Antibody drug conjugates comprising mutated Fc regions

Abstract

The present invention addresses the problem of desiring to develop a molecule which maintains anti-tumor activity and is excellent in terms of safety. Furthermore, it is desirable to develop an antibody drug conjugate that can be systemically administered and that can specifically deliver an STING agonist to a target cell or organ (e.g., a tumor site), and a therapeutic agent and / or a treatment method for diseases associated with STING agonist activity using the antibody drug conjugate. The diseases are, for example, diseases that can be treated by immunostimulation (e.g., cancer). The solution of the present invention is to provide an antibody drug conjugate in which a CDN derivative characterized by having a condensed tricyclic substituent and a specific antibody containing a mutant Fc region or a functional fragment of the antibody are bound via a linker, and an antibody containing a mutant Fc region.

Description

Technical Field

[0001] The present invention relates to a molecule that maintains antitumor activity and is excellent in safety, an antibody-drug conjugate in which a cyclic dinucleotide derivative having STING agonist activity is linked to an antibody comprising a mutant Fc region or a functional fragment of the antibody via a linker, a pharmaceutical composition containing the antibody-drug conjugate, an antibody comprising a mutant Fc region, a method for producing an Fc-containing molecule having an N297-linked sugar chain comprising a sugar chain derived from a sugar chain donor molecule, and the like. Background Art

[0002] The Fc region brings effector functions such as long plasma half-life, antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP) to antibodies. In the case where the Fc region is derived from a subclass called IgG used in most antibody medicines, the effector function depends on the binding of the Fc region to a family of receptors called Fcγ receptors (FcγR). However, with respect to FcγR binding activity, it is suggested that it is involved in safety risks such as infusion reactions (non-patent literature 1), so there are many cases where it is considered to be an undesirable property in antibody medicines.

[0003] There have been reports of amino acid substitutions in the Fc region of various IgG antibodies that reduce the binding activity to human FcγR (eg, Patent Documents 1 to 6 and Non-Patent Documents 2 and 3).

[0004] However, it has been reported that in antibody-drug conjugates in which a drug having an activity of activating immune cells (immunostimulant) is conjugated to an antibody, if the effector function of the antibody is reduced, the drug efficacy is also reduced (Non-Patent Documents 4 to 6, Patent Document 36).

[0005] STING (Stimulator of Interferon Genes) is a transmembrane adaptor protein that is locally present in the endoplasmic reticulum (Non-Patent Document 7). STING functions as a central molecule for the activation of innate immunity in mammals and is responsible for the frontline of defense against the entry of pathogens such as bacteria and viruses. It is known that the activation of STING is caused by signals when multiple cytoplasmic DNA sensors sense exogenous and endogenous DNA. Among the cytoplasmic DNA sensors, cGAS (Cyclic GMP-AMP Synthase) is considered to be an important DNA sensor. If cGAS senses DNA, cyclic dinucleotides (2', 3'-cGAMP) are produced, and the 2', 3'-cGAMP directly binds to STING, activating STING (Non-Patent Document 8). The activated STING moves to the Golgi apparatus and promotes the autophosphorylation of TBK1 (Tank-binding kinase 1) in the Golgi apparatus. TBK1 activated by autophosphorylation activates both the IRF3 (Interferon regulatory factor 3) transcription pathway (Non-patent document 9) and the NFκB transcription pathway (Non-patent document 10), increasing the production of interferons and inflammatory proteins called cytokines (type I IFN (Interferon), IL-6 (Interleukin-6), TNF-α (Tumor Necrosis Factor-α)). These proteins activate the acquired immune system including T cells that destroy pathogens and cancer cells through a complex cascade reaction.

[0006] According to recent studies, STING not only promotes host defense against microorganisms, but also promotes anti-tumor immunity. For example, when immunogenic tumors were transplanted into STING-deficient mice, the tumors proliferated more rapidly than in wild-type mice and TRIF-deficient mice. In addition, in STING-deficient mice, unlike TLR-like receptor, MyD88-deficient mice, and MAVS-deficient mice, spontaneous CD8 + The stimulation of T cells also disappeared. This suggests that the STING pathway, which is activated by cytoplasmic DNA sensing, is involved in the control of tumor proliferation (Non-patent Document 11). Other studies have also shown that STING is also necessary for the anti-tumor effects of radiotherapy (Non-patent Document 12) or anti-CD47 antibody therapy (Non-patent Document 13). DNA from dead tumor cells treated with radiation or anti-CD47 antibodies moves to the cytoplasm of dendritic cells and activates the cGAS-STING pathway, inducing IFN production and bridging innate immunity and acquired immunity. This study suggests that cross-sensitization involving dendritic cells activated via the STING pathway is important for inducing acquired immunity against tumors.

[0007] DMXAA, a flavonoid low molecular compound known as a vascular disruptor, induces type I IFN of macrophages and, therefore, shows strong anti-tumor activity in a mouse tumor model (non-patent document 14). DMXAA has excellent anti-tumor effects in preclinical settings, so it is expected to be an immunotherapy drug for non-small cell lung cancer, but it failed in human clinical trials (non-patent document 15). According to recent studies, it has been clarified that DMXAA is a specific agonist for STING in mice, but since human STING does not have species cross-reactivity, it cannot bind (non-patent document 16). DMXAA is ineffective for humans in terms of results, but according to studies in mouse models, it is suggested that low molecular weight drugs intervene in STING and effectively pre-activate (priming) CD8 + T cells, which can enhance anti-tumor immunity.

[0008] Cyclic dinucleotides (CDNs), which are other low molecular weight compounds, when administered to tumor-bearing mice showed an enhanced anti-tumor immune response caused by STING intervention, significantly inhibited tumor proliferation, and improved the survival rate of mice (Non-patent Document 17). CDNs are classified into CDNs (cyclic-di-GMP, cyclic-di-AMP, 3',3'-cGAMP) derived from bacteria with standard two 3'-5' phosphate bonds, and mixed-binding CDNs (2',3'-cGAMP) with non-standard 2'-5' phosphate bonds produced by cGAS in mammals. According to recent studies, mixed-binding CDNs are more able to activate diverse STINGs more generally than standard CDNs (Non-patent Document 18).

[0009] Natural CDNs are rapidly degraded by nucleases in the blood like most nucleic acid molecules and therefore cannot be administered in their original form. Therefore, synthetic low-molecular compounds having STING agonist activity in vivo have been developed (eg, Patent Documents 8 to 33).

[0010] MIW-815 (sometimes also called ADU-S100, MLRR-S2CDA or MLRR-CDA·2Na) is a STING agonist currently undergoing clinical trials as an anti-tumor agent. + ) is directly administered into the tumor. In the method of directly administering a STING agonist to a tumor, the drug can only be administered within a limited range within the tumor. In addition, it is difficult to directly administer to all of the multiple distant metastatic tumors, so there is a problem that the tumors that can be treated are limited. Non-patent document 19 records that the anti-tumor effect is shown by administration of MLRR-S2 CDA, but it is limited to administration within the tumor, and the anti-tumor effect of systemic administration (such as intravenous administration) is not shown. Non-patent document 20 records that if the STING agonist SB11285 is administered intravenously to a mouse tumor model, an anti-tumor effect will be shown, but it is not clear what kind of compound SB11285 specifically has. Patent document 21 records a conjugate containing an immunostimulatory compound, an antibody construct, and a linker, but does not record a specific example of a conjugate using a STING agonist as an immunostimulatory compound. Patent document 33 records a conjugate formed by combining a CDN having a specific structure with an antibody via a linker, but does not record an example of administering the conjugate in vivo, and the anti-tumor effect of the conjugate has not been confirmed. Patent Documents 34, 35, 37, and 38 describe conjugates formed by using a STING agonist as an immunostimulatory compound and binding it to an antibody via a linker, and describe examples of in vivo administration.

[0011] On the other hand, as a method for making the sugar chains added to medical antibodies or glycoprotein molecules including antibody Fc regions uniform, sugar chain transfer reactions using enzymes are known, and in particular, a one-pot method using two types of ENGase to directly transfer sugar chains to GlcNAc acceptors is known.

[0012] Prior art literature

[0013] Patent Literature

[0014] Patent Document 1: International Publication No. 1988 / 07089

[0015] Patent Document 2: International Publication No. 1999 / 51642

[0016] Patent Document 3: International Publication No. 2000 / 42072

[0017] Patent Document 4: International Publication No. 2013 / 092001

[0018] Patent Document 5: International Publication No. 2015 / 109131

[0019] Patent Document 6: International Publication No. 2020 / 086776

[0020] Patent Document 7: International Publication No. 2013 / 118858

[0021] Patent Document 8: International Publication No. 2014 / 099824

[0022] Patent Document 9: International Publication No. 2014 / 179335

[0023] Patent Document 10: International Publication No. 2014 / 189805

[0024] Patent Document 11: International Publication No. 2014 / 189806

[0025] Patent Document 12: International Publication No. 2015 / 074145

[0026] Patent Document 13: International Publication No. 2015 / 185565

[0027] Patent Document 14: International Publication No. 2016 / 096714

[0028] Patent Document 15: International Publication No. 2016 / 012305

[0029] Patent Document 16: International Publication No. 2016 / 145102

[0030] Patent Document 17: International Publication No. 2017 / 027646

[0031] Patent Document 18: International Publication No. 2017 / 027645

[0032] Patent Document 19: International Publication No. 2017 / 075477

[0033] Patent Document 20: International Publication No. 2017 / 093933

[0034] Patent Document 21: International Publication No. 2017 / 100305

[0035] Patent Document 22: International Publication No. 2017 / 123669

[0036] Patent Document 23: International Publication No. 2017 / 161349

[0037] Patent Document 24: International Publication No. 2017 / 175147

[0038] Patent Document 25: International Publication No. 2017 / 175156

[0039] Patent Document 26: International Publication No. 2018 / 009466

[0040] Patent Document 27: International Publication No. 2018 / 045204

[0041] Patent Document 28: International Publication No. 2018 / 060323

[0042] Patent Document 29: International Publication No. 2018 / 067423

[0043] Patent Document 30: International Publication No. 2018 / 065360

[0044] Patent Document 31: International Publication No. 2014 / 093936

[0045] Patent Document 32: International Publication No. 2018 / 009648

[0046] Patent Document 33: International Publication No. 2018 / 100558

[0047] Patent Document 34: International Publication No. 2021 / 202984

[0048] Patent Document 35: International Publication No. 2022 / 097117

[0049] Patent Document 36: International Publication No. 2019 / 084060

[0050] Patent Document 37: International Publication No. 2020 / 050406

[0051] Patent Document 38: International Publication No. 2021 / 177438

[0052] Non-patent literature

[0053] Non-patent literature 1: J Immnotoxicol; 5(1):11-5 (2008)

[0054] Non-patent literature 2: Protein Eng Des Sel.; 29(10):457-66(2016).

[0055] Non-patent document 3: J Biol Chem; 292(5):1865-75(2017)

[0056] Non-patent literature 4: Nature Cancer 2021, 2, 18-33

[0057] Non-patent literature 5: AACR 2021 Poster #1773

[0058] Non-patent document 6: Sean W. Smith, "SBT6050, a HER2-Directed TLR8ImmunoTACTMTherapeutic, is a Potent Human Myeloid Cell Agonist with Tumor-LocalizedActivity", World ADC 2020

[0059] Non-patent document 7: Nature 2008, 455, 674-678

[0060] Non-patent document 8: Mol. Cell, 2013, 51, 226-235

[0061] Non-patent literature 9: Science 2015a, 347, aaa2630

[0062] Non-patent document 10: J. Virol. 2014, 88, 5328-5341

[0063] Non-patent document 11: Immunity 2014, 41, 830-842

[0064] Non-patent document 12: Immunity 2014, 41, 843-852

[0065] Non-patent document 13: Nat. Med. 2015, 21, 1209-1215

[0066] Non-patent document 14: J. Immunol. 1994, 153, 4684-4693

[0067] Non-patent literature 15: J. Clin. Oncol. 2011, 29, 2965-2971

[0068] Non-patent document 16: J. Immunol. 2013, 190, 5216-5225

[0069] Non-patent literature 17: Sci.Rep.2016,6,19049

[0070] Non-patent document 18: Mol. Cell, 2015, 59, 891-903

[0071] Non-patent document 19: Cell Rep. 2015, 11, 1018-1030

[0072] Non-patent literature 20: AACR Tumor Immunology and Immunotherapy, 2017, Poster#A25 Summary of the invention

[0073] Problems to be solved by the invention

[0074] It is desirable to develop a molecule that maintains anti-tumor activity and is excellent in safety. In addition, it is desirable to develop an antibody drug conjugate, and a therapeutic agent and / or a therapeutic method for a disease associated with the STING agonist activity using the antibody drug conjugate, wherein the antibody drug conjugate can be systemically administered and can specifically deliver the STING agonist to target cells and organs (e.g., tumor sites), and the disease is, for example, a disease that can be treated by immune stimulation (e.g., cancer). Furthermore, it is desirable to establish a novel method for producing Fc-containing molecules (especially antibodies) having a uniform sugar chain structure, especially a method for improving the sugar chain transfer rate.

[0075] Solutions for solving problems

[0076] The present inventors conducted intensive research to solve the above-mentioned problems, and as a result, discovered an antibody-drug conjugate formed by binding a CDN derivative characterized by having a condensed tricyclic substituent to a specific antibody or a functional fragment of the antibody containing a mutant Fc region via a linker, and discovered that if the antibody-drug conjugate is systemically administered, it will show an anti-tumor effect in tumors expressing the antigen, and further discovered that it is excellent in safety, thereby completing the present invention. It was further discovered that the specific antibody containing a mutant Fc region is excellent in safety, and an improved method for producing Fc-containing molecules (especially antibodies) using a one-pot method was discovered, thereby completing the present invention.

[0077] That is, the present invention relates to the following, but is not limited thereto. [1]

[0079] An antibody-drug conjugate is represented by the following formula (II).

[0080]

[0081] (In the formula, m 1 is in the range of 1 to 10, Ab represents an antibody or a functional fragment of the antibody, the antibody or the functional fragment of the antibody optionally has a remodeled sugar chain, the amino acid residues at positions 234, 235, and 265 (all EU numbering) of the Fc region of the antibody or the functional fragment of the antibody are Ala, Ala, and Gly, respectively, herein, antibody represents an anti-EGFR antibody or an anti-CDH6 antibody, L represents a linker linking Ab and D, and Ab is optionally directly bound to L from its amino acid residue, or is bound to L from the sugar chain of Ab or a remodeled sugar chain. D represents a compound represented by the following formula (I).

[0082]

[0083] (Here, L and L 1 The hydroxyl groups contained in the 1 represents a group of the following formula (wherein the wavy line indicates the substitution position), Q and Q' each independently represent a hydroxyl group or a thiol group, and R 21 and R 22 Each independently represents a hydroxyl group or a fluorine atom, and W represents -NH- or a sulfur atom.

[0084] [2]

[0086] The antibody-drug conjugate according to [1], wherein D is represented by any one of the following two formulas (herein, L 1 , Q, Q' and W are as defined above).

[0087] [3]

[0089] The antibody-drug conjugate according to [1] or [2], wherein D is represented by any of the following two formulae (wherein the asterisk indicates binding to L, and Q, Q' and W are as defined above).

[0090] [4]

[0092] The antibody-drug conjugate according to any one of [1] to [3], wherein D is represented by any one of the following three formulae (wherein an asterisk indicates binding to L, and W is as defined above).

[0093] [5]

[0095] The antibody-drug conjugate according to any one of [1] to [4], wherein D is represented by any one of the following three formulae (where an asterisk indicates binding to L).

[0096] [6]

[0098] The antibody-drug conjugate according to any one of [1] to [4], wherein D is represented by any one of the following four formulae (where an asterisk indicates binding to L).

[0099] [7]

[0101] The antibody-drug conjugate according to any one of [1] to [4] or [6], wherein D is represented by the following formula (herein, an asterisk indicates binding to L).

[0102] [8]

[0104] The antibody drug conjugate according to any one of [1] to [7], wherein the linker L is represented by -Lb-La-Lp-Lc-*, wherein the asterisk indicates binding to the drug D, Lp represents a linker consisting of an amino acid sequence that can be cut in the target cell, or Lp is absent, and La represents any one selected from the following group: -C(=O)-(CH2CH2)n 2 -C(=O)-, -C(=O)-(CH2CH2)n 2 -CH2-C(=O)-, -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2)n 3 -C(=O)-, -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2)n 3 -CH2-C(=O)-, -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2O)n 3 -CH2-C(=O)-, -(CH2)n 4 -O-C(=O)-, and -(CH2)n9 -C(=O)-(here, n 2 Integer from 1 to 3, n 3 represents an integer from 1 to 5, n 4 represents an integer from 0 to 2, n 9 represents an integer of 2 to 7), Lb represents a spacer that binds La to the sugar chain or reconstructed sugar chain of Ab or a spacer that binds La to the cysteine ​​residue of Ab, and Lc represents -NH-CH2-, -NH-phenyl-CH2-O(C=O)- or -NH-heteroaryl-CH2-O(C=O)-, or Lc is absent. [9]

[0106] The antibody-drug conjugate according to [8], wherein Lc is -NH-CH2-.

[10]

[0108] The antibody-drug conjugate according to [8] or [9], wherein Lp is any one of -GGFG-, -GGPI-, -GGVA-, -GGFM-, -GGVCit-, -GGFCit-, -GGICit-, -GGPL-, -GGAQ-, or -GGPP-.

[11]

[0110] The antibody-drug conjugate according to

[10] , wherein Lp is -GGFG- or -GGPI-.

[12]

[0112] The antibody drug conjugate according to any one of [8] to

[11] , wherein La represents any one selected from the group consisting of -C(=O)-CH2CH2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)3-CH2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)4-CH2-C(=O)-, and -(CH2)5-C(=O)-.

[13]

[0114] The antibody-drug conjugate according to any one of [8] to

[12] , wherein Lb is represented by any one of the following formulae.

[0115]

[0116] (In the structural formula of Lb shown above, the asterisk indicates binding to La, and the wavy line indicates binding to the sugar chain or reconstructed sugar chain of Ab)

[14]

[0118] The antibody drug conjugate according to any one of [8] to

[12] , wherein Lb is -(succinimidyl-3-yl-N)-, wherein -(succinimidyl-3-yl-N)- represents the following structural formula.

[0119]

[0120] Here, the asterisk indicates binding to La, and the wavy line indicates binding to the side chain of the cysteine ​​residue of the antibody by forming a thioether.

[15]

[0122] The antibody-drug conjugate according to any one of [8] to

[13] , wherein the linker L is represented by -Lb-La-Lp-Lc-*, wherein the asterisk indicates binding to the drug D, Lp is -GGFG- or -GGPI-, La represents -C(=O)-CH2CH2-C(=O)-, Lb represents the following formula, and Lc represents -NH-CH2-.

[0123]

[0124] (In the structural formula of Lb shown above, the asterisk indicates binding to La, and the wavy line indicates binding to the sugar chain or reconstructed sugar chain of Ab)

[16]

[0126] The antibody-drug conjugate according to any one of [1] to

[15] , wherein the average number of drug-bound molecules per antibody molecule in the antibody-drug conjugate is in the range of 1 to 10.

[17]

[0128] The antibody-drug conjugate according to

[16] , wherein the average drug binding number per antibody molecule in the antibody-drug conjugate is in the range of 1 to 5.

[18]

[0130] The antibody-drug conjugate according to

[17] , wherein the average number of drug-bound molecules per antibody molecule in the antibody-drug conjugate is in the range of 1 to 3, or 3 to 5.

[19]

[0132] The antibody-drug conjugate according to any one of [1] to

[18] , wherein the antibody is bound to L via a sugar chain linked to Asn297 of the antibody (N297 sugar chain).

[20]

[0134] The antibody-drug conjugate according to

[19] , wherein the N297 sugar chain is a remodeled sugar chain. [twenty one]

[0136] The antibody-drug conjugate according to

[19] or

[20] , wherein the N297 sugar chain is N297-(Fuc)MSG1 or N297-(Fuc)SG having a structure represented by the following formula.

[0137]

[0138] In the formula, the wavy line indicates the connection with Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at the 2-position of the non-reducing end of the sialic acid at the 1-3 chain side of the branched chain of the β-Man of the N297 sugar chain, and the asterisk indicates the bond with the nitrogen atom at the 1-position or 3-position on the 1,2,3-triazole ring of Lb in the linker L, and n 5 An integer from 2 to 5.

[0139]

[0140] In the formula, the wavy line indicates the connection with Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at the 2-position of the sialic acid at both the 1-3 chain side and the 1-6 chain side of the β-Man branch of the N297 sugar chain, and the asterisk indicates the bond to the nitrogen atom at the 1-position or 3-position on the 1,2,3-triazole ring of Lb in the linker L, and n 5 An integer from 2 to 5. [twenty two]

[0142] The antibody-drug conjugate according to any one of

[19] to

[21] , which is represented by the following formula.

[0143]

[0144] (In the formula, m 2 represents an integer of 1 or 2, L is a linker linking the N297 sugar chain and D, and according to the above definition, Ab represents an antibody or a functional fragment of the antibody, the amino acid residues at positions 234, 235, and 265 (all EU numbering) of the Fc region of the antibody or the functional fragment of the antibody are Ala, Ala, and Gly, respectively, here, the antibody represents an anti-EGFR antibody or an anti-CDH6 antibody, and the N297 sugar chain represents N297-(Fuc)MSG1 or N297-(Fuc)SG having a structure shown in the following formula.

[0145]

[0146] In the formula, the wavy line indicates the connection with Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at the 2-position of the non-reducing end of the sialic acid at the 1-3 chain side of the branched chain of the β-Man of the N297 sugar chain, and the asterisk indicates the bond with the nitrogen atom at the 1-position or 3-position on the 1,2,3-triazole ring of Lb in the linker L, and n 5 Indicates an integer from 2 to 5.

[0147]

[0148] In the formula, the wavy line indicates the connection with Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at the 2-position of the sialic acid at both the 1-3 chain side and the 1-6 chain side of the β-Man branch of the N297 sugar chain, and the asterisk indicates the bond to the nitrogen atom at the 1-position or 3-position on the 1,2,3-triazole ring of Lb in the linker L, and n 5 Indicates an integer from 2 to 5.

[0149] D is represented by any of the following four formulas.

[0150]

[0151] Here, in the formula, the asterisk indicates binding with L) [twenty three]

[0153] The antibody-drug conjugate according to

[22] is selected from the following formula.

[0154]

[0155]

[0156]

[0157] In the above structural formulas, m 2 Represents an integer of 1 or 2, Ab represents an antibody or a functional fragment of the antibody, the amino acid residues at positions 234, 235, and 265 (all EU numbering) of the Fc region of the antibody or the functional fragment of the antibody are Ala, Ala, and Gly, respectively, here, antibody represents an anti-EGFR antibody or an anti-CDH6 antibody, and the N297 sugar chain represents either N297-(Fuc)MSG1 or N297-(Fuc)SG having a structure shown in the following formula.

[0158]

[0159] In the formula, the wavy line indicates the connection with Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at the 2-position of the non-reducing end of the sialic acid at the 1-3 chain side of the branched chain of the β-Man of the N297 sugar chain, and the asterisk indicates the bond with the nitrogen atom at the 1-position or 3-position on the 1,2,3-triazole ring of Lb in the linker L, and n 5 Indicates an integer from 2 to 5.

[0160]

[0161] In the formula, the wavy line indicates the connection with Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at the 2-position of the sialic acid at both the 1-3 chain side and the 1-6 chain side of the β-Man branch of the N297 sugar chain, and the asterisk indicates the bond to the nitrogen atom at the 1-position or 3-position on the 1,2,3-triazole ring of Lb in the linker L, and n 5 Indicates an integer from 2 to 5. [twenty four]

[0163] The antibody-drug conjugate according to

[23] is selected from the following formula.

[0164]

[0165]

[0166] In the above structural formulas, m 2 Represents an integer of 1 or 2, Ab represents an antibody or a functional fragment of the antibody, the amino acid residues at positions 234, 235, and 265 (all EU numbering) of the Fc region of the antibody or the functional fragment of the antibody are Ala, Ala, and Gly, respectively, here, antibody represents an anti-EGFR antibody or an anti-CDH6 antibody, and the N297 sugar chain represents either N297-(Fuc)MSG1 or N297-(Fuc)SG having a structure shown in the following formula.

[0167]

[0168] In the formula, the wavy line indicates the connection with Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5-CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at the 2-position of the non-reducing end of the sialic acid at the 1-3 chain side of the branched chain of the β-Man of the N297 sugar chain, and the asterisk indicates the bond with the nitrogen atom at the 1-position or 3-position on the 1,2,3-triazole ring of Lb in the linker L, and n 5 Indicates an integer from 2 to 5.

[0169]

[0170] In the formula, the wavy line indicates the connection with Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at the 2-position of the sialic acid at both the 1-3 chain side and the 1-6 chain side of the β-Man branch of the N297 sugar chain, and the asterisk indicates the bond to the nitrogen atom at the 1-position or 3-position on the 1,2,3-triazole ring of Lb in the linker L, and n 5 Indicates an integer from 2 to 5.

[25]

[0172] The antibody-drug conjugate according to any one of [1] to

[24] , wherein the antibody is an anti-EGFR antibody.

[26]

[0174] The antibody-drug conjugate according to any one of [1] to

[24] , wherein the antibody is an anti-CDH6 antibody.

[27]

[0176] The antibody-drug conjugate according to

[25] , wherein the antibody is an antibody comprising a light chain consisting of the amino acid sequence recorded in SEQ ID NO: 30 and a heavy chain consisting of the amino acid sequence recorded in SEQ ID NO: 32, or an antibody comprising a light chain consisting of the amino acid sequence recorded in SEQ ID NO: 30 and a heavy chain consisting of the amino acid sequence recorded in SEQ ID NO: 33, or an antibody comprising a heavy chain in which one or more amino acid residues at the carboxyl terminus are deleted in any of the above antibodies.

[28]

[0178] The antibody-drug conjugate according to

[26] , wherein the antibody is an antibody comprising a light chain consisting of the amino acid sequence recorded in SEQ ID NO: 44 and a heavy chain consisting of the amino acid sequence recorded in SEQ ID NO: 46, or an antibody comprising a heavy chain in which one or more amino acid residues at the carboxyl terminus are deleted.

[29]

[0180] The antibody-drug conjugate according to

[25] , wherein the antibody is an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence recorded in SEQ ID NO: 29 and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence recorded in SEQ ID NO: 31, and the amino acid residues at positions 234, 235, and 265 (all EU numbering) in the Fc region of the antibody are Ala, Ala, and Gly, respectively.

[30]

[0182] The antibody-drug conjugate according to

[26] , wherein the antibody is an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence recorded in SEQ ID NO: 43 and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence recorded in SEQ ID NO: 45, and the amino acid residues at positions 234, 235, and 265 (all EU numbering) in the Fc region of the antibody are Ala, Ala, and Gly, respectively.

[31]

[0184] The antibody-drug conjugate according to

[25] , wherein the antibody is an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence recorded in SEQ ID NO: 23, CDRL2 consisting of the amino acid sequence recorded in SEQ ID NO: 24, and CDRL3 consisting of the amino acid sequence recorded in SEQ ID NO: 25, and a heavy chain comprising CDRH1 consisting of the amino acid sequence recorded in SEQ ID NO: 26, CDRH2 consisting of the amino acid sequence recorded in SEQ ID NO: 27, and CDRH3 consisting of the amino acid sequence recorded in SEQ ID NO: 28, and the amino acid residues at positions 234, 235, and 265 (all EU numbering) in the Fc region of the antibody are Ala, Ala, and Gly, respectively.

[32]

[0186] The antibody-drug conjugate according to

[26] , wherein the antibody is an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence recorded in SEQ ID NO: 37, CDRL2 consisting of the amino acid sequence recorded in SEQ ID NO: 38, and CDRL3 consisting of the amino acid sequence recorded in SEQ ID NO: 39, and a heavy chain comprising CDRH1 consisting of the amino acid sequence recorded in SEQ ID NO: 40, CDRH2 consisting of the amino acid sequence recorded in SEQ ID NO: 41, and CDRH3 consisting of the amino acid sequence recorded in SEQ ID NO: 42, wherein the amino acid residues at positions 234, 235, and 265 (all EU numbering) in the Fc region of the antibody are Ala, Ala, and Gly, respectively.

[33]

[0188] An antibody-drug conjugate is represented by the following formula.

[0189]

[0190] In the formula, Ab represents any one selected from the following groups: an antibody comprising a light chain consisting of the amino acid sequence recorded in sequence number 30 and a heavy chain consisting of the amino acid sequence recorded in sequence number 32; an antibody comprising a light chain consisting of the amino acid sequence recorded in sequence number 30 and a heavy chain consisting of the amino acid sequence recorded in sequence number 33; an antibody comprising a light chain consisting of the amino acid sequence recorded in sequence number 44 and a heavy chain consisting of the amino acid sequence recorded in sequence number 46; and an antibody comprising a heavy chain in which one or more amino acid residues at the carboxyl terminus are deleted in any of the antibodies.

[0191] The N297 sugar chain is represented by the following formula.

[0192]

[0193] (Wherein, the wavy line indicates the connection with Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at the 2-position of the sialic acid at both the 1-3 chain side and the 1-6 chain side of the β-Man branch of the N297 sugar chain, and the asterisk indicates that it is bonded to the nitrogen atom at the 1-position or 3-position on the 1,2,3-triazole ring of Lb in the linker L, n 5 is 3, and m 2 For 2)

[34]

[0195] An antibody-drug conjugate is represented by the following formula.

[0196]

[0197]

[0198] In the formula, Ab represents any one selected from the following groups: an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence recorded in SEQ ID NO: 29 and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence recorded in SEQ ID NO: 31; and an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence recorded in SEQ ID NO: 43 and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence recorded in SEQ ID NO: 45, wherein the amino acid residues at positions 234, 235, and 265 (all EU numbering) in the Fc region of the antibody are Ala, Ala, and Gly, respectively, and the N297 sugar chain is represented by the following formula.

[0199]

[0200] (Wherein, the wavy line indicates the connection with Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at the 2-position of the sialic acid at both the 1-3 chain side and the 1-6 chain side of the β-Man branch of the N297 sugar chain, and the asterisk indicates that it is bonded to the nitrogen atom at the 1-position or 3-position on the 1,2,3-triazole ring of Lb in the linker L, n 5 is 3, and m 2 For 2)

[35]

[0202] An antibody-drug conjugate is represented by the following formula.

[0203]

[0204] In the formula, Ab represents any one selected from the following groups: an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence recorded in SEQ ID NO: 23, CDRL2 consisting of the amino acid sequence recorded in SEQ ID NO: 24, and CDRL3 consisting of the amino acid sequence recorded in SEQ ID NO: 25, and a heavy chain comprising CDRH1 consisting of the amino acid sequence recorded in SEQ ID NO: 26, CDRH2 consisting of the amino acid sequence recorded in SEQ ID NO: 27, and CDRH3 consisting of the amino acid sequence recorded in SEQ ID NO: 28; and an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence recorded in SEQ ID NO: 37, CDRL2 consisting of the amino acid sequence recorded in SEQ ID NO: 38, and CDRL3 consisting of the amino acid sequence recorded in SEQ ID NO: 39, and a heavy chain comprising CDRH1 consisting of the amino acid sequence recorded in SEQ ID NO: 40, CDRH2 consisting of the amino acid sequence recorded in SEQ ID NO: 41, and CDRH3 consisting of the amino acid sequence recorded in SEQ ID NO: 42. The amino acid residues at positions 234, 235, and 265 (all EU numbering) of the Fc region of this antibody are Ala, Ala, and Gly, respectively. The N297 sugar chain is represented by the following formula.

[0205]

[0206] (Wherein, the wavy line indicates the connection with Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5-CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at the 2-position of the sialic acid at both the 1-3 chain side and the 1-6 chain side of the β-Man branch of the N297 sugar chain, and the asterisk indicates that it is bonded to the nitrogen atom at the 1-position or 3-position on the 1,2,3-triazole ring of Lb in the linker L, n 5 is 3, and m 2 For 2)

[36]

[0208] An antibody-drug conjugate is represented by the following formula.

[0209]

[0210] In the formula, Ab represents any one selected from the following groups: an antibody comprising a light chain consisting of the amino acid sequence recorded in sequence number 30 and a heavy chain consisting of the amino acid sequence recorded in sequence number 32; an antibody comprising a light chain consisting of the amino acid sequence recorded in sequence number 30 and a heavy chain consisting of the amino acid sequence recorded in sequence number 33; an antibody comprising a light chain consisting of the amino acid sequence recorded in sequence number 44 and a heavy chain consisting of the amino acid sequence recorded in sequence number 46; and an antibody comprising a heavy chain in which one or more amino acid residues at the carboxyl terminal are deleted in any of the antibodies. The N297 sugar chain is represented by the following formula.

[0211]

[0212] (Wherein, the wavy line indicates the connection with Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at the 2-position of the sialic acid at both the 1-3 chain side and the 1-6 chain side of the β-Man branch of the N297 sugar chain, and the asterisk indicates that it is bonded to the nitrogen atom at the 1-position or 3-position on the 1,2,3-triazole ring of Lb in the linker L, n 5 is 3, and m 2 1)

[37]

[0214] An antibody-drug conjugate is represented by the following formula.

[0215]

[0216]

[0217] In the formula, Ab represents any one selected from the following groups: an antibody comprising a light chain comprising a light chain variable region consisting of an amino acid sequence recorded in SEQ ID NO: 29 and a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence recorded in SEQ ID NO: 31; and an antibody comprising a light chain comprising a light chain variable region consisting of an amino acid sequence recorded in SEQ ID NO: 43 and a heavy chain comprising a heavy chain variable region consisting of an amino acid sequence recorded in SEQ ID NO: 45. The amino acid residues at positions 234, 235, and 265 (all EU numbering) of the Fc region of the antibody are Ala, Ala, and Gly, respectively. The N297 sugar chain is represented by the following formula.

[0218]

[0219] (Wherein, the wavy line indicates the connection with Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at the 2-position of the sialic acid at both the 1-3 chain side and the 1-6 chain side of the β-Man branch of the N297 sugar chain, and the asterisk indicates that it is bonded to the nitrogen atom at the 1-position or 3-position on the 1,2,3-triazole ring of Lb in the linker L, n 5 is 3, and m 2 1)

[38]

[0221] An antibody-drug conjugate is represented by the following formula.

[0222]

[0223] In the formula, Ab represents any one selected from the following groups: an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence recorded in SEQ ID NO: 23, CDRL2 consisting of the amino acid sequence recorded in SEQ ID NO: 24, and CDRL3 consisting of the amino acid sequence recorded in SEQ ID NO: 25, and a heavy chain comprising CDRH1 consisting of the amino acid sequence recorded in SEQ ID NO: 26, CDRH2 consisting of the amino acid sequence recorded in SEQ ID NO: 27, and CDRH3 consisting of the amino acid sequence recorded in SEQ ID NO: 28; and an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence recorded in SEQ ID NO: 37, CDRL2 consisting of the amino acid sequence recorded in SEQ ID NO: 38, and CDRL3 consisting of the amino acid sequence recorded in SEQ ID NO: 39, and a heavy chain comprising CDRH1 consisting of the amino acid sequence recorded in SEQ ID NO: 40, CDRH2 consisting of the amino acid sequence recorded in SEQ ID NO: 41, and CDRH3 consisting of the amino acid sequence recorded in SEQ ID NO: 42. The amino acid residues at positions 234, 235, and 265 (all EU numbering) of the Fc region of this antibody are Ala, Ala, and Gly, respectively. The N297 sugar chain is represented by the following formula.

[0224]

[0225] (Wherein, the wavy line indicates the connection with Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at the 2-position of the sialic acid at both the 1-3 chain side and the 1-6 chain side of the β-Man branch of the N297 sugar chain, and the asterisk indicates that it is bonded to the nitrogen atom at the 1-position or 3-position on the 1,2,3-triazole ring of Lb in the linker L, n 5 is 3, and m 2 1)

[39]

[0227] A STING agonist comprising the antibody-drug conjugate described in any one of [1] to

[38] .

[40]

[0229] A pharmaceutical composition comprising the antibody-drug conjugate according to any one of [1] to

[38] .

