WO2019150985A1 - Antibody-drug complex and pharmaceutical composition containing same - Google Patents

Abstract

Provided is an antibody-drug complex that controls the binding of an antibody and a drug and exhibits an appropriate antitumor effect. An antibody-drug complex that is a complex in which an IgG1 antibody and an antitumor compound are bonded via a crosslinking agent-derived portion having an N oxy radical group or an N hydroxy group, and the N oxy radical group or N hydroxy group bonds covalently with the IgG1 antibody by reacting with a tryptophan residue of the IgG1 antibody.

Description

Antibody drug conjugate and pharmaceutical composition containing the same

The present invention relates to an antibody-drug conjugate (ADC).

An antibody-drug conjugate is a substance in which an antibody and a drug are bound via a linker, and has attracted attention in recent years because it can effectively act on a drug by utilizing high target cell selectivity by the antibody. . For example, Patent Document 1 discloses an antibody drug complex in which an anti-HER2 antibody and a camptothecin fusion are bound by a linker having a succinimido-3-yl group, and Patent Document 2 discloses an anti-ErbB2 antibody and a maytansinoid. Antibody drug conjugates are disclosed wherein are linked by a linker such as N-succinimidyl-4- (2-pyridylthio) pentanoate (SPP). In addition, cadsira (registered trademark of H. Hoffmann-La Roche AG) in which trastuzumab and emtansine (DM1) as an anti-HER2 antibody are bound is marketed.
Patent Document 3 describes that anti-amyloid β antibody and fluorescein methyl ester can be bound by a linker having an azabicyclo [3.3.1] nonane N-oxyl group.

Japanese Patent No. 6105183 Japanese Patent No. 4780633 International Publication No. 2017/154997

However, it is difficult to control the binding position and the number of bonds between the antibody and the drug or linker, and it is not possible to control even with the above-mentioned cadsila (registered trademark of H. Hoffmann-La Roche AG). The present invention has been made paying attention to the above-described circumstances, and an object of the present invention is to provide an antibody-drug conjugate that controls the binding between an antibody and a drug and exhibits an appropriate antitumor effect. .

The present invention is as follows.
(1) A complex in which an IgG1 antibody and an antitumor compound are bound via a cross-linking agent-derived moiety having an N oxy radical group or an N hydroxy group,
An antibody drug complex in which the N oxy radical group or N hydroxy group reacts with the tryptophan residue of the IgG1 antibody and is covalently bonded to the IgG1 antibody.
(2) The antibody drug conjugate according to (1), wherein the antitumor compound is bound to the constant region of the IgG1 antibody.
(3) The ratio of the antitumor compound bound in the constant region of the IgG1 antibody is 90% or more (on a molar basis) with respect to the total antitumor compound bound to the IgG1 antibody ( The antibody drug conjugate according to 1) or (2).
(4) The number of tryptophan residues present in the constant region of the IgG1 antibody is 6 to 18 per antibody molecule before binding to the cross-linking agent, and the number of tryptophan residues present in the variable region is 4. The antibody-drug conjugate according to any one of (1) to (3), wherein 4 to 16 molecules / antibody molecule is present before binding to the agent.
(5) When the IgG1 antibody does not contain a tryptophan residue in the variable region or contains a tryptophan residue in the variable region, the carbon atom at the 3-position of the indole ring in the side chain of the tryptophan residue is a protein. The antibody drug conjugate according to any one of (1) to (4), which is embedded in the molecule.
(6) The antibody drug conjugate according to any one of (1) to (5), wherein the IgG1 antibody is an anti-HER2 antibody.
(7) The antibody drug conjugate according to (6), wherein the anti-HER2 antibody is a humanized monoclonal antibody.
(8) The antibody-drug conjugate according to any one of (1) to (7), wherein the antitumor compound is maytansinoid.
(9) The crosslinking agent having an N oxy radical group or an N hydroxy group has a bicyclo structure represented by the following formula (b1a) or the following formula (b1b);

(Where
A ¹ , B ¹ , C ¹ , D ¹ , E ¹ and E ² are each independently CR ¹ R ² ; C = CR ³ R ⁴ ; C = O; C = S; C = NR ⁵ ; NR ⁵ SiR ⁶ R ⁷ ; an oxygen atom; or a hetero atom other than a nitrogen atom, a silicon atom and an oxygen atom;
F ¹ and G ¹ each independently represent CR ⁸ or a nitrogen atom,
R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ are each independently a hydrogen atom; a halogen atom; a heteroatom group; an optionally substituted carbon atom of 1 to 30 An alkyl group having 2 to 30 carbon atoms which may be substituted, an alkynyl group having 2 to 30 carbon atoms which may be substituted, an aryl group having 6 to 30 carbon atoms which may be substituted, An optionally substituted heteroaryl group having 4 to 30 carbon atoms, an optionally substituted aralkyl group having 7 to 30 carbon atoms, an optionally substituted cycloalkyl group having 3 to 30 carbon atoms; May be an alkyloxy group having 1 to 30 carbon atoms, an alkenyloxy group having 2 to 30 carbon atoms which may be substituted, an alkynyloxy group having 2 to 30 carbon atoms which may be substituted, or an optionally substituted group. Ants with 6 to 30 carbon atoms An aryloxy group, an optionally substituted heteroaryloxy group having 4 to 30 carbon atoms, an optionally substituted aralkyloxy group having 7 to 30 carbon atoms, and an optionally substituted cycloalkyl having 3 to 30 carbon atoms An oxy group; an optionally substituted polyalkyleneoxy group; or a reactive functional group,
However, R ⁵ is not a halogen atom,
E ¹ and E ² together may form an optionally substituted —CH (CH ₂ ) _m CH— group, m represents an integer from 0 to 12,
The antitumor compound is bound to at least one group of A ¹ , B ¹ , C ¹ , D ¹ , E ¹ and E ² directly or through an intermediate chain)
The bicyclo structure forms at least one of a bond represented by the following formula (B1), a bond represented by the following formula (B2), and a bond represented by the following formula (B3) with the tryptophan residue of the IgG1 antibody (1 The antibody-drug conjugate according to any one of (8) to (8).

(Wherein A ¹ , B ¹ , C ¹ , D ¹ , E ¹ , E ² , F ¹ and G ¹ have the same meaning as described above)
(10) The bicyclo structure of the cross-linking agent and the antitumor compound comprise N-succinimidyl-4- (2-pyridylthio) pentanoate (SPP), N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) Succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), dimethyl adipimidate HCL, disuccinimidyl suberate, glutaraldehyde, bis (p- Azidobenzoyl) hexanediamine, bis- (p-diazoniumbenzoyl) -ethylenediamine, or triene-2,6-diisocyanate, 1,5-difluoro-2,4-dinitrobenzene linked using (9) Antibody drug conjugate body.
(11) A pharmaceutical composition comprising the antibody drug conjugate according to any one of (1) to (10) and a pharmaceutically acceptable carrier.

According to the present invention, it is possible to obtain an antibody-drug conjugate that can control the binding between an antibody and a drug and has an excellent drug effect.

FIG. 1 is a chart showing MALDI-TOF-MS measurement results of Herceptin ^* -TrpADC (DM1), which corresponds to an example of the present invention. FIG. 2 is a chart showing MALDI-TOF-MS measurement results of Herceptin ^* , which corresponds to the prior art. FIG. 3 is a graph showing the survival rate of SK-BR-3 cells in the presence of Herceptin ^* -TrpADC (DM1). *: Herceptin is Gentech inc. Is a registered trademark.

1. IgG1 Antibody The present invention is a complex in which an IgG1 antibody and an antitumor compound (also referred to as an anticancer compound) are bound via a cross-linking agent-derived moiety having an N oxy radical group or an N hydroxy group. It has been found by the present inventors that a cross-linking agent having an N oxy radical group or an N hydroxy group selectively reacts with a specific tryptophan residue among tryptophan residues of an IgG1 antibody. Therefore, if the cross-linking agent is used, the binding between the antitumor compound and the IgG1 antibody can be highly controlled, and the antitumor effect of the obtained antibody drug conjugate can be made more appropriate.

