Title of the invention: A substance having an affinity for an antibody, and a compound having a bioorthogonal functional group or a salt thereof.
Technical field
[0001]
　The present invention relates to an affinity substance for an antibody, a compound having a bioorthogonal functional group, or a salt thereof.
Background technology
[0002]
　In recent years, research and development of antibody-drug conjugates (ADCs) have been actively carried out. As the name implies, ADC is a drug in which a drug (eg, an anticancer drug) is conjugated to an antibody, and has direct cell-killing activity against cancer cells and the like. A typical ADC is T-DM1 (trade name: Kadcyla (registered trademark)) (Non-Patent Documents 1 to 3).
[0003]
　The non-uniformity of ADCs such as T-DM1 has been a problem from the beginning of development. That is, since a small molecule drug is randomly reacted with Lys residues of about 70 to 80 in the antibody, the drug antibody ratio (Drug / Antibody Ratio: DAR) and the conjugation position are not constant. It is known that usually, in such a random conjugation method, the DA is in the range of 0 to 8, and a plurality of drugs having different numbers of drug bindings are produced. In recent years, it has been reported that the pharmacokinetics, drug release rate, and effect change when the number and position of binding of a drug in ADC are changed. For these reasons, next-generation ADCs are required to control the number and position of conjugating drugs. It is considered that if the number and position are constant, the problems of efficacy, variation of conjugation drug, and lot difference, so-called regulation, as expected can be solved (Non-Patent Document 4).
[0004]
　Regioselective modifications of antibodies have been studied all over the world, most of which are genetically engineered or enzymatically modified. Regarding the genetic engineering modification method, although the regioselectivity and the number selectivity can be controlled, problems such as a decrease in the expression efficiency of the antibody itself (a decrease in the total yield when preparing the ADC) have been pointed out. In addition, it takes a long time to construct an antibody expression system (Non-Patent Documents 5 to 7).
[0005]
　In addition, a method of chemically modifying a protein in a congested environment such as inside a cell using a small molecule probe has been reported in recent years. This method is used for receptor identification in imaging and repositioning of small molecule drugs. Further, in the field of chemical biology, an organic chemical protein modification method using a synthetic small molecule probe has attracted attention (Non-Patent Documents 8 to 11).
[0006]
　Recently, the CCAP (Chemical Conjugation by Affinity Peptide) method has been developed. This method is carried out by reacting a peptide reagent in which an NHS activating ester and a drug are linked to an affinity peptide with an antibody (that is, a method for producing an ADC via a linker containing a peptide moiety). We have succeeded in position-selective modification of the antibody. This method was the first in the world to succeed in regioselectively modifying the antibody Fc region with a drug by a chemically synthesized method, and the results were also good in practice [reaction time 30 minutes, yield 70%]. (In the case of DAR 1), regioselectivity is 100%] has been confirmed. It has been demonstrated that the DA can be controlled by 2 by adding about 5 equivalents of the peptide reagent, and it is epoch-making in that the modification position can also be controlled (Patent Document 1).
Prior art literature
Patent documents
[0007]
Patent Document 1: International Publication No. 2016/186206
Non-patent literature
[0008]
Non-Patent Document 1: Reichert JM et al. , Nat Biotechnol 2005; 23: 1073-8
Non-Patent Document 2: Kubota T et al. , Cancer Science 2009; 100: 1566-72
Non-Patent Document 3: Wu AM et al. , Nat Biotechnol 2005; 23: 1137-46
Non-Patent Document 4: Juntura JR et al. , Nat Biotechnol 2008; 26: 925-32
Non-Patent Document 5: Shen BQ et al. , Nat Biotechnol 2012; 30: 184-9
Non-Patent Document 6: Hofer T et al. , Biochemistry 2009; 48: 12047-57
Non-Patent Document 7: Liu W et al. , Nat Methods 2007; 4: 239-44
Non-Patent Document 8: S.A. T. Laughlin et al. , Science 2008; 320,664
Non-Patent Document 9: A. E. Spaces et al. , ChemBioChem 2004; 5,41
Non-Patent Document 10: Y. et al . Takaoka et al. , Angew. Chem. Int. Ed. 2013; 52,4088
Non-Patent Document 11: S.A. Fujishima et al. , J. Am. Chem. Soc, 2012; 134: 3961-64
Outline of the invention
Problems to be solved by the invention
[0009]
　An object of the present invention is to develop a technique that enables modification of an antibody, particularly, regioselective modification of an antibody.
Means to solve problems
[0010]
　As a result of diligent studies, the present inventors have conducted a structural unit called A-LE (where A is an affinity substance for an antibody and L is a divalent group containing a predetermined leaving group. , E is a divalent group containing (i) an electrophilic group linked to the leaving group and (ii) capable of reacting with a nucleophilic group in the antibody). Alternatively, they have found that the salt is useful for position-specific modification of the antibody. For example, a predetermined compound having an affinity substance for an antibody and a bioorthogonal functional group represented by the formula (I) has been found to be useful for position-specific modification of an antibody (eg, FIG. 1. Various examples). In addition, it has been found that a compound having an affinity substance for an antibody and a functional substance or a salt thereof, which is represented by the formula (IV), is useful for position-specific modification of the antibody (eg, Examples). 13, 14). The present inventors further prepare an antibody (antibody drug conjugate (ADC)) having a functional substance (eg, drug) regioselectively without containing a peptide moiety as a linker by using such a compound. I found out what I could do. Avoidance of the use of linkers containing peptide moieties that have potential immunogenicity and are susceptible to hydrolysis in the blood is desirable in clinical applications of ADCs. That is, according to the method developed by the present inventors, the antibody Fc region can be regioselectively modified with a drug by a chemically synthesized method and without using a linker containing a peptide moiety.
[0011]
　That is, the present invention is as follows.
[1] The following formula (I):
A-L-EB (I)
[In the formula,
　A is an affinity substance for an antibody,
　L is a divalent group containing a elimination group, and
　E. Is a divalent group containing (i) an electrophilic group linked to the desorbing group and (ii) capable of reacting with a nucleophilic group in the antibody, and
　B is bioorthogonal. It is a functional group, and the
　desorbing group has the ability to be cleaved from E and desorbed by the reaction between the nucleophilic group and the electrophilic group. ], A compound having an affinity for an antibody and a bioorthogonal functional group, or a salt thereof.
[2] The compound of [1] or a salt thereof, wherein the affinity substance is a peptide.
[3] The compound of [2] or a salt thereof, wherein the peptide is a peptide having a binding ability to a constant region of a monoclonal antibody.
[4] The compound of [2] or [3] or a salt thereof, wherein the peptide is a peptide having a binding ability to the Fc region of a monoclonal antibody.
[5] The compound of [4] or a salt thereof, wherein the peptide is a peptide having an ability to bind to the Fc region of IgG.
[6] The compound of any of [2] to [5] or a salt thereof, wherein the peptide has 10 to 40 amino acid residues.
[7] The peptide is
(A-1-1) In the amino acid sequence of FNMQQQRQRRFYEALHDPNLNEEQRNARIRSIRDD (SEQ ID NO: 11), or in the amino acid sequence of
(a-1-2) FNMQCQRRFYEALHDPNLNEQRNARIRSIRDDDC (SEQ ID NO: 12)
　, any one to three in the sequence. The amino acid residues may be the same or different and are replaced by one amino acid residue each selected from the group consisting of lysine residues, aspartic acid residues, and glutamate residues
(a-2-1). In the amino acid sequence of β-Ala-NMQQQRRFYEALHDPNLNEEQRNARIRSIRDD (SEQ ID NO: 13) or the amino acid sequence of
(a-2-2) β-Ala-NMQCQRRFYEALHDPNLNEEQRNARIRSIRDDDC (SEQ ID NO: 14)
　, any one to three amino acids remaining in the sequence. The groups may be the same or different, each being replaced by one amino acid residue selected from the group consisting of lysine residues, aspartic acid residues, and glutamate residues, and
(b) SEQ ID NOs: 11-14. A compound according to any one of [2] to [6] or a salt thereof, which comprises an amino acid sequence having 85% or more identity with respect to each of the above amino acid sequences.
[8] The peptide is represented by the
formula 1-1: 1 (X 0-3 ) a- C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-I-IWC- (X 0-3).) B (SEQ ID NO
: 15) Equation 1-2: (X 0-3 ) a- C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-IVW-C- (X 0-3 ) b ( SEQ ID NO
: 16) Equation 1-3: (X 0-3 ) a- C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-VVW-C- (X 0-3 ) b (SEQ ID NO: 17 ) )
Equation 1-4: (X 0-3 ) a- C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-AVVW-C- (X 0-3 ) b (SEQ ID NO: 18)
Equation 1 -5: (X 0-3 ) a- C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-LL-W-C- (X) 0-3 ) b (SEQ ID NO
: 19) Equation 1-6: (X 0-3 ) a- C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L-IWC- (X 0-3) ) B (SEQ ID NO
: 20) Equation 1-7: (X 0-3 ) a- C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-LVFC- (X 0-3 ) b ( SEQ ID NO
: 21) Equation 1-8: (X 0-3 ) a- C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Q-VWC- (X 0-3 ) b (SEQ ID NO: 22 ) )
Equation 1-9: (X 0-3 ) a-C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-EVW-C- (X 0-3 ) b (SEQ ID NO: 23)
[In the formula,
　(X 0-3 ) a is none, Arginine residue-glycine residue-asparagin residue, glycine residue-asparagin residue, asparaginic acid residue, or asparagin residue,
　(X 0-3 ) b is none, threonine residue-tyrosine residue 　-Histidine
　residue or threonine residue ,
Xaa1 is an alanine residue, Xaa2 is a tyrosine residue, tryptophan residue, or histidine residue, and
　Xaa3 is a histidine residue, phenylalanine residue, Tyrosine residue, tryptophan residue, arginine residue, or glycine residue,
　Xaa4 is a lysine residue, aspartic acid residue, or glutamate residue, and
　Xaa5 is a glycine residue, serine residue, asparagine. Residues, glutamine residues, aspartic acid residues, glutamic acid residues, phenylalanine residues, tyrosine residues, tryptophan residues, histidine residues, threonine residues, leucine residues, alanine residues, valine residues, isoleucine residues A group, or an arginine residue,
　Xaa6 is a glutamine residue, a glutamic acid residue, an asparagine residue, or an aspartic acid residue. ], Or
formula 2-1: (X 0-3 ') a- C- (Xaa1')-(Xaa2')-(Xaa3')-(Xaa4')-(Xaa5')-(Xaa6')-L -VWC- (X 0-3 ') b (SEQ ID NO: 24)
[In the formula,
　(X 0-3 ') a and (X 0-3 ') b are the above-mentioned (X 0-3 ', respectively. ) Same as a and (X 0-3 ) b, and
　Xaa1', Xaa2', Xaa3', Xaa4', Xaa5', Xaa6'are the same as Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, respectively. is there. ], The compound according to any one of [2] to [6] or a salt thereof.
[9] The leaving group is (1) -O-, -S-, -Se-, -SO. 2- O-, -SO 2- N (R)-, -SO 2- , -C≡C-CH 2- O-, -N (OR)-, -N (R)-, and -ON A group selected from the group consisting of (R)-(where R is a hydrogen atom or an alkyl having 1 to 6 carbon atoms), or (2) a heteroarylene, [1] to [8]. Any compound of or a salt thereof.
[10] The nucleophilic group is NH 2 in the side chain of the lysine residue, OH in the side chain of the tyrosine residue, OH in the side chain of the serine residue, OH in the side chain of the threonine residue, and the cysteine ​​residue. A compound according to any one of [1] to [9] or a salt thereof, which is a group selected from the group consisting of SH in the side chain of.
[11] The compound of any of [1] to [10], wherein the electrophilic group is a group selected from the group consisting of -C (= O)-, -SO 2- , and -CH 2-. That salt.
[12] The bioorthogonal functional group contains an azide residue, an aldehyde residue, a thiol residue, an alkin residue, an alkene residue, a halogen residue, a tetrazine residue, a nitron residue, a hydroxylamine residue, and a nitrile residue. Group, hydrazine residue, ketone residue, boronic acid residue, cyanobenzothiazole residue, allyl residue, phosphine residue, maleimide residue, disulfide residue, thioester residue, α-halocarbonyl residue, isonitrile residue A compound according to any one of [1] to [11] or a salt thereof, which is a group selected from the group consisting of a group, a sidonon residue, and a selenium residue.
[13] Any of [1] to [12], wherein the bioorthogonal functional group is a group selected from the group consisting of azide residues, thiol residues, alkyne residues, maleimide residues, and disulfide residues. The compound or its salt.
[14] The compound represented by the formula (I) is the following formula (I-1):
A-L 1- L 2- E 1- E 2- E 3- B (I-1)
[In the formula,
　A and B are the same as those of the above formula (I),
　L 1 is a binding or divalent group,
　L 2 is a leaving group, and
　E. Reference numeral 1 is (i) an electrophilic group linked to the desorbing group and (ii) capable of reacting with a nucleophilic group in the antibody, and
　E 2 is (a) -XY. -[Here, X bonded to E 1 is C (R 1 ) (R 2 ) (where R 1 and R 2 are independently hydrogen atoms or alkyl atoms having 1 to 6 carbon atoms, respectively. ), N (R 3 ) (where R 3 is a hydrogen atom or an alkyl having 1 to 6 carbon atoms), O, S, or Se, and Y bonded to E 3 is C. (R 4 ) (R 5 ) (where R 4 and R 5 are independently alkyl hydrogen atoms or 1 to 6 carbon atoms). ], Or (b) The following equation (i):
[Chemical formula 1]