[41]

[0231] An antitumor agent comprising the antibody-drug conjugate according to any one of [1] to

[38] .

[42]

[0233] The antitumor agent according to

[41] , wherein the tumor is lung cancer, kidney cancer, urothelial carcinoma, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, hepatocellular carcinoma, bladder cancer, gastric cancer, esophageal cancer, uterine cancer, testicular cancer, cervical cancer, placental choriocarcinoma, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, gastrointestinal stromal tumor (GIST), gallbladder cancer, bile duct cancer, adrenal cancer, squamous cell carcinoma, pharyngeal cancer, tongue cancer, auditory organ cancer, thymic cancer, small intestine cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, sarcoma, EGFR mutation-positive cancer or cancer with mutation in EGFR downstream signaling.

[0234] [42-2]

[0235] The antitumor agent according to

[41] , wherein the tumor is a CDH6-positive cancer.

[0236] [42-3]

[0237] The antitumor agent according to

[41] or [42-2], wherein the tumor is ovarian cancer or renal cancer.

[43]

[0239] A method for treating cancer, comprising administering any one selected from the group consisting of an antibody drug conjugate as described in any one of [1] to

[38] , a STING agonist as described in

[39] , a pharmaceutical composition as described in

[40] , and an anti-tumor agent as described in

[41] ,

[42] , [42-2] or [42-3].

[44]

[0241] The method described in

[43] , wherein the cancer is lung cancer, kidney cancer, urothelial carcinoma, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, hepatocellular carcinoma, bladder cancer, gastric cancer, esophageal cancer, uterine cancer, testicular cancer, cervical cancer, placental choriocarcinoma, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, gastrointestinal stromal tumor (GIST), gallbladder cancer, bile duct cancer, adrenal cancer, squamous cell carcinoma, pharyngeal cancer, tongue cancer, auditory organ cancer, thymus cancer, small intestine cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, sarcoma, EGFR mutation-positive cancer or cancer with mutation in EGFR downstream signaling.

[0242] [44-2]

[0243] The method according to

[43] , wherein the cancer is CDH6-positive cancer.

[0244] [44-3]

[0245] The method according to

[43] or [44-2], wherein the cancer is ovarian cancer or renal cancer.

[45]

[0247] An antibody, which is (A) antibody or (B) antibody.

[0248] (A) an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 23, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 24, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 25, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 26, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 27, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 28, wherein the amino acid residues at positions 234, 235, and 265 (all EU numbering) in the Fc region of the antibody are Ala, Ala, and Gly, respectively; (B) The antibody comprises a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 37, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 38, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 39, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 40, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 41, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 42, wherein the amino acid residues at positions 234, 235, and 265 (all EU numbering) in the Fc region of the antibody are Ala, Ala, and Gly, respectively.

[0249] [45-2]

[0250] An antibody, which is (A) antibody or (B) antibody.

[0251] (A) an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 23, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 24, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 25, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 26, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 27, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 28, wherein the amino acid residues at positions 234, 235, and 265 (all EU numbering) in the Fc region of the antibody are Ala, Ala, and Gly, respectively, and the antibody has binding activity to EGFR; (B) The antibody comprises a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 37, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 38, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 39, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 40, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 41, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 42, wherein the amino acid residues at positions 234, 235, and 265 (all EU numbering) in the Fc region of the antibody are Ala, Ala, and Gly, respectively, and the antibody has binding activity to CDH6.

[46]

[0253] An antibody, which is (A) antibody or (B) antibody.

[0254] (A) The antibody comprises a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 29 and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 31, wherein the amino acid residues at positions 234, 235, and 265 (all EU numbering) in the Fc region of the antibody are Ala, Ala, and Gly, respectively; (B) The antibody comprises a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 43 and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 45, wherein the amino acid residues at positions 234, 235, and 265 (all EU numbering) in the Fc region of the antibody are Ala, Ala, and Gly, respectively.

[0255] [46-2]

[0256] An antibody, which is (A) antibody or (B) antibody.

[0257] (A) an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 29 and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 31, wherein the amino acid residues at positions 234, 235, and 265 (all EU numbering) in the Fc region of the antibody are Ala, Ala, and Gly, respectively, and the antibody has binding activity to EGFR; (B) an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 43 and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 45, wherein the amino acid residues at positions 234, 235, and 265 (all EU numbering) in the Fc region of the antibody are Ala, Ala, and Gly, respectively, and the antibody has binding activity to CDH6.

[47]

[0259] An antibody which is (A) antibody, (B) antibody or (C) antibody.

[0260] (A) The antibody is an antibody comprising a light chain consisting of the amino acid sequence recorded in SEQ ID NO: 30 and a heavy chain consisting of the amino acid sequence recorded in SEQ ID NO: 32, or an antibody comprising a light chain consisting of the amino acid sequence recorded in SEQ ID NO: 30 and a heavy chain consisting of the amino acid sequence recorded in SEQ ID NO: 33; (B) The antibody is an antibody comprising a light chain consisting of the amino acid sequence recorded in SEQ ID NO: 44 and a heavy chain consisting of the amino acid sequence recorded in SEQ ID NO: 46; (C) The antibody is an antibody comprising a heavy chain in which one or more amino acid residues at the carboxyl terminus are deleted in any one of the antibodies (A) to (B).

[0261] [47-2]

[0262] An antibody which is (A) antibody, (B) antibody or (C) antibody.

[0263] (A) The antibody is an antibody comprising a light chain consisting of the amino acid sequence recorded in SEQ ID NO: 30 and a heavy chain consisting of the amino acid sequence recorded in SEQ ID NO: 32, or an antibody comprising a light chain consisting of the amino acid sequence recorded in SEQ ID NO: 30 and a heavy chain consisting of the amino acid sequence recorded in SEQ ID NO: 33 and having binding activity to EGFR; (B) The antibody is an antibody comprising a light chain consisting of the amino acid sequence recorded in SEQ ID NO: 44 and a heavy chain consisting of the amino acid sequence recorded in SEQ ID NO: 46 and having binding activity to CDH6; (C) The antibody is an antibody comprising a heavy chain in which one or more amino acid residues at the carboxyl terminus are deleted in any one of the antibodies (A) to (B).

[48]

[0265] The pharmaceutical composition of

[40] or the antitumor agent of

[41] ,

[42] , [42-2] or [42-3], which is administered in combination with other drugs.

[49]

[0267] The pharmaceutical composition according to

[40] , or the antitumor agent according to

[41] ,

[42] , [42-2] or [42-3], comprising another drug.

[50]

[0269] The method according to

[43] ,

[44] , [44-2] or [44-3], wherein any one selected from the group consisting of the antibody drug conjugate described in any one of [1] to

[38] , the STING agonist described in

[39] , the pharmaceutical composition described in

[40] and the anti-tumor agent described in

[41] ,

[42] , [42-2] or [42-3] is administered in combination with other drugs.

[51]

[0271] A polynucleotide encoding the amino acid sequence of the antibody described in any one of

[45] , [45-2],

[46] , [46-2],

[47] and [47-2].

[52]

[0273] An expression vector comprising the polynucleotide described in

[51] .

[53]

[0275] A host cell transformed with the expression vector as described in

[52] .

[54]

[0277] A culture method comprising culturing the host cell as described in

[53] .

[55]

[0279] A method for producing an antibody, comprising the step of culturing the host cell described in

[53] , and the step of collecting the target antibody from the culture obtained in the culturing step.

[56]

[0281] A method for producing an Fc-containing molecule having an N297-linked sugar chain including a sugar chain derived from a sugar chain donor molecule, the method comprising the following step 1.

[0282] (Step 1) is a step of reacting an acceptor molecule with a sugar chain donor molecule containing a sugar chain including GlcNAc with an unactivated reducing end in the presence of an enzyme-A, an enzyme-B, and an additive to obtain a reaction mixture, wherein the enzyme-A is an endo-β-N-acetylglucosaminidase that uses the N297-linked sugar chain of an Fc-containing molecule as a substrate, the enzyme-B is an endo-β-N-acetylglucosaminidase that uses the sugar chain of the sugar chain donor molecule as a substrate, the additive is selected from a monovalent salt, an organic solvent, a surfactant, a sugar, an amino acid, or any combination thereof, and the acceptor molecule is an Fc-containing molecule having a core GlcNAc to which fucose is optionally added as the N297-linked sugar chain.

[57]

[0284] The method described in

[56] further includes the following step 2.

[0285] (Step 2) is a step of contacting the reaction mixture with a cation exchange chromatography medium or a multimodal chromatography medium under acidic conditions to recover Fc-containing molecules having N297-linked sugar chains including sugar chains derived from sugar chain donor molecules.

[58]

[0287] The method according to

[56] or

[57] , wherein the Fc-containing molecule is an antibody or a functional fragment of the antibody, and the amino acid residues at positions 234, 235, and 265 (all EU numbering) of the Fc region of the antibody or the functional fragment of the antibody are Ala, Ala, and Gly, respectively.

[59]

[0289] The method according to any one of

[56] to

[58] , wherein the additive is a monovalent salt.

[60]

[0291] The method according to any one of

[56] to

[59] , wherein the additive is a monovalent alkali metal salt.

[61]

[0293] The method according to any one of

[56] to

[60] , wherein the additive is sodium chloride.

[62]

[0295] The method according to any one of

[56] to

[61] , wherein the additive is 50 to 1000 mM of a monovalent salt.

[63]

[0297] The method according to any one of

[56] to

[62] , wherein the additive is 500 mM or less sodium chloride.

[64]

[0299] The method according to any one of

[56] to

[63] , wherein the reaction time of step 1 is between 16 hours and 24 hours.

[65]

[0301] The method according to any one of

[56] to

[64] , wherein the Fc-containing molecule is an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 30 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 33.

[66]

[0303] The method according to any one of

[56] to

[64] , wherein the Fc-containing molecule is an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 44 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 46.

[0304] Effects of the Invention

[0305] According to the present invention, a molecule that maintains antitumor activity and is excellent in safety is provided. In addition, according to the present invention, a novel antibody-drug conjugate that can be systemically administered and exhibits antitumor effects in tumors expressing antigens is provided. Furthermore, according to the present invention, an improved method for producing Fc-containing molecules (particularly antibodies) using a one-pot method is provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0306] Figure 1 The antibody-drug conjugate ( ) obtained from the SG-type sugar chain-remodeled antibody is schematically shown as the antibody-drug conjugate (molecule (II)) of the present invention. Figure 1 A (II) molecule) and an antibody drug conjugate obtained by remodeling an antibody with a MSG-type sugar chain ( Figure 1 Figure 2 shows a molecule of (II) of B). (a) represents drug D, (b) represents linker L, (c) represents PEG linker (L(PEG)), and (d) represents N297 sugar chain (here, white circle represents NeuAc(Sia), white hexagon represents Man, black hexagon represents GlcNAc, white diamond represents Gal, and white inverted triangle represents Fuc). The white pentagon represents a triazole ring generated by the reaction of the alkyne derived from linker L and the azide group derived from the PEG linker. The Y-shape represents the antibody Ab. The PEG linker is linked to the carboxyl group at position 2 of the sialic acid at the non-reducing end via an amide bond. This representation method is applicable throughout this specification unless otherwise specified.

[0307] Figure 2 The present invention is a (Fucα1,6)GlcNAc-antibody ( Figure 2 A (III) molecule), SG type sugar chain remodeling antibody ( Figure 2 B (IV) molecules), and MSG-type sugar chain remodeling antibodies ( Figure 2 Schematic diagram of the structure of the molecule of C (IV). In all figures, the Y-shaped Figure 1 The same refers to the antibody Ab. Figure 2 In A, (e) represents an N297 sugar chain consisting of a disaccharide in which the 6-position of GlcNAc forms an α-glycosidic bond with the 1-position of Fuc. Figure 2 In B and C, (d) represents Figure 1 The same N297 sugar chain, (f) is a PEG linker with an azide group, and the terminal shows the azide group for linker L to bind. The binding pattern of the PEG linker with an azide group is similar to Figure 1 The same is true for the PEG linker.

[0308] Figure 3 This is a schematic diagram of the process of producing SG-type glycan-remodeled antibodies and MSG-type glycan-remodeled antibodies from antibodies produced in animal cells. Figure 2 Similarly, (III) represents a (Fucα1,6)GlcNAc-antibody, (IV) represents an SG-type sugar chain remodeled antibody or an MSG-type sugar chain remodeled antibody. The molecule of (V) is an antibody produced in animal cells, and the N297 sugar chain is a mixture of heterogeneous molecules. Figure 3 A represents a step of preparing a uniform (Fucα1,6)GlcNAc-antibody (III) by treating the heterogeneous N297 sugar chain (V) with a hydrolase such as EndoS. Figure 3 B represents a step of preparing the SG-type sugar chain remodeled antibody (IV) by transferring the sugar chain of the N297 sugar chain of the antibody (III) to the GlcNAc of the N297 sugar chain using a glycosyltransferase such as the EndoSD233Q / Q303L mutant using an SG-type sugar chain donor molecule. Figure 3 C and Figure 3 Similarly, B represents the process of preparing (IV) the MSG-type sugar chain remodeled antibody by transferring the sugar chain of the MSG-type sugar chain donor molecule to the antibody (III). The SG-type sugar chain donor molecule and the MSG-type sugar chain donor molecule used herein are each modified with a PEG linker having an azide group at the non-reducing end of the sialic acid. In the prepared SG-type N297 sugar chain remodeled antibody and the MSG-type N297 sugar chain remodeled antibody, Figure 2 As shown in B and C, the sialic acid at the non-reducing end is modified in the same way.

[0309] Figure 4The figure shows the anti-tumor effect of anti-EGFR antibody 1-CDN conjugate 1 and anti-EGFR antibody 3-CDN conjugate 1 achieved by intravenous administration. The average number of drug binding is about 4. The black quadrilateral line in the figure represents the vehicle group, the black triangle line represents the anti-EGFR antibody 3-CDN conjugate 1 administration group, and the black circle line represents the anti-EGFR antibody 1-CDN conjugate 1 administration group. The vertical axis represents the tumor volume (mm 3 ), the horizontal axis represents the days after tumor transplantation.

[0310] Figure 5 The figure shows the anti-tumor effect of anti-EGFR antibody 2-CDN conjugate 1 achieved by intravenous administration. The average number of drug binding is about 4. The black quadrilateral line in the figure represents the vehicle group, and the black circular line represents the anti-EGFR antibody 2-CDN conjugate 1 administration group. The vertical axis represents the tumor volume (mm 3 ), the horizontal axis represents the days after tumor transplantation.

[0311] Figure 6 The figure shows the anti-tumor effect of anti-EGFR antibody 1-CDN conjugate 2 and anti-EGFR antibody 3-CDN conjugate 2 by intravenous administration. The average number of drug binding is about 2. The black quadrilateral line in the figure represents the vehicle group, the black triangle line represents the anti-EGFR antibody 3-CDN conjugate 2 administration group, and the black circle line represents the anti-EGFR antibody 1-CDN conjugate 2 administration group. The vertical axis represents the tumor volume (mm 3 ), the horizontal axis represents the days after tumor transplantation.

[0312] Figure 7 The figure shows the anti-tumor effect of anti-EGFR antibody 2-CDN conjugate 2 achieved by intravenous administration. The average number of drug binding is about 2. The black quadrilateral line in the figure represents the vehicle group, and the black circular line represents the anti-EGFR antibody 2-CDN conjugate 2 administration group. The vertical axis represents the tumor volume (mm 3 ), the horizontal axis represents the days after tumor transplantation.

[0313] Figure 8 The figure shows the anti-tumor effect of anti-EGFR antibody 2-CDN conjugate 1 achieved by intravenous administration. The average number of drug binding is about 4. The black quadrilateral line in the figure represents the vehicle group, and the black circular line represents the anti-EGFR antibody 2-CDN conjugate 1 administration group. The vertical axis represents the tumor volume (mm 3 ), the horizontal axis represents the days after tumor transplantation.

[0314] Fig. 9The figure shows the anti-tumor effect of anti-EGFR antibody 2-CDN conjugate 1 achieved by intravenous administration. The average number of drug binding is about 4. The black quadrilateral line in the figure represents the vehicle group, and the black circular line represents the anti-EGFR antibody 2-CDN conjugate 1 administration group. The vertical axis represents the tumor volume (mm 3 ), the horizontal axis represents the days after tumor transplantation.

[0315] Fig.10 The figure shows the anti-tumor effect of anti-CDH6 antibody 1-CDN conjugate 1 and anti-CDH6 antibody 1-CDN conjugate 2 by intravenous administration. The black quadrilateral line in the figure represents the vehicle group, the white circular line represents the anti-CDH6 antibody 1-CDN conjugate 1 administration group, and the white inverted triangle line represents the anti-CDH6 antibody 1-CDN conjugate 2 administration group. The vertical axis represents the tumor volume (mm 3 ), the horizontal axis represents the days after tumor transplantation.

[0316] Fig.11 The figure shows the activity of anti-EGFR antibody 1-CDN conjugate 1, anti-EGFR antibody 3-CDN conjugate 1, anti-EGFR antibody 1-CDN conjugate 2, and anti-EGFR antibody 3-CDN conjugate 2 on reporter cells. The black triangle line in the figure shows the group with anti-EGFR antibody 3-CDN conjugate addition, the black circle line shows the group with anti-EGFR antibody 1-CDN conjugate addition, the dotted line shows CDN conjugate 2 with an average drug binding number of about 2, and the solid line shows CDN conjugate 1 with an average drug binding number of about 4. The vertical axis shows the count value, and the horizontal axis shows the concentration.

[0317] Fig.12 The figure shows the activity of anti-CDH6 antibody 1-CDN conjugate 1 and anti-CDH6 antibody 1-CDN conjugate 2 on reporter cells. The black triangle line in the figure shows the anti-CDH6 antibody 1-CDN conjugate addition group. The dotted line shows CDN conjugate 2 with an average drug binding number of about 2, and the solid line shows CDN conjugate 1 with an average drug binding number of about 4. The vertical axis shows the count value, and the horizontal axis shows the concentration.

[0318] Fig.13 The figure shows the anti-tumor effect of anti-EGFR antibody 1-CDN conjugate 1 in the tumor re-transplantation test. The vertical axis shows the tumor volume (mm 3 ).

[0319] Fig.14 The figure shows the anti-tumor effect of anti-EGFR antibody 1-CDN conjugate 2 in the tumor re-transplantation test. The vertical axis shows the tumor volume (mm 3 ).

[0320] Fig.15AThe cytokine production in the human whole blood test of each antibody-CDN conjugate is shown. The vertical axis represents the cytokine amount, and the horizontal axis represents the Fc site of each antibody-CDN conjugate. The cytokine amount is calculated using the standard line of the logarithmic scale.

[0321] Fig. 15B The cytokine production in the human whole blood test of each antibody-CDN conjugate is shown. The vertical axis represents the cytokine amount, and the horizontal axis represents the Fc site of each antibody-CDN conjugate. The cytokine amount is calculated using the standard line of the linear scale.

[0322] Fig.16A This is a graph evaluating the binding activity of an anti-EGFR antibody (IgG2) (anti-EGFR antibody B) which is an IgG2-type antibody, an Fc-substituted version of the antibody (IgG1 WT) (anti-EGFR antibody A) in which the Fc part of the antibody is replaced with an IgG1-type Fc, a mutant in which the L234A / L235A (LALA) mutation is introduced into the Fc part of IgG1 WT (modified anti-EGFR antibody 3), and a mutant in which the L234A / L235A / D265G (LALA-DG) mutation is introduced into the Fc part of IgG1 WT (modified anti-EGFR antibody 1) for human FcγRI by the SPR method. The horizontal axis represents the antibody concentration, and the vertical axis represents the RU (Response Unit) value obtained corresponding to the antibody concentration.

[0323] Fig. 16B It was evaluated using the SPR method. Fig.16A The graph shows the binding activity of the antibody group used in to human FcγRIIa. The horizontal axis represents the antibody concentration, and the vertical axis represents the RU value obtained corresponding to the antibody concentration.

[0324] Fig. 16C It was evaluated using the SPR method. Fig.16A The graph shows the binding activity of the antibody group used in to human FcγRIIb / c. The horizontal axis represents the antibody concentration, and the vertical axis represents the RU value obtained corresponding to the antibody concentration.

[0325] Fig.16D It was evaluated using the SPR method. Fig.16A The graph shows the binding activity of the antibody group used in to human FcγRIIIa. The horizontal axis represents the antibody concentration, and the vertical axis represents the RU value obtained corresponding to the antibody concentration.

[0326] Fig.16E It was evaluated using the SPR method. Fig.16A The horizontal axis represents the antibody concentration, and the vertical axis represents the RU value obtained corresponding to the antibody concentration.

[0327] Fig.17A It means that the SPR method was used to evaluate Fig.16A A graph showing the RU values ​​obtained at an antibody concentration of 4.7 μM for the antibody groups used in the assay and the antibody drug conjugates prepared by conjugating STING agonists to these antibody groups using DAR2 or DAR4 (DAR2: anti-EGFR antibody B-CDN conjugate 2, anti-EGFR antibody 3-CDN conjugate 2, modified anti-EGFR antibody 1-CDN conjugate 2; or DAR4: anti-EGFR antibody A-CDN conjugate 1, anti-EGFR antibody 3-CDN conjugate 1, modified anti-EGFR antibody 1-CDN conjugate 1) in terms of their binding activity to human FcγRI.

[0328] Fig. 17B It means that the SPR method was used to evaluate Fig.17A This is a graph showing the RU values ​​obtained at an antibody concentration of 4.7 μM when the antibody group used in the experiment had binding activity to human FcγRIIa.

[0329] Fig. 17C It means that the SPR method was used to evaluate Fig.17A This is a graph showing the RU values ​​obtained at an antibody concentration of 4.7 μM in the case of the binding activity of the antibody group used in to human FcγRIIb / c.

[0330] Fig.17D It means that the SPR method was used to evaluate Fig.17A This is a graph showing the RU values ​​obtained at an antibody concentration of 4.7 μM when the antibody group used in the experiment had binding activity to human FcγRIIIa.

[0331] Fig.17E It means that the SPR method was used to evaluate Fig.17A This is a graph showing the RU values ​​obtained at an antibody concentration of 4.7 μM when the antibody group used in the experiment had binding activity to human FcγRIIIb.

[0332] Fig.18 The T of anti-EGFR antibody A-CDN conjugate 1 (IgG1 WT), anti-EGFR antibody 3-CDN conjugate 1 (LALA), modified anti-EGFR antibody 1-CDN conjugate 1 (LALA-DG) is shown. m In addition, the T values ​​of anti-EGFR antibody A-CDN conjugate 1 and various anti-EGFR antibody-CDN conjugates 1 are also shown. m The difference between the values ​​(ΔT m ).

[0333] Fig.19The diagram shows (a) the amino acid sequence of the wild-type human STING (SEQ ID NO: 1), (b) the amino acid sequence of the REF mutant (R232H) of the human STING (SEQ ID NO: 3), and (c) the amino acid sequence of the HAQ mutant (R71H, G230A, R293Q) of the human STING (SEQ ID NO: 5).

[0334] Fig. 20 (a) the amino acid sequence of CDRL1 of an anti-EGFR antibody (modified anti-EGFR antibodies 1 to 3) (SEQ ID NO: 23), (b) the amino acid sequence of CDRL2 of an anti-EGFR antibody (modified anti-EGFR antibodies 1 to 3) (SEQ ID NO: 24), (c) the amino acid sequence of CDRL3 of an anti-EGFR antibody (modified anti-EGFR antibodies 1 to 3) (SEQ ID NO: 25), (d) the amino acid sequence of CDRH1 of an anti-EGFR antibody (modified anti-EGFR antibodies 1 to 3) (SEQ ID NO: 26), (e) the amino acid sequence of CDRH2 of an anti-EGFR antibody (modified anti-EGFR antibodies 1 to 3) (SEQ ID NO: 27), and (f) the amino acid sequence of CDRH3 of an anti-EGFR antibody (modified anti-EGFR antibodies 1 to 3) (SEQ ID NO: 28).

[0335] Figure 21-1 shows (a) the amino acid sequence of the light chain variable region of the anti-EGFR antibody (modified anti-EGFR antibodies 1 to 3) (SEQ ID NO: 29), (b) the amino acid sequence of the light chain of the anti-EGFR antibody (modified anti-EGFR antibodies 1 to 3) (SEQ ID NO: 30), and (c) the amino acid sequence of the heavy chain variable region of the anti-EGFR antibody (modified anti-EGFR antibodies 1 to 3) (SEQ ID NO: 31).

[0336] Figure 21-2 shows (d) the amino acid sequence of the heavy chain of the anti-EGFR antibody LALA-DG mutant (modified anti-EGFR antibody 1) (SEQ ID NO: 32), (e) the amino acid sequence of the heavy chain of the anti-EGFR antibody LALA-DG mutant (modified anti-EGFR antibody 2) (SEQ ID NO: 33), and (f) the amino acid sequence of the heavy chain of the anti-EGFR antibody A (SEQ ID NO: 34).

[0337] Figure 21-3 shows (g) the amino acid sequence of the heavy chain of anti-EGFR antibody B (Vectibix: panitumumab) (SEQ ID NO: 35), and (h) the amino acid sequence of the heavy chain of anti-EGFR antibody LALA mutant (modified anti-EGFR antibody 3) (SEQ ID NO: 36).

[0338] Fig. 22Represents (a) the amino acid sequence of CDRL1 of anti-CDH6 antibody (modified anti-CDH6 antibody 1) (sequence number 37), (b) the amino acid sequence of CDRL2 of anti-CDH6 antibody (modified anti-CDH6 antibody 1) (sequence number 38), (c) the amino acid sequence of CDRL3 of anti-CDH6 antibody (modified anti-CDH6 antibody 1) (sequence number 39), (d) the amino acid sequence of CDRH1 of anti-CDH6 antibody (modified anti-CDH6 antibody 1) (sequence number 40), (e) the amino acid sequence of CDRH2 of anti-CDH6 antibody (modified anti-CDH6 antibody 1) (sequence number 41), and (f) the amino acid sequence of CDRH3 of anti-CDH6 antibody (modified anti-CDH6 antibody 1) (sequence number 42).

[0339] Fig.23 Represents (a) the amino acid sequence of the light chain variable region of the anti-CDH6 antibody (modified anti-CDH6 antibody 1) (sequence number 43), (b) the amino acid sequence of the light chain of the anti-CDH6 antibody (modified anti-CDH6 antibody 1) (sequence number 44), (c) the amino acid sequence of the heavy chain variable region of the anti-CDH6 antibody (modified anti-CDH6 antibody 1) (sequence number 45), and (d) the amino acid sequence of the heavy chain of the anti-CDH6 antibody LALA-DG mutant (modified anti-CDH6 antibody 1) (sequence number 46).

[0340] Fig.24 The figure shows the anti-tumor effect of CT26.WT-hCDH6 re-implanted into the left axilla of mice whose tumors completely regressed after intravenous administration of anti-CDH6 antibody 1-CDN conjugate 1 and anti-CDH6 antibody 1-CDN conjugate 2. The vertical axis shows tumor volume (mm 3 ).

[0341] Fig.25 The graph shows the anti-tumor effects of anti-CDH6 antibody 1-CDN conjugate 1 and anti-CDH6 antibody 1-CDN conjugate 2 by intravenous administration. The vertical axis shows tumor volume (mm 3 ), the horizontal axis represents the days after tumor transplantation.

[0342] Fig.26 The graph shows the anti-tumor effect of anti-CDH6 antibody 1-CDN conjugate 1 by intravenous administration. The vertical axis shows tumor volume (mm 3 ), the horizontal axis represents the days after tumor transplantation.

[0343] Fig. 27 The graph shows the anti-tumor effect of anti-CDH6 antibody 1-CDN conjugate 2 by intravenous administration. The vertical axis shows tumor volume (mm 3 ), the horizontal axis represents the days after tumor transplantation.

[0344] Fig.28 The graph shows the anti-tumor effect of anti-CDH6 antibody 1-CDN conjugate 1 by intravenous administration. The vertical axis shows tumor volume (mm 3 ), the horizontal axis represents the days after tumor transplantation.

[0345] Fig.29 The graph shows the anti-tumor effect of anti-CDH6 antibody 1-CDN conjugate 2 by intravenous administration. The vertical axis shows tumor volume (mm 3 ), the horizontal axis represents the days after tumor transplantation.

[0346] Fig.30 The amino acid sequence of wild-type Endo-S is shown (SEQ ID NO: 47). Amino acid residues corresponding to the catalytic activity center of the enzyme are indicated in bold and underlined, and amino acid residues expected to control enzyme activity by mutation are indicated in bold.

[0347] Fig.31 The amino acid sequence of wild-type Endo-S2 (SEQ ID NO: 48) is shown. Amino acid residues corresponding to the catalytic activity center of the enzyme are indicated in bold and underlined, and amino acid residues expected to control enzyme activity by mutation are indicated in bold.

[0348] Fig.32 The amino acid sequence of wild-type Endo-Si (SEQ ID NO: 49) is shown. Amino acid residues corresponding to the catalytic active center of the enzyme are indicated in bold and underlined, and amino acid residues expected to control enzyme activity by mutation are indicated in bold.

[0349] Fig.33 The amino acid sequence of wild-type Endo-M is shown (SEQ ID NO: 50). Amino acid residues corresponding to the catalytic activity center of the enzyme are indicated in bold and underlined, and amino acid residues expected to control enzyme activity by mutation are indicated in bold.

[0350] Fig.34 The amino acid sequence of wild-type Endo-Rp (SEQ ID NO: 51) is shown. Amino acid residues corresponding to the catalytic activity center of the enzyme are indicated in bold and underlined, and amino acid residues expected to control enzyme activity by mutation are indicated in bold.

[0351] Fig.35 The amino acid sequence of the wild-type Endo-CC is shown (SEQ ID NO: 52). The amino acid residues corresponding to the catalytic activity center of the enzyme are indicated in bold and underlined, and the amino acid residues expected to control the enzyme activity by mutation are indicated in bold.

[0352] Fig.36The amino acid sequence of the wild-type Endo-Om (SEQ ID NO: 53) is shown. The amino acid residues corresponding to the catalytic activity center of the enzyme are indicated in bold and underlined, and the amino acid residues expected to control the enzyme activity by mutation are indicated in bold.

[0353] Fig.37 The amino acid sequence of the wild-type Endo-Sd is shown (SEQ ID NO: 54). The amino acid residues corresponding to the catalytic activity center of the enzyme are indicated in bold and underlined, and the amino acid residues expected to control the enzyme activity by mutation are indicated in bold.

[0354] Fig.38 The amino acid sequence of the wild-type Endo-Sz (SEQ ID NO: 55) is shown. The amino acid residues corresponding to the catalytic activity center of the enzyme are indicated in bold and underlined, and the amino acid residues expected to control the enzyme activity by mutation are indicated in bold.

[0355] Fig.39 The amino acid sequence of wild-type Endo-Se (SEQ ID NO: 56) is shown. Amino acid residues corresponding to the catalytic active center of the enzyme are indicated in bold and underlined, and amino acid residues expected to control enzyme activity by mutation are indicated in bold. DETAILED DESCRIPTION

[0356] In one embodiment, the present invention relates to a molecule comprising a target molecule binding portion, a mutant Fc region and an immunostimulatory agent, wherein the mutant Fc region comprises at least three amino acid substitutions compared to a wild-type Fc region, and the molecule has reduced binding to human Fcγ receptors, reduced inflammatory cytokine production capacity, and equivalent or enhanced in vivo anti-tumor activity compared to a corresponding molecule having a wild-type Fc region.

[0357] In this specification, "target molecule binding portion" refers to a portion (moiety) that binds to an arbitrary target molecule, preferably a target molecule involved in human diseases. As this portion, examples may include antibodies, functional fragments of antibodies, antigen-binding fragments of antibodies, non-immunoglobulin proteins or fragments thereof, receptor proteins, ligand proteins, proteins (polypeptides) such as scaffold proteins, aptamers, nucleic acid molecules such as antisense individuals, organic synthetic low molecular compounds, naturally occurring low molecular compounds, etc., preferably antibodies, functional fragments of antibodies, or antigen-binding fragments of antibodies can be listed, more preferably antibodies or functional fragments of antibodies can be listed, and antibodies can be particularly preferably listed, but are not limited thereto. In one embodiment of the present invention, in the case where the "target molecule binding portion" contains an Fc region (for example, in the case where the "target molecule binding portion" is an antibody), the Fc region contained in the target molecule binding portion can be a mutant Fc region. In other words, in one embodiment of the present invention, the mutant Fc region can be contained in the target molecule binding portion.

[0358] The terms “antibody,” “functional fragment of an antibody,” and “antigen-binding fragment of an antibody” will be described in detail later.

[0359] In the present invention, "immune stimulation" refers to activation of immune cells involved in anti-tumor immunity such as monocytes, macrophages, dendritic cells, T cells, B cells, NK cells, neutrophils, etc. in some form, such as cytokine and chemokine production, increased expression of immune activation markers, decreased expression of immunosuppressive markers, phosphorylation of intracellular signal transduction systems, changes in gene expression, and changes in the structure and function of all immune cells. In addition, it also includes changes caused by tumor cells that induce anti-tumor immunity, such as activation of immune cells or induction of the production of cytokines and chemokines that migrate, induction of hypersensitivity to immune cells, etc.

[0360] In the present invention, "immunostimulant" refers to all substances that bring about the immunostimulation specified above. In one embodiment of the present invention, the immunostimulant is preferably a STING agonist, a TLR1-10 agonist, a CIITA agonist, a NAIPs agonist, a NOD1 agonist, a NOD2 agonist, a NLRC3 agonist, a NLRP1-14 agonist, a DEC205 agonist, an MMR agonist, a Dectin1 agonist, a Dectin2 agonist, a Mincle agonist, a DC-SIGN agonist, a DNGR-1 agonist, an MBL agonist, an AIM2 agonist, an MDA-5 agonist, a RIG-I agonist, an AhR agonist, more preferably a TLR3 agonist, a TLR7 agonist, a TLR8 agonist, a TLR9 agonist, a RIG-I agonist, a STING agonist, and particularly preferably a STING agonist.

[0361] In this specification, the "Fc region" is the C-terminal region of the immunoglobulin heavy chain, and contains the CH2 domain and the CH3 domain. In one embodiment of the present invention, the "Fc region" includes at least a portion of the hinge domain. In another embodiment of the present invention, the "Fc region" includes at least a portion of the hinge domain, two CH2 domains and two CH3 domains. The "Fc region" generally refers to homologous and heterologous dimers or polymers in which the C-terminal regions of two or more immunoglobulin heavy chains are bound by S-S bonds, but it can also be a single-chain Fc region (scFc) in which they form a single chain.

[0362] In one embodiment, the "mutated Fc region" of the present invention comprises at least three amino acid substitutions compared to the wild-type Fc region. In one embodiment of the present invention, the "wild-type Fc region" and the "mutated Fc region" can be derived from all species, but preferably from humans, monkeys (including cynomolgus monkeys, rhesus monkeys, common marmosets, squirrel monkeys, etc., but not limited thereto), mice, rats, rabbits, more preferably from humans or monkeys, and particularly preferably from humans. In one embodiment of the present invention, the "wild-type Fc region" and the "mutated Fc region" can be derived from any one of the immunoglobulin molecules of IgG, IgE, IgM, IgD, IgA and IgY, preferably from IgG. In addition, as a subclass, it can be any one of IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, preferably IgG1, IgG2 or IgG4, more preferably IgG1 or IgG4, and particularly preferably IgG1. Therefore, in one embodiment of the present invention, the Fc region in the "wild-type Fc region" and the "mutant Fc region" is a human IgG1 Fc region or a human IgG4 Fc region, preferably a human IgG1 Fc region.

[0363] As described above, in one embodiment of the present invention, the mutant Fc region comprises at least three amino acid substitutions compared to the wild-type Fc region. The at least three amino acid substitutions may be present in any one or more of the hinge domain, CH2 domain, and CH3 domain of the mutant Fc region, preferably in the CH2 domain of the mutant Fc region. In one embodiment of the present invention, the at least three amino acid substitutions are present at the three positions of position 234, position 235, and position 265 (all based on EU numbering) of the mutant Fc region. In another embodiment of the present invention, the at least three amino acid substitutions preferably include 234Ala, 235Ala, and 265Gly (all based on EU numbering). In the present invention, the mutation in the Fc region comprising 234Ala, 235Ala, and 265Gly (all based on EU numbering) can be recorded as "LALA-DG", "L234A / L235A / D265G", "-L234A, L235A, D265G", etc.