As the IgG1 antibody, one having a tryptophan residue in the variable region as well as the constant region can be used. According to the present invention, the binding between the antibody and the antitumor compound can be controlled, and even when the variable region has tryptophan, the antitumor compound can be bound preferentially in the constant region. The number of tryptophan residues present in the constant region of the IgG1 antibody used in the present invention is, for example, 6-18 per antibody before binding with a cross-linking agent, preferably 8-16 per antibody. More preferably, 10 to 14 antibodies / molecule of antibody. For example, trastuzumab, which will be described later, has heavy regions Ala121 to Gly449 (containing 5 tryptophan residues) and light chain Arg108 to Cys214 (containing 1 tryptophan residue) as constant regions, and tryptophan present in the constant regions. The number of residues is 12 / molecule of antibody.

Further, the number of tryptophan residues present in the variable region is, for example, 4 to 16 per antibody molecule, preferably 6 to 14 per antibody molecule, and more preferably 8 to 1 before binding to the cross-linking agent. 12 / one antibody molecule. For example, trastuzumab, which will be described later, has a heavy chain Glu1 to Ser120 (containing 4 tryptophan residues) and a light chain Asp1 to Lys107 (containing 1 tryptophan residue), and the tryptophan present in the variable region. The number of residues is 10 / molecule of antibody.

In the IgG1 antibody, the number of tryptophan residues in the complementarity determining region (hypervariable region) is, for example, 10 / antibody molecule or less, preferably 6 / antibody molecule or less, and 2 / antibody. Most preferably, it is 1 molecule or less. The fewer tryptophan residues in the complementarity determining region, the more reliably the reaction of the cross-linking agent in the complementarity determining region can be prevented, and the production efficiency of the antibody drug conjugate can be increased. For example, trastuzumab described below includes heavy chain Asp31 to His35 (including 0 tryptophan residues), Arg50 to Gly66 (including 0 tryptophan residues), Trp99 to Tyr109 (including 1 tryptophan residue), and light chain Arg24 to Ala34 (including 0 tryptophan residues), Ser50 to Ser56 (including 0 tryptophan residues), Gln89 to Thr97 (including 0 tryptophan residues) are complementarity determining regions, and tryptophan in the complementarity determining region The number of residues is 2 / molecule of antibody.

Preferably, the IgG1 antibody does not contain a tryptophan residue in the complementarity determining region and its vicinity (for example, within 3 residues before and after), and the IgG1 antibody is in the complementarity determining region and its vicinity (within 3 residues before and after). When a tryptophan residue is included, it is preferable that the carbon atom at the 3-position of the indole ring in the side chain of the tryptophan residue in the complementarity determining region and the vicinity thereof is buried in the protein molecule. The carbon atom at the 3-position of the indole ring is a carbon atom numbered 3 in the formulas (B1) to (B3) described later.

The IgG1 antibody preferably does not contain a tryptophan residue in the variable region. When the IgG1 antibody contains a tryptophan residue in the variable region, the tryptophan residue in the variable region also has an indole ring on its side chain. The 3-position carbon atom is preferably buried inside the protein molecule. If the variable region contains multiple tryptophan residues, the carbon atom at the 3-position of the indole ring in the side chain of the tryptophan residues in all the variable regions need not be buried inside the protein molecule. Of all the tryptophan residues, for example, 40% or more, preferably 60% or more, more preferably 80% or more, and most preferably 100% of the carbon at the 3-position in the indole ring of the side chain. It suffices if atoms are buried inside the protein molecule.

On the other hand, it is preferable that at least two carbon atoms at the 3-position of the indole ring in the side chain of the tryptophan residue in one antibody molecule are exposed from the tryptophan residue present in the IgG1 antibody constant region. .

Whether the carbon atom at the 3-position of the indole ring in the side chain of the tryptophan residue is buried in the protein molecule or exposed on the surface of the protein molecule can be determined based on the X-ray structural analysis of the antibody. As long as the three-dimensional structure of the protein molecule is not affected, the X-ray structural analysis result of a part of the antibody (such as Fab region), a modified antibody, or a part of the modified antibody may be used. For example, whether or not the carbon atom at the 3-position of the indole ring in the side chain of the tryptophan residue of Trastuzumab Fab, which will be described later, is buried in the protein molecule or exposed on the surface is Ne- (o-azobenzoylcarbonyl) -L- Three-dimensional structure of fabric of trastuzumab modified with lysine (registered as 5XHG in Protein Data Bank (https://www.rcsb.org/)) and trastuzumab modified with p-azido-L-phenylalanine It can be determined using the result of the three-dimensional structure of the variable region (registered as 5XHF in the Protein Data Bank).
More specifically, the X-ray structural analysis result is displayed using a molecular graphics tool such as PyMOL (https://pymol.org/2/) to display the 3D view, and the side chain to be determined is exposed. Based on whether or not the carbon atom at the 3-position of the indole ring of the side chain is exposed on the surface of the protein molecule or whether it is buried inside.
In light of the above criteria, it is determined that the carbon atom at the 3-position of the indole ring in the side chain of all tryptophan residues in the variable region of trastuzumab is buried in the protein molecule.

The IgG1 antibody is preferably an antibody that specifically binds to a tumor cell, more preferably an antibody against the epidermal growth factor receptor family, and the anti-EGFR antibody (anti-ErbB antibody) is an anti-HER1 antibody (anti-ErbB antibody). Anti-ErbB1 antibody), anti-HER2 antibody (anti-ErbB2 antibody), anti-HER3 antibody (anti-ErbB3 antibody), anti-HER4 antibody (anti-ErbB4 antibody) and the like. By binding the anti-ErbB antibody and the antitumor compound, the effect of the antitumor compound can be appropriately exhibited. In the present invention, it is particularly preferable to bind an antitumor compound to an anti-HER2 antibody. HER2 is known to form heterodimers with homodimers or other EGF receptors HER1 (ErbB1), HER3 (ErbB3), HER4 (ErbB4), etc., and act on cell proliferation, differentiation and survival. By binding an anti-tumor compound to an anti-HER2 antibody, the drug effect of the anti-tumor compound can be caused to act on the tumor cells more efficiently.

The anti-HER2 antibody may be derived from any species, and preferably, mammals such as humans, rats, mice, and rabbits can be exemplified, and anti-HER2 antibodies derived from rats or mice are easily available. If the antibody is derived from a species other than human, it is preferably chimerized or humanized using well-known techniques.

Examples of the chimeric antibody include antibodies in which the variable region and constant region of the antibody are derived from different species, for example, a chimeric antibody in which the variable region of a rat or mouse-derived antibody is joined to the constant region derived from human (Proc. Natl. Acad.Sci.U.S.A., 81, 6851-6855, (1984)). Although there is no restriction | limiting in particular as a chimeric antibody of this invention, The chimeric antibody containing the variable region of mouse | mouth antibody 4D5 and the heavy chain constant region of human IgG1 or IgG2 is mentioned.

As a humanized antibody, an antibody in which only a complementarity determining region (CDR; complementarity determining region) is incorporated into a human-derived antibody (see Nature (1986) 321, p.522-525), a CDR sequence is obtained by CDR grafting. In addition to the above, an antibody obtained by transplanting amino acid residues of some frameworks into a human antibody (WO 90/07861), an antibody humanized using a gene conversion mutagenesis strategy (US patent) 5821337).

Human antibodies are not limited to human-derived anti-HER2 antibodies as long as they have only human chromosome-derived antibody gene sequences, for example, human antibody production having human chromosome fragments including human antibody heavy and light chain genes Method using mouse (Tomizuka, K. et al., Nature Genetics (1997) 16, p. 133-143; Kuroiwa, Y. et. Al., Nucl. Acids Res. (1998) 26, p. 3447- 3448; Yoshida, H. et.al., Animal Cell Technology: Basic and Applied Aspects vol.10, p.69-73 (Kitagawa, Y., Matsuda, T. and Iijima, S.eds.). cademic Publishers, 1999; Tomizuka, K.et.al., Proc.Natl.Acad.Sci.USA (2000) 97, may be an antibody obtained by reference) to p.722-727 like..