(Here, the ring Z is E 1The ring-constituting atom X'bonded to and all of the ring-constituting atoms adjacent to it are divalent ring groups that are carbon atoms, or the ring-constituting atom X'bonding to E 1 is a nitrogen atom and nitrogen. It is a divalent heterocyclic group in which the ring-constituting atoms on both sides of the atom are carbon atoms. ・ Is a bond. ),
　E 3 is a divalent group when E 2 is −XY—, and is bound or bonded when E 2 is a group represented by formula (i). a divalent group,
　wherein the leaving group, E by the reaction between the electrophilic group and the nucleophilic group 1 has the ability to desorption is disconnected from. ], Which is any compound of [1] to [13] or a salt thereof.
[15] The L 2 is
(a) ring PQ- [where ring P is an arylene which may be substituted with an electron-withdrawing group, a heteroarylene which may be substituted with an electron-withdrawing group. , May be fused 2,5-diketopyrrolidine, may be fused 2,6-diketopiperidine, may be fused 2-ketopyrrolidine, may be fused It is a group selected from the group consisting of 2-ketopiperidine and 2-pyrrolidine, and Q is -O-, -S-, -Se-, -SO 2- O-, -SO 2- N (R)-, -SO 2A group selected from the group consisting of-, -C≡C-CH 2- O-, -N (OR)-, -N (R)-, and -ON (R)-(where R is It is a hydrogen atom or an alkyl having 1 to 6 carbon atoms.) ],
(B) Heteroarylene, or
(c) -Q- [Here, Q is -O-, -S-, -Se-, -SO 2- O-, -SO 2- N (R)- , -SO 2- , -C≡C-CH 2- O-, -N (OR)-, -N (R)-, and -ON (R)-, a group selected from the group (here). , R is a hydrogen atom or an alkyl having 1 to 6 carbon atoms.). ], The compound of [14] or a salt thereof.
[16] The compound of [14] or [15] or a salt thereof, wherein L 2 is a group selected from the group consisting of the following structural formulas:
[Chemical formula 2]

(Here, EWG is an electron-withdrawing group. Yes,
　m is an integer of 0 to 4,
　n is an integer of 0 to 3,
　R is a hydrogen atom or an alkyl having 1 to 6 carbon atoms, and
　○ (white circle) is L 1. ● (black circle) is E It is a bond to 1 . ).
[17] The compound of any one of [1] to [16] or a salt thereof, wherein the main chain of L or L 1 to L 2 connecting A and E is composed of 20 or less atoms.
[18] The compound represented by the formula (I-1) is the following formula (I-2):
A-L 1- L 2- E 1 -XY-E 3- B (I-2)
[ In the formula,
　A, L 1 , X, Y, and B are the same as those in the above formula (I-1), and
　L 2 is
(a) ring PQ- [where ring P is ... An arylene which may be substituted with an electron-withdrawing group, a heteroarylene which may be substituted with an electron-withdrawing group, 2,5-diketopyrrolidine which may be fused, or a ring-fused 2 , 6-Diketopiperidine, 2-ketopyrrolidine which may be fused, 2-ketopiperidine which may be fused, 2-pyridone, which is a group selected from the group, and Q is -O-. , -S-, -Se-, -SO 2- O-, -SO 2From the group consisting of -N (R)-, -SO 2- , -C≡C-CH 2- O-, -N (OR)-, -N (R)-, and -ON (R)- The group of choice (where R is a hydrogen atom or an alkyl having 1 to 6 carbon atoms). ],
(B) Heteroarylene, or
(c) -Q- [Here, Q is -O-, -S-, -Se-, -SO 2- O-, -SO 2- N (R)- , -SO 2- , -C≡C-CH 2- O-, -N (OR)-, -N (R)-, and -ON (R)-, a group selected from the group (here). , R is a hydrogen atom or an alkyl having 1 to 6 carbon atoms.). ],
　E 1 is a group selected from the group consisting of -C (= O)-, -SO 2- , and -CH 2- , and
　E 3 is a divalent group. ], Which is any compound of [14] to [17] or a salt thereof.
[19] The compound represented by the formula (I-1) is the following formula (I-3):
[Chemical Formula 3].

[In the formula,
　A, L 1 , ring Z, and B are the same as those in the above formula (I-1), and
　L 2 is
(a) ring PQ- [where ring P is ... An arylene which may be substituted with an electron-withdrawing group, a heteroarylene which may be substituted with an electron-withdrawing group, 2,5-diketopyrrolidine which may be fused, or a ring-fused 2 , 6-Diketopiperidine, 2-ketopyrridine which may be fused, 2-ketopiperidine which may be fused, 2-pyridone, and Q is -O- , -S-, -Se-, -SO 2- O-, -SO 2- N (R)-, -SO 2- , -C≡C-CH 2- O-, -N (OR)-,- A group selected from the group consisting of N (R)-and -ON (R)-(where R is a hydrogen atom or an alkyl having 1 to 6 carbon atoms). ],
(B) Heteroarylene, or
(c) -Q- [Here, Q is -O-, -S-, -Se-, -SO 2- O-, -SO 2- N (R)- , -SO 2- , -C≡C-CH 2A group selected from the group consisting of -O-, -N (OR)-, -N (R)-, and -ON (R)-(where R is a hydrogen atom or 1 to 6 carbon atoms. It is an alkyl of.). ],
　E 1 is a group selected from the group consisting of -C (= O)-, -SO 2- , and -CH 2- , and
　E 3 is a binding or divalent group. ], Which is any compound of [14] to [17] or a salt thereof.
[20] The following formula (I):
A-L-EB (I)
[In the formula,
　A is an affinity substance for an antibody,
　L is a divalent group containing a elimination group, and
　E. Is a divalent group containing (i) an electrophilic group linked to the desorbing group and (ii) capable of reacting with a nucleophilic group in the antibody, and
　B is bioorthogonal. It is a functional group, and the
　desorbing group has the ability to be cleaved from E and desorbed by the reaction between the nucleophilic group and the electrophilic group. ], A reagent for regioselective modification of an antibody with a bioorthogonal functional group, which comprises a substance having an affinity for the antibody and a compound having a bioorthogonal functional group or a salt thereof.
[21] A method for producing an antibody having a bioorthogonal functional group or a salt thereof.
　The following formula (I):
A-LEB (I)
[In the formula,
　A is an affinity substance for an antibody,
　L is a divalent group containing a elimination group, and
　E is ( i) A divalent group containing an electrophilic group linked to the desorbing group and (ii) capable of reacting with the nucleophilic group in the antibody, where
　B is a bioorthogonal functional group.
　Yes , the desorbing group has the ability to be cleaved from E and desorbed by the reaction between the nucleophilic group and the electrophoryl group. ], A compound having an affinity for the antibody and a compound having a bioorthogonal functional group or a salt thereof is reacted with the antibody, and the following
　formula (II):
Ab-EB (II)
[in the formula,
　E and B are the same as those of the above formula (I), and
　Ab is an antibody. ], The method comprising producing an antibody having a bioorthogonal functional group or a salt thereof.
[22] A method for producing an antibody or a salt thereof having a functional substance, wherein
(1) the following formula (I):
ALEB (I)
[In the formula,
　A is an affinity substance for an antibody. And
　L is a divalent group containing a leaving group,
　E is a divalent group containing (i) an effervescent group linked to the leaving group and (ii) capable of reacting with a nucleophilic group in the antibody, and
　B is a bio-orthogonal group. It is a sex functional group, and the
　leaving group has the ability to be cleaved from E and removed by the reaction between the nucleophilic group and the electronically active group. ], A compound having an affinity for the antibody and a compound having a bioorthogonal functional group or a salt thereof is reacted with the antibody, and the following
　formula (II):
Ab-EB (II)
[in the formula,
　E and B are the same as those of the above formula (I), and
　Ab is an antibody. ] To produce an antibody having a bioorthogonal functional group or a salt thereof; and
(2) an antibody having a bioorthogonal functional group represented by the above formula (II) or a salt thereof. By reacting with a functional substance via an orthogonal functional group, the following
　formula (III):
Ab-EB'-F (III)
[In the formula,
　Ab is the same as that of the above formula (II). ,
　E are the same as those of the above formula (I),
　B'is a divalent group containing a moiety produced by the reaction between the functional group and the bioorthogonal functional group, and
　F is a divalent group . It is a functional substance. ], The method comprising producing an antibody having a functional substance or a salt thereof.
[23] The following formula (II-1):
Ab-E 1- E 2- E 3- B (II-1)
[In the formula,
　Ab is an antibody, and
　E 1 is a nucleophilic group in the antibody.
　E 2 is a linked electrophilic group, and E 2 is (a) -XY- [where X bonded to E 1 is C (R 1 ) (R 2 ) (here, R 1 and R 2 is independently an alkyl having a hydrogen atom or a carbon atom number of 1 to 6), N (R 3 ) (where R 3 is an alkyl having a hydrogen atom or a carbon atom number of 1 to 6). .), O, S or a Se, E, 3 Y that binds the, C (R 4 ) (R 5 ) (wherein, R 4And R 5 are independently alkyl hydrogen atoms or 1 to 6 carbon atoms. ). ], Or (b) the following formula (i):
[Chemical formula 4]

(Here, the ring Z is a divalent atom in which all of the ring-constituting atoms X'bonding to E 1 and the ring-constituting atoms on both sides thereof are carbon atoms. Is a divalent heterocyclic group in which the ring-constituting atom X'bonded to E 1 is a nitrogen atom and the ring-constituting atoms on both sides of the nitrogen atom are carbon atoms. It is a group represented by (hand),
　E 3 is a divalent group when E 2 is −XY—, and E 2 is a group represented by the formula (i). Is a bound or divalent group, and
　B is a bioorthogonal functional group. ], An antibody or a salt thereof having a bioorthogonal functional group regioselectively.
[24] The antibody of [23] or a salt thereof, wherein the antibody is an antibody having a bioorthogonal functional group only in a constant region of a monoclonal antibody.
[25] The antibody of [23] or [24] or a salt thereof, wherein the antibody is an antibody having a bioorthogonal functional group only in the Fc region of a monoclonal antibody.
[26] The antibody is a human IgG having a bioorthogonal functional group regioselectively in a region consisting of amino acid residues at positions 246 to 248 or 288 to 290 in the human IgG Fc region [23] to The antibody of any of [25] or a salt thereof.
[27] The antibody represented by the formula (II-1) is the following formula (II-2):
Ab-E 1- XYE 3- B (II-2)
[In the formula,
　Ab, X, Y, and B are the same as those of the above formula (II-1), and
　E 1 is selected from the group consisting of -C (= O)-, -SO 2- , and -CH 2-. It is a group, and
　E 3 is a divalent group. ], Which is an antibody having a bioorthogonal functional group regioselectively, any of the antibodies [23] to [26] or a salt thereof.
[28] The antibody represented by the formula (II-1) is the following formula (II-3):
[Chemical

formula 5] [In the formula,
　Ab, ring-constituting atom X', ring Z, and B are described above. Same as that of formula (II-1),
　E 1Is a group selected from the group consisting of -C (= O)-, -SO 2- , and -CH 2- , and
　E 3 is a binding or divalent group. ], Which is an antibody having a bioorthogonal functional group regioselectively, any of the antibodies [23] to [26] or a salt thereof.
[29] The following formula (III-1):
Ab-E 1- E 2- E 3- B'-F (III-1)
[In the formula,
　Ab is an antibody, and
　E 1 is a finding in the antibody. It is an electrophilic group linked to a nuclear group, and
　E 2 is (a) -XY- [where X bonded to E 1 is C (R 1 ) (R 2 ) (here, here, R 1 and R 2 are independently hydrogen atoms or alkyl having 1 to 6 carbon atoms.), N (R) 3 ) (Here, R 3 is a hydrogen atom or an alkyl having 1 to 6 carbon atoms), O, S, or Se, and Y bonded to E 3 is C (R 4 ) (R). 5 ) (Here, R 4 and R 5 are independently alkyl hydrogen atoms or 1 to 6 carbon atoms). ], Or (b) the following formula (i):
[Formula 6]