[0364] In one embodiment of the present invention, the molecule comprising a target molecule binding portion, a mutant Fc region and an immunostimulant may comprise an additional Fc region, which may be a mutant Fc region. In another embodiment of the present invention, the "mutant Fc region" comprises two CH2 domains, one or both of which comprise the at least three amino acid substitutions.

[0365] In one embodiment, the molecule of the present invention comprising a target molecule binding portion, a mutant Fc region, and an immunostimulatory agent has reduced binding to human Fcγ receptors compared to a corresponding molecule having a wild-type Fc region. In this specification, "a corresponding molecule having a wild-type Fc region" refers to a molecule that is identical or equivalent to the molecule of the present invention comprising a target molecule binding portion, a mutant Fc region, and an immunostimulatory agent, except that it comprises a wild-type Fc region instead of a mutant Fc region. In the present specification, "human Fcγ receptor" (also referred to as "human FcγR") refers to one or more receptors selected from the group consisting of FcγRI (CD64) (including isoforms FcγRIa, FcγRIb, and FcγRIc), FcγRII (CD32) (including isoforms FcγRIIa (including allotypes H131 (H type) and R131 (R type)), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc), and FcγRIII (CD16) (including isoforms FcγRIIIa (including allotypes V158 and F158), FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), and FcγRIIIc). In one embodiment of the present invention, "human Fcγ receptor" is at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all of the nine isotypes listed above, preferably one or more receptors selected from the group consisting of human FcγRI, human FcγRIIa, human FcγRIIb / c, human FcγRIIIa, and human FcγRIIIb, and more preferably human FcγRI. The binding of an analyte to a human Fcγ receptor can be determined using any known method, for example, by surface plasmon resonance (SPR) method. In one embodiment of the present invention, the binding property of the analyte to the human Fcγ receptor can be determined by the following method: after capturing the His-tagged human Fcγ receptor as a ligand on a sensor chip on which an anti-His antibody is immobilized, the analyte is added to the sensor chip and the ligand / analyte interaction is measured by the surface SPR method. Specifically, it can be determined using the method disclosed in Test Examples 15 to 20 of this specification.

[0366] In one embodiment, when the target molecule binding portion of the present invention is an antibody and the mutant Fc region is contained in the antibody, the binding of the molecule of the present invention comprising the target molecule binding portion, the mutant Fc region and the immunostimulant to human Fcγ receptor is reduced to the same degree as the parent antibody. In one embodiment, the reduction of binding to human Fcγ receptor means the reduction of effector function.

[0367] In one embodiment, the molecule of the present invention comprising a target molecule binding portion, a mutant Fc region, and an immunostimulant has a reduced ability to produce inflammatory cytokines compared to a corresponding molecule having a wild-type Fc region. In the present invention, "inflammatory cytokine" refers to a cytokine that causes or promotes an inflammatory reaction or inflammatory symptoms. In one embodiment of the present invention, an "inflammatory cytokine" is a cytokine that causes or promotes an inflammatory reaction or inflammatory symptoms, and its production is not expected or expected to be enhanced to a level higher than required by the test substance. For example, as described in Test Example 14 of this specification, a cytokine that is enhanced in the absence of a cancer cell that is a target of the target molecule binding portion is consistent with a cytokine that is not usually expected or expected to be enhanced to a level higher than required, and therefore can be included in the "inflammatory cytokine" of the present invention. In one embodiment of the present invention, "inflammatory cytokine" is an interleukin, TNF-α, TNF-β, interferon, chemokine, preferably TNF-α, IL-1, IL-6, IL-10, CCL2, CCL4, IFN-γ, and particularly preferably IL-6. The inflammatory cytokine production ability of the test substance (e.g., a molecule comprising a mutant Fc region of the present invention) can be determined using all known methods, for example, it can be determined by immunoassays such as ELISA. In one embodiment of the present invention, the inflammatory cytokine production ability of the test substance can be determined using the method disclosed in Test Example 14 of this specification.

[0368] In one embodiment, the molecules of the invention comprising a target molecule binding portion, a mutant Fc region, and an immunostimulatory agent have equivalent or enhanced in vivo tumor activity compared to corresponding molecules having a wild-type Fc region.

[0369] In this specification, "cancer", "cancer" and "tumor" are used with the same meaning.

[0370] In the present invention, "anti-tumor effect" means that the drug directly or indirectly affects tumor cells, thereby inducing a reduction or regression of the tumor. For example, the drug directly damages tumor cells, tumor cells activate anti-tumor immunity due to drug stimulation, and drugs delivered to tumor cells are released outside the cells to activate anti-tumor immunity around tumor cells, etc., which causes a reduction in the number of tumor cells and damage, or regression of the tumor. The above effects are called anti-tumor effects.

[0371] The in vivo antitumor activity of the test substance can be determined using any known method, for example, it can be determined using the methods disclosed in Test Examples 3 to 9 of the present specification.

[0372] In one embodiment, the molecule of the present invention comprising a target molecule binding portion, a mutant Fc region, and an immunostimulant has an equivalent or improved thermal stability, preferably an improved thermal stability, compared to a corresponding molecule having a wild-type Fc region. The thermal stability of the above-mentioned molecule can be determined using any known method, for example, by preparing a solution containing the above-mentioned molecule and investigating the heat capacity C of the solution when the temperature is raised. p (kcal / mol / °C) can be determined, specifically, it can be determined using the method disclosed in Test Example 21 of this specification. The peak of the curve representing the change in heat capacity represents the thermal denaturation of each domain of the antibody, and the peaks of thermal denaturation are usually observed in the order of CH2, Fab, and CH3 (Biochem. Biophys. Res. Commun. 355, 751-757 (2007)). Therefore, the thermal denaturation midpoint (T) of the domain into which the mutation is introduced is calculated based on the peak of thermal denaturation. m ), the molecule containing the mutant Fc region is compared with the corresponding molecule having the wild-type Fc region to determine the change in thermal stability. It should be noted that in this specification, the temperature of the peak of the curve representing the change in heat capacity is referred to as T m In this specification, if T m The change in value (ΔT m ) is less than 1°C, the thermal stability of the two is considered to be equal. m The change in value (ΔT m ) is increased by 1°C or more, the molecule comprising the mutant Fc region is judged to have improved thermal stability compared to the corresponding molecule having the wild-type Fc region. For example, in the case where the mutation is present in the CH2 domain, if the T of the peak in the CH2 domain is m The change in (ΔT m ) is less than 1°C, the thermal stabilities of the two are considered equal. The T m The change in (ΔT m ) is increased by 1°C or more, the molecule comprising the mutant Fc region is judged to have improved thermal stability compared to the corresponding molecule having the wild-type Fc region.

[0373] In one embodiment, when the mutant Fc region comprises 234Ala, 235Ala, and 265Gly (EU numbering) amino acid substitutions, the molecules comprising the target molecule binding portion, the mutant Fc region, and the immunostimulant of the present invention are also excellent in safety while maintaining the efficacy, and the effector function is reduced to the same extent as the parent antibody. In addition, in one embodiment, when the mutant Fc region comprises 234Ala, 235Ala, and 265Gly (EU numbering) amino acid substitutions, the molecules comprising the target molecule binding portion, the mutant Fc region, and the immunostimulant of the present invention are also excellent in thermal stability.

[0374] In one embodiment, the present invention relates to a pharmaceutical composition, which comprises the molecule comprising a target molecule binding portion, a mutant Fc region and an immunostimulant as described above. In one embodiment of the present invention, the pharmaceutical composition is an anti-tumor agent. The "pharmaceutical composition" will be described in detail later.

[0375] In one embodiment, the present invention relates to an antibody-drug conjugate comprising a CDN derivative having STING agonist activity and its use. The CDN derivative has STING agonist activity, activates immune cells and induces the production of interferons and cytokines. In addition, the CDN derivative exerts an anti-tumor effect through the activation of the immune cells. In one embodiment, the antibody-drug conjugate of the present invention is made by connecting the CDN derivative to an antibody that can recognize and bind to target cells (such as tumor cells or immune cells) via an arbitrary linker, and can be administered systemically. As specific examples of systemic administration, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes can be listed. It should be noted that the antibody-drug conjugate can be positioned as a scheme of the molecule comprising a target molecule binding portion, a mutant Fc region, and an immunostimulant described above.

[0376] STING (Stimulator of Interferon Genes) refers to a transmembrane adaptor protein that is localized in the endoplasmic reticulum. It is known that congenital polymorphism exists at a high frequency in STING (PLoS One, 2013 Oct, 21, 8 (10), e77846). As STING mutants, for example, the R232H mutation in which the amino acid at position 232 mutates from arginine (R) to histidine (H); the HAQ mutation in which the arginine (R) at position 71 mutates to histidine (H), the glycine (G) at position 230 mutates to alanine (A), and the arginine (R) at position 293 mutates to glutamine (Q). It is known that among such STING polymorphisms, there are differences in the intensity of responses such as the amount of cytokine production induced by STING agonist stimulation (Genes and Immunity, 2011, 12, 263-269). Therefore, in order for a STING agonist to stably act on humans, it is desirable that it has activity against each type of STING.

[0377] In the present invention, "cytotoxic activity" refers to pathological changes in cells in some form, not just direct trauma, but also damage to the structure and function of all cells, such as DNA cleavage, formation of base dimers, chromosome cleavage, damage to the cell division apparatus, and reduction in the activity of various enzymes.

[0378] In the present invention, "cells" also include cells in animal individuals and cultured cells.

[0379] <1.CDN derivatives>

[0380] The CDN derivative has a structure represented by the following formula (I).

[0381]

[0382] L 1 It is a group represented by the following formula.

[0383]

[0384] Q and Q' each independently represent a hydroxyl group or a thiol group. Preferably, Q and Q' are both thiol groups.

[0385] R 21 and R 22 Each independently represents a hydroxyl group or a fluorine atom. 21 Preferably it is a hydroxyl group. 22 Preferred is a fluorine atom.

[0386] W is -NH- or a sulfur atom. W is preferably -NH-.

[0387] The method for producing the CDN derivative is described in <3. Production method> described later.

[0388] <2. Antibody-drug conjugates>

[0389] In one embodiment, the antibody-drug conjugate of the present invention can be systemically administered as an antibody-drug conjugate formed by linking the above-mentioned CDN derivative and an antibody capable of recognizing and binding to target cells (e.g., tumor cells or immune cells) via an arbitrary linker. It should be noted that in this specification, when only "the antibody-drug conjugate of the present invention" is mentioned below, in principle, it refers to an antibody-drug conjugate comprising the above-mentioned CDN derivative as a drug part, but there are exceptions, and in addition to this, it sometimes refers to the above-mentioned "molecule comprising a target molecule binding part, a mutant Fc region, and an immunostimulant".

[0390] The antibody-drug conjugate of the present invention is represented by the following formula (II).

[0391]

[0392] m 1 is in the range of 1 to 10, indicating the number of drug binding per antibody molecule in the antibody-drug conjugate. Ab represents an antibody or a functional fragment of the antibody, L represents a linker linking Ab and D, and D represents the above-mentioned CDN derivative (in this specification, when a CDN derivative is used as a part of an antibody-drug conjugate, it is sometimes simply referred to as a "drug").

[0393] Drug D is a compound having the activity of activating immune cells, specifically, a compound having STING agonist activity. If part or all of the linker is cut off in the target cell (e.g., tumor cell or immune cell), drug D is free with its original structure and exerts an immunostimulatory effect. The target function is exerted by hypersensitivity of target cells to immune cells or by activation of immune cells of target cells. As the target function, there is no particular limitation as long as it is a function related to STING agonist activity, but anti-tumor activity is preferred. That is, drug D linked to an antibody targeting a tumor (e.g., anti-EGFR antibody, anti-CDH6 antibody) via an arbitrary linker is delivered to the target cell or tissue, part or all of the linker is cut off, and an anti-tumor effect is exerted by hypersensitivity of target cells to immune cells or by activation of immune cells of target cells (e.g., production of interferon, cytokine).

[0394] The drug D bound to the antibody-drug conjugate of the present invention is represented by the following formula (I).

[0395]

[0396] (Here, L1 ,Q,Q',R 21 , R 22 and W is as specified in the above <1. CDN derivatives>).

[0397] In addition, the drug D used in the antibody-drug conjugate of the present invention is preferably represented by the following two formulas.

[0398]

[0399] (Here, L 1 , Q, Q', and W are as specified in the above <1. CDN derivatives>).

[0400] In addition, the drug D used in the antibody-drug conjugate of the present invention is preferably represented by the following two formulas.

[0401]

[0402] (Here, the asterisk indicates bonding to L, and Q, Q', and W are as defined in the above <1. CDN derivatives>).

[0403] In addition, the drug D used in the antibody-drug conjugate of the present invention is preferably represented by the following three formulas.

[0404]

[0405] (Here, the asterisk indicates binding to L, and W is as defined in the above <1. CDN derivatives>).

[0406] In addition, the drug D used in the antibody-drug conjugate of the present invention is preferably represented by the following three formulas.

[0407]

[0408] (Here, the asterisk indicates binding to L).

[0409] In addition, the drug D used in the antibody-drug conjugate of the present invention is preferably represented by the following four formulas.

[0410]

[0411] (Here, the asterisk indicates binding to L).

[0412] Furthermore, the drug D used in the antibody-drug conjugate of the present invention is more preferably represented by the following formula.

[0413]

[0414] <2.1. Connector structure>

[0415] The structure of the linker that binds the drug to the antibody in the antibody drug conjugate of the present invention is described. The linker used in the antibody drug conjugate of the present invention is not particularly limited as long as it is understood by those skilled in the art as a linker that connects the antibody to the drug. As a linker for the antibody drug conjugate of the present invention, for example, a linker described in Protein Cell, 2018, 9 (1): 33-46, Pharm Res, 2015, 32: 3526-3540 or Int. J. Mol. Sci., 2016, 17, 561 can be cited, but it is not limited thereto. The linker can be a linker that will be cut off in vivo, or it can be a linker that will not be cut off in vivo, preferably a linker that will be cut off in vivo.

[0416] As the linker used for the antibody drug conjugate of the present invention, for example, it can be listed: a linker (for example, described in WO2018 / 003983) that binds the drug to the sugar chain or reconstructed sugar chain of the Fc part of the antibody (sometimes referred to as "sugar chain conjugation" in this specification) or a linker (for example, described in WO2014 / 057687) that binds the drug to any amino acid residue (for example, cysteine ​​residue or lysine residue) of the antibody, but is not limited thereto. As a linker that binds the drug to any amino acid residue of the antibody, preferably, it is listed: a case where a thioether bond is formed with the thiol group (SH group) of cysteine ​​of Ab (sometimes referred to as "cysteine ​​coupling" in this specification) or an amide bond is formed with the amino group (NH2 group) of lysine of Ab (sometimes referred to as "lysine coupling" in this specification), preferably cysteine ​​coupling.

[0417] A preferred linker L for use in the antibody drug conjugate of the present invention is represented by the following formula.

[0418] -Lb-La-Lp-Lc-*

[0419] (Here, the asterisk indicates binding to drug D).

[0420] First, Lp is described. Lp represents a linker consisting of an amino acid sequence that can be cut in vivo or in a target cell (hereinafter also referred to as a peptide linker in this specification), or Lp does not exist.

[0421] Lp is cut by the action of enzymes such as peptidases and esterases. Lp is a peptide composed of 2 to 7 (preferably 2 to 4) amino acids. Lp forms an amide bond with the carbonyl group at the right end of La described later at its N-terminus, and forms an amide bond with the amino group (-NH-) of Lc at the C-terminus. The amide bond on the C-terminal side of Lp is cut by enzymes such as the peptidase.

[0422] The amino acids constituting Lp are not particularly limited, and are, for example, L- or D-amino acids, preferably L-amino acids. In addition, in addition to α-amino acids, amino acids of structures such as β-alanine, ε-aminocaproic acid, and γ-aminobutyric acid may also be used, and further, for example, non-natural amino acids such as N-methylated amino acids may be used. The amino acid sequence of Lp is not particularly limited, and as the constituent amino acids, glycine (Gly; G), valine (Val; V), alanine (Ala; A), phenylalanine (Phe; F), glutamic acid (Glu; E), isoleucine (Ile; I), proline (Pro; P), citrulline (Cit), leucine (Leu; L), methionine (Met; M), serine (Ser; S), lysine (Lys; K), and aspartic acid (Asp; D) can be listed. Among them, glycine (Gly; G), valine (Val; V), alanine (Ala; A), phenylalanine (Phe: F), citrulline (Cit), isoleucine (Ile; I), and proline (Pro; P) are preferred. These amino acids may be repeated to have an amino acid sequence containing any selected amino acids. In addition, the pattern of drug release can be controlled by the type of amino acids.

[0423] Specific examples of Lp include -GGFG-, -GGPI-, -GGVA-, -GGFM-, -GGVCit-, -GGFCit-, -GGICit-, -GGPL-, -GGAQ-, and -GGPP-. The linker Lp is preferably -GGFG- or -GGPI-, and more preferably -GGFG-.

[0424] Next, La is described. La represents a group selected from the following: -C(=O)-(CH2CH2)n 2 -C(=O)-, -C(=O)-(CH2CH2)n 2 -CH2-C(=O)-, -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2)n 3 -C(=O)-, -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2)n 3 -CH2-C(=O)-, -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2O)n 3 -CH2-C(=O)-, -(CH2)n 4 -O-C(=O)-, and -(CH2)n 9 -C(=O)-. (Here, in the formula, n 2represents an integer of 1 to 3 (preferably 1 or 2), n 3 represents an integer of 1 to 5 (preferably an integer of 2 to 5, more preferably 3 or 4), n 4 represents an integer from 0 to 2 (preferably 0 or 1), n 9 represents an integer of 2 to 7 (preferably an integer of 2 to 5, more preferably 2, 3 or 5))

[0425] La preferably represents any one selected from the group consisting of -C(=O)-CH2CH2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)3-CH2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)4-CH2-C(=O)-, -C(=O)-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2)2-CH2-C(=O)-, -CH2-OC(=O)-, -OC(=O)-, and -(CH2)5-C(=O)-.

[0426] La is more preferably -C(=O)-CH2CH2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)3-CH2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)4-CH2-C(=O)-, or -(CH2)5-C(=O)-.

[0427] La is more preferably -C(=O)-CH2CH2-C(=O)-.

[0428] Next, Lb is described. Lb represents a spacer used in a sugar chain-coupled linker (also referred to as "a spacer of a sugar chain-coupled linker" in this specification) or a spacer used in a cysteine-coupled linker (also referred to as "a spacer of a cysteine-coupled linker" in this specification).

[0429] <When Lb is a "spacer of a linker for sugar chain coupling">

[0430] When Lb is a "spacer of a linker for sugar chain coupling", Lb is not particularly limited, and examples thereof include spacers represented by the following formulas.

[0431] or

[0432] In each of the structural formulas shown above, the asterisk (*) indicates binding to -(C=O)-, -CH2- or -OC(=O)- at the left end of La, and the wavy line indicates binding to the sugar chain or reconstructed sugar chain of Ab.

[0433] When Lb is selected from Lb-1 or Lb-3, the triazole ring portion has a geometrically isomeric structure, and one Lb contains either of the two structures, or contains a mixture thereof. The antibody-drug conjugate of the present invention can bind multiple drugs to one molecule of antibody. In the case of binding multiple drugs to one molecule of antibody, multiple Lb may exist (for example, refer to the schematic diagram (1e) of the antibody-drug conjugate shown in the E method of <3. Manufacturing method> described later). When Lb is selected from Lb-1 or Lb-3, there are multiple Lb relative to one molecule of antibody (for example, m 2 In the case where Lb is 1 or 2, the triazole ring portion in each Lb has a geometrically isomeric structure, and one Lb contains either of the two structures, or a mixture thereof.

[0434] <When Lb is a "spacer of a cysteine-coupled linker">

[0435] When Lb is a "spacer of a cysteine-coupled linker", Lb is not particularly limited, and examples thereof include -(succinimidyl-3-yl-N)-. In the present invention, "-(succinimidyl-3-yl-N)-" has a structure represented by the following formula.

[0436]

[0437] In the structural formula shown above, the asterisk indicates binding to La, and the wavy line indicates binding to the side chain of the cysteine ​​residue of the antibody by forming a thioether.

[0438] Next, Lc is described. Lc represents -NH-CH2-, -NH-phenyl-CH2-O(C=O)-, or -NH-heteroaryl-CH2-O(C=O)-, or Lc does not exist. Here, as a phenyl group, 1,4-phenyl is preferred, and as a heteroaryl group, 2,5-pyridyl, 3,6-pyridyl, 2,5-pyrimidyl, or 2,5-thienyl is preferred. Lc is preferably -NH-CH2-, or Lc does not exist.

[0439] When the drug-antibody binding mode is "sugar chain coupling", the more preferred linker L used in the antibody-drug conjugate of the present invention is -Z L1 -C(=O)-CH2CH2-C(=O)-GGFG-,-Z L1 -C(=O)-CH2CH2-C(=O)-GGVA-,-Z L1-C(=O)-CH2CH2-C(=O)-GGVCit-,-Z L1 -C(=O)-CH2CH2-C(=O)-GGFCit-,-Z L1 -C(=O)-CH2CH2-C(=O)-GGICit-,-Z L1 -C(=O)-CH2CH2-C(=O)-GGFM-,-Z L1 -C(=O)-CH2CH2-C(=O)-GGPI-,-Z L1 -C(=O)-CH2CH2-C(=O)-GGLM-,-Z L1 -C(=O)-CH2CH2-C(=O)-FG-, -Z L1 -C(=O)-CH2CH2-C(=O)-VA-, -Z L1 -C(=O)-CH2CH2-C(=O)-GGFG-NH-CH2-,-Z L1 -C(=O)-CH2CH2-C(=O)-GGVA-NH-CH2-,-Z L1 -C(=O)-CH2CH2-C(=O)-GGVCit-NH-CH2-,-Z L1 -C(=O)-CH2CH2-C(=O)-GGFCit-NH-CH2-,-Z L1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)3-CH2-C(=O)-, or -Z L1 -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)4-CH2-C(=O)-(Here, Z L1 The structural formula shown below represents the above-mentioned Lb).

[0440]

[0441] Alternatively, when the drug-antibody binding mode is "cysteine ​​coupling", a more preferred linker L for use in the antibody-drug conjugate of the present invention is -Z L2 -(CH2)5-C(=O)-GGFG-,-Z L2 -(CH2)5-C(=O)-GGVA-,-Z L2 -(CH2)5-C(=O)-GGVCit-,-Z L2 -(CH2)5-C(=O)-GGFCit-,-Z L2 -(CH2)5-C(=O)-GGICit-,-Z L2 -(CH2)5-C(=O)-GGFM-,-ZL2 -(CH2)5-C(=O)-GGPI-,-Z L2 -(CH2)5-C(=O)-GGLM-,-Z L2 -(CH2)5-C(=O)-FG-,-Z L2 -(CH2)5-C(=O)-VA-,-Z L2 -(CH2)5-C(=O)-GGFG-NH-CH2-,-Z L2 -(CH2)5-C(=O)-GGVA-NH-CH2-, -Z L2 -(CH2)5-C(=O)-GGVCit-NH-CH2-,-Z L2 -(CH2)5-C(=O)-GGFCit-NH-CH2-,-Z L2 -(CH2)5-C(=O)-NH-(CH2CH2O)3-CH2-C(=O)-, or -Z L2 -(CH2)5-C(=O)-NH-(CH2CH2O)4-CH2-C(=O)-, (here, Z L2 The above-mentioned Lb is represented by -(succinimide-3-yl-N)- represented by the structural formula shown below.

[0442]

[0443] As for the further preferred linker L used in the antibody drug conjugate of the present invention, the drug and antibody are bound in a "sugar chain coupling" manner, which is -Z L1 -C(=O)-CH2CH2-C(=O)-GGFG-NH-CH2-, or -Z L1 -C(=O)-CH2CH2-C(=O)-GGPI-NH-CH2-, (here, Z L1 The structural formula shown below represents the above-mentioned Lb).

[0444]

[0445] As for the more preferred linker L used in the antibody-drug conjugate of the present invention, the drug-antibody binding mode is "sugar chain coupling", which is -Z L1 -C(=O)-CH2CH2-C(=O)-GGFG-NH-CH2-(here, Z L1 The structural formula shown below represents the above-mentioned Lb).

[0446]

[0447] The right ends of the above-mentioned “preferred linker L”, “more preferred linker L”, “further preferred linker L” and “further preferred linker L” are bound to the drug D.

[0448] The "linker L-drug D" used in the antibody-drug conjugate of the present invention is preferably represented by the following four formulas.

[0449]

[0450]

[0451] (Here, the wavy line indicates binding to the sugar chain or remodeled sugar chain of Ab.)

[0452] The "linker L-drug D" used in the antibody drug conjugate of the present invention is more preferably represented by the following four formulas.

[0453]

[0454]

[0455] (Here, the wavy line indicates binding to the sugar chain of Ab or remodeled sugar chain)

[0456] The "linker L-drug D" used in the antibody-drug conjugate of the present invention is further preferably represented by the following formula.

[0457]

[0458]

[0459] (Here, the wavy line indicates binding to the sugar chain of Ab or remodeled sugar chain)

[0460] <2.2. Antibodies and their sugar chain modifications>

[0461] <2.2.1 Antibodies>

[0462] In the present specification, "gene" refers to a nucleotide or a nucleotide sequence or its complementary chain containing a nucleotide sequence encoding amino acids of a protein. For example, a polynucleotide, oligonucleotide, DNA, mRNA, cDNA, RNA, etc. that is a nucleotide sequence or its complementary chain containing a nucleotide sequence encoding amino acids of a protein is included in the meaning of "gene".

[0463] In this specification, "nucleotide", "polynucleotide" or "nucleotide sequence" has the same meaning as "nucleic acid". For example, DNA, RNA, probe, oligonucleotide, polynucleotide, primer, etc. are also included in the meaning of "nucleotide" or "nucleotide sequence".

[0464] In this specification, "polypeptide", "peptide" and "protein" are used without distinction.

[0465] In this specification, "functional fragments of antibodies" are also referred to as "antibody antigen-binding fragments", which refer to partial fragments of antibodies that have binding activity to antigens, including linear antibodies and multispecific antibodies formed by antibody fragments. Among them, as long as they have the ability to bind to antigens, they are not limited to these molecules. In addition, these antigen-binding fragments include not only molecules obtained by treating the full-length molecules of antibody proteins with appropriate enzymes, but also proteins produced in appropriate host cells using genetically engineered antibody genes.

[0466] The functional fragment of the antibody used in the antibody drug conjugate of the present invention includes: a functional fragment that retains asparagine (Asn297) and its surrounding amino acids that are modified by a highly conserved N-linked sugar chain in the Fc region of the IgG heavy chain and has the ability to bind to an antigen.

[0467] The antibody used in the antibody drug conjugate of the present invention refers to an immunoglobulin, which is a molecule containing an antigen binding site that immunospecifically binds to an antigen. As the antibody used in the antibody drug conjugate of the present invention, it can be any one of IgG, IgE, IgM, IgD, IgA and IgY, preferably IgG. In addition, as a subclass, it can be any one of IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, preferably IgG1, IgG2 or IgG4, more preferably IgG1 or IgG4, and particularly preferably IgG1 (an antibody having a mutation that affects ADCC and ADCP activity in the Fc region of the IgG heavy chain). Therefore, in one embodiment of the present invention, the antibody used in the antibody drug conjugate of the present invention is IgG1 or IgG4, preferably IgG1, and the functional fragment of the antibody used in the antibody drug conjugate of the present invention is a functional fragment of an antibody of IgG1 or IgG4, preferably a functional fragment of an antibody of IgG1.

[0468] The antibody or functional fragment of the antibody used in the antibody drug conjugate of the present invention comprises a mutant Fc region in which the amino acid residues at positions 234, 235, and 265 (all EU numbering) are Ala, Ala, and Gly, respectively. Here, in this specification, unless otherwise specified, the numbering of amino acid residues is based on the EU index or EU numbering (Proceedings of the National Academy of Sciences of the United States of America, Vol. 63, No. 1 (May 15, 1969), pp. 78-85).

[0469] In one embodiment of the present invention, when the antibody used in the antibody drug conjugate is IgG1 or the functional fragment of the antibody used in the antibody drug conjugate is a functional fragment of an IgG1 antibody, the mutant Fc region comprises an IgG1LALA-DG mutation (IgG1-L234A, L235A, D265G).

[0470] It has been known that the effector function can be adjusted by replacing part of the amino acid residues in the constant region (see WO88 / 07089, WO94 / 28027, WO94 / 29351). In particular, it is also known that by reducing the effector function, the possibility of activating immune cells other than the tumor site can be avoided. On the other hand, it has been reported that in a conjugate using a drug having the activity of activating immune cells, if the effector function of the antibody is reduced, the efficacy is also reduced (Non-Patent Documents 4 to 6).

[0471] In one embodiment of the present invention, when the antibody or functional fragment of the antibody used in the antibody-drug conjugate includes the mutation in the Fc region mentioned above, the effector function can be reduced while maintaining the efficacy. In addition, in one embodiment of the present invention, when the antibody or functional fragment of the antibody used in the antibody-drug conjugate includes the mutation in the Fc region mentioned above, the antibody-drug conjugate has reduced binding to human Fcγ receptors, reduced inflammatory cytokine production ability, and / or, equivalent or enhanced in vivo anti-tumor activity compared to the corresponding antibody-drug conjugate with a wild-type Fc region. Here, "corresponding antibody-drug conjugate with a wild-type Fc region" refers to a molecule that contains a wild-type Fc region instead of a mutant Fc region, and is otherwise the same or equivalent to the antibody-drug conjugate of the present invention.

[0472] Furthermore, in one embodiment of the present invention, when the antibody or the functional fragment of the antibody used in the antibody-drug conjugate contains the mutation in the Fc region mentioned above, the effector function of the antibody-drug conjugate is reduced to the same level as that of the antibody (parent antibody; antibody used for the preparation of the antibody-drug conjugate) containing the mutation in the Fc region mentioned above. Moreover, in one embodiment of the present invention, when the antibody or the functional fragment of the antibody used in the antibody-drug conjugate contains the mutation in the Fc region mentioned above, the antibody-drug conjugate has the same or improved thermal stability as the corresponding molecule having the wild-type Fc region, and in particular, when the antibody or the functional fragment of the antibody used in the antibody-drug conjugate contains the LALA-DG mutation, the antibody-drug conjugate has improved thermal stability as compared to the corresponding antibody-drug conjugate having the wild-type Fc region. It should be noted that it is generally known that IgG2 antibodies have lower effector functions than IgG1 antibodies (Front. Immunol. 2014, 5, 520).

[0473] In one embodiment of the present invention, when the mutant Fc region includes the LALA-DG mutation, the antibody drug conjugate of the present invention is also excellent in safety while maintaining the efficacy, and the effector function is reduced to the same degree as the parent antibody. In addition, in one embodiment, when the mutant Fc region includes the LALA-DG mutation, the antibody drug conjugate of the present invention is also excellent in thermal stability.

[0474] It is known that there are multiple allotypes in the constant region of antibodies. For example, regarding the IgG1 heavy chain, G1m17, G1m3, G1m1, and G1m2 can be listed. The constant region of the antibody used in the present invention is not particularly limited, but G1m17 or G1m3 is preferably used. The amino acid sequence of Fc without the Fab region is common to G1m17 and G1m3. In the LALA-DG mutant of Fc without the Fab region, compared with the LALA mutant, the weakening effect of FcγR binding activity and the improvement of thermal stability are shown (data not shown). Therefore, it is believed that the weakening effect of FcγR binding activity and the improvement effect of thermal stability possessed by the molecules of the present invention (especially antibodies, antibody-drug conjugates) are not dependent on the allotypes of G1m17 and G1m3.

[0475] It is known that there are three complementary determining regions (CDRs) in each of the heavy and light chains of antibody molecules. CDRs are also called hypervariable regions and are located in the variable regions of the heavy and light chains of antibodies. They are sites with particularly high mutability in the primary structure and are separated into three sites in the primary structure of the polypeptide chains of the heavy and light chains. In this specification, regarding the CDRs of antibodies, the CDRs of the heavy chain are denoted as CDRH1, CDRH2, and CDRH3 from the amino terminal side of the heavy chain amino acid sequence, and the CDRs of the light chain are denoted as CDRL1, CDRL2, and CDRL3 from the amino terminal side of the light chain amino acid sequence. These sites are close to each other in stereo structure and determine the specificity for the bound antigen.

[0476] In the present specification, the amino acid sequence of the CDR contained in the variable region of the antibody is determined according to the definition of KABAT (KABAT et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service National Institutes of Health, Bethesda, MD. (1991)).

[0477] The antibody can be derived from any species, preferably humans, rats, mice and rabbits are exemplified. In the case of species other than humans, chimerization or humanization is preferably performed using known techniques. The antibody used in the present invention can be a polyclonal antibody or a monoclonal antibody, preferably a monoclonal antibody. Monoclonal antibodies include monoclonal antibodies derived from non-human animals such as rat antibodies, mouse antibodies, and rabbit antibodies, chimeric antibodies, humanized antibodies, humanized antibodies, their functional fragments, or their modified bodies.

[0478] The antibody is preferably an antibody that targets tumor cells or immune cells, but is not limited thereto. The antibody is more preferably an antibody that targets tumor cells.

[0479] In the case of using an antibody targeting tumor cells, as an antibody, it is preferred to have one or more of the characteristics of being able to recognize tumor cells, being able to bind to tumor cells, being engulfed into tumor cells and internalized, and being able to damage tumor cells. The drug used in the antibody drug conjugate of the present invention has STING agonist activity. The drug activates the signal of interferon regulatory factor-3 (IRF3) to induce interferon. Therefore, it is believed that in the case of using an antibody targeting tumor cells in the antibody drug conjugate of the present invention, after the antibody drug conjugate is administered in vivo, it is delivered to the tumor site, and after being engulfed into the tumor cell, the linker part is cut by peptidase, etc., and the drug part is freed. The freed drug part increases the sensitivity of tumor cells to immune cells through STING agonist activity and activates anti-tumor immunity, thereby exerting an anti-tumor effect. Alternatively, it is also believed that even if the antibody drug conjugate accumulated in the tumor cell is not internalized, the tumor cell and / or the antibody drug conjugate is engulfed into the immune cell by phagocytosis, etc., and the anti-tumor immunity is activated by STING agonist activity, thereby exerting an anti-tumor effect.

[0480] The binding of antibodies to tumor cells can be confirmed by flow cytometry. The incorporation of antibodies into tumor cells can be confirmed by the following methods: (1) using a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody to visualize the antibody ingested into the cell using a fluorescence microscope (Cell Death and Differentiation (2008) 15, 751-761); (2) using a secondary antibody (fluorescently labeled) that binds to the therapeutic antibody to measure the amount of fluorescence ingested into the cell (Molecular Biology of the Cell Vol. 15, 5268-5282, December 2004); or (3) Mab-ZAP analysis, using an immunotoxin that binds to the therapeutic antibody, when ingested into the cell, the toxin is released to inhibit cell proliferation (Bio Techniques 28: 162-165, January 2000). As an immunotoxin, a recombinant complex protein of the catalytic domain of diphtheria toxin and protein G can also be used.

[0481] When an antibody targeting tumor cells is used in the antibody-drug conjugate of the present invention, it is preferred but not essential that the antibody itself has an anti-tumor effect.

[0482] The antitumor activity of a drug or antibody-drug conjugate refers to cytotoxic activity against tumor cells, anti-cellular effects, and reduction in tumor volume. The antitumor activity can be confirmed using a known in vitro or in vivo evaluation system.

[0483] The effects and immunostimulatory activity of drugs and antibody drug conjugates refer to the hypersensitivity of tumor cells to immune cells or the activation of immune cells via tumor cells. The effects and immunostimulatory activity of drugs and antibody drug conjugates can be confirmed using known in vitro or in vivo evaluation systems.