The anti-HER2 antibody may be either a polyclonal antibody or a monoclonal antibody, and is preferably a monoclonal antibody, more preferably a human antibody or a humanized monoclonal antibody.

The anti-HER2 antibody includes Coussens L, et al. , Science. 1985; 230 (4730): 1132-1139, a transmembrane receptor protein having a tyrosine kinase domain with a molecular weight of 185 kDa (its DNA sequence and amino acid sequence have been published on public databases, for example, M11730 (Genbank ), And can be referred to by an accession number such as NP_004439.2 (NCBI)). Specifically, mouse anti-HER2 antibody 4D5 (Fendly. Et al., Cancer Research 1990 (50): 1550- 1558, US Pat. No. 5,677,171), Trastuzumab (huMAb4D5-8, rhuMAb HER2, Herceptin (Genen), a recombinant humanized monoclonal antibody of the mouse anti-HER2 antibody 4D5 ech inc. registered trademark of)), such as pertuzumab (Pajeta (a registered trademark of H.Hoffmann-La Roche AG)) are known.

As long as the anti-HER2 antibody of the present invention can specifically bind to HER2, an amino acid residue in which a part of amino acid residues is substituted, deleted, or added to the specific examples of the anti-HER2 antibody shown above. It may have an array. Whether or not the binding is specific can be determined based on a dissociation constant (hereinafter referred to as a KD value). The KD value for the HER2 protein of a suitable antibody is 1 × 10 ⁻⁵ M or less, more preferably 1 × 10 ⁻⁷ M or less, even more preferably 1 × 10 ⁻⁸ M or less, and most preferably 1 × 10 ^-9 M or less. The binding between the HER2 protein and the antibody can be measured using a known method such as Surface Plasma Resonance method, ELISA method, or RIA method.

The amino acid residue substitution is preferably a conservative amino acid substitution. Conservative amino acid substitutions are those that take place within a group of amino acids that are related in their side chains. Suitable amino acid groups are as follows:
Acidic group: aspartic acid, glutamic acid Basic group: lysine, arginine, histidine Nonpolar group: alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan Uncharged polar group: glycine, asparagine, glutamine, cysteine, serine, Threonine, tyrosine Aliphatic hydroxy group: serine, threonine Amide-containing group: asparagine, glutamine Aliphatic group: alanine, valine, leucine, isoleucine Aromatic group: phenylalanine, tryptophan, tyrosine

It is preferable that the substitution, deletion, or addition of amino acid residues is performed so that the heavy chain amino acid sequence and the light chain amino acid sequence of the antibody have a sequence exhibiting high homology (identity). It is preferable that there is 80% or more homology, more preferably 90% or more, even more preferably 95% or more, and most preferably 99% or more.

The homology between the two types of antibodies (amino acid sequences) is Blast algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaeffer, Jinghui Zhang, ZhangWebbhan, ZhangWebbhan. J. Lipman (1997), “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25: 3389-3402). Blast algorithm can also be used by accessing www.ncbi.nlm.nih.gov/blast on the Internet.

In addition, the IgG1 antibody used in the present invention is a modified antibody, for example, a chemical compound, as long as it can bind to a cross-linking agent-derived moiety having an N oxy radical group or an N hydroxy group, such as a tryptophan residue remaining. It may be modified chemically or biologically. The chemical modification includes a modification with a compound capable of forming a covalent bond with the amino acid skeleton, for example, a modification with an N-linked or O-linked carbohydrate chain. Biological modifications include post-translational modifications (eg, glycosylation to N- or O-links, N- or C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation) And those in which a methionine residue is added to the N-terminus by expression using a prokaryotic host cell. Such modifications also include those labeled to enable detection or isolation of the antibody or antigen of the present invention, such as enzyme labels, fluorescent labels, and affinity labels.

2. Anti-tumor compound The anti-tumor compound that binds to the IgG1 antibody can be a compound that has an anti-tumor effect and can bind to a cross-linking agent. For example, a maytansin analog (maytansinoid); the following formula (2a) Exatecan ((1S, 9S) -1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H, 12H-benzo [de] pyrano [3 ′, 4 ': 6,7] Indolizino [1,2-b] quinoline-10,13 (9H, 15H) -dione) and other camptothecin derivatives; doxorubicin, daunorubicin, mitomycin C, bleomycin, cyclocytidine, vincristine, vinblastine, methotrexate , Platinum-based antitumor agents (cisplatin or derivatives thereof), paclitaxel (eg, taxo Taxol is a registered trademark of Bristol-Myers Squibb), pyrimidine analogues such as 5-FU, antiestrogens such as tamoxifen, auristatins such as MMAE and MMAF, and derivatives thereof, preferably maytansinoids and captos. A mycin derivative is mentioned, More preferably, a maytansinoid is mentioned.

Maytansinoid refers to a compound group developed using maytansine represented by the following formula (2b) as a lead compound, and is characterized by having a group represented by the following formula (2c). As the maytansinoid, a compound represented by the following formula (2d) is preferable.

(In the formula (2d), R is an alkanediyl group having 1 to 10 carbon atoms. The carbon number of R is preferably 1 to 6, more preferably 1 to 3. R is ethane-1 , 2-diyl is referred to as maytansinoid DM1, and R is 1,1-dimethylpropane-1,3-diyl (where SH is bonded to the 1-position) It is said to be maytansinoid DM4.)
In addition, the maytansinoid represented by the formula (2d) often reacts with a crosslinking agent at the SH portion.

3. Crosslinking agent The IgG1 antibody and the antitumor compound are bound together by a crosslinking agent having an N oxy radical group or an N hydroxy group. The N oxy radical group or N hydroxy group of the cross-linking agent reacts with the tryptophan residue of the IgG1 antibody to form a covalent bond.

The crosslinking agent having an N oxy radical group or an N hydroxy group preferably has a bicyclo structure (preferably an azabicyclononane (ABNO) structure) represented by the following formula (b1a) or the following formula (b1b).

(Where
A ¹ , B ¹ , C ¹ , D ¹ , E ¹ and E ² are each independently CR ¹ R ² ; C = CR ³ R ⁴ ; C = O; C = S; C = NR ⁵ ; NR ⁵ SiR ⁶ R ⁷ ; an oxygen atom; or a hetero atom other than a nitrogen atom, a silicon atom and an oxygen atom (for example, a sulfur atom);
F ¹ and G ¹ each independently represent CR ⁸ or a nitrogen atom,
R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ are each independently a hydrogen atom; a halogen atom; a heteroatom group; an optionally substituted carbon atom of 1 to 30 An alkyl group having 2 to 30 carbon atoms which may be substituted, an alkynyl group having 2 to 30 carbon atoms which may be substituted, an aryl group having 6 to 30 carbon atoms which may be substituted, An optionally substituted heteroaryl group having 4 to 30 carbon atoms, an optionally substituted aralkyl group having 7 to 30 carbon atoms, an optionally substituted cycloalkyl group having 3 to 30 carbon atoms; May be an alkyloxy group having 1 to 30 carbon atoms, an alkenyloxy group having 2 to 30 carbon atoms which may be substituted, an alkynyloxy group having 2 to 30 carbon atoms which may be substituted, or an optionally substituted group. Ants with 6 to 30 carbon atoms An aryloxy group, an optionally substituted heteroaryloxy group having 4 to 30 carbon atoms, an optionally substituted aralkyloxy group having 7 to 30 carbon atoms, and an optionally substituted cycloalkyl having 3 to 30 carbon atoms An oxy group; an optionally substituted polyalkyleneoxy group; or a reactive functional group,
However, R ⁵ is not a halogen atom,
E ¹ and E ² together may form an optionally substituted —CH (CH ₂ ) _m CH— group, m represents an integer from 0 to 12,
The antitumor compound is bound to at least one group of A ¹ , B ¹ , C ¹ , D ¹ , E ¹ and E ² directly or through an intermediate chain)

In the present specification, the “hetero atom” means a divalent or higher atom other than carbon and hydrogen.
In the present specification, the “heteroatom group” means a substituent having a heteroatom as part of the group.