(wherein, ring Z is, E 1 2 monovalent all ring members X 'and ring members both adjacent to bind is a carbon atom Is a divalent heterocyclic group in which the ring-constituting atom X'bonded to E 1 is a nitrogen atom and the ring-constituting atoms on both sides of the nitrogen atom are carbon atoms. It is a group represented by (hand),
　E 3 is a divalent group when E 2 is −XY—, and E 2 is a group represented by the formula (i). Is a bound or divalent group, and
　B'is a divalent group containing a moiety produced by the reaction between a functional substance and a bioorthogonal functional group.
　F is a functional substance. ], An antibody or a salt thereof having a functional substance regioselectively.
[30] The antibody of [29] or a salt thereof, wherein the antibody is an antibody having a bioorthogonal functional group only in a constant region of a monoclonal antibody.
[31] The antibody of [29] or [30] or a salt thereof, wherein the antibody is an antibody having a bioorthogonal functional group only in the Fc region of a monoclonal antibody.
[32] The antibody is a human IgG having a functional substance position-selectively in a region consisting of amino acid residues at positions 246 to 248 or 288 to 290 in the human IgG Fc region [29] to [31]. ] Any antibody or salt thereof.
[33] The antibody represented by the formula (III-1) is the following formula (III-2):
Ab-E 1 - XYE 3- B'-F (III-2)
[in the formula ,
　Ab, X, Y, B 'and F are the same as those of the formula
　(III-1), E 1 is -C (= O) -, - SO 2 -, and -CH 2 - from It is a group selected from the group of
　E 3Is an antibody having a functional substance regioselectively represented by [is a divalent group], any of the antibodies [29] to [32] or a salt thereof.
[34] The antibody represented by the formula (III-1) is the following formula (III-3):
[Chemical

