WO2022154116A1 - 化合物またはその塩、およびそれらにより得られる抗体 - Google Patents

化合物またはその塩、およびそれらにより得られる抗体 Download PDF

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Publication number
WO2022154116A1
WO2022154116A1 PCT/JP2022/001358 JP2022001358W WO2022154116A1 WO 2022154116 A1 WO2022154116 A1 WO 2022154116A1 JP 2022001358 W JP2022001358 W JP 2022001358W WO 2022154116 A1 WO2022154116 A1 WO 2022154116A1
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Prior art keywords
linker
salt
indicates
group
antibody
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PCT/JP2022/001358
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English (en)
French (fr)
Japanese (ja)
Inventor
紀子 畑田
慧 山田
豊 松田
友博 藤井
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味の素株式会社
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Priority to KR1020237024074A priority Critical patent/KR20230133294A/ko
Priority to CA3208296A priority patent/CA3208296A1/en
Priority to AU2022208654A priority patent/AU2022208654A1/en
Priority to JP2022575662A priority patent/JPWO2022154116A1/ja
Priority to EP22739521.7A priority patent/EP4279500A1/en
Priority to CN202280010420.0A priority patent/CN116829573A/zh
Publication of WO2022154116A1 publication Critical patent/WO2022154116A1/ja
Priority to US18/354,436 priority patent/US20240000965A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • 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
    • A61K47/6835—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • 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
    • A61K47/6835—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

  • the present invention relates to a compound or a salt thereof, an antibody obtained by the compound, and the like.
  • ADC antibody-drug conjugates
  • ADC is a drug in which a drug (eg, an anticancer drug) is conjugated 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.
  • ADCs such as T-DM1
  • DAR Drug Antibody Ratio
  • a C-CAP (Chemical Conjugation by Affinity Peptide) method that enables regioselective modification of an antibody by a chemically synthesized method (Patent Document 1).
  • This method has succeeded in position-selective modification of an antibody by reacting an affinity peptide with a peptide reagent in which an NHS-activated ester and a drug are linked to the antibody.
  • the ADC produced by this method the antibody and the drug are bound via a linker containing a peptide moiety.
  • the peptide moiety has potential immunogenicity and is susceptible to hydrolysis in the blood. Therefore, the ADC produced by this method has room for improvement in that it contains a peptide moiety in the linker.
  • an antibody having a functional substance (eg, a drug) that does not contain a peptide portion as a linker and has a functional substance (eg, a drug) in a position-selective manner is obtained by a chemical synthesis method using a predetermined compound containing an affinity peptide.
  • Techniques that can be prepared have been reported (Patent Documents 2 to 6). Avoiding the use of linkers containing peptide moieties is desirable in clinical applications.
  • An object of the present invention is to regioselectively modify an antibody with a functional substance and control the binding ratio between the antibody and the functional substance within a desired range.
  • the present inventors have selected the lysine residue at position 288/290 of the heavy chain in the immunoglobulin unit as the modification position of the antibody, and made it possible to modify the lysine residue in a position-specific manner.
  • the antibody can be position-selectively modified with a functional substance, and the average ratio of binding between the immunoglobulin unit and the functional substance (number of functional substances / immunoglobulin unit) is desired. It was found that it becomes easy to control highly to the range (1.5 to 2.5) of.
  • the total number of atoms constituting the main chain and the number of atoms constituting the main chain in the second linker are 5 to 7).
  • the present inventors have succeeded in developing a compound represented by the formula (I) or a salt thereof, and a reagent for antibody derivatization containing them.
  • the present inventors also use a compound represented by the formula (I) or a salt thereof to selectively modify a specific antibody, that is, the lysine residue at position 288/290 in the antibody with a modifying group.
  • an antibody antibody intermediate, thiol group introduction
  • an antibody in which the average ratio of binding between immunoglobulin units and modifying groups (number of modifying groups / immunoglobulin units) is highly controlled within a desired range (1.5 to 2.5).
  • [1] A compound represented by the formula (I) or a salt thereof.
  • [2] The compound of [1] or a salt thereof, wherein the leaving group is selected from the following: (A) RS (where R represents a hydrogen atom, a monovalent hydrocarbon group which may have a substituent, or a monovalent heterocyclic group which may have a substituent. , S represents a sulfur atom.); (B) RO (where R represents a hydrogen atom, a monovalent hydrocarbon group which may have a substituent, or a monovalent heterocyclic group which may have a substituent.
  • O represents an oxygen atom.
  • RA- ( RB- ) N (where RA and RB each have a hydrogen atom and a substituent independently of each other.) It represents a monovalent hydrocarbon group, or a monovalent heterocyclic group which may have a substituent, where N represents a nitrogen atom.); Or (d) a halogen atom.
  • the compound of [1] or [2] or a salt thereof, wherein the immunoglobulin unit is a human immunoglobulin unit.
  • affinity peptide is as follows: (A) Affinity peptide containing the amino acid sequence of CQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 1); An affinity peptide comprising an amino acid sequence containing a variation of selected 1-5 amino acid residues, where the lysine residue at position 27 and the two cysteine residues at positions 1 and 30 are maintained.
  • the affinity peptide of (B) is selected from the group consisting of the following, the compound of [6] or a salt thereof: (A) Affinity peptide containing the amino acid sequence of FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 2); (B) Affinity peptide containing the amino acid sequence of FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEDC (SEQ ID NO: 3); (C) Affinity peptide containing the amino acid sequence of FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC (SEQ ID NO: 4); (D) Affinity peptide containing the amino acid sequence of NMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC (SEQ ID NO: 5); (E) Affinity peptide comprising the amino acid sequence of MQCQRRFYEALHDPNLNEEQRNARIRSIKEEC (SEQ ID NO: 6);
  • the N-terminal and C-terminal amino acid residues in the affinity peptide may be protected, and the two thiol groups in the side chains of the two cysteine residues (C) in the affinity peptide are disulfide bonds.
  • the compound represented by the formula (I) is the compound represented by the formula (I') or a salt thereof according to any one of [1] to [8].
  • the compound represented by the formula (I') is the compound represented by the formula (I'') or a salt thereof.
  • a reagent for antibody derivatization which comprises a compound represented by the formula (I) or a salt thereof.
  • the reagent of [12], wherein the compound represented by the formula (I') is represented by the formula (I'').
  • the antibody intermediate of [14] or a salt thereof, wherein the antibody intermediate is a human antibody intermediate.
  • the antibody intermediate of [14] or [15] or a salt thereof, wherein the antibody intermediate is a human IgG intermediate.
  • [25] The conjugate of [24] or a salt thereof, wherein a specific amino acid residue other than the lysine residue present at the 288/290 position in the two heavy chains is further modified.
  • [26] The conjugate of [25] or a salt thereof, wherein the specific amino acid residue is a lysine residue present at position 246/248 in two heavy chains.
  • [27] The conjugate of any one of [24] to [26] or a salt thereof, wherein the structural unit represented by the formula (IV) is represented by the formula (IV').
  • [28] The conjugate of [27] or a salt thereof, wherein the structural unit represented by the formula (IV') is represented by the formula (IV ′′).
  • a compound represented by the formula (V) or a salt thereof is the compound represented by the formula (V') or a salt thereof.
  • the compound represented by the formula (V') is the compound represented by the formula (V'') or a salt thereof.
  • the compound of [31] or a salt thereof, wherein the compound represented by the formula (V'') is represented by the formula (V''-1) or (V''-2).
  • the leaving group having a higher ability to eliminate than the leaving group X is a pentafluorophenyloxy group, a tetrafluorophenyloxy group, a paranitrophenyloxy group, or an N-succinimidyloxy group.
  • the compound represented by the formula (VI) is the compound of the formula [33] or [34] or a salt thereof, which is represented by the formula (VI').
  • the compound represented by the formula (VI') is the compound represented by the formula (VI'') or a salt thereof.
  • a method for producing a thiol group-introduced antibody derivative or a salt thereof comprising reacting a compound represented by the formula (I) or a salt thereof with an antibody containing an immunoglobulin unit.
  • the antibody intermediate or a salt thereof is subjected to a cleavage reaction of a thioester to produce a thiol group-introduced antibody derivative or a salt thereof containing a structural unit represented by the formula (III); and (3) thiol.
  • a group-introduced antibody derivative or a salt thereof is reacted with a functional substance to form a conjugate of the antibody and the functional substance or a salt thereof, which comprises a structural unit represented by the formula (IV).
  • a method for producing a conjugate of a functional substance or a salt thereof is reacted with a functional substance to form a conjugate of the antibody and the functional substance or a salt thereof, which comprises a structural unit represented by the formula (IV).
  • the compound represented by the formula (VI) or a salt thereof is reacted with an affinity peptide having a binding region to the CH2 domain in the immunoglobulin unit containing two heavy chains and two light chains to react with the formula (').
  • a method for producing the represented compound or a salt thereof is reacted with an affinity peptide having a binding region to the CH2 domain in the immunoglobulin unit containing two heavy chains and two light chains.
  • a thiol group-introduced antibody derivative or a salt thereof containing a structural unit represented by the formula (II) is subjected to a cleavage reaction of a thioester to contain the structural unit represented by the formula (III).
  • a method for producing a thiol group-introduced antibody derivative or a salt thereof which comprises producing the salt thereof.
  • a method for producing a conjugate of an antibody and a functional substance or a salt thereof which comprises producing a conjugate of the sex substance or a salt thereof.
  • the average ratio of the bonds between the immunoglobulin unit and the affinity peptide-containing group is in a desired range (1.5 to 2).
  • the lysine residue at position 288/290 of the heavy chain in the immunoglobulin unit can be specifically and highly modified so as to be .5). Therefore, the compound represented by the formula (I) or a salt thereof is useful as a reagent for antibody derivatization.
  • the lysine residue at position 288/290 of the heavy chain in the immunoglobulin unit is specifically modified with an affinity peptide-containing group
  • the antibody intermediate represented by the formula (II) or an antibody intermediate thereof in which the average ratio of the bonds between the immunoglobulin unit and the affinity peptide-containing group (affinity peptide-containing group number / immunoglobulin unit) is highly controlled within a desired range. Salt can be provided.
  • an antibody prepared by using the antibody intermediate represented by the formula (II) or a salt thereof as a raw material can inherit the regioselectivity and the average ratio of binding of the antibody intermediate or the salt thereof.
  • a thiol group-introduced antibody derivative represented by the formula (III) or a salt thereof which has the above regioselectivity and an average ratio of binding, and a conjugation of the antibody and the functional substance represented by the formula (IV).
  • a gate or salt thereof can be provided.
  • compounds represented by formulas (V) and (VI) or salts thereof which are synthetic intermediates that enable efficient production of compounds represented by formula (I) or salts thereof.
  • FIG. 1 is a schematic diagram (No. 1) showing the concept of modification of immunoglobulin units with the compound of the present invention represented by the formula (I) or a salt thereof.
