg, age-related macular degeneration, diabetic retinopathy, Retinitis pigmentosa), bone and orthopedic diseases (eg, osteoarthritis), blood diseases (eg, leukemia, purpura), and other diseases (eg, diabetes, hyperlipidemia, etc.) , Liver disease, kidney disease, lung disease, cardiovascular disease, Organ system disease), and the like. The drug may be a drug that treats or prevents a given disease, or a drug that alleviates the side effects associated with the use of antibodies to the target protein of a given disease.
　より具体的には、薬物は、抗癌剤である。抗癌剤としては、例えば、化学療法剤、毒素、放射性同位体またはそれを含む物質が挙げられる。化学療法剤としては、例えば、ＤＮＡ損傷剤、代謝拮抗薬、酵素阻害剤、ＤＮＡインターカレート剤、ＤＮＡ切断剤、トポイソメラーゼ阻害剤、ＤＮＡ結合阻害剤、チューブリン結合阻害剤、細胞傷害性ヌクレオシド、白金化合物が挙げられる。毒素としては、例えば、細菌毒素（例、ジフテリア毒素）、植物毒素（例、リシン）が挙げられる。放射性同位体としては、例えば、水素原子の放射性同位体（例、３Ｈ）、炭素原子の放射性同位体（例、１４Ｃ）、リン原子の放射性同位体（例、３２Ｐ）、硫黄原子の放射性同位体（例、３５Ｓ）、イットリウムの放射性同位体（例、９０Ｙ）、テクネチウムの放射性同位体（例、９９ｍＴｃ）、インジウムの放射性同位体（例、１１１Ｉｎ）、ヨウ素原子の放射性同位体（例、１２３Ｉ、１２５Ｉ、１２９Ｉ、１３１Ｉ)、サマリウムの放射性同位体（例、１５３Ｓｍ）、レニウムの放射性同位体（例、１８６Ｒｅ）、アスタチンの放射性同位体（例、２１１Ａｔ）、ビスマスの放射性同位体（例、２１２Ｂｉ）が挙げられる。 More specifically, the drug is an anti-cancer agent. Examples of the anticancer agent include a chemotherapeutic agent, a toxin, a radioisotope, and a substance containing the same. Chemotherapeutic agents include, for example, DNA damaging agents, antimetabolites, enzyme inhibitors, DNA intercalating agents, DNA cleaving agents, topoisomerase inhibitors, DNA binding inhibitors, tubulin binding inhibitors, cytotoxic nucleosides, Platinum compounds may be mentioned. Examples of toxins include bacterial toxins (eg, diphtheria toxin) and plant toxins (eg, ricin). Examples of the radioactive isotope include a radioactive isotope of a hydrogen atom (eg, 3 H), a radioactive isotope of a carbon atom (eg, 14 C), a radioactive isotope of a phosphorus atom (eg, 32 P), and a radioactive isotope of a sulfur atom. Radioactive isotope (eg, 35 S ), radioactive isotope of yttrium (eg, 90 Y), radioactive isotope of technetium (eg, 99m Tc), radioactive isotope of indium (eg, 111 In), radioactivity of iodine atom Isotopes (eg, 123 I, 125 I, 129 I, 131 I), radioisotopes of samarium (eg, 153 Sm), radioisotopes of rhenium (eg, 186 Re), radioisotopes of astatine (eg, 211 At) and a radioisotope of bismuth (eg, 212 Bi).
　標識物質としては、例えば、酵素（例、ペルオキシダーゼ、アルカリホスファターゼ、ルシフェラーゼ、βガラクトシダーゼ）、親和性物質（例、ストレプトアビジン、ビオチン、ジゴキシゲニン、アプタマー）、蛍光物質（例、フルオレセイン、フルオレセインイソチオシアネート、ローダミン、緑色蛍光タンパク質、赤色蛍光タンパク質）、発光物質（例、ルシフェリン、エクオリン、アクリジニウムエステル、トリス（２，２’－ビピリジル）ルテニウム、ルミノール）、放射性同位体（例、上述したもの）またはそれを含む物質が挙げられる。 Examples of the labeling substance include enzymes (eg, peroxidase, alkaline phosphatase, luciferase, β-galactosidase), affinity substances (eg, streptavidin, biotin, digoxigenin, aptamer), fluorescent substances (eg, fluorescein, fluorescein isothiocyanate, rhodamine) , Green fluorescent protein, red fluorescent protein), luminescent substance (eg, luciferin, aequorin, acridinium ester, tris (2,2′-bipyridyl) ruthenium, luminol), radioisotope (eg, those described above) or And the like.
　機能性物質はまた、高分子化合物、中分子化合物、または低分子化合物であり、好ましくは、低分子化合物である。低分子化合物とは、分子量１５００以下の化合物をいう。低分子化合物は、天然化合物または合成化合物である。低分子化合物の分子量は、１２００以下、１０００以下、９００以下、８００以下、７００以下、６００以下、５００以下、４００以下、または３００以下であってもよい。低分子化合物の分子量はまた、３０以上、４０以上、または５０以上であってもよい。低分子化合物は、上述したような薬物または標識物質であってもよい。また、低分子化合物としては、例えば、アミノ酸、オリゴペプチド、ビタミン、ヌクレオシド、ヌクレオチド、オリゴヌクレオチド、単糖、オリゴ糖、脂質、脂肪酸、およびそれらの塩が挙げられる。 The functional substance is also a high molecular compound, a medium molecular compound, or a low molecular compound, and is preferably a low molecular compound. A low molecular compound is a compound having a molecular weight of 1500 or less. A low molecular compound is a natural compound or a synthetic compound. The molecular weight of the low molecular weight compound may be 1200 or less, 1000 or less, 900 or less, 800 or less, 700 or less, 600 or less, 500 or less, 400 or less, or 300 or less. The molecular weight of the low molecular weight compound may also be 30 or higher, 40 or higher, or 50 or higher. The low molecular weight compound may be a drug or a labeling substance as described above. Examples of the low-molecular compound include amino acids, oligopeptides, vitamins, nucleosides, nucleotides, oligonucleotides, monosaccharides, oligosaccharides, lipids, fatty acids, and salts thereof.
　機能性物質はさらに、ペプチド化合物であってもよい。ペプチド化合物は、上述したような抗癌剤等の薬物、標識物質、または低分子化合物であってもよい。 The functional substance may be a peptide compound. The peptide compound may be a drug such as an anticancer agent as described above, a labeling substance, or a low molecular compound.
　機能性物質は、その構造に応じた種々の官能基を有する。機能性物質が、生体直交性官能基と反応し易い官能基を有する場合、機能性物質の当該官能基と生体直交性官能基とを適宜反応させることができる。生体直交性官能基と反応し易い官能基は、生体直交性官能基の具体的な種類によっても異なり得る。当業者であれば、適切な官能基を、生体直交性官能基と反応し易い官能基として適宜選択することができる（例、Ｂｏｕｔｕｒｅｉｒａ　ｅｔ　ａｌ．，　Ｃｈｅｍ．　Ｒｅｖ．，２０１５，１１５，２１７４－２１９５）。生体直交性官能基と反応し易い官能基としては、例えば、生体直交性官能基がアルキン残基の場合はアジド残基が挙げられ、生体直交性官能基がアルデヒド残基またはケトン残基の場合はヒドラジン残基が挙げられ、生体直交性官能基がチオール残基の場合はマレイミド残基およびジスルフィド残基が挙げられるが、これらに限定されない。例えば、生体直交性官能基がアルキン残基であり、かつ生体直交性官能基と反応し易い官能基がアジド残基である場合（またはその逆である場合）、機能性物質と生体直交性官能基との間の反応により生成する部分を含む２価の基は、トリアゾール残基（他の環と縮合していても縮合していなくてもよい）を含む２価の基であってもよい；生体直交性官能基がアルデヒド残基またはケトン残基であり、かつ生体直交性官能基と反応し易い官能基がヒドラジン残基である場合（またはその逆である場合）、機能性物質と生体直交性官能基との間の反応により生成する部分を含む２価の基は、ヒドラゾン残基を含む２価の基であってもよい；生体直交性官能基がチオール残基であり、かつ生体直交性官能基と反応し易い官能基がマレイミド残基またはジスルフィド残基である場合（またはその逆である場合）、機能性物質と生体直交性官能基との間の反応により生成する部分を含む２価の基は、チオスクシンイミド残基を含む２価の基、またはジスルフィド残基を含む２価の基であってもよい（例、Ｂｏｕｔｕｒｅｉｒａ　ｅｔ　ａｌ．，　Ｃｈｅｍ．　Ｒｅｖ．，２０１５，１１５，２１７４－２１９５）。トリアゾール残基（他の環と縮合していても縮合していなくてもよい）を含む２価の基、ヒドラゾン残基を含む２価の基、チオスクシンイミド残基を含む２価の基、またはジスルフィド残基を含む２価の基は、機能性物質と生体直交性官能基との間の反応により生成する部分を含む２価の基の好ましい例である。 The functional substance has various functional groups according to its structure. When the functional substance has a functional group that easily reacts with the bio-orthogonal functional group, the functional group of the functional substance and the bio-orthogonal functional group can be appropriately reacted. The functional group that easily reacts with the bio-orthogonal functional group may vary depending on the specific type of the bio-orthogonal functional group. Those skilled in the art can appropriately select an appropriate functional group as a functional group that easily reacts with a bioorthogonal functional group (eg, Boutureira et al., Chem. Rev., 2015, 115, 2174-2195). ). Examples of the functional group that easily reacts with the bioorthogonal functional group include, for example, an azide residue when the bioorthogonal functional group is an alkyne residue, and an aldehyde or ketone residue when the bioorthogonal functional group is an aldehyde residue or a ketone residue. Is a hydrazine residue, and when the bioorthogonal functional group is a thiol residue, includes a maleimide residue and a disulfide residue, but is not limited thereto. For example, when the bioorthogonal functional group is an alkyne residue and the functional group that easily reacts with the bioorthogonal functional group is an azide residue (or vice versa), the functional substance and the bioorthogonal functional group The divalent group containing a moiety generated by the reaction with the group may be a divalent group containing a triazole residue (which may or may not be condensed with another ring). A case where the bioorthogonal functional group is an aldehyde residue or a ketone residue and the functional group which easily reacts with the bioorthogonal functional group is a hydrazine residue (or vice versa); The divalent group containing the moiety generated by the reaction between the orthogonal functional group and the orthogonal functional group may be a divalent group containing a hydrazone residue; the biological orthogonal functional group is a thiol residue, and Functional groups that readily react with the orthogonal functional groups Is a disulfide residue (or vice versa), a divalent group containing a moiety formed by a reaction between a functional substance and a bioorthogonal functional group is a divalent group containing a thiosuccinimide residue Or a divalent group containing a disulfide residue (eg, Boutureira et al., Chem. Rev., 2015, 115, 2174-2195). A divalent group containing a triazole residue (which may or may not be condensed with another ring), a divalent group containing a hydrazone residue, a divalent group containing a thiosuccinimide residue, or A divalent group containing a disulfide residue is a preferred example of a divalent group containing a moiety generated by a reaction between a functional substance and a bioorthogonal functional group.
　一方、機能性物質が、生体直交性官能基と反応し易い官能基を有しない場合、機能性物質として、所望の官能基を有するように誘導体化されたものを使用することができる。例えば、機能性物質が可溶性タンパク質である場合、可溶性タンパク質が天然に有しない官能基を有するように誘導体化されたものを使用することができる。 On the other hand, when the functional substance does not have a functional group that easily reacts with the bio-orthogonal functional group, a functional substance derivatized so as to have a desired functional group can be used. For example, when the functional substance is a soluble protein, a substance derivatized so that the soluble protein has a functional group which does not naturally exist can be used.
　誘導体化は、当該分野における技術常識である（例、国際公開第２００４／０１０９５７号、米国特許出願公開第２００６／００７４００８号明細書、米国特許出願公開第２００５／０２３８６４９号明細書）。例えば、誘導体化は、上述したような架橋剤を用いて行われてもよい。あるいは、誘導体化は、所望の官能基を有する特定のリンカーを用いて行われてもよい。例えば、このようなリンカーは、適切な環境（例、細胞内または細胞外）において機能性物質と抗体とをリンカーの切断により分離可能なものであってもよい。このようなリンカーとしては、例えば、特定のプロテアーゼ〔例、細胞内プロテアーゼ（例、リソソーム、またはエンドソーム中に存在するプロテアーゼ）、細胞外プロテアーゼ（例、分泌性プロテアーゼ）〕で分解されるペプチジルリンカー（例、米国特許第６，２１４，３４５号；Ｄｕｂｏｗｃｈｉｋ　ｅｔ　ａｌ．，Ｐｈａｒｍ．Ｔｈｅｒａｐｅｕｔｉｃｓ　８３：６７－１２３（１９９９））、生体内に存在する局所酸性部位で切断され得るリンカー（例、米国特許第５，６２２，９２９号、同第５，１２２，３６８号；同第５，８２４，８０５号）が挙げられる。リンカーは、自壊的（ｓｅｌｆ－ｉｍｍｏｌａｔｉｖｅ）であってもよい（例、国際公開第０２／０８３１８０号、国際公開第０４／０４３４９３号、国際公開第０５／１１２９１９号）。本発明では、誘導体化された機能性物質も、単に「機能性物質」と呼称することができる。 Derivatization is a common technical knowledge in the art (eg, WO 2004/010957, US Patent Application Publication No. 2006/0074008, US Patent Application Publication No. 2005/0238649). For example, derivatization may be performed using a crosslinking agent as described above. Alternatively, derivatization may be performed using a specific linker having a desired functional group. For example, such a linker may be capable of separating a functional substance and an antibody by cleavage of the linker in an appropriate environment (eg, inside or outside a cell). As such a linker, for example, a peptidyl linker (eg, a protease that is present in an intracellular protease (eg, a protease present in a lysosome or an endosome) or an extracellular protease (eg, a secretory protease)) (eg, a protease) For example, US Patent No. 6,214,345; Dubowchik et al., Pharm. Therapeutics 83: 67-123 (1999), a linker that can be cleaved at a local acidic site present in vivo (eg, US Patent No. 5). No. 5,622,929; No. 5,122,368; No. 5,824,805). The linker may be self-immortal (eg, WO 02/083180, WO 04/044933, WO 05/112919). In the present invention, the derivatized functional substance can also be simply referred to as “functional substance”.
　上記（１）（２）の工程により得られる、定常領域中のリジン残基の側鎖に機能性物質を有する修飾抗体は、機能性物質と生体直交性官能基との間の反応により生成する部分を含む２価の基を含む。このような２価の基は、例えば、トリアゾール残基、ヒドラゾン残基、チオスクシンイミド残基、ジスルフィド残基、アセタール残基、ケタール残基、エステル残基、カルバモイル残基、アルコキシアルキル残基、イミン残基、三級アルキルオキシカルバメート残基、シラン残基、ヒドラゾン含有残基、フォスフォルアミデート残基、アコニチル残基、トリチル残基、アゾ残基、ビシナルジオール残基、セレン残基、電子吸引基（例、ハロゲン原子、ボロン酸残基、メシル、トシル、トリフレート）を有する芳香族環含有残基、クマリン含有残基、スルホン含有残基、不飽和結合含有鎖残基、グリコシル残基からなる群より選ばれる残基を含む２価の基であってもよい。好ましくは、このような２価の基は、トリアゾール残基、ヒドラゾン残基、チオスクシンイミド残基、またはジスルフィド残基である。 The modified antibody having a functional substance on the side chain of a lysine residue in the constant region, obtained by the above steps (1) and (2), is produced by a reaction between the functional substance and a bio-orthogonal functional group. Includes divalent groups containing moieties. Such a divalent group includes, for example, a triazole residue, a hydrazone residue, a thiosuccinimide residue, a disulfide residue, an acetal residue, a ketal residue, an ester residue, a carbamoyl residue, an alkoxyalkyl residue, an imine residue. Residue, tertiary alkyloxycarbamate residue, silane residue, hydrazone-containing residue, phosphoramidate residue, aconityl residue, trityl residue, azo residue, vicinal diol residue, selenium residue, electron Aromatic ring-containing residue having a suction group (eg, halogen atom, boronic acid residue, mesyl, tosyl, triflate), coumarin-containing residue, sulfone-containing residue, unsaturated bond-containing chain residue, glycosyl residue Or a divalent group containing a residue selected from the group consisting of: Preferably, such a divalent group is a triazole residue, a hydrazone residue, a thiosuccinimide residue, or a disulfide residue.
　工程（２）の反応は、抗体の変性・分解（例、アミド結合の切断）を引き起こし得ない条件（温和な条件）下で適宜行うことができる。例えば、このような反応は、適切な反応系、例えば緩衝液（例、リン酸緩衝液）中において、常温（例えば約１５～３５℃、好ましくは約２０～３０℃）で行うことができる。緩衝液のｐＨは、例えば５～９であり、好ましくは５．５～８．５であり、より好ましくは６．０～８．０である。緩衝液は、ＡＴＰ等の適切な補因子を含んでいてもよい。反応時間は、例えば１０分～１５０時間、好ましくは２０分～１２０時間、より好ましくは３０分～１００時間、さらにより好ましくは６０分～８０時間である。Ｌｐｌの濃度は、適宜調製することができる。生体直交性官能基の反応は、周知である（例、Ｓｈａｒｐｌｅｓｓ　Ｋ．Ｂ．ｅｔ　ａｌ．，Ａｎｇｅｗ．Ｃｈｅｍ．Ｉｎｔ．Ｅｄ．４０，２００４（２０１５）；Ｂｅｒｔｏｚｚｉ　Ｃ．Ｒ．ｅｔ　ａｌ．，Ｓｃｉｅｎｃｅ　２９１，２３５７（２００１）；Ｂｅｒｔｏｚｚｉ　Ｃ．Ｒ．ｅｔ　ａｌ．，Ｎａｔｕｒｅ　Ｃｈｅｍｉｃａｌ　Ｂｉｏｌｏｇｙ　１，１３（２００５）を参照）。 反 応 The reaction in step (2) can be appropriately performed under conditions (mild conditions) that do not cause denaturation / decomposition of the antibody (eg, cleavage of an amide bond). For example, such a reaction can be carried out in a suitable reaction system, for example, a buffer (eg, a phosphate buffer) at normal temperature (for example, about 15 to 35 ° C., preferably about 20 to 30 ° C.). The pH of the buffer is, for example, 5 to 9, preferably 5.5 to 8.5, and more preferably 6.0 to 8.0. The buffer may contain a suitable cofactor such as ATP. The reaction time is, for example, 10 minutes to 150 hours, preferably 20 minutes to 120 hours, more preferably 30 minutes to 100 hours, and even more preferably 60 minutes to 80 hours. The concentration of Lpl can be appropriately adjusted. Reactions of bioorthogonal functional groups are well known (eg, Sharless @ KB et al., Angew. Chem. Int. Ed. 40, 2004 (2015); Bertozzi {CR et al., Science # 291). , 2357 (2001); Bertozzi CR et al., Nature Chemical Biology 1, 13 (2005)).
　反応系において、定常領域中のリジン残基の側鎖に生体直交性官能基を含む修飾部分を有する修飾抗体（Ｙ）に対する、機能性物質（Ｚ）のモル比率（Ｚ／Ｙ）は、生体直行性官能基、機能性物質、および抗体の種類、ならびに修飾されるべき抗体中の部位の数（例、ＤＡＲ）等に応じて変動することから特に限定されないが、例えば０．１～１００であり、好ましくは０．５～８０であり、より好ましくは１～７０であり、さらにより好ましくは２～５０であり、特に好ましくは３～３０である。 In the reaction system, the molar ratio (Z / Y) of the functional substance (Z) to the modified antibody (Y) having a modified portion containing a bioorthogonal functional group in the side chain of a lysine residue in the constant region is determined by It is not particularly limited since it varies depending on the type of the orthogonal functional group, the functional substance, and the antibody, and the number of sites in the antibody to be modified (eg, DAR). Yes, preferably from 0.5 to 80, more preferably from 1 to 70, even more preferably from 2 to 50, particularly preferably from 3 to 30.
　定常領域中のリジン残基の側鎖に機能性物質を有する修飾抗体の生成の確認は、その具体的な原料および生成物の分子量にもよるが、例えば、電気泳動法、クロマトグラフィー（例、ゲル濾過クロマトグラフィー、イオン交換クロマトグラフィー、逆相カラムクロマトグラフィー、ＨＰＬＣ）、または質量分析により、好ましくは、質量分析により行うことができる。抗体において修飾されたリジン残基の位置の確認は、例えば、ペプチドマッピングにより行うことができる。ペプチドマッピングは、例えば、プロテアーゼ（例、トリプシン、キモトリプシン、）処理および質量分析により行うことができる。プロテアーゼとしては、エンドプロテアーゼが好ましい。このようなエンドプロテアーゼとしては、例えば、トリプシン、キモトリプシン、Ｇｌｕ－Ｃ、Ｌｙｓ－Ｎ、Ｌｙｓ－Ｃ、Ａｓｐ－Ｎが挙げられる。定常領域中のリジン残基の側鎖に機能性物質を有する修飾抗体が保有する、機能性物質の個数の確認は、例えば、電気泳動法、クロマトグラフィー、または質量分析により、好ましくは、質量分析により行うことができる。定常領域中のリジン残基の側鎖に機能性物質を有する修飾抗体は、クロマトグラフィー（例、上述したクロマトグラフィー、およびアフィニティークロマトグラフィー）等の任意の方法により適宜精製することができる。 Confirmation of the generation of a modified antibody having a functional substance on the side chain of a lysine residue in the constant region depends on the specific raw material and the molecular weight of the product. For example, electrophoresis, chromatography (eg, Gel filtration chromatography, ion exchange chromatography, reverse phase column chromatography, HPLC) or mass spectrometry, preferably mass spectrometry. Confirmation of the position of the modified lysine residue in the antibody can be performed, for example, by peptide mapping. Peptide mapping can be performed, for example, by protease (eg, trypsin, chymotrypsin) treatment and mass spectrometry. As the protease, an endoprotease is preferable. Such endoproteases include, for example, trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, Asp-N. Confirmation of the number of functional substances carried by a modified antibody having a functional substance on the side chain of a lysine residue in the constant region is performed, for example, by electrophoresis, chromatography, or mass spectrometry, preferably by mass spectrometry. Can be performed. The modified antibody having a functional substance on the side chain of a lysine residue in the constant region can be appropriately purified by any method such as chromatography (eg, the above-described chromatography and affinity chromatography).
（２．修飾抗体）
　本発明はまた、定常領域中のリジン残基の側鎖に目的部分（修飾部分または機能性物質）を有する修飾抗体を提供する。 (2. Modified antibody)
The present invention also provides a modified antibody having a target portion (modified portion or functional substance) on the side chain of a lysine residue in the constant region.
　本発明の修飾抗体は、修飾部分を有するＣ４～Ｃ１０アルキル－カルボニルを、抗体特有のリジン残基の側鎖のみに有する。「抗体特有のリジン残基」とは、抗体の重鎖における可変領域（ＶＨ）、および定常領域（ＣＨ１、ＣＨ２、ＣＨ３）、ならびに抗体の軽鎖における可変領域（ＶＬ）、および定常領域（ＣＬ）を有する全長抗体またはその抗体断片中に存在する１以上のリジン残基をいう。したがって、抗体の所定の領域に所定のアミノ酸配列（例、タグ配列、スペーサー配列）が付加または挿入された構造を有する融合タンパク質である抗体において、修飾部分を有するＣ４～Ｃ１０アルキル－カルボニルを、付加または挿入された所定のアミノ酸配列に対応するリジン残基の側鎖に有する修飾抗体は、本発明の修飾抗体から除かれる。 The modified antibody of the present invention has a C 4 -C 10 alkyl-carbonyl having a modified portion only on the side chain of a lysine residue unique to the antibody. "An antibody-specific lysine residue" refers to the variable region (VH) and constant region (CH1, CH2, CH3) in the heavy chain of an antibody, and the variable region (VL) and constant region (CL) in the light chain of an antibody. ) Refers to one or more lysine residues present in a full length antibody or antibody fragment thereof. Therefore, in an antibody that is a fusion protein having a structure in which a predetermined amino acid sequence (eg, a tag sequence, a spacer sequence) is added or inserted into a predetermined region of the antibody, a C 4 -C 10 alkyl-carbonyl having a modified portion is substituted with A modified antibody having a side chain of a lysine residue corresponding to a predetermined amino acid sequence added or inserted is excluded from the modified antibody of the present invention.