The number of carbon atoms of the alkyl group in R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ is preferably 1-20. Specific examples of the alkyl group having 1 to 30 carbon atoms include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert, -Butyl group, n-pentyl group, n-hexyl group, n-octyl group, n-decyl group, n-dodecyl group, n-octadecyl group, n-icosyl group and the like.

The carbon number of the alkenyl group in R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ is preferably 2-15. Preferred examples of the alkenyl group having 2 to 30 carbon atoms include a vinyl group, an allyl group, a 3-butenyl group, a 4-pentenyl group, a 5-hexenyl group, a 6-heptenyl group, and a 7-octenyl group.

The number of carbon atoms of the alkynyl group in R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ is preferably 2-15. Preferred examples of the alkynyl group having 2 to 30 carbon atoms include ethynyl group, 2-propynyl group, 3-butynyl group, 4-pentynyl group, 5-hexynyl group, 6-heptynyl group, and 7-octynyl group. .

The number of carbon atoms of the aryl group in R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ is preferably 6-15. Preferred examples of the aryl group having 6 to 30 carbon atoms include phenyl group, 1-naphthyl group, 2-naphthyl group and the like.

The heteroaryl group in R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ preferably has 4 to 15 carbon atoms. Preferred examples of the heteroaryl group having 4 to 30 carbon atoms include 2-pyridyl group, 3-pyridyl group, 4-pyridyl group, 2-thiophenyl group, 2-furyl group and the like.

The number of carbon atoms of the aralkyl group in R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ is preferably 7-15. Preferred examples of the aralkyl group having 7 to 30 carbon atoms include benzyl group and 1-phenethyl group.

The number of carbon atoms of the cycloalkyl group in R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ is preferably 3-15. Preferred examples of the cycloalkyl group having 3 to 30 carbon atoms include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, and a cyclohexyl group.

^{Further, R 1, R 2, R} 3, R 4, R 5, R 6, alkyl group in R ⁷ and R ^8, alkenyloxy group, alkynyloxy group, an aryloxy group, heteroaryloxy group, an aralkyloxy group And an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an aralkyl group and a cycloalkyl group which are part of the cycloalkyloxy group, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , The alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, aralkyl group and cycloalkyl group in R ⁶ , R ⁷ and R ⁸ can be selected.

The polyalkyleneoxy group in R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ is preferably a polyethyleneoxy group, more preferably — (CH ₂ CH ₂ O ) A _-group (a is an integer from 1 to 10), more preferably-(CH ₂ CH ₂ O) _a -group (a is 4). At the free end of the polyoxyalkylene group, for example, a hydrogen atom, an optionally substituted alkyl group, an optionally substituted alkenyl group, an optionally substituted alkynyl group, or an optionally substituted aryl A group, an optionally substituted heteroaryl group, an optionally substituted aralkyl group and an optionally substituted cycloalkyl group may be bonded. Examples of the alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, aralkyl group and cycloalkyl group bonded to the free end of the polyoxyalkylene group include R ¹ , R ² , R ³ , R ⁴ , R ⁵ , The alkyl group, alkenyl group, alkynyl group, aryl group, heteroaryl group, aralkyl group and cycloalkyl group in R ⁶ , R ⁷ and R ⁸ can be selected.

Further, the substituents in R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ include a hydrogen atom; a halogen atom; a heteroatom group (preferably a hydroxyl group); An optionally substituted alkyl group having 1 to 30 carbon atoms, an optionally substituted alkenyl group having 2 to 30 carbon atoms, an optionally substituted alkynyl group having 2 to 30 carbon atoms, and an optionally substituted carbon number An aryl group having 6 to 30 carbon atoms, a heteroaryl group having 4 to 30 carbon atoms that may be substituted, an aralkyl group having 7 to 30 carbon atoms that may be substituted, and an alkyl group having 3 to 30 carbon atoms that may be substituted A cycloalkyl group; an optionally substituted alkyloxy group having 1 to 30 carbon atoms, an optionally substituted alkenyloxy group having 2 to 30 carbon atoms, and an optionally substituted alkynyloxy group having 2 to 30 carbon atoms Group, substituted An optionally substituted aryloxy group having 6 to 30 carbon atoms, an optionally substituted heteroaryloxy group having 4 to 30 carbon atoms, an optionally substituted aralkyloxy group having 7 to 30 carbon atoms, and a substituted group. A cycloalkyloxy group having 3 to 30 carbon atoms which may be substituted; a polyalkyleneoxy group which may be substituted; or a reactive functional group. Preferred examples include a reactive functional group described below and a halogen Atoms. The number of substitution groups and the substitution position are not particularly limited.

In addition, the reactive functional group as a group or a part of the group (substituent) in R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ is represented by the formula (b1a) or the formula It is preferable to select in consideration of imparting reactivity between the crosslinking agent having a bicyclo structure (b1b) and the antitumor compound or the compound forming the intermediate chain. A person skilled in the art can make an appropriate selection based on the description of “Bioconjugate Techniques, third edition” by Hermanson. Suitable examples of reactive functional groups include alcohol groups, epoxy groups, acetal groups, orthoester groups, ester groups, carbonyl groups, carboxyl groups, carboxylic anhydride groups, amide groups, imidate groups, amino groups, imino groups, Aziridine group, diazo group, azido group, amidino group, guanidyl group, hydrazyl group, hydrazone group, alkoxyamino group, oxime group, carbonate group, carbamate group, sulfhydryl group, ether group, imide group, thioester group, thioamide group, isothiocyano group Group, thioether group, disulfide group, halogen group, isocyano group, isocyanate group, oxazirine group, diaziridine group, sulfonyl group, sulfone group, sulfoxide group, sulfonimide group, seleno group, silyl group, boryl group, stannyl group, phosphine group Hosphi A functional group having an oxide group, a phosphate group, a phosphate ester group, a phosphate amide group, a methylene group, an alkenyl group, or an alkynyl group as a group or a part of the group is preferable, and an alcohol group, an epoxy group, or an ester is more preferable. Group, carbonyl group, carboxyl group, carboxylic anhydride group, amide group, amino group, azide group, hydrazone group, oxime group, carbonate group, carbamate group, sulfhydryl group, maleimide group, thioester group, thioamide group, isothiocyano group, thioether A functional group having a group, an isocyano group, an isocyanate group, an oxazirine group, an azide group, a methanesulfonyl group, a p-toluenesulfonyl group, a methylene group, an alkenyl group, an alkynyl group or a combination thereof as a group or a part of the group. As preferable examples of the reactive functional group having a methylene group, an alkenyl group or an alkynyl group, an alkyl group having 1 to 30 carbon atoms, an alkenyl group having 2 to 30 carbon atoms, an alkynyl group having 2 to 30 carbon atoms, carbon An aryl group having 6 to 30 carbon atoms, a heteroaryl group having 4 to 30 carbon atoms, an aralkyl group having 7 to 30 carbon atoms, a cycloalkyl group having 3 to 30 carbon atoms, an alkyloxy group having 1 to 30 carbon atoms, and 2 carbon atoms -30 alkenyloxy group, alkynyloxy group having 2-30 carbon atoms, aryloxy group having 6-30 carbon atoms, heteroaryloxy group having 4-30 carbon atoms, aralkyloxy group having 7-30 carbon atoms, carbon number It may have 3 to 30 cycloalkyloxy groups or the like as a group or a part of the group, and the present invention includes such an embodiment.

According to one preferred embodiment, any one of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ is an optionally substituted alkyl group having 1 to 30 carbon atoms. Or an optionally substituted alkenyl group having 2 to 30 carbon atoms, more preferably an n-butyl group, an n-pentyl group, an n-hexyl group, an allyl group, or a 3-butenyl group.