  • the compound of the present invention represented by the formula (I) or a salt thereof associates with the CH2 domain in the immunoglobulin unit via the affinity peptide (Y).
  • the compound of the present invention represented by the formula (I) or a salt thereof is a side chain of a specific amino acid residue in the CH2 domain via an activated carbonyl group having a elimination group (X) (Fig.). In it, it reacts with an amino group in the side chain of a lysine residue to produce an antibody intermediate or a salt thereof.
  • FIG. 2 is a schematic diagram (No.
  • FIG. 3 is a schematic diagram (No. 3) showing the concept of modification of immunoglobulin units with the compound of the present invention represented by the formula (I) or a salt thereof.
  • the reaction of a thiol group with a functional substance (Z) in a thiol group-introduced antibody derivative or a salt thereof produces a conjugate of the antibody and the functional substance or a salt thereof.
  • FIG. 4 is a diagram showing an outline of an embodiment of the present invention.
  • FIG. 5 is a diagram showing an outline of a preferred embodiment of the present invention.
  • FIG. 6 is a diagram showing an outline of a more preferable embodiment of the present invention.
  • FIG. 7 is a diagram showing ESI-TOFMS analysis of specific modification (number of introduced binding peptides) of trastuzumab (anti-HER2 IgG antibody) (Example 1).
  • FIG. 8 is a diagram showing ESI-TOFMS analysis of specific modification (heavy chain selectivity) of trastuzumab (Example 1).
  • FIG. 9 is a diagram showing (1) the amino acid sequence of the heavy chain of trastuzumab (SEQ ID NO: 8) and (2) the amino acid sequence of the light chain of trastuzumab (SEQ ID NO: 9).
  • FNWYVDGVEVHNAKTTKPR SEQ ID NO: 10
  • a peptide consisting of 18 amino acid residues containing a site of modification of trussumab to a lysine residue by trypsin digestion (a thiol-introduced product (+145.09 Da) subjected to carbamidometry with iodoacetamide).
  • MS spectrum measured value: m / z 577.03606, theoretical value: 577.03557, tetravalent
  • FIG. 11 shows the modification of the lysine residue at position 288/290 of the human IgG heavy chain in EU numbering, which is a product ion of m / z 682.13 (theoretical value: 682.01) corresponding to trivalent y16. It is a figure which shows the CID spectrum (Example 1-9-4).
  • FIG. 12 shows the results of searching for a peptide fragment containing a lysine residue-modified thiol-introduced product (+145.09Da) modified to a lysine residue (Carbamidemethylized with iodoacetamide) in a trypsin digested product of trastuzumab using BioPharma Finder.
  • FIG. 13 is a diagram showing ESI-TOFMS analysis of specific modification of trastuzumab (number of introduced binding peptides) (Example 2).
  • FIG. 14 is a diagram showing ESI-TOFMS analysis of specific modification (heavy chain selectivity) of trastuzumab (Example 2).
  • FNWYVDGVEVHNAKTTKPR SEQ ID NO: 10
  • a peptide consisting of 18 amino acid residues containing a site of modification of trussumab to a lysine residue by trypsin digestion a thiol-introduced product (+145.09 Da) subjected to carbamidometry with iodoacetamide.
  • MS spectrum measured value: m / z 577.03571, theoretical value: 577.03557, tetravalent
  • FIG. 16 shows the modification of the lysine residue at position 288/290 of the human IgG heavy chain in EU numbering, which is a product ion of m / z 682.41 (theoretical value: 682.01) corresponding to trivalent y16. It is a figure which shows the CID spectrum (Example 2-7-4).
  • FIG. 17 shows the results of searching for a peptide fragment containing a lysine residue-modified thiol-introduced product (+145.09Da) modified to a lysine residue by using a BioPharma Finder for a trypsin digest of trastuzumab. It is a figure (Example 2-7-4).
  • FIG. 18 is a diagram showing ESI-TOFMS analysis of specific modification of trastuzumab (number of introduced binding peptides) (Example 3).
  • FIG. 19 is a diagram showing ESI-TOFMS analysis of specific modification (heavy chain selectivity) of trastuzumab (Example 3).
  • FIG. 20 is a diagram showing ESI-TOFMS analysis of specific modification of trastuzumab (number of introduced binding peptides) (Example 4).
  • FIG. 21 is a diagram showing ESI-TOFMS analysis of specific modification (heavy chain selectivity) of trastuzumab (Example 4).
  • FIG. 18 is a diagram showing ESI-TOFMS analysis of specific modification of trastuzumab (number of introduced binding peptides) (Example 3).
  • FIG. 19 is a diagram showing ESI-TOFMS analysis of specific modification (heavy chain selectivity) of trastuzumab (Example
  • FIG. 22 is a diagram showing ESI-TOFMS analysis of specific modification of trastuzumab (number of introduced binding peptides) (Example 5).
  • FIG. 23 is a diagram showing ESI-TOFMS analysis of specific modification (heavy chain selectivity) of trastuzumab (Example 5).
  • FIG. 24 is a diagram showing ESI-TOFMS analysis of specific modification of trastuzumab (number of introduced binding peptides) (Example 6).
  • FIG. 25 is a diagram showing ESI-TOFMS analysis of specific modification (heavy chain selectivity) of trastuzumab (Example 6).
  • FIG. 23 is a diagram showing ESI-TOFMS analysis of specific modification of trastuzumab (number of introduced binding peptides) (Example 5).
  • FIG. 24 is a diagram showing ESI-TOFMS analysis of specific modification of trastuzumab (number of introduced binding peptides)
  • FIG. 26 is a diagram showing ESI-TOFMS analysis of specific modification of trastuzumab (number of introduced binding peptides) (Example 7).
  • FIG. 27 is a diagram showing ESI-TOFMS analysis of specific modification (heavy chain selectivity) of trastuzumab (Example 7).
  • FIG. 28 shows FNWYVDGVEVHNAKTTKPR (SEQ ID NO: 10), a peptide consisting of 18 amino acid residues containing a site of modification of trussumab to a lysine residue by trypsin digestion (a thiol-introduced product (+145.09 Da) subjected to carbamidometry with iodoacetamide).
  • FIG. 29 shows the modification of the lysine residue at position 288/290 of the human IgG heavy chain in EU numbering, showing the modification of the product ion of m / z 1022.21 (theoretical value: 1022.51) corresponding to divalent y16. It is a figure which shows the CID spectrum (Example 7-6-4).
  • FIG. 30 shows the results of searching for a peptide fragment containing a modification to a lysine residue (a thiol-introduced product (+145.09Da) subjected to Carbamidemethylization with iodoacetamide) in a trypsin digested product of trastuzumab using BioPharma Finder. It is a figure (Example 7-6-4). The horizontal axis shows the identified lysine residue, and the vertical axis shows the Integrity.
  • FIG. 31 is a diagram showing ESI-TOFMS analysis of specific modification of trastuzumab (number of introduced binding peptides) (Example 8).
  • FIG. 32 is a diagram showing ESI-TOFMS analysis of specific modification (heavy chain selectivity) of trastuzumab (Example 8).
  • FIG. 33 is a diagram showing ESI-TOFMS analysis of specific modification of trastuzumab (number of introduced binding peptides) (Example 9).
  • FIG. 34 is a diagram showing ESI-TOFMS analysis of specific modification (heavy chain selectivity) of trastuzumab (Example 9).
  • the term "antibody” is as follows.
  • the term "immunoglobulin unit” corresponds to a divalent monomeric unit which is a basic component of such an antibody, and is a unit containing two heavy chains and two light chains. .. Therefore, for immunoglobulin units, definitions, examples, and preferred examples of their origin, type (polyclonal or monoclonal, isotype, and full-length antibody or antibody fragment), antigen, lysine residue position, and regioselectivity are given below. Similar to that of the antibody described.
  • the origin of the antibody is not particularly limited, and may be derived from an animal such as a mammal, a bird (eg, a chicken), for example.
  • the immunoglobulin unit is derived from a mammal.
  • mammals include, for example, primates (eg, humans, monkeys, chimpanzees), rodents (eg, mice, rats, guinea pigs, hamsters, rabbits), pet animals (eg, dogs, cats), domestic animals. (Eg, cows, pigs, goats), servants (eg, horses, sheep), preferably primates or rodents, more preferably humans.
  • the type of antibody may be a polyclonal antibody or a monoclonal antibody.
  • the antibody may also be a divalent antibody (eg, IgG, IgD, IgE) or a tetravalent or higher valent antibody (eg, IgA antibody, IgM antibody).
  • the antibody is a monoclonal antibody.
  • the monoclonal antibody is modified so as to have, for example, a chimeric antibody, a humanized antibody, a human antibody, or an antibody to which a predetermined sugar chain is added (eg, a sugar chain binding consensus sequence such as an N-type sugar chain binding consensus sequence).
  • Antibodies bispecific antibodies, Fc region proteins, Fc fusion proteins.
  • Isotypes of monoclonal antibodies include, for example, IgG (eg, IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY.
  • a full-length antibody or an antibody fragment containing a variable region and CH1 domain and CH2 domain can be used as the monoclonal antibody, but a full-length antibody is preferable.
  • the antibody is preferably a human IgG monoclonal antibody, more preferably a human IgG full-length monoclonal antibody.
  • any antigen can be used as the antigen of the antibody.
  • antigens include proteins [oligopeptides, polypeptides. It may be a protein modified with a biomolecule such as sugar (eg, glycoprotein)], sugar chains, nucleic acids, small molecule compounds.
  • the antibody may be an antibody that uses a protein as an antigen.
  • proteins include cell membrane receptors, cell membrane proteins other than cell membrane receptors (eg, extracellular substrate proteins), ligands, and soluble receptors.
  • the protein that is the antigen of the antibody may be a disease target protein.
  • diseases target proteins include the following.
  • Amyloid AL Hereditary / rare diseases Amyloid AL, SEMA4D (CD100), insulin receptor, ANGPTL3, IL4, IL13, FGF23, corticostimulatory hormone, transthyretin, huntingtin
  • monoclonal antibodies include specific chimeric antibodies (eg, rituximab, baciliximab, infliximab, cetuximab, siltuximab, dinutuximab, altertoximab), specific humanized antibodies (eg, dacrizumab, paribizumab, trastuzumab, allenzumab, allenzumab).
  • specific chimeric antibodies eg, rituximab, baciliximab, infliximab, cetuximab, siltuximab, dinutuximab, altertoximab
  • specific humanized antibodies eg, dacrizumab, paribizumab, trastuzumab, allenzumab, allenzumab.
  • Efarizumab Efarizumab, Bebashizumab, Natarizumab (IgG4), Toshirizumab, Ekurizumab (IgG2), Mogamurizumab, Pertsuzumab, Obinutsuzumab, Bedorizumab, Penproridumab (IgG4), Mepolizumab, Erotsumub Human antibodies (eg, adalimumab (IgG1), panitumumab, golimumab, ustequinumab, canaquinumab, ofatumumab, denosumab (IgG2), ipilimumab, berimumab, rapiximab, lambsilmab, nibolumab, dupilumab (IgG4) Necitumumab, Brodalumab (IgG2), Oralatumab) can be mentioned (if the IgG subtype is
  • the lysine residue at position 288 corresponds to the 58th residue in the human IgG CH2 region
  • the lysine residue at position 290 corresponds to the residue at position 60 in the human IgG CH2 region.