formula 7] [In the formula,
　Ab, ring-constituting atom X', ring Z, B'and F are , The same as that of the above formula (III-1),
　E 1 is a group selected from the group consisting of -C (= O)-, -SO 2- , and -CH 2- , and
　E 3 is. , Bonded or divalent group. ], Which is an antibody having a functional substance regioselectively, any of the antibodies [29] to [32] or a salt thereof.
[35] The following formula (IV):
ALF (IV)
[In the formula,
　A is an affinity substance for an antibody,
　L is a divalent group containing a leaving group, and
　E. Is a divalent group containing (i) an electrophilic group linked to the leaving group and (ii) capable of reacting with the nucleophilic group in the antibody, and
　F is a functional substance. And
　The leaving group has the ability to be cleaved from E and eliminated by the reaction between the nucleophilic group and the electrophilic group. ], A compound having an affinity substance for an antibody and a functional substance, or a salt thereof.
[36] The following formula (IV):
ALF (IV)
[In the formula,
　A is an affinity substance for an antibody,
　L is a divalent group containing a leaving group, and
　E. Is a divalent group containing (i) an electrophilic group linked to the leaving group and (ii) capable of reacting with the nucleophilic group in the antibody, and
　F is a functional substance. The
　leaving group has the ability to be cleaved from E and removed by the reaction between the nucleophile and the electron-forming group. ], A reagent for regioselective modification of an antibody by a functional substance, which comprises a substance having an affinity for the antibody and a compound having a functional substance or a salt thereof.
[37] A method for producing an antibody having a functional substance or a salt thereof, wherein 　A is an affinity substance for the antibody according to the following
　formula (IV):
ALEF (IV)
[In the formula,
A is an affinity substance for the antibody.
　L is a divalent group containing a leaving group, and
　E is (i) a nucleophile linked to the leaving group and (ii) capable of reacting with a nucleophile in the antibody. It is a divalent group containing a group, and
　F is a functional substance.
　The leaving group has the ability to be cleaved from E and eliminated by the reaction between the nucleophilic group and the electrophilic group. ], A compound having an affinity substance for the antibody and a functional substance or a salt thereof is reacted with the antibody, and the following
　formula (III):
Ab-EF (III)
[In the formula,
　Ab is , Antibodies,
　E and F are the same as those of formula (IV) above. ], The method comprising producing an antibody having a functional substance or a salt thereof.
Effect of the invention
[0012]
　Compounds of the invention or salts thereof having affinity substances for antibodies and bioorthogonal functional groups or functional substances are useful, for example, for regioselective modification of antibodies.
　The antibody or salt thereof of the present invention having a bioorthogonal functional group regioselectively is useful as an intermediate in the preparation of an antibody or a salt thereof having a functional substance regioselectively, for example.
　The antibody or salt thereof of the present invention having a functional substance regioselectively is useful as, for example, a medicine or a reagent (eg, a diagnostic agent, a research reagent).
A brief description of the drawing
[0013]
FIG. 1 is a schematic view showing an outline of the present invention.
FIG. 2 is a diagram showing the results of analysis by SDS-PAGE in the synthesis of IgG antibody trastuzumab-peptide complex. Lanes 1, 3, 6, 8: Molecular weight marker; Lane 2: Unreacted IgG antibody trastuzumab (control, band with molecular weight around 50,000 indicates heavy chain, band with molecular weight around 25,000 indicates light chain); Lane 4: Complex of IgG antibody trastuzumab and compound 10 (The upper band with a molecular weight of around 50,000 indicates that compound 10 was conjugated to the heavy chain of trastuzumab. The lower band with a molecular weight of around 50,000 is an unreacted heavy chain. , A band with a molecular weight of around 25,000 indicates an unreacted light chain); Lane 5: A complex of IgG antibody trastuzumab and compound 11 (the upper band with a molecular weight of around 50,000 is a heavy chain of trastuzumab with compound 11 It indicates that it has been gated. The band below the molecular weight of about 50,000 indicates the unreacted heavy chain, and the band of the molecular weight of about 25,000 indicates the unreacted light chain); Lane 7: Conjugation with IgG antibody trastuzumab and compound 12. Post-gation reaction mixture (bands with a molecular weight of around 50,000 indicate unreacted heavy chains, bands with a molecular weight of around 25,000 indicate unreacted light chains, and no band with compound 12 conjugated).
FIG. 3 is a diagram showing the results of analysis by SDS-PAGE of a trastuzumab-peptide complex synthesized by introducing maleimide position-specifically into the IgG antibody trastuzumab and then conjugating it with a peptide reagent having a thiol. Lanes 1 and 9: Molecular weight marker. Lane 2: IgG antibody trastuzumab is treated with compound 22 (10 molar equivalents relative to the antibody), maleimide is introduced position-selectively, and then compound 25 is conjugated. The upper band with a molecular weight of around 50,000 indicates that maleimide was introduced into the heavy chain of trastuzumab and conjugated with compound 25. The lower band with a molecular weight of around 50,000 indicates an unreacted heavy chain, and the band with a molecular weight of around 25,000 indicates an unreacted light chain. Lane 3: A complex in which the IgG antibody trastuzumab was treated with compound 22 (20 molar equivalents relative to the antibody), maleimide was introduced position-selectively, and then compound 25 was conjugated. The upper band with a molecular weight of around 50,000 indicates that maleimide was introduced into the heavy chain of trastuzumab and conjugated with compound 25. The lower band with a molecular weight of around 50,000 indicates an unreacted heavy chain, and the band with a molecular weight of around 25,000 indicates an unreacted light chain. Lane 4: A complex in which the IgG antibody trastuzumab was treated with compound 22 (40 molar equivalents relative to the antibody), maleimide was introduced position-selectively, and then compound 25 was conjugated. The upper band with a molecular weight of around 50,000 indicates that maleimide was introduced into the heavy chain of trastuzumab and conjugated with compound 25. The lower band with a molecular weight of around 50,000 indicates an unreacted heavy chain, and the band with a molecular weight of around 25,000 indicates an unreacted light chain. Lane 5: A complex in which the IgG antibody trastuzumab was treated with compound 23 (10 molar equivalents relative to the antibody), maleimide was introduced position-selectively, and then compound 25 was conjugated. The upper band with a molecular weight of around 50,000 indicates that maleimide was introduced into the heavy chain of trastuzumab and conjugated with compound 25. The lower band with a molecular weight of around 50,000 is an unreacted heavy chain, and the band with a molecular weight of around 25,000 is an unreacted light chain. Shows a chain. Lane 6: IgG antibody trastuzumab was treated with compound 23 (20 molar equivalents relative to the antibody), maleimide was introduced position-selectively, and then compound 25 was conjugated. The upper band with a molecular weight of around 50,000 indicates that maleimide was introduced into the heavy chain of trastuzumab and conjugated with compound 25. The lower band with a molecular weight of around 50,000 indicates an unreacted heavy chain, and the band with a molecular weight of around 25,000 indicates an unreacted light chain. Lane 7: A complex obtained by treating IgG antibody trastuzumab with compound 23 (40 molar equivalents relative to the antibody), introducing maleimide position-selectively, and then conjugating compound 25. The upper band with a molecular weight of around 50,000 indicates that maleimide was introduced into the heavy chain of trastuzumab and conjugated with compound 25. The lower band with a molecular weight of around 50,000 indicates an unreacted heavy chain, and the band with a molecular weight of around 25,000 indicates an unreacted light chain. Lanes 8 and 10: Unreacted IgG antibody trastuzumab (control, band with molecular weight around 50,000 indicates heavy chain, band with molecular weight around 25,000 indicates light chain) Lane 11: IgG antibody trastuzumab as compound 24 (antibody) A reaction mixture to which Compound 25 was added after treatment with 10 molar equivalents. A band having a molecular weight of about 50,000 indicates an unreacted heavy chain, and a band having a molecular weight of about 25,000 indicates an unreacted light chain. Lane 12: A reaction mixture in which the IgG antibody trastuzumab was treated with Compound 24 (20 molar equivalents relative to the antibody) followed by Compound 25. A band having a molecular weight of about 50,000 indicates an unreacted heavy chain, and a band having a molecular weight of about 25,000 indicates an unreacted light chain. Lane 13: A reaction mixture in which the IgG antibody trastuzumab was treated with Compound 24 (40 molar equivalents relative to the antibody) followed by Compound 25. A band having a molecular weight of about 50,000 indicates an unreacted heavy chain, and a band having a molecular weight of about 25,000 indicates an unreacted light chain.
FIG. 4 is a diagram showing ESI-TOFMS under post-reduction conditions of the trastuzumab-maleimide modified product synthesized in (4-5-1). The upper row shows the measurement of unreacted trastuzumab, and the lower row shows the modified product.
FIG. 5 is a diagram showing ESI-TOFMS under post-reduction conditions of the trastuzumab-azide modified product (azide modified antibody 1) synthesized in (6-1-1). The lower row shows the measurement of unreacted trastuzumab, and the upper row shows the modified product.
FIG. 6 is a diagram showing ESI-TOFMS under post-reduction conditions of the trastuzumab-azide modified product (azide modified antibody 3) synthesized in (6-1-2). The lower row shows the measurement of unreacted trastuzumab, and the upper row shows the modified product.
FIG. 7 is a diagram showing ESI-TOFMS under post-reduction conditions of the trastuzumab-azide modified product (azide modified antibody 6) synthesized in (6-1-3). The upper row shows the measurement of unreacted trastuzumab, and the lower row shows the modified product.
FIG. 8 is a diagram showing ESI-TOFMS under post-reduction conditions of the trastuzumab-azide modified product (azide modified antibody 8) synthesized in (6-1-3). The upper row shows the measurement of unreacted trastuzumab, and the lower row shows the modified product.
FIG. 9 is a diagram showing ESI-TOFMS under post-reduction conditions of the trastuzumab-azide modified product (azide modified antibody 10) synthesized in (6-1-3). The upper row shows the measurement of unreacted trastuzumab, and the lower row shows the modified product.
FIG. 10 is a diagram showing ESI-TOFMS under post-reduction conditions of the adalimumab-azide modified product (azide modified antibody 28) synthesized in (6-1-4). The upper row shows the measured unreacted adalimumab, and the lower row shows the modified product.
FIG. 11 is a diagram showing ESI-TOFMS under post-reduction conditions of the denosumab-azide-modified antibody (azido-modified antibody 29) synthesized in (6-1-4). The upper row shows the measured unreacted denosumab, and the lower row shows the modified product.
FIG. 12 is a diagram showing ESI-TOFMS under post-reduction conditions of the dupilumab-azide-modified antibody (azido-modified antibody 30) synthesized in (6-1-4). The upper row shows the measured unreacted dupilumab, and the lower row shows the modified product.
FIG. 13 is a diagram showing ESI-TOFMS under post-reduction conditions of the rituximab-azide-modified antibody (azido-modified antibody 31) synthesized in (6-1-4). The lower row shows the measured unreacted rituximab, and the upper row shows the modified product.
FIG. 14 is a diagram showing ESI-TOFMS under post-reduction conditions of the trastuzumab-protected thiol modified product synthesized in (8-1-1). The upper row shows the measurement of unreacted trastuzumab, and the lower row shows the modified product.
FIG. 15 is a diagram showing ESI-TOFMS under post-reduction conditions of the trastuzumab-thiol modified product deprotected in (8-3-1). The upper row shows the measurement of unreacted trastuzumab, and the lower row shows the modified product.
FIG. 16 is a diagram showing ESI-TOFMS under post-reduction conditions of the trastuzumab-azide modified product synthesized in (9-1-1). The upper row shows the measurement of unreacted trastuzumab, and the lower row shows the modified product.
FIG. 17 is a diagram showing ESI-TOFMS under post-reduction conditions of the trastuzumab-azide modified product synthesized in (9-1-5). The upper row shows the measurement of unreacted trastuzumab, and the lower row shows the modified product.
FIG. 18 is a diagram showing ESI-TOFMS under post-reduction conditions of the trastuzumab-azide modified product synthesized in (9-2-2). The upper row shows the measurement of unreacted trastuzumab, and the lower row shows the modified product.
FIG. 19 is a diagram showing ESI-TOFMS of the trastuzumab-Cy3 complex synthesized in (10-1-1). The lower row shows the measurement of unreacted trastuzumab, the middle row shows the measurement of the trastuzumab-azide modified product synthesized in (6-1-1), and the upper row shows the trastuzumab-Cy3 complex.
FIG. 20 is a diagram showing ESI-TOFMS under reducing conditions of the trastuzumab-Cy3 complex treated in (10-1-2). The lower row shows the measurement of unreacted trastuzumab, the middle row shows the measurement of the trastuzumab-azide modified product synthesized in (6-1-1), and the upper row shows the trastuzumab-Cy3 complex.
FIG. 21 is a diagram showing ESI-TOFMS of the trastuzumab-peptide complex synthesized in (10-2-2). The lower row shows the measurement of unreacted trastuzumab, the middle row shows the measurement of the trastuzumab-azide modified product synthesized in (6-1-1), and the upper row shows the trastuzumab-Cy3 complex.
FIG. 22 is a diagram showing ESI-TOFMS under reducing conditions of the trastuzumab-Cy3 complex treated in (10-2-3). The lower row shows the measurement of unreacted trastuzumab, the middle row shows the measurement of the trastuzumab-azide modified product synthesized in (6-1-1), and the upper row shows the trastuzumab-peptide complex.
FIG. 23 is a diagram showing ESI-TOFMS under reducing conditions of the trastuzumab-maleimide compound complex treated in (10-3-1). The upper row shows the measurement of the thiol-introduced body of trastuzumab, and the lower row shows the trastuzumab-maleimide compound complex.
FIG. 24 is a diagram showing ESI-TOFMS under the reduction conditions of the reaction product treated in (10-3-2). The upper row shows the measurement of the thiol-introduced body of trastuzumab, and the lower row shows the trastuzumab-maleimide compound complex.
FIG. 25 is a diagram showing (1) the amino acid sequence of the heavy chain of trastuzumab (SEQ ID NO: 2) and (2) the amino acid sequence of the light chain of trastuzumab (SEQ ID NO: 4).
FIG. 26 is a diagram showing (1) the amino acid sequence of the heavy chain of denosumab (SEQ ID NO: 104) and (2) the amino acid sequence of the light chain of denosumab (SEQ ID NO: 105).
FIG. 27 is a diagram showing (1) the amino acid sequence of the heavy chain of dupilumab (SEQ ID NO: 106) and (2) the amino acid sequence of the light chain of dupilumab (SEQ ID NO: 107).
FIG. 28 is a diagram showing the MS spectrum of a peptide fragment of THTCPPCPAPELLLGGPSVFPFPPKPKDTLMISS (SEQ ID NO: 11) containing a site of modification to a lysine residue by trypsin digestion of azide benzoic acid modified trastuzumab.
FIG. 29 shows the CID spectrum of a peptide fragment of THTCPPCPAPELLLGGPSVFPFPPKPKDTLMISS (SEQ ID NO: 11) containing a site of modification to a lysine residue by trypsin digestion of azide benzoic acid modified trastuzumab.
FIG. 30 is a diagram showing the MS spectrum of a peptide fragment of FNWYVDGVEVHNAKTTKPR (SEQ ID NO: 12) containing a site of modification to a lysine residue by trypsin digestion of azide benzoic acid modified trastuzumab.
FIG. 31 shows the CID of a peptide fragment of FNWYVDGVEVHNAKTKPR (SEQ ID NO: 12) containing a site modified to a lysine residue by trypsin digestion of azide benzoic acid modified trastuzumab (amine benzoic acid introducer (+119.037 Da)). It is a figure which shows the spectrum.
FIG. 32 shows the MS spectrum of a peptide fragment of THTCPPCPAPELLLGGPSVFLFPPKPKDTLMISS (SEQ ID NO: 11) containing a site of modification to a lysine residue by trypsin digestion of mercaptopropionic acid-added maleimide-modified trastuzumab.
FIG. 33 shows the CID spectrum of a peptide fragment of THTCPPCPAPELLLGGPSVFLFPPKPKDTLMISS (SEQ ID NO: 11) containing a site for modification of maleimide MPA-modified trastuzumab to a lysine residue by trypsin digestion.
FIG. 34 is a diagram showing the MS spectrum of a peptide fragment of THTCPPCPAPELLLGGPSVFLFPPKPKDTLMISS (SEQ ID NO: 11) containing a site of modification of alkyl azide-modified trastuzumab to a lysine residue by trypsin digestion.
FIG. 35 shows the CID spectrum of a peptide fragment of THTCPPCPAPELLLGGPSVFLFPPKPKDTLMISS (SEQ ID NO: 11) containing a site of modification of alkyl azide-modified trastuzumab to a lysine residue by trypsin digestion.
FIG. 36 is a diagram showing the MS spectrum of a peptide fragment of VVSVLTVLLHQDWLNGKEYK (SEQ ID NO: 101) containing a site of modification to a lysine residue by trypsin digestion of azide benzoic acid modified trastuzumab.
FIG. 37 shows a CID spectrum of a peptide fragment of VVSVLTVLLHQDWLNGKEYK (SEQ ID NO: 101) containing a site of modification to a lysine residue by trypsin digestion of azide benzoic acid modified trastuzumab.
FIG. 38 is a diagram showing the results of analysis by BioPharma Finder that the lysine residue at position 317 of azide benzoic acid-modified trastuzumab is highly selectively modified.
FIG. 39 is a diagram showing the MS spectrum of a peptide fragment of FNWYVDGVEVHNAKTTKPR (SEQ ID NO: 12) containing a site of modification to a lysine residue by trypsin digestion of azide benzoic acid modified trastuzumab.
FIG. 40 shows the CID spectrum of a peptide fragment of FNWYVDGVEVHNAKTTKPR (SEQ ID NO: 12) containing a site of modification to a lysine residue by trypsin digestion of azide benzoic acid modified trastuzumab.
FIG. 41 is a diagram showing the results of analysis by BioPharma Finder that the lysine residue at position 288 or 290 of azide benzoic acid-modified trastuzumab is highly selectively modified.
FIG. 42 shows the MS spectrum of a peptide fragment of THTCPPCPAPELLLGGPSVFPFPPKPKDTLMISS (SEQ ID NO: 11) containing a site of modification to a lysine residue by trypsin digestion of azide benzoic acid modified trastuzumab.
FIG. 43 shows the CID spectrum of a peptide fragment of THTCPPCPAPELLLGGPSVFPFPPKPKDTLMISS (SEQ ID NO: 11) containing a site of modification to a lysine residue by trypsin digestion of azide benzoic acid modified trastuzumab.
FIG. 44 is a diagram showing the results of analysis by BioPharma Finder that the lysine residue at position 246 or 248 of azide benzoic acid-modified trastuzumab is highly selectively modified.
FIG. 45 is a diagram showing the MS spectrum of a peptide fragment of THTCPPCPAPELLLGGPSVFLFPPKPKDTLMISS (SEQ ID NO: 11) containing a site of modification of acetylthiol-modified trastuzumab to a lysine residue by trypsin digestion.
FIG. 46 shows the CID spectrum of a peptide fragment of THTCPPCPAPELLLGGPSVFLFPPKPKDTLMISS (SEQ ID NO: 11) containing a site of modification of acetylthiol-modified trastuzumab to a lysine residue by trypsin digestion.
FIG. 47 is a diagram showing the results of analysis by BioPharma Finder that the lysine residue at position 246 or 248 of acetylthiol-modified trastuzumab is highly selectively modified.
FIG. 48 shows the MS spectrum of a peptide fragment of THTCPPCPAPELLLGGPSVFPFPPKPKDTLMISS (SEQ ID NO: 11) containing a site of modification to a lysine residue by trypsin digestion of azide benzoic acid modified trastuzumab.
FIG. 49 shows the CID spectrum of a peptide fragment of THTCPPCPAPELLLGGPSVFPFPPKPKDTLMISS (SEQ ID NO: 11) containing a site of modification of azide benzoic acid modified trastuzumab to a lysine residue by trypsin digestion.
FIG. 50 is a diagram showing the MS spectrum of a peptide fragment of FNWYVDGVEVHNAKTTKPR (SEQ ID NO: 12) containing a site of modification to a lysine residue by trypsin digestion of azide benzoic acid modified trastuzumab.
FIG. 51 shows the CID spectrum of a peptide fragment of FNWYVDGVEVHNAKTTKPR (SEQ ID NO: 12) containing a site of modification to a lysine residue by trypsin digestion of azide benzoic acid modified trastuzumab.
FIG. 52 shows the MS spectrum of a peptide fragment of THTCPPCPAPELLLGGPSVFLFPPKPKDTLMISS (SEQ ID NO: 11) containing a site of modification to a lysine residue by trypsin digestion of acetylthiol and azide benzoic acid modified trastuzumab.
FIG. 53 shows the CID spectrum of a peptide fragment of THTCPPCPAPELLLGGPSVFLFPPKPKDTLMISS (SEQ ID NO: 11) containing a site of modification to a lysine residue by trypsin digestion of acetylthiol and azide benzoic acid modified trastuzumab.
FIG. 54 is a diagram showing the MS spectrum of a peptide fragment of FNWYVDGVEVHNAKTKPR (SEQ ID NO: 12) containing a site of modification to a lysine residue by trypsin digestion of acetylthiol and azide benzoic acid modified trastuzumab.
FIG. 55 shows the CID spectrum of a peptide fragment of FNWYVDGVEVHNAKTKPR (SEQ ID NO: 12) containing a site of modification to a lysine residue by trypsin digestion of acetylthiol and azide benzoic acid modified trastuzumab.
FIG. 56 shows the results of analysis by BioPharma Finder that the lysine residues at positions 246 or 248 and 288 or 290 of acetylthiol and azide benzoic acid modified trastuzumab are highly selectively modified. Is.
FIG. 57 shows the MS spectrum of a peptide fragment of CCVECPPCPAPVAGPSVFLFPPKPKDTLMISS (SEQ ID NO: 102) containing a site of modification of benzoic acid modified denosumab to a lysine residue by trypsin digestion.
FIG. 58 shows the CID spectrum of a peptide fragment of CCVECPPCPAPVAGPSVFLFPPKPKDTLMISS (SEQ ID NO: 102) containing a site of modification of benzoic acid modified denosumab to a lysine residue by trypsin digestion.
FIG. 59 is a diagram showing the results of analysis by BioPharma Finder that the lysine residue at position 247 or 249 of benzoic acid-modified denosumab is highly selectively modified.
FIG. 60 is a diagram showing the MS spectrum of a peptide fragment of YGPPPCPPCPAPEFLGGGPSVFLFPPKPKDTLMISS (SEQ ID NO: 103) containing a site of modification of benzoic acid-modified dupilumab to a lysine residue by trypsin digestion.
FIG. 61 shows the CID spectrum of a peptide fragment of YGPPPCPPCPAPEFLGGGPSVFLFPPKPKDTLMISS (SEQ ID NO: 103) containing a site of modification of benzoic acid-modified dupilumab to a lysine residue by trypsin digestion.
FIG. 62 is a diagram showing the results of analysis by BioPharma Finder that the lysine residue at position 251 or 253 of benzoic acid-modified dupilumab is highly selectively modified.
FIG. 63 is a diagram showing the analysis results of the trastuzumab-DM1 conjugate synthesized in (12-1-1) by ESI-TOFMS (non-reducing conditions).
FIG. 64 is a diagram showing the analysis results of the trastuzumab-DM1 conjugate synthesized in (12-1-1) by ESI-TOFMS (reduction conditions).
FIG. 65 is a diagram showing the analysis results of the trastuzumab-MMAE conjugate synthesized in (12-2-1) by ESI-TOFMS (non-reducing conditions).
FIG. 66 is a diagram showing the analysis results of the trastuzumab-MMAE conjugate synthesized in (12-2-1) by ESI-TOFMS (reduction conditions).
FIG. 67 is a diagram showing the analysis results of the rituximab-DM1 conjugate synthesized in (12-3-1) by ESI-TOFMS (non-reducing conditions).
FIG. 68 is a diagram showing the analysis results of the rituximab-DM1 conjugate synthesized in (12-3-1) by ESI-TOFMS (reduction conditions).
FIG. 69 is a diagram showing the analysis results of the rituximab-DM1 conjugate synthesized in (12-4-1) by ESI-TOFMS (non-reducing conditions).
FIG. 70 is a diagram showing the analysis results of the rituximab-DM1 conjugate synthesized in (12-4-1) by ESI-TOFMS (reduction conditions).
[Fig. 71] Fig. 71 shows (1) the consensus amino acid sequence of the Fc region and the IgG1 Fc region (SEQ ID NO: 1) in the heavy chain of trusszumab, and (2) the amino acid sequence of the IgG1 Fc region (SEQ ID NO: 3). It is a figure which shows.
Mode for carrying out the invention
[0014]
1. 1. A substance having an affinity for an antibody and a compound having a bioorthogonal functional group or a salt thereof
1-1. Overview The
　present invention provides a compound having an affinity for an antibody and a bioorthogonal functional group represented by the formula (I) or a salt thereof.
A-LEB (I)
[In the formula,
　A is an affinity substance for an antibody,
　L is a divalent group containing a elimination group, and
　E is (i) the elimination group. (Ii) is a divalent group containing an electrophilic group capable of reacting with a nucleophilic group in the antibody,
　B is a bioorthogonal functional group, and the
　elimination group is Has the ability to be cleaved and desorbed from E by the reaction between the nucleophilic group and the electron nucleophilic group. ]
[0015]
　In the notations such as formula (I) and other formulas presented in connection with the present invention,-(hyphen) indicates that two units existing on both sides thereof are covalently bonded. Therefore, in formula (I), A is covalently bonded to L, L is covalently bonded to A and E, E is covalently bonded to L and B, and B is covalently bonded to E. Covalently bonded. The antibody-affinitive substance and the compound having a bioorthogonal functional group or a salt thereof represented by the formula (I) are structural units in which the antibody-affinitive substance (A) has LEB. It is shown to be included via a covalent bond between A and L. Therefore, in the formula (I), the affinity substance (A) for the antibody is one structural unit having LEB, or a plurality of substances (for example, 2 to 5, preferably 2) having LEB. It may have up to 4, more preferably 2 or 3 structural units (same or different).
　Also in other formulas, it is shown that the affinity substance (A) or antibody (Ab) for an antibody contains a specific structural unit (structural unit excluding A or Ab) in the formula via a covalent bond. Therefore, also in other formulas, the affinity substance (A) or antibody (Ab) for the antibody is one specific structural unit or a plurality (for example, 2 to 5, preferably 2 to 4, more preferably 2). Or 3) specific structural units (same or different).
[0016]
1-2. Affinity substance for antibody (A) In
　formula (I), A is an affinity substance for antibody. An affinity substance for an antibody is a substance having a non-covalent binding ability to the antibody.
[0017]
　The affinity substances used in the present invention target antibodies. The antibody may be an antibody modified with a biomolecule (eg, sugar) or an antibody not modified with a biomolecule. As the antibody, any antibody against any component such as a biological component, a virus-derived component, and a component found in the environment can be used, but an antibody against a biological component or a virus-derived component is preferable. Examples of biological components include components derived from animals such as mammals and birds (eg, chickens), insects, microorganisms, plants, fungi, and fish (eg, proteins). Preferably, the biological component is a mammalian-derived component. Mammals include, for example, primates (eg humans, monkeys, chimpanzees), rodents (eg mice, rats, guinea pigs, hamsters, rabbits), pets (eg dogs, cats), domestic animals (eg dogs, cats) Examples include cows, pigs, goats) and working animals (eg horses, sheep). The biological component is more preferably a primate or rodent-derived component (eg, protein), and even more preferably a human-derived component (eg, protein) from the viewpoint of clinical application of the present invention. is there. Examples of the virus-derived component include components derived from influenza virus (eg, tri-influenza virus, swine flu virus), AIDS virus, Ebola virus, and phage virus (eg, protein).
[0018]
　The antibody is also a polyclonal antibody or a monoclonal antibody, preferably a monoclonal antibody. As the monoclonal antibody, for example, it is modified to have a sugar chain binding consensus sequence such as a chimeric antibody, a humanized antibody, a human antibody, and an antibody to which a predetermined sugar chain is added (eg, an N-type sugar chain binding consensus sequence). Antibodies), bispecific antibodies, scFv antibodies, Fab antibodies, F (ab') 2 antibodies, VHH antibodies, Fc region proteins, Fc fusion proteins. The antibody may also be a divalent antibody (eg, IgG, IgD, IgE) or a tetravalent or higher valent antibody (eg, IgA antibody, IgM antibody).
[0019]
　The antibody that is the target of the affinity substance may also be composed of any amino acid residue, but is preferably composed of 20 natural L-α-amino acid residues that normally constitute a protein. Examples of such amino acid residues include L-alanine (A), L-asparagine (N), L-cysteine ​​(C), L-glutamine (Q), L-isoleucine (I), and L-leucine ( L), L-methionine (M), L-phenylalanine (F), L-proline (P), L-serine (S), L-threonine (T), L-tryptophane (W), L-tyrosine (Y) ), L-valine (V), L-aspartic acid (D), L-glutamic acid (E), L-arginine (R), L-histidine (H), or L-lysine (K), and glycine (G). ) (Hereinafter, the notation of L is omitted). The antibody may be composed of, for example, 100 or more, preferably 120 or more, more preferably 150 or more, even more preferably 180 or more, and particularly preferably 200 or more amino acid residues. The number of antibodies may also be, for example, 1000 or less, preferably 900 or less, more preferably 800 or less, even more preferably 700 or less, and particularly preferably 600 or less. More specifically, the antibody has, for example, 100 to 1000 amino acids, preferably 120 to 900, more preferably 150 to 800, even more preferably 180 to 700 or more, and particularly preferably 200 to 600 amino acid residues. It may be composed of groups. When the antibody is an antibody (eg, a monoclonal antibody as described above), the above number of amino acid residues may correspond to the amino acid residues of the heavy chain of the antibody.
[0020]
　The antibody that is the target of the affinity substance further comprises one specific amino acid residue having a side chain or terminal (N-terminal and / or C-terminal), preferably a side chain, with which a bioorthogonal functional group can react, as described below. A protein contained at one or more positions (preferably multiple positions). Examples of such a specific amino acid residue include amino acid residues as described later, but preferably consist of a lysine residue, a tyrosine residue, a serine residue, a threonine residue, and a cysteine ​​residue. Amino acid residues selected from the group. Considering that the compound of the present invention can regioselectively modify an antibody, an antibody containing such a specific amino acid residue at a plurality of positions is preferable. The plurality of positions are not particularly limited as long as they are two or more positions, but for example, three or more positions, preferably five or more positions, more preferably ten or more positions, and even more preferably 20 or more positions, particularly preferable. May be in more than 30 positions. The plurality of positions may also be, for example, 200 or less positions, preferably 180 or less positions, more preferably 150 or less positions, even more preferably 120 or less positions, and particularly preferably 100 or less positions. More specifically, the plurality of positions are, for example, 3 to 200 positions, preferably 5 to 180 positions, more preferably 10 to 150 positions, even more preferably 20 to 120 positions, and particularly preferably 30 to. It may be in the 100 position. Even in an antibody containing such a specific amino acid residue at a plurality of positions, the compound of the present invention regioselectively modifies one or two specific amino acid residues existing in a specific region. Can be done. For example, the number of lysine residues in human IgG1 is generally said to be about 70 to 90, although it depends on the amino acid composition in the variable region. The present invention has succeeded in regioselectively modifying one or two lysine residues present in such a specific region of human IgG1.
[0021]
　More specifically, the present invention modifies amino acid residues present at specific target sites in an antibody while preserving antibody function (ie, maintaining native folding without denaturing the antibody). From this point of view, position-selective modification of amino acid residues exposed on the surface of the antibody is preferable. For example, in human IgG such as human IgG1, the exposed lysine residue and the exposed tyrosine residue are present at the following positions (according to EU numbering; http: //www.imgt.org/IMGTScientificChart / Numbering / Hu_IGHGnber.html. reference).
(1) Exposed lysine residue
CH2 domain (246th, 248th, 274th, 288th, 290th, 317th, 320th, 322nd, 338th)
CH3 domain (360th, 414th, 439th)
( 2) Exposed tyrosine residue
CH2 domain (278th, 296th, 300th)
CH3 domain (436th)
(3) Exposed serine residue
CH2 domain (254th, 267th, 298th, 324th)
CH3 domain (375 ) Position, 400, 415, 440, 442)
(4) Exposed threonine residue
CH2 domain (256, 289, 307)
CH3 domain (335, 359, 393, 437)
Therefore, when human IgG such as human IgG1 is modified with a lysine residue or a tyrosine residue, the modification at the above position is preferable.
[0022]
　Preferably, when a human IgG such as human IgG1 is modified with a lysine residue, a tyrosine residue, a serine residue or a threonine residue, the exposure to the surface among the positions (1) to (4) above is preferable. The lysine residue, tyrosine residue, serine residue or threonine residue present at the following positions where the value is high may be modified.
(1') Exposed lysine residue
CH2 domain (246th, 248th, 274th, 288th, 290th, 317th, 320th, 322nd)
CH3 domain (360th, 414th, 439th)
(2' ) ) Exposed tyrosine residue
CH2 domain (278th, 296th, 300th)
CH3 domain (436th)
(3') Exposed serine residue
CH2 domain (254th, 267th, 298th)
CH3 domain (400th, 415) Position 440)
(4') Exposed threonine residue
CH2 domain (256th position, 289th position)
CH3 domain (335th position, 359th position)
Therefore, human IgG such as human IgG1 has lysine residue, tyrosine residue, and serine residue. When modified with a group or threonine residue, modification at the above positions is more preferred.
[0023]
　More preferably, when human IgG such as human IgG1 is modified with a lysine residue, a predetermined position (eg,) in the CH2 domain that can be efficiently modified in the present invention among the positions in (1) above. The lysine residues present at positions 246, 248, 288, 290, and 317) may be modified.
[0024]
　In certain embodiments, an antibody that is the target of an affinity substance is specified in a target region consisting of 1 to 50 consecutive amino acid residues when it contains a particular amino acid residue at multiple positions as described above. It may contain one or more amino acid residues of the above, and may contain five or more specific amino acid residues in a non-target region other than the target region. The target region is preferably 1 to 30, more preferably 1 to 20, even more preferably 1 to 10, 1 to 5, or 1 to 3 (ie, 1, 2, or 3). ) May consist of amino acid residues. Particularly preferably, the target region may be a region consisting of a specific amino acid residue present at a specific position. Such a specific position varies depending on the type of target protein and affinity substance, and is, for example, a specific position in a specific region (eg, CH1, CH2, CH3) in the constant region of an antibody. It may also be preferably the position in CH2 of the antibody. More specifically, the target region may be the following residues according to
Eunumbering in human IgG Fc: (1) Lys248 residues (hereinafter, also simply referred to as "Lys248" herein, human IgG CH2. The 16th residue of the human IgG CH2 region (SEQ ID NO: 1), which corresponds to the 18th residue of the region (SEQ ID NO: 1) or the Lys246 residue (hereinafter, also simply referred to as "Lys246" in the present specification).
( Corresponding to): (2) Lys288 residue (hereinafter, also simply referred to as "Lys288" in the present specification, corresponding to the 58th residue of the human IgG CH2 region (SEQ ID NO: 1)) or Lys290 residue (corresponding to) Hereinafter, it is also simply referred to as “Lys290” in the present specification, and corresponds to the 60th residue of the human IgG CH2 region (SEQ ID NO: 1));
(3) Lys317 residue (hereinafter, also simply referred to as “Lys317” in the present specification, corresponds to the 87th residue of the human IgG CH2 region (SEQ ID NO: 1)).
[0025]
　According to the present invention, a specific amino acid residue in the target region can be modified highly regioselectively. Such regioselectivity is, for example, 30% or more, preferably 40% or more, more preferably 50% or more, even more preferably 60% or more, particularly preferably 70% or more, 80% or more, 90% or more, 95. It may be% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.
[0026]
　The target region also has a specific amino acid residue present at a specific position with respect to the N-terminal side and the C-terminal side, respectively, centered on the specific position (where a is an arbitrary 1 to 10). In the region up to the remote position of the amino acid residue (which is an integer of), the amino acid residue of the same type as the specific amino acid residue is not contained other than the specific amino acid residue existing at the specific position. May be good. a is preferably an integer of 1 to 5, more preferably an integer of 1 to 3, even more preferably 1 or 2, and particularly preferably 1.
[0027]
　In a preferred embodiment, the antibody is a monoclonal antibody. Examples of antibody isotypes such as monoclonal antibodies include IgG (eg, IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, and IgY. The monoclonal antibody is a full-length antibody or an antibody fragment (eg, F (ab') 2 , Fab', Fab, Fv, single-chain antibody), but a full-length antibody is preferable. Particularly preferably, the antibody is a human antibody, a humanized antibody, or a chimeric antibody having human IgG (eg, IgG1, IgG2, IgG3, IgG4) in a constant region.
[0028]
　Antibodies are antibodies against any antigen. For example, such an antigen may be a component found in an organism or virus as described above. Such antigens also include, for example, proteins [oligopeptides, polypeptides. Proteins modified with biomolecules such as sugars (eg, glycoproteins)], sugar chains, nucleic acids, low molecular weight compounds can be mentioned.
[0029]
　Preferably, the antibody may be an antibody that uses a protein as an antigen. Examples of proteins include cell membrane receptors, cell membrane proteins other than cell membrane receptors (eg, extracellular substrate proteins), ligands, and soluble receptors.
[0030]
　More specifically, the protein that is the antigen of the antibody may be a disease target protein. Examples of disease target proteins include the following.
[0031]
(1) Cancer area
　PD-L1, GD2, PDGFRα (Plotlet-derived growth factor receptor), CD22, HER2, phosphatidylserine (PS), EpCAM, fibronectin, PD-1, VEGFR-2, CD33, HGF, gpNMB, CD27, DEC-205, Folic acid receptor, CD37, CD19, Trop2, CEACAM5, S1P, HER3, IGF-1R, DLL4, TNT-1 / B, CPAAs, PSMA, CD20, CD105 (endoglin), ICAM-1, CD30, CD16A, CD38, MUC1, EGFR, KIR2DL1,2, NKG2A, tenascin-C, IGF (Insulin-like growth factor), CTLA-4, meshothelin, CD138, c-Met, Ang2, VEGF-A, CD79b, ENPD3, folic acid receptor , TEM-1, GM2, Gripican 3, macrophage inhibitory factor, CD74, Notch1, Notch2, Notch3, CD37, TLR-2, CD3, CSF-1R, FGFR2b, HLA-DR, GM-CSF, EphA3, B7- CD123, gpA33, Frizzled7 receptor, DLL4, VEGF, RSPO, LIV-1, SLITRK6, Nectin-4, CD70, CD40, CD19, SEMA4D (CD100), CD25, MET, Tissue Factor, IL-8, EGFR, cMet, KIR3DL2, Bst1 (CD157), P-cadherin, CEA, GITR, TAM (tumor associated macrophage), CEA, DLL4, Ang2, CD73, FGFR2, CXCR4, LAG-3, GITR, FucosylG CSF-1R, FGFR3, OX40, BCMA, ErbB3, CD137 (4-1BB), PTK7, EFNA4, FAP, DR5, CEA, Ly6E, CA6, CEACAM5, LAMP1, thisset factor,EPHA2, DR5, B7-H3, FGFR4, FGFR2, α2-PI, A33, GDF15, CAIX, CD166, ROR1, GITR, BCMA, TBA, LAG-3, EphA2, TIM-3, CD-200, EGFRvIII, CD16A, CD32B, PIGF, Axl, MICA / B, Thomasen-Friendenrich, CD39, CD37, CD73, CLEC12A, Lgr3, transferrin receptor, TGFβ, IL-17, 5T4, RTK, Immunosuppressor Protein, NaPi2b, Lewis blood group A34, Lysil-Oxidase, DLK-1, TROP-2, α9 integrin, TAG-72 (CA72-4), CD70
[0032]
(2) Autoimmune diseases / inflammatory diseases
　IL-17, IL-6R, IL-17R, INF-α, IL-5R, IL-13, IL-23, IL-6, ActRIIB, β7-Integrin, IL- 4αR, HAS, Eotaxin-1, CD3, CD19, TNF-α, IL-15, CD3ε, Fibronectin, IL-1β, IL-1α, IL-17, TSLP (Thematic Structural Lymphopoietin), LAMP (Alpha4 Integrin) , IL-23, GM-CSFR, TSLP, CD28, CD40, TLR-3, BAFF-R, MAdCAM, IL-31R, IL-33, CD74, CD32B, CD79B, IgE (immunoglobulin E), IL-17A, IL-17F, C5, FcRn, CD28, TLR4, MCAM, B7RP1, CXCR1, Litens, IL-21, Cadherin-11, CX3CL1, CCL20, IL-36R, IL-10R, CD86, TNF-α, IL-7R , Kv1.3, α9 integrin, LIFEHT
[0033]
(3) Neurological diseases
　CGRP, CD20, β-amyloid, β-amyloid protofibrin, Calcitonin Gene-Related Peptide Receptor, LINGO (Ig Domain Contining1), α-synuclein, extracellular tau, CD52, insulin receptor Tau, CD52, insulin receptor t , SOD1, TauC3, JC virus
[0034]
(4) Infectious diseases
　Clostridium difficile toxin B, cytomegalovirus, RS virus, LPS, S. d. Aureus Alpha-toxin, M2e protein, Psl, PcrV, S. a. Aureus toxin, Influenza A, Alginate, Staphylococcus aureus, PD-L1, Influenza B, Acinetobacter, F-protein, Env, CD3, Pathogenic Escherichia coli, Klebsiella, Streptococcus pneumoniae
[0035]
(5) Hereditary / rare diseases
　Amyloid AL, SEMA4D (CD100), insulin receptor, ANGPTL3, IL4, IL13, FGF23, adrenocorticotropic hormone, transthyretin, huntingtin
[0036]
(6) Eye diseases
　Factor D, IGF-1R, PGDFR, Ang2, VEGF-A, CD-105 (Endoglin), IGF-1R, β-amyloid
[0037]
(7) Bone and Orthopedic Surgery Area
Sclerostin, Myostatin, Dickkopf-1, GDF8, RNAKL, HAS, SIGLEC-15
[0038]
(8) Blood diseases
　vWF, Factor IXa, Factor X, IFNγ, C5, BMP-6, Ferroportin, TFPI
[0039]
(9) Other diseases
　BAFF (B cell activating factor), IL-1β, PCSK9, NGF, CD45, TLR-2, GLP-1, TNFR1, C5, CD40, LPA, prolactin receptor, VEGFR-1, CB1, Endoglin, PTH1R, CXCL1, CXCL8, IL-1β, AT2-R, IAPP
[0040]
　In a more preferred embodiment, the affinity for the antibody is an affinity for the monoclonal antibody. The isotype of the monoclonal antibody is similar to that described above for the antibody, but IgG (eg, IgG1, IgG2, IgG3, IgG4) is preferred. Preferably, the monoclonal antibody is a full-length monoclonal antibody.
[0041]
　In an even more preferred embodiment, the affinity for the antibody is a chimeric antibody, a humanized antibody, or an affinity for a human antibody (eg, IgG such as IgG1, IgG2, IgG3, IgG4), which is a full-length monoclonal antibody.
[0042]
　In a particularly preferred embodiment, the amino acid-affinitive substance is an amino acid-affinitive substance containing any one Fc region protein selected from the group consisting of the following (A) to (C) and having an antigen-binding ability. :
(A) Fc region protein containing the amino acid sequence of SEQ ID NO : 1;
(B) Contains an amino acid sequence in which one or several amino acid residues are inserted, added, deleted or substituted in the amino acid sequence of SEQ ID NO: 1. Fc region protein; or
(C) an Fc region protein containing an amino acid sequence showing 90% or more identity with the amino acid sequence of SEQ ID NO: 1.
[0043]
　The amino acid sequence of SEQ ID NO: 1 is an Fc region protein. Such Fc region proteins are known to have secretory capacity. Therefore, the Fc region proteins (A) to (C) can have a secretory capacity. In addition, an antibody containing such an Fc region protein can have an antigen-binding ability. The amino acid residue at position 18 in SEQ ID NO: 1 is an arbitrary amino acid residue, but is preferably a neutral amino acid residue, and more preferably an amino acid residue having a non-polar side chain as described later. Even more preferably, it is leucine, isoleucine or alanine, and particularly preferably leucine or alanine. The amino acid residue at position 19 in SEQ ID NO: 1 is an arbitrary amino acid residue, preferably a neutral amino acid residue or an acidic amino acid residue, and more preferably an amino acid residue or an acidic amino acid having a non-polar side chain. It is an amino acid residue, and even more preferably leucine or glutamic acid. The amino acid residue at position 21 in SEQ ID NO: 1 is an arbitrary amino acid residue, but is preferably a neutral amino acid residue, more preferably an amino acid residue having a non-polar side chain, and even more preferably. Glycine or alanine. The amino acid residue at position 140 in SEQ ID NO: 1 is an arbitrary amino acid residue, but is preferably an acidic amino acid residue, and more preferably glutamic acid or aspartic acid. The amino acid residue at position 142 in SEQ ID NO: 1 is an arbitrary amino acid residue, but is preferably a neutral amino acid residue, more preferably an amino acid residue having a non-polar side chain, and even more preferably. It is methionine, leucine or isoleucine, and particularly preferably methionine or leucine. The amino acid residue at position 177 in SEQ ID NO: 1 is an arbitrary amino acid residue, but is preferably a neutral amino acid residue, and more preferably an amino acid residue having an uncharged polar side chain as described later. Amino acid residues with non-polar side chains, even more preferably threonine, alanine or glycine
[0044]
　In a preferred embodiment, the amino acid sequence of SEQ ID NO: 1 may be an amino acid sequence consisting of amino acid residues at positions 220 to 449 in the amino acid sequence of SEQ ID NO: 2.
[0045]
　In another preferred embodiment, the amino acid sequence of SEQ ID NO: 1 may be an amino acid sequence consisting of amino acid residues at positions 7 to 236 in the amino acid sequence of SEQ ID NO: 3.
[0046]
　In certain embodiments, the antibody comprising an Fc region protein comprising an amino acid sequence as described above may be an Fc region protein comprising an amino acid sequence as described above and an antibody comprising a constant region of the antibody. The constant region of such an antibody may be a constant region of a chimeric antibody, a humanized antibody, or a human antibody (eg, IgG such as IgG1, IgG2, IgG3, IgG4).
[0047]
　In the Fc region protein (B), one or several amino acid residues are modified by 1, 2, 3 or 4 mutations selected from the group consisting of deletion, substitution, addition and insertion of amino acid residues. obtain. Mutations in amino acid residues may be introduced into one region of the amino acid sequence, or may be introduced into a plurality of different regions. The term "one or several" refers to a number that does not significantly impair the activity of the protein. The number indicated by the term "1 or several" is, for example, 1 to 100, preferably 1 to 80, more preferably 1 to 50, 1 to 30, 1 to 20, 1 to 10 or 1 to 5 (eg, 1, 2, 3, 4, or 5).
[0048]
　In the Fc region protein (C), the% identity with the amino acid sequence of SEQ ID NO: 1 is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93. % Or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. In the present invention, the calculation of% identity of a peptide or polypeptide (protein) can be performed by the algorithm blastp. More specifically, the calculation% of the identity of the polypeptide is determined by the default setting of Scoring Parameters (Matrix: BLOSUM62; Gap Costs: Existence) in the algorithm blastp provided in the National Center for Biotechnology Information (NCBI). = 1; Composional Adjustments: Conditional Compostional score matrix adaptation) can be used. Moreover, the calculation of the identity% of a polynucleotide (gene) can be performed by the algorithm blastn. More specifically, the calculation of the percent identity of a polynucleotide is performed using the default setting of Scoring Parameters (Match / Mismatch Scores = 1, -2; Gap Costs = Linear) in the algorithm blastn provided by NCBI. It can be carried out.
[0049]
　Secretory in secretory capacity is synonymous with secretory protein secretion (so-called soluble). Therefore, "having a secretory capacity" means functioning as an antibody in the same manner as a normal antibody.
[0050]
　An antibody containing the Fc region protein may have a mutation introduced at a specific site as long as it retains the desired properties (eg, secretory ability, antigen-binding ability). The positions of amino acid residues into which mutations may be introduced that can retain the properties of interest are apparent to those of skill in the art. Specifically, those skilled in the art will 1) compare the amino acid sequences of multiple proteins with similar properties and 2) identify relatively conserved and relatively unconserved regions. Then, 3) from the relatively conserved area and the relatively unconserved area, it is possible to predict the area that can play an important role in the function and the area that cannot play an important role in the function, respectively. Can recognize the correlation between structure and function. Therefore, those skilled in the art can identify the position of an amino acid residue in which a mutation may be introduced in the amino acid sequence of the antibody containing the Fc region protein.