According to another preferred embodiment, any one of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ is a polyalkyleneoxy group whose free end may be an alkoxy group. Yes, more preferably, R ⁵ is a polyalkyleneoxy group whose free end may be an alkoxy group. Further, the polyalkyleneoxy group whose free end side may be an alkoxy group is preferably substituted. Suitable examples of the substituent include an allyl group, a vinyl group, a phenyl group, a naphthyl group, and an azide group. , Pyridyl group, triazole group and the like.

According to a preferred embodiment, in the crosslinking agent having the bicyclo structure of the formula (b1a) or the formula (b1b), A ¹ , B ¹ , C ¹ and D ¹ are each independently CR ¹ R ² , Is CH ₂ or CHR ¹ . Here, in CHR ¹ represented by A ¹ , B ¹ , C ¹ and D ¹ , R ¹ is a reactive functional group, more preferably an alcohol group, an epoxy group, an acetal group, an orthoester group, an ester group, Carbonyl group, carboxyl group, carboxylic anhydride group, amide group, imidate group, amino group, imino group, aziridine group, diazo group, azido group, amidino group, guanidyl group, hydrazyl group, hydrazone group, alkoxyamino group, oxime group , Carbonate group, carbamate group, sulfhydryl group, ether group, imide group, thioester group, thioamide group, isothiocyano group, thioether group, disulfide group, halogen group, isocyano group, isocyanate group, oxazirine group, diaziridine group, sulfonyl group, sulfone Group, sulfoxide group, sulfonimide group Having a seleno group, silyl group, boryl group, stannyl group, phosphine group, phosphine oxide group, phosphate group, phosphate ester group, phosphate amide group, methylene group, alkenyl group, alkynyl group as a group or part of a group Functional group, more preferably alcohol group, epoxy group, ester group, carbonyl group, carboxyl group, carboxylic anhydride group, amide group, amino group, azide group, hydrazone group, oxime group, carbonate group, carbamate group, sulfhydryl Group, maleimide group, thioester group, thioamide group, isothiocyano group, thioether group, isocyano group, isocyanate group, oxazirine group, azide group, methanesulfonyl group, p-toluenesulfonyl group, methylene group, alkenyl group, alkynyl group or their Based on a combination or It is a functional group that is part of the group.

According to another preferred embodiment, in the cross-linking agent having the bicyclo structure of the formula (b1a) or the formula (b1b), E ¹ and E ² are each independently CR ¹ R ² , C = CR ³ R ⁴ , C = O, C = S, C = NR ⁵ , NR ⁵ , SiR ⁶ R ⁷ , an oxygen atom, or a heteroatom other than a nitrogen atom, a silicon atom and an oxygen atom (for example, a sulfur atom), preferably C = O, C = NR ⁵ , CHR ¹ , CH ₂ or an oxygen atom. Here, in C═NR ⁵ and CHR ¹ represented by E ¹ and E ² , R ¹ and R ⁵ are preferably polyoxyalkylene groups having an alkyl group having a reactive functional group on the free end side; or reaction More preferably, the reactive functional group is an alcohol group, epoxy group, acetal group, orthoester group, ester group, carbonyl group, carboxyl group, carboxylic anhydride group, amide group, imidate group, amino group. Group, imino group, aziridine group, diazo group, azido group, amidino group, guanidyl group, hydrazyl group, hydrazone group, alkoxyamino group, oxime group, carbonate group, carbamate group, sulfhydryl group, ether group, imide group, thioester group , Thioamide group, isothiocyano group, thioether group, disulfide group, halogen group, Cyano group, isocyanate group, oxazirine group, diaziridine group, sulfonyl group, sulfone group, sulfoxide group, sulfonimide group, seleno group, silyl group, boryl group, stannyl group, phosphine group, phosphine oxide group, phosphoric acid group, phosphoric acid A functional group having an ester group, a phosphoric acid amide group, a methylene group, an alkenyl group or an alkynyl group as a group or a part of the group, more preferably an alcohol group, an epoxy group, an ester group, a carbonyl group, a carboxyl group, or an anhydrous carboxylic acid Acid group, amide group, amino group, azide group, hydrazone group, oxime group, carbonate group, carbamate group, sulfhydryl group, maleimide group, thioester group, thioamide group, isothiocyano group, thioether group, isocyano group, isocyanate group, oxadiline group Azide , Methanesulfonyl group, a functional group having p- toluenesulfonyl group, a methylene group, an alkenyl group, an alkynyl group, or a combination thereof as a group or part of a group.

E ¹ and E ² may be taken together to form a —CH (CH ₂ ) _m CH— group. Here, m is preferably an integer of 0 to 12, more preferably an integer of 0 to 9. The —CH (CH ₂ ) _m CH— group may preferably have a substituent, and the substituent in the —CH (CH ₂ ) _m CH— group is preferably a reactive functional group. More preferably, alcohol group, epoxy group, acetal group, orthoester group, ester group, carbonyl group, carboxyl group, carboxylic anhydride group, amide group, imidate group, amino group, imino group, aziridine group, diazo group, Azido group, amidino group, guanidyl group, hydrazyl group, hydrazone group, alkoxyamino group, oxime group, carbonate group, carbamate group, sulfhydryl group, ether group, imide group, thioester group, thioamide group, isothiocyano group, thioether group, disulfide Group, halogen group, isocyano group, isocyanate group, oxazirine group, dia Lysine group, sulfonyl group, sulfone group, sulfoxide group, sulfonimide group, seleno group, silyl group, boryl group, stannyl group, phosphine group, phosphine oxide group, phosphate group, phosphate ester group, phosphate amide group, methylene Group, an alkenyl group, a functional group having an alkynyl group or a part of the group, more preferably an alcohol group, an epoxy group, an ester group, a carbonyl group, a carboxyl group, a carboxylic anhydride group, an amide group, an amino group, Azide group, hydrazone group, oxime group, carbonate group, carbamate group, sulfhydryl group, maleimide group, thioester group, thioamide group, isothiocyano group, thioether group, isocyano group, isocyanate group, oxazirine group, azide group, methanesulfonyl group, p -Toluenesulfonyl group, Styrene group, an alkenyl group, a functional group having an alkynyl group, or a combination thereof as a group or part of a group.

In the crosslinking agent having a bicyclo structure represented by the formula (b1a) or the formula (b1b), A ¹ , B ¹ , C ¹ and D ¹ all represent CH ₂ , and F ¹ and G ¹ both represent CH In view of ensuring covalent bond formation reactivity with the antitumor compound or the compound forming the intermediate chain, at least one of E ¹ and E ² is CR ¹ R ² (R ¹ , R ² is at least one reactive functional group), C═O, C═S, C═NR ⁵ , NR ⁵ , SiR ⁶ R ⁷ , or E ¹ and E ² together , —CH (CH ₂ ) _m CH— group, and at least one hydrogen on the —CH (CH ₂ ) _m CH— group is preferably substituted with a reactive functional group. In the crosslinking agent having the bicyclo structure of the formula (b1a) or the formula (b1b), when E ¹ and E ² are both CH ₂ and F ¹ and G ¹ are both CH, A ¹ , B ¹ , C ¹ and D ¹ are preferably CHR ¹ .

According to another aspect, A ¹ , B ¹ , C ¹ and D ¹ all represent CH ₂ , F ¹ and G ¹ both represent CH, and ^one group of E ¹ and E ² Represents CH ₂ , an oxygen atom or a sulfur atom, the other group of E ¹ and E ² represents C═O, C═NH, C═NOH, NH, C═NR ⁵ or NR ⁵ (provided that C = R ⁵ of .NR ⁵ R ⁵ is other than a hydrogen atom and the alcohol group (hydroxy group) of the NR ⁵ except a hydrogen atom).