  • the notation at position 288/290 indicates that the lysine residue at position 288 or 290 is the subject.
  • the lysine residue at position 288/290 in the antibody can be regioselectively modified.
  • regioselective or “regioselectivity” refers to binding to a specific amino acid residue in an antibody even though the specific amino acid residue is not unevenly distributed in a specific region in the antibody. It means that a predetermined structural unit that can be formed is unevenly distributed in a specific region in an antibody. Therefore, expressions related to regioselectivity, such as “regioselectively possessed”, “regioselectively bound”, and “regioselectively bound”, are target regions containing one or more specific amino acid residues.
  • the retention or binding rate of a given structural unit in the target region is significantly higher than the retention or binding rate of the structural unit in a non-target region containing a plurality of amino acid residues that are the same species as the specific amino acid residue in the target region. It means that it is high at a high level.
  • Such regioselectivity is 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, particularly preferably 90% or more, 95% or more, 96% or more, It may be 97% or more, 98% or more, 99% or more, 99.5% or more, or 100%.
  • the lysine residue at position 288/290 can be regioselectively modified without utilizing a linker containing a peptide.
  • the peptide moiety has potential immunogenicity and is susceptible to hydrolysis in the blood. Therefore, avoiding the use of linkers containing peptide moieties is desirable in clinical applications.
  • a specific amino acid residue at another position may be further regioselectively modified.
  • methods for regioselectively modifying a particular amino acid residue at a given position in an antibody are described in WO/2018/199337, WO 2019/240288, WO 2019/240287, and International. It is described in Publication No. 2020/090979.
  • Such specific amino acid residues include amino acid residues (eg, lysine residue, aspartic acid residue) having side chains that are easily modified (eg, amino group, carboxy group, amide group, hydroxy group, thiol group).
  • Glutamic acid residue asparagine residue, glutamine residue, threonine residue, serine residue, tyrosine residue, cysteine residue
  • a lysine residue having a side chain containing an amino group hydroxy A tyrosine residue having a side chain containing a group, a serine residue, and a threonine residue, or a cysteine residue having a side chain containing a thiol group, more preferably a lysine residue, and even more preferably.
  • It is a lysine residue at position 246/248 or a lysine residue at position 317, and is particularly preferably a lysine residue at position 246/248.
  • the notation at position 246/248 indicates that the lysine residue at position 246 or 248 is the subject.
  • salt includes, for example, a salt with an inorganic acid, a salt with an organic acid, a salt with an inorganic base, a salt with an organic base, and a salt with an amino acid.
  • the salt with the inorganic acid include salts with hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, and nitric acid.
  • salts with organic acids include formic acid, acetic acid, trifluoroacetic acid, lactic acid, tartaric acid, fumaric acid, oxalic acid, maleic acid, citric acid, succinic acid, malic acid, benzenesulfonic acid, and p-toluenesulfonic acid. Salt is mentioned.
  • Salts with inorganic bases include, for example, alkali metals (eg, sodium, potassium), alkaline earth metals (eg, calcium, magnesium), and other metals such as zinc, aluminum, and salts with ammonium. ..
  • Examples of the salt with an organic base include salts with trimethylamine, triethylamine, propylenediamine, ethylenediamine, pyridine, ethanolamine, monoalkylethanolamine, dialkylethanolamine, diethanolamine, and triethanolamine.
  • salts with amino acids include salts with basic amino acids (eg, arginine, histidine, lysine, ornithine) and acidic amino acids (eg, aspartic acid, glutamic acid).
  • the salt is preferably a salt with an inorganic acid (eg, hydrogen chloride) or a salt with an organic acid (eg, trifluoroacetic acid).
  • the present invention provides a compound represented by the following formula (I) or a salt thereof.
  • X indicates a leaving group
  • Y represents an affinity peptide having a binding region to the CH2 domain in an immunoglobulin unit containing two heavy chains and two light chains.
  • O indicates an oxygen atom
  • S represents a sulfur atom
  • W represents an oxygen atom or a sulfur atom
  • La indicates the first linker
  • Lb indicates a second linker
  • the total number of atoms constituting the main chain in the first linker and the number of atoms constituting the main chain in the second linker are 5 to 7.
  • the leaving group represented by X is a group that can be eliminated by a reaction between a carbon atom of a carbonyl group adjacent to X and an amino group.
  • Those skilled in the art can appropriately set such a leaving group.
  • Examples of such leaving groups include: (A) RS (where R represents a hydrogen atom, a monovalent hydrocarbon group which may have a substituent, or a monovalent heterocyclic group which may have a substituent. , S represents a sulfur atom.); (B) RO (where R represents a hydrogen atom, a monovalent hydrocarbon group which may have a substituent, or a monovalent heterocyclic group which may have a substituent.
  • O represents an oxygen atom.
  • C RA- ( RB- ) N
  • RA and RB are independent hydrogen atoms, monovalent hydrocarbon groups which may have substituents, or substituents, respectively. Indicates a monovalent heterocyclic group which may have, where N represents a nitrogen atom.
  • the leaving group represented by X may be: (A) RS (where R represents a hydrogen atom, a monovalent hydrocarbon group which may have a substituent, or a monovalent heterocyclic group which may have a substituent. , S represents a sulfur atom.); (B) RO (where R represents a hydrogen atom, a monovalent hydrocarbon group which may have a substituent, or a monovalent heterocyclic group which may have a substituent. , O represent an oxygen atom.); Or (c) RA- ( RB- ) N (where RA and RB each independently have a hydrogen atom and a substituent. It may be a monovalent hydrocarbon group, or a monovalent heterocyclic group which may have a substituent.);
  • the leaving group represented by X may be: (A) RS (where R represents a hydrogen atom, a monovalent hydrocarbon group which may have a substituent, or a monovalent heterocyclic group which may have a substituent. , S represents a sulfur atom.); Or (b) RO (where R has a hydrogen atom, a monovalent hydrocarbon group which may have a substituent, or a substituent. It represents a monovalent heterocyclic group which may be present, and O represents an oxygen atom.)
  • the leaving group represented by X may be: (A) RS (where R is a monovalent aromatic hydrocarbon group which may have a substituent or a monovalent aromatic heterocyclic group which may have a substituent. Shown, S represents a sulfur atom.).
  • the leaving group represented by X may be: (A') RS (where R represents a monovalent aromatic hydrocarbon group (eg, phenyl) which may have a substituent and S represents a sulfur atom).
  • halogen atom examples include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
  • Examples of the monovalent hydrocarbon group include a monovalent chain hydrocarbon group, a monovalent alicyclic hydrocarbon group, and a monovalent aromatic hydrocarbon group.
  • the monovalent chain hydrocarbon group means a hydrocarbon group composed of only a chain structure and does not contain a cyclic structure in the main chain. However, the chain structure may be linear or branched. Examples of the monovalent chain hydrocarbon group include alkyl, alkenyl and alkynyl. Alkyl, alkenyl, and alkynyl may be linear or branched.
  • alkyl an alkyl having 1 to 12 carbon atoms is preferable, an alkyl having 1 to 6 carbon atoms is more preferable, and an alkyl having 1 to 4 carbon atoms is further preferable.
  • the carbon atom number of the substituent does not include the carbon atom number of the substituent.
  • alkyl having 1 to 12 carbon atoms include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl. , Dodecyl.
  • an alkenyl having 2 to 12 carbon atoms is preferable, an alkenyl having 2 to 6 carbon atoms is more preferable, and an alkenyl having 2 to 4 carbon atoms is further preferable.
  • the carbon atom number of the substituent does not include the carbon atom number of the substituent. Examples of the alkenyl having 2 to 12 carbon atoms include vinyl, propenyl, and n-butenyl.
  • alkynyl having 2 to 12 carbon atoms is preferable, alkynyl having 2 to 6 carbon atoms is more preferable, and alkynyl having 2 to 4 carbon atoms is even more preferable.
  • the carbon atom number of the substituent does not include the carbon atom number of the substituent. Examples of alkynyl having 2 to 12 carbon atoms include ethynyl, propynyl, and n-butynyl.
  • alkyl is preferable.
  • the monovalent alicyclic hydrocarbon group means a hydrocarbon group containing only an alicyclic hydrocarbon as a ring structure and not containing an aromatic ring, and the alicyclic hydrocarbon is either monocyclic or polycyclic. It may be. However, it does not have to be composed only of alicyclic hydrocarbons, and a chain structure may be contained in a part thereof.
  • Examples of the monovalent alicyclic hydrocarbon group include cycloalkyl, cycloalkenyl, and cycloalkynyl, which may be monocyclic or polycyclic.
  • cycloalkyl a cycloalkyl having 3 to 12 carbon atoms is preferable, a cycloalkyl having 3 to 6 carbon atoms is more preferable, and a cycloalkyl having 5 to 6 carbon atoms is further preferable.
  • the carbon atom number of the substituent does not include the carbon atom number of the substituent.
  • Examples of cycloalkyl having 3 to 12 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
  • cycloalkenyl a cycloalkenyl having 3 to 12 carbon atoms is preferable, a cycloalkenyl having 3 to 6 carbon atoms is more preferable, and a cycloalkenyl having 5 to 6 carbon atoms is further preferable.
  • the carbon atom number of the substituent does not include the carbon atom number of the substituent.
  • examples of the cycloalkenyl having 3 to 12 carbon atoms include cyclopropenium, cyclobutenyl, cyclopentenyl, and cyclohexenyl.
  • cycloalkynyl having 3 to 12 carbon atoms is preferable, cycloalkynyl having 3 to 6 carbon atoms is more preferable, and cycloalkynyl having 5 to 6 carbon atoms is even more preferable.
  • the carbon atom number of the substituent does not include the carbon atom number of the substituent.
  • Examples of cycloalkynyl having 3 to 12 carbon atoms include cyclopropynyl, cyclobutynyl, cyclopentinyl, and cyclohexynyl.
  • cycloalkyl is preferable.
  • the monovalent aromatic hydrocarbon group means a hydrocarbon group containing an aromatic ring structure. However, it does not have to be composed of only an aromatic ring, and a chain structure or an alicyclic hydrocarbon may be contained in a part thereof, and the aromatic ring may be either a monocyclic ring or a polycyclic ring. good.
  • the monovalent aromatic hydrocarbon group an aryl having 6 to 12 carbon atoms is preferable, an aryl having 6 to 10 carbon atoms is more preferable, and an aryl having 6 carbon atoms is further preferable.
  • the carbon atom number of the substituent does not include the carbon atom number of the substituent. Examples of the aryl having 6 to 12 carbon atoms include phenyl and naphthyl.