The scope of the claims
[Claim 1]
　The following formula (I):
A-LEB (I)
[In the formula,
　A is an affinity substance for an antibody,
　L is a divalent group containing a elimination group, and
　E is ( i) A divalent group containing an electrophilic group linked to the desorbing group and (ii) capable of reacting with the nucleophilic group in the antibody, where
　B is a bioorthogonal functional group.
　Yes , the desorbing group has the ability to be cleaved from E and desorbed by the reaction between the nucleophilic group and the electrophoryl group. ], A compound having an affinity for an antibody and a bioorthogonal functional group, or a salt thereof.
[Claim 2]
　The compound according to claim 1, or a salt thereof, wherein the affinity substance is a peptide.
[Claim 3]
　The compound according to claim 2, or a salt thereof, wherein the peptide is a peptide having an ability to bind to a constant region of a monoclonal antibody.
[Claim 4]
　The compound according to claim 2 or 3, or a salt thereof, wherein the peptide is a peptide having an ability to bind to the Fc region of a monoclonal antibody.
[Claim 5]
　The compound according to claim 4 or a salt thereof, wherein the peptide is a peptide having an ability to bind to the Fc region of IgG.
[Claim 6]
　The compound according to any one of claims 2 to 5, or a salt thereof, wherein the peptide has 10 to 40 amino acid residues.
[Claim 7]
　The peptide is
any of the amino acid sequences of (a) (a-1-1) FNMQQQRRRFYEALHDPNLNEEQRNARIRSIRDD (SEQ ID NO: 11) or
(a-1-2) FNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDDC (SEQ ID NO: 12)
　. One to three amino acid residues may be the same or different and are replaced by one amino acid residue each selected from the group consisting of lysine residues, aspartic acid residues, and glutamate residues
(a-). 2-1) In the amino acid sequence of β-Ala-NMQQQRRFYEALHDPNLNEEQRNARIRSIRDD (SEQ ID NO: 13) or the amino acid sequence of
(a-2-2) β-Ala-NMQCQRRFYEALHDPNLNEEQRNARIRSIRDDDC (SEQ ID NO: 14)
　, any one of 1 to 2 in the sequence. The three amino acid residues may be the same or different, each being replaced by one amino acid residue selected from the group consisting of a lysine residue, an aspartic acid residue, and a glutamate residue, and
(b) sequence. The compound according to any one of claims 2 to 6, or a salt thereof, which comprises an amino acid sequence having 85% or more identity with respect to each of the amino acid sequences of Nos. 11 to 14.
[Claim 8]
　The peptide is represented by the
formula 1-1: 1 (X 0-3 ) a- C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-I-IWC- (X 0-3 ) b (SEQ ID NO: 15 ). )
Equation 1-2: (X 0-3 ) a- C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-IVW-C- (X 0-3 ) b (SEQ ID NO: 16)
Equation 1 -3: (X 0-3 ) a- C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-VVW-C- (X 0-3 ) b (SEQ ID NO
: 17) Equation 1-4: (X 0-3 ) a- C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-AVW-C- (X 0-3 ) b (SEQ ID NO: 18)
expression 1-5: (X 0-3 ) a -C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa @ 5-Xaa @ 6-L-L-W-C-(X 0-3 ) b (SEQ No. 19)
Equation 1-6: (X 0-3 ) a- C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L-IWC- (X 0-3 ) b (SEQ ID NO: 20)
Formula 1-7: (X 0-3 ) a- C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-L-VFC- (X 0-3 ) b (SEQ ID NO: 21)
Formula 1- 8: (X 0-3 ) a- C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Q-V-WC- (X 0-3)) B (SEQ ID NO
: 22) Equation 1-9: (X 0-3 ) a- C-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-EVW-C- (X 0-3 ) b ( SEQ ID NO: 23)
[In the formula,
　(X 0-3 ) a is none, arginine residue-glycine residue-asparagin residue, aspartic acid residue, glycine residue-asparagin residue, or asparagine residue. ,
　(X 0-3 ) b is none, threonine residue-tyrosine residue-histidine residue, or threonine residue
　,
　Xaa1 is an alanine residue, and Xaa2 is a tyrosine residue, tryptophan residue. , Or histidine residue,
　Xaa3 is histidine residue, phenylalanine residue, tyrosine residue, tryptophan residue, arginine residue, or glycine residue, and
　Xaa4 is lysine residue, aspartic acid residue, Or a glutamate residue,
　Xaa5 contains glycine residue, serine residue, asparagine residue, glutamine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, tyrosine residue, tryptophan residue, histidine residue, threonine residue, leucine residue. A group, alanine residue, valine residue, isoleucine residue, or arginine residue, and
　Xaa6 is a glutamine residue, glutamic acid residue, asparagine residue, or aspartic acid residue. ], Or
formula 2-1: (X 0-3 ') a- C- (Xaa1')-(Xaa2')-(Xaa3')-(Xaa4')-(Xaa5')-(Xaa6')-L -VWC- (X 0-3 ') b (SEQ ID NO: 24)
[In the formula,
　(X 0-3 ') a and (X 0-3 ') b are the above-mentioned (X 0-3 ', respectively. ) Same as a and (X 0-3 ) b ,
　Xaa1', Xaa2', Xaa3', Xaa4', Xaa5', and Xaa6'are the same as Xaa1, Xaa2, Xaa3, Xaa4, Xaa5, and Xaa6, respectively. ], The compound according to any one of claims 2 to 6, or a salt thereof, which comprises the amino acid sequence of any one of claims.
[Claim 9]
　The leaving group is (1) -O-, -S-, -Se-, -SO 2- O-, -SO 2- N (R)-, -SO 2- , -C≡C-CH 2 A group selected from the group consisting of -O-, -N (OR)-, -N (R)-, and -ON (R)-(where R is a hydrogen atom or 1 to 6 carbon atoms. The compound according to any one of claims 1 to 8, or a salt thereof, which is an alkyl (is) or (2) heteroarylene.
[Claim 10]
　The nucleophilic groups are NH 2 in the side chain of the lysine residue, OH in the side chain of the tyrosine residue, OH in the side chain of the serine residue, OH in the side chain of the threonine residue, and the side chain of the cysteine ​​residue. The compound according to any one of claims 1 to 9, or a salt thereof, which is a group selected from the group consisting of SH in.
[Claim 11]
The compound according to any one of claims 1 to 10, or a salt thereof 　, wherein the electrophilic group is a group selected from the group consisting of -C (= O)-, -SO 2- , and -CH 2-. ..
[Claim 12]
　The bioorthogonal functional groups are azide residues, aldehyde residues, thiol residues, alkin residues, alkene residues, halogen residues, tetrazine residues, nitron residues, hydroxylamine residues, nitrile residues, and hydrazine. Residues, ketone residues, boronic acid residues, cyanobenzothiazole residues, allyl residues, phosphine residues, maleimide residues, disulfide residues, thioester residues, α-halocarbonyl residues, isonitrile residues, sidonone The compound according to any one of claims 1 to 11, or a salt thereof, which is a group selected from the group consisting of residues and selenium residues.
[Claim 13]
　The invention according to any one of claims 1 to 12, wherein the bioorthogonal functional group is a group selected from the group consisting of an azide residue, a thiol residue, an alkyne residue, a maleimide residue, and a disulfide residue. A compound or a salt thereof.
[Claim 14]
　The compound represented by the formula (I) is the following formula (I-1):
A-L 1- L 2- E 1- E 2- E 3- B (I-1)
[
　A and B in the formula. Is the same as that of the formula (I),
　L 1 is a binding or divalent group,
　L 2 is a leaving group, and
　E 1 is (i) linked to the leaving group. And (ii) an electrophilic group capable of reacting with the nucleophilic group in the antibody,
　E 2 is (a) -XY- [where X that binds to E 1 is , C (R 1 ) (R 2 ) (where R 1 and R 2)Are each independently a hydrogen atom or an alkyl having 1 to 6 carbon atoms. ), N (R 3 ) (where R 3 is a hydrogen atom or an alkyl having 1 to 6 carbon atoms), O, S, or Se, and Y bonded to E 3 is C ( R 4 ) (R 5 ) (where R 4 and R 5 are independently alkyl hydrogen atoms or 1 to 6 carbon atoms). ], Or (b) the following formula (i):
[Chemical formula 1]