According to another preferred embodiment, in the crosslinking agent having the bicyclo structure of the formula (b1a) or the formula (b1b), A ¹ , B ¹ , C ¹ and D ¹ all represent CH ₂ and F ¹ And G ¹ both represent CH, ^one of E ¹ and E ² represents CH ₂ or an oxygen atom, and the other represents C═O, CHNH ₂ , CH (CO) NH _2, or C═NOH. In yet another preferred embodiment, one of E ¹ and E ² preferably represents CH ₂ . In this preferred embodiment, the crosslinking agent having the bicyclo structure of the formula (b1a) or the formula (b1b) can be said to have the structure of the following formula (b2a) or the formula (b2b), and binds an antitumor compound or an intermediate chain. Depending on the compound that forms, a compound in which the structure of the formula (b2a) or the formula (b2b) is appropriately changed can be used.

The E ¹ moiety (carbon atom) and Ex moiety (single bond or double bond) in formula (b2a) or formula (b2b) may be a part or all of C═O, amide, amine, oxime. Good.

Examples of the cross-linking agent having a bicyclo structure represented by the formula (b1a) or the formula (b1b) include those described in Sonobe, T .; Oisaki, K .; Kanai, M .; Chem. Sci. 2012, 3, 1572-1576, Lauber, M.M. B. Stahl, S .; S. ACS Catal. 2013, 3, 2612-2616, Hayashi, M .; Sasano, Y .; Nagasawa, S .; Shibuya, M .; Iwabuchi, Y .; Chem. Pharm. Bull 2011, 59, 1570, Sasano, Y.B. ; Nishiyama, T .; Tomizawa, M .; Shibuya, M .; Iwabuchi, Y .; According to Heterocycles 2013, 87, 2109, etc., Greg T. It can be obtained by compound modification according to “Bioconjugate Technologies, third edition” by Hermanson. Moreover, as long as the structure of Formula (b1a) or Formula (b1b) is satisfy | filled, you may use a commercial item.

The cross-linking agent having the bicyclo structure of the formula (b1a) or the formula (b1b) is a tryptophan residue of an IgG1 antibody, a bond represented by the following formula (B1), a bond represented by the following formula (B2), and a formula (B3 It is preferable to form at least one of the bonds represented by

(Wherein A ¹ , B ¹ , C ¹ , D ¹ , E ¹ , E ² , F ¹ and G ¹ have the same meaning as described above)

The number of bonds of the cross-linking agent per molecule of IgG1 antibody is, for example, 1 to 10 / antibody molecule, preferably 2 to 8 / antibody molecule, as an arithmetic average. Furthermore, the cross-linking agent is preferably bound in the constant region of the IgG1 antibody, and the ratio of the cross-linking agent bound in the constant region of the IgG1 antibody is, for example, It is 90% or more (on a molar basis), preferably 95% or more (on a molar basis), more preferably 99% or more (on a molar basis), and may be 100% (on a molar basis). The cross-linking agent is preferably bound in the constant region of IgG1 antibody. According to the combination of the antibody and the crosslinking agent of the present invention, the number of bonds of the crosslinking agent, its accuracy, the binding site, etc. can be controlled to a higher degree.

In addition, usually, one antitumor compound binds to an antibody via one crosslinker. Therefore, the number of binding of the cross-linking agent per antibody molecule, its standard deviation, and the binding site are in the same range as the number of binding of the antitumor compound per antibody molecule, its standard deviation and binding site, respectively.

4). Compound forming intermediate chain The cross-linking agent and the antitumor compound may be bonded directly or via an intermediate chain. As the compound forming the intermediate chain, various compounds (bifunctional protein coupling agents) used as a linker between an antibody and a drug in conventional antibody-drug conjugates can be used, and N-succinimidyl-4- (2-pyridylthio) pentanoate (SPP), N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT) ), Dimethyl adipimidate HCL, disuccinimidyl suberate, glutaraldehyde, bis (p-azidobenzoyl) hexanediamine, bis- (p-diazoniumbenzoyl) -ethylenediamine, or triene-2,6- Gii Cyanate, and the like 1,5-difluoro-2,4-dinitrobenzene. Preferably N-succinimidyl-4- (2-pyridylthio) pentanoate (SPP), N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxyl Rate (SMCC), more preferably succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC).

5). Method for Producing Antibody Drug Complex The antibody drug complex of the present invention can be produced without applying a transition metal catalyst or non-biocompatible conditions. Specifically, an IgG1 antibody, a crosslinking agent having an N oxyradical group or an N hydroxy group, and an antitumor compound may be mixed in an appropriate order, and an IgG1 antibody-crosslinking agent bond and a crosslinking agent-antitumor may be mixed. The order of formation of the bond between the active compounds is not limited. For example, after binding an IgG1 antibody and a cross-linking agent having an N oxy radical group or an N hydroxy group, an anti-tumor compound may be bound to the cross-linking agent, and a cross-linking agent bound with an anti-tumor compound and An IgG1 antibody may be bound. Furthermore, an antibody drug complex may be prepared by mixing an IgG1 antibody, a cross-linking agent having an N oxy radical group or an N hydroxy group, and an antitumor compound.
In the case of using a compound that forms an intermediate chain, the order of formation of an IgG1 antibody-crosslinking agent bond, a crosslinking agent-intermediate chain-forming compound bond, and an intermediate chain-forming compound-antitumor compound bond is not limited. The order of formation is adopted. In the case of focusing on the reaction of the intermediate chain forming compound, the compound forming the intermediate chain was reacted with a cross-linking agent (which may be bound to the antibody or may be bound to the antibody). Later, it may be conjugated with an anti-tumor compound, and a compound that forms an intermediate chain is conjugated with an anti-tumor compound, and then a cross-linking agent (which may be conjugated with an antibody or conjugated with an antibody). Or a cross-linking agent (which may be bound to an antibody or may be bound to an antibody), a compound forming an intermediate chain, and an antitumor compound (Furthermore, antibodies may be mixed as necessary) to obtain these conjugates. In the case of forming an intermediate chain, a product obtained by previously reacting an intermediate chain forming compound with a crosslinking agent having an N oxy radical group or an N hydroxy group (the product obtained by this reaction is hereinafter referred to as a composite linker) It is preferable to mix the IgG1 antibody and the antitumor compound in an appropriate order, and it is more preferable to react the composite linker with the IgG1 antibody and then react the antitumor compound.

The formation reaction of the IgG1 antibody-crosslinking agent bond can be performed, for example, in an aqueous solvent or a water-water-soluble organic solvent mixed solvent. From the viewpoint of convenience and safety, the reaction is performed in an aqueous solvent. Is preferred. Since the crosslinking agent having an N oxy radical group or an N hydroxy group has high solubility in water and can be used for a modification reaction in water, it is useful for a reaction excellent in biocompatibility.

Examples of the water-soluble organic solvent include alcohol solvents, ketone solvents, ether solvents, nitrile solvents, amide solvents, and sulfoxide solvents. Specific examples of these solvents include, but are not limited to, methanol, ethanol, propanol, ethylene glycol, acetone, dioxane, tetrahydrofuran, acetonitrile, dimethylformamide, dimethyl sulfoxide and the like.

Further, in the IgG1 antibody-crosslinking agent formation reaction, for example, nitrite, Bronsted acid, metal catalyst, photocatalyst, peracid, oxygen, or the like is used as an activator / oxidant for generating active species. Preferably, nitrite is used. When nitrite is used in the reaction, it is preferably used in an amount of 0.6 to 3 equivalents per equivalent of the crosslinking agent. When a Bronsted acid is used, it is preferably used in an amount such that the reaction solution has a pH of 2.0 to 4.5, more preferably in an amount such that the pH is 3.0 to 4.0. In the case of using a metal catalyst / photocatalyst, it is preferably used in an amount of 1 equivalent or less that can function as a catalyst with respect to 1 equivalent of the crosslinking agent. When using a peracid, preferably 1 equivalent or more with respect to 1 equivalent of the cross-linking agent, and when using oxygen, it is preferably used at normal pressure. The temperature and reaction time of the IgG1 antibody-crosslinking agent bond formation reaction may be appropriately adjusted by those skilled in the art depending on the type of catalyst, the amount of each reaction component, etc., but when nitrite is used in the reaction, The temperature is, for example, −10 to 60 ° C., preferably 20 to 40 ° C. The reaction time can be, for example, about 1 minute to 24 hours.