[0484] As the in vitro or in vivo evaluation system that can be used in the present invention, examples include: the BALB / c mouse system described in Experimental Examples 3, 4, 5 and 6, in which CT26.WT-hEGFR cells, in which the human EGFR gene is introduced into the mouse colorectal cancer cell line CT26.WT, are subcutaneously transplanted; the BALB / c-nu mouse system described in Experimental Example 7, in which human lung cancer cell line PC-9 cells are subcutaneously transplanted; the BALB / c-nu mouse system described in Experimental Example 8, in which human lung cancer cell line NCI-H358 is subcutaneously transplanted; the BALB / c-nu mouse system described in Experimental Example 9, in which CT26-hEGFR cells are introduced into the mouse colorectal cancer cell line CT26.WT, are subcutaneously transplanted; .WT by subcutaneously transplanting CT26.WT-hCDH6 cells into which the human CDH6 gene has been introduced; the co-culture analysis system of THP1 reporter cells and the human lung cancer cell line HCC827 described in Experimental Example 10; the co-culture analysis system of THP1 reporter cells and the human ovarian cancer cell line OVCAR4 described in Experimental Example 11; the BALB / c mouse system obtained by subcutaneously transplanting CT26.WT cells again as described in Experimental Examples 12 and 13; the analysis system using human whole blood as described in Experimental Example 14, etc., but are not limited to these.

[0485] Examples of the antibody used in the present invention include anti-CDH6 antibodies and anti-EGFR antibodies.

[0486] The antibodies used in the present invention can be obtained by immunizing animals with polypeptides that will become antigens using methods commonly used in the field, collecting antibodies produced in the organism and purifying them. The source of the antigen is not limited to humans, and animals can also be immunized with antigens derived from animals other than mice and rats. In this case, antibodies that can be applied to human diseases can be selected by testing the cross-reactivity of the obtained antibodies that bind to heterologous antigens and human antigens.

[0487] Alternatively, monoclonal antibodies can be obtained by fusing antibody-producing cells that produce antibodies against an antigen with myeloma cells to establish hybridomas according to known methods (e.g., Kohler and Milstein, Nature (1975) 256, p. 495-497, Kennett, R. ed., Monoclonal Antibodies, p. 365-367, Plenum Press, NY (1980)).

[0488] It should be noted that antigens can be obtained by genetically manipulating host cells to produce genes encoding antigenic proteins.

[0489] The antibody used in the antibody-drug conjugate of the present invention can be obtained according to known methods (eg, Proc. Natl. Acad. Sci. USA, 81, 6851-6855, (1984), Nature (1986) 321, p. 522-525, WO90 / 07861).

[0490] For example, anti-EGFR antibodies (WO1998 / 050433, WO2002 / 092771, etc.) and anti-CDH6 antibodies (WO2018 / 212136, etc.) can be obtained by known means.

[0491] The anti-EGFR antibody used in the present invention is not particularly limited, but preferably has the following properties, for example.

[0492] (1) An anti-EGFR antibody that specifically binds to EGFR.

[0493] (2) The antibody according to (1) above, wherein the antibody binds to the extracellular domain of human EGFR.

[0494] (3) The antibody according to (1) or (2) above, wherein the antibody is a monoclonal antibody.

[0495] (4) The antibody according to any one of (1) to (3) above, wherein the antibody is a mouse monoclonal antibody, a chimeric monoclonal antibody, a human monoclonal antibody, or a humanized monoclonal antibody.

[0496] (5) The antibody according to (1) to (4) above, wherein the heavy chain constant region is a heavy chain constant region of human IgG1 and comprises a mutation that reduces ADCC and ADCP activities.

[0497] (6) The antibody according to (4) above, wherein the heavy chain constant region is that of human IgG1, and the amino acid residues at positions 234, 235, and 265 (all EU numbering) in the Fc region are Ala, Ala, and Gly, respectively.

[0498] (7) The antibody according to (6) above, wherein the antibody is a humanized or human monoclonal antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 32 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 30.

[0499] (8) The antibody according to (6) above, wherein the antibody is a humanized or human monoclonal antibody comprising a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 33 and a light chain consisting of the amino acid sequence described in SEQ ID NO: 30.

[0500] (9) The antibody according to (6) above, wherein the antibody is a humanized or human monoclonal antibody comprising a heavy chain variable region contained in a heavy chain consisting of the amino acid sequence recorded in sequence number 32 or 33 and a light chain variable region contained in a light chain consisting of the amino acid sequence recorded in sequence number 30.

[0501] (10) The antibody according to (6) above, wherein the antibody is a humanized or human monoclonal antibody comprising the following light chains and heavy chains: a light chain comprising CDRL1 consisting of the amino acid sequence recorded in sequence number 23, CDRL2 consisting of the amino acid sequence recorded in sequence number 24, and CDRL3 consisting of the amino acid sequence recorded in sequence number 25; and a heavy chain comprising CDRH1 consisting of the amino acid sequence recorded in sequence number 26, CDRH2 consisting of the amino acid sequence recorded in sequence number 27, and CDRH3 consisting of the amino acid sequence recorded in sequence number 28.

[0502] (11) The antibody according to any one of (1) to (10) above, wherein the antibody lacks one or two amino acids at the carboxyl terminus of the heavy chain.

[0503] (12) An antibody obtained by a method for producing the antibody, the method comprising: a step of culturing a host cell transformed with an expression vector containing a polynucleotide encoding the antibody according to any one of (1) to (11) above; and a step of collecting the target antibody from the culture obtained in the step.

[0504] Examples of the anti-EGFR antibody include panitumumab, nimotuzumab, cetuximab, ametumumab (SY-101), SYN-004, SCT-200, tomozutuximab, GC-1118, GR-1401, depatuxizumab (ABT-806), serclutamab, AMG595, and matuzumab, and preferably panitumumab and ABT806 are included.

[0505] The anti-CDH6 antibody used in the present invention is not particularly limited, but preferably has the following properties, for example.

[0506] (1) An anti-CDH6 antibody that specifically binds to CDH6.

[0507] (2) The antibody according to (1) above, wherein the antibody binds to the extracellular domain of human CDH6.

[0508] (3) The antibody according to (1) or (2) above, wherein the antibody is a monoclonal antibody.

[0509] (4) The antibody according to any one of (1) to (3) above, wherein the antibody is a mouse monoclonal antibody, a chimeric monoclonal antibody, a human monoclonal antibody, or a humanized monoclonal antibody.

[0510] (5) The antibody according to (1) to (4) above, wherein the heavy chain constant region is a heavy chain constant region of human IgG1 and comprises a mutation that reduces ADCC and ADCP activities.

[0511] (6) The antibody according to (5) above, wherein the heavy chain constant region is that of human IgG1, and the amino acid residues at positions 234, 235, and 265 (all EU numbering) in the Fc region are Ala, Ala, and Gly, respectively.

[0512] (7) The antibody according to (6) above, wherein the antibody is a humanized or human monoclonal antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 46 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 44.

[0513] (8) The antibody according to (6) above, wherein the antibody is a humanized or human monoclonal antibody comprising a heavy chain variable region contained in a heavy chain consisting of the amino acid sequence recorded in sequence number 46 and a light chain variable region contained in a light chain consisting of the amino acid sequence recorded in sequence number 44.

[0514] (9) The antibody according to (6) above, wherein the antibody is a humanized or human monoclonal antibody comprising the following light chains and heavy chains: a light chain comprising CDRL1 consisting of the amino acid sequence recorded in sequence number 37, CDRL2 consisting of the amino acid sequence recorded in sequence number 38, and CDRL3 consisting of the amino acid sequence recorded in sequence number 39; and a heavy chain comprising CDRH1 consisting of the amino acid sequence recorded in sequence number 40, CDRH2 consisting of the amino acid sequence recorded in sequence number 41, and CDRH3 consisting of the amino acid sequence recorded in sequence number 42.

[0515] (10) The antibody according to any one of (1) to (9) above, wherein the antibody lacks one or two amino acids at the carboxyl terminus of the heavy chain.

[0516] (11) An antibody obtained by a method for producing the antibody, the method comprising: culturing a host cell transformed with an expression vector containing a polynucleotide encoding the antibody according to any one of (1) to (10) above; and collecting the target antibody from the culture obtained in the step.

[0517] As anti-CDH6 antibodies, for example, there can be listed: the antibody described in WO2018212136 having a light chain consisting of the amino acid sequence recorded at positions 21 to 233 of sequence number 61 and a heavy chain consisting of the amino acid sequence recorded at positions 20 to 471 of sequence number 69 (antibody X), NOV0712, and LTV977, and preferably antibody X can be listed.

[0518] The antibody used in the present invention can be an antibody having 80% to 99% amino acid identity compared to the heavy chain and / or light chain of the above-mentioned antibody. Here, the term "identity" has the usual definition used in the art. The % of identity refers to the percentage of the number of identical amino acids relative to the total number of amino acids (including gaps) when two amino acid sequences are aligned (compared) in a manner that maximizes the consistency of amino acids. Such identity is usually more than 80% identity, preferably 90%, 91%, 92%, 93% or 94% identity, more preferably 95%, 96%, 97% or 98% identity, and more preferably 99% identity. In addition, by combining the amino acid sequences with one to several amino acid residues substituted, deleted and / or added in the amino acid sequence of the heavy chain and / or light chain, antibodies with various effects equivalent to the above-mentioned antibodies can also be selected. The number of amino acid residues substituted, deleted and / or added is usually less than 10 amino acid residues, preferably less than 5 to 6 amino acid residues, more preferably less than 2 to 3 amino acid residues, and more preferably 1 amino acid residue.

[0519] It should be noted that in the antibodies produced by mammalian cultured cells, it is known that the lysine residue at the carboxyl terminus of the heavy chain is deleted (Journal of Chromatography A, 705: 129-134 (1995)), and it is also known that the two amino acid residues of glycine and lysine at the carboxyl terminus of the heavy chain are deleted, and the proline residue newly located at the carboxyl terminus is amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, the deletion or modification of these heavy chain sequences will not affect the antigen binding ability and effector function (complement activation, antibody-dependent cellular cytotoxicity, etc.) of the antibody. Therefore, the antibodies used in the present invention also include antibodies that undergo the above-mentioned deletions or modifications and functional fragments of the antibodies. Therefore, in one embodiment of the present invention, the antibody is an antibody comprising a heavy chain in which one or more amino acid residues at the carboxyl terminus are deleted. In this context, "one or several amino acid residues" refers to preferably 1 to 10 amino acid residues, 1 to 9 amino acid residues, 1 to 8 amino acid residues, 1 to 7 amino acid residues, 1 to 6 amino acid residues, 1 to 5 amino acid residues, 1 to 4 amino acid residues, 1 to 3 amino acid residues, 1 or 2 amino acid residues, or 1 amino acid residue. In one embodiment of the present invention, the antibody used in the present invention also includes a deletion in which 1 or 2 amino acids are missing at the carboxyl terminus of the heavy chain, and the amidated deletion (for example, a heavy chain in which the proline residue at the carboxyl terminal is amidated). Among them, as long as the deletion or modification (amidation) of the carboxyl terminus does not have a significant effect on the antigen binding ability and effector function, the deletion of the carboxyl terminus of the heavy chain of the antibody used in the present invention is not limited to the above-mentioned types. The two heavy chains constituting the antibody used in the present invention can be any one of the heavy chains selected from the group consisting of the full length and the above-mentioned deletion, or any two of them can be combined. The quantitative ratio of each deletion form may be affected by the type and culture conditions of the mammalian culture cells producing the antibodies used in the present invention. Preferred antibodies used in the present invention include antibodies having a deletion of one amino acid residue at the carboxyl terminus of both heavy chains.

[0520] <2.2.2 Polynucleotides, etc.>

[0521] In one embodiment of the present invention, a polynucleotide is provided that encodes the amino acid sequence of the antibody described above. For example, in one embodiment of the present invention, a polynucleotide is provided that encodes the amino acid sequence of the antibody described in any one of

[45] to

[47] (including [45-2], [46-2] and [47-2]).

[0522] In one embodiment of the present invention, an expression vector is provided, which contains the above-mentioned polynucleotide. As the expression vector, there is no particular limitation, and all plasmids, viruses and other vectors that can express the above-mentioned polynucleotide can be used. As a method for constructing such an expression vector, all known methods can be used, for example, an example can be shown: using an animal cell expression vector containing a gene of a wild-type Fc region as a template, using KOD-Plus-Mutagenesis Kit (TOYOBO) to introduce a desired effector-free mutation into the Fc region by PCR; or, synthesizing a gene of an Fc region containing a desired effector-free mutation, and incorporating it into an animal cell expression vector by a ligation reaction.

[0523] In one embodiment of the present invention, a host cell is provided, which is transformed by the above-mentioned expression vector. As the host cell, there is no particular limitation, and all kinds of eukaryotic cells and prokaryotic cells that can express the polynucleotide contained in the above-mentioned expression vector can be used. In one embodiment of the present invention, the host cell of the present invention is a eukaryotic cell, for example, animal cells, plant cells, eukaryotic microorganisms. In particular, as animal cells, mammalian cells can be listed, for example, COS cells (Cell (1981) 23, p.175-182, ATCC CRL-1650) as monkey cells, mouse fibroblasts NIH3T3 (ATCC No.CRL-1658), dihydrofolate reductase-deficient strains of Chinese hamster ovary cells (CHO cells, ATCC CCL-61) (Proc. Natl. Acad. Sci. USA (1980) 77, p.4126-4220), FreeStyle 293F cells (Invitrogen), lymphocytes, myeloma, etc. In another embodiment of the present invention, the host cell of the present invention is a prokaryotic cell, examples of which include Escherichia coli and Bacillus subtilis.

[0524] In one embodiment of the present invention, a culture method is provided, which comprises culturing the host cell. As the culture method, there is no particular limitation as long as the host cell can express the target antibody, and the culture conditions can be appropriately determined according to the type of host cell and expression vector used.

[0525] In one embodiment of the present invention, a method for producing an antibody is provided, which includes a process of culturing the above-mentioned host cells and a process of collecting the target antibody from the culture obtained from the culturing process. As the above-mentioned "process of culturing host cells", as long as the above-mentioned host cells can express the target antibody, there is no particular restriction, and the culture conditions, etc. can be appropriately determined according to the type of host cells and expression vectors used. In addition, as the process of "collecting the target antibody from the culture obtained from the culturing process", there is no particular restriction, and it can be appropriately determined according to the type of host cells, expression vectors, etc. used, the properties of the target antibody, etc. As a method for producing the antibody, as mentioned in Examples 13 and 14 of this specification, the manufacturing method described in the review result report of panitumumab intravenous drip 100mg (March 5, 2019, Medical and Food Bureau Review and Management Section) and the manufacturing method described in WO2018 / 212136 can be cited, but it is not limited thereto.

[0526] <2.2.3 Sugar chain remodeling of antibodies>

[0527] In recent years, a method has been reported to uniformly introduce sugar chains with functional groups by remodeling the sugar chains of non-uniform antibodies through enzyme reactions (ACS Chem. Biol. 2012, 7, 110-122, ACS Med. Chem. Lett. 2016, 7, 1005-1008). Attempts have also been made to use this sugar chain reconstruction technology to site-specifically introduce drugs and synthesize uniform ADCs (Bioconjugate Chem. 2015, 26, 2233-2242, Angew. Chem. Int. Ed. 2016, 55, 2361-2367, US2016361436).

[0528] Sugar chain reconstruction first uses a hydrolase to remove the uneven sugar chains added to proteins (antibodies, etc.) leaving only the terminal GlcNAc, and prepares a uniform protein part (hereinafter referred to as the "acceptor") to which GlcNAc is added. Next, an arbitrary sugar chain prepared separately (hereinafter referred to as the "donor") is prepared, and the acceptor and the donor are linked using a glycosyltransferase. In this way, a uniform glycoprotein with an arbitrary sugar chain structure can be synthesized.

[0529] In the present invention, "sugar chain" refers to a structural unit formed by two or more monosaccharides linked by glycosidic bonds. Specific monosaccharides and sugar chains are sometimes labeled with abbreviations such as "GlcNAc-" and "SG-". When these abbreviations are used in structural formulas, the oxygen atom or nitrogen atom at the reducing end that belongs to the glycosidic bond with another structural unit is expressed in a form not included in the abbreviation representing the sugar chain, except for cases with special definitions.

[0530] In the present invention, the monosaccharide that is the basic unit of the sugar chain is recorded as the carbon atom that is bonded to the oxygen atom that constitutes the ring and directly bonded to the hydroxyl group (or the oxygen atom belonging to the glycosidic bond) in the ring structure as position 1 (only in sialic acid, position 2). The names of the example compounds are named according to the overall chemical structure, and this rule does not necessarily apply.

[0531] In the present invention, when a sugar chain is described as a symbol (e.g., SG, MSG, GlcNAc, etc.), unless otherwise defined, the carbon to the reducing terminal is deemed to be included in the symbol, and N or O belonging to the N- or O-glycosidic bond is deemed to be excluded in the symbol.

[0532] The antibody-drug conjugate of the present invention is represented by the following formula.

[0533]

[0534] The antibody Ab or its functional fragment binds to L directly through the side chain of its amino acid residue (eg, cysteine, lysine, etc.), or binds to L through the sugar chain or remodeled sugar chain of Ab.

[0535] The sugar chain of Ab in the present invention is an N-linked sugar chain or an O-linked sugar chain, and is preferably an N-linked sugar chain.

[0536] The N-linked sugar chain is linked to the amino acid side chain of the antibody through an N-glycosidic bond, and the O-linked sugar chain is linked to the amino acid side chain of the antibody through an O-glycosidic bond.

[0537] The Ab in the present invention is IgG, preferably IgG1, IgG2 or IgG4, more preferably IgG1 or IgG4, and particularly preferably IgG1.

[0538] It is known that IgG has a highly conserved N-linked sugar chain (hereinafter referred to as "Asn297 sugar chain or N297 sugar chain") at the 297th asparagine residue in the Fc region of its heavy chain, which contributes to the activity, dynamics, etc. of the antibody molecule (Eon-Duval, A. et al, Biotechnol. Prog. 2012, 28, 608-622, Sanglier-Cianferani, S., Anal. Chem. 2013, 85, 715-736).

[0539] The amino acid sequence in the constant region of IgG is highly conserved, and in the report of Edelman et al. (Proc. Natl. Acad. Sci. USA, 63, 78-85, (1969)), each amino acid is identified by EU numbering (EU INDEX). For example, Asn297 added to the N-linked sugar chain in the Fc region corresponds to position 297 in the EU numbering, and even if the actual amino acid position changes due to molecular fragmentation or region deletion, the amino acid is unambiguously specified by indicating it by EU numbering.

[0540] The figure below shows the binding of the N297 sugar chain of the antibody or its functional fragment to L in the antibody-drug conjugate of the present invention.

[0541]

[0542] In addition, an antibody having such a remodeled sugar chain is referred to as a sugar chain remodeled antibody.

[0543] SGP (α2,6-SGP) is the abbreviation of Sialylglycopeptide, which is a representative glycopeptide of N-linked glycopeptides. SGP can be isolated and purified from egg yolks according to the method described in WO2011 / 027868, for example. In addition, refined SGP is sold by Tokyo Chemical Industry and Fushimi Pharmaceutical. In this specification, the sugar chain part of SGP is denoted as SG, and the sugar chain with one GlcNAc missing at the reducing end of SG is denoted as SG (10). SG (10), for example, can be prepared by enzymatic hydrolysis of SGP with reference to the report of Umekawa et al. (Biochim. Biophys. Acta 2010, 1800, 1203-1209). In addition, SG (10) can also be purchased from Tokyo Chemical Industry and Fushimi Pharmaceutical.

[0544] In this specification, a sugar chain structure having a sialic acid deficiency at the non-reducing terminal only on one of the β-Man branches of SG (10) is referred to as MSG (9), a structure having sialic acid only on the 1st to 3rd sugar chains of the branch is referred to as MSG1, and a structure having sialic acid only on the 1st to 6th sugar chains of the branch is referred to as MSG2.

[0545] The reconstructed sugar chain used in the antibody drug conjugate of the present invention is N297-(Fuc)SG, N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture of N297-(Fuc)MSG1 and N297-(Fuc)MSG2, preferably N297-(Fuc)SG, N297-(Fuc)MSG1 or N297-(Fuc)MSG2, and more preferably N297-(Fuc)SG or N297-(Fuc)MSG1.

[0546] N297-(Fuc)SG is represented by the following structural formula or sequence formula.

[0547]

[0548] In the above formula, the wavy line indicates the connection with Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at the 2-position of the sialic acid at both the 1-3 chain side and the 1-6 chain side of the β-Man branch of the N297 sugar chain, and the asterisk indicates that it is bonded to the nitrogen atom at the 1-position or 3-position on the 1,2,3-triazole ring of Lb in the linker L, in particular, n 5 It is an integer of 2-10, and preferably an integer of 2-5.

[0549] N297-(Fuc)MSG1 is represented by the following structural formula or sequence formula.

[0550]

[0551] In the above formula, the wavy line indicates the connection with Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of L(PEG) forms an amide bond with the carboxyl group at the 2-position of the non-reducing end of the sialic acid at the 1-3 chain side of the branched chain of β-Man of the N297 sugar chain, and the asterisk indicates that it is bonded to the nitrogen atom at the 1-position or 3-position on the 1,2,3-triazole ring of Lb in the linker L, in particular, n 5 It is an integer of 2-10, and preferably an integer of 2-5.

[0552] N297-(Fuc)MSG2 is represented by the following structural formula or sequence formula.

[0553]

[0554] In the above formula, the wavy line indicates the connection with Asn297 of the antibody, and L(PEG) represents -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at the 2-position of the sialic acid at the non-reducing end of the 1-6 chain side of the β-Man branch of the N297 sugar chain, and the asterisk indicates that it is bonded to the nitrogen atom at the 1-position or 3-position on the 1,2,3-triazole ring of the linker L, especially Lb in the linker L. Here, n 5 It is an integer of 2-10, and preferably an integer of 2-5.

[0555] When the N297 sugar chain of the antibody in the antibody-drug conjugate of the present invention is N297-(Fuc)SG, one antibody molecule has two heavy chains, and thus the antibody-drug conjugate becomes a molecule to which four linkers L and four drugs D are bound (the above m 2 =2) (refer to Figure 1 That is, the number of drug D bound to each heavy chain in the antibody drug conjugate is 2.

[0556] When the N297 sugar chain of the antibody in the antibody-drug conjugate of the present invention is N297-(Fuc)MSG1 or N297-(Fuc)MSG2 or a mixture thereof, one antibody molecule has two heavy chains, and thus the antibody-drug conjugate becomes a molecule to which two linkers L and two drugs D are bound (the above m 2 =1) (refer to Figure 1 B). That is, the number of drug D bound to each heavy chain in the antibody-drug conjugate is 1.

[0557] The N297 sugar chain is preferably N297-(Fuc)SG, N297-(Fuc)MSG1, or N297-(Fuc)MSG2, more preferably N297-(Fuc)SG or N297-(Fuc)MSG1, and even more preferably N297-(Fuc)SG.

[0558] When the N297 sugar chain of the antibody in the antibody-drug conjugate of the present invention is N297-(Fuc)SG, N297-(Fuc)MSG1, or N297-(Fuc)MSG2, an antibody-drug conjugate with high uniformity can be obtained.

[0559] <3. Manufacturing method>

[0560] The representative production method of the antibody-drug conjugate or its production intermediate containing the CDN derivative of the present invention is described. It should be noted that the numbers of the compounds shown in each reaction formula are used to represent the compounds below. That is, they are referred to as "compounds of formula (1)", "compound (1)", etc. In addition, the same description is also given to compounds with other numbers.

[0561] In the following methods A to E, the substituent L 1 The substituent L has the same meaning as above. 2 represents a group selected from the following (i) or (ii). (i) When combined with L, L 2represents -NHR', hydroxy C1-C6 alkyl or amino C1-C6 alkyl, wherein R' represents a hydrogen atom, a C1-C6 alkyl, a C2-C6 alkenyl, a C2-C6 alkynyl or a C3-C6 cycloalkyl, and the C1-C6 alkyl, C2-C6 alkenyl or C2-C6 alkynyl is optionally substituted by 1 to 6 halogen atoms; or (ii) when not combined with L, L 2 represents a hydrogen atom or a halogen atom. 1 represents -NH- or a sulfur atom. Substituent W 2 Represents -CH=. Substituent Z 1 ~Z 3 They all represent -CH2-CH2-CH2-. The substituent R 1 ~R 3 Each independently represents a hydrogen atom, a halogen atom, -OR', -OC(=O)R', -N3, -NHR', -NR'R" or -NHC(=O)R' (herein, R' is as defined above, and R" represents a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group or a C3-C6 cycloalkyl group). Substituent R 4 Represents a hydrogen atom. 5 , in W 1 When it is a nitrogen atom, R 5 represents hydrogen atoms, in W 1 When it is an oxygen atom, R 5 Not present. Substituent R a , R c , R e and R g It represents the side chain of natural α-amino acid. For example, it is methyl, isopropyl, sec-butyl, isobutyl, benzyl, etc. PRO 1 It represents a protecting group for a primary alcohol. It is preferably 4,4'-dimethoxytrityl, 4-methoxytrityl, etc. PRO 2 、PRO 3 、PRO 7 、PRO 8 represents a protecting group for a secondary alcohol. Preferred are tert-butyldimethylsilyl, triisopropylsilyloxymethyl, benzoyl, 2-nitrobenzyl, 4-methoxytetrahydropyran-4-yl, and the like. PRO 6 represents a protecting group of a carboxylic acid. Preferably, it is tert-butyl, benzyl, etc. 5 、PRO 9 Represents a protecting group for an amine. 5 Preferred are tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl, and the like. 9 Preferred is 9-fluorenylmethyloxycarbonyl or 2-(trimethylsilyl)ethoxycarbonyl. 4represents a protecting group for alcohol or amine. In the case of alcohol, it is preferably tert-butyldimethylsilyl, benzoyl, etc., and in the case of amine, it is preferably 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, tert-butyloxycarbonyl, etc. a represents an oxygen atom or a sulfur atom, Q b represents a hydroxyl group or a thiol group. a’ and Q b’ Each independently represents a negatively charged oxygen atom (O － ) or sulfur atom (S － ). x and R y Each independently represents a halogen atom or -O-PRO 2 . n represents an integer from 1 to 3.

[0562] A Method

[0563] The CDN derivative shown in (1) used in the antibody-drug conjugate of the present invention can be produced by the method A described below.

[0564]

[0565] The present production method is a method for producing a compound represented by the general formula (1). Steps A-1 to A-5 of the present production method can be synthesized in one pot, which can be carried out with reference to the report of Gaffney et al. (Org. Lett. 2010, 12, 3269-3271).

[0566]

[0567] (A-1 process)

[0568] This step is a step of preparing a compound of formula (2a) by continuously performing a hydrolysis reaction and removing a cyanoethyl group on a compound of formula (1a) using a known organic chemical method. The hydrolysis reaction is carried out by treating compound (1a) with water and an acid (pyridine trifluoroacetate, 4,5-dicyanoimidazole, 1H-tetrazole, etc.) in a solvent (acetonitrile, tetrahydrofuran, N,N-dimethylformamide or a mixed solvent thereof) at -10°C to the boiling point of the solvent used in the reaction, preferably at 15°C to 35°C. Water is used in an amount of 2 mol to an excess mol, preferably 2 mol to 10 mol, and the acid is used in an amount of 1 mol to an excess mol, preferably 1 mol to 5 mol, relative to 1 mol of compound (1a). The reaction time is 1 minute to 3 hours, preferably 5 minutes to 30 minutes. Next, a base (tert-butylamine, etc.) is added to the reaction solution to remove the cyanoethyl group. The base is used in an excess mol, preferably 30 mol to 50 mol, relative to 1 mol of compound (1a). The reaction time is 5 minutes to 6 hours, preferably 15 minutes to 1 hour. The reaction solution is concentrated under reduced pressure to obtain a crude product of compound (2a). The crude product of compound (2a) can be transferred to the next step without purification.

[0569] (A-2 process)

[0570] This step is a step of removing the protective group of the hydroxyl group of the compound of formula (2a) by a known organic chemical method to produce a compound of formula (3a). Before starting the reaction of this step, the crude product of formula (2a) is dried by azeotropy with acetonitrile once to three times as needed. 1 In the case of 4,4'-dimethoxytrityl, the 4,4'-dimethoxytrityl group is removed by treating compound (2a) with water and an acid (dichloroacetic acid, trifluoroacetic acid, etc.) in a solvent (dichloromethane, chloroform, dichloroethane, etc.) at -10°C to the boiling point of the solvent used in the reaction, preferably at 15°C to 35°C. Water is used in excess moles, preferably 10 to 20 moles, relative to 1 mole of compound (2a). The acid is diluted to 1% to 50% (v / v), preferably 5% to 10% (v / v) with the solvent used in the reaction, and this diluted solution is used in excess moles, preferably 5 to 15 moles. The reaction time is 1 minute to 3 hours, preferably 5 minutes to 30 minutes. Pyridine is added to the reaction solution to terminate the reaction. Pyridine is used in an amount sufficient to neutralize the acid used, preferably 2 to 10 moles relative to 1 mole of the acid. The reaction solution is concentrated under reduced pressure to obtain a crude product of compound (3a). The crude product of compound (3a) was azeotroped 3 to 5 times with dehydrated acetonitrile. Acetonitrile was left in the final azeotropy to obtain a 0.01M to 1M acetonitrile solution of compound (3a). The obtained acetonitrile solution was directly used in the next step.

[0571] (A-3 process)

[0572] This step is a step of preparing a compound of formula (5a) by continuously performing a coupling reaction with a compound of formula (4a) and a sulfidation reaction of the obtained coupling product on a compound of formula (3a) using a known organic chemical method. Before starting the reaction of this step, compound (4a) is azeotroped 3 to 5 times with dehydrated acetonitrile. Acetonitrile is left in the final azeotropy to prepare a 0.01M to 1M acetonitrile solution of compound (4a). A desiccant (powdered or granular molecular sieve 3A or molecular sieve 4A) is added to the solution and stored under a nitrogen or argon atmosphere until the solution is used. The coupling reaction is carried out by adding an acetonitrile solution of compound (4a) dried by azeotropy to the acetonitrile solution of compound (3a) at 5°C to 35°C. The reaction time is 1 minute to 24 hours, preferably 5 minutes to 6 hours. Next, a sulfiding agent (N,N-dimethyl-N'-(3-sulfanylidene-3H-1,2,4-dithiazol-5-yl)formamidine, 3H-1,2-benzodithiol-3-one, etc.) is added to the reaction solution to carry out a sulfiding reaction. The sulfiding agent is used in an amount of 1 to 5 mol, preferably 1 to 2 mol, relative to 1 mol of compound (3a). The reaction time is 5 minutes to 24 hours, preferably 30 minutes to 6 hours. The reaction solution is concentrated under reduced pressure to obtain a crude product of compound (5a). The crude product of compound (5a) obtained is directly subjected to the next step.

[0573] (A-4 process)

[0574] This step is a step of removing the protective group of the hydroxyl group of the compound of formula (5a) using a known organic chemical method to produce a compound of formula (6a). 1 In the case of 4,4'-dimethoxytrityl group, the 4,4'-dimethoxytrityl group is removed by treating the compound (5a) with water and an acid (dichloroacetic acid, trifluoroacetic acid, etc.) in a solvent (dichloromethane, chloroform, dichloroethane, etc.) at -10°C to the boiling point of the solvent used in the reaction, preferably at 15°C to 35°C. Water is used in excess moles, preferably 10 to 20 moles, relative to 1 mole of compound (5a). The acid is diluted to 1% to 50% (v / v), preferably 5% to 10% (v / v) with the solvent used in the reaction, and the diluted solution is used in excess moles, preferably 5 to 15 moles. The reaction time is 1 minute to 3 hours, preferably 5 minutes to 30 minutes. Pyridine is added to the reaction solution to terminate the reaction. Pyridine is used in an amount sufficient to neutralize the acid used, preferably 10 to 200 moles relative to 1 mole of the acid. The reaction solution is concentrated under reduced pressure to obtain a crude product of compound (6a). The obtained crude product of compound (6a) is directly used for the next step.

[0575] (A-5 process)

[0576] This step is a step of preparing a compound of formula (7a) by continuously performing a cyclization reaction and a sulfidation reaction on a compound of formula (6a) using a known organic chemical method. Compound (6a) is dissolved in pyridine and concentrated under reduced pressure to prepare a 0.01M to 0.5M pyridine solution. A dehydrating condensation agent (2-chloro-5,5-dimethyl-1,3,2λ) is added to the pyridine solution at 5°C to 35°C. 5 -dioxaphosphinane-2-one, etc.) to carry out the cyclization reaction. The dehydration condensation agent is used in an amount of 1 mol to an excess amount, preferably 3 mol to 5 mol, relative to 1 mol of compound (6a). The reaction time is 1 minute to 6 hours, preferably 5 minutes to 1 hour. Next, water and a sulfiding agent (3H-1,2-benzodithiol-3-one, N,N-dimethyl-N'-(3-sulfylidene-3H-1,2,4-dithiazol-5-yl)formamidine, etc.) are added to the reaction solution to carry out the sulfidation reaction. With respect to 1 mol of compound (6a), water is used in an excess amount, preferably 30 mol to 50 mol, and the sulfiding agent is used in an amount of 1 mol to 5 mol, preferably 1 mol to 2 mol. The reaction time is 5 minutes to 12 hours, preferably 30 minutes to 3 hours. The reaction solution is added to an aqueous sodium bicarbonate solution (0.1 M to 1 M), stirred for 15 minutes to 24 hours, and the reaction is stopped. After extracting the reaction solution 1 to 5 times with an organic solvent (ethyl acetate, diethyl ether, toluene or a mixed solvent thereof), the combined extracts are dried with anhydrous salt (anhydrous sodium sulfate or anhydrous magnesium sulfate). The desiccant is filtered out and the filtrate is concentrated under reduced pressure. The residue obtained is purified by silica gel column chromatography [dichloromethane / methanol, ethyl acetate / methanol, hexane / ethyl acetate, etc.], C18 silica gel column chromatography [buffer / acetonitrile] or a combination thereof to obtain compound (7a) in the form of a mixture of two or more diastereomers or two or more pure diastereomers. In this step, two diastereomers are obtained in most cases, but depending on the raw materials (1a) and (4a), one or two diastereomers may be further obtained. Even if the obtained compound (7a) is a mixture of multiple diastereomers, it can enter the next step without further purification.

[0577] (A-6 process)

[0578] This step is a step of simultaneously removing the cyanoethyl group and all acyl protecting groups from the compound of formula (7a) using a known organic chemical method to produce a compound of formula (8a). This step can be carried out in an autoclave or a sealed tube as required. 4In the case of a benzoyl group, the cyanoethyl group and the benzoyl group are removed by treating the compound (7a) with 28% (v / v) ammonia water at 5°C to the boiling point of the solvent used in the reaction in a solvent (methanol, ethanol, tetrahydrofuran or a mixed solvent thereof). Relative to 1 mol of compound (7a), an excess mole of ammonia is used, preferably 300 to 3000 mol. The reaction time is 30 minutes to 96 hours, preferably 2 hours to 48 hours. The reaction solution is concentrated as needed, and the residue is refined by preparative HPLC [buffer / acetonitrile, buffer / methanol, etc.], C18 silica gel column chromatography [buffer / acetonitrile, buffer / methanol, etc.] or a combination thereof to obtain compound (8a). Even if the obtained compound (8a) is a mixture of diastereomers, it can proceed to the next step without further refining. In addition, in this step, it is also possible to proceed directly to the next step without refining.

