Antibody-drug conjugate including mutant fc region

Abstract

[Problem] It is desired to develop a molecule that maintains antitumor activity and offers excellent safety. It is also desired to develop an antibody-drug conjugate that can be administered systemically and delivers a STING agonist specifically to target cells or an organ (for example, a tumor site), and a therapeutic agent and / or therapeutic method that uses the antibody-drug conjugate and is for diseases related to STING agonist activity, for example, diseases (for example, cancers) to which immunostimulation therapy can be applied. [Solution] Provided are: an antibody-drug conjugate in which a CDN derivative characterized by having a fused tricyclic substituent is joined via a linker with a specific antibody including a mutant Fc region, or with a functional fragment of the antibody; and an antibody including a mutant Fc region.

Description

Antibody drug conjugates containing mutant Fc regions

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

[0002] The Fc region provides antibodies with a long blood half-life and effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). When the Fc region is derived from a subclass called IgG, which is used in the majority of antibody drugs, the effector function depends on the binding of the Fc region to a family of receptors called Fcγ receptors (FcγR). However, FcγR binding activity has been suggested to be involved in safety risks such as infusion reactions (Non-Patent Document 1), and is therefore often considered an undesirable property in antibody drugs.

[0003] To date, various amino acid substitutions in the Fc region of IgG antibodies that attenuate binding activity to human FcγR have been reported (for example, 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 an antibody is conjugated with a drug (immunopotentiator) that has the activity of activating immune cells, the efficacy of the antibody decreases when the effector function of the antibody is reduced (Non-Patent Documents 4 to 6, Patent Document 36).

[0005] STING (Stimulator of Interferon Genes) is a transmembrane adaptor protein localized in the endoplasmic reticulum (Non-Patent Document 7). STING functions as a central molecule in innate immune activation in mammals and serves as the first line of defense against the invasion of pathogens such as bacteria and viruses. It is known that STING activation is triggered by signals generated when multiple cytoplasmic DNA sensors sense exogenous and endogenous DNA. Among these cytoplasmic DNA sensors, cGAS (cyclic GMP-AMP synthase) is considered to be an important DNA sensor. When cGAS senses DNA, a cyclic dinucleotide (2',3'-cGAMP) is produced, which then directly binds to STING and activates it (Non-Patent Document 8). Activated STING translocates to the Golgi apparatus, where it promotes the autophosphorylation of TBK1 (Tank-binding kinase 1). Autophosphorylated and activated TBK1 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 inflammatory proteins called interferons and cytokines (type I IFN (Interferon), IL-6 (Interleukin-6), and TNF-α (Tumor Necrosis Factor-α)). These proteins trigger the adaptive immune system, including T cells, which destroy pathogens and cancer cells through a complex cascade.

[0006] Recent studies have demonstrated that STING not only promotes host defense against microorganisms but also promotes antitumor immunity. For example, when immunogenic tumors are transplanted into STING-deficient mice, the tumors grow more rapidly than in wild-type mice or TRIF (Toll / Interleukin-1 (IL-1) receptor domain containing adaptor-inducing interferon-β)-deficient mice. Furthermore, STING-deficient mice, unlike TLR (Toll-like receptor), MyD88 (Myeloid differentiation primary response 88), and MAVS (Mitochondrial antiviral-signaling protein)-deficient mice, do not spontaneously express CD8 against tumors. ＋ T cell priming was also abolished. This suggests that the STING pathway, triggered by cytoplasmic DNA sensing, is involved in the control of tumor growth (Non-Patent Document 11). Other studies have also shown that STING is required for the antitumor effects of radiation therapy (Non-Patent Document 12) or anti-CD47 antibody therapy (Non-Patent Document 13). After treatment with radiation or anti-CD47 antibody, DNA from dead tumor cells migrates into the cytoplasm of dendritic cells, activates the cGAS-STING pathway, and then induces IFN production, bridging the gap between innate and adaptive immunity. This study suggests that cross-priming mediated by dendritic cells activated by the STING pathway is important for triggering adaptive immunity against tumors.