　本発明の修飾抗体は、上述した本発明の方法で用いられる、修飾部分を有するリポ酸アナログの種類に応じて、異なる構造を有する抗体として生成される。例えば、修飾部分を有するリポ酸アナログが、修飾部分を有するＣ４～Ｃ１０アルキル－カルボン酸である場合、定常領域中のリジン残基の側鎖に修飾部分を有する抗体として、修飾部分を有するＣ４～Ｃ１０アルキル－カルボニルを、定常領域中のリジン残基の側鎖に有する抗体が生成される。修飾部分を有するリポ酸アナログが、修飾部分を有するＣ５～Ｃ９アルキル－カルボン酸である場合、定常領域中のリジン残基の側鎖に修飾部分を有する抗体として、修飾部分を有するＣ５～Ｃ９アルキル－カルボニルを、定常領域中のリジン残基の側鎖に有する抗体が生成される。修飾部分を有するリポ酸アナログが、修飾部分を有するＣ６～Ｃ８アルキル－カルボン酸である場合、定常領域中のリジン残基の側鎖に修飾部分を有する抗体として、修飾部分を有するＣ６～Ｃ８アルキル－カルボニルを、定常領域中のリジン残基の側鎖に有する抗体が生成される。修飾部分を有するリポ酸アナログが、修飾部分を有するＣ７アルキル－カルボン酸である場合、定常領域中のリジン残基の側鎖に修飾部分を有する抗体として、修飾部分を有するＣ７アルキル－カルボニルを、定常領域中のリジン残基の側鎖に有する抗体が生成される。 The modified antibody of the present invention is produced as an antibody having a different structure depending on the type of the lipoic acid analog having a modified moiety used in the above-described method of the present invention. For example, when the lipoic acid analog having a modified moiety is a C 4 -C 10 alkyl-carboxylic acid having a modified moiety, the antibody having the modified moiety on the side chain of a lysine residue in the constant region has the modified moiety. Antibodies are generated that have a C 4 -C 10 alkyl-carbonyl in the side chain of a lysine residue in the constant region. Lipoic acid analogs having modified parts, C 5 ~ C 9 alkyl with the modifying moiety - If a carboxylic acid, as an antibody having a modifying moiety to the side chain of a lysine residue in the constant region, C 5 having a modifying moiety Antibodies are generated that have ～ C 9 alkyl-carbonyl in the side chain of a lysine residue in the constant region. When the lipoic acid analog having a modified moiety is a C 6 -C 8 alkyl-carboxylic acid having a modified moiety, an antibody having a modified moiety on the side chain of a lysine residue in the constant region may be a C 6 having a modified moiety. Antibodies are generated that have ～ C 8 alkyl-carbonyl in the side chain of a lysine residue in the constant region. When the lipoic acid analog having a modified moiety is a C 7 alkyl-carboxylic acid having a modified moiety, an antibody having a modified moiety on the side chain of a lysine residue in the constant region may be used as a C 7 alkyl-carbonyl having a modified moiety. On the side chain of a lysine residue in the constant region.
　上述した本発明の方法によれば、特定の定常領域（重鎖のＣＨ１領域、軽鎖のＣＬ領域）中のリジン残基の側鎖に修飾部分を有する修飾抗体を生成することができる。本発明者らが把握する限り、このような簡便な構造でこのような特定の位置のリジン残基を高度に修飾することに成功した事例は知られていない。したがって、本発明の修飾抗体は、重鎖のＣＨ１領域中のリジン残基の側鎖に修飾部分を有する修飾抗体である。本発明の修飾抗体はまた、重鎖のＣＨ１領域および軽鎖のＣＬ領域の双方に存在するリジン残基の側鎖に修飾部分を有する修飾抗体であってもよい。好ましくは、本発明の修飾抗体は、ヒトＩｇＧ重鎖における１３３位のリジン残基、およびヒトＩｇＧ軽鎖における１６９位のリジン残基の双方の側鎖に修飾部分を有する修飾抗体である。なお、本発明では、軽鎖のアミノ酸残基の位置に関してはアミノ酸配列番号１５中の位置で示し、重鎖のアミノ酸配列番号１４における１２０番目以前のアミノ酸残基の位置に関してはアミノ酸配列番号１４中の位置で示し、重鎖のアミノ酸配列番号１４における１２１番目以降のアミノ酸残基に関してはＥＵ　ｎｕｍｂｅｒｉｎｇの位置で示す（したがって、ヒトＩｇＧ重鎖における１３３位のリジン残基、およびヒトＩｇＧ軽鎖における１６９位のリジン残基の位置は、ヒトＩｇＧにおけるＥｕ　ｎｕｍｂｅｒｉｎｇに従う。本明細書中で同様。）。重鎖のＣＨ１領域および軽鎖のＣＬ領域は、定常領域のなかでも抗原結合部位の近傍に存在し、そしてこれらの領域に存在する所望の修飾は、修飾や抗原を含む標的の種類にもよるものの、抗原を含む標的に対して強く相互作用できる可能性がある。また、これらの領域に存在する所望の修飾は、抗体との結合部位を有する細胞に対して修飾物（例、薬剤）を効率的に導入できる可能性がある。したがって、ＣＨ１領域およびＣＬ領域において所望の修飾を有する本発明の修飾抗体は、医薬または試薬として有用であり得る。本発明の修飾抗体は、２個の重鎖のＣＨ１領域における２個のリジン残基（例、１３３位のリジン残基２個）の側鎖に修飾部分を有することができる。本発明の修飾抗体はまた、２個の軽鎖のＣＬ領域における２個のリジン残基（例、１６９位のリジン残基２個）の側鎖に修飾部分を有することができる。本発明の修飾抗体は、このようなリジン残基が高度に修飾されたものであり得る。例えば、本発明の修飾抗体では、ヒトＩｇＧ　ＣＨ１全体の修飾に占める１３３位のリジン残基の修飾割合は、３０％以上、好ましくは４０％以上、より好ましくは５０％以上、さらにより好ましくは６０％以上、特に好ましくは７０％以上であってもよい。一方、ヒトＩｇＧ　ＣＨ１全体の修飾に占めるヒトＩｇＧ　ＣＨ１中の１３３位以外の各位置のリジン残基の修飾割合は、低度（例、３０％未満、２０％未満、１０％未満、または５％未満）であり得る。また、ヒトＩｇＧ　ＣＬ全体の修飾に占める１６９位のリジン残基の修飾割合は、３０％以上であってもよい。一方、ヒトＩｇＧ　ＣＬ全体の修飾に占めるヒトＩｇＧ　ＣＬ中の１６９位以外の各位置のリジン残基の修飾割合は、低度（例、３０％未満、２０％未満、１０％未満、または５％未満）であり得る。 According to the method of the present invention described above, a modified antibody having a modified portion in the side chain of a lysine residue in a specific constant region (CH1 region of a heavy chain, CL region of a light chain) can be produced. As far as the present inventors understand, there has been no known case in which such a simple structure has successfully modified a lysine residue at such a specific position to a high degree. Therefore, the modified antibody of the present invention is a modified antibody having a modified portion on the side chain of a lysine residue in the CH1 region of the heavy chain. The modified antibody of the present invention may also be a modified antibody having a modified portion on the side chain of a lysine residue present in both the CH1 region of the heavy chain and the CL region of the light chain. Preferably, the modified antibody of the present invention is a modified antibody having a modified portion in the side chain of both the lysine residue at position 133 in the human IgG heavy chain and the lysine residue at position 169 in the human IgG light chain. In the present invention, the position of the amino acid residue in the light chain is indicated by the position in amino acid sequence number 15, and the position of the amino acid residue before the 120th position in the amino acid sequence number 14 in the heavy chain is defined by the position in amino acid sequence number 14. And the amino acid residues from position 121 onwards in the amino acid sequence number 14 of the heavy chain are shown at the position of EU numbering (therefore, the lysine residue at position 133 in the human IgG heavy chain and 169 in the human IgG light chain). The position of the lysine residue at position follows Eu @ numbering in human IgG, as in the present specification.). The CH1 region of the heavy chain and the CL region of the light chain are amongst the constant regions near the antigen binding site, and the desired modifications present in these regions also depend on the type of modification and the target containing the antigen. However, it may be able to interact strongly with targets, including antigens. In addition, desired modifications existing in these regions may allow efficient introduction of a modification (eg, a drug) into cells having an antibody binding site. Therefore, the modified antibodies of the present invention having desired modifications in the CH1 region and CL region may be useful as medicines or reagents. The modified antibody of the present invention can have a modified portion in the side chain of two lysine residues (eg, two lysine residues at position 133) in the CH1 region of two heavy chains. The modified antibodies of the present invention can also have a modified moiety in the side chain of two lysine residues (eg, two lysine residues at position 169) in the CL regions of the two light chains. The modified antibody of the present invention may be one in which such a lysine residue is highly modified. For example, in the modified antibody of the present invention, the modification ratio of the lysine residue at position 133 in the total modification of human IgG CH1 is 30% or more, preferably 40% or more, more preferably 50% or more, and even more preferably 60% or more. %, Particularly preferably 70% or more. On the other hand, the modification ratio of lysine residues at positions other than position 133 in human IgG @ CH1 in the modification of human IgG @ CH1 is low (eg, less than 30%, less than 20%, less than 10%, or 5%). Less than). Further, the modification ratio of the lysine residue at position 169 in the modification of the whole human IgG @ CL may be 30% or more. On the other hand, the modification ratio of lysine residues at positions other than position 169 in human IgG @ CL in the total modification of human IgG @ CL is low (eg, less than 30%, less than 20%, less than 10%, or 5%). Less than).
　本発明の修飾抗体はまた、抗体の重鎖における他の位置のリジン残基〔例、定常領域中のリジン残基（例、２２２位のリジン残基）、または可変領域中のリジン残基〕が修飾されていてもよい。例えば、本発明の修飾抗体では、ヒトＩｇＧ　ＣＨ２全体の修飾に占める２２２位のリジン残基の修飾割合は、３０％以上であってもよい。一方、本発明の修飾抗体では、ヒトＩｇＧ　ＣＨ２全体の修飾に占めるヒトＩｇＧ　ＣＨ２中の２２２位以外の各位置のリジン残基の修飾割合は、低度（例、３０％未満、２０％未満、１０％未満、または５％未満）であり得る。 The modified antibodies of the present invention also include lysine residues at other positions in the heavy chain of the antibody (eg, lysine residues in the constant region (eg, lysine residue at position 222) or lysine residues in the variable region). May be modified. For example, in the modified antibody of the present invention, the modification ratio of the lysine residue at position 222 in the modification of human IgGＩCH2 may be 30% or more. On the other hand, in the modified antibody of the present invention, the modification ratio of lysine residues at positions other than position 222 in human IgG @ CH2 in the modification of human IgG @ CH2 is low (eg, less than 30%, less than 20%, (Less than 10%, or less than 5%).
　本発明の修飾抗体はまた、ポリクローナル抗体またはモノクローナル抗体である。本発明の修飾抗体はまた、生体分子（例、糖）で修飾された抗体（例、糖タンパク質）であっても、生体分子で未修飾の抗体であってもよい。本発明の修飾抗体は、生物由来成分、ウイルス由来成分、および環境中に見出される成分等の任意の成分に対する任意の抗体であってもよいが、生物由来成分またはウイルス由来成分に対する抗体が好ましい。生物由来成分としては、例えば、哺乳動物、鳥類（例、ニワトリ）等の動物、昆虫、微生物、植物、菌類、および魚類由来の成分（例、タンパク質）が挙げられる。好ましくは、生物由来成分は、哺乳動物由来の成分である。哺乳動物としては、例えば、霊長類（例、ヒト、サル、チンパンジー）、齧歯類（例、マウス、ラット、モルモット、ハムスター、ウサギ）、愛玩動物（例、イヌ、ネコ）、家畜（例、ウシ、ブタ、ヤギ）、使役動物（例、ウマ、ヒツジ）が挙げられる。生物由来成分は、より好ましくは、霊長類または齧歯類由来の成分（例、タンパク質）であり、さらにより好ましくは、本発明の臨床応用の観点から、ヒト由来の成分（例、タンパク質）である。ウイルス由来成分としては、例えば、インフルエンザウイルス（例、トリインフルエンザウイルス、ブタインフルエンザウイルス）、エイズウイルス、エボラウイルス、ファージウイルスに由来する成分（例、タンパク質）が挙げられる。 修飾 The modified antibody of the present invention is also a polyclonal antibody or a monoclonal antibody. The modified antibody of the present invention may be an antibody (eg, glycoprotein) modified with a biomolecule (eg, sugar) or an antibody not modified with a biomolecule. The modified antibody of the present invention may be any antibody against any component such as a biological component, a virus-derived component, and a component found in the environment, but is preferably an antibody against a biological-derived component or a virus-derived component. Examples of the biological component include components (eg, proteins) derived from animals such as mammals and birds (eg, chickens), insects, microorganisms, plants, fungi, and fish. Preferably, the biological component is a component derived from a mammal. Examples of mammals include primates (eg, humans, monkeys, chimpanzees), rodents (eg, mice, rats, guinea pigs, hamsters, rabbits), companion animals (eg, dogs, cats), livestock (eg, Cattle, pigs, goats) and working animals (eg, horses, sheep). The biological component is more preferably a primate or rodent-derived component (eg, a protein), and even more preferably, from the viewpoint of clinical application of the present invention, a human-derived component (eg, a protein). is there. Examples of virus-derived components include components (eg, proteins) derived from influenza virus (eg, avian influenza virus, swine influenza virus), AIDS virus, Ebola virus, and phage virus.
　本発明の修飾抗体はまた、任意の抗原に対する抗体である。例えば、このような抗原は、上述したような生物またはウイルスにおいて見出される成分であってもよい。このような抗原としてはまた、例えば、タンパク質〔オリゴペプチド、ポリペプチドを含む。糖等の生体分子で修飾されたタンパク質（例、糖タンパク質）であってもよい〕、糖鎖、核酸、低分子化合物が挙げられる。 修飾 The modified antibody of the present invention is also an antibody against any antigen. For example, such an antigen may be a component found in an organism or virus as described above. Such antigens also include, for example, proteins [including oligopeptides and polypeptides. It may be a protein modified with a biomolecule such as sugar (eg, glycoprotein)], a sugar chain, a nucleic acid, and a low molecular compound.
　好ましくは、本発明の修飾抗体は、タンパク質を抗原とする抗体であってもよい。タンパク質としては、例えば、細胞膜受容体、細胞膜受容体以外の細胞膜タンパク質（例、細胞外基質タンパク質）、リガンド、可溶性受容体が挙げられる。 Preferably, the modified antibody of the present invention may be an antibody having a protein as an antigen. Examples of proteins include cell membrane receptors, cell membrane proteins other than cell membrane receptors (eg, extracellular matrix proteins), ligands, and soluble receptors.
　より具体的には、本発明の修飾抗体の抗原であるタンパク質は、疾患標的タンパク質であってもよい。疾患標的タンパク質としては、例えば、（１）がん領域、（２）自己免疫疾患・炎症性疾患、（３）脳神経疾患、（４）感染症、（５）遺伝性・希少疾患、（６）眼疾患、（７）骨・整形外科領域、（８）血液疾患、（９）その他の疾患における上述したようなタンパク質が挙げられる。 More specifically, the protein that is the antigen of the modified antibody of the present invention may be a disease target protein. Examples of disease target proteins include (1) cancer regions, (2) autoimmune diseases / inflammatory diseases, (3) cranial nerve diseases, (4) infectious diseases, (5) hereditary / rare diseases, and (6) Examples include the above-mentioned proteins in eye diseases, (7) bone and orthopedic fields, (8) blood diseases, and (9) other diseases.
　好ましい実施形態では、本発明の修飾抗体は、上述したような、天然ポリペプチド鎖構造を有する。 In a preferred embodiment, the modified antibodies of the present invention have a native polypeptide chain structure, as described above.
　別の好ましい実施形態では、本発明の修飾抗体は、モノクローナル抗体である。モノクローナル抗体としては、例えば、キメラ抗体、ヒト化抗体、ヒト抗体、所定の糖鎖が付加された抗体（例、Ｎ型糖鎖結合コンセンサス配列等の糖鎖結合コンセンサス配列を有するように改変された抗体）、二重特異性抗体、ｓｃＦｖ抗体、Ｆａｂ抗体、Ｆ（ａｂ‘）２抗体、ＶＨＨ抗体、Ｆｃ領域タンパク質、Ｆｃ融合タンパク質が挙げられる。モノクローナル抗体等の抗体のアイソタイプとしては、例えば、ＩｇＧ、ＩｇＭ、ＩｇＡ、ＩｇＤ、ＩｇＥ、およびＩｇＹが挙げられる。抗体はまた、２価の抗体（例、ＩｇＧ、ＩｇＤ、ＩｇＥ）、または４価以上の抗体（例、ＩｇＡ抗体、ＩｇＭ抗体）であってもよい。 In another preferred embodiment, the modified antibodies of the present invention are monoclonal antibodies. As the monoclonal antibody, for example, a chimeric antibody, a humanized antibody, a human antibody, an antibody to which a predetermined sugar chain is added (for example, modified to have a sugar chain binding consensus sequence such as an N-type sugar chain binding consensus sequence) Antibodies), bispecific antibodies, scFv antibodies, Fab antibodies, F (ab ') 2 antibodies, VHH antibodies, Fc region proteins, and Fc fusion proteins. Isotypes of antibodies such as monoclonal antibodies include, for example, IgG, IgM, IgA, IgD, IgE, and IgY. The antibody may also be a bivalent antibody (eg, IgG, IgD, IgE) or a tetravalent or higher-valent antibody (eg, IgA antibody, IgM antibody).
　さらに別の好ましい実施形態では、本発明の修飾抗体は、全長抗体、または抗体断片（例、Ｆａｂ、Ｆ（ａｂ’）２、Ｆａｂ’、Ｆｖ、単鎖抗体）であってもよい。抗体断片は、重鎖のＣＨ１領域または軽鎖のＣＬ領域のいずれかを有することが好ましく、重鎖のＣＨ１領域および軽鎖のＣＬ領域の双方を有することがより好ましい。 In yet another preferred embodiment, the modified antibodies of the present invention may be full length antibodies, or antibody fragments (eg, Fab, F (ab ') 2 , Fab', Fv, single chain antibodies). The antibody fragment preferably has either the CH1 region of the heavy chain or the CL region of the light chain, and more preferably has both the CH1 region of the heavy chain and the CL region of the light chain.
　さらに別の好ましい実施形態では、本発明の修飾抗体は、ヒト抗体またはヒト化抗体である。ヒト抗体およびヒト化抗体の具体例は、上述したものと同じである。 で は In yet another preferred embodiment, the modified antibodies of the present invention are human or humanized antibodies. Specific examples of the human antibody and the humanized antibody are the same as those described above.
　さらに別の好ましい実施形態では、本発明の修飾抗体は、ＩｇＧである。ＩｇＧとしては、例えば、ＩｇＧ１、ＩｇＧ２、ＩｇＧ３、ＩｇＧ４が挙げられる。ＩｇＧとしては、ヒトＩｇＧが好ましい。 で は In yet another preferred embodiment, the modified antibodies of the invention are IgG. Examples of IgG include IgG1, IgG2, IgG3, and IgG4. As IgG, human IgG is preferable.
　さらに別の好ましい実施形態では、本発明の修飾抗体は、Ｆａｂ、またはＦ（ａｂ’）２である。 In yet another preferred embodiment, the modified antibodies of the invention are Fab, or F (ab ') 2 .
　一実施形態では、本発明の修飾抗体は、定常領域中のリジン残基の側鎖に修飾部分を有する修飾抗体である。 In one embodiment, the modified antibody of the present invention is a modified antibody having a modification portion on the side chain of a lysine residue in the constant region.
　好ましくは、定常領域中のリジン残基の側鎖に修飾部分を有する本発明の修飾抗体は、下記式（ＩＩ）で表される部分を抗体特有のリジン残基の側鎖のみに有する：
Ｒ－ＣｎＨ２ｎ－ＣＯ－ＮＨ－　　　（ＩＩ）
〔式中、
　Ｒは、修飾部分であり、
　ｎは、４～１０の整数であり、
　ＮＨ－は、定常領域中のリジン残基の側鎖に存在する基である。〕 Preferably, the modified antibody of the present invention having a modified portion on the side chain of a lysine residue in the constant region has a portion represented by the following formula (II) only on the side chain of the antibody-specific lysine residue:
R—C n H 2n —CO—NH— (II)
(In the formula,
R is a modifying moiety;
n is an integer of 4 to 10,
NH- is a group present on the side chain of a lysine residue in the constant region. ]
　上記式（ＩＩ）において、修飾部分は、上述のものと同じである。 に お い て In the above formula (II), the modifying moiety is the same as described above.
　上記式（ＩＩ）において、ｎは、好ましくは５～９の整数、より好ましくは６～８の整数、特に好ましくは７である。 Ｎ In the above formula (II), n is preferably an integer of 5 to 9, more preferably an integer of 6 to 8, and particularly preferably 7.
　特定の実施形態では、本発明の修飾抗体における修飾部分は、生体直交性官能基または機能性物質を含む。このような本発明の修飾抗体は、上述した本発明の方法（抗体を、リポ酸タンパク質リガーゼＡの存在下において、生体直交性官能基を含む修飾部分を有するリポ酸アナログと反応させて、定常領域中のリジン残基の側鎖に生体直交性官能基を含む修飾部分を有する修飾抗体を生成すること）により得ることができる。このようにして得られる本発明の修飾抗体（修飾部分として生体直交性官能基を含むもの）は、機能性物質を有する抗体の合成のための中間体として有用である。また、このようにして得られる本発明の修飾抗体（修飾部分として機能性物質を含むもの）は、医薬、または試薬（例、診断薬、研究用試薬）として有用である。 で は In certain embodiments, the modifying moiety in the modified antibodies of the present invention comprises a bioorthogonal functional group or a functional substance. Such a modified antibody of the present invention can be prepared by reacting the antibody with a lipoic acid analog having a modified portion containing a bioorthogonal functional group in the presence of lipoic acid protein ligase A as described above. Producing a modified antibody having a modified moiety containing a bioorthogonal functional group on the side chain of a lysine residue in the region. The modified antibody of the present invention (containing a bioorthogonal functional group as a modifying moiety) thus obtained is useful as an intermediate for the synthesis of an antibody having a functional substance. The thus obtained modified antibody of the present invention (containing a functional substance as a modifying moiety) is useful as a medicine or a reagent (eg, a diagnostic agent, a research reagent).
　特定の好ましい実施形態では、本発明の修飾抗体における修飾部分は、生体直交性官能基を含む。このようにして得られる本発明の修飾抗体（修飾部分として生体直交性官能基を含むもの）は、種々の機能性物質を有する抗体の合成のための中間体として有用である（特に、修飾部分として機能性物質を有するリポ酸アナログが、機能性物質のサイズ等に起因して、Ｌｐｌの基質として作用できない場合、またはＬｐｌの基質として作用できるものの、その酵素としての反応速度が遅い場合）。 で は In certain preferred embodiments, the modifying moiety in the modified antibodies of the present invention comprises a bioorthogonal functional group. The modified antibody of the present invention (containing a bioorthogonal functional group as a modified moiety) thus obtained is useful as an intermediate for the synthesis of antibodies having various functional substances (in particular, the modified moiety). Lipoic acid analogs having a functional substance cannot function as an Lpl substrate due to the size of the functional substance, or can function as an Lpl substrate but have a low reaction rate as an enzyme).
　別の実施形態では、本発明の修飾抗体は、定常領域中のリジン残基の側鎖に機能性物質を有する修飾抗体である。 In another embodiment, the modified antibody of the present invention is a modified antibody having a functional substance on the side chain of a lysine residue in the constant region.
　好ましくは、定常領域中のリジン残基の側鎖に機能性物質を有する本発明の修飾抗体は、下記式（ＩＩＩ）で表される部分を抗体特有のリジン残基の側鎖のみに有する：
Ｆ－Ｒ’－ＣｎＨ２ｎ－ＣＯ－ＮＨ－　　　（ＩＩＩ）
〔式中、
　Ｆは、機能性物質であり、
　Ｒ’は、機能性物質と生体直交性官能基との間の反応により生成する部分を含む２価の基であり、
　ｎは、４～１０の整数であり、
　ＮＨ－は、定常領域中のリジン残基の側鎖に存在する基である。〕 Preferably, the modified antibody of the present invention having a functional substance on the side chain of a lysine residue in the constant region has a portion represented by the following formula (III) only in the side chain of the antibody-specific lysine residue:
F-R'-C n H 2n -CO-NH- (III)
(In the formula,
F is a functional substance,
R ′ is a divalent group including a moiety generated by a reaction between a functional substance and a bioorthogonal functional group,
n is an integer of 4 to 10,
NH- is a group present on the side chain of a lysine residue in the constant region. ]
　上記式（ＩＩＩ）において、機能性物質、生体直交性官能基、および機能性物質と生体直交性官能基との間の反応により生成する部分を含む２価の基は、上述のものと同じである。 In the above formula (III), the functional substance, the bio-orthogonal functional group, and the divalent group containing the moiety generated by the reaction between the functional substance and the bio-orthogonal functional group are the same as those described above. is there.
　上記式（ＩＩＩ）において、ｎは、好ましくは５～９の整数、より好ましくは６～８の整数、特に好ましくは７である。 に お い て In the above formula (III), n is preferably an integer of 5 to 9, more preferably an integer of 6 to 8, and particularly preferably 7.
　定常領域中のリジン残基の側鎖に機能性物質を有する本発明の修飾抗体は、上述した工程（１）および（２）を含む本発明の方法により得ることができる。このようにして得られる本発明の修飾抗体は、医薬、または試薬（例、診断薬、研究用試薬）として有用である。 修飾 The modified antibody of the present invention having a functional substance on the side chain of a lysine residue in the constant region can be obtained by the method of the present invention including the above-described steps (1) and (2). The modified antibody of the present invention thus obtained is useful as a medicine or a reagent (eg, a diagnostic agent, a research reagent).