[0579] (A-7 process)

[0580] This step is a step of simultaneously removing all silyl-based protecting groups from the compound of formula (8a) using a known organic chemical method to produce a compound of formula (9a). 2 and PRO 3 In the case of a tert-butyldimethylsilyl group, the tert-butyldimethylsilyl group is removed by directly treating the compound (8a) with triethylamine trihydrofluoride at 5°C to 100°C, preferably at 35°C to 60°C. The triethylamine trihydrofluoride is used in excess mole relative to 1 mole of the compound (8a), preferably 100 to 200 moles. The reaction time is 30 minutes to 24 hours, preferably 2 hours to 12 hours. After the reaction solution is cooled to room temperature, a mixed solution of 3:1 to 10:1 (v / v) of an ice-cold 1M triethylammonium bicarbonate aqueous solution and triethylamine is injected little by little into the reaction solution to stop the reaction. If necessary, the reaction solution can also be injected into a mixed solution of an ice-cold 1M triethylammonium bicarbonate aqueous solution and triethylamine. In this case, the reaction container is cleaned with acetonitrile and water. Triethylamine is used in an amount sufficient to change the liquid property of the reaction solution to weak alkalinity, preferably about 2 moles of triethylamine are used relative to 1 mole of triethylamine trihydrofluoride. After the organic solvent component of the reaction solution is distilled off under reduced pressure, the residual aqueous solution is purified by preparative HPLC [buffer / acetonitrile, buffer / methanol, etc.], C18 silica gel column chromatography [buffer / acetonitrile, buffer / methanol, etc.], or a combination thereof to obtain compound (9a) as a single diastereomer.

[0581] (A-8 process)

[0582] This step is a step of producing a compound of formula (1) by subjecting a compound of formula (9a) to ion exchange using a known organic chemical method. A cation exchange resin (BT AG (registered trademark) 50W-X2 resin, 100-200 mesh, hydrogen type) is suspended in pure water and filled into an empty column cartridge. The cation exchange resin is used in an amount of 10 to 50 times the weight ratio of compound (9a). After allowing excess pure water to flow naturally, a 1M sodium hydroxide aqueous solution is allowed to flow naturally for 3 column volumes, and then 6 column volumes of pure water are allowed to flow naturally. Compound (9a) is dissolved in about 3 column volumes of pure water and loaded into a chromatography column. When the compound is poorly soluble in pure water, a mixed solution with a small amount of an organic solvent (acetonitrile, methanol, etc.) can be used. After the naturally flowing solution is separated, it is further eluted with 6 column volumes of pure water to separate the fractions. The fractions containing the target compound are combined and lyophilized to obtain compound (1) as a single diastereomer.

[0583] A' Method

[0584] The CDN derivative represented by (1′) used in the antibody-drug conjugate of the present invention can be produced by the following method A′.

[0585]

[0586] This production method is a method for producing a compound represented by the general formula (1') by modifying a part of the method A. Specifically, the compound represented by the general formula (1') can be produced by modifying the step A-5 of the method A to the step A'-5 shown below. x and R y When both are halogen atoms, step A-7 can be omitted.

[0587]

[0588] (A'-5 process)

[0589] This step is a step of preparing a compound of formula (7a') by continuously performing a cyclization reaction and an oxidation reaction on the compound of formula (6a') using a known organic chemical method. Compound (6a') is dissolved in pyridine and concentrated under reduced pressure to prepare a 0.01M to 0.5M pyridine solution. A dehydrating condensation agent (2-chloro-5,5-dimethyl-1,3,2λ 5-dioxaphosphinane-2-one, etc.) to carry out cyclization reaction. Relative to 1 mol of compound (6a'), the dehydration condensation agent is used in an amount of 1 mol to an excess mol, preferably 3 mol to 5 mol. The reaction time is 1 minute to 6 hours, preferably 5 minutes to 1 hour. Next, water and an oxidant (iodine, etc.) are added to the reaction solution to carry out oxidation reaction. Relative to 1 mol of compound (6a'), water is used in an amount of 0 mol to an excess mol, preferably 30 mol to 50 mol, and the oxidant is used in an amount of 2 mol to 10 mol, preferably 3 mol to 5 mol. The reaction time is 5 minutes to 12 hours, preferably 30 minutes to 3 hours. The reaction solution is added to an aqueous sodium bicarbonate solution (0.1 M to 1 M), stirred for 15 minutes to 24 hours, and the reaction is stopped. After the reaction solution is extracted 1 to 5 times with an organic solvent (ethyl acetate, diethyl ether, toluene or a mixed solvent thereof), the extracts are combined and dried with an anhydrous salt (anhydrous sodium sulfate or anhydrous magnesium sulfate). The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography [dichloromethane / methanol, ethyl acetate / methanol, hexane / ethyl acetate, etc.], C18 silica gel column chromatography [buffer / acetonitrile] or a combination thereof to obtain compound (7a').

[0590] A" method

[0591] The CDN derivative shown in (1") used in the antibody-drug conjugate of the present invention can be produced by the method A" described below.

[0592]

[0593] The present production method is a method for producing a compound represented by the general formula (1") by modifying a part of the A method. Specifically, the compound represented by the general formula (1") can be produced by modifying the A-3 step of the A method to the A"-3 step shown below. x and R y When both are halogen atoms, step A-7 can be omitted.

[0594]

[0595] (A”-3 steps)

[0596] This step is a step of producing a compound of formula (5a") by continuously performing a coupling reaction of a compound of formula (3a") with a compound of formula (4a") and an oxidation reaction of the resulting coupled product using a known organic chemical method. Prior to starting the reaction of this step, compound (4a") is azeotroped 3 to 5 times with dehydrated acetonitrile. Acetonitrile is left in the final azeotropy to prepare a 0.01M to 1M acetonitrile solution of compound (4a"). A desiccant (powdered or granular molecular sieve 3A or molecular sieve 4A) is added to the solution and stored under a nitrogen or argon atmosphere until the solution is used. The coupling reaction is carried out by adding an acetonitrile solution of compound (4a") that has been dried by azeotropy to the acetonitrile solution of compound (3a") at 5°C to 35°C. The reaction time is 1 minute to 24 hours, preferably 5 minutes to 6 hours. Next, an oxidant (tert-butyl hydroperoxide, etc.) is added to the reaction solution to carry out an oxidation reaction. Relative to 1 mole of compound (3a"), 1 mole to 5 moles of the oxidant are used, preferably 2 moles to 3 moles are used. The reaction time is 5 minutes to 24 hours, preferably 30 minutes to 6 hours. A saturated aqueous solution of sodium thiosulfate is added to the reaction solution, stirred for 10 minutes to 12 hours, and the reaction is stopped. After extracting the reaction solution 1 to 5 times with an organic solvent (a mixed solvent of dichloromethane and methanol, etc.), the combined extracts are dried with anhydrous salt (anhydrous sodium sulfate or anhydrous magnesium sulfate). The desiccant is filtered out, and the filtrate is concentrated under reduced pressure to obtain a crude product of compound (5a"). The crude product of compound (5a") obtained is directly subjected to the next step.

[0597] A' method

[0598] The CDN derivative represented by (1''") used in the antibody-drug conjugate of the present invention can be produced by the A'' method described below.

[0599]

[0600] The present production method is a method for producing a compound represented by the general formula (1'") by modifying a portion of the A method. Specifically, the compound of the general formula (1'") can be produced by modifying the A-3 step of the A method to the A"-3 step, and modifying the A-5 step to the A'-5 step. In addition, in the substituent R x and R y When both are halogen atoms, step A-7 can be omitted.

[0601]

[0602] Method B: Coupling Precursor (Sugar Chain Coupling)

[0603] The conjugate precursor shown in (2) used in the antibody-drug conjugate of the present invention can be produced by the method B described below.

[0604]

[0605] The manufacturing method is used to manufacture L 1 A method for coupling the precursor (2) in the case where -NH2 is substituted at any position.

[0606]

[0607] (B-1 process)

[0608] This step is a step of producing a compound of formula (2b) by removing the protecting group from the compound of formula (1b) using a known organic chemical method. 5 In the case of tert-butyloxycarbonyl, the protecting group is removed by treating compound (1b) with trifluoroacetic acid in a solvent (dichloromethane, dioxane, acetonitrile, ethyl acetate, tetrahydrofuran, or a mixed solvent thereof) at -10°C to the boiling point of the solvent used in the reaction, preferably at 15°C to 35°C. Trifluoroacetic acid is used in excess mole, preferably 20 to 50 moles, relative to 1 mole of compound (1b). The reaction time is 5 minutes to 24 hours, preferably 30 minutes to 6 hours. After concentrating the reaction solution under reduced pressure, it is suspended in toluene and concentrated again under reduced pressure. This operation is repeated 2 to 5 times. After adding a solvent (diethyl ether, diisopropyl ether, hexane, dichloromethane, ethyl acetate, or a mixed solvent thereof) to make a slurry, the solid is filtered out to obtain a crude product of compound (2b). The crude product of compound (2b) enters the next step without further purification.

[0609] (B-2 process)

[0610] This step is a step of preparing a compound of formula (4b) by amidating a compound of formula (2b) with a compound of formula (3b) using a known organic chemical method. Amidation is carried out by reacting compound (2b) with a base (triethylamine, N,N-diisopropylethylamine, etc.) and compound (3b) in a solvent (N,N-dimethylformamide, N-methylpyrrolidone, N,N-dimethylacetamide, acetonitrile, etc.) at 5°C to 35°C. The base is used in an amount of 1 to 5 moles, and the compound (3b) is used in an amount of 0.5 to 1.5 moles per mole of compound (2b). The reaction time is 10 minutes to 72 hours, preferably 1 hour to 24 hours. The reaction solution is poured into two layers of an organic solvent (dichloromethane, chloroform, ethyl acetate, methanol or a mixed solvent thereof) and water or an acidic aqueous solution (0.1M to 1M hydrochloric acid, citric acid aqueous solution, etc.), and extraction is performed with the organic solvent once to five times. The combined extracts are washed with saturated brine and dried with anhydrous salt (anhydrous sodium sulfate or anhydrous magnesium sulfate). The desiccant is filtered off and the filtrate is concentrated under reduced pressure. It should be noted that the above-mentioned liquid separation operation can also be omitted, and the reaction solution can be directly concentrated under reduced pressure and enter the next step of silica gel column refining. The obtained residue is refined by silica gel column chromatography [dichloromethane / methanol, ethyl acetate / methanol, etc.] to obtain compound (4b). As needed, the obtained compound (4b) can be dissolved in a good solvent (ethyl acetate, acetonitrile, dichloromethane, methanol or a mixed solvent thereof), and then a poor solvent (diethyl ether, diisopropyl ether, hexane, etc.) is added for reprecipitation, and the purity is improved by filtering out the solid.

[0611] (B-3 process)

[0612] This step is a step of preparing a compound of formula (5b) by esterifying a compound of formula (4b) using a known organic chemical method. The esterification is carried out by reacting compound (4b) with N-hydroxysuccinimide and a condensing agent (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, etc.) in a solvent (N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, acetonitrile, etc.) at 5°C to 35°C. 1 to 3 mol of each of N-hydroxysuccinimide and the condensing agent are used relative to 1 mol of compound (4b). The reaction time is 30 minutes to 72 hours, preferably 2 hours to 24 hours. After diluting the reaction solution with an organic solvent (dichloromethane, chloroform, ethyl acetate or a mixed solvent thereof), wash with ice water 3 to 5 times. The organic layer is dried using an anhydrous salt (anhydrous sodium sulfate or anhydrous magnesium sulfate). After filtering and removing the drying agent, the filtrate is concentrated under reduced pressure to obtain a crude product of compound (5b). If necessary, the obtained compound (5b) can be purified by C18 silica gel column chromatography [acetonitrile only]. In addition, the obtained compound (5b) is dissolved in a good solvent (ethyl acetate, acetonitrile, dichloromethane or a mixed solvent thereof), and then a poor solvent (diethyl ether, diisopropyl ether, hexane, etc.) is added for reprecipitation, and the purity is improved by filtering out the solid.

[0613] (B-4 process)

[0614] This step is a step of producing a compound of formula (2) by subjecting a compound of formula (5b) to a condensation reaction with a compound of formula (6b) using a known organic chemical method. The condensation reaction is carried out by reacting compound (6b) with a base (triethylamine, N,N-diisopropylethylamine, etc.) and compound (5b) in a solvent (N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, acetonitrile, etc.) at -10°C to 100°C, preferably at 15°C to 35°C. The base is used in an amount of 2 to 5 moles, and the compound (5b) is used in an amount of 1 to 2 moles, relative to 1 mole of compound (6b). The reaction time is 5 minutes to 24 hours, preferably 1 hour to 6 hours. Benzylamine is added to the reaction solution to terminate the reaction. Benzylamine is used in an amount of 4 to 10 moles, relative to 1 mole of compound (6b). If necessary, the reaction solution is partially concentrated under reduced pressure, and the residual solution is purified by preparative HPLC [buffer / acetonitrile, buffer / methanol, etc.], C18 silica gel column chromatography [buffer / acetonitrile, buffer / methanol, etc.], or a combination thereof to obtain compound (2).

[0615] B' Method: Precursor coupling (cysteine ​​coupling)

[0616] The conjugate precursor represented by (2') used in the antibody-drug conjugate of the present invention can be produced by the following method B'.

[0617]

[0618] The manufacturing method is used to manufacture L 1 A method for coupling the precursor (2') in the case where -NH2 is substituted at any position.

[0619]

[0620] (B-5 process)

[0621] This step is a step of preparing a compound of formula (8b) by amidating a compound of formula (2b') with a compound of formula (7b) using a known organic chemical method. Compound (8b) is obtained by the method described in step B-2 of method B except that no base is used.

[0622] (B-6 process)

[0623] This step is a step of producing a compound of formula (9b) by removing the protecting group from the compound of formula (8b) using a known organic chemical method. 6 In the case of a tert-butyl group, the compound (9b) was obtained according to the procedure described in step B-1 of method B, except that silica gel column chromatography [dichloromethane / methanol] was used for the purification operation.

[0624] (B-7 process)

[0625] This step is a step of producing a compound of formula (10b) by esterifying a compound of formula (9b) using a known organic chemical method. Compound (10b) is obtained by the method described in step B-3 of method B.

[0626] (B-8 process)

[0627] This step is a step of producing a compound of formula (2') by subjecting the compound of formula (6b) to a condensation reaction with a compound of formula (10b) using a known organic chemical method. Compound (2') is obtained by the method described in step B-4 of method B.

[0628] C Method

[0629] The conjugate precursor shown in (3) used in the antibody-drug conjugate of the present invention can be produced by the method C described below.

[0630]

[0631] The manufacturing method is used to manufacture L 1 A method for coupling the precursor (3) in the case where a hydroxyl group is substituted at any position.

[0632]

[0633] (C-1 process)

[0634] This step is a step of preparing a compound of formula (3c) by amidating the compound of formula (1c) with a compound of formula (2c) using a known organic chemical method. Compound (3c) is obtained by the method described in step B-2 of method B.

[0635] (C-2 process)

[0636] This step is a step of producing a compound of formula (4c) by esterifying the compound of formula (3c) using a known organic chemical method. Compound (4c) is obtained by the method described in step B-3 of method B.

[0637] (C-3 process)

[0638] This step is a step of producing a compound of formula (7c) by successively performing a coupling reaction (aminomethylidene formation) with a compound of formula (6c) and deprotecting the resulting coupled product using a known organic chemical method. 9 In the case of 9-fluorenylmethyloxycarbonyl, the aminomethylidene reaction is carried out by reacting the compound (5c) with the compound (6c) and an acid (p-toluenesulfonic acid, etc.) in tetrahydrofuran at 5°C to 35°C. The compound (6c) is used in an amount of 1 to 20 mol, preferably 2 to 10 mol, and the acid is used in an amount of 0.05 to an excess amount, preferably 0.1 to 3 mol, relative to 1 mol of the compound (5c). The reaction time is 30 minutes to 72 hours, preferably 2 to 24 hours. Next, a base (such as 1,8-diazabicyclo[5.4.0]-7-undecene) is added to the reaction solution to carry out deprotection. When the reaction solution is in a suspended state, a solvent (such as N,N-dimethylformamide) may be added as needed to dissolve the solution and then the reaction is carried out. The base is used in an excess amount, preferably 5 to 20 mol, relative to 1 mol of the compound (5c). The reaction time is 10 minutes to 24 hours, preferably 2 hours to 12 hours. Water is added to the reaction solution, and the mixture is directly purified by C18 silica gel column chromatography [buffer / acetonitrile, etc.] to obtain compound (7c).

[0639] (C-4 process)

[0640] This step is a step of producing a compound of formula (8c) by removing the protecting group from the compound of formula (7c) using a known organic chemical method. 7 and PRO 8In the case of tert-butyldimethylsilyl, compound (8c) can be obtained according to the procedure described in step A-7 of method A.

[0641] (C-5 process)

[0642] This step is a step of producing a compound of formula (3) by subjecting the compound of formula (8c) to a condensation reaction with a compound of formula (4c) using a known organic chemical method. Compound (3) is obtained by the method described in step B-4 of method B.

[0643] C' Method

[0644] The conjugate precursor represented by (3') used in the antibody-drug conjugate of the present invention can be produced by the C' method described below.

[0645]

[0646] The manufacturing method is used to manufacture L 1 A method for coupling the precursor (3') in the case where a hydroxyl group is substituted at any position.

[0647]

[0648] (C'-1 process)

[0649] This step is a step of producing a compound of formula (2C') by continuously hydrolyzing the compound of formula (1C') and removing the cyanoethyl group using a known organic chemical method. Compound (2C') is obtained by the method described in step A-1 of method A.

[0650] (C'-2 process)

[0651] This step is a step of producing a compound of formula (3C') by removing the protective group of the hydroxyl group of the compound of formula (2C') using a known organic chemical method. Compound (3C') is obtained by the method described in step A-2 of method A.

[0652] (C'-3 process)

[0653] This step is a step of producing a compound of formula (5C') by continuously performing a coupling reaction of the compound of formula (3C') with a compound of formula (4C') and a sulfidation reaction or oxidation reaction of the resulting coupled product using a known organic chemical method. Compound (5C') is obtained by the method described in step A-3 of method A or step A"-3 of method A".

[0654] (C'-4 process)

[0655] This step is a step of removing the protective group of the hydroxyl group of the compound of formula (5C') using a known organic chemical method to produce a compound of formula (6C'). Compound (6C') is obtained by the method described in step A-4 of method A.

[0656] (C'-5 process)

[0657] This step is a step of preparing a compound of formula (7C') by continuously subjecting the compound of formula (6C') to cyclization and sulfidation or oxidation using known organic chemical methods. Compound (7C') is obtained by the method described in step A-5 of method A or step A'-5 of method A'.

[0658] (C'-6 process)

[0659] This step is a step of preparing a compound of formula (8C') by simultaneously removing the cyanoethyl group and all acyl protecting groups from the compound of formula (7C') using a known organic chemical method. Compound (8C') is obtained by the method described in step A-6 of method A.

[0660] (C'-7 process)

[0661] This step is a step of simultaneously removing all silyl-based protecting groups from the compound of formula (8C') using a known organic chemical method to produce a compound of formula (9C'). 9 In the case of 2-(trimethylsilyl)ethoxycarbonyl, the 2-(trimethylsilyl)ethoxycarbonyl group is removed by treating compound (8C') with a tetrabutylammonium fluoride solution in tetrahydrofuran at 5°C to 100°C, preferably at 35°C to 60°C. Tetrabutylammonium fluoride is used in excess moles, preferably 10 to 30 moles, relative to 1 mole of compound (8C'). The reaction time is 1 hour to 48 hours, preferably 4 hours to 24 hours. After adding a buffer to the reaction solution for dilution, the organic solvent component is distilled off under reduced pressure as needed. The residue is purified by preparative HPLC [buffer / acetonitrile, buffer / methanol, etc.], C18 silica gel column chromatography [buffer / acetonitrile, buffer / methanol, etc.] or a combination thereof to obtain compound (9C').

[0662] (C'-8 process)

[0663] This step is a step of producing a compound of formula (3') by subjecting the compound of formula (9C') to a condensation reaction with a compound of formula (4c) using a known organic chemical method. Compound (3') is obtained by the method described in step B-4 of method B.

[0664] Method D: Production of Glycan Remodeled Antibodies

[0665] Sugar chain remodeled antibodies can be produced by the method shown in the following formula according to the methods described in, for example, WO2018 / 003983, WO2020 / 050406, WO2021 / 177438, WO2022 / 050300, PLos ONE 2018, 13, e0193534, etc.

[0666]

[0667] (D-1 process)

[0668] This step is a step of producing a sugar chain cleavage antibody by hydrolyzing and cleaving the glycosidic bond between GlcNAcβ1-4GlcNAc of the reducing terminal chitobiose structure of the N-linked sugar chain (N297 linked sugar chain) linked to the asparagine at position 297 of the amino acid sequence of the antibody heavy chain using a known enzyme reaction. The target antibody (1d) (10 mg / mL) is subjected to a hydrolysis reaction of the glycosidic bond between GlcNAcβ1 and 4GlcNAc of the chitobiose structure of the reducing terminal in a buffer (phosphate buffer, etc.) at 0°C to 40°C using a hydrolase such as wild-type EndoS enzyme. The reaction time is 10 minutes to 72 hours, preferably 1 hour to 6 hours. With respect to 100 mg of the antibody (1d), 0.1 mg to 10 mg of the wild-type EndoS enzyme is used, preferably 0.1 mg to 3 mg. After the reaction, the residue was purified by affinity chromatography (HiTrap rProtein A FF (5 ml) (manufactured by GE HEALTH CARE)) and / or hydroxyapatite column (Bio-Scale Mini CHT Type I tube (5 ml) (manufactured by BIO-RAD)) to obtain (Fucα1,6)GlcNAc antibody (2d).

[0669] (D-2 process)

[0670] This step is a step for producing a sugar chain remodeled antibody (3d) by binding an SG-type or MSG (MSG1, MSG2)-type sugar chain oxazoline compound having an azide-containing PEG linker (hereinafter referred to as "azido sugar chain oxazoline compound") to the (Fucα1,6)GlcNAc antibody (2d) obtained in step D-1 using a known enzyme reaction.

[0671] In a buffer (phosphate buffer, etc.), at 0°C to 40°C, in the presence of a glycosyltransferase such as EndoS (D233Q / Q303L), the antibody (2d) is reacted with an azido sugar chain oxazoline body to perform a sugar chain transfer reaction. The reaction time is 10 minutes to 72 hours, preferably 1 hour to 6 hours. Relative to 100 mg of the antibody, 1 mg to 10 mg of the EndoS enzyme (D233Q / Q303L) is used, preferably 1 mg to 3 mg, and 2 equivalents to an excess of equivalents of the azido sugar chain oxazoline body are used, preferably 4 equivalents to 20 equivalents. After the reaction is completed, it is purified by affinity chromatography (HiTrap rProtein A FF (5 ml) (manufactured by GE HEALTH CARE)) and a hydroxyapatite column (Bio-Scale Mini CHT Type I tube (5 ml) (manufactured by BIO-RAD)) to obtain a sugar chain remodeled antibody (3d).

[0672] In the preparation of the above-mentioned sugar chain remodeled antibody, concentration of the antibody aqueous solution, concentration measurement, and buffer exchange can be performed according to the common procedures A to C described below.

[0673] It should be noted that the SG-type azidosugar chain oxazoline is synthesized according to the method described in WO2018 / 003983. As an example, the synthesis method of [N3-PEG(3)]2-SG(10)-Ox (compound 1-10 described in WO2018 / 003983) is shown in the following formula.

[0674]

[0675] MSG-type azidosugar chain oxazolines are also synthesized according to the method described in WO2018 / 003983. As an example, the following formula shows a method for synthesizing [N3-PEG(3)]-MSG1(9)-Ox (compound 1-11 described in WO2018 / 003983).

[0676]

[0677] (D-3 process)

[0678] This step is a step for producing a sugar chain remodeled antibody (3d) by using two Endo enzymes to carry out a sugar chain transfer reaction on the (Fucα1,6)GlcNAc antibody (2d) obtained in step D-1. By using two enzymes simultaneously, SGP, (SG)Asn, etc., which are not activated at the reducing end, can be used as sugar chain donors to directly transfer sugar chains to the N297 sugar chain of the antibody.

[0679] As for the two Endo enzymes used, enzyme-B (EndoM-like enzyme) and enzyme-A (EndoS-like enzyme) may be appropriately combined.

[0680] Examples of enzyme-B include EndoM, EndoOm, EndoCC, and EndoM mutants, EndoOm mutants, EndoCC mutants having reduced hydrolysis activity, etc. Preferred enzymes as enzyme-B are EndoM N175Q, EndoCC N180H, and EndoOm N194Q.

[0681] Examples of enzyme-A include EndoS, EndoS2 (EndoS49), EndoS mutants having reduced hydrolytic activity thereof, EndoS2 (EndoS49) mutants, etc. Preferred enzymes as enzyme-A include EndoSD233Q, EndoSD233Q / Q303L, EndoSD233Q / E350A, EndoS D233Q / E350Q, EndoSD233Q / E350D, EndoSD233Q / E350N, EndoS D233Q / D405A, EndoS2 D184M, and EndoS2 T138Q.

[0682] As the sugar chain donor, ([N3-PEG(3)]2-SG)-Asn-PEG(3)-N3, [N3-PEG(3)]-MSG1-Asn-PEG(3)-N3, [N3-PEG(3)]-MSG2-Asn-PEG(3)-N3, etc. can be used.

[0683] In a buffer (Tris buffer, etc.), in the presence of glycosyltransferases such as enzyme-B (EndoM enzyme) and enzyme-A (EndoS enzyme), the antibody (2d) is reacted with ([N3-PEG(3)]2-SG)-Asn-PEG(3)-N3 to carry out a sugar chain transfer reaction. The reaction temperature can be appropriately selected according to the optimal temperature of the enzyme used, and is generally 15 to 50°C, preferably 25 to 40°C. The reaction time can be appropriately selected between 2 hours and 48 hours. After the reaction is completed, a purification method (affinity chromatography, hydroxyapatite column, etc.) and an ultrafiltration method (ultrafiltration membrane) suitable for the reaction scale can be selected to obtain a sugar chain reconstructed antibody (3d).

[0684] In the preparation of the above-mentioned sugar chain remodeled antibody, concentration of the antibody aqueous solution, concentration measurement, and buffer exchange can be performed according to the common procedures A to C described below.

[0685] It should be noted that ([N3-PEG(3)]2-SG)-Asn-PEG(3)-N3 was synthesized according to the method described in WO2018 / 003983. In step 1-2A described in WO2018 / 003983, a free Fmoc-(SG-)Asn prepared from Fmoc-(SG-)Asn (1S2S-11NC-Asn-Fmoc, manufactured by Sugar Chain Engineering Research Institute) can be reacted with 11-azido-3,6,9-trioxaundecane-1-amine to obtain ([N3-PEG(3)]2-SG)-Asn-PEG(3)-N3 (compound 1-13 described in WO2018 / 003983).

[0686] MSG-type sugar chain donors [N3-PEG(3)]-MSG1-Asn-PEG(3)-N3 and [N3-PEG(3)]-MSG2-Asn-PEG(3)-N3 can also be synthesized according to the method described in steps 1 to 3 of Example 154 of WO2019065964.

[0687] In addition, step D-3 can also be performed according to the description of <5. Method for producing Fc-containing molecule> described later.

[0688] Method E: Conjugation of Antibodies to Drugs (Sugar Chain Conjugation 1)

[0689]

[0690] (Here, the two asterisks (*) on the left side of the antibody drug conjugate (1e) indicate the drug linker moiety indicated by the asterisk on the right side.)

[0691] The present production method is a method for producing an antibody-drug conjugate (1e) by combining the sugar chain remodeled antibody (3d) obtained in step D-2 of method D with the conjugation precursor (2) obtained in step B-4 of method B through a SPAAC (strain-promoted azide-alkyne cycloaddition: J.Am.Chem.Soc.2004,126,15046-15047) reaction.

[0692] (E-1 process)

[0693] The SPAAC reaction is carried out by mixing a buffer solution (phosphate buffer, acetate buffer, borate buffer, etc.) of the sugar chain remodeled antibody (3d) with a solution prepared by dissolving the coupling precursor (2) in an appropriate solvent (dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, propylene glycol or a mixed solvent thereof). The coupling precursor (2) is 2 mol to excess mol, preferably 4 mol to 30 mol, relative to 1 mol of the sugar chain remodeled antibody (3d), and the ratio of the organic solvent is preferably 1% to 200% (v / v) relative to the buffer solution of the antibody. The reaction temperature is 0°C to 37°C, preferably 15°C to 25°C, and the reaction time is 1 hour to 150 hours, preferably 6 hours to 72 hours. The pH of the reaction solution is preferably 5 to 9. The reaction solution is purified according to the method described in the common operation D described later to obtain an antibody drug conjugate (1e).

[0694] E' method: conjugation of antibodies to drugs (cysteine ​​conjugation)

[0695] The antibody-drug conjugate of the present invention having cysteine ​​coupling can be produced according to the method described in WO2014 / 057687 etc. using the target antibody prepared in Reference Example 3 etc. and the coupling precursor (2') having a maleimide group obtained in step B-8 of the B' method.

[0696] E" method: conjugation of antibodies and drugs (sugar chain conjugation 2)

[0697] In method E, the antibody drug conjugate (1e") represented by the following formula is obtained by replacing the conjugation precursor (2) with the conjugation precursor (3') obtained in step C'-8 of method C'.

[0698]

[0699] (Here, the two asterisks (*) on the left side of the antibody drug conjugate (1e") 1 ) indicates the drug linker moiety indicated by the asterisk on the right. )

[0700] The antibody-drug conjugate can be identified by buffer exchange, purification, determination of antibody concentration, and determination of the average number of drug bound per antibody molecule through the common operations D to G described below.

[0701] Method F: Production of antibodies containing mutated Fc regions

[0702] Antibodies containing a mutant Fc region can be produced by known methods using an Fc effector-less mutant animal cell expression vector. Examples of methods for constructing an Fc effector-less mutant animal cell expression vector include the following methods.

[0703] Construction of animal cell expression vector for Fc effector-free mutant

[0704] Using an animal cell expression vector containing a wild-type Fc region gene as a template, the desired effector-free mutation is introduced into the Fc region by PCR using KOD-Plus-Mutagenesis Kit (TOYOBO). Alternatively, a gene containing a desired effector-free mutation is synthesized and incorporated into an animal cell expression vector by ligation.

[0705] It should be noted that the antibody-drug conjugate or its manufacturing intermediate comprising a CDN derivative of the present invention can also be manufactured with reference to WO2022163846. In addition, regarding the molecules comprising a target molecule binding portion, a mutant Fc region, and an immunostimulant in the present invention and comprising an immunostimulant other than the above-mentioned CDN derivative, it can be manufactured by appropriately modifying the manufacturing method described above with reference to a known method or by referring to a known method.

[0706] Common operation A: Concentration of antibody aqueous solution

[0707] The antibody or antibody-drug conjugate solution was added to an Amicon (registered trademark) Ultra centrifugal filter device (50000 NMWL, Merck Millipore Ltd.), and the antibody and antibody-drug conjugate solution was concentrated by centrifugation (centrifugation at 2000 G to 4000 G for 5 to 20 minutes) using a centrifuge (Allegra X-15R, Beckman Coulter, Inc.).

[0708] Common procedure B: Antibody concentration determination

[0709] The antibody concentration was measured using a UV analyzer (Nanodrop 1000, Thermo Fisher Scientific, Inc.) according to the manufacturer's method. At this time, different absorbances at 280 nm (1.3 mL mg －1 cm －1 Up to 1.8 mL mg －1 cm －1 ).

[0710] Common procedure C: Antibody buffer exchange

[0711] A buffer (phosphate buffered saline (pH 6.0), phosphate buffer (pH 6.0), etc.) is added to the antibody aqueous solution, and the solution is concentrated according to the method described in common procedure A. After the operation is performed several times, the antibody concentration is measured according to the method described in common procedure B. An appropriate buffer (phosphate buffered saline (pH 6.0), phosphate buffer (pH 6.0), etc.) is added to the antibody buffer solution to prepare an antibody buffer solution of a target concentration (e.g., about 10 mg / mL).

[0712] Common procedure D: Purification of antibody-drug conjugates (gel filtration chromatography)

[0713] The NAP column (NAP-5, NAP-10, NAP-25 (manufactured by GE HEALTH CARE)) is equilibrated with an acetate buffer (10 mM acetate buffer, 5% sorbitol, pH 5.5; referred to as ABS in this specification) or an appropriate buffer other than the above. The antibody-drug conjugate reaction solution is loaded into the NAP column, and the amount of buffer specified by the manufacturer is allowed to flow down naturally, and the antibody fraction is separated. The fraction is loaded into the NAP column again, and the amount of buffer specified by the manufacturer is allowed to flow down naturally, and the antibody fraction is separated. This operation is repeated 2 to 3 times in total, thereby obtaining an antibody-drug conjugate from which unbound drug linkers, dimethyl sulfoxide, and propylene glycol are removed. The concentration of the antibody-drug conjugate solution is adjusted by common operations A and C as needed.

[0714] Common procedure E: Determination of antibody concentration in antibody-drug conjugates and average number of drug binding per antibody molecule (UV method)

[0715] The concentration of bound drug in the antibody-drug conjugate can be calculated by measuring the absorbance of the antibody-drug conjugate aqueous solution at two wavelengths of 280 nm and 250 nm using an absorptiophotometer (UV / VIS Spectrometer Lambda 25, PerkinElmer, Inc.) and then performing the following calculation. The total absorbance at a certain wavelength is equal to the sum of the absorbances of all absorbing chemical species present in the system (additivity of absorbance), so if it is assumed that the molar absorptivity of the antibody and the drug does not change before and after the conjugation of the antibody and the drug, the antibody concentration and the drug concentration in the antibody-drug conjugate are expressed by the following relationship.

[0716] A 280 =A D，280 +A A，280 =ε D，280 C D +ε A，280 C A ...Formula (I)

[0717] A 250 =AD，250 +A A，250 =ε D，250 C D +ε A，250 C A …Formula (II)

[0718] Here, A 280 A represents the absorbance of the antibody-drug conjugate aqueous solution at 280 nm. 250 A represents the absorbance of the antibody-drug conjugate aqueous solution at 250 nm. A，280 represents the absorbance of the antibody at 280 nm, A A，250 represents the absorbance of the antibody at 250 nm, A D，280 represents the absorbance of the conjugate precursor at 280 nm, A D，250 represents the absorbance of the conjugate precursor at 250 nm, ε A，280 represents the molar absorption coefficient of the antibody at 280 nm, ε A，250 represents the molar absorption coefficient of the antibody at 250nm, ε D， 280 represents the molar absorption coefficient of the conjugate precursor at 280 nm, ε D，250 represents the molar absorption coefficient of the conjugate precursor at 250 nm, C A represents the antibody concentration in the antibody drug conjugate, C D represents the drug concentration in the antibody-drug conjugate. Here, ε A，280 , ε A，250 , ε D，280 , ε D，250 Use the values ​​prepared in advance (calculated values ​​or measured values). For example, ε A，280 It can be estimated based on the amino acid sequence of the antibody by a known calculation method (Protein Science, 1995, vol. 4, 2411-2423). A，250 Using the measured value obtained by UV measurement of the antibody and ε A，280 In the examples, the molar absorption coefficient of the anti-EGFR antibody 1 is calculated using ε A，280 =203460 and ε A，250 =70151 or 63837. The molar absorption coefficient of anti-EGFR antibody 2 is expressed as ε A，280 =203460 and ε A，250 =63051 or 63370. The molar absorption coefficient of anti-EGFR antibody 3 is expressed as ε A，280 =203460 and ε A，250 =62692 or 63853. The molar absorption coefficient of anti-EGFR antibody A is expressed as ε A，280 =203460 and ε A，250=69640. The molar absorption coefficient of anti-EGFR antibody B is expressed as ε A，280 =197750 and ε A，250 =66256. The molar absorption coefficient of anti-CDH6 antibody 1 was calculated using ε A，280 =223400 and ε A，250 =74671 or 71530. D，280 and ε D，250 The absorbance of the solution obtained by dissolving the conjugate precursor to a certain molar concentration can be determined according to the Lambert-Beer law (absorbance = molar concentration × molar absorption coefficient × slot optical path length). The molar absorption coefficient of the conjugate precursor in the example is obtained by each UV measurement. 280 and A 250 Substituting these values ​​into equations (I) and (II) and solving the simultaneous equations, we can find C A and C D . Then through C D Divide by C A , the average number of drug binding to each antibody molecule can be calculated.