(Here, the ring Z is a divalent atom in which all of the ring-constituting atoms X'bonding to E 1 and the ring-constituting atoms on both sides thereof are carbon atoms. Is a divalent heterocyclic group in which the ring-constituting atom X'bonded to E 1 is a nitrogen atom and the ring-constituting atoms on both sides of the nitrogen atom are carbon atoms. It is a group represented by (hand),
　E 3 is a divalent group when E 2 is −XY—, and E 2 is a group represented by the formula (i). Is a binding or divalent group,
　The leaving group, E by the reaction between the electrophilic group and the nucleophilic group 1 has the ability to desorption is disconnected from. ], Which is the compound according to any one of claims 1 to 13, or a salt thereof.
[Claim 15]
　The L 2 is
(a) ring PQ- [where ring P is an arylene which may be substituted with an electron-withdrawing group, a heteroarylene which may be substituted with an electron-withdrawing group, or a condensed ring. 2,5-Diketopyrrolidine, which may be fused, 2,6-diketopiperidine which may be fused, 2-ketopyrrolidine which may be fused, 2-keto which may be fused. It is a group selected from the group consisting of piperidin and 2-pyridone, and Q is -O-, -S-, -Se-, -SO 2- O-, -SO 2- N (R)-, -SO 2. A group selected from the group consisting of-, -C≡C-CH 2- O-, -N (OR)-, -N (R)-, and -ON (R)-(where R is It is an alkyl having a hydrogen atom or a carbon atom number of 1 to 6). ],
(B) Heteroarylene, or
(c) -Q- [Here, Q is -O-, -S-, -Se-, -SO 2- O-, -SO 2- N (R)- , -SO 2- , -C≡C-CH 2- O-, -N (OR)-, -N (R)-, and -ON (R)-, a group selected from the group (here). , R is a hydrogen atom or an alkyl having 1 to 6 carbon atoms.). ], The compound according to claim 14, or a salt thereof.
[Claim 16]
　The compound according to claim 14 or 15, or a salt thereof, wherein L 2 is a group selected from the group consisting of the following structural formulas:
[Chemical formula 2]