  • Phenyl is preferable as the monovalent aromatic hydrocarbon group.
  • alkyl, cycloalkyl, and aryl are preferable as the monovalent hydrocarbon group.
  • a monovalent heterocyclic group is a group obtained by removing one hydrogen atom from the heterocycle of a heterocyclic compound.
  • the monovalent heterocyclic group is a monovalent aromatic heterocyclic group or a monovalent non-aromatic heterocyclic group.
  • the hetero atom constituting the heterocyclic group preferably contains at least one selected from the group consisting of an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom and a silicon atom, and preferably contains an oxygen atom, a sulfur atom and a nitrogen atom. It is more preferable to include one or more selected from the group consisting of atoms.
  • an aromatic heterocyclic group having 1 to 15 carbon atoms is preferable, an aromatic heterocyclic group having 1 to 9 carbon atoms is more preferable, and an aromatic heterocyclic group having 1 to 6 carbon atoms is more preferable.
  • Group heterocyclic groups are more preferred.
  • the carbon atom number of the substituent does not include the carbon atom number of the substituent.
  • Examples of the monovalent aromatic heterocyclic group include pyrrolyl, furanyl, thiophenyl, pyridinyl, pyrariainyl, pyrimidinyl, pyrazinyl, triazinyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, isooxazolyl, triazolyl, tetrazolyl, indolyl, prynyl and anthraquinolyl. , Carbazonyl, fluorenyl, quinolinyl, isoquinolinyl, quinazolinyl, and phthalazinyl.
  • a non-aromatic heterocyclic group having 2 to 15 carbon atoms is preferable, a non-aromatic heterocyclic group having 2 to 9 carbon atoms is more preferable, and a non-aromatic heterocyclic group having 2 to 9 carbon atoms is more preferable.
  • the non-aromatic heterocyclic group of 6 is more preferred.
  • the carbon atom number of the substituent does not include the carbon atom number of the substituent.
  • Examples of the monovalent non-aromatic heterocyclic group include oxylanyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, dihydrofuranyl, tetrahydrofuranyl, dioxolanyl, tetrahydrothiophenyl, pyrolinyl, imidazolidinyl, oxazolidinyl, piperidinyl and dihydropyranyl.
  • Tetrahydropyranyl Tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, piperazinyl, dihydrooxadinyl, tetrahydrooxadinyl, dihydropyrimidinyl, and tetrahydropyrimidinyl.
  • a 5-membered or 6-membered heterocyclic group is preferable.
  • halogen atoms, monovalent hydrocarbon groups, and monovalent heterocyclic groups in the substituents are the monovalent hydrocarbons described in R , RA , and RB, respectively. It is similar to that of a group and a monovalent heterocyclic group.
  • Arylkill means arylalkyl. Definitions, examples and preferred examples of aryl and alkyl in arylalkyl are as described above.
  • aralkyl aralkyl having 3 to 15 carbon atoms is preferable. Examples of such aralkyl include benzoyl, phenethyl, naphthylmethyl, and naphthylethyl.
  • the affinity peptide indicated by Y has a binding region in the CH2 domain in the immunoglobulin unit containing two heavy chains and two light chains.
  • any peptide having a binding region in the CH2 domain in the immunoglobulin unit can be used.
  • Amino acid residues eg, lysine residues, proline residues, etc.
  • are amino acid residues containing a side chain containing a moiety (eg, amino group, hydroxy group) capable of binding to a carbonyl group (C O) adjacent to Y.
  • affinity peptides include International Publication No. 2016/186206, International Publication No. 2018/199337, International Publication No. 2009240288, International Publication No. 2019/240287, and International Publication No. 2020/090979.
  • affinity peptides disclosed in the literature cited in these international publications can be used.
  • the affinity peptide may be: (A) Affinity peptide containing the amino acid sequence of CQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 1); Amino acid sequences containing variations of 1 to 5 selected amino acid residues (ie, 1, 2, 3, 4, or 5) (where the lysine residue at position 27, as well as the 1 and 1 and Affinity peptide containing (the two cysteine residues at position 30 are maintained). Since the affinity peptide containing the amino acid sequence of SEQ ID NO: 1 is a suitable peptide having a binding region in the CH2 domain in the immunoglobulin unit, it is preferable to use such an affinity peptide in the present invention.
  • the N-terminal and C-terminal amino acid residues of the affinity peptide may be protected. Further, the thiol groups of the side chains of the two cysteine residues (C) in the affinity peptide may be linked by a disulfide bond or via a linker.
  • the affinity peptide of (B) may be selected from the group consisting of: (A) Affinity peptide containing the amino acid sequence of FNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 2); (B) Affinity peptide containing the amino acid sequence of FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEDC (SEQ ID NO: 3); (C) Affinity peptide containing the amino acid sequence of FNMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC (SEQ ID NO: 4); (D) Affinity peptide containing the amino acid sequence of NMQCQRRFYEALHDPNLNEEQRNARIRSIKEEC (SEQ ID NO: 5); (E) Affinity peptide comprising the amino acid sequence of MQCQRRFYEALHDPNLNEEQRNARIRSIKEEC (SEQ ID NO: 6); and (f) Affinity peptide containing
  • the N-terminal and C-terminal amino acid residues of the affinity peptide may be protected. Further, the thiol groups of the side chains of the two cysteine residues (C) in the affinity peptide may be linked by a disulfide bond or via a linker.
  • At least two separated cysteine residues in each amino acid sequence of the affinity peptide can form a cyclic peptide by a disulfide bond.
  • the thiol groups in the two cysteine residues may be linked by a carbonyl group-containing linker represented by the following.
  • the broken line portion of the carbonyl group-containing linker represented above means the bonding portion with the thiol group.
  • the linker is more stable to reduction reactions and the like than ordinary disulfide bonds.
  • Such peptides can be prepared, for example, by the methods described in WO 2016/186206.
  • the amino acids constituting the affinity peptide may be either L-form or D-form, but L-form is preferable (in the examples, all the amino acid residues constituting the peptide are L-form).
  • the affinity peptide may be linked to the compound of formula (I) or a salt thereof by modifying a specific amino acid residue with a cross-linking agent. Examples of such a specific amino acid residue include a lysine residue, an aspartic acid residue, and a glutamate residue, but a lysine residue is preferable.
  • cross-linking agent for example, a cross-linking agent containing two or more succinimidyl groups such as DSG (disuccinidily glutarate, disuccinimidyl sulrate) and DSS (disuccinidily sublate), DMA (dimethylate).
  • DSG disuccinidily glutarate, disuccinimidyl sulrate
  • DSS disuccinidily sublate
  • DMA dimethylate
  • Imidic acid moieties such as 2HCl, dimethyl dihydrochloride adipimide), DMP (dimethyl pimelimidate 2 HCl, dimethyl dimethylidate pimellimidate), and DMS (dimethyl suberimide 2 HCl, dimethyl dihydrochloride suberimide) are preferred.
  • Cross-linking agent containing 2 or more and DTBP (dimethyl 3,3'-dithiobispropionimide ⁇ 2HCl, 3,3'-dithiobispropionimide acid dimethyl dihydrochloride) and DSP (dithiobis (succinimimidyl tropionate), dithiobiscus cinmidyl).
  • DTBP dimethyl 3,3'-dithiobispropionimide ⁇ 2HCl, 3,3'-dithiobispropionimide acid dimethyl dihydrochloride
  • DSP dithiobis (succinimimidyl tropionate), dithiobiscus cinmidyl).
  • cross-linking agents having SS bonds such as (acid) (eg, International Publication No. 2016/186206).
  • the affinity peptide may have an amino group and a carboxy group at the ends protected.
  • the protective group for the N-terminal amino group include an alkylcarbonyl group (acyl group) (eg, a butoxycarbonyl group such as an acetyl group, a propoxy group, a tert-butoxycarbonyl group) and an alkyloxycarbonyl group (eg, fluore).
  • Nylmethoxycarbonyl group aryloxycarbonyl group, arylalkyl (aralkyl) oxycarbonyl group (eg, benzyloxycarbonyl group).
  • an acetyl group is preferable.
  • the protected N-terminal glutamic acid may have a cyclic structure of pyroglutamic acid. Further, when the N-terminal amino acid is glutamine, the protected N-terminal glutamine may have a pyroglutamic acid type cyclic structure.
  • Protecting groups for the C-terminal carboxy group include, for example, groups capable of forming esters or amides.
  • Groups capable of forming esters or amides include, for example, alkyloxy groups (eg, methyloxy, ethyloxy, propyloxy, butyloxy, pentyloxy, hexyloxy), aryloxy groups (eg, phenyloxy, naphthyloxy), aralkyl. Examples include an oxy group (eg, benzyloxy) and an amino group. As the protecting group for the C-terminal carboxy group, an amino group is preferable.
  • the first and second linkers are divalent groups, as can be seen from the chemical structure of formula (I).
  • the total number of atoms constituting the main chain in the first linker and the number of atoms constituting the main chain in the second linker are 5 to 7.
  • the lysine residue at the 288/290 position of the heavy chain in the immunoglobulin unit can be position-selectively modified with an affinity peptide-containing group, and the antibody. It becomes easy to highly control the average ratio of the binding between the antibody and the affinity peptide-containing group to a desired range (1.5 to 2.5).
  • the average ratio of the binding between the antibody and a predetermined group eg, affinity peptide-containing group
  • the number of atoms constituting the main chain in the first linker is The number of atoms is 1 to 6, and the number of atoms constituting the main chain in the second linker is 1 to 6. More specifically, the relationship between the number of atoms constituting the main chain in the first linker and the second linker is as follows.
  • the main chains in the first linker and the second linker are composed of a chain structure, a cyclic structure, or a structure containing a combination thereof.
  • the number of atoms in the main chain can be determined by counting the number of atoms in the chain structure.
  • the main chain has a structure including a cyclic structure, it can be determined by counting a predetermined number of atoms constituting the cyclic structure as the number of atoms in the main chain.
  • the number of atoms in the main chain in the cyclic structure can be determined by counting the number of atoms in the shortest path connecting the two bonds in the cyclic structure (for example, (a) to (d) below). ) Bold route).
  • the main chain is a structure containing a combination of a chain structure and a cyclic structure
  • the number of atoms in the main chain communicates the number of atoms in the chain structure not including the cyclic structure with the two bonds in the cyclic structure. It can be determined by adding up with the number of atoms in the shortest path. ⁇ Is a bond.
  • the divalent linear hydrocarbon group is a linear alkylene, a linear alkenylene, or a linear alkynylene.
  • the linear alkylene is a linear alkylene having 1 to 6 carbon atoms, and a linear alkylene having 1 to 4 carbon atoms is preferable.
  • Examples of the linear alkylene include methylene, ethylene, n-propylene, n-butylene, n-pentylene and n-hexylene.
  • the linear alkenylene is a linear alkenylene having 2 to 6 carbon atoms, and a linear alkenylene having 2 to 4 carbon atoms is preferable.