[0719] Common procedure F: Determination of antibody concentration in antibody-drug conjugates and the average number of drug-bound molecules per antibody molecule (reversed phase high performance liquid chromatography: RP-HPLC)

[0720] The antibody concentration in the antibody-drug conjugate and the average number of drug bound per antibody molecule can be determined by high performance liquid chromatography analysis using the following method in addition to the above-mentioned common procedure E.

[0721] [F-1. Preparation of samples for HPLC analysis (reduction of antibody-drug conjugate)]

[0722] The antibody drug conjugate solution (about 1 mg / mL, 60 μL) was mixed with a dithiothreitol (DTT) aqueous solution (100 mM, 15 μL). The mixture was incubated at 37°C for 30 minutes to cut the disulfide bond between the L chain and the H chain of the antibody drug conjugate. The reaction solution was directly used for HPLC analysis.

[0723] [F-2.HPLC analysis]

[0724] Representative analysis conditions are described below.

[0725] HPLC system: Agilent 1290 HPLC system (Agilent Technologies).

[0726] Detector: UV absorbance meter (measurement wavelength: 280 nm).

[0727] Column: Acquity BEH Phenyl (2.1×50 mm, 1.7 μm, manufactured by Waters).

[0728] Column temperature: 75℃.

[0729] Flow rate: 0.8mL / min.

[0730] Sample injection volume: 10 μL.

[0731] Mobile phase A: 0.1% trifluoroacetic acid (TFA), 15% isopropanol in water.

[0732] Mobile phase B: 0.075% TFA, 15% isopropanol in acetonitrile.

[0733] Gradient program (mobile phase B): 14%-36% (0 min-15 min), 36%-80% (15-17 min), 80%-14% (17 min-17.1 min), 14%-14% (17.1 min-23 min).

[0734] [F-3. Data analysis]

[0735] [F-3-1] In the case of sugar chain coupling in the SPAAC reaction, the H chain bound to the drug (H chain bound to one drug: H1, H chain bound to two drugs: H2) increases in hydrophobicity and retention time in proportion to the number of bound drugs, relative to the L chain (L0) and H chain (H0) of the antibody to which the drug is not bound. Therefore, in principle, they are eluted in the order of L0, H0, H1, and H2. By comparing the retention time with L0 and H0, the detection peak can be assigned to any of L0, H0, H1, and H2. The same is true for cysteine ​​coupling. The L chain bound to the drug (L chain bound to one drug: L1) and the H chain bound to the drug (H chain bound to one drug: H1, H chain bound to two drugs: H2, H chain bound to three drugs: H3) increase in hydrophobicity and retention time in proportion to the number of bound drugs, so in principle they are eluted in the order of L0, L1, H0, H1, H2, and H3. By comparing the retention time with L0 and H0, the detection peak can be assigned to any of L0, L1, H0, H1, H2, and H3.

[0736] [F-3-2] The drug linker has UV absorption, so in the case of sugar chain coupling in the SPAAC reaction, the peak area is corrected according to the following formula using the molar absorption coefficients of the H chain and the drug linker according to the number of drug linkers. In the case of coupling where the drug is also bound to the cysteine ​​of the L chain, the peak area is corrected similarly for the L chain.

[0737]

[0738] Here, the molar absorption coefficients (280 nm) of the L chain and the H chain in each antibody were estimated values ​​calculated by the known calculation method described in the common operation E. In the case of anti-EGFR antibodies 1, 2, 3, and A, 23232 was used as the molar absorption coefficient of the L chain, and 78498 was used as the molar absorption coefficient of the H chain. In the case of anti-EGFR antibody B, 23232 was used as the molar absorption coefficient of the L chain, and 75642 was used as the molar absorption coefficient of the H chain. In the case of anti-CDH6 antibody 1, 31712 was used as the molar absorption coefficient of the L chain, and 79988 was used as the molar absorption coefficient of the H chain. The molar absorption coefficient (280 nm) of the drug linker was the measured value of the coupling precursor in the case of sugar chain coupling in the SPAAC reaction, and the measured value of the compound obtained by reacting the coupling precursor with mercaptoethanol or N-acetylcysteine ​​to convert the maleimide group to succinimide thioether in the case of cysteine ​​coupling.

[0739] [F-3-3] The peak area ratio (%) of each chain relative to the total of the peak area corrected value was calculated according to the following formula.

[0740]

[0741] [F-3-4] The average drug binding number (DAR) per antibody molecule in the antibody-drug conjugate was calculated according to the following formula.

[0742]

[0743] [F-3-5] Calculate the antibody concentration in the antibody-drug conjugate according to the following formula.

[0744]

[0745] Here, the absorbance (280 nm) of the antibody drug conjugate uses the measured value of the antibody drug conjugate aqueous solution. The dilution factor indicates how many times the antibody drug conjugate aqueous solution is diluted when measuring the absorbance, and is usually diluted 4 times. The molar absorptivity (280 nm) of the antibody uses an estimated value calculated by the known calculation method described in common operation E. The average drug binding number uses the value obtained in [F-3-4]. For the molar absorptivity (280 nm) of the drug linker, in the case of sugar chain coupling in the SPAAC reaction, the measured value of the coupling precursor is used, and in the case of cysteine ​​coupling, the measured value of the compound in which the maleimide group is converted to succinimide thioether by reacting the coupling precursor with mercaptoethanol or N-acetylcysteine ​​is used.

[0746] Common procedure G: Determination of antibody concentration in antibody-drug conjugates and the average number of drug-bound molecules per antibody molecule (hydrophobic interaction-high performance liquid chromatography: HI-HPLC)

[0747] The antibody concentration in the antibody-drug conjugate and the average number of drug bound per antibody molecule can be determined by high performance liquid chromatography analysis using the following method in addition to the above-mentioned common procedures E and F.

[0748] [G-1. Preparation of samples for HPLC analysis]

[0749] The antibody drug conjugate solution (about 1 mg / mL, 60 μL) was directly used for HPLC analysis.

[0750] [G-2.HPLC analysis]

[0751] Representative analysis conditions are described below.

[0752] HPLC system: SHIMADZU CBM-20A (Shimadzu Corporation).

[0753] Detector: UV absorbance meter (measurement wavelength: 280 nm).

[0754] Column: TSK-gel Butyl-NPR (4.6×100 mm, 2.5 μm, manufactured by TOSOH).

[0755] Column temperature: constant temperature around 25°C.

[0756] Mobile phase A: 25 mM phosphate buffer (pH = 7.0) containing 1.5 M ammonium sulfate.

[0757] Mobile phase B: 25 mM phosphate buffer (pH=7.0) / isopropanol mixture (3:1).

[0758] Flow rate: 0.8mL / min.

[0759] Sample injection volume: 15 μL.

[0760] Gradient program (mobile phase B): 10%-15% (0 min-5 min), 15%-65% (5 min-20 min).

[0761] or:

[0762] HPLC system: SHIMADZU CBM-20A (Shimadzu Corporation).

[0763] Detector: UV absorbance meter (measurement wavelength: 280 nm).

[0764] Column: PolyPROPYL A (4.6×100mm, 3μm, PolyLC).

[0765] Column temperature: constant at around 40°C.

[0766] Mobile phase A: 20 mM phosphate buffer (pH=7.4) containing 1.5 M ammonium sulfate.

[0767] Mobile phase B: 20 ​​mM phosphate buffer (pH=7.4).

[0768] Flow rate: 0.8mL / min.

[0769] Sample injection volume: 15 μL.

[0770] Gradient program (mobile phase B): 40% - 80% (0 min - 20 min).

[0771] [G-3. Data analysis]

[0772] [G-3-1] As the hydrophobicity and retention time increase in proportion to the number of drugs bound to the antibody, in principle, in the case of sugar chain coupling in the SPAAC reaction, it is eluted in the order of DAR=0, DAR=2, and DAR=4. By comparing the retention time with DAR=0, the detection peak can be assigned to either DAR=2 or DAR=4. Depending on the type of antibody and drug linker, peaks of DAR=1 and DAR=3 may be detected. The DAR of the detection peak is sometimes estimated by measuring the mass spectrum after dividing the peak by HI-HPLC.

[0773] [G-3-2] Since the drug linker has UV absorption, the peak area value was corrected according to the following formula using the molar absorption coefficients of the antibody and the drug linker according to the number of drug linkers bound.

[0774]

[0775] Here, the molar absorption coefficient (280 nm) of the antibody was an estimated value calculated by the known calculation method described in Common Operation E. The molar absorption coefficient (280 nm) of the drug linker was an actually measured value of the conjugation precursor.

[0776] [G-3-3] The antibody peak area ratio (%) relative to the total peak area corrected value was calculated according to the following formula.

[0777]

[0778] [G-3-4] Calculate the average number of drug bound per antibody molecule in the antibody-drug conjugate according to the following formula.

[0779]

[0780] [G-3-5] The antibody concentration in the antibody-drug conjugate was calculated according to the formula described in [F-3-5]. At this time, the average number of drug binding was the value obtained in [G-3-4].

[0781] The antibody-drug conjugate of the present invention or its production intermediates may sometimes contain stereoisomers or optical isomers derived from asymmetric carbon atoms, geometric isomers, tautomers, or optical isomers such as d-isomers, l-isomers, and atropisomers, and these isomers, optical isomers, and mixtures thereof are all included in the present invention.

[0782] In the antibody-drug conjugate of the present invention, the number of drugs combined with one molecule of antibody is an important factor affecting its effectiveness and safety. The manufacture of antibody-drug conjugates is implemented by specifying the reaction conditions such as the amount of raw materials and reagents to be reacted in a fixed number of drug combinations, but unlike the chemical reaction of low molecular weight compounds, it is usually obtained in the form of a mixture of drugs with different numbers. The number of drugs combined with one molecule of antibody can be specified in the form of an average value, that is, the average drug combination number (DAR). The number of cyclic dinucleotide derivatives combined with antibody molecules can be controlled, and as the average drug combination number of each antibody, cyclic dinucleotide derivatives in the range of 1 to 10 can be combined, but preferably 1 to 8, more preferably 1 to 5, and further preferably 2 to 4. In the present invention, the average drug combination number and the average drug combination number have the same meaning and can be substituted for each other.

[0783] In the antibody-drug conjugate of the present invention, when the antibody Ab is bound to L from the remodeled sugar chain of the antibody Ab, the drug binding number m of each antibody heavy chain in the antibody-drug conjugate is 2 is an integer of 1 or 2. When the sugar chain is an N297 sugar chain or an N297-(Fuc)SG sugar chain, m 2 is 2, and DAR is in the range of 3 to 5 (preferably in the range of 3.2 to 4.8, and more preferably in the range of 3.5 to 4.2). When the N297 sugar chain is N297-(Fuc)MSG1, N297-(Fuc)MSG2, or a mixture of N297-(Fuc)MSG1 and N297-(Fuc)MSG2, m 2 The value is 1, and the DAR is in the range of 1 to 3 (preferably in the range of 1.0 to 2.5, and more preferably in the range of 1.2 to 2.2).

[0784] It should be noted that a person skilled in the art can design a reaction for binding an antibody to a desired number of drugs based on the description of the examples of the present application, and can obtain an antibody with a controlled number of cyclic dinucleotide derivatives bound thereto.

[0785] It should be noted that the antibody-drug conjugate of the present invention or its production intermediate may absorb moisture due to being left in the air or recrystallized, and may become a hydrate with adsorbed water. The above-mentioned compounds and salts containing water are also included in the present invention.

[0786] In the case where the antibody drug conjugate of the present invention or its manufacturing intermediate has a basic group such as an amino group, a pharmaceutically acceptable salt can be prepared as desired. As such salts, for example, hydrohalide salts such as hydrochloride and hydroiodide; inorganic acid salts such as nitrate, perchlorate, sulfate, and phosphate; lower alkyl sulfonates such as methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate; aryl sulfonates such as benzenesulfonate and p-toluenesulfonate; organic acid salts such as formate, acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts such as ornithine, glutamate, and aspartate.

[0787] The antibody drug conjugate of the present invention contains a phosphate group and / or a thiophosphate group in its structure, and therefore can usually form a base addition salt. In addition, when the manufacturing intermediate has an acidic group such as a carboxyl group, a base addition salt can usually be formed. As a pharmaceutically acceptable salt, for example, alkali metal salts such as sodium salts, potassium salts, and lithium salts; alkaline earth metal salts such as calcium salts and magnesium salts; inorganic salts such as ammonium salts; dibenzylamine salts, morpholine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methyl reduced glucosamine salts, diethylamine salts, triethylamine salts, cyclohexylamine salts, dicyclohexylamine salts, N, N'-dibenzylethylenediamine salts, diethanolamine salts, N-benzyl-N-(2-phenylethoxy)amine salts, piperazine salts, tetramethylammonium salts, tris (hydroxymethyl) aminomethane salts and other organic amine salts, etc.

[0788] The antibody drug conjugate of the present invention and its manufacturing intermediate sometimes exist in the form of a hydrate by absorbing moisture in the air. As the solvate of the present invention, there is no particular limitation as long as it is a pharmaceutically acceptable solvate, and specifically, it is preferably a hydrate, an ethanolate, a 2-propanolate, etc. In addition, when a nitrogen atom exists in the antibody drug conjugate of the present invention and its manufacturing intermediate, it can also become an N-oxide, and these solvates and N-oxides are also included in the scope of the present invention. In addition, when a sulfur atom exists in the antibody drug conjugate of the present invention and its manufacturing intermediate, it can also become a sulfoxide, and these solvates and sulfoxides are also included in the scope of the present invention.

[0789] In addition, the present invention also includes compounds labeled with various radioactive or non-radioactive isotopes. One or more atoms constituting the antibody drug conjugate of the present invention and its manufacturing intermediates may also contain non-natural ratios of atomic isotopes. As atomic isotopes, for example, deuterium (2H), tritium (3H), iodine-125 (125I) or carbon-14 (14C) can be listed. In addition, the compounds of the present invention can be radiolabeled with radioactive isotopes such as tritium (3H), iodine-125 (125I) or carbon-14 (14C). Radiolabeled compounds are useful as therapeutic or preventive agents, research reagents (such as analytical reagents) and diagnostic agents (such as in vivo imaging diagnostic agents). All isotopic variants of the antibody drug conjugate of the present invention, whether or not they are radioactive, are included in the scope of the present invention.

[0790] <4.Medicine>

[0791] The antibody-drug conjugate of the present invention exhibits anti-tumor immune activity or cytotoxic activity against cancer cells and is also excellent in safety, so it can be used as a medicine, in particular, as a therapeutic and / or preventive agent for cancer, or an anti-tumor agent. Furthermore, the antibody-drug conjugate of the present invention can be used as a medicine, as a therapeutic and / or preventive agent for infectious diseases. In addition, in one embodiment of the present invention, the antibody-drug conjugate of the present invention can be used as a medicine with high thermal stability.

[0792] As the types of cancers applicable to the antibody-drug conjugate of the present invention, there can be listed: lung cancer (non-small cell lung cancer, small cell lung cancer, etc.), renal cancer, urothelial carcinoma, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer (surface epithelial tumors, stromal tumors, germ cell tumors, etc.), pancreatic cancer, breast cancer, melanoma, liver cancer, hepatocellular carcinoma, bladder cancer, gastric cancer, esophageal cancer, uterine endometrial cancer, testicular cancer (seminoma, non-seminoma), cervical cancer, placental choriocarcinoma, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, gastrointestinal stromal tumor (Gastrointestinal Stromal Tumor, GIST), gallbladder cancer, bile duct cancer, adrenal cancer, pharyngeal cancer, tongue cancer, auditory organ cancer, thymic cancer, small intestine cancer, squamous cell carcinoma, leukemia, malignant lymphoma, plasma cell tumor, myeloma, sarcoma, etc. In addition, the antibody-drug conjugate of the present invention can be applied to EGFR-positive cancer or CDH6-positive cancer. EGFR-positive cancer refers to cancer that expresses EGFR, and examples thereof include lung cancer (non-small cell lung cancer, small cell lung cancer, etc.), renal cancer, urothelial carcinoma, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer (superficial epithelial tumors, stromal tumors, germ cell tumors, etc.), pancreatic cancer, breast cancer, melanoma, liver cancer, hepatocellular carcinoma, bladder cancer, gastric cancer, esophageal cancer, uterine cancer, cervical cancer, brain tumors, head and neck cancer, thyroid cancer, mesothelioma, gastrointestinal stromal tumors (GIST), gallbladder cancer, bile duct cancer, squamous cell carcinoma, pharyngeal cancer, tongue cancer, thymic cancer, small intestine cancer, etc. CDH6-positive cancer refers to cancer that expresses CDH6, and examples thereof include lung cancer (non-small cell lung cancer, small cell lung cancer, etc.), renal cancer, urothelial cancer, ovarian cancer (surface epithelial tumors, stromal tumors, germ cell tumors, etc.), pancreatic cancer, uterine cancer, cervical cancer, thyroid cancer, mesothelioma, gallbladder cancer, bile duct cancer, sarcoma, etc. Furthermore, the antibody-drug conjugate of the present invention can be applied to EGFR signal mutation-positive cancer. EGFR signal mutation-positive cancer includes cancers with mutations in EGFR (EGFR mutation-positive cancer) and cancers with mutations in EGFR signaling molecules (cancers with mutations in EGFR downstream signals). As cancers with mutations in EGFR, examples thereof include lung cancer, head and neck cancer, etc., which contain at least one mutation in exons 18 to 21, preferably lung cancer with mutations in exon 19. As cancers with mutations in EGFR signaling molecules, examples thereof include cancers with mutations in at least one of RAS, RAF, PI3K, etc. Preferred examples include RAS mutant cancers such as lung cancer, colorectal cancer, and pancreatic cancer in which at least one of RAS (KRAS, NRAS, and HRAS) is mutated, more preferably lung cancer or colorectal cancer having a KRAS mutation, and even more preferably lung cancer having a KRAS mutation.Cancers to which the antibody-drug conjugate of the present invention is applicable are not limited to these cancers as long as they express a protein that can be recognized by the antibody in the antibody-drug conjugate.

[0793] The antibody-drug conjugate of the present invention can be preferably administered to a mammal, more preferably a human.

[0794] The dosage and concentration of substances used in the pharmaceutical composition comprising the antibody-drug conjugate of the present invention can be appropriately selected from pharmaceutical additives and the like commonly used in the field.

[0795] The antibody drug conjugate of the present invention can be administered in the form of a pharmaceutical composition comprising one or more pharmaceutically compatible ingredients. For example, the pharmaceutical composition typically comprises one or more pharmaceutical carriers (e.g., a sterilized liquid (e.g., comprising water and oil (oil of petroleum, animal, plant, or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.))). In the case where the pharmaceutical composition is administered intravenously, water is a more representative carrier. In addition, saline solutions, glucose (dextrose) aqueous solutions, and glycerol aqueous solutions can also be used as liquid carriers, particularly for injection solutions. Suitable pharmaceutical excipients are well known in the art. In addition, as long as desired, the composition can contain a trace amount of a wetting agent or emulsifier, or a pH buffering agent. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EWMartin. Its prescription corresponds to the regimen of administration.

[0796] Various delivery systems are well known and can be used to administer the antibody drug conjugate of the present invention. As the introduction method, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes can be listed, but are not limited thereto. Administration can be performed, for example, by infusion or bolus injection. In a specific preferred embodiment, the administration of the above-mentioned antibody drug conjugate is performed by infusion. Parenteral administration is a preferred route of administration.

[0797] In a representative embodiment, the pharmaceutical composition comprising the above-mentioned antibody drug conjugate is prescribed as a pharmaceutical composition suitable for intravenous administration to humans according to conventional procedures. Representatively, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. Where necessary, the above-mentioned pharmaceutical composition may also include a solubilizer and a local anesthetic (e.g., lidocaine) for relieving pain at the injection site. Typically, the above-mentioned ingredients are supplied separately in the form of a lyophilized powder or anhydrous concentrate in a sealed and sealed container such as an ampoule or a sachet showing the amount of the active agent, or are mixed together in a unit dosage form and supplied. In the case where the above-mentioned pharmaceutical composition is intended to be administered by infusion, it can be administered, for example, by an injection bottle containing sterilized pharmaceutical grade water or saline. In the case where the above-mentioned pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline can be provided in a manner such that the above-mentioned ingredients can be mixed before administration. The above-mentioned pharmaceutical composition is sometimes provided in the form of a solution.

[0798] The pharmaceutical composition of the present invention may be a pharmaceutical composition comprising only the antibody drug conjugate of the present invention, or may be a pharmaceutical composition comprising the antibody drug conjugate of the present invention and other medicines (including cancer therapeutic agents). The antibody drug conjugate of the present invention may also be administered together (simultaneously, separately or one after the other) with one or more other medicines (including cancer therapeutic agents but not limited thereto), or administered in combination, thereby enhancing the anti-tumor effect or improving safety. Other medicines used for such purposes may be administered to an individual simultaneously, separately, or continuously with the antibody drug conjugate, or may be administered by changing the respective administration intervals. Examples of such other cancer therapeutic agents include hormone regulating drugs (such as leuprolide, goserelin, estramustine, and estrogen antagonists such as tamoxifen and raloxifene), aromatase inhibitors (such as anastrozole, letrozole, and exemestane), kinase inhibitors, PARP inhibitors, bone destruction inhibitors, bone formation promoters, metastasis inhibitors, anti-regulatory T cell drugs (such as anti-GITR antibodies, anti-GARP antibodies, anti-TIGIT antibodies, and anti-CCR8 antibodies), immunostimulators (such as anti-4-1BB antibodies, anti-OX40 antibodies, anti-CD40 antibodies, anti-CD3 antibodies, anti-CD28 antibodies, IL-2 analogs, cytokines, and TLR agonists), immunomodulators (such as anti-CD47 antibodies, anti-SIRPα antibodies, and suppressive bone marrow regulating agents), and drugs having ADCC (antibody dependent cellular cytotoxicity) activity and ADCP (antibody dependent cellular phagocytosis). Phagocytosis) activity or complement activity of antibody medicines, BiTE (bispecific T cell engagers), ADC combined with photodynamic therapy, etc., as well as anti-tumor vaccines, anti-tumor cell therapy (CAR-T, TCR-T, dendritic cells, NK cells, etc.), anti-tumor bacterial therapy, anti-tumor viral therapy, etc., but there is no limitation as long as it is an agent with anti-tumor activity. Furthermore, the antibody drug conjugate of the present invention can be administered together with other antibody drug conjugates with anti-tumor activity of the present invention, thereby enhancing the anti-tumor effect. In addition, the antibody drug conjugate of the present invention can enhance the anti-tumor effect not only by using it in combination with drugs, but also by using it in combination with treatments that bring about anti-tumor effects. The above-mentioned treatments are, for example, radiation, heavy particle radiation, surgery, bone marrow transplantation, etc., as long as it is a treatment with anti-tumor effect, there is no limitation.

[0799] Such pharmaceutical composition preparations can be prepared into preparations, lyophilized preparations or liquid preparations with selected composition and required purity. When the preparation is prepared into lyophilized preparations, it can be a preparation comprising the appropriate preparation additives used in this area. In addition, for liquid preparations, it can also be prepared into liquid preparations comprising the various preparation additives used in this area.

[0800] The composition and concentration of the pharmaceutical composition vary depending on the method of administration, but the antibody drug conjugate contained in the pharmaceutical composition of the present invention has a higher affinity (lower Kd value) for the antigen, i.e., the dissociation constant (Kd value) for the antigen. The drug effect can be exerted with a small amount of administration. Therefore, when determining the dosage of the antibody drug conjugate, the dosage can be set based on the affinity of the antibody drug conjugate to the antigen. When the antibody drug conjugate of the present invention is administered to a human, for example, about 0.001 to 100 mg / kg can be administered once or multiple times at intervals of 1 to 180 days.

[0801] <5. Method for producing Fc-containing molecules>

[0802] In one aspect of the present invention, there is provided a method for producing an Fc-containing molecule having an N297-linked sugar chain including a sugar chain derived from a sugar chain donor molecule.

[0803] In one aspect of the present invention, there is provided a method for producing an Fc-containing molecule having an N297-linked sugar chain including a sugar chain derived from a sugar chain donor molecule, the method comprising the following step 1.

[0804] (Step 1) is a step of reacting an acceptor molecule with a sugar chain donor molecule containing a sugar chain including GlcNAc with an unactivated reducing end in the presence of enzyme-A, enzyme-B and an additive to obtain a reaction mixture, wherein the acceptor molecule is an Fc-containing molecule having a core GlcNAc optionally added with fucose as an N297-linked sugar chain, the enzyme-A is an endo-β-N-acetylglucosaminidase using the N297-linked sugar chain of the Fc-containing molecule as a substrate, the enzyme-B is an endo-β-N-acetylglucosaminidase using the sugar chain of the sugar chain donor molecule as a substrate, and the additive is selected from a monovalent salt, an organic solvent, a surfactant, a sugar, an amino acid, or any combination thereof.

[0805] <N297 linked sugar chain>

[0806] In one embodiment of the present invention, "N297-linked sugar chain" refers to an N-linked sugar chain in a state of being linked to the side chain of Asn at position 297 of the IgG heavy chain. In addition, even if the addition position of the N-linked sugar chain of the IgG heavy chain is changed by any method, as long as the N-linked sugar chain is affected by enzyme-A, it is also considered to be included in the N297-linked sugar chain. For example, in one embodiment of the present invention, when the IgG heavy chain is fragmented, even a sugar chain in a state of being linked to the corresponding Asn in a peptide fragment containing the Asn is included in the N297-linked sugar chain. Generally, the N297-linked sugar chain in IgG produced by animals and the like has a basic structure comprising the structure of the following formula (I) or (II), and its non-reducing end can be further chemically modified, for example, Gal or sialic acid can be added.

[0807]

[0808] Most of the N297-linked sugar chains of IgG produced by cells have a diversity of sugar chain structures, including structures in which sugar chains are further bonded to the GlcNAc (core GlcNAc) at the reducing end, the non-reducing end, the branched sugar, etc. in the basic structure. The N297-linked sugar chain can have a structure ((Fucα1,6)GlcNAc) containing fucose (Fuc) and a core fucose at the 6th position of the core GlcNAc forming an α1,6 bond. In Man as a branched sugar, a triantennary sugar chain is sometimes formed in which a sugar chain containing GlcNAc is further bonded to its 5th position. In the GlcNAc at the non-reducing end, sugar chains containing galactose (Gal) and sialic acid (Sia) are sometimes further bonded.

[0809] <Sugar chain donor molecule>

[0810] In one embodiment of the present invention, a "sugar chain donor molecule" (also referred to as a "sugar chain donor") refers to a molecule that donates a sugar chain to an acceptor molecule.

[0811] In the case of sugar chain reconstruction for the purpose of drug development, it is preferred to use a sugar chain donor molecule having a human sugar chain or a human-adapted sugar chain that has few problems when applied to humans. Such sugar chains are known to be non-antigenic in the human body. Among N-linked sugar chains, high mannose type, hybrid type, and complex type are known. These three have a common basic structure. The high mannose type is a sugar chain having a mannose-rich structure in which multiple mannoses are continuous in two branches (1-3 chain, 1-6 chain) branching from mannose (β-mannose) located near the reducing end. The hybrid type refers to a structure in which one of the two branches (1-3 chain, 1-6 chain) branching from mannose (β-mannose) located near the reducing end has GlcNAc. The complex type refers to a structure in which two branches (1-3 chains, 1-6 chains) branching from mannose (β-mannose) located near the reducing end have GlcNAc, and has various structures including the presence or absence of galactose, the presence or absence of sialic acid, and the bonding isomerism and positional isomerism of these. As complex sugar chains, biantennary, triantennary, or tetraantennary are known.

[0812] Examples of high mannose type, hybrid type and complex type structures are shown below. It should be noted that their common basic structure is shown in the dotted box (in the dotted box, the structure containing GlcNAc (bisecting GlcNAc) introduced into the 4th position of β-mannose and the structure not containing this GlcNAc are mixed, but in this specification, they all refer to the common basic structure).

[0813]

[0814] In one embodiment of the present invention, the sugar chain part in the sugar chain donor molecule can be any one of a high mannose sugar chain, a hybrid sugar chain, and a complex sugar chain, but is preferably a high mannose sugar chain or a complex sugar chain, and is particularly preferably a complex sugar chain. Therefore, in one embodiment of the present invention, the sugar chain donor molecule comprises a complex sugar chain. In addition, in the present invention, the complex sugar chain can be any one of a biantennary type, a triantennary type, or a tetraantennary type, and preferably, the sugar chain donor molecule of the present invention comprises a biantennary complex sugar chain.

[0815] In one embodiment of the present invention, the sugar chain donor molecule is a sugar chain-containing molecule having GlcNAc at the reducing end of the sugar chain that is not activated, and preferably includes SGP, (SG-)Asn, (MSG1-)Asn, (MSG2-)Asn or a mixture of (MSG1-)Asn and (MSG2-)Asn. Their sugar chain parts contain sialic acid polysaccharide (Sialyl Glycan, hereinafter referred to as "SG"), which is a representative of N-linked sugar chains and is formed by the structure shown in the following structural formula and sequence formula. The connection pattern of the sugar chain on the reducing end side can be changed from N-linked to O-linked. It should be noted that in this specification, unless otherwise specified, the partial structure of the side chain of the amino acid in the case of linking to the sugar chain is represented by the side chain part in brackets, as described above, for example, "(SG-)Asn".

[0816]

[0817] (In the formula, “-(N / O)” means that a glycosidic bond is formed with a N atom or an O atom.)

[0818] Specific examples of the sugar chain portion of the sugar chain donor molecule that can be used in the present invention include: AG(9) (the following structural formula and sequence formula AG(9)) in which NeuAc at both non-reducing terminals is deleted by treating SG with neuraminidase, AG(7) (the following structural formula and sequence formula AG(7)) in which Gal at both non-reducing terminals is deleted by treating AG(9) with β-galactosidase, and the like.

[0819]

[0820] (In the formula, “-(N / O)” means that a glycosidic bond is formed with a N atom or an O atom.)

[0821]

[0822] (In the formula, “-(N / O)” means that a glycosidic bond is formed with a N atom or an O atom.)

[0823] In the present invention, the sugar chain donor molecule may be chemically modified, for example, the non-reducing terminal may be chemically modified.

[0824] Examples of the sugar chain portion of the sugar chain donor molecule whose non-reducing terminus can be chemically modified and can be used in the present invention include derivatives (SG type) in which the site on the wild-type sialic acid is chemically modified with an arbitrary substituent.

[0825]

[0826] (wherein, X represents a substituent introduced into the sialic acid at the non-reducing ends of the 1-3 chain side and the 1-6 chain side of the β-Man side chain, and is a substituent that can be synthesized by various methods known in the field of organic synthetic chemistry. The substituents used in the conjugation method for antibody-drug conjugate synthesis are used as a reference (Bioconjugate Chem. 2015, 26, 2198-2215). For example, ethynyl, azido (N3-), dibenzylcyclooctenyl (DBCO), bicyclo[6.1.0]non-4-enyl (BCN), a structure containing a 1,2,4,5-tetrazine ring, an aldehyde group, an SH group, a methylsulfonyltriazolyl group, etc. are listed. Preferably, ethynyl, azido ( In addition, as a "substituent having an azido group (N3-)", for example, a polyethylene glycol linker having N3, that is, the upper limit of the number of ethylene glycol units (-CH2-CH2-O-) contained in the linker structure is about 20 or less, preferably about 11 or less, and more preferably about 7 or less. As a "substituent having an azido group (N3-)", for example, a polyethylene glycol linker having N3 (for example, 35-azido-3,6,9,12,15,18,21,24,27,30,33-undeca-pentatriacontane-1-amino, 32-azido-3,6,9,12,15 , 18,21,24,27,30-decaoxatriacontane-1-amino, 29-azido-3,6,9,12,15,18,21,24,27-nonaoxanonacosane-1-amino, 26-azido-3,6,9,12,15,18,21,24-octaoxahexacosane-1-amino, 23-azido-3,6,9,12,15,18,21-heptaoxatricosane-1-amino, 20-azido-3,6,9,12,15,18-hexaoxaicosane-1-amino, 17-azido-3,6,9,12,15-pentaoxaheptadecan-1-amino, 14-azido-3,6,9,12-tetraoxatetradecane-1-amino, 11-azido- The present invention may be a linear substituent such as 3,6,9-trioxaundecane-1-amino, 2-[2-(2-azidoethoxy)ethoxy]ethylamino, 2-(azidoethoxy)ethylamino, 2-azidoethylamino, 2-[2-[2-(2-azidoethoxy)ethoxy]-N-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]ethyl]ethylamino; or a branched substituent such as 1,3-bis[2-[2-(2-azidoethoxy)ethoxy]ethoxy]propane-2-amino, 3-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]-2-[2-[2-(2-azidoethoxy)ethoxy]ethoxymethyl]propane-1-amino, but is not limited thereto.In the formula, "-(N / O)" means forming a glycosidic bond with a N atom or an O atom. ).

[0827] Among the X-SG type structures shown here, X-MSG1 type having a structure lacking chemically modified sialic acid on the 1-6 chain side of the β-Man branch chain and X-MSG2 type having a structure lacking chemically modified sialic acid on the 1-3 chain side can also be cited as examples of the sugar chain portion in the sugar chain donor molecule whose non-reducing terminal can be chemically modified.

[0828] Furthermore, sites other than sialic acid may be considered as sites that can be chemically modified, and examples thereof include the following AG(9) type derivatives and AG(7) type derivatives.

[0829]

[0830] (In the formula, X represents a substituent introduced into the galactose at the non-reducing terminal of both the 1-3 chain side and the 1-6 chain side of the β-Man branch, and specifically, has the same meaning as defined above. In the formula, "-(N / O)" represents the formation of a glycosidic bond with an N atom or an O atom.)

[0831]

[0832] (In the formula, X represents a substituent introduced into the GlcNAc at the non-reducing terminal of both the 1-3 chain side and the 1-6 chain side of the β-Man branch, and specifically, has the same meaning as defined above. In the formula, "-(N / O)" represents the formation of a glycosidic bond with an N atom or an O atom.)

[0833] The X-AG(9) type structure shown here in which the chemically modified galactose on the 1-6 chain side of the β-Man side chain is missing, or the chemically modified galactose on the 1-3 chain side is missing, or the X-AG(7) type structure in which the chemically modified GlcNAc on the 1-6 chain side of the β-Man side chain is missing, or the chemically modified GlcNAc on the 1-3 chain side is missing can be cited as examples of the sugar chain portion in the donor molecule.

[0834] Preferred examples of the sugar chain donor molecule of the present invention include SGP, AG(9)-P, AG(7)-P, SG-Asn, AG(9)-Asn, AG(7)-Asn, SG-OR, AG(9)-OR, AG(7)-OR, (MSG1)-Asn, (MSG2)-Asn, a mixture of (MSG1)-Asn and (MSG2)-Asn, MSG1-OR, MSG2-OR, or a mixture of MSG1-OR and MSG2-OR, wherein the non-reducing terminus thereof may be chemically modified, or examples thereof include sugar chain donor molecules having the structures shown below.

[0835]

[0836] It should be noted that in the above structural formula, "P" represents a peptide portion derived from SGP, and "X" has the same meaning as defined above. As the sugar chain donor molecule of the present invention, more preferably, as a molecule containing an N-linked sugar chain, ([N3-PEG(3)]2-SG)-P-PEG(3)-N3, ([N3-PEG(3)]2-SG)-Asn-PEG(3)-N3, ([N3-PEG(3)]-MSG1)-Asn-PEG(3)-N3, ([N3-PEG(3)]-MSG2)-Asn-PEG(3)-N3, or ([N3-PEG(3)]-MSG1 )-Asn-PEG(3)-N3 and ([N3-PEG(3)]-MSG2)-Asn-PEG(3)-N3. Examples of molecules containing an O-linked sugar chain include ([N3-PEG(3)]2-SG)-OR, ([N3-PEG(3)]-MSG1)-OR, ([N3-PEG(3)]-MSG2)-OR, or a mixture of ([N3-PEG(3)]-MSG1)-OR and ([N3-PEG(3)]-MSG2)-OR.