　次に実施例を示して本発明をさらに詳細に説明するが、本発明は以下の実施例に限定されるものではない。 Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.
（実施例１）ＩｇＧ抗体の修飾活性の探索
（１－１）Ｅ．ｃｏｌｉ　ＬｐｌＡの発現と精製
　リポ酸タンパク質リガーゼ（Ｌｐｌ）としては、Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ由来のＬｐｌＡ（ＥｃＬｐｌＡ）とグルタチオン－Ｓ－トランスフェラーゼ（ＧＳＴ）との融合タンパク質を用いた。ＥｃＬｐｌＡとＧＳＴとの融合タンパク質を発現するプラスミド（以下、単に「ＥｃＬｐｌＡ発現プラスミド」と呼ぶ）を得るため、ＬｐｌＡ全長配列をコードする塩基配列をｐＣｏｌｄ　ＧＳＴ　ＤＮＡベクター（ＴａＫａＲａ社）にクローニングして、ｐＣｏｌｄ－ＬｐｌＡ（Ｅｃ）を構築した。なお、ｐＣｏｌｄ－ｌｐｌＡ（Ｅｃ）にはベクター由来のヒスチジンタグが付加してある。 Example 1 Search for Modification Activity of IgG Antibody (1-1) Expression and purification of E. coli LplA As lipoic acid protein ligase (Lpl), a fusion protein of LplA (EcLplA) derived from Escherichia coli and glutathione-S-transferase (GST) was used. In order to obtain a plasmid that expresses a fusion protein of EcLplA and GST (hereinafter simply referred to as “EcLplA expression plasmid”), the base sequence encoding the full-length LplA sequence is cloned into a pCold GST DNA vector (TaKaRa), and pCold -LplA (Ec) was constructed. A histidine tag derived from a vector is added to pCold-IplA (Ec).
　ｐＣｏｌｄ－ＬｐｌＡ（Ｅｃ）は、以下の方法により構築した。Ｅ．ｃｏｌｉ　ＭＧ１６５５株のゲノムＤＮＡを鋳型鎖として、ｌｐｌＡ（Ｅｃ）　ｆｗ（配列番号９）およびｌｐｌＡ（Ｅｃ）　ｒｖ（配列番号１０）をプライマーとして用いたＰＣＲによって、ＥｃＬｐｌＡをコードする塩基配列を含むＤＮＡ断片を取得した。ＰＣＲにはＰｒｉｍｅＳｔａｒポリメラーゼ（ＴａＫａＲａ社）を用い、添付のプロトコールに記載の反応組成で行った。ＰＣＲサイクルは９４℃５分の後、９８℃５秒、５５℃１０秒、７２℃２分を３０サイクル、最後に４℃保温の条件で行った。また、ｐＣｏｌｄ　ＧＳＴ　ＤＮＡ（配列番号１１）をテンプレートＤＮＡとし、配列番号１２および１３のオリゴヌクレオチドをプライマーとして用いたＰＣＲによって、ｐＣｏｌｄ　ＧＳＴ　ＤＮＡのＤＮＡ断片を得た。ＰＣＲにはＰｒｉｍｅＳｔａｒポリメラーゼを用い、添付のプロトコールに記載の反応組成で行い、ＰＣＲサイクルは９４℃５分の後、９８℃５秒、５５℃１０秒、７２℃５分を３０サイクル、最後に４℃保温の条件で行った。得られた両ＤＮＡ断片をＩｎ－Ｆｕｓｉｏｎ　ＨＤクローニングキット（クロンテック社）を用いて連結し、ＥｃＬｐｌＡ発現プラスミドｐＣｏｌｄ－ｌｐｌＡ（Ｅｃ）を構築した。ｌｐｌＡ（Ｅｃ）の塩基配列およびそれがコードするアミノ酸配列を、それぞれ配列番号１および２に示す。 PCold-LplA (Ec) was constructed by the following method. E. FIG. A DNA fragment containing a base sequence encoding EcLplA by PCR using genomic DNA of E. coli strain MG1655 as a template chain and lplA (Ec) fw (SEQ ID NO: 9) and lplA (Ec) rv (SEQ ID NO: 10) as primers I got PCR was performed using PrimeStar polymerase (TaKaRa) with the reaction composition described in the attached protocol. The PCR cycle was performed at 94 ° C for 5 minutes, followed by 30 cycles of 98 ° C for 5 seconds, 55 ° C for 10 seconds, and 72 ° C for 2 minutes, and finally, at 4 ° C. In addition, a DNA fragment of pCold \ GST \ DNA was obtained by PCR using pCold \ GST \ DNA (SEQ ID NO: 11) as a template DNA and oligonucleotides of SEQ ID NOS: 12 and 13 as primers. The PCR was performed using PrimeStar polymerase with the reaction composition described in the attached protocol. The PCR cycle was 94 ° C for 5 minutes, followed by 30 cycles of 98 ° C for 5 seconds, 55 ° C for 10 seconds and 72 ° C for 5 minutes, and finally 4 cycles. The test was performed under the condition of keeping the temperature at ℃. The obtained DNA fragments were ligated using an In-Fusion @ HD cloning kit (Clontech) to construct an EcLplA expression plasmid pCold-lplA (Ec). The nucleotide sequence of lplA (Ec) and the amino acid sequence encoded thereby are shown in SEQ ID NOs: 1 and 2, respectively.
　ＥｃＬｐｌＡ発現プラスミドｐＣｏｌｄ－ｌｐｌＡ（Ｅｃ）をＥ．ｃｏｌｉ　ＪＭ１０９株（ＴａＫａＲａ社）へヒートショックを用いた形質転換法により導入し、ＪＭ１０９／ｐＣｏｌｄ－ｌｐｌＡ（Ｅｃ）株を得た。ヒートショックを用いた形質転換法は添付のプロトコールに記載の方法で行った。この株をＬＢ培地（１．０％（ｗ／ｖ）ペプトン、０．５０％（ｗ／ｖ）酵母エキス、１．０％（ｗ／ｖ）ＮａＣｌ）に１００μｇ／ｍＬアンピシリンを添加した培地に植菌し、３７℃で一晩振盪培養を行った。その後、培養液を坂口フラスコに１００ｍＬ張りこんだＬＢ培地中に終濃度１％となるよう植菌した。３７℃にて３時間振とう培養後、イソプロピル－β－Ｄ－チオガラクトピラノシド（ＩＰＴＧ）を終濃度１．０ｍＭとなるよう添加した後、培養温度を１５℃に低下した条件にてさらに１６時間培養を行った。 を The EcLplA expression plasmid pCold-lplA (Ec) was coli JM109 strain (TaKaRa) was introduced by a transformation method using heat shock to obtain a JM109 / pCold-lplA (Ec) strain. Transformation using heat shock was performed according to the protocol described in the attached protocol. This strain was transformed into an LB medium (1.0% (w / v) peptone, 0.50% (w / v) yeast extract, 1.0% (w / v) NaCl) supplemented with 100 μg / mL ampicillin. The cells were inoculated and cultured with shaking at 37 ° C. overnight. Thereafter, the culture solution was inoculated to a final concentration of 1% in an LB medium in which 100 mL of the medium was inserted into a Sakaguchi flask. After culturing with shaking at 37 ° C. for 3 hours, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1.0 mM, and the culture temperature was further reduced to 15 ° C. Culture was performed for 16 hours.
　培養液を全て９，２００ｇ、５分間遠心分離して菌体を回収し、２０ｍＭ　Ｔｒｉｓ－ＨＣｌ（ｐＨ７．６）緩衝液１０ｍＬにより２回洗浄し、同緩衝液５０ｍＬにて懸濁した。得られた懸濁液を超音波破砕機（久保田商事）にて破砕処理（１７０Ｗ、４℃、１０分間）した後、９，１００ｇにて１０分間遠心分離を行い、得られた上清を菌体抽出液とした。 All the cultures were centrifuged at 9,200 g for 5 minutes to recover the cells, washed twice with 10 mL of 20 mM Tris-HCl (pH 7.6) buffer, and suspended in 50 mL of the same buffer. The resulting suspension was crushed (170 W, 4 ° C., 10 minutes) using an ultrasonic crusher (Kubota Shoji), and then centrifuged at 9,100 g for 10 minutes. It was a body extract.
　得られた菌体抽出液４５ｍＬを平衡化バッファー（２０ｍＭ　Ｔｒｉｓ（ｐＨ７．６）、３００ｍＭ　ＮａＣｌ、５．０ｍＭイミダゾール）により平衡化したカラム（Ｈｉｓ　ＴＡＬＯＮ　Ｓｕｐｅｒｆｌｏｗ　Ｃａｒｔｒｉｄｇｅ、５．０ｍＬ、クロンテック社）に通液してＥｃＬｐｌＡをカラムに吸着させた。その後、カラムをカラムレジン容量の４倍量の平衡化バッファーにて洗浄した。さらに終濃度３００ｍＭのイミダゾールを添加した平衡化バッファー（溶出バッファー）をカラムに通液してＥｃＬｐｌＡが含まれるフラクションを分取し、０．６３ｍｇ／ｍＬのフラクション１．０ｍＬを精製ＥｃＬｐｌＡとした。 45 mL of the obtained cell extract was passed through a column (His TALON Superflow Cartridge, 5.0 mL, Clontech) equilibrated with an equilibration buffer (20 mM Tris (pH 7.6), 300 mM NaCl, 5.0 mM imidazole). Then, EcLplA was adsorbed on the column. Thereafter, the column was washed with 4 times the volume of the column resin in equilibration buffer. Further, an equilibration buffer (elution buffer) to which imidazole having a final concentration of 300 mM was added was passed through the column to collect a fraction containing EcLplA, and 1.0 mL of a 0.63 mg / mL fraction was used as purified EcLplA.
（１－２）各種酵素のＩｇＧ抗体修飾活性の評価
　各種酵素のＩｇＧ抗体修飾活性を評価した。酵素としては、Ｌｐｌ、４’－ホスホパンテテイニルトランスフェラーゼ（４’－ｐｈｏｓｐｈｏｐａｎｔｅｔｈｅｉｎｙｌ　ｔｒａｎｓｆｅｒａｓｅ、ＰＰＴａｓｅ）、およびビオチンリガーゼを用いた。ＩｇＧ抗体としては、抗ＨＥＲ２　ＩｇＧ抗体トラスツズマブ（中外製薬）を用いた。 (1-2) Evaluation of IgG antibody modifying activities of various enzymes IgG antibody modifying activities of various enzymes were evaluated. As the enzymes, Lpl, 4'-phosphopantetheinyl transferase (4'-phosphopantheinyl transferase, PPTase), and biotin ligase were used. As an IgG antibody, an anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical) was used.
　Ｌｐｌによる抗体修飾反応は、次のとおり行なった。Ｌｐｌとして（１－１）で調製したＥｃＬｐｌＡを用いた。修飾物として８－アジドオクタン酸を用いた。反応組成は次のとおりである。トラスツズマブ５００μｇを１．０ｍＭリン酸ナトリウムバッファー（ｐＨ６．０）２５０μＬに溶解させて用いた。終濃度でトラスツズマブ１．０ｍｇ／ｍＬ、ＡＴＰ２．５ｍＭ、８－アジドオクタン酸（Ｓｕｎｄｉａ）０．５０ｍＭ、硫酸マグネシウム３．２ｍＭ、リン酸ナトリウムバッファー２５ｍＭ（ｐＨ７．０）を反応液組成とした混合液に、終濃度０．０５０ｍｇ／ｍＬのＥｃＬｐｌＡを加えて総量１００μＬの反応液を調製し、３０℃で１５時間反応させて、修飾抗体として８－アジドオクタン酸修飾トラスツズマブを生成させた。反応液にＥＤＴＡ－２Ｎａ溶液２μＬを添加（終濃度１０ｍＭ）し反応を停止した。 The antibody modification reaction with に よ る Lpl was performed as follows. EcLplA prepared in (1-1) was used as Lpl. 8-Azidooctanoic acid was used as a modification. The reaction composition is as follows. 500 μg of trastuzumab was dissolved in 250 μL of 1.0 mM sodium phosphate buffer (pH 6.0) and used. A mixed solution having a final concentration of 1.0 mg / mL trastuzumab, ATP 2.5 mM, 8-azidooctanoic acid (Sundia) 0.50 mM, magnesium sulfate 3.2 mM, and sodium phosphate buffer 25 mM (pH 7.0). Then, EcLplA having a final concentration of 0.050 mg / mL was added thereto to prepare a reaction solution having a total volume of 100 μL, and reacted at 30 ° C. for 15 hours to produce 8-azidooctanoic acid-modified trastuzumab as a modified antibody. 2 μL of an EDTA-2Na solution was added to the reaction solution (final concentration: 10 mM) to stop the reaction.
　ＰＰＴａｓｅによる抗体修飾反応は、次のとおり行なった。ＰＰＴａｓｅとしてＳＦＰ　ｓｙｎｔｈａｓｅ（Ｎｅｗ　Ｅｎｇｌａｎｄ　Ｂｉｏｌａｂｓ）を用いた。修飾物としてＣｏＡリチウム塩（シグマ・アルドリッチ）を用いた。反応組成は次のとおりである。終濃度でトラスツズマブ１．０ｍｇ／ｍＬ、ＣｏＡリチウム塩（シグマ・アルドリッチ）０．５０ｍＭ、硫酸マグネシウム２．０ｍＭ、Ｈｅｐｅｓ－ＫＯＨバッファー５０ｍＭ（ｐＨ７．５）を反応液組成とした混合液に、終濃度１．０μＭのＳＦＰ　ｓｙｎｔｈａｓｅを加えて総量１００μＬとし、３０℃で１５時間反応させて、修飾抗体としてＣｏＡ修飾トラスツズマブを生成させた。反応液にＥＤＴＡ－２Ｎａ溶液２μＬを添加（終濃度１０ｍＭ）し反応を停止した。 抗体 Antibody modification reaction with PPTase was performed as follows. SFP @ synthase (New @ England @ Biolabs) was used as the PPTase. CoA lithium salt (Sigma-Aldrich) was used as a modifier. The reaction composition is as follows. A final concentration of a mixture of trastuzumab 1.0 mg / mL, CoA lithium salt (Sigma-Aldrich) 0.50 mM, magnesium sulfate 2.0 mM, Hepes-KOH buffer 50 mM (pH 7.5) as a reaction solution was added to the final concentration. 1.0 μM SFP @ synthase was added to make a total volume of 100 μL, and reacted at 30 ° C. for 15 hours to generate CoA-modified trastuzumab as a modified antibody. 2 μL of an EDTA-2Na solution was added to the reaction solution (final concentration: 10 mM) to stop the reaction.
　ビオチンリガーゼよる抗体修飾反応は、次のとおり行なった。ビオチンリガーゼとしてＢｉｒＡ（Ａｂｃａｍ）を用いた。修飾物としてビオチンを用いた。反応組成は次のとおりである。終濃度でトラスツズマブ１．０ｍｇ／ｍＬ、ＡＴＰ１．０ｍＭ、ビオチン０．５０ｍＭ、硫酸マグネシウム１．０ｍＭ、Ｔｒｉｓ塩酸バッファー５０ｍＭ（ｐＨ８．３）を反応液組成とした混合液に、終濃度０．０５０ｍｇ／ｍＬのＢｉｒＡを加えて総量１００μＬとし、３０℃で１５時間反応させて、修飾抗体としてビオチン修飾トラスツズマブを生成させた。反応液にＥＤＴＡ－２Ｎａ溶液２μＬを添加（終濃度１０ｍＭ）し反応を停止した。 抗体 Antibody modification with biotin ligase was performed as follows. BirA (Abcam) was used as biotin ligase. Biotin was used as a modification. The reaction composition is as follows. A final concentration of 1.050 mg / mL of trastuzumab, 1.0 mM of ATP, 0.50 mM of biotin, 1.0 mM of magnesium sulfate, and 50 mM of Tris-HCl buffer (pH 8.3) was added to a mixed solution having a final concentration of 0.050 mg / mL. mL of BirA was added to make a total volume of 100 μL, and reacted at 30 ° C. for 15 hours to generate biotin-modified trastuzumab as a modified antibody. 2 μL of an EDTA-2Na solution was added to the reaction solution (final concentration: 10 mM) to stop the reaction.
（１－３）反応物の精製
　Ｐｒｏｔｅｉｎ　Ａカラム（Ｐｒｏｅｉｎ　Ａ　ＨＰ　ＭｕｌｔｉＴｒａｐ（９６ｗｅｌｌ）、ＧＥヘルスケア）により修飾抗体を精製した。
　Ｐｒｏｔｅｉｎ　Ａカラムは２０ｍＭ　Ｔｒｉｓ（ｐＨ７．６）、２０％（ｖ／ｖ）エタノールにて平衡化したものを用いた。通液には遠心分離機を用い、以降は各段階で９００ｇにて１分間遠心操作した。トラスツズマブ５００μｇを含む酵素反応液５００μＬを２５０μＬずつ２つのウェルに分割しアプライ後、３００μＬの平衡化バッファーにて２回洗浄した。さらに１００ｍＭグリシンバッファー（ｐＨ２．７）２００μＬを添加して溶出した後、ただちに１．０Ｍ　Ｔｒｉｓ－ＨＣｌを１５μＬ添加しｐＨを８．５付近に調整し、修飾抗体サンプルとした。 (1-3) Purification of the reaction product The modified antibody was purified using a Protein A column (Protein A HP MultiTrap (96 well), GE Healthcare).
The Protein A column used was equilibrated with 20 mM Tris (pH 7.6) and 20% (v / v) ethanol. A centrifugal separator was used to pass the solution, and centrifugation was performed at 900 g for 1 minute at each stage. 500 μL of the enzyme reaction solution containing 500 μg of trastuzumab was divided into two wells of 250 μL each, applied, and washed twice with 300 μL of equilibration buffer. Further, 200 μL of 100 mM glycine buffer (pH 2.7) was added and eluted. Immediately, 15 μL of 1.0 M Tris-HCl was added to adjust the pH to around 8.5 to obtain a modified antibody sample.
（１－４）蛍光修飾
　（１－３）で精製した修飾抗体サンプルのうち、Ｌｐｌ反応物およびＰＰＴａｓｅ反応物については修飾体分析のため蛍光標識をした。Ｌｐｌ反応物は終濃度０．１０ｍＭ　ＤＢＣＯ－Ｃｙ３（ライフテクノロジーズ）、ＰＰＴａｓｅ反応物は終濃度０．１０ｍＭ　Ｃｙ３－マレイミド（Ｌｕｍｉｐｒｏｂｅ）を添加し、３０℃で３時間標識反応した。 (1-4) Fluorescence modification Among the modified antibody samples purified in (1-3), the Lpl reaction product and the PPTase reaction product were labeled with a fluorescent compound for analysis of the modified product. The Lpl reaction product was added with a final concentration of 0.10 mM DBCO-Cy3 (Life Technologies), and the PPTase reaction product was added with a final concentration of 0.10 mM Cy3-maleimide (Lumiprobe), followed by labeling reaction at 30 ° C. for 3 hours.
（１－５）反応物の分析
　Ｌｐｌ反応物およびＰＰＴａｓｅ反応物については、以下の方法により抗体への修飾を評価した。（１－４）で蛍光標識した修飾抗体サンプルを（１－３）と同様の方法で精製した。蛍光標識後の精製サンプル中の蛍光標識量を定量することにより抗体への修飾を評価した。全ての分析は９６穴プレート上で行い、蛍光は励起波長５２０ｎｍ、蛍光波長５８０～６４０ｎｍにて検出した。
　ビオチンリガーゼ反応物については、以下の方法により抗体への修飾を評価した。（１－３）で精製した修飾抗体サンプル中の側鎖ビオチン濃度をＦｌｕｏｒｅｓｃｅｎｃｅ　Ｂｉｏｔｉｎ　Ｑｕａｎｔｉｔａｔｉｏｎ　Ｋｉｔ（サーモフィッシャーサイエンティフィック製）により定量することにより抗体への修飾を評価した。Ｆｌｕｏｒｅｓｃｅｎｃｅ　Ｂｉｏｔｉｎ　Ｑｕａｎｔｉｔａｔｉｏｎ　Ｋｉｔは溶液中の側鎖ビオチン濃度を蛍光標識にて定量することが可能であり、０～６．０μＭまで定量性を確認している。側鎖ビオチン濃度の定量においては励起波長４７５ｎｍ、蛍光波長５００～５５０ｎｍにて検出した。プレートリーダーとしてＧｒｏＭＡＸ（プロメガ）を用いた。
　分析の結果、Ｌｐｌ反応物のみに抗体への修飾が観測された（表２）。 (1-5) Analysis of reaction product The Lpl reaction product and the PPTase reaction product were evaluated for antibody modification by the following method. The modified antibody sample fluorescently labeled in (1-4) was purified in the same manner as in (1-3). The modification to the antibody was evaluated by quantifying the amount of the fluorescent label in the purified sample after the fluorescent labeling. All analyzes were performed on a 96-well plate, and fluorescence was detected at an excitation wavelength of 520 nm and a fluorescence wavelength of 580-640 nm.
The biotin ligase reaction product was evaluated for antibody modification by the following method. The modification to the antibody was evaluated by quantifying the concentration of side chain biotin in the modified antibody sample purified in (1-3) using a Fluorescence Biotin Quantitation Kit (manufactured by Thermo Fisher Scientific). The Fluorescence Biotin Quantitation Kit is capable of quantifying the concentration of side-chain biotin in a solution by fluorescent labeling, and the quantification has been confirmed from 0 to 6.0 μM. In the determination of the concentration of side chain biotin, detection was performed at an excitation wavelength of 475 nm and a fluorescence wavelength of 500 to 550 nm. GroMAX (Promega) was used as a plate reader.
As a result of the analysis, modification to the antibody was observed only in the Lpl reaction product (Table 2).
　この結果から、Ｌｐｌ、ＰＰＴａｓｅ、およびビオチンリガーゼのうちＬｐｌのみが抗体修飾能を有することが示された。 The results showed that of Lpl, PPTase, and biotin ligase, only Lpl had the ability to modify antibodies.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
（実施例２）ＬｐｌＡによるＩｇＧ抗体の修飾とＥＳＩ－ＭＳによる解析
（２－１）酵素反応とＭＳによる解析
　ＩｇＧ抗体およびＬｐｌＡとして、抗ＨＥＲ２　ＩｇＧ抗体トラスツズマブ（中外製薬）および（１－１）で調製したＥｃＬｐｌＡを用いた。反応組成は次のとおりである。トラスツズマブ５００μｇを１ｍＭリン酸ナトリウムバッファー（ｐＨ６．０）２５０μＬに溶解させて用いた。終濃度でトラスツズマブ１．０ｍｇ／ｍＬ、ＡＴＰを２．５ｍＭ、オクタン酸０．５ｍＭ、硫酸マグネシウム３．２ｍＭ、リン酸ナトリウムバッファー２５ｍＭ（ｐＨ７．０）を反応液組成とした混合液に、終濃度０．０５ｍｇ／ｍＬのＥｃＬｐｌＡを加えて総量５００μＬの反応液を調製し、３０℃で６４時間反応させて、修飾抗体としてオクタン酸修飾トラスツズマブを生成させた。反応液にＥＤＴＡ－２Ｎａ溶液１０μＬを添加（終濃度１０ｍＭ）し反応を停止した。 (Example 2) Modification of IgG antibody by LplA and analysis by ESI-MS (2-1) Enzyme reaction and analysis by MS As IgG antibody and LplA, anti-HER2 IgG antibody trastuzumab (Chugai Pharmaceutical) and (1-1) The prepared EcLplA was used. The reaction composition is as follows. 500 μg of trastuzumab was dissolved in 250 μL of 1 mM sodium phosphate buffer (pH 6.0) and used. A final concentration of 1.0 mg / mL of trastuzumab, 2.5 mM of ATP, 0.5 mM of octanoic acid, 3.2 mM of magnesium sulfate, and 25 mM of sodium phosphate buffer (pH 7.0) in the final concentration was added to the final mixture. A total of 500 μL of a reaction solution was prepared by adding 0.05 mg / mL EcLplA, and reacted at 30 ° C. for 64 hours to produce octanoic acid-modified trastuzumab as a modified antibody. The reaction was terminated by adding 10 μL of an EDTA-2Na solution (final concentration: 10 mM) to the reaction solution.
　ＥＳＩ－ＭＳにより質量を測定したところ、原料のトラスツズマブは１４５１６２にピークが観測され、生成物にはオクタン酸が１個から１１個導入された１４５２９０から１４６５５４にピークが観測された。 When the mass was measured by ESI-MS, a peak of trastuzumab as a raw material was observed at 145162, and a peak was observed at 145290 to 146554 where 1 to 11 octanoic acids were introduced in the product.
　この結果から、ＬｐｌＡは抗体修飾能を有し、抗体当たり１～１０個の修飾物で抗体を修飾することが示された。 The results show that LplA has the ability to modify antibodies, and modifies antibodies with 1 to 10 modifications per antibody.