The cross-linking agent-antitumor compound bond forming reaction, the cross-linking agent-intermediate chain forming compound bond forming reaction, or the intermediate chain forming compound-anti-tumor compound bond forming reaction is the IgG1 antibody-crosslinking agent bond forming reaction. When the reaction is performed at the same timing as the above, or when the IgG1 antibody-crosslinking agent bond forming reaction is performed in one pot, it is preferably performed in the same solvent as the IgG1 antibody-crosslinking agent bond forming reaction.

Conversely, the cross-linking agent-antitumor compound-binding reaction, the cross-linking agent-intermediate-chain-forming compound bond-forming reaction, or the intermediate chain-forming compound-anti-tumor compound-binding, in addition to the IgG1 antibody-crosslinking agent-binding reaction. When performing the formation reaction, it may be performed in the same solvent as the IgG1 antibody-crosslinking agent bond formation reaction, or in a solvent different from the IgG1 antibody-crosslinking agent bond formation reaction, for example, in an organic solvent, The organic solvent may be water-soluble or water-insoluble.

Examples of the water-soluble or water-insoluble organic solvent include alcohol solvents, ketone solvents, ether solvents, ester solvents, nitrile solvents, amide solvents, sulfoxide solvents, halogen solvents, hydrocarbon solvents, and the like. Specific examples include methanol, n-hexanol, t-butyl alcohol, ethylene glycol, acetone, methyl ethyl ketone, cyclohexanone, dioxane, tetrahydrofuran, diethyl ether, ethyl acetate, acetonitrile, methylformamide, dimethylformamide, dimethylacetamide, Examples include dimethyl sulfoxide, methylene chloride, n-hexane, toluene, xylene and the like.

6). Pharmaceutical composition The antibody-drug conjugate of the present invention is purified to a predetermined purity, and then mixed with a pharmaceutically acceptable carrier and, if necessary, an excipient, a stabilizer, or the like. The preservative dosage form may be a freeze-dried preparation, an aqueous solution, or the like.
Carriers, excipients, stabilizers, etc. include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride, hexametho About 10 residues; such as nium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol, resorcinol, cyclohexanol; 3-pentanol and m-cresol) The following polypeptides; proteins such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; glycine, glutamine, asparagine, histidine, arginine or rigid Amino acids such as glucose; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metals Exemplified are non-ionic surfactants such as complexes (eg Zn-protein complexes); and / or TWEEN, PLURONICS or polyethylene glycol (PEG).

The pharmaceutical composition may be a sustained-release preparation. Sustained release formulations include semi-permeable materials of solid hydrophobic polymers containing antibodies, which are preferably in the form of molded articles such as films or microcapsules. Examples of formulation materials used in sustained release formulations include polyesters, hydrogels (eg, poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides (US Pat. No. 3,773,919). ), Copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, LUPRONDEPOT (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D- And degradable lactic acid-glycolic acid copolymers such as (−)-3-hydroxybutyric acid.

This application claims the benefit of priority based on Japanese Patent Application No. 2018-015404 filed on Jan. 31, 2018. The entire contents of the specification of Japanese Patent Application No. 2018-015404 filed on January 31, 2018 are incorporated herein by reference.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited by the following examples, but may be appropriately modified within a range that can meet the purpose described above and below. Of course, it is possible to implement them, and they are all included in the technical scope of the present invention.

Production Example 1
(Process 1)

tert-Butyl (9-hydroxy-9-azabicyclo [3.3.1] nonan-3-yl) carbamate (Compound 5 in the above formula) was prepared according to M.C. C. Franz, E .; M.M. Skoda, J .; R. Sacher, M.M. W. Epperly, J.M. P. Goff, J. et al. S. Greenberger and P.M. Wipf, Org. Biomol. Chem. , 2013, 11, pp 4147-4153. Manufactured on the basis of

(Process 2)

Compound 5 (245 mg, 0.96 mmol), tert-butyldimethylsilyl chloride (TBSCl, 1.45 g, 9.6 mmol) and imidazole (1.30 g, 19.2 mmol) were dissolved in dimethyl sulfoxide (DMSO, 10 mL). Stir at room temperature overnight. The resulting reaction solution was extracted three times with ethyl acetate / hexane = 4/1, and the organic layer was washed with a saturated aqueous sodium chloride solution and then dried over sodium sulfate. The desiccant (sodium sulfate) was removed with a filter, and the remaining solvent was removed under reduced pressure. Residual DMSO was completely removed by freeze-drying, and then purified by silica gel chromatography (CH ₂ Cl ₂ / MeOH = 50/1) to obtain Compound 6 in the above formula as a white solid (301 mg, 85%).

(Process 3)

Compound 6 (185 mg, 0.5 mmol) obtained in Step 2, 2,6-di-tert-butylpyridine (1.1 mL, 5 mmol) and trimethylsilyl trifluoromethanesulfonate (720 μL, 4 mmol) were dissolved in 5 mL of dichloromethane. Stir at room temperature overnight. Saturated ammonium chloride was added to the reaction solution, followed by extraction three times with ethyl acetate, and the organic layer was washed with a saturated aqueous sodium chloride solution and dried over sodium sulfate to obtain a yellow oily compound. To the obtained yellow oily compound was added succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC, 250 mg, 0.75 mmol), triethylamine (207 μL, 1.5 mmol), and 5 mL of dichloromethane. For 8 hours. Saturated ammonium chloride was added to the reaction mixture, and the mixture was extracted 3 times with ethyl acetate. The organic layer was washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, and silica gel chromatography (ethyl acetate / hexane = 1/2). Purification gave compound 7 in the above formula as a white solid (150 mg, 61%).

(Process 4)

Compound 7 (0.49 mg, 1 μmol) obtained in step 3, THF (4.3 μL), H ₂ O (1.4 μL), acetic acid (4.3 μL) was added to an Eppendorf tube (Note: Eppendorf is a registered trademark of Eppendorf) ) And stirred at room temperature for 8 hours to obtain Compound 8 in the above formula as a composite linker (MCC-ABNO-Linker). The obtained MCC-ABNO-Linker (8) was used in the subsequent steps as it was.

Example 1 (Synthesis of Herceptin-TrpADC (DM1))
Herceptin (registered trademark; Genentech Inc.) (0.53 mg, 3.6 nmol), MCC-ABNO-Linker (8) obtained in Production Example 1 (35 nmol, H ₂ O / THF / AcOH = 1/3/3) 0.35 μL), sodium nitrite (21 nmol, 2.1 μL as an aqueous solution) and H ₂ O (68.3 μL) were added to an Eppendorf tube and stirred at room temperature for 60 minutes. Thereafter, maytansinoid DM1 (0.03 mg, 42 nmol, 50 mM, 70 μL dissolved in NH ₄ HCO ₃ aqueous solution / DMSO = 4/1) was added and stirred overnight at room temperature. The obtained compound was subjected to size exclusion chromatography (chromatography system AKTA pure M1 (F9-R, PC set) / manufactured by GE Healthcare Japan, column: Superdex 200 Increase 10/100 GL, eluent: 30 mM NH ₄ HCO ₃ aq./CH ₃ CN = 4/1). Residual solvent and ammonium bicarbonate were removed by lyophilization to obtain 0.1 mg of the target compound, Herceptin-TrpADC (DM1).

About the obtained Herceptin-TrpADC (DM1), the mass of the sample component was measured using MALDI-TOF-MS (manufactured by Shimadzu Corporation: AXIMA-TOF2, matrix: 3,5-dimethoxy-4-hydroxycinnamic acid). The result shown in FIG. 1 was obtained. The peaks at 151 and 804 in FIG. 1 correspond to Herceptin-TrpADC (DM1). The result of measuring MALDI-TOF-MS under the same conditions for Herceptin before the reaction is as shown in FIG. 2, and 149 and 202 in FIG. 2 correspond to Herceptin. From FIG. 1 and FIG. 2, it can be seen that in Herceptin-TrpADC (DM1), two molecules of ABNO-MCC-DM1 are introduced on average.