  • linear alkenylene examples include ethyleneylene, n-propinylene, n-butenylene, n-pentenylene, and n-hexenylene.
  • the linear alkynylene is a linear alkynylene having 2 to 6 carbon atoms, and a linear alkynylene having 2 to 4 carbon atoms is preferable.
  • Examples of the linear alkynylene include ethynylene, n-propinylene, n-butynylene, n-pentinylene, and n-hexinylene.
  • divalent linear hydrocarbon group a linear alkylene is preferable.
  • the divalent cyclic hydrocarbon group is an arylene or a divalent non-aromatic cyclic hydrocarbon group.
  • a divalent cyclic hydrocarbon group By appropriately setting two bonds in such a divalent cyclic hydrocarbon group, it is possible to set the number of atoms constituting the main chain as described above to be 1 to 6 (hereinafter, described below). The same applies to groups having a cyclic structure).
  • the arylene an arylene having 6 to 14 carbon atoms is preferable, an arylene having 6 to 10 carbon atoms is more preferable, and an arylene having 6 carbon atoms is particularly preferable.
  • the arylene include phenylene, naphthylene, and anthraceneylene.
  • divalent non-aromatic cyclic hydrocarbon group a monocyclic or polycyclic divalent non-aromatic cyclic hydrocarbon group having 3 to 12 carbon atoms is preferable, and a simple monocyclic hydrocarbon group having 4 to 10 carbon atoms is preferable.
  • a divalent non-aromatic cyclic hydrocarbon group having a cyclic or polycyclic type is more preferable, and a divalent non-aromatic cyclic hydrocarbon group having a monocyclic number of 5 to 8 carbon atoms is particularly preferable.
  • divalent non-aromatic cyclic hydrocarbon group examples include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene and cyclooctylene.
  • divalent cyclic hydrocarbon group arylene is preferable.
  • the divalent heterocyclic group is a divalent aromatic heterocyclic group or a divalent non-aromatic heterocyclic group.
  • the hetero atom constituting the heterocycle preferably contains at least one selected from the group consisting of an oxygen atom, a sulfur atom, a nitrogen atom, a phosphorus atom, a boron atom and a silicon atom, and preferably contains an oxygen atom, a sulfur atom and a nitrogen atom. It is more preferable to include one or more selected from the group consisting of.
  • a divalent aromatic heterocyclic group having 3 to 15 carbon atoms is preferable, a divalent aromatic heterocyclic group having 3 to 9 carbon atoms is more preferable, and a carbon atom.
  • a divalent aromatic heterocyclic group having the number 3 to 6 is particularly preferable.
  • the divalent aromatic heterocyclic group include pyrrol diyl, frangyl, thiophene diyl, pyridine diyl, pyridazine diyl, pyrimidine diyl, pyrazine diyl, triazine diyl, pyrazole diyl, imidazole diyl, thiazole diyl, isothiazole diyl, and oxazole diyl.
  • Isoxazole diyl triazole diyl, tetrazole diyl, indole diyl, purine diyl, anthraquinone diyl, carbazole diyl, full orange yl, quinoline diyl, isoquinolin diyl, quinazoline diyl, and phthalazine diyl.
  • a non-aromatic heterocyclic group having 3 to 15 carbon atoms is preferable, a non-aromatic heterocyclic group having 3 to 9 carbon atoms is more preferable, and a non-aromatic heterocyclic group having 3 to 9 carbon atoms is more preferable.
  • the non-aromatic heterocyclic group of 6 is particularly preferred.
  • divalent non-aromatic heterocyclic group examples include pyrroldionediyl, pyrrolinedionediyl, oxylandiyl, aziridinediyl, azetidinediyl, oxetandiyl, thietandiyl, pyrrolidinediyl, dihydrofurandiyl, tetrahydrofurandiyl, dioxorandiyl and tetrahydrothiophene.
  • Diyl Pyrroline Diyl, Imidazolidine Diyl, Oxazolidine Diyl, Piperidine Diyl, Dihydropyran Diyl, Tetrahydropyran Diyl, Tetrahydropyran Diyl, Morpholine Diyl, Thiomorpholindiyl, Piperazine Diyl, Dihydrooxazine Diyl, Tetrahydropyran Diyl, Dihydropyrandiyl, And tetrahydropyrandiyl.
  • a divalent aromatic heterocyclic group is preferable.
  • W represents an oxygen atom or a sulfur atom, preferably an oxygen atom.
  • La and Lb indicate a first linker and a second linker, respectively.
  • the main chain in the first linker and the second linker may be composed of only carbon atoms, or may be composed of a combination of carbon atoms and heteroatoms (eg, oxygen atom, nitrogen atom, sulfur atom). However, from the viewpoint of easiness of organic synthesis and improvement of stability, it may be composed of only carbon atoms.
  • the main chain in the first linker represented by La is preferably set so that the number of atoms constituting the main chain as described above is 2 to 4.
  • the main chain in the second linker represented by Lb is preferably set so that the number of atoms constituting the main chain as described above is 1 to 5.
  • the main chain in the first linker represented by La is preferably set so that the number of atoms constituting the main chain as described above is two.
  • the main chain in the second linker represented by Lb is preferably set so that the number of atoms constituting the main chain as described above is 3 to 5.
  • La is a portion contained in the thiol group-introduced antibody derivative and has a low steric hindrance so as not to interfere with the reaction between the thiol group of the thiol group-introduced antibody derivative and the functional substance, and organic synthesis is carried out.
  • the main chain in the first linker represented by La is composed of a divalent linear hydrocarbon group having 2 to 4 carbon atoms.
  • the main chain in the first linker represented by La is even more preferably an ethylene group, a propylene group, or a butylene group, and particularly preferably an ethylene group.
  • this second linker can be decomposed in the production without any problem. That is, the main chain in the second linker is different from the main chain in the first linker, and its stability is less likely to be a problem.
  • the number of atoms constituting the main chain in the second linker is preferably 1 to 5, and more preferably 3 to 5.
  • the number of atoms constituting the main chain in the second linker is preferably 1 to 5, and more preferably 3 to 5.
  • the number of atoms constituting the main chain in the second linker is preferably 1 to 5, and more preferably 3 to 5.
  • the number of atoms constituting the main chain in the second linker is preferably 1 to 5, and more preferably 3 to 5.
  • the main chain in the second linker represented by Lb may be propylene or m-phenylene.
  • the groups constituting the main chain in the first linker and the second linker may have, for example, 1 to 5, preferably 1 to 3, and more preferably 1 or 2 substituents. Further, the Rd, which is one of the groups constituting the main chain in the first linker and the second linker, may indicate a substituent as described above.
  • halogen atoms examples, and preferred examples of halogen atoms, monovalent hydrocarbon groups, aralkyl, and monovalent heterocyclic groups in the substituents are R , RA , and RB, respectively, and (i) to (i) above. This is similar to that of the halogen atom, monovalent hydrocarbon group, aralkyl, and monovalent heterocyclic group described in (iv).
  • the compound represented by the formula (I) may be a compound represented by the following formula (I').
  • X, Y, O, S and W are the same as those in formula (I).
  • Lb represents a second linker having 3 to 5 atoms constituting the main chain.
  • R 1 , R 2 , R 3 and R 4 each independently represent a hydrogen atom or substituent.
  • examples and preferred examples of substituents represented by R1 , R2 , R3 , and R4 in formula (I') may be possessed by the groups that make up the backbone of the first linker. It is the same as that of the above-mentioned substituent.
  • the compound represented by the formula (I') may be a compound represented by the following formula (I ′′).
  • X, Y, O, S and W are the same as those in formula (I).
  • Lb is the same as that of the formula (I').
  • the compound of the present invention represented by the formulas (I) to (I ′′) or a salt thereof is, for example, the Y portion of the compound represented by the formulas (I) to (I ′′) or a salt thereof is eliminated.
  • Obtained by synthesizing a compound or a salt thereof substituted with a group (a leaving group having a higher leaving ability than X), and then reacting the synthesized compound or a salt thereof with an affinity peptide. can be done.
  • a reaction can be carried out in a suitable reaction system (eg, in an organic solvent or aqueous solution or a mixed solvent thereof) at a suitable temperature (eg, about 15 to 200 ° C.).
  • the reaction system may include a suitable catalyst.
  • the reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours.
  • the Y moiety is replaced with a leaving group (a leaving group having a higher ability to remove than X) or a compound thereof.
  • the salt may be a compound represented by the following formula (VI) or a salt thereof.
  • O indicates an oxygen atom
  • S represents a sulfur atom
  • W represents an oxygen atom or a sulfur atom
  • La indicates the first linker
  • Lb indicates a second linker
  • the total number of atoms constituting the main chain in the first linker and the number of atoms constituting the main chain in the second linker are 5 to 7.
  • the leaving group represented by X' which has a higher leaving ability than the leaving group X, is not particularly limited as long as it is a leaving group having a higher leaving ability than the leaving group X, for example, pentafluoro. Examples thereof include a phenyloxy group, a tetrafluorophenyloxy group, a paranitrophenyloxy group, and an N-succinimidyloxy group.
  • the compound represented by the formula (VI) or a salt thereof is useful as, for example, a synthetic intermediate for efficiently producing the compound represented by the formula (I) or a salt thereof.
  • the compound represented by the formula (VI) may be a compound represented by the following formula (VI').
  • X, X', O, S and W are the same as those of equation (VI) and Lb represents a second linker having 3 to 5 atoms constituting the main chain.
  • R 1 , R 2 , R 3 and R 4 each independently represent a hydrogen atom or substituent.
  • the compound represented by the formula (VI') may be a compound represented by the following formula (VI ′′).
  • X, X', O, S and W are the same as those of equation (VI) and Lb is the same as that of the formula (VI').
  • the compound represented by the formula (VI ′′) may be a compound represented by the following formula (VI ′′ -1) or (VI ′′ -2).
  • O, S and W are the same as those of equation (VI) and F is a fluorine atom.
  • the compound represented by the formula (VI) or a salt thereof can be produced from the compound represented by the following formula (V) or a salt thereof.
  • X indicates a leaving group
  • O indicates an oxygen atom
  • OH indicates a hydroxy group
  • S represents a sulfur atom
  • W represents an oxygen atom or a sulfur atom
  • La indicates the first linker
  • Lb indicates a second linker
  • the total number of atoms constituting the main chain in the first linker and the number of atoms constituting the main chain in the second linker are 5 to 7.
  • the compound represented by the formula (VI) or a salt thereof can be obtained by reacting the compound represented by the formula (V) or a salt thereof with a carboxyl group modifying reagent.
  • carboxyl group modifying reagent include a pentafluorophenylation reagent (eg, pentafluorophenyl trifluoroacetate), a tetrafluorophenylation reagent (eg, trifluoroacetate tetrafluorophenyl), and a paranitrophenylation reagent (eg, tri).