（分析装置）
ＵＨＰＬＣ：ＡＣＱＵＩＴＹ　ＵＰＬＣ（ウォーターズ）
質量分析計：トライブリッド質量分析計Ｏｒｂｉｔｒａｐ　Ｆｕｓｉｏｎ（サーモフィッシャーサイエンティフィック） (Analysis equipment)
UHPLC: ACQUITY UPLC (Waters)
Mass spectrometer: Tribrid mass spectrometer Orbitrap Fusion (Thermo Fisher Scientific)
（ＵＨＰＬＣ分析条件）
分析カラム：ＡＣＱＵＩＴＹ　ＵＰＬＣ　Ｐｒｏｔｅｉｎ　ＢＥＨ　Ｃ４（３００Å、２．１×１００ｍｍ、１．７μｍ（ウォーターズ））
　移動相Ａ：０．１％ギ酸水溶液
　移動相Ｂ：０．１％ギ酸、アセトニトリル溶液
　流速：１００μＬ／ｍｉｎ
　サンプル注入量：１０μＬ
　グラジエント条件（Ｂ％）：９％→９０％（０．０－３．５分）、９０％→９０％（３．５－５．０分）、９０％→９％（５－５．１分）、９％→９％（５．１分－１０分） (UHPLC analysis conditions)
Analysis column: ACQUITY UPLC Protein BEH C4 (300 mm, 2.1 × 100 mm, 1.7 μm (Waters))
Mobile phase A: 0.1% formic acid aqueous solution Mobile phase B: 0.1% formic acid, acetonitrile solution Flow rate: 100 μL / min
Sample injection volume: 10 μL
Gradient conditions (B%): 9% → 90% (0.0-3.5 minutes), 90% → 90% (3.5-5.0 minutes), 90% → 9% (5-5.1 minutes) Minutes), 9% → 9% (5.1 minutes-10 minutes)
（質量分析計分析条件）
　イオン化法：ＥＳＩ，Ｐｏｓｉｔｉｖｅモード
　Ｏｒｂｉｔｒａｐ　Ｒｅｓｏｌｕｔｉｏｎ：１５０００
　ＡＧＣ　Ｔａｒｇｅｔ：１．００Ｅ＋０６
　Ｓｏｕｒｃｅ　Ｆｒａｇｍｅｎｔａｔｉｏｎ７０（Ｖ）
　データの取得は付属ソフトであるＸｃａｌｉｂｕｒ　３．０（サーモフィッシャーサイエンティフィック）およびＴｈｅｒｍｏ　Ｏｒｂｉｔｒａｐ　Ｆｕｓｉｏｎ　Ｔｕｎｅ　Ａｐｐｌｉｃａｔｉｏｎ　２．０（サーモフィッシャーサイエンティフィック）を用いて行った。 (Mass spectrometer analysis conditions)
Ionization method: ESI, positive mode Orbitrap Resolution: 15000
AGC Target: 1.00E + 06
Source Fragmentation 70 (V)
Data acquisition was performed using attached software Xcalibur 3.0 (Thermo Fisher Scientific) and Thermo Orbitrap Fusion Tune Application 2.0 (Thermo Fisher Scientific).
（トラスツズマブの修飾部位の解析条件）
　ＬＣ／ＭＳ／ＭＳ測定結果に対する修飾部位解析については、ＢｉｏＰｈａｒｍａ　Ｆｉｎｄｅｒ　２．０（サーモフィッシャーサイエンティフィック）を用いて行った。
　ＢｉｏＰｈａｒｍａ　Ｆｉｎｄｅｒでの解析は、Ｍｉｎｉｍｕｍ　Ａｄｊａｃｅｎｔ　Ｃｈａｒｇｅｓを６から１０に設定し、Ｎｏｉｓｅ　Ｒｅｊｅｃｔｉｏｎに関しては９５％Ｃｏｎｆｉｄｅｎｃｅとした。また、Ｔａｒｇｅｔ　Ｍａｓｓは１４６０００Ｄａとし、Ｍａｓｓ　Ｔｏｌｅｒａｎｃｅは２０ｐｐｍとした。Ｄｙｎａｍｉｃ　Ｍｏｄｉｆｉｃａｔｉｏｎｓについては、ＣＨＯ細胞由来の糖鎖修飾、メチオニンの酸化（＋１５．９９５Ｄａ）およびリジン残基への修飾体（オクタン酸導入体（＋１２６．１０５Ｄａ））を設定した。さらに、Ｐｅｐｔｉｄｅ　ＣｏｎｆｉｄｅｎｃｅがＨｉｇｈのもののみとなるようフィルターをかけた。 (Analysis conditions for modification site of trastuzumab)
The modification site analysis for the LC / MS / MS measurement results was performed using BioPharma Finder 2.0 (Thermo Fisher Scientific).
Analysis by BioPharma Finder set Minimum Adjuvant Charges from 6 to 10, and 95% Confidence for Noise Rejection. Further, Target Mass was set to 146000 Da, and Mass Tolerance was set to 20 ppm. Regarding Dynamic Modifications, glycan modification derived from CHO cells, oxidation of methionine (+15.995 Da), and modification to lysine residue (octanoic acid-introduced substance (+126.105 Da)) were set. Further, a filter was applied so that Peptide Confidence was only High.
　また、修飾部位の検索対象のアミノ酸配列のデータとして、図１に示される（１）および（２）（配列番号１４および１５）を統合したものを用いた。 In addition, as the data of the amino acid sequence to be searched for the modification site, those obtained by integrating (1) and (2) (SEQ ID NOS: 14 and 15) shown in FIG. 1 were used.
（実施例３）各種ＬｐｌＡによるＩｇＧの修飾とＥＳＩ－ＭＳによる解析
（３－１）各種ＬｐｌＡの発現と精製
　実施例１に記載した以外のＬｐｌＡとして、Ｂａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓ由来ＬｐｌＡ（ＢｓＬｐｌＡ）、Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ　ｇｌｕｔａｍｉｃｕｍ由来ＬｐｌＡ（ＣｇＬｐｌＡ）、Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ　ｅｐｉｄｅｒｍｉｄｅｓ由来ＬｐｌＡ（ＳｅＬｐｌＡ）を利用した。各種ＬｐｌＡ全長配列をコードする塩基配列をｐＣｏｌｄ　ＧＳＴ　ＤＮＡベクターにクローニングし、ｌｐｌＡの発現プラスミドｐＣｏｌｄ－ｌｐｌＡ（Ｂｓ）、ｐＣｏｌｄ－ｌｐｌＡ（Ｃｇ）、ｐＣｏｌｄ－ｌｐｌＡ（Ｓｅ）を構築した。 (Example 3) Modification of IgG with various LplAs and analysis by ESI-MS (3-1) Expression and purification of various LplAs As other LplAs described in Example 1, LplA derived from Bacillus subtilis (BsLplA) and Corynebacterium glutamicum. LplA (CgLplA) and LplA derived from Staphylococcus epidermides (SeLplA) were used. The nucleotide sequences encoding various LplA full-length sequences were cloned into a pCold GST DNA vector, and lplA expression plasmids pCold-lplA (Bs), pCold-lplA (Cg), and pCold-lplA (Se) were constructed.
　各ｌｐｌＡの遺伝子を人工遺伝子合成した（ユーロフィンジェノミクス社）。両末端に付加したＢａｍＨＩおよびＨｉｎｄＩＩＩサイトを用い、同制限酵素にて処理したｐＣｏｌｄ　ＧＳＴ　ＤＮＡのＢａｍＨＩ－ＨｉｎｄＩＩＩサイトに挿入し、各種ｌｐｌＡ遺伝子がクローニングされたプラスミドｐＣｏｌｄ－ｌｐｌＡ（Ｂｓ）、ｐＣｏｌｄ－ｌｐｌＡ（Ｃｇ）、ｐＣｏｌｄ－ｌｐｌＡ（Ｓｅ）を構築した。ｌｐｌＡ（Ｂｓ）、ｌｐｌＡ（Ｃｇ）、ｌｐｌＡ（Ｓｅ）の塩基配列（５’－および３’－末端にそれぞれ６塩基のＢａｍＨＩおよびＨｉｎｄＩＩＩ認識配列が付加されている）ならびに、ｌｐｌＡ（Ｂｓ）、ｌｐｌＡ（Ｃｇ）、ｌｐｌＡ（Ｓｅ）のアミノ酸配列を、配列番号３、５、および７ならびに配列番号４、６、および８に示す。 (4) Artificial gene synthesis of each lplA gene (Eurofin Genomics). Using the BamHI and HindIII sites added to both ends, the plasmids were inserted into the BamHI-HindIII site of pCold {GST} DNA treated with the same restriction enzymes, and plasmids pCold-lplA (Bs) and pCold-lplA (Bs) in which various lplA genes were cloned. Cg), pCold-lplA (Se) was constructed. lplA (Bs), lplA (Cg), and lplA (Se) base sequences (6 bases each having a BamHI and HindIII recognition sequence added to the 5′- and 3′-ends), and lplA (Bs), lplA The amino acid sequence of (Cg), lplA (Se) is shown in SEQ ID NOs: 3, 5, and 7, and SEQ ID NOs: 4, 6, and 8.
　ｐＣｏｌｄ－ｌｐｌＡ（Ｂｓ）、ｐＣｏｌｄ－ｌｐｌＡ（Ｃｇ）、ｐＣｏｌｄ－ｌｐｌＡ（Ｓｅ）をＥ．ｃｏｌｉ　ＪＭ１０９株へ導入した。（１－１）に記載の方法で形質転換体を培養し調製した菌体抽出液より精製酵素液を調製した。 を pCold-lplA (Bs), pCold-lplA (Cg) and pCold-lplA (Se) were purchased from E. coli. coli JM109 strain. A purified enzyme solution was prepared from a cell extract prepared by culturing the transformant by the method described in (1-1).
（３－２）酵素反応
　（１－２）と同様の方法で抗ＨＥＲ２　ＩｇＧ抗体トラスツズマブと各精製酵素を反応させた。終濃度でトラスツズマブ１ｍｇ／ｍＬ、ＡＴＰ２．５ｍＭ、８－アジドオクタン酸（Ｓｕｎｄｉａ社）０．５０ｍＭ、硫酸マグネシウム３．２ｍＭ、リン酸ナトリウムバッファー２５ｍＭ（ｐＨ７．０）となるように調製した反応液に、各酵素液を終濃度で０．０１０ｍｇ／ｍＬとなるよう加えて総量５００μＬの反応液とし３０℃で２４時間反応させた。反応液にＥＤＴＡ－２Ｎａ溶液１０μＬを添加（終濃度１０ｍＭ）し反応を停止後、（１－３）同様の方法でＰｒｏｔｅｉｎ　Ａカラムにより修飾抗体を精製し、精製修飾抗体サンプルを調製した。 (3-2) Enzyme reaction The anti-HER2 IgG antibody trastuzumab was reacted with each purified enzyme in the same manner as in (1-2). The reaction solution was adjusted to have a final concentration of trastuzumab 1 mg / mL, ATP 2.5 mM, 8-azidooctanoic acid (Sundia) 0.50 mM, magnesium sulfate 3.2 mM, sodium phosphate buffer 25 mM (pH 7.0). Each enzyme solution was added to a final concentration of 0.010 mg / mL to give a total reaction volume of 500 μL, and reacted at 30 ° C. for 24 hours. After adding 10 μL of EDTA-2Na solution (final concentration: 10 mM) to the reaction solution to stop the reaction, the modified antibody was purified by a Protein A column in the same manner as in (1-3) to prepare a purified and modified antibody sample.
（３－３）ＤＩＢＡＣ－ＨＨＨＨＨＨＧ－ＯＨペプチド（配列番号２８）の合成
　クリック反応によりアジド基と結合することができる機能性物質として、ＤＩＢＡＣ－ＨＨＨＨＨＨＧ－ＯＨペプチド（配列番号２８）を、下記の反応スキームにより合成した。
Figure JPOXMLDOC01-appb-C000004
 　ペプチド合成装置はＢｉｏｔａｇｅ社製Ｉｎｉｔｉａｔｏｒ＋　Ａｌｓｔｒａを用いた。試薬は全て渡辺化学工業製のものを用いた。レジンはＨ－Ｇｌｙ－Ｔｒｔ（２－Ｃｌ）－Ｒｅｓｉｎ　（０．８９　ｍｍｏｌ／ｇ）を用いた。固相合成終了後、少量レジンをとり９５％ＴＦＡ／２．５％ＴＩＳ／２．５％Ｈ２Ｏで切り出しを行い、ＬＣ－ＭＳにて目的物のペプチドの分子量を確認した。続いて、Ｎ末端にシクロアルキンを導入するため、ＤＭＦ（４ｍＬ）、１－ヒドロキシベンゾトリアゾール（２．０ｅｑ）、１－エチル－３－（３－ジメチルアミノプロピル）カルボジイミド塩酸塩（２．０ｅｑ）、トリエチルアミン（２．０ｅｑ）、ＤＩＢＡＣ　ａｃｉｄ（２．２ｅｑ）を随時添加し、１９時間攪拌。反応後、同様に少量レジンをとり９５％ＴＦＡ／２．５％ＴＩＳ／２．５％Ｈ２Ｏで切り出しを行い、ＬＣ－ＭＳにて分析し目的物（４）のピークを確認した。酸によるシクロアルキンの分解が懸念されたため、切り出し後のろ液にはＤＩＢＡＣが分解しないように、トリエチルアミンを加え中和を行った。切り出し後レジンをフィルトレーションにより除去し、トリフルオロ酢酸を除去した。生成した結晶中にジエチルエーテルを加えエーテル沈殿を行い、生成した白色結晶をフィルトレーションにより回収した。これを０．１０％トリフルオロ酢酸水溶液に溶解し、オクタドデシル基化学結合型シリカゲルを充填剤とする逆相高速液体クロマトグラフィーに付し、トリフルオロ酢酸を０．１０％含有する水とアセトニトリルの混合溶液で溶出し、各フラクションをＥＳＩ－ＭＳにより確認した。生成物が含まれるフラクションを回収し、減圧下濃縮することによりアセトニトリルのみ除去した後、凍結乾燥を行った。ＬＣ－ＭＳにて確認を行い、目的物のピークを確認した。ＤＩＢＡＣ－ＨＨＨＨＨＨＧ－ＯＨ（配列番号２８）を、収量１０ｍｇ（収率９％）の固体として得た。 (3-3) Synthesis of DIBAC-HHHHHHG-OH peptide (SEQ ID NO: 28) DIBAC-HHHHHHG-OH peptide (SEQ ID NO: 28) as a functional substance capable of binding to an azide group by a click reaction was prepared by the following reaction It was synthesized according to the scheme.
Figure JPOXMLDOC01-appb-C000004
 The peptide synthesizer used was Initiator + Alstra manufactured by Biotage. All reagents used were made by Watanabe Chemical Industry. As the resin, H-Gly-Trt (2-Cl) -Resin (0.89 mmol / g) was used. After completion of the solid phase synthesis, a small amount of resin was taken out, cut out with 95% TFA / 2.5% TIS / 2.5% H 2 O, and the molecular weight of the target peptide was confirmed by LC-MS. Subsequently, DMF (4 mL), 1-hydroxybenzotriazole (2.0 eq), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (2.0 eq) to introduce cycloalkyne into the N-terminal. , Triethylamine (2.0 eq) and DIBAC acid (2.2 eq) were added as needed, and the mixture was stirred for 19 hours. After the reaction, similarly, a small amount of resin was taken out, cut out with 95% TFA / 2.5% TIS / 2.5% H 2 O, and analyzed by LC-MS to confirm the peak of the target product (4). Since there was concern about the decomposition of cycloalkyne by the acid, triethylamine was added to the filtrate after cutting out so that DIBAC was not decomposed, and the filtrate was neutralized. After cutting out, the resin was removed by filtration to remove trifluoroacetic acid. Diethyl ether was added to the generated crystals to perform ether precipitation, and the generated white crystals were collected by filtration. This was dissolved in a 0.10% aqueous trifluoroacetic acid solution, and subjected to reversed-phase high-performance liquid chromatography using octadodecyl group chemically bonded silica gel as a filler, to obtain water containing 0.10% trifluoroacetic acid and acetonitrile. The fraction was eluted with the mixed solution, and each fraction was confirmed by ESI-MS. The fraction containing the product was collected, concentrated under reduced pressure to remove only acetonitrile, and then freeze-dried. Confirmation was performed by LC-MS, and a peak of the target substance was confirmed. DIBAC-HHHHHHG-OH (SEQ ID NO: 28) was obtained as a solid in a yield of 10 mg (9%).
（３－４）クリック反応とＭＳによる解析
　（３－２）にて調製した精製修飾抗体サンプルに（３－３）で合成したＤＩＢＡＣ－ＨＨＨＨＨＨＧ－ＯＨ（配列番号２８）　２．６μＬを終濃度５０μＭとなるよう添加し、３０℃で１８時間反応させた。 (3-4) Click Reaction and Analysis by MS To the purified and modified antibody sample prepared in (3-2), 2.6 μL of DIBAC-HHHHHHG-OH (SEQ ID NO: 28) synthesized in (3-3) was added to a final concentration of 50 μM. And reacted at 30 ° C. for 18 hours.
　クリック反応後の反応液を（１－３）と同様にＰｒｏｔｅｉｎ　Ａカラムにより抗体を精製した。溶出した抗体を含むフラクションを限外ろ過ユニット（Ａｍｉｃｏｎ　Ｕｌｔｒａ　０．５ｍＬ　１０ｋＤａ、　Ｍｅｒｃｋ　Ｍｉｌｌｉｐｏｒｅ社）を用い１，０００倍容量の２０ｍＭ酢酸アンモニウム溶液（ｐＨ６．４）で溶媒交換した後、濃縮しＩｇＧ濃度０．５０ｍｇ／ｍＬ以上となったのを確認し、ＭＳによる解析を行った。 The antibody was purified from the reaction solution after the click reaction using a Protein A column in the same manner as in (1-3). The fraction containing the eluted antibody was solvent-exchanged with a 1,000-fold volume of a 20 mM ammonium acetate solution (pH 6.4) using an ultrafiltration unit (Amicon Ultra 0.5 mL 10 kDa, Merck Millipore), and then concentrated to concentrate IgG. After confirming that the concentration was 0.50 mg / mL or more, analysis by MS was performed.
　ＥＳＩ－ＭＳの測定条件として、以下のものを用いた。
　分析装置：Ａｇｉｌｅｎｔ　１２９０　Ｉｎｆｉｎｉｔｙ　ＩＩ　ａｎｄ　６５３０　Ａｃｃｕｒａｔｅ－Ｍａｓｓ
　Ｑ－ＴＯＦ条件：
・ＬＣ：カラム無し
・溶媒：７０：３０の０．１％ギ酸水溶液：０．１％ギ酸、アセトニトリル溶液
・流速：０．３５ｍＬ／ｍｉｎ
・ＭＳ：イオン化法；ｄｕａｌ　ＡＪＳ　ＥＳＩ
・ガス温度：３５０℃
・ドライガス流速：１１Ｌ／ｍｉｎ
・ネブライザー：３５　ｐｓｉｇ
・ｓｈｅａｔｈガス温度：３００℃
・ｓｈｅａｔｈガス流速：１２Ｌ／ｍｉｎ
・Ｖｃａｐ：３０００Ｖ
・フラグメンター：３５０Ｖ
・スキマ―：６５Ｖ
・Ｏｃｔ　１　ＲＦ　Ｖｐｐ：７５０Ｖ
・サンプル濃度：０．５０ｍｇ／ｍＬ抗体５μＬを１０μＬの超純水で希釈し、７．０ｍＭ　ＴＣＥＰ　４．０μＬを添加し室温にて１５ｍｉｎ還元処理をした後、ＬＣ－ＭＳに供した。 The following conditions were used as the measurement conditions of ESI-MS.
Analyzer: Agilent 1290 Infinity II and 6530 Accurate-Mass
Q-TOF conditions:
-LC: No column-Solvent: 70:30 0.1% formic acid aqueous solution: 0.1% formic acid, acetonitrile solution-Flow rate: 0.35 mL / min
MS: ionization method; dual AJS ESI
・ Gas temperature: 350 ° C
・ Dry gas flow rate: 11 L / min
・ Nebulizer: 35 psig
・ Sheath gas temperature: 300 ° C
・ Sheath gas flow rate: 12 L / min
・ Vcap: 3000V
・ Fragmenter: 350V
-Skimmer: 65V
・ Oct 1 RF Vpp: 750V
Sample concentration: 5 μL of 0.50 mg / mL antibody was diluted with 10 μL of ultrapure water, added with 4.0 μL of 7.0 mM TCEP, reduced at room temperature for 15 minutes, and then subjected to LC-MS.
　ＥＳＩ－ＭＳにより質量を測定したところ、原料のトラスツズマブは１４５１７８にピークが観測され、生成物には８－アジドオクタン酸及びＤＢＣＯ－ヒスチジンタグが一個導入された１４６５４１付近に全てのサンプルにおいてピークが観測された。 When the mass was measured by ESI-MS, a peak of trastuzumab as a raw material was observed at 145178, and a peak was observed in all samples near 146541 where 8-azidooctanoic acid and one DBCO-histidine tag were introduced into the product. Was done.
　この結果から、本実施例で用いた各種ＬｐｌＡのすべてが抗体修飾能を有すること、および、ＬｐｌＡおよびクリック反応を用いて抗体を修飾できることが示された。 From these results, it was shown that all of the various LplAs used in this example have antibody modifying ability, and that the antibodies can be modified using LplA and click reaction.
（実施例４）ペプチドマッピングによる修飾位置の同定
（４－１）ペプチドマッピングによる修飾位置の同定
　１．５ｍＬ低吸着マイクロテストチューブに、実施例２で得られたサンプル溶液を１０μＬ、５０ｍＭ炭酸水素アンモニウム緩衝液、４０％トリフルオロエタノールに溶解した２０ｍＭのジチオスレイトール水溶液１０μＬを加えて６５℃で１時間加温後、５０ｍＭのヨードアセトアミド水溶液１０μＬを添加し、遮光下室温で３０分間３００ｒｐｍで振盪し反応させた。反応後、５０ｍＭ炭酸水素アンモニウム緩衝液を４０μＬ加えて撹拌し、２０ｎｇ／μＬのトリプシン水溶液を１０μＬ添加して３７℃で１８時間酵素消化した。消化後、２０％トリフルオロ酢酸水溶液を２μＬ添加し反応を止め、ＬＣ／ＭＳ／ＭＳ測定を実施した。 (Example 4) Identification of Modification Position by Peptide Mapping (4-1) Identification of Modification Position by Peptide Mapping 10 μL of the sample solution obtained in Example 2 in 50 mL low-adsorption microtest tube, 50 mM ammonium bicarbonate 10 μL of a 20 mM dithiothreitol aqueous solution dissolved in a buffer solution and 40% trifluoroethanol was added, and the mixture was heated at 65 ° C. for 1 hour. Reacted. After the reaction, 40 μL of a 50 mM ammonium bicarbonate buffer was added and stirred, and 10 μL of a 20 ng / μL aqueous trypsin solution was added, followed by enzyme digestion at 37 ° C. for 18 hours. After the digestion, 2 μL of a 20% aqueous trifluoroacetic acid solution was added to stop the reaction, and LC / MS / MS measurement was performed.
（４－２）トラスツズマブのＬＣ／ＭＳ／ＭＳ測定条件
（分析装置）
ナノＨＰＬＣ：ＥＡＳＹ－ｎＬＣ　１０００（サーモフィッシャーサイエンティフィック）
質量分析計：トライブリッド質量分析計Ｏｒｂｉｔｒａｐ　Ｆｕｓｉｏｎ（サーモフィッシャーサイエンティフィック） (4-2) LC / MS / MS measurement conditions of trastuzumab (analyzer)
Nano HPLC: EASY-nLC 1000 (Thermo Fisher Scientific)
Mass spectrometer: Tribrid mass spectrometer Orbitrap Fusion (Thermo Fisher Scientific)
（ＨＰＬＣ分析条件）
　トラップカラム：Ａｃｃｌａｉｍ　ＰｅｐＭａｐ（登録商標）　１００，７５μｍｘ２ｃｍ（サーモフィッシャーサイエンティフィック）
分析カラム：ＥＳＩ－ｃｏｌｕｍｎ（ＮＴＣＣ－３６０／７５－３－１２５，７５μｍ×１２．５ｃｍ，３μｍ（日京テクノス株式会社））
　移動相Ａ：０．１％ギ酸水溶液
　移動相Ｂ：０．１％ギ酸、アセトニトリル溶液
　ローディング溶液：０．１％トリフルオロ酢酸水溶液
　流速：３００ｎＬ／ｍｉｎ
　サンプル注入量：１μＬ
　グラジエント条件（Ｂ％）：２％（０．０－０．５分）、２％→３０％（０．５－２３．５分）、３０％→７５％（２３．５－２５．５分）、７５％（２５．５－３５.０分） (HPLC analysis conditions)
Trap column: Acclaim PepMap (registered trademark) 100, 75 μm × 2 cm (Thermo Fisher Scientific)
Analytical column: ESI-column (NTCC-360 / 75-3-125, 75 μm × 12.5 cm, 3 μm (Nikyo Technos Co., Ltd.))