(where EWG is an electron-withdrawing group and
　m is is an integer of 0 ~ 4,
　n is an integer of 0 ~ 3,
　R is a hydrogen atom or an alkyl carbon atoms 1 ~ 6,,
　○ (white circle) is L 1 in bond for Yes, ● (black circle) is, E 1 is a bond against.).
[Claim 17]
The compound according to any one of claims 1 to 16, or a salt thereof, wherein the main chain of 　L or L 1 to L 2 connecting A and E is composed of 20 or less atoms.
[Claim 18]
　The compound represented by the formula (I-1) is the following formula (I-2):
A-L 1- L 2- E 1- XYE 3- B (I-2)
[in the formula,
　A, L 1 , X, Y, and B are the same as those of the above formula (I-1), and
　L 2 is
(a) ring PQ- [where ring P is electron-withdrawing. Allylenes may be substituted with a group, heteroarylenes may be substituted with an electron-withdrawing group, 2,5-diketopyrrolidine may be fused, 2,6-may be fused. It is a group selected from the group consisting of diketopiperidine, 2-ketopyrrolidine which may be fused, 2-ketopyperidine which may be fused, and 2-pyridone, and Q is -O-, -S. -, -Se-, -SO 2- O-, -SO 2- N (R)-, -SO 2- , -C≡C-CH 2- O-, -N (OR)-, -N (R) )-, A group selected from the group consisting of -ON (R)-(where R is a hydrogen atom or an alkyl having 1 to 6 carbon atoms). ],
(B) Heteroarylene, or
(c) -Q- [Here, Q is -O-, -S-, -Se-, -SO 2- O-, -SO 2- N (R)-,- A group selected from the group consisting of SO 2- , -C≡C-CH 2- O-, -N (OR)-, -N (R)-, and -ON (R)-(here, R). Is a hydrogen atom or an alkyl having 1 to 6 carbon atoms.). ],
　E 1 is a group selected from the group consisting of -C (= O)-, -SO 2- , and -CH 2- , and
　E 3 is a divalent group. ], Which is the compound according to any one of claims 14 to 17, or a salt thereof.
[Claim 19]
　The compound represented by the formula (I-1) is the following formula (I-3):
[Chemical