  • N-succinimidylating reagent eg, trifluoroacetate N-succinimidyl
  • a suitable temperature eg, about
  • a suitable organic solvent system eg, an organic solvent containing an alkyl halide (eg, methyl halide) such as CH 2 Cl 2 and an amine such as triethylamine.
  • the reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours.
  • the compound represented by the formula (V) or a salt thereof is useful as, for example, a synthetic intermediate for efficiently producing the compound represented by the formula (VI) or a salt thereof.
  • the compound represented by the formula (V) may be a compound represented by the following formula (V').
  • V' represents a second linker having 3 to 5 atoms constituting the main chain.
  • R 1 , R 2 , R 3 and R 4 each independently represent a hydrogen atom or substituent.
  • the compound represented by the formula (V') may be a compound represented by the following formula (V ′′).
  • V ′′ a compound represented by the following formula (V ′′).
  • the compound represented by the formula (V ′′) may be a compound represented by the following formula (V ′′ -1) or (V ′′ -2). [During the ceremony, O, OH, S and W are the same as those of the formula (V). ]
  • the present invention provides an antibody intermediate or a salt thereof containing a structural unit represented by the following formula (II).
  • Ig represents an immunoglobulin unit containing two heavy chains and two light chains, and is an amino in the side chain of a lysine residue present at position 288/290 in the two heavy chains according to Eunumbering. An amide bond is formed with the carbonyl group adjacent to Ig via the group.
  • Y represents an affinity peptide having a binding region to the CH2 domain in the immunoglobulin unit.
  • O indicates an oxygen atom
  • S represents a sulfur atom
  • W represents an oxygen atom or a sulfur atom
  • La indicates the first linker
  • Lb indicates a second linker
  • the total number of atoms constituting the main chain in the first linker and the number of atoms constituting the main chain in the second linker are 5 to 7.
  • the average ratio r of the amide bonds per two heavy chains is 1.5 to 2.5. ]
  • the immunoglobulin unit indicated by Ig is as described above. Definitions, examples, and preferred examples of symbols, terms and expressions such as Y (affinity peptide), W (oxygen atom or sulfur atom), La (first linker), Lb (second linker) in formula (II) , The same as that of the above formula.
  • the average ratio (r) of the amide bond per two heavy chains is the average ratio of the bonds between the immunoglobulin unit and the affinity peptide-containing group (affinity peptide-containing group number / immunoglobulin unit). Is shown. Such an average ratio is 1.5 to 2.5. Such an average ratio may be preferably 1.6 or more, more preferably 1.7 or more, even more preferably 1.8 or more, and particularly preferably 1.9 or more. Such an average ratio may also be preferably 2.4 or less, more preferably 2.3 or less, even more preferably 2.2 or less, and particularly preferably 2.1 or less. More specifically, such an average ratio is preferably 1.6 to 2.4, more preferably 1.7 to 2.3, even more preferably 1.8 to 2.2, and particularly preferably 1. It may be .9 to 2.1.
  • the structural unit represented by the formula (II) may be the structural unit represented by the following formula (II').
  • Ig, Y, O, S, W and r are the same as those of formula (II).
  • Lb represents a second linker having 3 to 5 atoms constituting the main chain.
  • R 1 , R 2 , R 3 and R 4 each independently represent a hydrogen atom or substituent.
  • It may be a structural unit represented by.
  • the structural unit represented by the formula (II') may be a structural unit represented by the following formula (II ′′).
  • Ig, Y, O, S, W and r are the same as those of formula (II).
  • Lb is the same as that of formula (II').
  • the antibody intermediate of the present invention or a salt thereof can be obtained by reacting the compound of the present invention or a salt thereof with an antibody containing the above-mentioned immunoglobulin unit.
  • the compound of the present invention or a salt thereof is mixed with the antibody. This allows the compounds of the invention or salts thereof to associate with the antibody via an affinity peptide that has an affinity for the antibody.
  • the amino group and the carbon atom of the carbonyl group are bonded, and the leaving group (X) is eliminated from the carbonyl group to obtain the antibody intermediate of the present invention or a salt thereof.
  • the molar ratio of the compound of the present invention or a salt thereof (the compound of the present invention or a salt thereof / antibody) to the antibody in the reaction varies depending on factors such as the compound of the present invention or a salt thereof and the type of antibody. Although not particularly limited, for example, it is 1 to 100, preferably 2 to 80, more preferably 4 to 60, even more preferably 5 to 50, and particularly preferably 6 to 30.
  • Such a reaction can be appropriately carried out under conditions (mild conditions) that cannot cause protein denaturation / decomposition (eg, cleavage of amide bond).
  • a reaction can be carried out at room temperature (eg, about 15-30 ° C.) in a suitable reaction system, eg buffer.
  • 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 catalyst.
  • the reaction time is, for example, 1 minute to 20 hours, preferably 10 minutes to 15 hours, more preferably 20 minutes to 10 hours, and even more preferably 30 minutes to 8 hours.
  • Confirmation of the formation of an antibody intermediate or a salt thereof depends on the specific raw material and the molecular weight of the product, but for example, electrophoresis, chromatography (eg, gel filtration chromatography, ion exchange chromatography, reverse phase). It can be done by column chromatography, HPLC), or mass spectrometry. Confirmation of regioselectivity can be performed, for example, by peptide mapping. Peptide mapping can be performed, for example, by protease treatment and mass spectrometry. As the protease, endoprotease is preferable.
  • affinity peptides introduced can be confirmed, for example, by electrophoresis, chromatography, or mass spectrometry, preferably mass spectrometry.
  • the antibody intermediate or a salt thereof can be appropriately purified by any method such as chromatography (eg, chromatography described above, and affinity chromatography).
  • a thiol group-introduced antibody derivative or a salt thereof which comprises a structural unit represented by the following formula (III).
  • Ig represents an immunoglobulin unit containing two heavy chains and two light chains, and is an amino in the side chain of a lysine residue present at position 288/290 in the two heavy chains according to Eunumbering.
  • An amide bond is formed with the carbonyl group adjacent to Ig via the group.
  • O indicates an oxygen atom
  • SH indicates a thiol group
  • La indicates the first linker
  • the number of atoms constituting the main chain in the first linker is 2 to 4, and the number of atoms is 2 to 4.
  • the average ratio r of the amide bonds per two heavy chains is 1.5 to 2.5.
  • the immunoglobulin unit indicated by Ig is as described above. Definitions, examples, and preferred examples of symbols, terms and expressions such as La (first linker) in formula (III) are the same as those in the above formula.
  • the average ratio (r) of the amide bond per two heavy chains indicates the average ratio of the bonds between the immunoglobulin unit and the thiol-containing group (thiol-containing group number / immunoglobulin unit).
  • Such an average ratio is 1.5 to 2.5.
  • Such an average ratio may be preferably 1.6 or more, more preferably 1.7 or more, even more preferably 1.8 or more, and particularly preferably 1.9 or more.
  • Such an average ratio may also be preferably 2.4 or less, more preferably 2.3 or less, even more preferably 2.2 or less, and particularly preferably 2.1 or less. More specifically, such an average ratio is preferably 1.6 to 2.4, more preferably 1.7 to 2.3, even more preferably 1.8 to 2.2, and particularly preferably 1. It may be .9 to 2.1.
  • the structural unit represented by the formula (III) may be the structural unit represented by the following formula (III').
  • Ig, O, SH and r are the same as those of formula (III).
  • R 1 , R 2 , R 3 and R 4 each independently represent a hydrogen atom or substituent.
  • examples and preferred examples of substituents represented by R1 , R2 , R3, and R4 in formula ( III ') may be possessed by the groups that make up the backbone of the first linker. It is the same as that of the above-mentioned substituent.
  • the structural unit represented by the formula (III') may be the structural unit represented by the following formula (III ′′). [During the ceremony, Ig, O, SH and r are the same as those in formula (III). ]
  • the thiol group-introduced antibody derivative of the present invention or a salt thereof can be obtained by subjecting the antibody intermediate of the present invention or a salt thereof to a thioester cleavage reaction.
  • the thioester cleavage reaction can be carried out under conditions that cannot cause denaturation / degradation of proteins (immunoglobulin / antibody) (eg, cleavage of amide bonds) (mild conditions as described above). More specifically, it can be cleaved by stirring for 1 hour in a hydroxylamine hydrochloride solution in the range of pH 4.0 to pH 8.0, 10 mM to 10 M (eg, Vance, Net al., Bioconjugate Chem. 2019, 30, 148-160.).
  • Confirmation of the formation of a thiol group-introduced antibody derivative or a salt thereof depends on the specific raw material and the molecular weight of the product, and is, for example, electrophoresis, chromatography (eg, gel filtration chromatography, ion exchange chromatography, etc.). It can be done by reverse phase column chromatography, HPLC), or mass spectrometry, preferably by mass spectrometry.
  • Confirmation of regioselectivity 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, endoprotease is preferable.
  • Examples of such an endoprotease include trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, and Asp-N.
  • the number of thiol groups introduced can be confirmed, for example, by electrophoresis, chromatography, or mass spectrometry, preferably by mass spectrometry.
  • the thiol group-introduced antibody derivative or a salt thereof can be appropriately purified by any method such as chromatography (eg, chromatography described above, and affinity chromatography).
  • conjugates of antibodies and functional substances or salts thereof which contain structural units represented by the following formula (IV).
  • Ig represents an immunoglobulin unit containing two heavy chains and two light chains, and is an amino in the side chain of a lysine residue present at position 288/290 in the two heavy chains according to Eunumbering.
  • An amide bond is formed with the carbonyl group adjacent to Ig via the group.
  • O indicates an oxygen atom
  • S represents a sulfur atom
  • Z indicates a functional substance
  • La indicates the first linker
  • the number of atoms constituting the main chain in the first linker is 2 to 4, and the number of atoms is 2 to 4.
  • the average ratio r of the amide bonds per two heavy chains is 1.5 to 2.5.
  • the immunoglobulin unit indicated by Ig is as described above. Definitions, examples, and preferred examples of symbols, terms and expressions such as La (first linker) in formula (IV) are similar to those in formula (IV) above.
  • the functional substance is not particularly limited as long as it is a substance that imparts an arbitrary function to the antibody, and examples thereof include a drug, a labeling substance, and a stabilizer, but a drug or a labeling substance is preferable.
  • 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, cancers (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, etc.
  • Brain tumor, melanoma autoimmune and inflammatory diseases (eg, allergic disease, rheumatoid arthritis, systemic erythematosus), neurological diseases (eg, cerebral infarction, Alzheimer's disease, Parkinson's disease, muscular atrophic lateral sclerosis), Infectious diseases (eg, bacterial infections, viral infections), hereditary and rare diseases (eg, hereditary globular erythropathy, non-dystrophic myotension), eye diseases (eg, age-related luteal degeneration, diabetic retinopathy, etc.) Retinal pigment degeneration), diseases in the field of bone and orthopedics (eg, osteoarthritis), blood diseases (eg, leukemia, purpura), and other diseases (eg, diabetes, hyperlipidemia, etc.) , Liver disease, kidney disease, lung disease, cardiovascular disease, digestive system disease).