Mobile phase A: 0.1% formic acid aqueous solution Mobile phase B: 0.1% formic acid, acetonitrile solution Loading solution: 0.1% trifluoroacetic acid aqueous solution Flow rate: 300 nL / min
Sample injection volume: 1 μL
Gradient conditions (B%): 2% (0.0-0.5 min), 2% → 30% (0.5-23.5 min), 30% → 75% (23.5-25.5 min) ), 75% (25.5-35.0 minutes)
（質量分析計分析条件）
　イオン化法：ＥＳＩ，　Ｐｏｓｉｔｉｖｅモード
　スキャンタイプ：Ｄａｔａ　Ｄｅｐｅｎｄｅｎｔ　Ａｑｕｉｓｉｔｉｏｎ
　Ａｃｔｉｖａｔｉｏｎ　Ｔｙｐｅ：Ｃｏｌｌｉｓｉｏｎ　Ｉｎｄｕｃｅｄ　Ｄｉｓｓｏｃｉａｔｉｏｎ（ＣＩＤ）
　データの取得は付属ソフトであるＸｃａｌｉｂｕｒ　３．０（サーモフィッシャーサイエンティフィック）およびＴｈｅｒｍｏ　Ｏｒｂｉｔｒａｐ　Ｆｕｓｉｏｎ　Ｔｕｎｅ　Ａｐｐｌｉｃａｔｉｏｎ　２．０（サーモフィッシャーサイエンティフィック）を用いて行った。 (Mass spectrometer analysis conditions)
Ionization method: ESI, Positive mode Scan type: Data Dependent Aquisition
Activation Type: Collision Induced Dissociation (CID)
Data acquisition was performed using attached software Xcalibur 3.0 (Thermo Fisher Scientific) and Thermo Orbitrap Fusion Tune Application 2.0 (Thermo Fisher Scientific).
（４－３）トラスツズマブの修飾部位の解析条件
　ＬＣ／ＭＳ／ＭＳ測定結果に対する修飾部位解析については、Ｐｒｏｔｅｏｍｅ　Ｄｉｓｃｏｖｅｒｅｒ　ｖｅｒｓｉｏｎ　１．４（サーモフィッシャーサイエンティフィック）を用いて行った。 (4-3) Analysis conditions for modification site of trastuzumab Modification site analysis for the results of LC / MS / MS was performed using Proteome Discoverer version 1.4 (Thermo Fisher Scientific).
　Ｐｒｏｔｅｏｍｅ　Ｄｉｓｃｏｖｅｒｅｒでの解析は、Ｓｅｑｕｅｓｔ　ＨＴを検索エンジンとして使用し、プリカーサーイオンの範囲を３５０～５０００Ｄａとした。また、トリプシンを消化酵素として設定し、Ｍａｘｉｍｕｍ　Ｍｉｓｓｅｄ　Ｃｌｅａｖａｇｅ　Ｓｉｔｅｓは３とした。また、プリカーサーおよびフラグメントイオンのＭａｓｓ　Ｔｏｌｅｒａｎｃｅは、それぞれ５ｐｐｍおよび０．５Ｄａとした。Ｓｔａｔｉｃ　Ｍｏｄｉｆｉｃａｔｉｏｎにはヨードアセトアミドによるシステイン残基の修飾として、Ｃａｒｂａｍｉｄｏｍｅｔｈｙｌ（＋５７．０２１Ｄａ）を設定した。Ｄｙｎａｍｉｃ　Ｍｏｄｉｆｉｃａｔｉｏｎｓについては、メチオニンの酸化（＋１５．９９５Ｄａ）およびリジン残基への修飾体（オクタン酸導入体（＋１２６．１０５Ｄａ））を設定した。さらに、Ｐｅｐｔｉｄｅ　ＣｏｎｆｉｄｅｎｃｅがＨｉｇｈのもののみとなるようフィルターをかけた。 In the analysis with \\ Proteome \ Discoverer, Sequence \ HT was used as a search engine, and the range of the precursor ion was set to 350 to 5000 Da. In addition, trypsin was set as a digestive enzyme, and Maximum \ Missed \ Clearage \ Sites was set to 3. In addition, the Mass @ Tolerance of the precursor and the fragment ion were set to 5 ppm and 0.5 Da, respectively. In Static @ Modification, Carbamidomethyl (+57.021 Da) was set as a modification of cysteine residue with iodoacetamide. For Dynamic @ Modifications, oxidation of methionine (+15.995 Da) and modification to lysine residue (octanoic acid-introduced substance (+126.105 Da)) were set. Further, a filter was applied so that Peptide @ Confidence was only High.
　また、修飾部位の検索対象のアミノ酸配列のデータとして、図１に示される（１）および（２）（配列番号１４および１５）を統合したものを用いた。 In addition, as the data of the amino acid sequence to be searched for the modification site, those obtained by integrating (1) and (2) (SEQ ID NOS: 14 and 15) shown in FIG. 1 were used.
（４－４）トラスツズマブのＬＣ／ＭＳ／ＭＳによる修飾部位の解析結果
　ＬＣ／ＭＳ／ＭＳを用いた解析の結果、実施例２により得られた被修飾トラスツズマブのトリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含むアミノ酸１９残基からなるペプチド、ＬＳＣＡＡＳＧＦＮＩＫＤＴＹＩＨＷＶＲ（配列番号１６）のペプチドフラグメントのＭＳスペクトル（実測値：ｍ／ｚ　１１８２．６１４３７、理論値１１８２．６１２４２、２価）が観測され（図２）、ＣＩＤスペクトルより重鎖のアミノ酸配列番号１４における３０位のリジン残基の修飾を示す、１価のｙ９に相当するｍ／ｚ　１３４３．７９（理論値：１３４３．７５）のプロダクトイオンが確認された（図３）。また、トリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含むアミノ酸２０残基からなるペプチド、ＦＴＩＳＡＤＴＳＫＮＴＡＹＬＱＭＮＳＬＲ（配列番号１７）のペプチドフラグメントのＭＳスペクトル（実測値：ｍ／ｚ　７９６．４１５３６、理論値：７９６．４１４５９、３価）が観測され（図４）、ＣＩＤスペクトルより同じく重鎖の７６位のリジン残基の修飾を示す、１価のｂ１２に相当するｍ／ｚ　１３６３．７１（理論値：１３６３．７１）のプロダクトイオンが確認された（図５）。また、トリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含むアミノ酸２６残基からなるペプチド、ＧＰＳＶＦＰＬＡＰＳＳＫＳＴＳＧＧＴＡＡＬＧＣＬＶＫ（配列番号１８）のペプチドフラグメントのＭＳスペクトル（実測値：ｍ／ｚ　６５４．６０９４９、理論値：６５４．６０８７２、４価）が観測され（図６）、ＣＩＤスペクトルより同じく重鎖のＥＵ　ｎｕｍｂｅｒｉｎｇにおける１３３位のリジン残基の修飾を示す、２価のｙ１７に相当するｍ／ｚ　８７５．６７（理論値：８７５．４７）のプロダクトイオンが確認された（図７）。また、トリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含むアミノ酸３０残基からなるペプチド、ＳＣＤＫＴＨＴＣＰＰＣＰＡＰＥＬＬＧＧＰＳＶＦＬＦＰＰＫＰＫ（配列番号１９）のペプチドフラグメントのＭＳスペクトル（実測値：ｍ／ｚ　１１５４．２５５７２、理論値：１１５４．２５３８１、３価）が観測され（図８）、ＣＩＤスペクトルより同じく重鎖の２２２位のリジン残基の修飾を示す、３価のｂ１２に相当するｍ／ｚ　５２３．３１（理論値：５２３．２３）のプロダクトイオンが確認された（図９）。さらに、トリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含むアミノ酸２６残基からなるペプチド、ＴＨＴＣＰＰＣＰＡＰＥＬＬＧＧＰＳＶＦＬＦＰＰＫＰＫ（配列番号２０）のペプチドフラグメントのＭＳスペクトル（実測値：ｍ／ｚ　１４８５．７８５５２、理論値：１４８５．７８４７４、２価）が観測され（図１０）、ＣＩＤスペクトルより同じく重鎖の２４６位もしくは２４８位のリジン残基の修飾を示す、１価のｙ５に相当するｍ／ｚ　６９２．４６（理論値：６９２．４７）のプロダクトイオンが確認された（図１１）。また、トリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含むアミノ酸１２残基からなるペプチド、ＶＳＮＫＡＬＰＡＰＩＥＫ（配列番号２１）のペプチドフラグメントのＭＳスペクトル（実測値：ｍ／ｚ　６９６．９２８１２、理論値：６９６．９２７２４、２価）が観測され（図１２）、ＣＩＤスペクトルより同じく重鎖の３２６位のリジン残基の修飾を示す、１価のｂ４に相当するｍ／ｚ　５５５．３８（理論値：５５５．３５）のプロダクトイオンが確認された（図１３）。また、トリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含むアミノ酸２６残基からなるペプチド、ＥＰＱＶＹＴＬＰＰＳＲＥＥＭＴＫＮＱＶＳＬＴＣＬＶＫ（配列番号２２）のペプチドフラグメントのＭＳスペクトル（実測値：ｍ／ｚ　７９４．１７１９７、理論値：７９４．１７０５２、４価）が観測され（図１４）、ＣＩＤスペクトルより同じく重鎖の３６０位のリジン残基の修飾を示す、２価のｂ１８に相当するｍ／ｚ　１１２８．２３（理論値：１１２８．０７）のプロダクトイオンが確認された（図１５）。 (4-4) Results of analysis of trastuzumab modification site by LC / MS / MS As a result of analysis using LC / MS / MS, modification of trastuzumab to lysine residue by trypsin digestion of modified trastuzumab obtained in Example 2 MS spectrum (actual value: m / z 1182.61437, theoretical value 1182.61242) of a peptide fragment of LSCAASGFNIKDTYIHWVR (SEQ ID NO: 16) comprising 19 amino acid residues including a site (octanoic acid-introduced compound (+126.105 Da)) (Divalent) was observed (FIG. 2), and the CID spectrum shows the modification of the lysine residue at position 30 in amino acid sequence number 14 of the heavy chain, m / z 1343.79 corresponding to monovalent y9 (theoretical value). : 1343.75) was confirmed (FIG. 3). Further, an MS spectrum of a peptide fragment of FTISADTSKNTAYLQMNSLR (SEQ ID NO: 17) comprising 20 amino acids including a site for modification to lysine residues by digestion with trypsin (octanoic acid-introduced compound (+126.105 Da)) (actual measurement: m / Z 796.41536, theoretical value: 796.41459, trivalent) (FIG. 4), and also shows a modification of the lysine residue at position 76 of the heavy chain from the CID spectrum, which corresponds to monovalent b12. A product ion of / z 1363.71 (theoretical value: 1363.71) was confirmed (FIG. 5). MS spectrum of peptide fragment of GPSVFPLAPSSSKSTSGGTAALGCLVK (SEQ ID NO: 18) consisting of a peptide consisting of 26 amino acids including a modification site to lysine residue (octanoic acid-introduced compound (+126.105 Da)) by trypsin digestion (actual measurement: m / Z 654.60949, theoretical value: 654.60872, 4 valences) (FIG. 6). From the CID spectrum, the divalent y17 also shows the modification of the lysine residue at position 133 in EU numbering of the heavy chain. A corresponding product ion of m / z 875.67 (theoretical value: 875.47) was confirmed (FIG. 7). In addition, MS spectrum of peptide fragment of SCDKTHTCPPCPAPELLGGPSVFLFPPPKPK (SEQ ID NO: 19), which is a peptide consisting of 30 amino acids including a site for modification to lysine residue (octanoic acid-introduced substance (+126.105 Da)) by trypsin digestion (actual value: m / Z 1154.252572, theoretical value: 1154.25381; trivalent) (FIG. 8), and the CID spectrum also shows modification of the lysine residue at position 222 of the heavy chain, which corresponds to trivalent b12. A product ion of / z 523.31 (theoretical value: 523.23) was confirmed (FIG. 9). Further, an MS spectrum of a peptide fragment of THTCPPCPAPELLGGPSVFFLPPKPK (SEQ ID NO: 20) including a peptide consisting of 26 amino acids including a site for modification to a lysine residue (octanoic acid-introduced compound (+126.105 Da)) by trypsin digestion (actual measurement: m / Z 1485.78252, theoretical value: 1485.77844, divalent) (FIG. 10). From the CID spectrum, the monovalent y5 also shows the modification of the lysine residue at position 246 or 248 of the heavy chain. A corresponding product ion of m / z 692.46 (theoretical value: 692.47) was confirmed (FIG. 11). In addition, an MS spectrum (actual measurement: m / Z 696.92812, theoretical value: 696.992724, divalent) was observed (FIG. 12), and the CID spectrum also shows modification of the lysine residue at position 326 of the heavy chain, m corresponding to monovalent b4. A product ion of / z 555.38 (theoretical value: 555.35) was confirmed (FIG. 13). MS spectrum of peptide fragment of EPQVYTLPPPSREEMTKNQVSLTCLVK (SEQ ID NO: 22) consisting of a peptide consisting of 26 amino acids including a modification site to lysine residue by lysine digestion (octanoic acid-introduced substance (+126.105 Da)) (actual value: m / Z 794.17197, theoretical value: 794.17052, 4 valences) (FIG. 14), and the CID spectrum shows a modification of the lysine residue at the 360-position of the heavy chain, which corresponds to divalent b18. A product ion of / z 1128.23 (theoretical value: 1128.07) was confirmed (FIG. 15).
　また、トリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含むアミノ酸２１残基からなるペプチド、ＡＳＱＤＶＮＴＡＶＡＷＹＱＱＫＰＧＫＡＰＫ（配列番号２３）のペプチドフラグメントのＭＳスペクトル（実測値：ｍ／ｚ　１２０７．１４９９４、理論値：１２０７．１４７９２、２価）が観測され（図１６）、ＣＩＤスペクトルより軽鎖のアミノ酸配列番号１５における４２位のリジン残基の修飾を示す、１価のｙ５に相当するｍ／ｚ　６２６．４１（理論値：６２６．４２）のプロダクトイオンが確認された（図１７）。また、トリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含むアミノ酸３４残基からなるペプチド、ＶＤＮＡＬＱＳＧＮＳＱＥＳＶＴＥＱＤＳＫＤＳＴＹＳＬＳＳＴＬＴＬＳＫ（配列番号２４）のペプチドフラグメントのＭＳスペクトル（実測値：ｍ／ｚ　１２４９．２７８１３、理論値：１２４９．２７６６８、３価）が観測され（図１８）、ＣＩＤスペクトルより同じく軽鎖の１６９位のリジン残基の修飾を示す、２価のｂ２２に相当するｍ／ｚ　１２２３．７２（理論値：１２２３．５６）のプロダクトイオンが確認された（図１９）。また、トリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含むアミノ酸７残基からなるペプチド、ＡＤＹＥＫＨＫ（配列番号２５）のペプチドフラグメントのＭＳスペクトル（実測値：ｍ／ｚ　５０８．７７５０１、理論値：５０８．７７４７７、２価）が観測され（図２０）、ＣＩＤスペクトルより同じく軽鎖の１８８位のリジン残基の修飾を示す、１価のｙ３に相当するｍ／ｚ　５３８．３６（理論値：５３８．３７）のプロダクトイオンが確認された（図２１）。また、トリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含むアミノ酸１９残基からなるペプチド、ＨＫＶＹＡＣＥＶＴＨＱＧＬＳＳＰＶＴＫ（配列番号２６）のペプチドフラグメントのＭＳスペクトル（実測値：ｍ／ｚ　１１３４．０９８４４、理論値：１１３４．０９６６０、２価）が観測され（図２２）、ＣＩＤスペクトルより同じく軽鎖の１９０位のリジン残基の修飾を示す、１価のｂ２に相当するｍ／ｚ　３９２．３２（理論値：３９２．２７）のプロダクトイオンが確認された（図２３）。さらに、トリプシン消化によるリジン残基への修飾部位（オクタン酸導入体（＋１２６．１０５Ｄａ））を含むアミノ酸２３残基からなるペプチド、ＨＫＶＹＡＣＥＶＴＨＱＧＬＳＳＰＶＴＫＳＦＮＲ（配列番号２７）のペプチドフラグメントのＭＳスペクトル（実測値：ｍ／ｚ　９２４．４８３５３、理論値：９２４．４８１８４、３価）が観測され（図２４）、ＣＩＤスペクトルより同じく軽鎖の２０７位のリジン残基の修飾を示す、１価のｙ８に相当するｍ／ｚ　１０７４．５８（理論値：１０７４．６３）のプロダクトイオンが確認された（図２５）。 In addition, MS spectrum of a peptide fragment of ASQDVNTAVAWYQQKPGKAPK (SEQ ID NO: 23) comprising a peptide consisting of 21 amino acids including a modification site to lysine residue by lysine digestion (octanoic acid-introduced substance (+126.105 Da)) (actual value: m / Z 1207.14994, theoretical value: 1207.14792, divalent) (FIG. 16), and the CID spectrum shows the modification of the lysine residue at position 42 in amino acid SEQ ID NO: 15 of the light chain. The product ion of m / z 626.41 (theoretical value: 626.42) corresponding to was obtained (FIG. 17). MS spectrum (actual value: m) of a peptide fragment of VDNALQSGNSQESVTEQDSKDSTYSLSLSTLTLSK (SEQ ID NO: 24), which is a peptide consisting of 34 amino acids including a site for modification to a lysine residue (octanoic acid-introduced substance (+126.105 Da)) by trypsin digestion. / Z 1249.227813, theoretical value: 1249.276768, trivalent) (FIG. 18), and the CID spectrum shows a modification of the lysine residue at position 169 of the light chain, which corresponds to divalent b22. A product ion of / z 1223.72 (theoretical value: 1223.56) was confirmed (FIG. 19). In addition, the MS spectrum of a peptide fragment of ADYEKHK (SEQ ID NO: 25), a peptide consisting of 7 amino acids including a site for modification to a lysine residue by digestion with trypsin (introduced octanoic acid (+126.105 Da)) (actual measurement: m / Z 508.77751, theoretical value: 508.77747, bivalent) was observed (FIG. 20), and the CID spectrum also shows modification of the lysine residue at position 188 of the light chain, m corresponding to monovalent y3. A product ion of /z（538.36 (theoretical value: 538.37) was confirmed (FIG. 21). In addition, MS spectrum of peptide fragment of HKVYACEVTHQGLSSPVTK (SEQ ID NO: 26) consisting of 19 amino acid residues including a modification site to lysine residue by lysine digestion (octanoic acid-introduced body (+126.105 Da)) (actual measurement: m / Z 1134.0844, theoretical value: 1134.09666, divalent) (FIG. 22), and the CID spectrum also shows a modification of the lysine residue at position 190 of the light chain, m corresponding to monovalent b2. A product ion of /z（392.32 (theoretical value: 392.27) was confirmed (FIG. 23). Furthermore, an MS spectrum of a peptide fragment of HKVYACEVTHQGLSSPVTKSFNR (SEQ ID NO: 27) comprising a 23 amino acid residue containing a site for modification to a lysine residue (octanoic acid-introduced compound (+126.105 Da)) by trypsin digestion (actual measurement: m / Z 924.48353, theoretical value: 924.48184, trivalent) (FIG. 24), and the CID spectrum also shows a modification of the lysine residue at position 207 of the light chain, which corresponds to monovalent y8. A product ion of /z（1074.58 (theoretical value: 1074.63) was confirmed (FIG. 25).
　この結果から、ＬｐｌＡが、重鎖ＣＨ１領域の１３３位および軽鎖ＣＬ領域の１６９位のリジン残基において抗体を修飾することができることが示された。 {These results indicate that LplA can modify the antibody at the lysine residue at position 133 in the heavy chain CH1 region and at position 169 in the light chain CL region.
（実施例５）
　実施例１により得られた被修飾トラスツズマブのトリプシン消化物のペプチド－スペクトル－マッチ（Ｐｓｍ）を指標にして、トラスツズマブにおいて高度に修飾される位置の割合を算出したところ、重鎖ＣＨ１領域の約７９％が１３３位のリジン残基への修飾であり、かつ軽鎖ＣＬ領域の約３３％が１６９位のリジン残基への修飾であることが確認された（表３）。また、重鎖ＣＨ２領域では、２２２位のリジン残基への修飾が約３２％であった（表３）。
Figure JPOXMLDOC01-appb-T000005
 (Example 5)
Using the peptide-spectrum match (Psm) of the tryptic digest of the modified trastuzumab obtained in Example 1 as an index, the ratio of highly modified positions in trastuzumab was calculated to be about 79% in the heavy chain CH1 region. % Was a modification to the lysine residue at position 133, and about 33% of the light chain CL region was confirmed to be a modification to the lysine residue at position 169 (Table 3). In the heavy chain CH2 region, modification to the lysine residue at position 222 was about 32% (Table 3).
Figure JPOXMLDOC01-appb-T000005
（実施例６）ＬｐｌＡによるＦａｂの修飾とＥＳＩ－ＭＳによる解析
（６－１）トラスツズマブ由来Ｆａｂの調製
　抗ＨＥＲ２　ＩｇＧ抗体トラスツズマブをパパインにより部分消化した。リン酸ナトリウムバッファー２０ｍＭ、ＥＤＴＡ－２Ｎａ　１０ｍＭ、Ｌ－システイン２０ｍＭにてビーズ固定化パパイン（Ｉｍｍｏｂｉｌｉｚｅｄ　Ｐａｐａｉｎ，Ｐｉｅｒｃｅ）を平衡化した。終濃度でＩｇＧ抗体トラスツズマブ０．７５ｍｇ／ｍＬ、リン酸ナトリウムバッファー２０ｍＭ、ＥＤＴＡ－２Ｎａ　１０ｍＭ、Ｌ－システイン２０ｍＭの溶液１．５ｍＬにビーズ固定化パパイン（Ｉｍｍｏｂｉｌｉｚｅｄ　Ｐａｐａｉｎ，Ｐｉｅｒｃｅ）を０．５ｍＬ添加し、２．０ｍＬとした後３７℃で穏やかに振盪しながら６．５時間湯浴中で消化した。その後、（１－３）同様に平衡化したＰｒｏｔｅｉｎ　Ａカラムにアプライし素通り画分にＦａｂ断片、溶出画分にＦｃ断片を確認した。Ｆａｂ断片を限外ろ過ユニットにより濃縮し、３．０ｍｇ／ｍＬのＦａｂを調製した。 (Example 6) Modification of Fab with LplA and analysis by ESI-MS (6-1) Preparation of trastuzumab-derived Fab The anti-HER2 IgG antibody trastuzumab was partially digested with papain. Bead-immobilized papain (Immobilized Papain, Pierce) was equilibrated with 20 mM sodium phosphate buffer, 10 mM EDTA-2Na, and 20 mM L-cysteine. At a final concentration of 0.75 mg / mL of the IgG antibody trastuzumab, 20 mM of sodium phosphate buffer, 10 mM of EDTA-2Na, and 1.5 mL of a solution of 20 mM L-cysteine, 0.5 mL of bead-immobilized papain (Immobilized Papain, Pierce) was added. After adjusting to 2.0 mL, the mixture was digested in a water bath for 6.5 hours with gentle shaking at 37 ° C. Then, the mixture was applied to a Protein A column equilibrated in the same manner as in (1-3), and the Fab fragment was confirmed in the flow-through fraction, and the Fc fragment was confirmed in the eluted fraction. The Fab fragment was concentrated by an ultrafiltration unit to prepare a 3.0 mg / mL Fab.
（６－２）酵素反応とＭＳによる解析
　（１－１）で調製したＥｃＬｐＡを用い（６－１）で調製したＦａｂとの反応を行った。終濃度でＦａｂ　２ｍｇ／ｍＬ、ＡＴＰ　２．５ｍＭ、８－アジドオクタン酸０．５ｍＭ、硫酸マグネシウム３．２ｍＭ、リン酸ナトリウムバッファー２５ｍＭ（ｐＨ７．０）となるように調製した反応液に、ＥｃＬｐｌＡを終濃度で０．０５ｍｇ／ｍＬとなるよう加えて総量５００μＬの反応液とし、３０℃で２４時間反応させた。反応液にＥＤＴＡ－２Ｎａ溶液１０μＬを添加（終濃度１０ｍＭ）し反応を停止後、Ｐｒｏｔｅｉｎ　Ｇカラム（ＨｉＴｒａｐ　Ｐｒｏｔｅｉｎ　Ｇ　ＨＰ　Ｃｏｌｕｍｎｓ、１ｍＬ、ＧＥヘルスケア社）によりＦａｂを精製した。
　Ｐｒｏｔｅｉｎ　Ｇカラムは２０ｍＭ　Ｔｒｉｓ（ｐＨ７．６）、２０％（ｖ／ｖ）エタノールにて平衡化したものを用いた。
　Ｆａｂ　１ｍｇを含む酵素反応液５００μＬをアプライ後、４ｍＬの平衡化バッファーにて洗浄した。さらに１００ｍＭ　グリシンバッファー（ｐＨ２．７）２ｍＬを添加して溶出した後、ただちに１Ｍ　Ｔｒｉｓ－ＨＣｌを１５０μＬ添加しｐＨを８．５付近に調整し、精製Ｆａｂサンプルとした。 (6-2) Enzyme Reaction and Analysis by MS Using EcLpA prepared in (1-1), a reaction with Fab prepared in (6-1) was performed. EcLplA was added to a reaction solution prepared so that the final concentration was 2 mg / mL Fab, 2.5 mM ATP, 0.5 mM 8-azidooctanoic acid, 3.2 mM magnesium sulfate, and 25 mM sodium phosphate buffer (pH 7.0). The reaction solution was added to a final concentration of 0.05 mg / mL to give a total volume of 500 μL, and reacted at 30 ° C. for 24 hours. After the reaction was stopped by adding 10 μL of an EDTA-2Na solution (final concentration: 10 mM) to the reaction solution, Fab was purified by a Protein G column (HiTrap Protein G HP Columns, 1 mL, GE Healthcare).