[0837] In the above chemical formula, "R" represents an arbitrary substituent linked to an oxygen atom via at least one carbon atom, preferably a C1-C6 alkyl group or an aryl group that may be substituted. "C1-C6 alkyl" refers to a straight-chain or branched alkyl group having 1 to 6 carbon atoms, and examples thereof include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, etc., but are not limited thereto. In addition, as "aryl", examples thereof include phenyl, benzyl, indenyl, naphthyl, fluorenyl, anthracenyl, phenanthrenyl, etc., but are not limited thereto. As substituents of the aryl group, C1-C6 alkoxy (e.g., methoxy, ethoxy, isopropoxy, etc.), C1-C6 alkyl (e.g., methyl, ethyl, etc.), and halogen groups may be listed, but are not limited thereto. As substituted aryl groups, p-methoxyphenyl, p-methylphenyl, etc. may be listed, but are not limited thereto. In one embodiment of the present invention, "R" is selected from the group consisting of PMP, Me, A-Mor, iPr, nPr, PrOMe and A-PEG-N3 (A represents the acetyl group in the glycolic acid unit, that is, the part sandwiched by the anomeric hydroxyl group and the amino group in Mor or PEG).

[0838] Furthermore, in one embodiment of the present invention, preferred examples of the sugar chain donor molecule include the following sugar chain donor molecules.

[0839]

[0840]

[0841]

[0842]

[0843] The sugar chain donor molecule of the present invention is preferably appropriately selected depending on various conditions such as the combination of enzyme-A and enzyme-B to be used.

[0844] In one embodiment of the present invention, the sugar chain donor molecule includes a high mannose type sugar chain. Examples of such sugar chain donor molecules include Man9-GlcNAc2-Asn ("M9-Asn") having the following structure, Man8-GlcNAc2-Asn ("M8-Asn") having a structure in which one, two or three mannoses are missing from M9-Asn, Man7-GlcNAc2-Asn ("M7-Asn"), and Man6-GlcNAc2-Asn ("M6-Asn"), but are not limited thereto.

[0845]

[0846] The sugar chain donor molecule can be appropriately purchased from a commercial product, or produced by utilizing or applying a known method.

[0847] <Fc-containing molecules>

[0848] In one embodiment of the present invention, "Fc-containing molecule" (sometimes also referred to as "Fc region-containing molecule") means a molecule containing an Fc region, and examples thereof include antibodies (immunoglobulins), Fc-fusion proteins, Fc fragments, etc. (or containing Fc fragments). Fc-containing fragments other than antibodies include CLCH, such as Fc fragments of IgG (especially IgG-type monoclonal antibodies), CLCH consisting only of a constant region (Patent Document 1), and bispecific antibodies containing Fc fragments (Expert Opin Ther Pat. 201828, 251-276), but are not limited thereto. In one embodiment of the present invention, the Fc-containing molecule is an antibody or a functional fragment of the antibody, and as the antibody or functional fragment, all of the molecules mentioned above can be listed. Thus, for example, in one embodiment of the present invention, the Fc-containing molecule is an antibody or a functional fragment of the antibody, wherein the amino acid residues at positions 234, 235, and 265 (all EU numbering) of the Fc region are Ala, Ala, and Gly, respectively. In another embodiment of the present invention, the Fc-containing molecule is an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 30 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 33. In yet another embodiment of the present invention, the Fc-containing molecule is an antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 44 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 46.

[0849] <What are receptor molecules>

[0850] In one embodiment of the present invention, "acceptor molecule" refers to a molecule that accepts a sugar chain from a sugar chain donor molecule. As an acceptor molecule, it is typically an IgG or Fc fragment thereof that is derived from a monoclonal antibody and has an N297-linked sugar chain consisting only of a core GlcNAc that is optionally bound to core Fuc. It should be noted that in this specification, "core Fuc" (or "core fucose") refers to fucose bound to the above-mentioned core GlnNAc. In the core GlcNAc, depending on the antibody from which it is derived or its production method, it may or may not be bound to core Fuc. Therefore, in one embodiment of the present invention, the acceptor molecule is an Fc-containing molecule (e.g., an antibody or Fc-containing fragment thereof) having a core GlcNAc to which fucose is optionally added as an N297-linked sugar chain, and preferably, an Fc-containing molecule (e.g., an antibody or Fc-containing fragment thereof) having an N297-linked sugar chain consisting of GlcNAc or (Fucα1,6)GlcNAc can be listed. As a source of receptor molecules, various monoclonal antibodies or sugar chain-containing molecules or Fc region-containing molecules (e.g., Fc, CLCH composed of a combination of CH consisting of a constant region consisting of a heavy chain with a variable region deleted and CL consisting of a light chain constant region, etc.) can be used. Preferred receptor molecules of the present invention include (Fucα1,6)-GlcNAc-IgG (e.g., (Fucα1,6)GlcNAc-mAb1), (Fucα1,6)-GlcNAc-Fc, (Fucα1,6)-GlcNAc-CLCH, GlcNAc-mAb4, etc.

[0851] In one embodiment of the invention, the Fc-containing molecule having an N297-linked sugar chain may be derived from an antibody.

[0852] The receptor molecule can be appropriately purchased from a commercial product, or prepared by utilizing or applying a known method. Typically, the receptor molecule can be prepared by treating the N297-linked sugar chain of an Fc-containing molecule (typically, an IgG-type monoclonal antibody or an Fc fragment of the antibody, an Fc-containing molecule such as CLCH consisting only of a constant region) that is the object of sugar chain modification (also referred to as sugar chain reconstruction) with ENGase, wherein the ENGase retains the activity of specifically hydrolyzing the 1,4-glycosidic bond (GlcNAcβ1-4GlcNAc) between GlcNAc in the core chitobiose structure of the N297-linked sugar chain. In this case, various ENGases represented by Endo-Si WT, Endo-A, Endo-D, Endo-E, Endo-F3, Endo-H, Endo-S, Endo-S2, and Endo-Si can be used as ENGase. Therefore, in one embodiment of the present invention, by reacting an enzyme that is the same as or different from the enzyme-A used in step 1 (an enzyme having an activity of transferring sugar chains in sugar chain donor molecules to acceptor molecules) with the raw material Fc-containing molecule (the Fc-containing molecule to be the target of sugar chain modification, typically, an Fc-containing molecule having N297-linked sugar chains derived from host cells, non-uniform N297-linked sugar chains, or non-uniform N297-linked sugar chains derived from host cells), the acceptor molecule can be prepared in a step independent of the sugar chain transfer reaction in step 1. In addition, as a feature of the enzyme-A that can be used in step 1, it has "residual hydrolysis activity". Therefore, by allowing the enzyme-A to act on the Fc-containing molecule having non-uniform sugar chains derived from host cells instead of pre-treatment with these ENGases, the acceptor molecule can be prepared in situ. Therefore, in one embodiment of the present invention, the Fc-containing molecule that becomes the raw material (the Fc-containing molecule that becomes the object of sugar chain modification, typically the Fc-containing molecule having N297-linked sugar chains derived from host cells, uneven N297-linked sugar chains, or uneven N297-linked sugar chains derived from host cells) is allowed to act on enzyme-A to prepare the receptor molecule in situ and / or in one pot. In addition, the IgG or Fc-containing molecule used for sugar chain modification (sugar chain reconstruction) is preferably derived from an IgG heavy chain composed of the same amino acid sequence, and is produced in the form of a molecule having an N297-linked sugar chain. The production method is not limited, and IgG produced by a generally known monoclonal antibody production method, CLCH of IgG, or Fc fragments obtained by enzymatic treatment thereof, etc. can be used. In addition, such IgG or Fc fragments can be a mixture of samples obtained by different production methods and different batches.

[0853] <Endo-β-N-acetylglucosaminidase>

[0854] "Endo-β-N-acetylglucosaminidase" refers to a glycolytic enzyme known as ENGase: Endo-β-N-acetylglucosaminidase, which is responsible for the cutting and bond formation of the chitobiose structure in the sugar chain structure. In addition, the names differ depending on their sources. In this specification, the endo-β-N-acetylglucosaminidase that uses the N297-linked sugar chain containing the Fc molecule as a substrate is referred to as "enzyme-A", and the endo-β-N-acetylglucosaminidase that uses the sugar chain of the sugar chain donor molecule, such as the sugar chain of the sugar chain raw material without fucose (such as SGP) as a substrate is referred to as "enzyme-B". The following describes Enzyme-A and Enzyme-B in the present invention.

[0855] <Enzyme-A>

[0856] "Enzyme-A" refers to an endo-β-N-acetylglucosaminidase that uses the N297-linked sugar chain of an Fc-containing molecule (such as an antibody) as a substrate. Enzyme-A usually includes a domain with high affinity for Fc-containing molecules, a domain with high affinity for sugar chains, and a sugar chain transfer reaction catalytic domain. Typically, it uses the N297-linked sugar chain of an Fc-containing molecule (such as an antibody) as a substrate and has both hydrolysis activity and sugar chain transfer activity. However, in the present invention, it is important to transfer the sugar chain in the sugar chain donor molecule (especially the sugar chain activated by chemical or enzymatic methods) to the acceptor molecule, so the hydrolysis activity may also be weakened or eliminated. On the contrary, an enzyme that maintains strong hydrolysis activity may hydrolyze the core GlcNAc sugar chain transferred to the acceptor molecule due to the sugar chain transfer activity as a substrate, so the hydrolysis activity of enzyme-A in the present invention is preferably relatively reduced. It should be noted that, as described above, when the enzyme-A has residual hydrolysis activity, the following advantages can be enjoyed: the acceptor molecule in step 1 does not need to be prepared separately, and can be prepared in situ in the same reaction tank in step 1. Therefore, in one embodiment of the present invention, the enzyme-A has an activity that can act on the sugar chain containing the Fc molecule (particularly, the sugar chain transfer activity using the N297-linked sugar chain containing the Fc molecule as a substrate), and in another embodiment of the present invention, the enzyme-A has an activity that can act on the sugar chain containing the Fc molecule and has residual hydrolysis activity or the hydrolysis activity has disappeared.

[0857] The hydrolytic activity of enzyme-A refers to the activity of specifically hydrolyzing the β1,4 glycosidic bond contained in the core chitobiose in the common basic structure of the sugar chain, and particularly refers to the activity of specifically hydrolyzing the β1,4 glycosidic bond contained in the core chitobiose in the common basic structure of the N297-linked sugar chain containing the Fc molecule.

[0858] The sugar chain transfer activity of enzyme-A refers to an activity for forming a glycosidic bond between the reducing end of the sugar chain donor molecule and an acceptor molecule comprising an Fc site having only core GlcNAc (core fucose may or may not be added) at N297.

[0859] Examples of the enzyme-A in the present invention include Endo-S (an enzyme derived from Streptococcus pyogenes) (see Collin M and Olsen A., EMBO J. 2001, 20, 3046-3055, and Goodfellow JJ, et al., J Am Chem Soc. 2012, 134, 8030-8033), EndoS2 or EndoS49 (see Sjogren J, et al., Biochem J. 2013, 455, 107-118, and Shivatare SS, et al., Chem Commun (Camb). 2018, 54, 6161-6164), and Endo-F3 (an enzyme derived from Flavobacterium meningosepticum) (see Huang W, et al., 2014). al., Chembiochem. 2011, 12, 932-941, and Giddens JP, et al., J Biol Chem. 2016, 291, 9356-9370, or their mutant enzymes. In addition, the enzyme-A in the present invention includes: endo-β-N-acetylglucosaminidase (hereinafter referred to as "Endo-Si" derived from Streptococcus iniae (see Pier GB and Madin SH., Int J Syst Bacteriol. 1976 26, 545-553), whose amino acid sequence (SEQ ID NO: 49) is shown in Fig.32 ), or a mutant enzyme thereof. Furthermore, the enzyme-A in the present invention includes an enzyme whose sequence is identified based on genome information of the genus Streptococcus (see Vincent P. Richard, et al., Genome Biol. Evol. 2014, 6, 741-753), or a mutant enzyme thereof.

[0860] As long as the enzyme-A of the present invention has the above-mentioned characteristics, it is not limited to the specific enzyme used in the examples, and can be an enzyme isolated from nature, or an enzyme artificially produced or modified based on the sequence information of the enzyme of the present invention. In the case of isolation from nature, the biological species as the isolation source is not particularly limited, and preferably bacteria.

[0861] The active domain and carbohydrate-binding module (CBM) of Endo-Si are estimated to be the regions of amino acid numbers 106 to 447 and amino acid numbers 762 to 897 of sequence number 49, respectively, based on sequence comparison with EndoS (B. Trastoy et al., PNAS (2014) vol111, No.18, pp6714-6719), which has undergone crystal structure analysis. These two regions are considered to be important sites for hydrolysis activity and / or transfer activity and interaction with antibodies. Therefore, the enzyme-A of the present invention includes: a polypeptide comprising the amino acid sequence recorded in amino acid numbers 106 to 447 and / or amino acid numbers 762 to 897 of SEQ ID NO: 49, preferably comprising the amino acid sequence recorded in amino acid numbers 106 to 897 of SEQ ID NO: 49, more preferably comprising the amino acid sequence recorded in amino acid numbers 106 to 928 of SEQ ID NO: 49, and further preferably comprising the amino acid sequence recorded in amino acid numbers 34 to 928 of SEQ ID NO: 49 and exhibiting sugar chain transfer activity.

[0862] As described above, endo-β-N-acetylglucosaminidase generally has both hydrolytic activity and sugar chain transfer activity, but enzymes that maintain strong hydrolytic activity sometimes hydrolyze the sugar chain of the core GlcNAc that is transferred to the acceptor molecule (antibody or Fc region molecule containing it with core GlcNAc as N297-linked sugar chain) due to the sugar chain transfer activity as a substrate, and the desired sugar chain transfer product cannot be properly obtained. Therefore, what is useful in the synthesis of sugar chain remodeling antibodies or sugar chain modified compounds is a mutant enzyme that relatively reduces the hydrolytic activity compared to the sugar chain transfer activity. Therefore, in one embodiment of the present invention, enzyme-A is a mutant enzyme of Endo-S, EndoS-2, Endo-Si, Endo-Sd, Endo-Se, or Endo-Sz, and is a mutant enzyme with lower hydrolytic activity than its corresponding wild-type enzyme, preferably appropriately substituted Figure 30 to Figure 32 and Figure 37 to Figure 39Particularly preferred mutant enzymes having amino acid residues indicated in bold include Endo-SD233Q, Endo-SD233Q / Q303L, Endo-SD233Q / E350A, Endo-S D233Q / E350Q, Endo-SD233Q / E350D, Endo-SD233Q / E350N, Endo-S D233Q / D405A, Endo-Si D241Q, Endo-Si D241Q / Q311L, Endo-SiD241Q / E360Q, Endo-Si D241M, Endo-Si D241M / Q311L, Endo-Si D241M / E360Q, Endo-Si T190Q, Endo-Si T190 / D241Q, Endo-Si T190Q / D241M, Endo-Si D241X1 (X1 represents any amino acid residue other than K, R and D, specifically, represents A, N, C, Q, E, G, H, I, L, M, F, P, S, T, W, Y or V, preferably represents A, Q, E, H, I, L, M, F, P, S, T, W, Y or V), Endo-Si T190X2 (X2 represents an amino acid residue of F, H, K, M, Q, R, W or Y), Endo-Si Q311X3 (X3 represents an amino acid residue of F, N or Y), Endo-Si E360K, Endo-S2D184M, Endo-S2 T138Q, Endo-S2 D184Q, Endo-S2 D184Q / Q250L, Endo-S2 D184Q / E289Q, Endo-S2 Endo-Sd D232Q D232M / Q302L, Endo-Se D233Q, Endo-Se D233M, Endo-SeD233Q / Q303L, Endo-Se D233M / Q303L, Endo-Sz D234Q, Endo-Sz D234M, Endo-SzD234Q / Q304L, or Endo-Sz D234M / Q304L, but not limited to this.In addition, in addition to the specific mutations mentioned above, enzyme-A can also be subjected to further mutations, for example, with reference to patent literature (International Publication No. 2022 / 050300), etc., within the range that does not affect the activity of the enzyme, there may be 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1 amino acid residue substitutions, deletions, insertions and / or additions. In addition, as the amino acid sequence of enzyme-A, an amino acid sequence having at least 80% or more, preferably 85% or more, more preferably 90% or more, further preferably 95%, 96%, 97%, 98% or 99% or more homology or identity with respect to amino acid residues other than specific mutant amino acid residues can be cited with reference to patent literature (International Publication No. 2022 / 050300), etc.

[0863] <Enzyme-B>

[0864] "Enzyme-B" refers to an endo-β-N-acetylglucosaminidase that uses the sugar chain of a sugar chain donor molecule as a substrate, and particularly refers to an endo-β-N-acetylglucosaminidase that uses the sugar chain of a sugar chain donor molecule as a substrate but does not use the N297-linked sugar chain as a substrate (in other words, the reactivity to the N297-linked sugar chain is low, preferably extremely low). The sugar chain portion on the sugar chain donor molecule that is the substrate of enzyme-B can be any of a high mannose type sugar chain, a hybrid type sugar chain, and a complex type sugar chain, preferably a high mannose type sugar chain or a complex type sugar chain, and particularly preferably a complex type sugar chain. Therefore, in one embodiment of the present invention, the sugar chain portion on the sugar chain donor molecule that is the substrate of enzyme-B is a high mannose type sugar chain or a complex type sugar chain, preferably a complex type sugar chain. Furthermore, in the present invention, the complex sugar chain may be any of a biantennary type, a triantennary type, or a tetraantennary type, and preferably, the sugar chain portion on the sugar chain donor molecule as the substrate of the enzyme-B is a biantennary type complex sugar chain. In one embodiment of the present invention, the enzyme-B is an endo-β-N-acetylglucosaminidase that uses a sugar chain raw material (glycopeptide, glycoprotein such as SGP) without fucose as a substrate, and preferably an endo-β-N-acetylglucosaminidase that uses a sugar chain raw material containing a biantennary type complex sugar chain without fucose as a substrate.

[0865] The enzyme-B of the present invention is preferably an enzyme having the following properties: by acting on a sugar chain donor molecule containing a sugar chain including GlcNAc whose reducing end is not activated in the presence of enzyme-A, an activated intermediate is generated in which the reducing end is in a state that can be used for a sugar chain transfer reaction, thereby promoting the sugar chain transfer reaction of the sugar chain derived from the sugar chain donor molecule to the acceptor molecule caused by enzyme-A. Here, the sugar chain transfer activity of enzyme-B itself is considered to be related to the ability to activate sugar chain donor molecules, so in one embodiment of the present invention, enzyme-B can be selected from endo-β-N-acetylglucosaminidase that uses sugar chains of sugar chain donor molecules containing GlcNAc with unactivated reducing ends (e.g., SGP) as substrates but does not use N297-linked sugar chains as substrates, using the sugar chain transfer activity to acceptors having GlcNAc (e.g., GlnNAc derivatives such as 1-methylethyl-2-(acetylamino)-2-deoxy-β-D-pyranoglucoside and 2-(acetylamino)-2-deoxy-β-D-pyranoglucoside) as an index. Therefore, in one embodiment of the present invention, enzyme-B is an enzyme having sugar chain transfer activity from SGP to GlcNAc derivatives.

[0866] The activation of sugar chain donor molecules by enzyme-B is an action that acts on sugar chain donor molecules containing GlcNAc whose reducing ends are not activated, and generates an activated intermediate in which the reducing ends are in a state that can be used for sugar chain transfer reactions. However, when the hydrolysis activity caused by enzyme-B is high, the activated intermediate does not react with the acceptor molecule via enzyme-A, but reacts with H2O molecules present in the reaction system, and there is a possibility that many free sugar chains are generated. Therefore, enzyme-B in the present invention preferably has a relatively lower hydrolysis activity than the sugar chain activation action.

[0867] Examples of the enzyme-B of the present invention include Endo-M (an enzyme derived from Mucor hiemalis) (see Yamamoto K, et al., Biochem Biophys Res Commun. 1994, 203, 244-252, and Umekawa M, et al., J. Biol Chem. 2021, 285, 511-521), Endo-CC (an enzyme derived from Coprinopsis cinerea) (see Eshima Y, et al., PLoS One. 2015, 10, e0132859), Endo-Om (an enzyme derived from methylotrophic yeast (Ogataea minuta)) (see Murakami S, et al., Glycobiology. 2013, 23, 736-744), Endo-Rp (an enzyme derived from Rhizomucor microsporus) (see Yamamoto K, et al., Biochem Biophys Res Commun. 1994, 203, 244-252, and Umekawa M, et al., J. Biol Chem. 2021, 285, 511-521), pusillus) (WO2018101454), or their mutant enzymes (preferably mutant enzymes with reduced hydrolysis activity compared to the wild type). In one embodiment of the present invention, enzyme-B is a mutant enzyme of Endo-M, Endo-Om, Endo-CC, or Endo-Rp, and is a mutant enzyme with lower hydrolysis activity than its corresponding wild-type enzyme, preferably Figure 33 to Figure 36The mutant enzyme in which the amino acid residues indicated in bold are appropriately substituted is particularly preferably selected from the group consisting of Endo-Rp N172Q, Endo-Rp N172H, Endo-Rp N172A, Endo-Rp N172C, Endo-Rp N172D, Endo-Rp N172E, Endo-Rp N172G, Endo-Rp N172I, Endo-Rp N172L, Endo-Rp N172M, Endo-Rp N172P, Endo-Rp N172S, Endo-Rp N172T, Endo-Rp N172V, Endo-Rp W278F / S216V, Endo-Rp W278F / N246D, Endo-Rp W278F / D276N, Endo-Rp The enzymes are selected from the group consisting of Endo-Rp W278F / A310D, Endo-Rp W278F / N172D / F307Y, Endo-Rp W278F / N172D / F307H, Endo-Rp W278F / N172D / A310D, Endo-Rp W214F / F307Y / L306I, Endo-M N175Q, Endo-M N175Q / Y217F, Endo-CC N180H, and Endo-Om N194Q, but are not limited thereto. In addition, in addition to the specific mutations mentioned above, enzyme-B may also be subjected to further mutations, for example, with reference to patent literature (International Publication No. 2022 / 050300), etc., within the range that does not affect the activity of the enzyme, there may be 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1 amino acid residue substitutions, deletions, insertions and / or additions. In addition, as the amino acid sequence of enzyme-B, an amino acid sequence having at least 80% or more, preferably 85% or more, more preferably 90% or more, further preferably 95%, 96%, 97%, 98% or 99% or more homology or identity with respect to amino acid residues other than specific mutant amino acid residues can be cited with reference to patent literature (International Publication No. 2022 / 050300), etc.

[0868] In a typical embodiment of the production method of the present invention, enzyme-A and enzyme-B are added to step 1 as different molecules, respectively. As long as enzyme-A and enzyme-B play the desired role, they can also be added to the reaction system in a state of direct or indirect bonding. Therefore, in one embodiment of the present invention, enzyme-A and enzyme-B can be added to the reaction system in a state of direct or indirect chemical bonding. For example, the two molecules can also be added to the reaction system in the state of an immobilized enzyme fixed to any solid phase carrier, or a fusion protein bonded by any amino acid, polymer such as PEG, or oligomer linker. In other words, in one embodiment of the present invention, enzyme-A and enzyme-B are directly or indirectly chemically bonded (= enzyme-A and enzyme-B are fused), for example, they can be added to the reaction system in the state of an immobilized enzyme fixed to any solid phase carrier, or a fusion protein bonded by any amino acid, polymer such as PEG, or oligomer linker, preferably an amino acid linker. It should be noted that, in the present invention, even if enzyme-A and enzyme-B are functional fragments rather than full-length molecules without deletions, as long as the functional fragment has the desired function, the fragment is also regarded as one embodiment of enzyme-A and enzyme-B. Therefore, enzyme-A and enzyme-B in the present invention also include, for example, "enzyme-A" and "enzyme-B", "enzyme fragment having the function of enzyme-A" and "enzyme fragment having the function of enzyme-B", "enzyme-A" and "enzyme fragment having the function of enzyme-B", and "enzyme fragment having the function of enzyme-A" and "enzyme-B" are directly or indirectly chemically bonded. Examples of the "enzyme fragment having the function of enzyme-A" include fragments having the following amino acid sequences: the amino acid sequence is composed of the above-mentioned enzyme-A or Endo-BI1 (GenBank Accession No.: ACJ53522.1), Endo-BI2 (GenBank Accession No.: BAJ71450.1), Endo-BT-3987 (GenBank Accession No.: AAO79092.1), Endo-E (GenBank Accession No.: AAR20477.1), Endo- The full-length sequences of Endo-F (GenBank Accession No.: AAA24922.1), Endo-F2 (GenBank Accession No.: AAA24923.1), Endo-F3 (GenBank Accession No.: AAA24924.1), Endo-CoM (GenBank Accession No.: XP006673222.1), and Endo-SB (GenBank Accession No.: BBB35949.1) are fragmented into amino acid sequences of specific functional sequences and specific mutations can be applied.In addition, as "enzyme fragments having the function of enzyme-B", fragments having the following amino acid sequences can be listed: the amino acid sequence is composed of the catalytic activity domain of the above-mentioned enzyme-B or Endo-A (GenBank Accession No.: AAD10851.1), Endo-CE (GenBank Accession No.: BAB84821.1), Endo-Tsp1006 (GenBank Accession No.: CCY37287.1), Endo-Tsp1263 (GenBank Accession No.: CDD88945.1), Endo-Tsp1457 (GenBank Accession No.: CDD89351.1), Endo-BB (GenBank Accession No.: The full-length sequences of Endo-BH (GenBank Accession No.: BAB04504.1), Endo-BN (GenBank Accession No.: WP_007484749.1), Endo-CC1 (GenBank Accession No.: XP_001839402.1), Endo-CC2 (GenBank Accession No.: XP_002911817.1), Endo-D (GenBank Accession No.: AAK74656.1), and Endo-Pm (GenBank Accession No.: ADK97032.1) are fragmented into amino acid sequences of specific functional sequences and specific mutations can be applied. For example, with respect to enzyme-A, enzyme-B, and various functional sequences, with reference to patent documents (International Publication No. 2022 / 050300), etc., 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1 amino acid residue substitutions, deletions, insertions and / or additions may be present within the range that does not affect the above functions. In addition, as respective amino acid sequences, reference to patent documents (International Publication No. 2022 / 050300), etc., with respect to amino acid residues other than specific amino acid residues that affect activity, amino acid sequences having at least 80% or more, preferably 85% or more, more preferably 90% or more, and further preferably 95%, 96%, 97%, 98% or 99% or more homology or identity can be cited.

[0869] As a combination of enzyme A and enzyme B, any combination may be used as long as enzyme A and enzyme B can exert their respective functions. For example, any combination of enzyme A and enzyme B described in the following table may be used.

[0870] Enzyme-A Enzyme-B A-1 Endo-Si D241M Q311L B-1 Endo-Rp N172H A-2 Endo-Si D241Q B-2 Endo-Rp N172Q A-3 Endo-Si D241Q E360Q B-3 Endo-Rp N172V A-4 Endo-Si D241M B-4 Endo-M N175Q A-5 Endo-Si D241Q Q31 1L B-5 Endo-M N175Q Y217F A-6 Endo-Si D241M E360Q A-7 Endo-Si WT A-8 Endo-SD2330E350Q A-9 Endo-S WT A-10 Endo-S2 D184M A-11 Endo-S2 T138Q A-12 Endo-S2 WT

[0871] Enzyme-A Enzyme-A A-13 Endo-Si T190F A-26 Endo-Si D241L A-14 Endo-Si T190H A-27 Endo-Si D241P A-15 Endo-Si T190K A-28 Endo-Si D241R A-16 Endo-Si T190M A-29 Endo-Si D241S A-17 Endo-Si T190Q A-30 Endo-Si D241T A-18 Endo-Si T190R A-31 Endo-Si D241V A-19 Endo-Si T190W A-32 Endo-Si D241w A-20 Endo-Si T190Y A-33 Endo-Si D241Y A-21 Endo-Si D241A A-34 Endo-Si Q311F A-22 Endo-Si D241E A-35 Endo-Si Q311K A-23 Endo-Si D241F A-36 Endo-Si Q311N A-24 Endo-Si D241H A-37 Endo-Si Q311Y A-25 Endo-Si D241I A-38 Endo-Si E360K

[0872] Enzyme-A A-39 Endo-S2 D184M / Q250L A-40 Endo-S2 D184Q / 0250L A-41 Endo-Sd WT A-42 Endo-Se WT A-43 Endo-Se D233M A-44 Endo-Sz WT A-45 Endo-Sz D234M A-46 Endo-Sz D234M / Q304L

[0873] In addition, as described above, when enzyme-A and enzyme-B are fused, any combination can be used as long as the enzyme-A and enzyme-B exert their respective functions. More specifically, for example, enzyme-A is an Endo-Si mutant and enzyme-B is an Endo-Rp mutant, for example, enzyme-A is Endo-Si D241M / Q311L or Endo-Si D241Q / Q311L and enzyme-B is Endo-Rp N172H or Endo-Rp N172V. As another specific embodiment, the fusion enzyme is a fusion protein of Endo-Rp N172H and Endo-Si D241M / Q311L, a fusion protein of Endo-Rp N172V and Endo-Si D241Q / Q311L, or Endo-Si D241Q / Q311L and Endo-Rp N172V, for example, [Enzyme-A]-[Enzyme-B] fusion protease and [Enzyme-B]-[Enzyme-A] fusion protease described in the following table.

[0874]

[0875] The fusion protein of enzyme-A and enzyme-B can be prepared according to conventional methods with reference to non-patent literature (Joaquim, et al., Processes. 2022, 10, 494.), for example, by binding enzyme-A and enzyme-B to any solid phase carrier through physical or chemical interaction. Alternatively, with reference to non-patent literature (F Grueninger-Leitch, et al., Protein Sci. 1996, 12, 2617-22, and Emily MK, et al., Protein Eng Des Sel. 2005, 10, 497-501) and patent literature (WO2017137459), the fusion protein can be produced by gene manipulation in host cells. That is, after combining the genes encoding enzyme-A and enzyme-B, the host cell can be made to produce a single enzyme having functions derived from enzyme-A and enzyme-B through gene manipulation. In the case where the host cell produces a fusion enzyme by genetic manipulation, it is not necessary to prepare enzyme-A and enzyme-B separately, which can reduce the number of enzymes to be prepared, thereby reducing the production cost and labor of the enzyme. In the case where enzyme-A and enzyme-B are combined through a linker composed of amino acids, in one embodiment, the linker is composed of one or more amino acids, and with reference to non-patent literature (Chen, et al., Adv Drug Deliv Rev. 2013 Oct 15; 65 (10): 1357-1369.), etc., a linker with typical properties known in the art can be used, which can be a soft or rigid linker, a linker with a property that can be cut or not cut, and a long or short linker can be used. In one embodiment, as an amino acid sequence fragment, the linker may include (SGGS)n, (GGGS)n, (GGGGS)n, (G)n, (EAAAK)n, (EF)n, or (GGS)n or a combination thereof (n is an integer of 1 to 30, preferably an integer of 1 to 20, more preferably an integer of 1 to 10, and more preferably an integer of 1 to 6). In addition, the linker may include amino acid sequences commonly used as purification tags such as polyhistidine tags, HA tags, FLAG tags, CBD tags, Fc tags, and GST tags. In one embodiment, the purification tag is a polyhistidine tag, HA tag, FLAG tag, Fc tag, and GST tag, preferably a polyhistidine tag, HA tag, FLAG tag, and CBD tag, more preferably a polyhistidine tag and FLAG tag, and more preferably a polyhistidine tag.

[0876] <Additives>

[0877] In one embodiment of the present invention, the above-mentioned step 1 is a step of reacting the acceptor molecule with the sugar chain donor molecule in the presence of enzyme-A, enzyme-B and a specific additive to obtain a reaction mixture. The additive has a positive effect on the sugar chain transfer rate in the one-pot reaction by means of electrostatic interaction, hydrophobic interaction, solvent effect, etc. In particular, when there is a mutation in the Fc region of an Fc-containing molecule (typically an antibody), the three-dimensional structure of the Fc region is sometimes changed, and the sugar chain transfer rate changes. For example, when there is a certain mutation in the Fc region of an antibody used as an acceptor molecule, the sugar chain transfer rate may decrease compared to the case without the mutation. Additives are useful for regulating the sugar chain transfer rate of an Fc-containing molecule (typically an antibody) used as an acceptor molecule, and are particularly useful for the case where there is a mutation in the Fc region of an Fc-containing molecule (typically an antibody) used as an acceptor molecule and the sugar chain transfer rate may decrease due to the mutation in the Fc region.

[0878] In one embodiment of the present invention, the additive used in process 1 is a monovalent salt. In this specification, "monovalent salt" refers to a salt containing a monovalent anion or a monovalent cation. As monovalent salts, there can be listed: inorganic salts (sodium chloride, lithium chloride, potassium chloride, potassium iodide, sodium iodide, lithium iodide, potassium bromide, sodium fluoride, and alkali metal salts such as potassium fluoride; ammonium salts such as ammonium fluoride, ammonium chloride, and ammonium sulfate); organic salts (ammonium acetate, sodium acetate, potassium acetate, etc.), but are not limited to this. In one embodiment of the present invention, the additive used in process 1 is sodium chloride, potassium acetate, ammonium acetate, lithium chloride or sodium acetate, preferably sodium chloride, ammonium acetate or sodium acetate. In another embodiment of the present invention, the additive used in process 1 is a monovalent alkali metal salt, preferably sodium chloride, potassium acetate, lithium chloride or sodium acetate, and more preferably sodium chloride.

[0879] In one embodiment of the present invention, the additive used in step 1 is an organic solvent. Examples of the organic solvent that can be used in step 1 include polar protic solvents such as tetrahydrofuran, acetone, acetonitrile, N,N-dimethylformamide, and dimethyl sulfoxide, and polar protic solvents such as 1-butanol, 2-propanol, 1-propanol, ethanol, and methanol, but are not limited thereto. In one embodiment of the present invention, the organic solvent used in step 1 is ethanol or dimethyl sulfoxide.

[0880] In one embodiment of the present invention, the additive used in step 1 is a surfactant. Examples of the surfactant that can be used in step 1 include anionic surfactants, cationic surfactants, nonionic surfactants, and amphoteric surfactants, but are not limited thereto. Examples of anionic surfactants include carboxylic acid type surfactants such as sodium octanoate and sodium caprate; sulfonic acid type surfactants such as sodium 1-hexane sulfonate and sodium 1-octane sulfonate; sulfate type surfactants such as sodium lauryl sulfate and sodium myristyl sulfate; and phosphate type surfactants such as lauryl phosphoric acid and sodium lauryl phosphate. Examples of cationic surfactants include quaternary ammonium salt type surfactants such as tetramethylammonium chloride and hexadecyltrimethylammonium bromide; alkylamine salt type surfactants such as monomethylamine hydrochloride and dimethylamine hydrochloride; and pyridine ring-containing surfactants such as butylpyridinium chloride and hexadecylpyridinium chloride. Examples of nonionic surfactants include ester type surfactants such as lauric acid glyceryl ester and sorbitan fatty acid ester; polyethylene glycol (polyethylene glycol 2000, polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 11000, polyethylene glycol 20000, etc.) ), ether type such as polyoxyethylene alkylphenyl ether; ester ether type such as polyoxyethylene glycerol fatty acid ester, polyoxyethylene anhydrous sorbitan fatty acid ester (polysorbate 20, polysorbate 60, polysorbate 65, polysorbate 80, etc.); alkanolamide type such as lauric acid diethanolamide and oleic acid diethanolamide; alkyl glycoside type such as octyl glucoside and decyl glucoside; and higher alcohol type surfactants such as cetyl alcohol and stearyl alcohol. As amphoteric surfactants, there can be listed: alkyl betaine type such as lauryl dimethylaminoacetic acid betaine; fatty acid amide propyl betaine type such as cocamidopropyl betaine and cocamidopropyl hydroxysulfonate; amino acid type such as sodium lauroyl glutamate and potassium lauroyl glutamate; and amine oxide type surfactants such as lauryl dimethylamine N-oxide and oleoyl dimethylamine N-oxide. In one embodiment of the present invention, the surfactant used in step 1 is a nonionic surfactant. In another embodiment of the present invention, the surfactant used in step 1 is an ether-type or ester-ether-type nonionic surfactant. In yet another embodiment of the present invention, the surfactant used in step 1 is polyethylene glycol or polyoxyethylene sorbitan fatty acid ester, preferably polyethylene glycol 6000 or polysorbate 20.