formula 3] [In the formula,
　A, L 1 , ring Z, and B are of the formula (I-1).
　L 2 is the same as that of
(a) ring PQ- [where ring P may be substituted with an electron-withdrawing group, an arylene, or an electron-withdrawing group. Heteroarylene, may be fused 2,5-diketopyrrolidine, may be fused 2,6-diketopiperidine, may be fused 2-ketopyrrolidine, fused It is a group selected from the group consisting of 2-ketopiperidine and 2-pyridone, and Q is -O-, -S-, -Se-, -SO 2- O-, -SO 2- N (R). A group selected from the group consisting of-, -SO 2- , -C≡C-CH 2 --O- , -N (OR)-, -N (R)-, and -ON (R)-(here). And R is an alkyl having a hydrogen atom or a carbon atom number of 1 to 6). ],
(B) Heteroarylene, or
(c) -Q- [Here, Q is -O-, -S-, -Se-, -SO 2- O-, -SO 2From the group consisting of -N (R)-, -SO 2- , -C≡C-CH 2- O-, -N (OR)-, -N (R)-, and -ON (R)- The group of choice (where R is a hydrogen atom or an alkyl having 1 to 6 carbon atoms). ],
　E 1 is a group selected from the group consisting of -C (= O)-, -SO 2- , and -CH 2- , and
　E 3 is a binding or divalent group. ], Which is the compound according to any one of claims 14 to 17, or a salt thereof.
[Claim 20]
　The following formula (I):
A-LEB (I)
[In the formula,
　A is an affinity substance for an antibody,
　L is a divalent group containing a elimination group, and
　E is ( i) A divalent group containing an electrophilic group linked to the desorbing group and (ii) capable of reacting with the nucleophilic group in the antibody, where
　B is a bioorthogonal functional group.
　Yes , the desorbing group has the ability to be cleaved from E and desorbed by the reaction between the nucleophilic group and the electrophoryl group. ], A reagent for regioselective modification of an antibody with a bioorthogonal functional group, which comprises a substance having an affinity for the antibody and a compound having a bioorthogonal functional group or a salt thereof.
[Claim 21]
　A method for producing an antibody having a bioorthogonal functional group or a salt thereof, wherein 　A is an affinity substance for the antibody and 　L is the following
　formula (I):
A-LEB (I)
[In the formula,
A is an affinity substance for the antibody.
Is a divalent group containing a elimination group, and
　E is an electrophilic group which is (i) linked to the elimination group and (ii) capable of reacting with a nucleophilic group in the antibody.
　Is a divalent group containing, B is a bioorthogonal functional group, and the
　desorbing group has the ability to be cleaved from E and desorbed by the reaction between the nucleophilic group and the electrophilic group. Has. ], A compound having an affinity for the antibody and a compound having a bioorthogonal functional group or a salt thereof is reacted with the antibody, and the following
　formula (II):
Ab-EB (II)
[in the formula,
　E and B are the same as those of the above formula (I), and
　Ab is an antibody. ], The method comprising producing an antibody having a bioorthogonal functional group or a salt thereof.
[Claim 22]
　A method for producing an antibody or a salt thereof having a functional substance, wherein
(1) the following formula (I):
A-LEB (I)
[In the formula,
　A is an affinity substance for the antibody, and
　L is a divalent group containing a desorbing group, and
　E is (i) an electrophilic group linked to the desorbing group and (ii) capable of reacting with a nucleophilic group in the antibody. It is a divalent group containing a group,
　B is a bioorthogonal functional group, and the
　desorbing group is cleaved from E and desorbed by a reaction between the nucleophilic group and the electrophilic group. Have the ability. ], A compound having an affinity for the antibody and a compound having a bioorthogonal functional group or a salt thereof is reacted with the antibody, and the following
　formula (II):
Ab-EB (II)
[in the formula,
　E and B are the same as those of the above formula (I), and
　Ab is an antibody. ] To produce an antibody or a salt thereof having a bioorthogonal functional group; and
(2) an antibody or a salt thereof having a bioorthogonal functional group represented by the above formula (II). By reacting with a functional substance via an orthogonal functional group, the following
　formula (III):
Ab-EB'-F (III)
[in the formula,
　Ab is the same as that of the formula (II),
　E is the same as that of the formula (I),
　and B'is generated by the reaction between the functional substance and the bioorthogonal functional group. It is a divalent group containing a portion to which
　F is a functional substance. ], The method comprising producing an antibody having a functional substance or a salt thereof.
[Claim 23]
　The following formula (II-1):
Ab-E 1- E 2- E 3- B (II-1)
[In the formula,
　Ab is an antibody, and
　E 1 is linked to the nucleophilic group in the antibody. a are electrophilic
　groups, E 2 is, (a) -X-Y- [wherein, E 1 X that binds the, C (R 1 ) (R 2 ) (wherein, R 1 and R 2 are Independently, they are alkyl hydrogen atoms or 1 to 6 carbon atoms.), N (R 3 ) (where R 3 is an alkyl hydrogen atom or 1 to 6 carbon atoms), O, S or a Se, E, 3 Y that binds the, C (R 4 ) (R 5 ) (wherein, R 4And R 5 are independently alkyl hydrogen atoms or 1 to 6 carbon atoms. ). ], Or (b) the following formula (i):
[Chemical formula 4]

(Here, the ring Z is a divalent atom in which all of the ring-constituting atoms X'bonding to E 1 and the ring-constituting atoms on both sides thereof are carbon atoms. Is a divalent heterocyclic group in which the ring-constituting atom X'bonded to E 1 is a nitrogen atom and the ring-constituting atoms on both sides of the nitrogen atom are carbon atoms. It is a group represented by (hand), and
　E 3 is a divalent group when E 2 is −XY—, and E 2 is a group represented by the formula (i). Is a bound or divalent group, and
　B is a bioorthogonal functional group. ], An antibody or a salt thereof having a bioorthogonal functional group regioselectively.
[Claim 24]
　The antibody according to claim 23 or a salt thereof, wherein the antibody is an antibody having a bioorthogonal functional group only in a constant region of a monoclonal antibody.
[Claim 25]
　The antibody according to claim 23 or 24 or a salt thereof, wherein the antibody is an antibody having a bioorthogonal functional group only in the Fc region of the monoclonal antibody.
[Claim 26]
　Any of claims 23-25, wherein the antibody is a human IgG having a bioorthogonal functional group regioselectively in a region consisting of amino acid residues at positions 246 to 248 or 288 to 290 in the human IgG Fc region. The antibody or salt thereof according to the above item.
[Claim 27]
　The antibody represented by the formula (II-1) is the following formula (II-2):
Ab-E 1 -XY-E 3- B (II-2)
[In the formula,
　Ab, X, Y , And B are the same as those of the above formula (II-1), and
　E 1 is a group selected from the group consisting of -C (= O)-, -SO 2- , and -CH 2- . ,
　E 3 is a divalent group. ], The antibody according to any one of claims 23 to 26 or a salt thereof, which is an antibody having a bioorthogonal functional group regioselectively.
[Claim 28]
　The antibody represented by the formula (II-1) is the following formula (II-3):
[Chemical

formula 5] [In the formula,
　Ab, ring-constituting atom X', ring Z, and B are the formula (II-3). -1) is the same,
　E 1 is a group selected from the group consisting of -C (= O)-, -SO 2- , and -CH 2- , and
　E 3 is a binding or divalent group. Is the basis of. ], The antibody according to any one of claims 23 to 26 or a salt thereof, which is an antibody having a bioorthogonal functional group regioselectively.
[Claim 29]
　The following formula (III-1):
Ab-E 1- E 2- E 3- B'-F (III-1)
[In the formula,
　Ab is an antibody, and
　E 1 is a nucleophile in the antibody.
　E 2 is a linked electrophilic group, and E 2 is (a) -XY- [where X bonded to E 1 is C (R 1 ) (R 2 ) (here, R 1 and R 2 is independently an alkyl having a hydrogen atom or a carbon atom number of 1 to 6), N (R 3 ) (where R 3 is an alkyl having a hydrogen atom or a carbon atom number of 1 to 6). ), O, S, or Se, and Y binding to E 3 is C (R 4 ) (R 5 ) (where R ). 4 and R 5 are independently alkyl hydrogen atoms or 1 to 6 carbon atoms. ). ], Or (b) the following formula (i):
[Chemical formula 6]

(Here, the ring Z is a divalent atom in which all of the ring-constituting atoms X'bonding to E 1 and the ring-constituting atoms on both sides thereof are carbon atoms. Is a divalent heterocyclic group in which the ring-constituting atom X'bonded to E 1 is a nitrogen atom and the ring-constituting atoms on both sides of the nitrogen atom are carbon atoms. It is a group represented by (hand), and
　E 3 is a divalent group when E 2 is −XY—, and E 2 is a group represented by the formula (i). Is a bound or divalent group, B'is a divalent group
　containing a moiety produced by the reaction between a functional substance and a bioorthogonal functional group, and
　F is a functional substance. .. ], An antibody or a salt thereof having a functional substance regioselectively.
[Claim 30]
　The antibody according to claim 29 or a salt thereof, wherein the antibody is an antibody having a bioorthogonal functional group only in a constant region of a monoclonal antibody.
[Claim 31]
　The antibody or a salt thereof according to claim 29 or 30, wherein the antibody is an antibody having a bioorthogonal functional group only in the Fc region of a monoclonal antibody.
[Claim 32]
　Any one of claims 29 to 31, wherein the antibody is a human IgG having a functional substance position-selectively in a region consisting of amino acid residues at positions 246 to 248 or 288 to 290 in the human IgG Fc region. The antibody or salt thereof according to the item.
[Claim 33]
　The antibody represented by the formula (III-1) is the following formula (III-2):
Ab-E 1 - XYE 3- B'-F (III-2)
[In the formula,
　Ab, X, Y, B'and F are the same as those of the above formula (III-1), and
　E 1 is from the group consisting of -C (= O)-, -SO 2- , and -CH 2-. a group selected,
　E 3 is represented by a is] divalent group is an antibody having a functional substance on the regioselective, the antibodies of any one of claims 29 to 32, or a salt.
[Claim 34]
　The antibody represented by the formula (III-1) is the following formula (III-3):
[Chemical

formula 7] [In the formula,
　Ab, ring-constituting atom X', ring Z, B'and F are the above-mentioned formulas. Same as that of (III-1),
　E 1 is a group selected from the group consisting of -C (= O)-, -SO 2- , and -CH 2- , and
　E 3 is a binding or It is a divalent group. ], The antibody or salt thereof according to any one of claims 29 to 32, which is an antibody having a functional substance regioselectively.
[Claim 35]
　Formula (IV):
A-LEF (IV)
[In the formula,
　A is an affinity substance for an antibody,
　L is a divalent group containing a leaving group, and
　E is ( i) A divalent group containing a nucleophilic group linked to the leaving group and (ii) capable of reacting with the nucleophilic group in the antibody, where
　F is a functional substance.
　The leaving group has the ability to be cleaved from E and desorbed by the reaction between the nucleophile and the electron-forming group. ], A compound having an affinity substance for an antibody and a functional substance, or a salt thereof.
[Claim 36]
　Formula (IV):
A-LEF (IV)
[In the formula,
　A is an affinity substance for an antibody,
　L is a divalent group containing a leaving group, and
　E is ( i) A divalent group containing a nucleophilic group linked to the leaving group and (ii) capable of reacting with the nucleophilic group in the antibody, where
　F is a functional substance.
　The leaving group has the ability to be cleaved from E and desorbed by the reaction between the nucleophile and the electron-forming group. ], A reagent for regioselective modification of an antibody by a functional substance, which comprises a substance having an affinity for the antibody and a compound having a functional substance or a salt thereof.
[Claim 37]
　A manufacturing method of an antibody or its salt with a functional material,
　the following formula
(IV): A-L-E-F (IV)
wherein,
　A is an affinity substance for the antibody,
　L is A divalent group containing a leaving group, where
　E comprises an electrophilic group that is (i) linked to the leaving group and (ii) capable of reacting with a nucleophile in the antibody. A divalent group,
　F is a functional substance, and the
　leaving group has the ability to be cleaved from E and desorbed by the reaction between the nucleophile and the electrophile. ], A compound having an affinity substance for the antibody and a functional substance or a salt thereof is reacted with the antibody, and the following
　formula (III):
Ab-EF (III)
[In the formula,
　Ab is , Antibodies,
　E and F are the same as those of formula (IV) above. ], The method comprising producing an antibody having a functional substance or a salt thereof.