As the Protein G column, a column equilibrated with 20 mM Tris (pH 7.6) and 20% (v / v) ethanol was used.
After applying 500 μL of the enzyme reaction solution containing 1 mg of Fab, the plate was washed with 4 mL of equilibration buffer. Further, 2 mL of a 100 mM glycine buffer (pH 2.7) was added and eluted. Immediately, 150 μL of 1 M Tris-HCl was added to adjust the pH to around 8.5 to obtain a purified Fab sample.
　精製Ｆａｂを（３－４）同様の方法で溶媒交換、濃縮し、ＥＳＩ－ＭＳにより質量を測定したところ、原料のＦａｂは４７６３８にピークが観測され、精製物にはオクタン酸アジドが１個導入された４７８０５および２個導入された４７９７１にピークが観測された。 The purified Fab was subjected to solvent exchange and concentration in the same manner as in (3-4), and the mass was measured by ESI-MS. As a result, a peak was observed at 47638 for the raw Fab, and one octanoic acid azide was introduced into the purified product. Peaks were observed at 47805 introduced and 47971 introduced at two sites.
　この結果から、ＬｐｌＡはＦａｂ修飾能を有し、Ｆａｂ当たり１～２個のＤＡＲでＦａｂを修飾することが示された。 The results show that LplA has the ability to modify Fab, and modifies Fab with 1-2 DARs per Fab.
（実施例７）ＬｐｌＡ変異バリアントの構築および試験
（７－１）修飾ＬｐｌＡバリアント：Ｒ１２１（Ａ，Ｖ，Ｓ，Ｔ）＊Ｙ１４０（Ａ，Ｖ）＊Ｅ１４２（Ａ，Ｖ，Ｓ，Ｔ）＊Ｋ１７６（Ｋ，Ａ，Ｖ，Ｓ，Ｔ）＊Ｉ１７８（Ａ，Ｖ）（合計３２０個のバリアント）のコンビナトリアルライブラリをコードするＤＮＡ断片の構築
　使用した全ての制限酵素、Ｔ４　ＤＮＡリガーゼの反応は、他に特定して記載しない限り、Ｆｅｒｍｅｎｔａｓ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ）より入手したプロトコールに従った。また、他に特定して記載しない限り、全てのＰＣＲはＫＡＰＡ　ＨｉＦｉ　ＨｏｔＳｔａｒｔ　ＲｅａｄｙＭｉｘ　ＰＣＲキット（Ｋａｐａ　Ｂａｉｏｓｙｓｔｅｍｓ）を用い、プロトコールに従い全ＰＣＲの容量は５０μＬで行った。 Example 7 Construction and Testing of LplA Mutant Variant (7-1) Modified LplA Variant: R121 (A, V, S, T) * Y140 (A, V) * E142 (A, V, S, T) * Construction of DNA Fragments Encoding a Combinatorial Library of K176 (K, A, V, S, T) * I178 (A, V) (Total of 320 Variants) All of the restriction enzymes used, T4 DNA ligase, Unless otherwise specified, the protocol obtained from Fermentas (Thermo Fisher Scientific) was followed. Unless otherwise specified, all PCRs were performed using KAPA HiFi HotStart ReadyMix PCR kit (Kapa Biosystems) in a total PCR volume of 50 μL according to the protocol.
（７－１－１）ｐＥＴ１５ｂ－ｗｔ－ｌｐｌＡプラスミドの構築
　プライマーＰ１（配列番号３８）およびＰ２（配列番号３９）を用いた。
　ＰＣＲ１：プライマーＰ１，Ｐ２；ＤＮＡ鋳型　Ｅ．ｃｏｌｉ　ＭＧ１６５５株（ＡＴＣＣ４７０７６）の染色体ＤＮＡ。プロトコール：９５℃，３分；９８℃，２０秒；６０℃，１５秒；７２℃，３０秒；２５サイクル。
　得られたＤＮＡ断片を、エンドヌクレアーゼＸｂａＩおよびＢａｍＨＩ（Ｆｅｒｍｅｎｔａｓ）を用いて消化し、同エンドヌクレアーゼで消化したベクターｐＥＴ１５ｂ（Ｎｏｖａｇｅｎ）に連結した。このようにして、ｐＥＴ１５ｂ－ｗｔ－ｌｐｌＡプラスミドを得た。 (7-1-1) Construction of pET15b-wt-lplA plasmid Primers P1 (SEQ ID NO: 38) and P2 (SEQ ID NO: 39) were used.
PCR1: primers P1, P2; DNA template coli chromosomal DNA of MG1655 strain (ATCC 47076). Protocol: 95 ° C, 3 minutes; 98 ° C, 20 seconds; 60 ° C, 15 seconds; 72 ° C, 30 seconds; 25 cycles.
The obtained DNA fragment was digested with the endonucleases XbaI and BamHI (Fermentas), and ligated to the vector pET15b (Novagen) digested with the endonuclease. Thus, the pET15b-wt-lplA plasmid was obtained.
（７－１－２）ｐＥＴ１５ｂ－ｗｔ－ｌｐｌＡ（ＳａｃＩ）プラスミドの構築
　プライマーＰ３（配列番号４０），Ｐ４（配列番号４１），Ｐ５（配列番号４２），およびＰ６（配列番号４３）を用いた：
　ＰＣＲ１：プライマーＰ４，Ｐ５；ＤＮＡ鋳型　ｐＥＴ１５－ｗｔ－ｌｐｌＡ。プロトコール：９５℃，３分；９８℃，２０秒；６０℃，１５秒；７２℃，１５秒；２５サイクル。その結果、ＤＮＡ断片Ｆ１が得られた。
　ＰＣＲ２：プライマーＰ３，Ｐ６；ＤＮＡ鋳型　ｐＥＴ１５－ｗｔ－ｌｐｌＡ。プロトコール：９５℃，３分；９８℃，２０秒；６０℃，１５秒；７２℃，１５秒；２５サイクル。その結果、ＤＮＡ断片Ｆ２が得られた。
　ｐＥＴ１５ｂベクターをＸｂａＩおよびＢａｍＨＩで消化し、ＸｂａＩ－ＳａｃＩで消化したＤＮＡ断片Ｆ１、およびＳａｃＩ－ＢａｍＨＩで消化したＤＮＡ断片Ｆ２と連結した。その結果、ｐＥＴ１５ｂ－ｗｔ－ｌｐｌＡ（ＳａｃＩ）プラスミドが構築された。 (7-1-2) Construction of pET15b-wt-lplA (SacI) Plasmid Primers P3 (SEQ ID NO: 40), P4 (SEQ ID NO: 41), P5 (SEQ ID NO: 42), and P6 (SEQ ID NO: 43) were used. :
PCR1: primers P4, P5; DNA template pET15-wt-lplA. Protocol: 95 ° C, 3 minutes; 98 ° C, 20 seconds; 60 ° C, 15 seconds; 72 ° C, 15 seconds; 25 cycles. As a result, a DNA fragment F1 was obtained.
PCR2: primers P3, P6; DNA template pET15-wt-lplA. Protocol: 95 ° C, 3 minutes; 98 ° C, 20 seconds; 60 ° C, 15 seconds; 72 ° C, 15 seconds; 25 cycles. As a result, a DNA fragment F2 was obtained.
The pET15b vector was digested with XbaI and BamHI, and ligated to a DNA fragment F1 digested with XbaI-SacI and a DNA fragment F2 digested with SacI-BamHI. As a result, a pET15b-wt-lplA (SacI) plasmid was constructed.
（７－１－３）修飾ＬｐｌＡバリアント：Ｒ１２１（Ａ，Ｖ，Ｓ，Ｔ）をコードするＤＮＡ断片混合物の構築
　プライマーＰ７（配列番号４４），Ｐ８（配列番号４５），Ｐ９（配列番号４６），Ｐ１０（配列番号４７），Ｐ１１（配列番号４８），Ｐ１２（配列番号４９），Ｐ１３（配列番号５０），およびＰ１４（配列番号５１）を用いた：
　ＰＣＲ１：プライマーＰ７，Ｐ８；ＤＮＡ鋳型　ｐＥＴ１５－ｌｐｌＡ（ＳａｃＩ）。プロトコール：９５℃，３分；９８℃，２０秒；６５℃，１５秒；７２℃，１分；２０サイクル。
　ＰＣＲ２：プライマーＰ９，Ｐ１１；ＤＮＡ鋳型　得られたＰＣＲ１混合液１μＬ。プロトコール：９５℃，３分；９８℃，２０秒；６４℃，１５秒；７２℃，３０秒；２５サイクル。結果として、ＤＮＡ断片Ｆ２が得られた。
　ＰＣＲ３：プライマーＰ１０，Ｐ１２；ＤＮＡ鋳型　ｐＥＴ１５－ｌｐｌＡ（ＳａｃＩ）。プロトコール：９５℃，３分；９８℃，２０秒；６４℃，１５秒；７２℃，３０秒；２５サイクル。結果として、ＤＮＡ断片Ｆ３が得られた。
　ＰＣＲ４：プライマーＰ１３，Ｐ１４；ＤＮＡ鋳型　Ｆ２およびＦ３　ＤＮＡ断片の等モル混合物（それぞれ０．３μＭ）。プロトコール：９５℃，３分；９８℃，２０秒；６４℃，１５秒；７２℃，１分；２５サイクル。結果として、４つの修飾ＬｐｌＡバリアント（Ｒ１２１Ａ，Ｖ，Ｓ，Ｔ）をコードするＤＮＡ断片混合物（ライブラリ１）が得られた。 (7-1-3) Modified LplA Variant: Construction of DNA Fragment Mixture Encoding R121 (A, V, S, T) Primers P7 (SEQ ID NO: 44), P8 (SEQ ID NO: 45), P9 (SEQ ID NO: 46) , P10 (SEQ ID NO: 47), P11 (SEQ ID NO: 48), P12 (SEQ ID NO: 49), P13 (SEQ ID NO: 50), and P14 (SEQ ID NO: 51) were used:
PCR1: primers P7, P8; DNA template pET15-lplA (SacI). Protocol: 95 ° C, 3 minutes; 98 ° C, 20 seconds; 65 ° C, 15 seconds; 72 ° C, 1 minute; 20 cycles.
PCR2: primers P9 and P11; DNA template 1 μL of the obtained PCR1 mixture. Protocol: 95 ° C, 3 minutes; 98 ° C, 20 seconds; 64 ° C, 15 seconds; 72 ° C, 30 seconds; 25 cycles. As a result, a DNA fragment F2 was obtained.
PCR3: primers P10, P12; DNA template pET15-lplA (SacI). Protocol: 95 ° C, 3 minutes; 98 ° C, 20 seconds; 64 ° C, 15 seconds; 72 ° C, 30 seconds; 25 cycles. As a result, a DNA fragment F3 was obtained.
PCR4: primers P13, P14; equimolar mixture of DNA templates F2 and F3 DNA fragments (0.3 μM each). Protocol: 95 ° C, 3 minutes; 98 ° C, 20 seconds; 64 ° C, 15 seconds; 72 ° C, 1 minute; 25 cycles. As a result, a DNA fragment mixture (library 1) encoding four modified LplA variants (R121A, V, S, T) was obtained.
（７－１－４）修飾ＬｐｌＡバリアント：Ｒ１２１（Ａ，Ｖ，Ｓ，Ｔ）＊Ｙ１４０（Ａ，Ｖ）＊Ｅ１４２（Ａ，Ｖ，Ｓ，Ｔ）（合計３２個）のコンビナトリアルライブラリをコードするＤＮＡ断片の構築
　プライマーＰ１５（配列番号５２），Ｐ１６（配列番号５３），Ｐ１７（配列番号５４），Ｐ１８（配列番号５５），Ｐ１９（配列番号５６），Ｐ２０（配列番号５７），Ｐ２１（配列番号５８），およびＰ２２（配列番号５９）を用いた。
　ＰＣＲ１：プライマーＰ１５，Ｐ１６；ＤＮＡ鋳型　ライブラリ１　ＤＮＡ混合液１μＬ。プロトコール：９５℃，３分；９８℃，２０秒；６５℃，１５秒；７２℃，１分；２０サイクル。
　ＰＣＲ２：プライマーＰ１７，Ｐ１９；ＤＮＡ鋳型　得られたＰＣＲ１混合液１μＬ。プロトコール：９５℃，３分；９８℃，２０秒；６４℃，１５秒；７２℃，３０秒；２５サイクル。結果として、ＤＮＡ断片Ｆ２が得られた。
　ＰＣＲ３：プライマーＰ１８，Ｐ２０；ＤＮＡ鋳型　ライブラリ１　ＤＮＡ混合液１μＬ。プロトコール：９５℃，３分；９８℃，２０秒；６４℃，１５秒；７２℃，３０秒；２５サイクル。結果として、ＤＮＡ断片３が得られた。
　ＰＣＲ４：プライマーＰ２１，Ｐ２２；ＤＮＡ鋳型　Ｆ２およびＦ３　ＤＮＡ断片の等モル混合液（それぞれ０．３μＭ）。プロトコール：９５℃，３分；９８℃，２０秒；６４℃，１５秒；７２℃，１分；２５サイクル。結果として、修飾ＬｐｌＡバリアント：Ｒ１２１（Ａ，Ｖ，Ｓ，Ｔ）＊Ｙ１４０（Ａ，Ｖ）＊Ｅ１４２（Ａ，Ｖ，Ｓ，Ｔ）のコンビナトリアルライブラリをコードするＤＮＡ断片の混合物が得られた。 (7-1-4) Modified LplA variant: encodes a combinatorial library of R121 (A, V, S, T) * Y140 (A, V) * E142 (A, V, S, T) (total of 32) Construction of DNA fragment Primers P15 (SEQ ID NO: 52), P16 (SEQ ID NO: 53), P17 (SEQ ID NO: 54), P18 (SEQ ID NO: 55), P19 (SEQ ID NO: 56), P20 (SEQ ID NO: 57), P21 (SEQ ID NO: 57) No. 58) and P22 (SEQ ID NO: 59) were used.
PCR1: primers P15, P16; 1 μL of DNA template library 1 DNA mixture. Protocol: 95 ° C, 3 minutes; 98 ° C, 20 seconds; 65 ° C, 15 seconds; 72 ° C, 1 minute; 20 cycles.
PCR2: primers P17 and P19; DNA template 1 μL of the obtained PCR1 mixture. Protocol: 95 ° C, 3 minutes; 98 ° C, 20 seconds; 64 ° C, 15 seconds; 72 ° C, 30 seconds; 25 cycles. As a result, a DNA fragment F2 was obtained.
PCR3: primers P18, P20; 1 μL of DNA template library 1 DNA mixture. Protocol: 95 ° C, 3 minutes; 98 ° C, 20 seconds; 64 ° C, 15 seconds; 72 ° C, 30 seconds; 25 cycles. As a result, DNA fragment 3 was obtained.
PCR4: Primers P21 and P22; DNA templates F2 and F3 equimolar mixtures of DNA fragments (0.3 μM each). Protocol: 95 ° C, 3 minutes; 98 ° C, 20 seconds; 64 ° C, 15 seconds; 72 ° C, 1 minute; 25 cycles. As a result, a mixture of DNA fragments encoding a combinatorial library of the modified LplA variant: R121 (A, V, S, T) * Y140 (A, V) * E142 (A, V, S, T) was obtained.
（７－１－５）修飾ＬｐｌＡバリアント：Ｒ１２１（Ａ，Ｖ，Ｓ，Ｔ）＊Ｙ１４０（Ａ，Ｖ）＊Ｅ１４２（Ａ，Ｖ，Ｓ，Ｔ）＊Ｋ１７６（Ｋ，Ａ，Ｖ，Ｓ，Ｔ）＊Ｉ１７８（Ａ，Ｖ）（合計３２０個のバリアント）のコンビナトリアルライブラリをコードするＤＮＡ断片の構築
　プライマーＰ２３（配列番号６０），Ｐ２４（配列番号６１），Ｐ２５（配列番号６１），Ｐ２６（配列番号６３），Ｐ２７（配列番号６４），Ｐ２８（配列番号６５），Ｐ２９（配列番号６６），Ｐ３０（配列番号６７），およびＰ３１（配列番号６８）を用いた。
　ＰＣＲ１：プライマーＰ２３，Ｐ２４，Ｐ２５；ＤＮＡ鋳型　ライブラリ２　ＤＮＡ混合液１μＬ。プロトコール：９５℃，３分；９８℃，２０秒；６５℃，１５秒；７２℃，１分；２０サイクル。
　ＰＣＲ２：プライマーＰ２６，Ｐ２８；ＤＮＡ－ｔｅｍｐｌａｔｅｓ，得られたＰＣＲ１混合液１μＬ。プロトコール：９５℃，３秒；９８℃，２０秒；６４℃，１５秒；７２℃，３０秒；２５サイクル。結果として、ＤＮＡ断片Ｆ２が得られた。
　ＰＣＲ３：プライマーＰ２７，Ｐ２８；ＤＮＡ鋳型　ライブラリ２　ＤＮＡ混合液１μＬ。プロトコール：９５℃，３分；９８℃，２０秒；６４℃，１５秒；７２℃，３０秒；２５サイクル。結果として、ＤＮＡ断片Ｆ３が得られた。
　ＰＣＲ４：プライマーＰ３０，Ｐ３１；ＤＮＡ鋳型　Ｆ２およびＦ３の等モル混合液。プロトコール：９５℃，３分；９８℃，２０秒；６４℃，１５秒；７２℃，１分；２５サイクル。
　結果として、修飾ＬｐｌＡバリアント：Ｒ１２１（Ａ，Ｖ，Ｓ，Ｔ）＊Ｙ１４０（Ａ，Ｖ）＊Ｅ１４２（Ａ，Ｖ，Ｓ，Ｔ）＊Ｋ１７６（Ａ，Ｖ，Ｓ，Ｔ）＊１７８（Ａ，Ｖ）のコンビナトリアルライブラリをコードするＤＮＡ断片の混合物（ライブラリ３）が得られた。 (7-1-5) Modified LplA variant: R121 (A, V, S, T) * Y140 (A, V) * E142 (A, V, S, T) * K176 (K, A, V, S, Construction of DNA fragment encoding combinatorial library of T) * I178 (A, V) (total of 320 variants) Primers P23 (SEQ ID NO: 60), P24 (SEQ ID NO: 61), P25 (SEQ ID NO: 61), P26 ( SEQ ID NO: 63), P27 (SEQ ID NO: 64), P28 (SEQ ID NO: 65), P29 (SEQ ID NO: 66), P30 (SEQ ID NO: 67), and P31 (SEQ ID NO: 68) were used.
PCR1: primers P23, P24, P25; DNA template library 2 1 μL of DNA mixture. Protocol: 95 ° C, 3 minutes; 98 ° C, 20 seconds; 65 ° C, 15 seconds; 72 ° C, 1 minute; 20 cycles.
PCR2: primers P26, P28; DNA-templates, 1 μL of the resulting PCR1 mixture. Protocol: 95 ° C, 3 seconds; 98 ° C, 20 seconds; 64 ° C, 15 seconds; 72 ° C, 30 seconds; 25 cycles. As a result, a DNA fragment F2 was obtained.
PCR3: primers P27, P28; 1 μL of DNA template library 2 DNA mixture. Protocol: 95 ° C, 3 minutes; 98 ° C, 20 seconds; 64 ° C, 15 seconds; 72 ° C, 30 seconds; 25 cycles. As a result, a DNA fragment F3 was obtained.
PCR4: primers P30, P31; an equimolar mixture of DNA templates F2 and F3. Protocol: 95 ° C, 3 minutes; 98 ° C, 20 seconds; 64 ° C, 15 seconds; 72 ° C, 1 minute; 25 cycles.
As a result, the modified LplA variant: R121 (A, V, S, T) * Y140 (A, V) * E142 (A, V, S, T) * K176 (A, V, S, T) * 178 (A , V) (mixture of DNA fragments encoding the combinatorial library) (library 3).
（７－２）酵素活性（オクタン酸アジドによるＩｇＧの標識）が野生型ＬｐｌＡのものよりも高い修飾ｍ（ｉ）ＬｐｌＡバリアントのスクリーニング
（７－２－１）修飾ＬｐｌＡバリアントのコンビナトリアルライブラリをコードする遺伝子を担持するｐＥＴ１５ｂ－ｍ（ｉ）－ｌｐｌＡ（ｉ=１～３２０）プラスミドのライブラリの構築
　ＤＮＡ断片ライブラリ３を、エンドヌクレアーゼＸｂａＩおよびＳａｃＩを用いて消化し、同エンドヌクレアーゼで消化したベクターｐＥＴ１５ｂ－ｗｔ－ｌｐｌＡ（ＳａｃＩ）に連結した。得られた連結混合液を、ｐＥＴ１５ｂ－ｍ（ｉ）－ｌｐｌＡプラスミドのライブラリとした。 (7-2) Screening of modified m (i) LplA variants with higher enzymatic activity (labeling of IgG with octanoic azide) than that of wild-type LplA (7-2-1) Encoding a combinatorial library of modified LplA variants Construction of library of pET15b-m (i) -lplA (i = 1 to 320) plasmid carrying gene DNA fragment library 3 was digested with endonucleases XbaI and SacI, and digested with the endonuclease. It was ligated to wt-lplA (SacI). The resulting ligation mixture was used as a library for the pET15b-m (i) -lplA plasmid.
（７－２－２）修飾ＬｐｌＡバリアントのコンビナトリアルライブラリを発現するＢＬ２１（ＤＥ３）／ｐＥＴ１５ｂ－ｍ（ｉ）－ｌｐｌＡ株のライブラリの構築
　Ｅ．ｃｏｌｉ　ＢＬ２１（ＤＥ３）株（ＮＥＷ　ＥＮＧＬＡＮＤ　ＢｉｏＬａｂｓ）を、ｐＥＴ１５ｂ－ｍ（ｉ）－ｌｐｌＡプラスミドのライブラリで形質転換した。その結果、１０００個のアンピシリン耐性コロニーが得られた。各クローンにおけるｍ（ｉ）ｌｐｌＡ遺伝子を担持するプラスミドの存在を、プライマーＰ１およびＰ２を用いＰＣＲにより検証した。その結果、ｐＥＴ１５ｂ－ｍ（ｉ）－ｌｐｌＡプラスミドを担持する７００個のコロニーが選択された。
　また、ｗｔ－ＬｐｌＡを発現できるＢＬ２１（ＤＥ３）／ｐＥ１５ｂ－ｗｔ－ｌｐｌＡ株、ｗｔ－ｌｐｌＡ遺伝子の１染色体コピーを発現できるＢＬ２１（ＤＥ３）／ｐＥＴ１５ｂ株を構築した。 (7-2-2) Construction of library of BL21 (DE3) / pET15b-m (i) -lplA strain expressing a combinatorial library of modified LplA variants E. coli strain BL21 (DE3) (NEW ENGLAND BioLabs) was transformed with the library of plasmid pET15b-m (i) -lplA. As a result, 1000 ampicillin-resistant colonies were obtained. The presence of the plasmid carrying the m (i) lplA gene in each clone was verified by PCR using primers P1 and P2. As a result, 700 colonies carrying the pET15b-m (i) -lplA plasmid were selected.
In addition, a BL21 (DE3) / pE15b-wt-lplA strain capable of expressing wt-LplA and a BL21 (DE3) / pET15b strain capable of expressing one chromosome copy of the wt-lplA gene were constructed.