Example 2
Herceptin (registered trademark; Genentech Inc.) (1 mg, 6.8 nmol), MCC-ABNO-Linker (8) obtained in Production Example 1 (272 nmol, dissolved in H ₂ O / THF / AcOH = 1/3/3) 2.72 μL), sodium nitrite (162 nmol, 16.2 μL as an aqueous solution) and H ₂ O (117 μL) were added to an Eppendorf tube and stirred at room temperature for 60 minutes. After stirring, maytansinoid DM1 (0.25 mg, 340 nmol, 135 μL dissolved in H ₂ O / DMSO = 4/1) was added and stirred overnight at room temperature.
After the reaction, the remaining substrate and by-products are precipitated by centrifugation (10,000 rpm, 15 minutes), and the supernatant fraction is subjected to size exclusion chromatography (chromatography system AKTA pure M1 (F9-R, PC set) / GE). The product was manufactured by Healthcare Japan Co., Ltd., and purified with column: Superdex 200 Increase 10/100 GL, eluent: PBS buffer (pH 7.4)). The target product fraction was concentrated by ultrafiltration (Amicon (registered trademark) Ultra-15 Centrifugal Filter Devices (Ultracel (registered trademark) -50k)) to obtain the target compound, Herceptin-TrpADC (DM1). The concentration of the target compound was quantified by absorbance at 280 nm and used in the subsequent experiments.
Using the obtained Herceptin-TrpADC (DM1), Teemu T. et al. Junttila, Guangmin Li, Kathlyn Parsons, Gal Lewis Phillips, Mark X. The cytotoxic activity to HER2 antigen positive cells was evaluated with reference to Sliwowski Breast Cancer Res Treat (2011) 128: 347-356.
Specifically, SK-BR-3 cells, which are HER2 antigen-positive human breast cancer lines, were cultured, and 100 μL each was added to a 96-well microplate so as to be 5 × 10 ³ cells / well 100 μL, followed by overnight culture.
On the next day, discard 50 μL of each medium from each well, and add 50 μL of Herceptin-TrpADC (DM1) dissolved in the above PBS buffer to a dilution of 0.002, 0.02, 0.2, 2 and 20 nM, respectively. Added in increments. In addition, 50 μL of Herceptin-TrpADC (DM1) -free medium was added to the control group. After culturing at 37 ° C. under 5% CO ₂ for 5 days, the microplate was removed from the incubator and allowed to stand at room temperature for 30 minutes. CellTiter-Glo Luminescent Cell Viability Assay (Promega) equivalent to the culture solution was added and stirred for 2 minutes. After standing at room temperature for 10 minutes, the amount of luminescence was measured with a luminometer (Promega), and the cell viability was calculated by the amount of luminescence (RLU: Relative Light Unit) relative to the control group.
As a comparative experiment, an example similar to the above was performed except that Herceptin was used instead of Herceptin-TrpADC (DM1), and the results were compared with the results of Herceptin-TrpADC (DM1).
The results are shown in FIG. As is clear from FIG. 3, Herceptin-TrpADC (DM1) showed higher cytotoxic activity than Herceptin.

The antibody-drug conjugate of the present invention is useful as a medicine for treating humans and animals (mammals).

Claims (11)

1. An IgG1 antibody and an antitumor compound are combined through a cross-linking agent-derived part having an N oxy radical group or an N hydroxy group,
   An antibody drug complex in which the N oxy radical group or N hydroxy group reacts with the tryptophan residue of the IgG1 antibody and is covalently bonded to the IgG1 antibody.
2. The antibody-drug conjugate according to claim 1, wherein the antitumor compound is bound to the constant region of the IgG1 antibody.
3. The ratio of the antitumor compound bound to the constant region of the IgG1 antibody is 90% or more (on a molar basis) with respect to the total antitumor compound bound to the IgG1 antibody. 3. The antibody drug conjugate according to 2.
4. The number of tryptophan residues present in the constant region of the IgG1 antibody is 6 to 18 per antibody before binding to the cross-linking agent, and the number of tryptophan residues present in the variable region is The antibody-drug conjugate according to any one of claims 1 to 3, which is 4 to 16 molecules / antibody molecule before binding.
5. The IgG1 antibody does not contain a tryptophan residue in the variable region,
   Or the antibody according to any one of claims 1 to 4, wherein when the variable region contains a tryptophan residue, the carbon atom at the 3-position of the indole ring in the side chain of the tryptophan residue is buried in the protein molecule. Drug complex.
6. 6. The antibody drug conjugate according to claim 1, wherein the IgG1 antibody is an anti-HER2 antibody.
7. The antibody-drug conjugate according to claim 6, wherein the anti-HER2 antibody is a humanized monoclonal antibody.
8. The antibody drug conjugate according to any one of claims 1 to 7, wherein the antitumor compound is maytansinoid.
9. The crosslinking agent having an N oxy radical group or an N hydroxy group has a bicyclo structure represented by the following formula (b1a) or the following formula (b1b):
   

   

   
   (Where
   A ¹ , B ¹ , C ¹ , D ¹ , E ¹ and E ² are each independently CR ¹ R ² ; C = CR ³ R ⁴ ; C = O; C = S; C = NR ⁵ ; NR ⁵ SiR ⁶ R ⁷ ; an oxygen atom; or a hetero atom other than a nitrogen atom, a silicon atom and an oxygen atom;
   F ¹ and G ¹ each independently represent CR ⁸ or a nitrogen atom,
   R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ and R ⁸ are each independently a hydrogen atom; a halogen atom; a heteroatom group; an optionally substituted carbon atom of 1 to 30 An alkyl group having 2 to 30 carbon atoms which may be substituted, an alkynyl group having 2 to 30 carbon atoms which may be substituted, an aryl group having 6 to 30 carbon atoms which may be substituted, An optionally substituted heteroaryl group having 4 to 30 carbon atoms, an optionally substituted aralkyl group having 7 to 30 carbon atoms, an optionally substituted cycloalkyl group having 3 to 30 carbon atoms; May be an alkyloxy group having 1 to 30 carbon atoms, an alkenyloxy group having 2 to 30 carbon atoms which may be substituted, an alkynyloxy group having 2 to 30 carbon atoms which may be substituted, or an optionally substituted group. Ants with 6 to 30 carbon atoms An aryloxy group, an optionally substituted heteroaryloxy group having 4 to 30 carbon atoms, an optionally substituted aralkyloxy group having 7 to 30 carbon atoms, and an optionally substituted cycloalkyl having 3 to 30 carbon atoms An oxy group; an optionally substituted polyalkyleneoxy group; or a reactive functional group,
   However, R ⁵ is not a halogen atom,
   E ¹ and E ² together may form an optionally substituted —CH (CH ₂ ) _m CH— group, m represents an integer from 0 to 12,
   The antitumor compound is bound to at least one group of A ¹ , B ¹ , C ¹ , D ¹ , E ¹ and E ² directly or through an intermediate chain)
   The bicyclo structure forms at least one of a bond represented by the following formula (B1), a bond represented by the following formula (B2), and a bond represented by the following formula (B3) with the tryptophan residue of the IgG1 antibody. 9. The antibody drug conjugate according to any one of 1 to 8.
   

   

   
   (Wherein A ¹ , B ¹ , C ¹ , D ¹ , E ¹ , E ² , F ¹ and G ¹ have the same meaning as described above)
10. The bicyclo structure of the cross-linking agent and the antitumor compound comprise N-succinimidyl-4- (2-pyridylthio) pentanoate (SPP), N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl- 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), dimethyl adipimidate HCL, disuccinimidyl suberate, glutaraldehyde, bis (p-azidobenzoyl) The antibody drug according to claim 9, which is linked using hexanediamine, bis- (p-diazoniumbenzoyl) -ethylenediamine, or triene-2,6-diisocyanate, 1,5-difluoro-2,4-dinitrobenzene. Complex.
11. A pharmaceutical composition comprising the antibody drug conjugate according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

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