  • the drug may be a prophylactic or therapeutic drug for a disease or a palliative drug for side effects.
  • the drug is an anti-cancer agent.
  • Anti-cancer agents include, for example, chemotherapeutic agents, toxins, radioisotopes or substances containing them.
  • Chemotherapeutic agents include, for example, DNA damaging agents, metabolic antagonists, enzyme inhibitors, DNA intercalating agents, DNA cleavage agents, topoisomerase inhibitors, DNA binding inhibitors, tubulin binding inhibitors, cytotoxic nucleosides, etc. Platinum compounds can be mentioned.
  • Examples of toxins include bacterial toxins (eg, diphtheria toxins) and plant toxins (eg, ricin).
  • Radioisotopes include, for example, a radioisotope of a hydrogen atom (eg, 3H ), a radioisotope of a carbon atom (eg, 14C ), a radioisotope of a phosphorus atom (eg, 32P ), and a sulfur atom.
  • Radioisotopes eg, 35 S
  • yttrium radioisotopes eg 90 Y
  • technetium radioisotopes eg 99 MTc
  • indium radioisotopes eg 111 In
  • iodine atom radioactivity Isotopes eg 123 I, 125 I, 129 I, 131 I
  • samarium radioisotopes eg 153 Sm
  • renium radioisotopes eg 186 Re
  • asstatin radioisotopes eg 156 Re
  • 211 At a radioisotope of bismuth
  • a radioisotope of bismuth eg, 212 Bi
  • auristatin MMAE, MMAF
  • maitansine DM1, DM4
  • PBD pyrrolobenzodiazepine
  • IGN camptothecin analog
  • calicheamicin duocalmycin
  • eribulin anthracycline
  • dmDNA31 tubricin.
  • the labeling substance is a substance that enables detection of a target (eg, tissue, cell, substance).
  • Labeling substances include, for example, enzymes (eg, peroxidase, alkaline phosphatase, luciferase, ⁇ -galactosidase), affinity substances (eg, streptavidin, biotin, digoxygenin, aptamer), fluorescent substances (eg, fluorescein, fluorescein isothiocyanate, rhodamine).
  • Green fluorescent protein Green fluorescent protein, red fluorescent protein
  • luminescent substances eg, luciferin, equolin, acridinium ester, tris (2,2'-bipyridyl) ruthenium, luminol
  • radioactive isotopes eg, those mentioned above
  • examples include substances containing it.
  • Stabilizer is a substance that enables the stabilization of antibodies.
  • Stabilizers include, for example, diols, glycerin, nonionic surfactants, anionic surfactants, natural surfactants, saccharides, and polyols.
  • Functional substances may also be peptides, proteins, nucleic acids, small molecule organic compounds, sugar chains, lipids, high molecular polymers, metals (eg gold), killer.
  • the peptide include a cell membrane penetrating peptide, a blood-brain barrier penetrating peptide, and a peptide drug.
  • proteins include enzymes, cytokines, fragment antibodies, lectins, interferons, serum albumin, and antibodies.
  • nucleic acids include DNA, RNA, and artificial nucleic acids. Nucleic acids also include, for example, RNA interference-inducing nucleic acids (eg, siRNA), aptamers, antisense.
  • small molecule organic compounds include proteolysis-inducing chimeric molecules, dyes, and photodegradable compounds.
  • the functional substance may be derivatized so as to have such a functional group.
  • Derivatization is common technical knowledge in the art (eg, International Publication No. 2004/010957, US Patent Application Publication No. 2006/0074008, US Patent Application Publication No. 2005/02386649).
  • derivatization may be carried out using any cross-linking agent.
  • the derivatization may be carried out using a specific linker having the desired functional group.
  • such a linker may be one in which the functional substance and the antibody can be separated by cleavage of the linker in an appropriate environment (eg, intracellular or extracellular).
  • linkers include, for example, peptidyl linkers that are degraded by specific proteases [eg, intracellular proteases (eg, proteases present in lysosomes, or endosomes), extracellular proteases (eg, secretory proteases)].
  • proteases eg, intracellular proteases (eg, proteases present in lysosomes, or endosomes), extracellular proteases (eg, secretory proteases)
  • a linker that can be cleaved at a locally acidic site present in the living body eg, US Pat. No. 5, US Pat. No. 5).
  • the linker may be self-destructive (eg, WO 02/083180, WO 04/043493, WO 05/1192919).
  • the derivatized functional substance is also simply referred to as "functional substance”.
  • the average ratio (r) of the amide bond per two heavy chains is the average ratio of the bonds between the immunoglobulin unit and the functional substance-containing group (functional substance-containing group number / immunoglobulin unit). Is shown. Such an average ratio is 1.5 to 2.5. Such an average ratio may be preferably 1.6 or more, more preferably 1.7 or more, even more preferably 1.8 or more, and particularly preferably 1.9 or more. Such an average ratio may also be preferably 2.4 or less, more preferably 2.3 or less, even more preferably 2.2 or less, and particularly preferably 2.1 or less. More specifically, such an average ratio is preferably 1.6 to 2.4, more preferably 1.7 to 2.3, even more preferably 1.8 to 2.2, and particularly preferably 1. It may be .9 to 2.1.
  • the structural unit represented by the formula (IV) may be the structural unit represented by the following formula (IV').
  • formula (IV') [During the ceremony, Ig, O, S, Z and r are the same as those of formula (IV).
  • R 1 , R 2 , R 3 and R 4 each independently represent a hydrogen atom or substituent. ]
  • the structural unit represented by the formula (IV') may be the structural unit represented by the following formula (IV ′′). [During the ceremony, Ig, O, S, Z and r are the same as those of formula (IV). ]
  • the conjugate of the present invention or a salt thereof can be obtained by reacting the thiol group-introduced antibody derivative of the present invention or a salt thereof with a functional substance.
  • a reaction can be carried out under conditions that cannot cause denaturation / degradation of a protein (immunoglobulin / antibody) (eg, cleavage of an amide bond) (mild conditions as described above).
  • a functional substance a substance having an arbitrary functional group capable of reacting with a thiol group under mild conditions can be used, but it is preferable to use a functional group that easily reacts with the thiol group.
  • a functional group examples include a maleimide group, a disulfide group, an ⁇ -haloketone, an ⁇ -haloamide, a benzyl bromide, and an iodoalkyl group.
  • the functional substance derivatized as described above can be used.
  • the molar ratio of the functional substance to the thiol group-introduced antibody derivative or its salt is determined by the type of the thiol group-introduced antibody derivative or its salt and the functional substance.
  • reaction time since it varies depending on factors such as reaction time, it is not particularly limited, but is, for example, 2 or more, preferably 3 or more, and more preferably 5 or more.
  • a sufficient amount (eg, excess amount) of the functional substance with respect to the thiol group-introduced antibody derivative or a salt thereof is used. be able to.
  • Confirmation of the formation of a conjugate or salt thereof depends on the specific raw material and the molecular weight of the product, but for example, electrophoresis, chromatography (eg, gel filtration chromatography, ion exchange chromatography, reverse phase column). It can be done by chromatography, HPLC), or mass spectrometry, preferably by mass spectrometry.
  • Confirmation of regioselectivity 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, endoprotease is preferable.
  • Examples of such an endoprotease include trypsin, chymotrypsin, Glu-C, Lys-N, Lys-C, and Asp-N.
  • the number of functional substances introduced can be confirmed by, for example, electrophoresis, chromatography, or mass spectrometry, preferably mass spectrometry.
  • the conjugate or salt thereof can be appropriately purified by any method such as chromatography (eg, chromatography described above, and affinity chromatography).
  • the compounds of the present invention or salts thereof can, for example, regioselectively modify the lysine residue at position 288/29 of an antibody. Therefore, the present invention provides a reagent for antibody derivatization, which comprises the compound of the present invention or a salt thereof.
  • the reagent of the present invention may be provided in the form of a composition further containing other components.
  • other components include solutions, stabilizers (eg, antioxidants, preservatives).
  • an aqueous solution is preferable.
  • the aqueous solution include water (eg, distilled water, sterile distilled water, purified water, physiological saline), buffer solution (eg, phosphoric acid aqueous solution, Tris-hydrochloride buffer solution, carbonic acid-bicarbonate buffer solution, boric acid aqueous solution). , Glycine-sodium hydroxide buffer, citrate buffer), but a buffer is preferred.
  • the pH of the solution is, for example, 5.0 to 9.0, preferably 5.5 to 8.5.
  • the reagents of the present invention can be provided in liquid or powder form (eg, lyophilized powder).
  • the antibody intermediate of the present invention or a salt thereof, and the thiol group-introduced antibody derivative of the present invention or a salt thereof are useful as, for example, a conjugate of an antibody and a functional substance or an intermediate for preparing a salt thereof.
  • the conjugate of the present invention or a salt thereof is useful as, for example, a drug or a reagent (eg, a diagnostic agent, a reagent for research).
  • the present invention is regioselectively modified with a functional substance, and the average ratio of binding between the antibody and the functional substance is highly controlled within a desired range (1.5 to 2.5).
  • the conjugate or salt thereof is useful as a medicine. It has been reported that changing the number and position of binding of a drug in an antibody-drug conjugate (ADC) changes the pharmacokinetics, the release rate of the drug, and the effect. For these reasons, next-generation ADCs are required to control the number and position of drugs to be conjugated. It is thought that if the number and position are constant, the problems of efficacy, variation of conjugation drug, and lot difference, so-called regulation, as expected will be solved. Therefore, the conjugates of the present invention or salts thereof can solve such regulation problems.
  • ADC antibody-drug conjugate
  • the conjugate of the present invention or a salt thereof may be provided in the form of a pharmaceutical composition.
  • a pharmaceutical composition may contain a pharmaceutically acceptable carrier in addition to the conjugate of the present invention or a salt thereof.
  • Pharmaceutically acceptable carriers include, for example, excipients such as sucrose, starch, mannit, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, cellulose, methyl cellulose, hydroxypropyl cellulose, polypropylpyrrolidone.
  • Gelatin gum arabic, polyethylene glycol, sucrose, starch and other binders, starch, carboxymethyl cellulose, hydroxypropyl starch, sodium hydrogen carbonate, calcium phosphate, calcium citrate and other disintegrants, magnesium stearate, aerodyl, talc, lauryl Lubricants such as sodium sulfate, citric acid, menthol, glycyrrhizin / ammonium salt, glycine, fragrances such as orange powder, preservatives such as sodium benzoate, sodium hydrogen sulfite, methylparaben, propylparaben, citric acid, sodium citrate, acetic acid Stabilizers such as, methyl cellulose, polyvinylpyrrolidone, suspending agents such as aluminum stearate, dispersants such as surfactants, diluents such as water, physiological saline, orange juice, cocoa butter, polyethylene glycol, white kerosene, etc.