（７－２－３）ｍ（ｉ）－ＬｐｌＡバリアントの発現
　選択した７００個の形質転換体およびＢＬ２１（ＤＥ３）／ｐＥＴ１５ｂ－ｗｔ－ｌｐｌＡ株をそれぞれ、アンピシリン（２００ｍｇ／Ｌ）を添加したＬＢブロス１ｍＬを各ウェルに含有する８個のポリプロピレンスクエア９６ディープウェルマイクロプレート（ＤｕｅｔｚＳｙｓｔｅｍ，（Ｋｕｈｎｅｒ社）（登録商標））に接種し、サンドイッチカバーで被覆した。全てのプレートでバックグランドＬｐｌＡ活性の測定のため、１０個のウェルにＢＬ２１（ＤＥ３）／ｐＥＴ１５ｂ株を接種した。また、全てのプレートで、プラスミドで発現させたｗｔＬｐｌＡ活性を測定するため、１０個のウェルにＢＬ２１（ＤＥ３）／ｐＥＴ１５ｂ－ｗｔＬｐｌＡ株を接種した。
　次いで、振盪機（Ｌａｂ－Ｔｈｅｒｍ　ＬＴ－Ｘ，Ｋｕｈｎｅｒ（登録商標））を用い、３００ＲＰＭ、３７℃で約２時間、マイクロプレートを培養した。ＯＤ５９５が約１に到達した時点で、ＩＰＴＧ（イソプロピルβ－Ｄ－１－チオガラクトピラノシド）を各ウェルに１ｍＭの濃度になるまで添加して、約２時間培養することにより、タンパク質合成を誘導した。Ｓ－４ｘユニバーサル－Ｌローターを取り付けたエッペンドルフ５９２０Ｒ遠心分離機により、４℃、４０００ＲＰＭ、１０分での遠心分離し、菌体を回収し、上清を除去した。
　各ウェルの菌体を、以下を含有する細胞溶解用緩衝液６０μＬに再懸濁した：５０ｍＭ　Ｔｒｉｓ－ＨＣｌ　ｐＨ７．５，５％グリセロール（ｖ／ｖ），１ｍＭ　ＤＴＴ（ジチオスレイトール），１ｍＭ　ＰＭＳＦ（フェニルメチルスルホニルフルオリド）。その後、リゾチームを終濃度３００μｇ／ｍＬとなるよう各ウェルに添加し、Ｋｕｈｎｅｒ振盪機を用い。１０℃にて４時間１００ｒｐｍで細胞懸濁液をインキュベートした。次いで、５ｍＭ　ＭｇＣｌ２および１μｇ／ｍＬとなるようＤＮａｓｅＩ（Ｓｉｇｍａ）をに添加し、反応混合液を１０℃にて３０分間１００ｒｐｍでインキュベートした。得られた粗細胞溶解物を、９６ウェルフィルタープレート（Ｃｏｒｎｉｎｇ；０．２μｍ，ＰＤＶＦ疎水性膜）を用いて濾過し、フロースルー画分を、ＬｐｌＡタンパク質を含む無細胞抽出液として調製した。 (7-2-3) Expression of m (i) -LplA variant 700 selected transformants and BL21 (DE3) / pET15b-wt-lplA strain were added to LB broth supplemented with ampicillin (200 mg / L), respectively. One mL was inoculated into eight polypropylene square 96 deep well microplates (DuetzSystem, (Kuhner) (registered trademark)) containing each well and covered with a sandwich cover. BL21 (DE3) / pET15b strain was inoculated into 10 wells for measurement of background LplA activity on all plates. In addition, BL21 (DE3) / pET15b-wtLplA strain was inoculated into 10 wells to measure wtLplA activity expressed in the plasmid on all plates.
Then, the microplate was cultured at 300 RPM and 37 ° C. for about 2 hours using a shaker (Lab-Therm LT-X, Kuhner (registered trademark)). When the OD 595 reaches about 1, IPTG (isopropyl β-D-1-thiogalactopyranoside) is added to each well to a concentration of 1 mM, and cultured for about 2 hours to obtain protein synthesis. Was induced. The cells were centrifuged at 4 ° C., 4000 RPM for 10 minutes using an Eppendorf 5920R centrifuge equipped with an S-4x Universal-L rotor, and the cells were collected and the supernatant was removed.
Cells in each well were resuspended in 60 μL of cell lysis buffer containing: 50 mM Tris-HCl pH 7.5, 5% glycerol (v / v), 1 mM DTT (dithiothreitol), 1 mM PMSF (Phenylmethylsulfonyl fluoride). Thereafter, lysozyme was added to each well to a final concentration of 300 μg / mL, and a Kuhner shaker was used. The cell suspension was incubated at 100 rpm for 4 hours at 10 ° C. Then, 5 mM MgCl 2 and 1 μg / mL of DNase I (Sigma) were added and the reaction mixture was incubated at 10 ° C. for 30 minutes at 100 rpm. The obtained crude cell lysate was filtered using a 96-well filter plate (Corning; 0.2 μm, PDVF hydrophobic membrane), and the flow-through fraction was prepared as a cell-free extract containing the LplA protein.
（７－２－４）粗細胞溶解物のＬｐｌＡ活性（オクタン酸アジドによるＩｇＧの標識）アッセイ
　以下を含有する反応液（２０μＬ）を調製した：ＮａＰｉ緩衝液，２０ｍＭ（ｐＨ７）；ＡＴＰ　１ｍＭ，（ｐＨ７）；ＭｇＳＯ４，２ｍＭ；オクタン酸アジド，２０μＭ；ＩｇＧ，２μＭ；ＬｐｌＡタンパク質調製物，１４．５μＬ。この反応液を３０℃で約１６時間（一晩）インキュベートした。
　次いで、ＤＢＣＯ－Ｃｙ３（Ｓｉｇｍａ）を終濃度３０μＭになるまで各反応液に添加し、３０℃で３０時間インキュベートした。標識反応後、２００μＬのＰＢＳ（リン酸緩衝化生理食塩水）緩衝液を各反応に添加し、得られた溶液を、同緩衝液で平衡化したプロテインＡ　ＨＰ　ＭｕｌｔｉＴｒａｐ９６ウェルプレート（ＧＥ　ｈｅａｌｔｈｃａｒｅ）にアプライした。プレートを、２０％　ＥｔＯＨを添加した同緩衝液により３回洗浄した。１００ｍＭ　Ｇｌｙ－ＨＣｌ（ｐＨ２．７）を用いて標識ＩｇＧを溶出させ、回収した。次いで、プレート蛍光測定機を用いることにより、溶出画分の蛍光強度（λｅｘ＝５５４ｎｍ／λｅｍ＝５６８ｎｍ）を測定した。各ｍＬｐｌＡの活性を、以下のようにノーマライズして算出した：［Ｆ（ｉ）－Ｆ（Ｎ）］／Ｆ（Ｐ），式中、Ｆ（ｉ）はｉ画分の蛍光であり，Ｆ（Ｎ）はＢＬ２１（ＤＥ３）／ｐＥＴ１５ｂ株の粗無細胞抽出液を用いた反応の１０個の反応の平均蛍光強度であり、Ｆ（Ｐ）はＢＬ２１（ＤＥ３）／ｐＥＴ１５ｂ－ｗｔ－ｌｐｌＡ（ＳａｃＩ）株の無細胞抽出液を用いた反応の１０個の反応からの平均蛍光強度である。
　その結果、野生型ＬｐｌＡよりも活性が２～３倍高い４つの変異型ＬｐｌＡが見いだされた。これらのバリアントを、ｍ２３１、ｍ７４６、ｍ７６６、およびｍ８７６と命名した。
　ｍ２３１、ｍ７４６、ｍ７６６、およびｍ８７６　ＬｐｌＡバリアントを担持するｐＥＴ１５ｂ－ｍ２３１－ｌｐｌＡ、ｐＥＴ１５ｂ－ｍ７４６－ｌｐｌＡ、ｐＥＴ１５ｂ－ｍ７６６－ｌｐｌＡ、およびｐＥＴ１５ｂ－ｍ８７６－ｌｐｌＡプラスミドの配列を解析し、４種の配列が決定された。
　得られたＤＮＡ配列を翻訳して、ＬｐｌＡの各修飾バリアントのアミノ酸置換を推定した。推定アミノ酸配列のアライメントを図２６に示す。推定アミノ酸置換を表４に要約する。
Figure JPOXMLDOC01-appb-T000006
 (7-2-4) LplA activity (labeling of IgG with octanoic acid azide) assay of crude cell lysate A reaction solution (20 μL) containing the following was prepared: NaPi buffer, 20 mM (pH 7); ATP 1 mM, ( pH 7); MgSO 4 , 2 mM; octanoic acid azide, 20 μM; IgG, 2 μM; LplA protein preparation, 14.5 μL. The reaction was incubated at 30 ° C. for about 16 hours (overnight).
Next, DBCO-Cy3 (Sigma) was added to each reaction solution until the final concentration became 30 μM, and the mixture was incubated at 30 ° C. for 30 hours. After the labeling reaction, 200 μL of PBS (phosphate buffered saline) buffer was added to each reaction, and the resulting solution was applied to a Protein A HP MultiTrap 96-well plate (GE healthcare) equilibrated with the same buffer. did. Plates were washed three times with the same buffer supplemented with 20% EtOH. The labeled IgG was eluted with 100 mM Gly-HCl (pH 2.7) and collected. Next, the fluorescence intensity (λex = 554 nm / λem = 568 nm) of the eluted fraction was measured by using a plate fluorometer. The activity of each mLplA was calculated by normalizing as follows: [F (i) -F (N)] / F (P), where F (i) is the fluorescence of the i fraction and F (i) (N) is the average fluorescence intensity of 10 reactions using the crude cell-free extract of BL21 (DE3) / pET15b strain, and F (P) is BL21 (DE3) / pET15b-wt-lplA (SacI). A) Average fluorescence intensity from ten reactions of cell-free extracts of the strain.
As a result, four mutant LplAs having 2-3 times higher activity than the wild-type LplA were found. These variants were named m231, m746, m766, and m876.
The sequences of the pET15b-m231-lplA, pET15b-m746-lplA, pET15b-m766-lplA, and pET15b-m876-lplA plasmids carrying the m231, m746, m766, and m876 LplA variants were analyzed and the four sequences determined. Was done.
The resulting DNA sequence was translated to estimate the amino acid substitution of each modified variant of LplA. FIG. 26 shows the alignment of the deduced amino acid sequence. Putative amino acid substitutions are summarized in Table 4.
Figure JPOXMLDOC01-appb-T000006
（７－３）Ｎ－Ｔａｇ６Ｈｉｓ－Ｘ－ＬｐｌＡバリアント（Ｘ＝ｗｔ，ｍ２３１，ｍ７４６，ｍ７６６，ｍ８７６）の発現、精製、および活性アッセイ
（７－３－１）ｐＥＴ１５ｂ－Ｎ－Ｔａｇ６Ｈｉｓ－Ｘ－ｌｐｌＡプラスミド（Ｘ＝ｗｔ，ｍ２３１，ｍ７４６，ｍ７６６，ｍ８７６）の構築
　プライマーＰ３２（配列番号７７）およびＰ３３（配列番号７８）を用いた。
　ＰＣＲ１：プライマーＰ３２，Ｐ３３；ＤＮＡ鋳型　ｐＥＴ１５ｂ－Ｘ－ｌｐｌＡ（Ｘ＝ｗｔ，２３１，ｍ７４６，ｍ７６６，ｍ８７６）。プロトコール：９５℃，３分；９８℃，２０秒；６４℃，１５秒；７２℃，１分；２５サイクル。
　得られたＤＮＡ断片を、ＮｃｏＩ，ＢａｍＨＩ制限酵素で消化し、同制限酵素で消化されたｐＥＴ１５ｂプラスミドと連結した。その結果、５個のｐＥＴ１５ｂ－Ｎ－Ｔａｇ６Ｈｉｓ－Ｘ－ｌｐｌＡプラスミド（Ｘ＝ｗｔ，ｍ２３１，ｍ７４６，ｍ７６６，ｍ８７６）が構築された。 (7-3) Expression, purification, and activity assay of N-Tag6His-X-LplA variant (X = wt, m231, m746, m766, m876) (7-3-1) pET15b-N-Tag6His-X-lplA Construction of plasmid (X = wt, m231, m746, m766, m876) Primers P32 (SEQ ID NO: 77) and P33 (SEQ ID NO: 78) were used.
PCR1: primers P32 and P33; DNA template pET15b-X-lplA (X = wt, 231, m746, m766, m876). Protocol: 95 ° C, 3 minutes; 98 ° C, 20 seconds; 64 ° C, 15 seconds; 72 ° C, 1 minute; 25 cycles.
The obtained DNA fragment was digested with NcoI and BamHI restriction enzymes, and ligated with the pET15b plasmid digested with the same restriction enzymes. As a result, five pET15b-N-Tag6His-X-IplA plasmids (X = wt, m231, m746, m766, m876) were constructed.
（７－３－２）Ｎ－Ｔａｇ６Ｈｉｓ－ｍ－ＬｐｌＡバリアントの発現および精製
　構築したｐＥＴ１５－Ｎ－Ｔａｇ６Ｈｉｓ－Ｘ－ＬｐｌＡプラスミド（Ｘ＝ｗｔ，ｍ２３１，ｍ７４６，ｍ７６６，ｍ８７６）を、Ｅ．ｃｏｌｉ　ＢＬ２１（ＤＥ３）株に導入した。得られたプラスミド株を、アンピシリン（２００μｇ／ｍＬ）を添加したＬＢブロス５０ｍＬを含む１２５ｍＬフラスコに接種し、ＯＤ６００＝１．０～１．２に到達するまで、３７℃、２５０ＲＰＭで振とう培養した。次いで、終濃度が１ｍＭとなるようＩＰＴＧを添加し、続けて３７℃で２時間培養した。
　得られた菌体を、遠心分離により回収し、０．９％　ＮａＣｌ溶液５０ｍＬで２回洗浄し、使用時まで－２０℃で凍結した。溶解させた菌体を、緩衝液Ａ（２０ｍＭ　Ｔｒｉｓ－ＨＣｌ，２０ｍＭ　イミダゾール，５００ｍＭ　ＮａＣｌ，ｐＨ７．４）　４ｍＬに再懸濁させ、懸濁液を５個の１．５バイアルに０．８ｍＬずつ分け、超音波処理により細胞を破壊した。破砕液の不溶性画分を、１３０００ＲＰＭで約２０分間遠心分離することにより除去した。得られた無細胞抽出液を、１ｍＬ　ＨｉＴｒａｐ　Ｃｈｅｌａｔｉｎｇカラム（ＧＥ　ｈｅａｌｔｈｃａｒｅ）にアプライし、標準的なプロトコールに従って精製した。
　次いでＮ－Ｔａｇ６Ｈｉｓ－ｍ－ＬｐｌＡ含有溶出画分の緩衝液を、５ｍＬ　ＨｉＴｒａｐ脱塩カラム（ＧＥ　Ｈｅａｌｔｈｃａｒｅ）を用い、５０ｍＭ　ＮａＰｉ緩衝液（ｐＨ７）に交換した。得られた溶液を、タンパク質終濃度が０．５～１ｍｇ／ＬになるまでＡｍｉｃｏｎ　１０Ｋメンブランにより濃縮した。 (7-3-2) Expression and Purification of N-Tag6His-m-LplA Variant The constructed pET15-N-Tag6His-X-LplA plasmid (X = wt, m231, m746, m766, m876) was isolated from E. coli. coli BL21 (DE3) strain. The obtained plasmid strain is inoculated into a 125 mL flask containing 50 mL of LB broth supplemented with ampicillin (200 μg / mL), and cultured with shaking at 37 ° C. and 250 RPM until OD 600 = 1.0 to 1.2 is reached. did. Next, IPTG was added to a final concentration of 1 mM, and the cells were cultured at 37 ° C. for 2 hours.
The obtained cells were collected by centrifugation, washed twice with 50 mL of 0.9% NaCl solution, and frozen at −20 ° C. until use. The lysed cells were resuspended in 4 mL of buffer A (20 mM Tris-HCl, 20 mM imidazole, 500 mM NaCl, pH 7.4), and the suspension was divided into five 1.5 vials by 0.8 mL. The cells were destroyed by sonication. The insoluble fraction of the lysate was removed by centrifugation at 13000 RPM for about 20 minutes. The resulting cell-free extract was applied to a 1 mL HiTrap Chelating column (GE healthcare) and purified according to standard protocols.
Next, the buffer of the eluted fraction containing N-Tag6His-m-LplA was exchanged with a 50 mM NaPi buffer (pH 7) using a 5 mL HiTrap desalting column (GE Healthcare). The resulting solution was concentrated on an Amicon 10K membrane until the final protein concentration was 0.5-1 mg / L.
（７－３－３）精製Ｎ－Ｔａｇ６ｔＨｉｓ－ｍ－ＬｐｌＡバリアントのＬｐｌＡ活性（オクタン酸アジドによるＩｇＧの標識）アッセイ
　以下を含有する反応混合液（１６０μＬ）を調製した：ＮａＰｉ緩衝液，２５ｍＭ（ｐＨ７）；ＡＴＰ　５ｍＭ（ｐＨ７）；ＭｇＳＯ４，３ｍＭ；オクタン酸アジド，５０μＭ；ＩｇＧ（ヒトミエローマ由来κ；Ｓｉｇｍａ　Ｌｏｔ＃ＳＬＢＲ０５００Ｖ），０．５ｍｇ／ｍｌ；精製ｍＬｐｌＡ，５０μｇ／ｍｌ。ＬｐｌＡを除く全ての成分を含有する反応液を、ネガティブコントロールとした。反応液を、３０℃で４８時間インキュベートし、経時的に反応混合液２０μＬをサンプリングした。ＥＤＴＡ（１０ｍＭ）の添加により反応を停止させ、分析時まで凍結保存した（－２０℃）。
　この反応液にＤＢＣＯ－Ｃｙ３　１００μＭとなるよう添加し、３０℃で５時間インキュベートした。標識後の反応液を、Ａｍｉｃｏｎ　Ｕｌｔｒａ　０．５ｍｌ　１０ｋＤａフィルターにアプライし、エタノール（２０％）を添加したＰＢＳ　０．３５ｍＬで３回洗浄した。この最終サンプルをＳＤＳ－ＰＡＧＥで分離し、ゲルを４０％エタノール，７％酢酸溶液中でインキュベートすることにより「固定」し、続いてＴｙｐｈｏｏｎ　ｖａｒｉａｂｌｅ　ｍｏｄｅ　Ｉｍａｇｅｒ　９２１０（ＧＥ　Ｈｅａｌｔｈｃａｒｅ）：蛍光モード，Ｅｘ５３２／Ｅｍ５８０でＣｙ３標識タンパクのバンドを蛍光で検出した。蛍光スキャンの際には過剰露光効果を回避するようにＰＭＴ電圧および感度を調整した。その後、ＰＡＧをクーマシーＲ２５０で染色し、タンパク質のバンドを検出し、バンド強度より定量した。
　得られた蛍光スキャン・クーマシー染色ＰＡＧＥを、図２７に示す。蛍光スキャン・クーマシー染色ＰＡＧの定量解析を表５～７に要約する。 (7-3-3) LplA Activity (Labeling IgG with Azide Octanoate) Assay of Purified N-Tag6tHis-m-LplA Variant A reaction mixture (160 μL) containing the following was prepared: NaPi buffer, 25 mM (pH 7) ATP 5 mM (pH 7); MgSO 4 , 3 mM; octanoic acid azide, 50 μM; IgG (κ from human myeloma; Sigma Lot # SLBR0500V), 0.5 mg / ml; purified mLplA, 50 μg / ml. A reaction solution containing all components except for LplA was used as a negative control. The reaction was incubated at 30 ° C. for 48 hours, and 20 μL of the reaction mixture was sampled over time. The reaction was stopped by the addition of EDTA (10 mM) and stored frozen (−20 ° C.) until analysis.
To this reaction solution, DBCO-Cy3 was added at 100 μM, and incubated at 30 ° C. for 5 hours. The reaction solution after labeling was applied to an Amicon Ultra 0.5 ml 10 kDa filter, and washed three times with 0.35 mL of PBS to which ethanol (20%) was added. The final sample was separated by SDS-PAGE and the gel was "fixed" by incubating it in a 40% ethanol, 7% acetic acid solution, followed by Typhoon variable mode imager 9210 (GE Healthcare): Fluorescence mode, Ex532 / Em580 The fluorescence of Cy3-labeled protein band was detected. During fluorescence scanning, the PMT voltage and sensitivity were adjusted to avoid overexposure effects. Thereafter, the PAG was stained with Coomassie R250, a protein band was detected and quantified based on the band intensity.
The resulting fluorescence scan Coomassie stained PAGE is shown in FIG. Quantitative analysis of fluorescence scan Coomassie stained PAGs is summarized in Tables 5-7.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
　本発明は、例えば、位置選択的に修飾された抗体の製造に有用である。 The present invention is useful, for example, for producing regioselectively modified antibodies.
　配列番号１および２はそれぞれ、Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ由来ＬｐｌＡ（ＥｃＬｐｌＡ）の塩基配列およびそれがコードするアミノ酸配列を示す。
　配列番号３および４はそれぞれ、Ｂａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓ由来ＬｐｌＡ（ＢｓＬｐｌＡ）の塩基配列（５’－および３’－末端にそれぞれ６塩基のＢａｍＨＩおよびＨｉｎｄＩＩＩ認識配列が付加されている）およびＢｓＬｐｌＡのアミノ酸配列を示す。
　配列番号５および６はそれぞれ、Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ　ｇｌｕｔａｍｉｃｕｍ由来ＬｐｌＡ（ＣｇＬｐｌＡ）の塩基配列（５’－および３’－末端にそれぞれ６塩基のＢａｍＨＩおよびＨｉｎｄＩＩＩ認識配列が付加されている）およびＣｇＬｐｌＡのアミノ酸配列を示す。
　配列番号７および８はそれぞれ、Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ　ｅｐｉｄｅｒｍｉｄｅｓ由来ＬｐｌＡ（ＳｅＬｐｌＡ）の塩基配列（５’－および３’－末端にそれぞれ６塩基のＢａｍＨＩおよびＨｉｎｄＩＩＩ認識配列が付加されている）およびＢｓＬｐｌＡのアミノ酸配列を示す。
　配列番号９および１０はそれぞれ、ＥｃＬｐｌＡ増幅用ＰＣＲプライマー（ｌｐｌＡ（Ｅｃ）　ｆｗおよびｌｐｌＡ（Ｅｃ）　ｒｖ）の塩基配列を示す。
　配列番号１１は、プラスミドベクターｐＣｏｌｄ　ＧＳＴ　ＤＮＡの塩基配列を示す。
　配列番号１２および１３はそれぞれ、ｐＣｏｌｄ　ＧＳＴ　ＤＮＡ増幅用ＰＣＲプライマー（ｐＣｏｌｄ　ＧＳＴ　ｆｗおよびｐＣｏｌｄ　ＧＳＴ　ｒｖ）の塩基配列を示す。
　配列番号１４および１５はそれぞれ、糖鎖をＰＮＧａｓｅで切断したトラスツズマブの重鎖のアミノ酸配列、およびトラスツズマブの軽鎖のアミノ酸配列を示す。
　配列番号１６～２２は、トラスツズマブのトリプシン消化による重鎖領域のペプチドフラグメントのアミノ酸配列を示す。
　配列番号２３～２７は、トラスツズマブのトリプシン消化による軽鎖領域のペプチドフラグメントのアミノ酸配列を示す。
　配列番号２８は、実施例３でトラスツズマブを修飾する機能性物質のモデルペプチドのアミノ酸配列を示す。
　配列番号２９～３７は、公知のＬｐｌＡ認識アミノ酸配列を示す。
　配列番号３８～６８は、それぞれ、プライマーＰ１～Ｐ３１の塩基配列を示す。
　配列番号６９および７０は、それぞれ、修飾ＬｐｌＡバリアントｍ２３１の塩基配列およびそれがコードするアミノ酸配列を示す。
　配列番号７１および７２は、それぞれ、修飾ＬｐｌＡバリアントｍ７４６の塩基配列およびそれがコードするアミノ酸配列を示す。
　配列番号７３および７４は、それぞれ、修飾ＬｐｌＡバリアントｍ７６６の塩基配列およびそれがコードするアミノ酸配列を示す。
　配列番号７５および７６は、それぞれ、修飾ＬｐｌＡバリアントｍ８７６の塩基配列およびそれがコードするアミノ酸配列を示す。
　配列番号７７および７８は、それぞれ、プライマーＰ３２およびＰ３３の塩基配列を示す。 SEQ ID NOs: 1 and 2 show the nucleotide sequence of LplA (EcLplA) derived from Escherichia coli and the amino acid sequence encoded thereby, respectively.
SEQ ID NOS: 3 and 4 show the amino acid sequences of Bacillus subtilis-derived LplA (BsLplA) (having 6 bases of BamHI and HindIII recognition sequences added at the 5′- and 3′-ends) and BsLplA, respectively. .
SEQ ID NOs: 5 and 6 show the amino acid sequences of Corynebacterium glutamicum-derived LplA (CgLplA) (having 6 bases of BamHI and HindIII recognition sequences at the 5′- and 3′-ends) and CgLplA, respectively. .
SEQ ID NOs: 7 and 8 show the amino acid sequences of Staphylococcus epidermides-derived LplA (SeLplA) (having 6 bases of BamHI and HindIII recognition sequences added at the 5′- and 3′-ends) and BsLplA, respectively. .
SEQ ID NOS: 9 and 10 show the nucleotide sequences of PCR primers for amplification of EcLplA (lplA (Ec) fw and lplA (Ec) rv), respectively.
SEQ ID NO: 11 shows the nucleotide sequence of plasmid vector pCold GST DNA.
SEQ ID NOs: 12 and 13 show the nucleotide sequences of PCR primers for pCold GST DNA amplification (pCold GST fw and pCold GST rv), respectively.
SEQ ID NOS: 14 and 15 show the amino acid sequence of the heavy chain of trastuzumab and the amino acid sequence of the light chain of trastuzumab, respectively, in which the sugar chain is cleaved with PNGase.
SEQ ID NOs: 16 to 22 show the amino acid sequences of peptide fragments of the heavy chain region obtained by trypsin digestion of trastuzumab.
SEQ ID NOs: 23 to 27 show the amino acid sequences of peptide fragments of the light chain region obtained by trypsin digestion of trastuzumab.
SEQ ID NO: 28 shows the amino acid sequence of a model peptide of a functional substance that modifies trastuzumab in Example 3.