[0881] In one embodiment of the present invention, the additive used in step 1 is a sugar. Examples of the sugar that can be used in step 1 include monosaccharides, disaccharides, oligosaccharides (in this specification, sugars with trisaccharides or more are referred to as oligosaccharides), deoxy sugars, uronic acids, amino sugars, sugar alcohols, lactones, and polysaccharides, but are not limited thereto. Examples of the monosaccharides used as additives in step 1 include: trioses such as glyceraldehyde; tetroses such as erythrose and threose; pentoses such as ribose and xylose; aldohexoses such as glucose, mannose, and galactose; pyruvoses such as dihydroxyacetone; tetroses such as erythrulose; ketopentoses such as xylulose and ribulose; and ketohexoses such as fructose and sorbose. It should be noted that when Endo-Rp enzyme (including mutants) is used as enzyme-B in step 1, glucose may become a receptor for sugar chains activated by Endo-Rp enzyme, and thus the sugar chain transfer rate may be reduced. Therefore, in one embodiment of the present invention, the sugar used as an additive in step 1 does not contain glucose. Examples of the disaccharide used as an additive in step 1 include trehalose, isotrehalose, kojibiose, sophorose, nigerobiose, laminaribiose, maltose, cellobiose, isomaltose, gentiobiose, lactose, and sucrose. Examples of oligosaccharides include fructooligosaccharides and galacto-oligosaccharides. Examples of deoxy sugars include deoxyribose and fucose. Examples of uronic acids include glucuronic acid and galacturonic acid. Examples of amino sugars include glucosamine and aminogalactose. Examples of sugar alcohols include glycerol, xylitol, and sorbitol. Examples of lactones include ascorbic acid and glucuronolactone. Examples of polysaccharides include starch, amylose, amylopectin, glycogen, cellulose, pectin, and glucomannan. In one embodiment of the present invention, the sugar used in step 1 is a monosaccharide, a disaccharide or an amino sugar. In another embodiment of the present invention, the sugar used in step 1 is a disaccharide or an amino sugar. In yet another embodiment of the present invention, the sugar used in step 1 is trehalose or sorbitol.

[0882] In one embodiment of the present invention, the additive used in step 1 is an amino acid. The amino acid that can be used as an additive in step 1 is not particularly limited as long as it is an organic compound having two functional groups of an amino group and a carboxyl group. Preferred examples include: basic amino acids such as arginine, histidine, and lysine; acidic amino acids such as glutamic acid and aspartic acid; neutral polar amino acids such as asparagine, glutamine, serine, threonine, tyrosine, and cysteine; neutral non-polar amino acids such as isoleucine, leucine, valine, methionine, phenylalanine, tryptophan, alanine, glycine, and proline. In one embodiment of the present invention, the amino acid used as an additive in step 1 is a basic amino acid, preferably arginine, histidine, or lysine, and more preferably arginine or histidine. In addition, the amino acid that can be used as an additive in step 1 can be added in the form of a salt, for example, arginine, histidine, or lysine, which are basic amino acids, can be added in the form of hydrochloride. Therefore, in one embodiment of the present invention, the amino acid used as the additive in step 1 is arginine hydrochloride, histidine hydrochloride or lysine hydrochloride, and more preferably arginine hydrochloride or histidine hydrochloride.

[0883] The concentration of the additive used in step 1 can be arbitrarily set as long as the Fc-containing molecules and enzymes are not precipitated by the addition of the additive, that is, as long as the reactivity of the one-pot reaction remains, for example, it can be set by using or applying the method described in Example 15-3 of this specification. In one embodiment of the present invention, when the additive used in step 1 is a monovalent salt (e.g., sodium chloride), its concentration is 1500 mM or less, 1000 mM or less, 900 mM or less, 800 mM or less, 700 mM or less, 600 mM or less, 500 mM or less, 450 mM or less, 400 mM or less, 350 mM or less, 300 mM or less, 250 mM or less, 200 mM or less, 150 mM or less, 100 mM or less, or 50 mM or less. In another embodiment of the present invention, when the additive used in step 1 is a monovalent salt (e.g., sodium chloride), its concentration is 1 mM or more, 5 mM or more, 10 mM or more, 20 mM or more, 30 mM or more, 450 mM or less, 400 mM or less, 350 mM or less, 300 mM or less, 250 mM or less, 200 mM or less, 150 mM or less, 100 mM or less, or 50 mM or less. 0mM or more, 50mM or more, 100mM or more, 150mM or more, 200mM or more, 250mM or more, 300mM or more, 350mM or more, 400mM or more, 450mM or more, 500mM or more, 600mM or more, 700mM or more, 800mM or more, 900mM or more or 1000mM or more. In another embodiment of the present invention, when the additive used in step 1 is a monovalent salt (such as sodium chloride), its concentration can be set within any concentration range sandwiched between any of the upper and lower limits exemplified above, for example, it can be set within the range of 1 to 700mM, 10 to 600mM, 100 to 550mM, or 50 to 1000mM.

[0884] In one embodiment of the present invention, the additive may be contained in advance or may be added in step 1.

[0885] In one embodiment of the present invention, when the additive used in step 1 is an organic solvent (e.g., ethanol, DMSO), its concentration is 0.1 to 20% v / v, preferably 0.2 to 10% v / v, more preferably 0.3 to 5% v / v, further preferably 0.4 to 3% v / v, particularly preferably 0.5 to 2% v / v, and most preferably about 1% v / v.

[0886] In one embodiment of the present invention, when the additive used in step 1 is a surfactant (e.g., polyethylene glycol 6000, polysorbate 20), its concentration is 0.1 to 20% v / v, preferably 0.2 to 10% v / v, more preferably 0.3 to 5% v / v, further preferably 0.4 to 3% v / v, particularly preferably 0.5 to 2% v / v, and most preferably about 1% v / v.

[0887] In one embodiment of the present invention, when the additive used in step 1 is a sugar (such as trehalose or sorbitol), its concentration is 1mM to 10M, preferably 5 to 1000mM, more preferably 10 to 500mM, further preferably 15 to 200mM, particularly preferably 20 to 100mM, and most preferably about 40mM.

[0888] In one embodiment of the present invention, when the additive used in step 1 is an amino acid (e.g., arginine hydrochloride, histidine hydrochloride), its concentration is 1 mM to 10 M, preferably 5 to 1000 mM, more preferably 10 to 500 mM, further preferably 15 to 200 mM, particularly preferably 20 to 100 mM, and most preferably about 40 mM.

[0889] <Process 1>

[0890] In one embodiment of the present invention, step 1 is a step of reacting an acceptor molecule with a sugar chain donor molecule in the presence of enzyme-A, enzyme-B, and a specific additive to obtain a reaction mixture. The reaction conditions of step 1 can be appropriately selected according to the reaction conditions of a known sugar chain transfer reaction using GlcNAc whose reducing terminal is not activated as a sugar chain donor molecule, or by utilizing or applying the reaction conditions of a known sugar chain transfer reaction.

[0891] The reaction of step 1 is preferably carried out in a buffer, and is preferably carried out under conditions that do not promote the decomposition of the sugar chain donor molecule containing GlcNAc whose reducing end is not activated. From this viewpoint, in one embodiment of the present invention, the pH in the reaction system of step 1 can be appropriately selected between 5.8 and 9.5, preferably 6.2 to 8.5, more preferably 6.5 to 8.0, and further preferably in the range of 6.5 to 7.5. The buffer can be appropriately selected from phosphate buffer (pH 6.0 to 7.5), MOPS-NaOH buffer (pH 6.5 to 8.0), tris-hydrochloric acid buffer (pH 7.0 to 9.0), etc., preferably tris-hydrochloric acid buffer (pH 7.0 to 9.0). For the purpose of stabilizing the enzyme, other additives that do not hinder the enzyme reaction can be added to the reaction solution.

[0892] The reaction temperature can be appropriately selected within the range of 4°C to 50°C, preferably 15°C to 45°C, more preferably 20°C to 40°C, and further preferably 25°C to 40°C.

[0893] The reaction time can be appropriately selected between 10 minutes and 96 hours, preferably 0.5 hours to 80 hours, more preferably 2 hours to 70 hours, further preferably 4 hours to 60 hours, particularly preferably 6 to 48 hours or 8 to 30 hours, and most preferably 16 to 24 hours. A small amount of reaction liquid can be collected over time to confirm the progress of the sugar chain transfer reaction and determine the end of the reaction. Generally, the progress of the sugar chain transfer reaction can be monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis, microchip electrophoresis, fully automatic electrophoresis system, or liquid chromatography mass spectrometry (LC-MS). For example, after the sugar chain reconstructed antibody fragments are divided into heavy chains and light chains, the retention time can be monitored by using a fully automatic electrophoresis system to confirm that the change only occurs on the heavy chain side to which the N297-linked sugar chain is added.

[0894] Step 1 can be performed in a one-pot method by directly transferring the sugar chain of the sugar chain donor molecule to an acceptor molecule, which is an antibody having a core GlcNAc optionally with fucose added as an N297-linked sugar chain or a molecule containing the Fc region thereof.

[0895] In the sugar chain reconstruction using the sugar chain donor molecule containing the sugar chain of oxazoline at the reducing end, the glycosylation (Glycation) as a chemical reaction of non-Endo enzyme mediation type is easy to be carried out on Lys in the acceptor molecule, so the antibody concentration in the reaction solution is usually set low. On the other hand, in the case of using the sugar chain donor molecule containing the unactivated GlnNAc at the reducing end, since the chemically synthesized oxazoline solution as the cause of glycosylation is not added, there is no need to worry about glycosylation, therefore, a higher concentration of acceptor molecule concentration can be used. On the contrary, it is effective to add a high concentration of acceptor molecules from the viewpoint of increasing the frequency of contact between molecules. Therefore, the acceptor molecule concentration in step 1 can be appropriately set according to a known method. For example, the upper limit of the final acceptor molecule concentration in the reaction system of step 1 may be 200 mg / mL, preferably 160 mg / mL, more preferably 120 mg / mL, further preferably 100 mg / mL, and particularly preferably 80 mg / mL. The lower limit may be 10 mg / mL, preferably 14 mg / mL, more preferably 18 mg / mL, and further preferably 20 mg / mL. The concentration range may be any combination of the upper and lower limits mentioned above or 10 mg / mL to 200 mg / mL, preferably 14 mg / mL to 160 mg / mL, more preferably 18 mg / mL to 120 mg / mL, further preferably 20 mg / mL to 100 mg / mL, and particularly preferably 20 mg / mL to 80 mg / mL, but is not limited thereto.

[0896] The amount of the sugar chain donor molecule added in step 1 can be appropriately set by utilizing or applying a known method. For example, in one embodiment of the present invention, the lower limit of the amount of the sugar chain donor molecule added (used) in step 1 is 2 equivalents, 3 equivalents, 4 equivalents, 5 equivalents, 6 equivalents, 7 equivalents, 8 equivalents, 9 equivalents, 10 equivalents, 11 equivalents, 12 equivalents, 13 equivalents, 14 equivalents, 15 equivalents, 16 equivalents, 17 equivalents, 18 equivalents, 19 equivalents or 20 equivalents relative to 1 equivalent of the acceptor molecule, and the upper limit of the amount of the sugar chain donor molecule is 300 equivalents, 200 equivalents, 150 equivalents, 100 equivalents, 90 equivalents, 80 equivalents, 75 equivalents, 70 equivalents, 65 equivalents, 60 equivalents, 55 equivalents, 45 equivalents, 40 equivalents, 35 equivalents, 30 equivalents, 25 equivalents or 20 equivalents, but is not limited thereto. In addition, as the range of the amount of the sugar chain donor molecule added (used) in step 1, any combination of the upper limit and the lower limit mentioned above can be listed, or 5 to 80 equivalents, preferably 7 to 60 equivalents, more preferably 8 to 50 equivalents, and particularly preferably 10 to 40 equivalents relative to 1 equivalent of the acceptor molecule can be listed, but it is not limited to these.

[0897] In one embodiment of the present invention, the sugar chain transfer rate in step 1 can be set to any value, for example, it can be set to 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. On the other hand, it can be set to 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, or 60% or less. In one embodiment of the present invention, the sugar chain transfer rate in step 1 exceeds 80%, preferably exceeds 85%, and particularly preferably exceeds 90%. It should be noted that the above-mentioned "sugar chain transfer rate" can be determined according to conventional methods, for example, it can be determined using the fully automatic electrophoresis system disclosed in Example 15-2B. It should be noted that the timing of measuring the sugar chain transfer rate varies depending on the enzyme used, and is 1 minute or more and within 100 hours, preferably 1 hour or more and within 80 hours, and more preferably 2 hours or more and within 72 hours.

[0898] It should be noted that the acceptor molecule used in step 1 can be prepared in another preparation method independent of step 1. Therefore, in one embodiment of the present invention, the method of the present invention further includes a step of prep...

Claims

1. An antibody-drug conjugate, represented by the following formula (II): In formula (II), m 1 is in the range of 1 to 10, Ab represents an antibody or a functional fragment of the antibody, and the antibody or the functional fragment of the antibody optionally has a reconstructed sugar chain, and the amino acid residues at positions 234, 235, and 265 of the Fc region of the antibody or the functional fragment of the antibody are Ala, Ala, and Gly, respectively, and all numberings are EU numberings. Herein, antibody represents an anti-EGFR antibody or an anti-CDH6 antibody, L represents a linker connecting Ab and D, Ab is optionally bound to L directly from its amino acid residues, or is bound to L from the sugar chain or reconstructed sugar chain of Ab, D represents a compound represented by the following formula (I), In formula (I), L vs L 1 The hydroxyl groups contained in L 1 represents a group of the following formula, in which the wavy line represents the substitution position, Q and Q' each independently represent a hydroxyl group or a thiol group, R 21 and R 22 each independently represents a hydroxyl group or a fluorine atom, W represents -NH- or a sulfur atom.

2. The antibody-drug conjugate according to claim 1, wherein: D is represented by either of the following two formulas, Here, L 1 , Q, Q' and W are as defined above.

3. The antibody-drug conjugate according to claim 1 or 2, wherein: D is represented by either of the following two formulas, Here, the asterisk indicates a combination with L, and Q, Q' and W are as defined above.

4. The antibody-drug conjugate according to any one of claims 1 to 3, wherein D is represented by any of the following three formulas, Here, the asterisk indicates binding to L, and W is as defined above.

5. The antibody-drug conjugate according to any one of claims 1 to 4, wherein D is represented by any of the following three formulas, Here, the asterisk indicates binding to L.

6. The antibody-drug conjugate according to any one of claims 1 to 4, wherein D is represented by any of the following four formulas, Here, the asterisk indicates binding to L.

7. The antibody-drug conjugate according to any one of claims 1 to 4 or 6, wherein D is represented by the following formula, Here, the asterisk indicates binding to L.

8. The antibody-drug conjugate according to any one of claims 1 to 7, wherein: The linker L is represented by -Lb-La-Lp-Lc-*. In the formula, the asterisk indicates binding with drug D. Lp represents a linker consisting of an amino acid sequence that can be cut in the target cell, or Lp does not exist, La represents any one selected from the following group: -C(=O)-(CH2CH2)n 2 -C(=O)-、 -C(=O)-(CH2CH2)n 2 -CH2-C(=O)-、 -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2)n 3 -C(=O)-、 -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2)n 3 -CH2-C(=O)-、 -C(=O)-(CH2CH2)n 2 -C(=O)-NH-(CH2CH2O)n 3 -CH2-C(=O)-、 -(CH2)n 4 -O-C(=O)-, and -(CH2)n 9 -C(=O)-, Here, n 2 Integer from 1 to 3, n 3 Integer from 1 to 5, n 4 Integer from 0 to 2, n 9 represents an integer from 2 to 7, Lb represents a spacer that binds La to the sugar chain or remodeled sugar chain of Ab or a spacer that binds La to the cysteine ​​residue of Ab, and, Lc represents -NH-CH2-, -NH-phenyl-CH2-O(C=O)-, or -NH-heteroaryl-CH2-O(C=O)-, or Lc is absent.

9. The antibody-drug conjugate according to claim 8, wherein: Lc is -NH-CH2-.

10. The antibody-drug conjugate according to claim 8 or 9, wherein: Lp is any one of -GGFG-, -GGPI-, -GGVA-, -GGFM-, -GGVCit-, -GGFCit-, -GGICit-, -GGPL-, -GGAQ-, or -GGPP-.

11. The antibody-drug conjugate according to claim 10, wherein: Lp is -GGFG- or -GGPI-.

12. The antibody-drug conjugate according to any one of claims 8 to 11, wherein La represents any one selected from the group consisting of -C(=O)-CH2CH2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)3-CH2-C(=O)-, -C(=O)-CH2CH2-C(=O)-NH-(CH2CH2O)4-CH2-C(=O)-, and -(CH2)5-C(=O)-.

13. The antibody-drug conjugate according to any one of claims 8 to 12, wherein: Lb is represented by any of the following formulas, In the structural formula of Lb shown above, the asterisk indicates binding to La, and the wavy line indicates binding to the sugar chain or reconstructed sugar chain of Ab.

14. The antibody-drug conjugate according to any one of claims 8 to 12, wherein: Lb is -(succinimidyl-3-yl-N)-, Here, -(succinimidyl-3-yl-N)- represents the following structural formula, Here, the asterisk indicates binding to La, and the wavy line indicates binding to the side chain of the cysteine ​​residue of the antibody by forming a thioether.

15. The antibody-drug conjugate according to any one of claims 8 to 13, wherein The linker L is represented by -Lb-La-Lp-Lc-*. In the formula, the asterisk indicates binding with drug D. Lp is -GGFG-, or -GGPI-, La represents -C(=O)-CH2CH2-C(=O)-, Lb represents the following formula, In the structural formula of Lb shown above, the asterisk indicates binding to La, and the wavy line indicates binding to the sugar chain or reconstructed sugar chain of Ab. Lc represents -NH-CH2-.

16. The antibody-drug conjugate according to any one of claims 1 to 15, wherein: The average number of drug binding per antibody molecule in the antibody drug conjugate ranges from 1 to 10.

17. The antibody-drug conjugate according to claim 16, wherein: The average number of drug binding per antibody molecule in the antibody drug conjugate ranges from 1 to 5.

18. The antibody-drug conjugate according to claim 17, wherein: The average number of drug binding per antibody molecule in the antibody drug conjugate is in the range of 1 to 3, or 3 to 5.

19. The antibody-drug conjugate according to any one of claims 1 to 18, wherein: The antibody binds to L via the sugar chain linked to Asn297 of the antibody, namely, the N297 sugar chain.

20. The antibody-drug conjugate according to claim 19, wherein: The N297 sugar chain is a remodeled sugar chain.

21. The antibody drug conjugate according to claim 19 or 20, wherein: The N297 sugar chain is N297-(Fuc)MSG1 or N297-(Fuc)SG having the structure shown in the following formula: In the formula, the wavy line indicates the connection with Asn297 of the antibody, L(PEG) stands for -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at position 2 of the sialic acid at the non-reducing end of the 1-3 chain side of the β-Man branch of the N297 sugar chain, The asterisk indicates that the linker L is bound to the nitrogen atom at position 1 or position 3 on the 1,2,3-triazole ring of Lb, and n 5 is an integer from 2 to 5; In the formula, the wavy line indicates the connection with Asn297 of the antibody, L(PEG) stands for -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at position 2 of the sialic acid at both the 1-3 chain side and the 1-6 chain side of the non-reducing end of the β-Man branch of the N297 sugar chain, The asterisk indicates that the linker L is bound to the nitrogen atom at position 1 or position 3 on the 1,2,3-triazole ring of Lb, and n 5 An integer from 2 to 5.

22. The antibody drug conjugate according to any one of claims 19 to 21, which is represented by the following formula: In the formula, m 2 represents an integer of 1 or 2, L is a linker connecting the N297 sugar chain to D, and according to the above definition, Ab represents an antibody or a functional fragment of the antibody, and the amino acid residues at positions 234, 235, and 265 of the Fc region of the antibody or the functional fragment of the antibody are Ala, Ala, and Gly, respectively, and all numberings are EU numberings. Here, antibody represents an anti-EGFR antibody or an anti-CDH6 antibody, The N297 sugar chain represents N297-(Fuc)MSG1 or N297-(Fuc)SG having the structure shown in the following formula: In the formula, the wavy line indicates the connection with Asn297 of the antibody, L(PEG) stands for -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at position 2 of the sialic acid at the non-reducing end of the 1-3 chain side of the β-Man branch of the N297 sugar chain, The asterisk indicates that the linker L is bound to the nitrogen atom at position 1 or position 3 on the 1,2,3-triazole ring of Lb, and n 5 Indicates an integer from 2 to 5; In the formula, the wavy line indicates the connection with Asn297 of the antibody, L(PEG) stands for -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at position 2 of the sialic acid at both the 1-3 chain side and the 1-6 chain side of the non-reducing end of the β-Man branch of the N297 sugar chain, The asterisk indicates that the linker L is bound to the nitrogen atom at position 1 or position 3 on the 1,2,3-triazole ring of Lb, and n 5 represents an integer from 2 to 5, D is represented by any of the following four formulas, Here, in the formula, the asterisk indicates binding with L.

23. The antibody drug conjugate according to claim 22, which is selected from the following formula, In the above structural formulas, m 2 represents an integer of 1 or 2, Ab represents an antibody or a functional fragment of the antibody, and the amino acid residues at positions 234, 235, and 265 of the Fc region of the antibody or the functional fragment of the antibody are Ala, Ala, and Gly, respectively, and all numberings are EU numberings. Here, antibody represents an anti-EGFR antibody or an anti-CDH6 antibody, The N297 sugar chain represents either N297-(Fuc)MSG1 or N297-(Fuc)SG having a structure represented by the following formula: In the formula, the wavy line indicates the connection with Asn297 of the antibody, L(PEG) stands for -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at position 2 of the sialic acid at the non-reducing end of the 1-3 chain side of the β-Man branch of the N297 sugar chain, The asterisk indicates that the linker L is bound to the nitrogen atom at position 1 or position 3 on the 1,2,3-triazole ring of Lb, and n 5 Indicates an integer from 2 to 5; In the formula, the wavy line indicates the connection with Asn297 of the antibody, L(PEG) stands for -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at position 2 of the sialic acid at both the 1-3 chain side and the 1-6 chain side of the non-reducing end of the β-Man branch of the N297 sugar chain, The asterisk indicates that the linker L is bound to the nitrogen atom at position 1 or position 3 on the 1,2,3-triazole ring of Lb, and n 5 Indicates an integer from 2 to 5.

24. The antibody drug conjugate according to claim 23, which is selected from the following formula, In the above structural formulas, m 2 represents an integer of 1 or 2, Ab represents an antibody or a functional fragment of the antibody, and the amino acid residues at positions 234, 235, and 265 of the Fc region of the antibody or the functional fragment of the antibody are Ala, Ala, and Gly, respectively, and all numberings are EU numberings. Here, antibody represents an anti-EGFR antibody or an anti-CDH6 antibody, The N297 sugar chain represents either N297-(Fuc)MSG1 or N297-(Fuc)SG having a structure represented by the following formula: In the formula, the wavy line indicates the connection with Asn297 of the antibody, L(PEG) stands for -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at position 2 of the sialic acid at the non-reducing end of the 1-3 chain side of the β-Man branch of the N297 sugar chain, The asterisk indicates that the linker L is bound to the nitrogen atom at position 1 or position 3 on the 1,2,3-triazole ring of Lb, and n 5 Indicates an integer from 2 to 5; In the formula, the wavy line indicates the connection with Asn297 of the antibody, L(PEG) stands for -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at position 2 of the sialic acid at both the 1-3 chain side and the 1-6 chain side of the non-reducing end of the β-Man branch of the N297 sugar chain, The asterisk indicates that the linker L is bound to the nitrogen atom at position 1 or position 3 on the 1,2,3-triazole ring of Lb, and n 5 Indicates an integer from 2 to 5.

25. The antibody drug conjugate according to any one of claims 1 to 24, wherein The antibody is an anti-EGFR antibody.

26. The antibody-drug conjugate according to any one of claims 1 to 24, wherein: The antibody is an anti-CDH6 antibody.

27. The antibody drug conjugate according to claim 25, wherein: The antibody is an antibody comprising a light chain consisting of the amino acid sequence recorded in sequence number 30 and a heavy chain consisting of the amino acid sequence recorded in sequence number 32, or an antibody comprising a light chain consisting of the amino acid sequence recorded in sequence number 30 and a heavy chain consisting of the amino acid sequence recorded in sequence number 33, or an antibody comprising a heavy chain in which one or more amino acid residues at the carboxyl terminus are deleted in any of the antibodies.

28. The antibody drug conjugate according to claim 26, wherein The antibody is an antibody comprising a light chain consisting of the amino acid sequence recorded in SEQ ID NO: 44 and a heavy chain consisting of the amino acid sequence recorded in SEQ ID NO: 46, or an antibody comprising a heavy chain in which one or several amino acid residues at the carboxyl terminus are deleted.

29. The antibody drug conjugate according to claim 25, wherein: The antibody is an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence recorded in SEQ ID NO: 29 and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence recorded in SEQ ID NO: 31, and the amino acid residues at positions 234, 235, and 265 in the Fc region of the antibody are Ala, Ala, and Gly, respectively, and all numbering is EU numbering.

30. The antibody drug conjugate according to claim 26, wherein: The antibody is an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence recorded in SEQ ID NO: 43, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence recorded in SEQ ID NO: 45, and the amino acid residues at positions 234, 235, and 265 in the Fc region of the antibody are Ala, Ala, and Gly, respectively, and all numbering is EU numbering.

31. The antibody drug conjugate according to claim 25, wherein: The antibody comprises a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 23, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 24, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 25, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 26, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 27, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 28, wherein the amino acid residues at positions 234, 235, and 265 of the Fc region of the antibody are Ala, Ala, and Gly, respectively, and all numbering is EU numbering.

32. The antibody drug conjugate according to claim 26, wherein: The antibody comprises a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 37, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 38, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 39, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 40, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 41, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 42, wherein the amino acid residues at positions 234, 235, and 265 of the Fc region of the antibody are Ala, Ala, and Gly, respectively, and all numbering is EU numbering.

33. An antibody drug conjugate represented by the following formula: In the formula, Ab represents any one selected from the following groups: An antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 30 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 32; An antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 30 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 33; An antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 44 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 46; and An antibody comprising a heavy chain in which one or more amino acid residues at the carboxyl terminus are deleted in any of the antibodies, The N297 sugar chain is represented by the following formula, In the formula, the wavy line indicates the connection with Asn297 of the antibody, L(PEG) stands for -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at position 2 of the sialic acid at both the 1-3 chain side and the 1-6 chain side of the non-reducing end of the β-Man branch of the N297 sugar chain, The asterisk indicates that the linker L is bound to the nitrogen atom at position 1 or position 3 on the 1,2,3-triazole ring of Lb. n 5 is 3, and m 2 is 2.

34. An antibody drug conjugate represented by the following formula: In the formula, Ab represents any one selected from the following groups: An antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 29 and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 31; and An antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 43 and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 45, The amino acid residues at positions 234, 235, and 265 of the Fc region of the antibody are Ala, Ala, and Gly, respectively, and all numberings are EU numberings. The N297 sugar chain is represented by the following formula, In the formula, the wavy line indicates the connection with Asn297 of the antibody, L(PEG) stands for -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at position 2 of the sialic acid at both the 1-3 chain side and the 1-6 chain side of the non-reducing end of the β-Man branch of the N297 sugar chain, The asterisk indicates that the linker L is bound to the nitrogen atom at position 1 or position 3 on the 1,2,3-triazole ring of Lb. n 5 is 3, and m 2 is 2.

35. An antibody drug conjugate represented by the following formula: In the formula, Ab represents any one selected from the following groups, An antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 23, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 24, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 25, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 26, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 27, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 28; and An antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 37, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 38, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 39, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 40, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 41, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 42, The amino acid residues at positions 234, 235, and 265 of the Fc region of the antibody are Ala, Ala, and Gly, respectively, and all numberings are EU numberings. The N297 sugar chain is represented by the following formula, In the formula, the wavy line indicates the connection with Asn297 of the antibody, L(PEG) stands for -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at position 2 of the sialic acid at both the 1-3 chain side and the 1-6 chain side of the non-reducing end of the β-Man branch of the N297 sugar chain, The asterisk indicates that the linker L is bound to the nitrogen atom at position 1 or position 3 on the 1,2,3-triazole ring of Lb. n 5 is 3, and m 2 is 2.

36. An antibody drug conjugate represented by the following formula: In the formula, Ab represents any one selected from the following groups: An antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 30 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 32; An antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 30 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 33; An antibody comprising a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 44 and a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 46; and An antibody comprising a heavy chain in which one or more amino acid residues at the carboxyl terminus are deleted in any of the antibodies, The N297 sugar chain is represented by the following formula, In the formula, the wavy line indicates the connection with Asn297 of the antibody, L(PEG) stands for -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at position 2 of the sialic acid at both the 1-3 chain side and the 1-6 chain side of the non-reducing end of the β-Man branch of the N297 sugar chain, The asterisk indicates that the linker L is bound to the nitrogen atom at position 1 or position 3 on the 1,2,3-triazole ring of Lb. n 5 is 3, and m 2 is 1.

37. An antibody drug conjugate represented by the following formula: In the formula, Ab represents any one selected from the following groups: An antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 29 and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 31; and An antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 43 and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 45; The amino acid residues at positions 234, 235, and 265 of the Fc region of the antibody are Ala, Ala, and Gly, respectively, and all numberings are EU numberings. The N297 sugar chain is represented by the following formula, In the formula, the wavy line indicates the connection with Asn297 of the antibody, L(PEG) stands for -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at position 2 of the sialic acid at both the 1-3 chain side and the 1-6 chain side of the non-reducing end of the β-Man branch of the N297 sugar chain, The asterisk indicates that the linker L is bound to the nitrogen atom at position 1 or position 3 on the 1,2,3-triazole ring of Lb. n 5 is 3, and m 2 is 1.

38. An antibody drug conjugate represented by the following formula: In the formula, Ab represents any one selected from the following groups: An antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 23, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 24, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 25, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 26, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 27, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 28; and An antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 37, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 38, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 39, and a heavy chain comprising CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 40, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 41, and CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 42; The amino acid residues at positions 234, 235, and 265 of the Fc region of the antibody are Ala, Ala, and Gly, respectively, and all numberings are EU numberings. The N297 sugar chain is represented by the following formula, In the formula, the wavy line indicates the connection with Asn297 of the antibody, L(PEG) stands for -(CH2-CH2-O)n 5 -CH2-CH2-NH-, indicating that the amino group at the right end of the L(PEG) forms an amide bond with the carboxyl group at position 2 of the sialic acid at both the 1-3 chain side and the 1-6 chain side of the non-reducing end of the β-Man branch of the N297 sugar chain, The asterisk indicates that the linker L is bound to the nitrogen atom at position 1 or position 3 on the 1,2,3-triazole ring of Lb. n 5 is 3, and m 2 is 1.

39. A STING agonist comprising the antibody-drug conjugate according to any one of claims 1 to 38.

40. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1 to 38.

41. An antitumor agent comprising the antibody-drug conjugate according to any one of claims 1 to 38.

42. The antitumor agent according to claim 41, wherein The tumor is lung cancer, kidney cancer, urothelial carcinoma, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, hepatocellular carcinoma, bladder cancer, gastric cancer, esophageal cancer, uterine cancer, testicular cancer, cervical cancer, placental choriocarcinoma, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, gastrointestinal stromal tumor (GIST), gallbladder cancer, bile duct cancer, adrenal cancer, squamous cell carcinoma, pharyngeal cancer, tongue cancer, auditory organ cancer, thymus cancer, small intestine cancer, leukemia, malignant lymphoma, plasma cell tumor, myeloma, sarcoma, EGFR mutation-positive cancer or cancer with mutation in EGFR downstream signal.

43. A method for treating cancer, comprising administering any one selected from the group consisting of the antibody drug conjugate according to any one of claims 1 to 38, the STING agonist according to claim 39, the pharmaceutical composition according to claim 40, and the anti-tumor agent according to claim 41 or 42.

44. The method of claim 43, wherein: The cancer is lung cancer, kidney cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, hepatocellular carcinoma, bladder cancer, gastric cancer, esophageal cancer, uterine cancer, testicular cancer, cervical cancer, placental choriocarcinoma, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, gastrointestinal stromal tumor (GIST), gallbladder cancer, bile duct cancer, adrenal cancer, squamous cell carcinoma, pharyngeal cancer, tongue cancer, auditory organ cancer, thymus cancer, small intestine cancer, leukemia, malignant lymphoma, plasma cell tumor, myeloma, sarcoma, EGFR mutation-positive cancer or cancer with mutation in EGFR downstream signal.

45. An antibody, which is an A antibody or a B antibody, The A antibody is an antibody comprising a light chain comprising CDRL1 consisting of the amino acid sequence recorded in SEQ ID NO: 23, CDRL2 consisting of the amino acid sequence recorded in SEQ ID NO: 24, and CDRL3 consisting of the amino acid sequence recorded in SEQ ID NO: 25, and a heavy chain comprising CDRH1 consisting of the amino acid sequence recorded in SEQ ID NO: 26, CDRH2 consisting of the amino acid sequence recorded in SEQ ID NO: 27, and CDRH3 consisting of the amino acid sequence recorded in SEQ ID NO: 28, wherein the amino acid residues at positions 234, 235, and 265 of the Fc region of the antibody are Ala, Ala, and Gly, respectively, and all numbering is EU numbering; The B antibody is an antibody comprising a light chain including CDRL1 consisting of the amino acid sequence recorded in sequence number 37, CDRL2 consisting of the amino acid sequence recorded in sequence number 38, and CDRL3 consisting of the amino acid sequence recorded in sequence number 39, and a heavy chain including CDRH1 consisting of the amino acid sequence recorded in sequence number 40, CDRH2 consisting of the amino acid sequence recorded in sequence number 41, and CDRH3 consisting of the amino acid sequence recorded in sequence number 42, and the amino acid residues at positions 234, 235, and 265 of the Fc region of the antibody are Ala, Ala, and Gly, respectively, and all numbering is EU numbering.

46. ​​An antibody, which is an A antibody or a B antibody, The A antibody is an antibody comprising a light chain comprising a light chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 29 and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 31, wherein the amino acid residues at positions 234, 235, and 265 of the Fc region of the antibody are Ala, Ala, and Gly, respectively, and all numbering is based on EU numbering; The B antibody is an antibody comprising a light chain including a light chain variable region consisting of the amino acid sequence recorded in sequence number 43 and a heavy chain including a heavy chain variable region consisting of the amino acid sequence recorded in sequence number 45, and the amino acid residues at positions 234, 235, and 265 in the Fc region of the antibody are Ala, Ala, and Gly, respectively, and all numbers are EU numbers.

47. An antibody, which is an A antibody, a B antibody or a C antibody, The A antibody is an antibody comprising a light chain consisting of the amino acid sequence recorded in SEQ ID NO: 30 and a heavy chain consisting of the amino acid sequence recorded in SEQ ID NO: 32, or an antibody comprising a light chain consisting of the amino acid sequence recorded in SEQ ID NO: 30 and a heavy chain consisting of the amino acid sequence recorded in SEQ ID NO: 33; The B antibody is an antibody comprising a light chain consisting of the amino acid sequence recorded in SEQ ID NO: 44 and a heavy chain consisting of the amino acid sequence recorded in SEQ ID NO: 46; The C antibody is an antibody comprising a heavy chain in which one or several amino acid residues at the carboxyl terminal are deleted in any one of the A antibody to the B antibody.

48. The pharmaceutical composition according to claim 40 or the antitumor agent according to claim 41 or 42, which is administered in combination with other drugs.

49. The pharmaceutical composition according to claim 40 or the antitumor agent according to claim 41 or 42, comprising other drugs.

50. The method of claim 43 or 44, wherein: Any one selected from the group consisting of the antibody drug conjugate according to any one of claims 1 to 38, the STING agonist according to claim 39, the pharmaceutical composition according to claim 40, and the anti-tumor agent according to claim 41 or 42 is administered in combination with other drugs.

Images