  • examples include,
  • Suitable formulations for oral administration are solutions in which an effective amount of ligand is dissolved in a diluted solution such as water, physiological saline, or orange juice, capsules containing an effective amount of ligand as solids or granules, sachets, or sachets.
  • a diluted solution such as water, physiological saline, or orange juice
  • capsules containing an effective amount of ligand as solids or granules, sachets, or sachets include tablets, suspensions in which an effective amount of the active ingredient is suspended in an appropriate dispersion medium, and emulsions in which a solution in which an effective amount of the active ingredient is dissolved is dispersed in an appropriate dispersion medium and emulsified.
  • the pharmaceutical composition is suitable for parenteral administration (eg, intravenous injection, subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration).
  • Pharmaceutical compositions suitable for such parenteral administration include aqueous and non-aqueous isotonic sterile injectable solutions, which include antioxidants, buffers, antibacterial agents, isotonic agents. Etc. may be included. Examples thereof include aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like.
  • the dose of the pharmaceutical composition varies depending on the type / activity of the active ingredient, the severity of the disease, the animal species to be administered, the drug acceptability of the administration target, the body weight, the age, etc., but can be appropriately set.
  • Example 1 Synthesis of a compound having an affinity substance for a soluble protein, a cleaving moiety and a reactive group (peptide thioester linker conjugate-thiophenol activator), and modification of the anti-HER2 antibody trastuzumab using the compound. And its analysis]
  • (1-1) Synthesis of IgG1 Fc-binding peptide
  • As the peptide synthesizer Liberty Blue manufactured by CEM was used.
  • a 2M aqueous trifluoroacetic acid solution was added to the reaction solution to stop the reaction, this was dissolved in a 0.05% aqueous trifluoroacetic acid solution, and subjected to reverse phase high-speed liquid chromatography using octadodecyl group chemically bound silica gel as a filler. , Elution with a mixed solution of water containing 0.05% trifluoroacetic acid and acetonitrile, and each fraction was confirmed by LC-MS. The fraction containing the product was collected and concentrated under reduced pressure to remove acetonitrile, and then freeze-dried to obtain the desired product (20.0 mg, 4.70 ⁇ mol).
  • Ig represents an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and the lysine residue present at position 288/290 in the two heavy chains according to Eunumbering.
  • An amide bond is formed with the carbonyl group adjacent to Ig via the amino group in the side chain of the group.
  • Y represents the affinity peptide represented by the amino acid sequence of SEQ ID NO: 2. The average ratio r of the amide bonds per two heavy chains is 2.0.
  • Ig represents an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and the lysine residue present at position 288/290 in the two heavy chains according to Eunumbering.
  • An amide bond is formed with the carbonyl group adjacent to Ig via the amino group in the side chain of the group.
  • the average ratio r of the amide bonds per two heavy chains is 2.0.
  • the heavy chain VH domain and the number on the light chain are the numbers in the sequence (that is, the N-terminal amino acid is the first; the same applies hereinafter), and the heavy chains CH1, CH2, and CH3 are used. On the domain, it is described using EU numbering.
  • (1) and (2) shown in FIG. 9 were used as the data of the amino acid sequence to be searched for the modification site.
  • Example 2 Synthesis of a compound having an affinity substance for a soluble protein, a cleaving moiety and a reactive group (peptide thioester linker conjugate-thiophenol activator), and modification of the anti-HER2 antibody trastuzumab using the compound. And its analysis]
  • (2-1) Synthesis of thioester linker (2-1-1)
  • Ig represents an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and the lysine residue present at position 288/290 in the two heavy chains according to Eunumbering.
  • An amide bond is formed with the carbonyl group adjacent to Ig via the amino group in the side chain of the group.
  • Y represents the affinity peptide represented by the amino acid sequence of SEQ ID NO: 2. The average ratio r of the amide bonds per two heavy chains is 2.0.
  • Example 3 Synthesis of a compound having an affinity substance for a soluble protein, a cleaving moiety and a reactive group (peptide thioester linker conjugate-thiophenol activator), and modification of the anti-HER2 antibody trastuzumab using the compound. And its analysis] (3-1) Synthesis of thioester linker (3-1-1)
  • Ig represents an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and the lysine residue present at position 288/290 in the two heavy chains according to Eunumbering.
  • An amide bond is formed with the carbonyl group adjacent to Ig via the amino group in the side chain of the group.
  • Y represents the affinity peptide represented by the amino acid sequence of SEQ ID NO: 2. The average ratio r of the amide bonds per two heavy chains is 1.9.
  • Example 4 Synthesis of a compound having an affinity substance for a soluble protein, a cleaving moiety and a reactive group (peptide thioester linker conjugate-thiophenol activator), and modification of the anti-HER2 antibody trastuzumab using the compound. And its analysis] (4-1) Synthesis of thioester linker (4-1-1)
  • Ig represents an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and the lysine residue present at position 288/290 in the two heavy chains according to Eunumbering.
  • An amide bond is formed with the carbonyl group adjacent to Ig via the amino group in the side chain of the group.
  • Y represents the affinity peptide represented by the amino acid sequence of SEQ ID NO: 2. The average ratio r of the amide bonds per two heavy chains is 1.8.
  • Example 5 Synthesis of a compound having an affinity substance for a soluble protein, a cleaving moiety and a reactive group (peptide thioester linker conjugate-thiophenol activator), and modification of the anti-HER2 antibody trastuzumab using the compound. And its analysis]
  • (5-1) Binding of peptide and linker
  • Both of the above two amino acid sequences are the amino acid sequences of SEQ ID NO: 3.
  • Ig represents an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and the lysine residue present at position 288/290 in the two heavy chains according to Eunumbering.
  • An amide bond is formed with the carbonyl group adjacent to Ig via the amino group in the side chain of the group.
  • Y represents the affinity peptide represented by the amino acid sequence of SEQ ID NO: 3. The average ratio r of the amide bonds per two heavy chains is 2.0.
  • Example 6 Synthesis of a compound having an affinity substance for a soluble protein, a cleaving moiety and a reactive group (peptide thioester linker conjugate-thiophenol activator), and modification of the anti-HER2 antibody trastuzumab using the compound. And its analysis] (6-1) Binding of peptide and linker Both of the above two amino acid sequences are the amino acid sequences of SEQ ID NO: 4.
  • Ig represents an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and the lysine residue present at position 288/290 in the two heavy chains according to Eunumbering.
  • An amide bond is formed with the carbonyl group adjacent to Ig via the amino group in the side chain of the group.
  • Y represents the affinity peptide represented by the amino acid sequence of SEQ ID NO: 4. The average ratio r of the amide bonds per two heavy chains is 2.0.
  • Example 7 Synthesis of a compound having an affinity substance for a soluble protein, a cleaving moiety and a reactive group (peptide thioester linker conjugate-thiophenol activator), and modification of the anti-HER2 antibody trastuzumab using the compound. And its analysis] (7-1) Binding of peptide and linker Both of the above two amino acid sequences are the amino acid sequences of SEQ ID NO: 5.
  • Ig represents an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and the lysine residue present at position 288/290 in the two heavy chains according to Eunumbering.
  • An amide bond is formed with the carbonyl group adjacent to Ig via the amino group in the side chain of the group.
  • Y represents the affinity peptide represented by the amino acid sequence of SEQ ID NO: 5. The average ratio r of the amide bonds per two heavy chains is 2.0.
  • Example 8 Synthesis of a compound having an affinity substance for a soluble protein, a cleaving moiety and a reactive group (peptide thioester linker conjugate-thiophenol activator), and modification of the anti-HER2 antibody trastuzumab using the compound. And its analysis] (8-1) Binding of peptide and linker Both of the above two amino acid sequences are the amino acid sequences of SEQ ID NO: 6.
  • Ig represents an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and the lysine residue present at position 288/290 in the two heavy chains according to Eunumbering.
  • An amide bond is formed with the carbonyl group adjacent to Ig via the amino group in the side chain of the group.
  • Y represents the affinity peptide represented by the amino acid sequence of SEQ ID NO: 6. The average ratio r of the amide bonds per two heavy chains is 2.0.
  • Example 9 Synthesis of a compound having an affinity substance for a soluble protein, a cleaving moiety and a reactive group (peptide thioester linker conjugate-thiophenol activator), and modification of the anti-HER2 antibody trastuzumab using the compound. And its analysis] (9-1) Binding of peptide and linker Both of the above two amino acid sequences are the amino acid sequences of SEQ ID NO: 7.
  • Ig represents an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and the lysine residue present at position 288/290 in the two heavy chains according to Eunumbering.
  • An amide bond is formed with the carbonyl group adjacent to Ig via the amino group in the side chain of the group.
  • Y represents the affinity peptide represented by the amino acid sequence of SEQ ID NO: 7. The average ratio r of the amide bonds per two heavy chains is 2.0.
  • the total number of atoms constituting the main chain in the first linker and the number of atoms constituting the main chain in the second linker correspond to less than 4 or 9 or more atoms. Showed an average PAR of 0.5 or less.
  • Example 10 Regioselective modification of different target regions of IgG1 Fc with IgG1 Fc-affinity peptide reagent, and synthesis of antibody-drug conjugate] (10-1) Production of thiol group-introduced antibody derivative by cleavage of thioester group following position-selective modification of anti-HER2 antibody trastuzumab The following peptide reagent described in the previous report (WO2019 / 240287A1) was dissolved in dimethylformamide to obtain 10 mM.
  • Anti-HER2 antibody trastuzumab (Chugai Pharmaceutical) 500 ⁇ g is dissolved in 20 mM sodium acetate buffer (pH 5.5) 200 ⁇ L (20 ⁇ M), and 10 mM of the above peptide reagent is added 3.38 ⁇ L (10 equivalents to the antibody) at room temperature. The mixture was stirred for 1 hour. Subsequently, a hydroxylamine solution was added according to the previous report (WO2019 / 240287A1), and the mixture was allowed to stand at room temperature for 1 hour. When the mass of the obtained thiol-introduced antibody was measured by ESI-TOFMS, a peak was confirmed at 148760 where the cleavage reaction by hydroxylamine proceeded.
  • the following peptide reagent is a reagent that enables modification of the lysine residue at position 246/248 of the IgG heavy chain
  • the lysine residue at position 246/248 of the IgG heavy chain is modified with the following peptide reagent. It is thought that it was.
  • the amino acid sequence is the amino acid sequence of SEQ ID NO: 11.
  • Ig represents an immunoglobulin unit (IgG) containing two heavy chains and two light chains, and the lysine residue present at position 288/290 in the two heavy chains according to Eunumbering.
  • An amide bond is formed with the carbonyl group adjacent to Ig via the amino group in the side chain of the group.
  • an amide bond is formed with the carbonyl group adjacent to Ig via the amino group in the side chain of the lysine residue present at the 246/248 position in the two heavy chains according to Eunumbering.
  • the average ratios r (288/290 positions) and n (246/248 positions) of the amide bonds per two heavy chains were 1.9 and 2.0, respectively, as a result of evaluation.

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