SEQ ID NOs: 29 to 37 show known LplA recognition amino acid sequences.
SEQ ID NOs: 38 to 68 show the nucleotide sequences of primers P1 to P31, respectively.
SEQ ID NOS: 69 and 70 show the base sequence of the modified LplA variant m231 and the amino acid sequence encoded thereby, respectively.
SEQ ID NOs: 71 and 72 show the nucleotide sequence of the modified LplA variant m746 and the amino acid sequence encoded thereby, respectively.
SEQ ID NOS: 73 and 74 show the nucleotide sequence of the modified LplA variant m766 and the amino acid sequence encoded thereby, respectively.
SEQ ID NOs: 75 and 76 show the base sequence of the modified LplA variant m876 and the amino acid sequence encoded thereby, respectively.
SEQ ID NOs: 77 and 78 show the nucleotide sequences of primers P32 and P33, respectively.

Claims (40)

  1. 　抗体を、リポ酸タンパク質リガーゼの存在下において、修飾部分を有するリポ酸アナログと反応させて、定常領域中のリジン残基の側鎖に修飾部分を有する修飾抗体を生成することを含む、修飾部分を有する修飾抗体の製造方法。 Reacting the antibody with a lipoic acid analog having a modifying moiety in the presence of a lipoic acid protein ligase to produce a modified antibody having a modifying moiety on the side chain of a lysine residue in the constant region. A method for producing a modified antibody having
  2. 　抗体が天然ポリペプチド鎖構造を有する、請求項１記載の方法。 方法 The method of claim 1, wherein the antibody has a native polypeptide chain structure.
  3. 　抗体がモノクローナル抗体である、請求項１または２記載の方法。 The method according to claim 1 or 2, wherein the antibody is a monoclonal antibody.
  4. 　抗体が全長抗体または抗体断片である、請求項１～３のいずれか一項記載の方法。 The method according to any one of claims 1 to 3, wherein the antibody is a full-length antibody or an antibody fragment.
  5. 　抗体がヒト抗体またはヒト化抗体である、請求項１～４のいずれか一項記載の方法。 The method according to any one of claims 1 to 4, wherein the antibody is a human antibody or a humanized antibody.
  6. 　抗体がＩｇＧである、請求項１～５のいずれか一項記載の方法。 The method according to any one of claims 1 to 5, wherein the antibody is an IgG.
  7. 　抗体がＦａｂまたはＦ（ａｂ’）２である、請求項１～６のいずれか一項記載の方法。 The method according to any one of claims 1 to 6, wherein the antibody is Fab or F (ab ') 2 .
  8. 　修飾部分を有するリポ酸アナログが、修飾部分を有するＣ４～Ｃ１０アルキル－カルボン酸であり、
    　定常領域中のリジン残基の側鎖に修飾部分を有する修飾抗体が、修飾部分を有するＣ４～Ｃ１０アルキル－カルボニルを、定常領域中のリジン残基の側鎖に有する抗体である、請求項１～７のいずれか一項記載の方法。     The lipoic acid analog having a modifying moiety is a C 4 -C 10 alkyl-carboxylic acid having a modifying moiety;
    The modified antibody having a modified portion in the side chain of a lysine residue in the constant region is an antibody having a C 4 to C 10 alkyl-carbonyl having a modified portion in the side chain of a lysine residue in the constant region. Item 8. The method according to any one of Items 1 to 7.
  9. 　修飾部分を有するリポ酸アナログが、下記式（Ｉ）：
    Ｒ－ＣｎＨ２ｎ－ＣＯＯＨ　　　（Ｉ）
    〔式中、
    　Ｒは、修飾部分であり、
    　ｎは、４～１０の整数である。〕で表されるものであり、かつ
    　定常領域中のリジン残基の側鎖に修飾部分を有する修飾抗体が、下記式（ＩＩ）：
    Ｒ－ＣｎＨ２ｎ－ＣＯ－ＮＨ－　　　（ＩＩ）
    〔式中、
    　Ｒおよびｎは、式（Ｉ）のものと同じであり、
    　ＮＨ－は、定常領域中のリジン残基の側鎖に存在する基である。〕で表される部分を定常領域中のリジン残基の側鎖に有する修飾抗体である、
    　請求項１～８のいずれか一項記載の方法。     A lipoic acid analog having a modifying moiety is represented by the following formula (I):
    R—C n H 2n —COOH (I)
    (In the formula,
    R is a modifying moiety;
    n is an integer of 4 to 10. And a modified antibody having a modified portion on the side chain of a lysine residue in the constant region is represented by the following formula (II):
    R—C n H 2n —CO—NH— (II)
    (In the formula,
    R and n are the same as in formula (I);
    NH- is a group present on the side chain of a lysine residue in the constant region. Is a modified antibody having a portion represented by the side chain of a lysine residue in the constant region,
    The method according to any one of claims 1 to 8.
  10. 　ｎが７である、請求項９記載の方法。 The method of claim 9, wherein n is 7.
  11. 　定常領域中のリジン残基の側鎖に修飾部分を有する修飾抗体が、修飾部分を抗体特有のリジン残基の側鎖のみに有する、請求項１～１０のいずれか一項記載の方法。 The method according to any one of claims 1 to 10, wherein the modified antibody having a modified portion on the side chain of a lysine residue in the constant region has the modified portion only on the side chain of a lysine residue specific to the antibody.
  12. 　定常領域中のリジン残基がＣＨ１領域中のリジン残基である、請求項１～１１のいずれか一項記載の方法。 The method according to any one of claims 1 to 11, wherein the lysine residue in the constant region is a lysine residue in the CH1 region.
  13. 　定常領域中のリジン残基が、重鎖のＣＨ１領域および軽鎖のＣＬ領域の双方に存在するリジン残基である、請求項１～１２のいずれか一項記載の方法。 The method according to any one of claims 1 to 12, wherein the lysine residue in the constant region is a lysine residue present in both the CH1 region of the heavy chain and the CL region of the light chain.
  14. 　重鎖のＣＨ１領域および軽鎖のＣＬ領域の双方に存在するリジン残基が、ヒトＩｇＧ重鎖における１３３位のリジン残基、およびヒトＩｇＧ軽鎖における１６９位のリジン残基である、請求項１３記載の方法。 The lysine residue present in both the CH1 region of the heavy chain and the CL region of the light chain is a lysine residue at position 133 in a human IgG heavy chain and a lysine residue at position 169 in a human IgG light chain. 13. The method according to 13.
  15. 　リポ酸タンパク質リガーゼが、微生物に由来する、請求項１～１４のいずれか一項記載の方法。 The method according to any one of claims 1 to 14, wherein the lipoic acid protein ligase is derived from a microorganism.
  16. 　リポ酸タンパク質リガーゼが、エシェリヒア（Ｅｓｃｈｅｒｉｃｈｉａ）属細菌、バチルス（Ｂａｃｉｌｌｕｓ）属細菌、コリネバクテリウム（Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ）属細菌、またはスタフィロコッカス（Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ）属細菌に由来する、請求項１～１５のいずれか一項記載の方法。 The lipoic acid protein ligase is derived from a bacterium belonging to the genus Escherichia, a bacterium belonging to the genus Bacillus, a bacterium belonging to the genus Corynebacterium, or a bacterium belonging to the genus Staphylococcus. The method of claim 1.
  17. 　リポ酸タンパク質リガーゼが、エシェリヒア・コリ（Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ）、バチルス・ズブチリス（Ｂａｃｉｌｌｕｓ　ｓｕｂｔｉｌｉｓ）、コリネバクテリウム・グルタミカム（Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ　ｇｌｕｔａｍｉｃｕｍ）、またはスタフィロコッカス・エピダーミス（Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ　ｅｐｉｄｅｒｍｉｄｉｓ）に由来する、請求項１～１６のいずれか一項記載の方法。 The lipoic acid protein ligase is derived from Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum or Corynebacterium glutamicum, or Staphylococcus epidermis from Stichrococcus epidermis. The method according to any one of claims 16 to 18.
  18. 　リポ酸タンパク質リガーゼが、下記（Ａ）～（Ｃ）からなる群より選ばれるタンパク質である、請求項１～１７のいずれか一項記載の方法：
    （Ａ）配列番号２、４、６、および８からなる群から選ばれるアミノ酸配列を含むタンパク質；
    （Ｂ）配列番号２、４、６、および８からなる群から選ばれるアミノ酸配列において、１もしくは数個のアミノ酸の置換、欠失、挿入、もしくは付加を含むアミノ酸配列を含み、かつ、リポ酸タンパク質リガーゼ活性を有するタンパク質；ならびに
    （Ｃ）配列番号２、４、６、および８からなる群から選ばれるアミノ酸配列に対して９０％以上の同一性を有するアミノ酸配列を含み、かつ、リポ酸タンパク質リガーゼ活性を有するタンパク質。     The method according to any one of claims 1 to 17, wherein the lipoic acid protein ligase is a protein selected from the group consisting of the following (A) to (C):
    (A) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8;
    (B) an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8, including an amino acid sequence containing substitution, deletion, insertion, or addition of one or several amino acids, and lipoic acid A protein having protein ligase activity; and (C) a lipoic acid protein comprising an amino acid sequence having 90% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, and 8. A protein having ligase activity.
  19. 　リポ酸タンパク質リガーゼが、下記（Ａ’）～（Ｃ’）からなる群より選ばれるタンパク質である、請求項１～１７のいずれか一項記載の方法：
    （Ａ’）配列番号２のアミノ酸配列において、下記（ｉ）～（ｖｉｉ）からなる群より選ばれる１個以上の変異を有するアミノ酸配列を含み、かつ、リポ酸タンパク質リガーゼ活性を有するタンパク質：
    （ｉ）１２１位のアルギニン残基
    　アラニン残基、またはスレオニン残基による置換；
    （ｉｉ）１３６位のセリン残基
    　ロイシン残基による置換；
    （ｉｉｉ）１４０位のチロシン残基
    　アラニン残基、またはバリン残基による置換；
    （ｉｖ）１４２位のグルタミン酸
    　セリン残基、スレオニン残基、またはバリン残基への置換；
    （ｖ）１４９位のヒスチジン残基
    　欠失；
    （ｖｉ）１７６位のリジン残基
    　アラニン残基、セリン残基、またはスレオニン残基による置換；
    （ｖｉｉ）１７８位のイソロイシン残基
    　アラニン残基、またはバリン残基による置換；
    （Ｂ’）（Ａ’）のアミノ酸配列において、１もしくは数個のアミノ酸の置換、欠失、挿入、もしくは付加を含むアミノ酸配列を含み、かつ、リポ酸タンパク質リガーゼ活性を有するタンパク質；ならびに
    （Ｃ’）（Ａ’）のアミノ酸配列に対して９０％以上の同一性を有するアミノ酸配列を含み、かつ、リポ酸タンパク質リガーゼ活性を有するタンパク質。     The method according to any one of claims 1 to 17, wherein the lipoic acid protein ligase is a protein selected from the group consisting of the following (A ') to (C'):
    (A ′) a protein having an amino acid sequence having one or more mutations selected from the group consisting of the following (i) to (vii) in the amino acid sequence of SEQ ID NO: 2, and having lipoic acid protein ligase activity:
    (I) substitution with an arginine residue at position 121, an alanine residue or a threonine residue;
    (Ii) substitution of a serine residue at position 136 with a leucine residue;
    (Iii) substitution with a tyrosine residue at position 140, an alanine residue or a valine residue;
    (Iv) substitution at position 142 of a glutamic acid serine residue, threonine residue, or valine residue;
    (V) deletion of histidine residue at position 149;
    (Vi) substitution with a lysine residue at position 176 by an alanine residue, a serine residue or a threonine residue;
    (Vii) substitution with an isoleucine residue at position 178, an alanine residue, or a valine residue;
    (B ′) a protein comprising an amino acid sequence containing substitution, deletion, insertion or addition of one or several amino acids in the amino acid sequence of (A ′) and having lipoic acid protein ligase activity; ') A protein comprising an amino acid sequence having 90% or more identity to the amino acid sequence of (A'), and having lipoic acid protein ligase activity.
  20. 　修飾部分が生体直交性官能基を含む、請求項１～１９のいずれか一項記載の方法。 20. The method according to any one of claims 1 to 19, wherein the modifying moiety comprises a bioorthogonal functional group.
  21. 　生体直交性官能基が、アジド残基、アルデヒド残基、チオール残基、アルキン残基、アルケン残基、ハロゲン残基、テトラジン残基、ニトロン残基、ヒドロキシルアミン残基、ニトリル残基、ヒドラジン残基、ケトン残基、ボロン酸残基、シアノベンゾチアゾール残基、アリル残基、ホスフィン残基、マレイミド残基、ジスルフィド残基、チオエステル残基、α―ハロカルボニル残基、イソニトリル残基、シドノン残基、およびセレン残基からなる群より選ばれる、請求項２０記載の方法。 The bioorthogonal functional group is azide residue, aldehyde residue, thiol residue, alkyne residue, alkene residue, halogen residue, tetrazine residue, nitrone residue, hydroxylamine residue, nitrile residue, hydrazine residue. Group, ketone residue, boronic acid residue, cyanobenzothiazole residue, allyl residue, phosphine residue, maleimide residue, disulfide residue, thioester residue, α-halocarbonyl residue, isonitrile residue, sydnon residue 21. The method according to claim 20, which is selected from the group consisting of a group and a selenium residue.
  22. 　以下（１）および（２）を含む、機能性物質を有する修飾抗体の製造方法：
    （１）抗体を、リポ酸タンパク質リガーゼＡの存在下において、生体直交性官能基を含む修飾部分を有するリポ酸アナログと反応させて、定常領域中のリジン残基の側鎖に生体直交性官能基を含む修飾部分を有する修飾抗体を生成すること；ならびに
    （２）定常領域中のリジン残基の側鎖に生体直交性官能基を含む修飾部分を有する修飾抗体を、生体直交性官能基を介して機能性物質と反応させて、定常領域中のリジン残基の側鎖に機能性物質を有する修飾抗体を生成すること。     A method for producing a modified antibody having a functional substance, comprising the following (1) and (2):
    (1) An antibody is reacted with a lipoic acid analog having a modified portion containing a bioorthogonal functional group in the presence of lipoic acid protein ligase A, so that the side chain of a lysine residue in the constant region has a bioorthogonal function. (2) generating a modified antibody having a modification moiety containing a bioorthogonal functional group on the side chain of a lysine residue in the constant region, To generate a modified antibody having a functional substance on the side chain of a lysine residue in the constant region.
  23. 　生体直交性官能基を含む修飾部分を有するリポ酸アナログが、生体直交性官能基を含む修飾部分を有するＣ４～Ｃ１０アルキル－カルボン酸であり、
    　定常領域中のリジン残基の側鎖に生体直交性官能基を含む修飾部分を有する修飾抗体が、生体直交性官能基を含む修飾部分を有するＣ４～Ｃ１０アルキル－カルボニルを、定常領域中のリジン残基の側鎖に有する抗体であり、
    　定常領域中のリジン残基の側鎖に機能性物質を有する修飾抗体が、機能性物質およびそれと反応した生体直交性官能基を含む修飾部分を有するＣ４～Ｃ１０アルキル－カルボニルを、定常領域中のリジン残基の側鎖に有する抗体である、請求項２０記載の方法。     The lipoic acid analog having a modifying moiety comprising a bioorthogonal functional group is a C 4 -C 10 alkyl-carboxylic acid having a modifying moiety comprising a bioorthogonal functional group;
    A modified antibody having a modified moiety containing a bioorthogonal functional group on the side chain of a lysine residue in the constant region is used to convert a C 4 -C 10 alkyl-carbonyl having a modified moiety containing a bioorthogonal functional group into the constant region. An antibody having a side chain of a lysine residue of
    A modified antibody having a functional substance on the side chain of a lysine residue in the constant region is characterized in that a C 4 to C 10 alkyl-carbonyl having a functional substance and a modified portion containing a bioorthogonal functional group reacted therewith is substituted with a constant region. 21. The method according to claim 20, which is an antibody having a side chain of a lysine residue therein.
  24. 　生体直交性官能基を含む修飾部分を有するリポ酸アナログが、下記式（Ｉ）：
    Ｒ－ＣｎＨ２ｎ－ＣＯＯＨ　　　（Ｉ）
    〔式中、
    　Ｒは、生体直交性官能基を含む修飾部分であり、
    　ｎは、４～１０の整数である。〕で表されるものであり、かつ
    　定常領域中のリジン残基の側鎖に生体直交性官能基を含む修飾部分を有する修飾抗体が、下記式（ＩＩ）：
    Ｒ－ＣｎＨ２ｎ－ＣＯ－ＮＨ－　　　（ＩＩ）
    〔式中、
    　Ｒおよびｎは、式（Ｉ）のものと同じであり、
    　ＮＨ－は、定常領域中のリジン残基の側鎖に存在する基である。〕で表される部分を定常領域中のリジン残基の側鎖に有する修飾抗体であり、
    　定常領域中のリジン残基の側鎖に機能性物質を有する修飾抗体が、下記式（ＩＩＩ）：
    Ｆ－Ｒ’－ＣｎＨ２ｎ－ＣＯ－ＮＨ－　　　（ＩＩＩ）
    〔式中、
    　ｎは、式（Ｉ）のものと同じであり、
    　ＮＨ－は、式（ＩＩ）のものと同じであり、
    　Ｆは、機能性物質であり、
    　Ｒ’は、機能性物質と生体直交性官能基との間の反応により生成する部分を含む２価の基である。〕で表される部分を定常領域中のリジン残基の側鎖に有する修飾抗体である、請求項２２または２３記載の方法。     A lipoic acid analog having a modifying moiety containing a bioorthogonal functional group is represented by the following formula (I):
    R—C n H 2n —COOH (I)
    (In the formula,
    R is a modifying moiety containing a bioorthogonal functional group,
    n is an integer of 4 to 10. And a modified antibody having a modified portion containing a bioorthogonal functional group on the side chain of a lysine residue in the constant region is represented by the following formula (II):
    R—C n H 2n —CO—NH— (II)
    (In the formula,
    R and n are the same as in formula (I);
    NH- is a group present on the side chain of a lysine residue in the constant region. A modified antibody having a portion represented by a side chain of a lysine residue in the constant region,
    A modified antibody having a functional substance on the side chain of a lysine residue in the constant region is represented by the following formula (III):
    F-R'-C n H 2n -CO-NH- (III)
    (In the formula,
    n is the same as that of formula (I);
    NH- is the same as that of formula (II),
    F is a functional substance,
    R ′ is a divalent group including a moiety generated by a reaction between a functional substance and a bioorthogonal functional group. 24. The method according to claim 22, wherein the antibody is a modified antibody having a portion represented by the following formula: in the side chain of a lysine residue in the constant region.
  25. 　機能性物質が薬物または標識物質である、請求項２０～２４のいずれか一項記載の方法。 方法 The method according to any one of claims 20 to 24, wherein the functional substance is a drug or a labeling substance.
  26. 　機能性物質が低分子化合物である、請求項２０～２５のいずれか一項記載の方法。 The method according to any one of claims 20 to 25, wherein the functional substance is a low molecular compound.
  27. 　機能性物質がペプチド化合物である、請求項２０～２６のいずれか一項記載の方法。 27. The method according to claim 20, wherein the functional substance is a peptide compound.
  28. 　定常領域中のリジン残基の側鎖に修飾部分を有する修飾抗体であって、
    　修飾部分を有するＣ４～Ｃ１０アルキル－カルボニルを、抗体特有のリジン残基の側鎖のみに有する、修飾抗体。     A modified antibody having a modified portion in a side chain of a lysine residue in a constant region,
    A modified antibody having a C 4 -C 10 alkyl-carbonyl having a modifying portion only on the side chain of a lysine residue unique to the antibody.
  29. 　定常領域中のリジン残基の側鎖に修飾部分を有する修飾抗体が、下記式（ＩＩ）：
    Ｒ－ＣｎＨ２ｎ－ＣＯ－ＮＨ－　　　（ＩＩ）
    〔式中、
    　Ｒは、修飾部分であり、
    　ｎは、４～１０の整数であり、
    　ＮＨ－は、定常領域中のリジン残基の側鎖に存在する基である。〕で表される部分を抗体特有のリジン残基の側鎖のみに有する、請求項２８記載の修飾抗体。     A modified antibody having a modified portion on the side chain of a lysine residue in the constant region is represented by the following formula (II):
    R—C n H 2n —CO—NH— (II)
    (In the formula,
    R is a modifying moiety;
    n is an integer of 4 to 10,
    NH- is a group present on the side chain of a lysine residue in the constant region. 29. The modified antibody according to claim 28, wherein the modified antibody has a portion represented by the following formula only in the side chain of a lysine residue unique to the antibody.
  30. 　ｎが７である、請求項２８または２９記載の修飾抗体。 30. The modified antibody according to claim 28 or 29, wherein n is 7.
  31. 　修飾部分が、生体直交性官能基を含む、請求項２８～３０のいずれか一項記載の修飾抗体。 The modified antibody according to any one of claims 28 to 30, wherein the modified moiety comprises a bioorthogonal functional group.
  32. 　定常領域中のリジン残基がＣＨ１領域中のリジン残基である、請求項２８～３１のいずれか一項記載の修飾抗体。 The modified antibody according to any one of claims 28 to 31, wherein the lysine residue in the constant region is a lysine residue in the CH1 region.
  33. 　定常領域中のリジン残基が、重鎖のＣＨ１領域および軽鎖のＣＬ領域の双方に存在するリジン残基である、請求項２８～３２のいずれか一項記載の修飾抗体。 The modified antibody according to any one of claims 28 to 32, wherein the lysine residue in the constant region is a lysine residue present in both the CH1 region of the heavy chain and the CL region of the light chain.
  34. 　重鎖のＣＨ１領域および軽鎖のＣＬ領域の双方に存在するリジン残基が、ヒトＩｇＧ重鎖における１３３位のリジン残基、およびヒトＩｇＧ軽鎖における１６９位のリジン残基である、請求項３３記載の修飾抗体。 The lysine residue present in both the CH1 region of the heavy chain and the CL region of the light chain is a lysine residue at position 133 in a human IgG heavy chain and a lysine residue at position 169 in a human IgG light chain. 34. The modified antibody according to 33.
  35. 　定常領域中のリジン残基の側鎖に機能性物質を有する修飾抗体であって、
    　機能性物質およびそれと反応した生体直交性官能基を含む修飾部分を有するＣ４～Ｃ１０アルキル－カルボニルを、抗体特有のリジン残基の側鎖のみに有する、修飾抗体。     A modified antibody having a functional substance on a side chain of a lysine residue in a constant region,
    A modified antibody comprising a functional substance and a C 4 -C 10 alkyl-carbonyl having a modifying moiety containing a bioorthogonal functional group reacted therewith only in the side chain of a lysine residue unique to the antibody.
  36. 　定常領域中のリジン残基の側鎖に機能性物質を有する修飾抗体が、下記式（ＩＩＩ）：
    Ｆ－Ｒ’－ＣｎＨ２ｎ－ＣＯ－ＮＨ－　　　（ＩＩＩ）
    〔式中、
    　Ｆは、機能性物質であり、
    　Ｒ’は、機能性物質と生体直交性官能基との間の反応により生成する部分を含む２価の基であり、
    　ｎは、４～１０の整数であり、
    　ＮＨ－は、定常領域中のリジン残基の側鎖に存在する基である。〕で表される部分を抗体特有のリジン残基の側鎖のみに有する、請求項３５記載の修飾抗体。     A modified antibody having a functional substance on the side chain of a lysine residue in the constant region is represented by the following formula (III):
    F-R'-C n H 2n -CO-NH- (III)
    (In the formula,
    F is a functional substance,
    R ′ is a divalent group including a moiety generated by a reaction between a functional substance and a bioorthogonal functional group,
    n is an integer of 4 to 10,
    NH- is a group present on the side chain of a lysine residue in the constant region. 36. The modified antibody according to claim 35, wherein the modified antibody has a portion represented by the following formula only in the side chain of a lysine residue unique to the antibody.
  37. 　ｎが７である、請求項３５または３６記載の修飾抗体。 The modified antibody according to claim 35 or 36, wherein Δn is 7.
  38. 　定常領域中のリジン残基がＣＨ１領域中のリジン残基である、請求項３５～３７のいずれか一項記載の修飾抗体。 The modified antibody according to any one of claims 35 to 37, wherein the lysine residue in the constant region is a lysine residue in the CH1 region.
  39. 　定常領域中のリジン残基が、重鎖のＣＨ１領域および軽鎖のＣＬ領域の双方に存在するリジン残基である、請求項３５～３８のいずれか一項記載の修飾抗体。 The modified antibody according to any one of claims 35 to 38, wherein the lysine residue in the constant region is a lysine residue present in both the CH1 region of the heavy chain and the CL region of the light chain.
  40. 　重鎖のＣＨ１領域および軽鎖のＣＬ領域の双方に存在するリジン残基が、ヒトＩｇＧ重鎖における１３３位のリジン残基、およびヒトＩｇＧ軽鎖における１６９位のリジン残基である、請求項３９記載の修飾抗体。 The lysine residue present in both the CH1 region of the heavy chain and the CL region of the light chain is a lysine residue at position 133 in a human IgG heavy chain and a lysine residue at position 169 in a human IgG light chain. 39. The modified antibody according to 39.
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