[0007] The flavonoid-based small molecule compound DMXAA, known as a vascular disrupting agent, has been shown to have potent antitumor activity in mouse tumor models due to its ability to induce type I IFN in macrophages (Non-Patent Document 14). Due to its excellent preclinical antitumor activity, DMXAA was expected to be a promising immunotherapeutic agent for non-small cell lung cancer, but human clinical trials have failed (Non-Patent Document 15). Recent studies have revealed that DMXAA is a specific agonist for mouse STING and cannot bind to human STING due to its lack of species cross-reactivity (Non-Patent Document 16). Although DMXAA was ultimately ineffective in humans, studies in mouse models have demonstrated that small molecule drugs can effectively inhibit CD8 through STING. ＋ It was suggested that T cells could be primed to enhance anti-tumor immunity.

[0008] Another small molecule, cyclic dinucleotides (CDNs), have been shown to enhance STING-mediated antitumor immune responses, significantly inhibit tumor growth, and improve survival in tumor-bearing mice (NPL 17). CDNs are classified into bacterial CDNs with canonical two 3'-5' phosphate linkages (cyclic-di-GMP, cyclic-di-AMP, 3',3'-cGAMP) and mixed-linkage CDNs with non-canonical 2'-5' phosphate linkages (2',3'-cGAMP) produced by mammalian cGAS. Recent studies have shown that mixed-linkage CDNs are more capable of universally activating diverse STINGs than canonical CDNs (NPL 18).

[0009] Natural CDNs, like many nucleic acid molecules, are rapidly degraded by nucleases in the blood and cannot be administered as is. Therefore, synthetic small molecule compounds with in vivo STING agonist activity have been developed (e.g., Patent Documents 8 to 33).

[0010] MIW-815 (ADU-S100, ML RR-S2 CDA or ML-RR-CDA·2Na), a STING agonist, is currently undergoing clinical trials as an antitumor agent. ＋(sometimes referred to as "STING agonist") is administered directly into the tumor. Direct administration of a STING agonist into a tumor limits the drug to a limited area within the tumor, and it is difficult to administer it directly to all distant metastatic tumors, limiting the number of tumors that can be treated. Non-Patent Document 19 describes that administration of ML RR-S2 CDA demonstrated antitumor effects, but only intratumoral administration; systemic administration (e.g., intravenous administration) has not demonstrated antitumor effects. Non-Patent Document 20 describes that intravenous administration of the STING agonist SB11285 to a mouse tumor model demonstrated antitumor effects, but the specific structure of SB11285 is not disclosed. Patent Document 21 describes a conjugate containing an immunostimulatory compound, an antibody construct, and a linker, but does not describe a specific example of a conjugate using a STING agonist as the immunostimulatory compound. Patent Document 33 describes a conjugate in which a CDN having a specific structure is linked to an antibody via a linker, but does not describe any examples of in vivo administration of the conjugate, and the antitumor effect of the conjugate has not been confirmed. Patent Documents 34, 35, 37, and 38 describe conjugates in which a STING agonist is used as an immunostimulatory compound and is linked to an antibody via a linker, and describe examples of in vivo administration.

[0011] On the other hand, as a method for uniformly adding sugar chains to glycoprotein molecules containing therapeutic antibodies or antibody Fc regions, enzymatic glycosylation reactions are known, and in particular, a one-pot method is known in which sugar chains are directly transferred to GlcNAc acceptors using two types of ENGase.

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[0014] There is a need for the development of molecules that are excellent in terms of safety while maintaining antitumor activity. There is also a need for the development of antibody-drug conjugates that can be administered systemically and that can deliver STING agonists specifically to target cells or organs (e.g., tumor sites), as well as therapeutic agents and / or methods using the antibody-drug conjugates for diseases associated with STING agonist activity, such as diseases treatable by immunostimulation (e.g., cancer). There is also a need for the establishment of novel methods for producing Fc-containing molecules (particularly antibodies) with uniform glycan structures, particularly methods with improved glycosylation rates.

[0015] As a result of intensive research to solve the above-mentioned problems, the present inventors discovered an antibody-drug conjugate in which a CDN derivative characterized by having a fused tricyclic substituent is linked via a linker to a specific antibody or functional fragment of the antibody containing a mutant Fc region, and found that systemic administration of the antibody-drug conjugate exhibits an antitumor effect on tumors expressing the antigen and is also excellent in terms of safety, thereby completing the present invention.Furthermore, the present inventors found that the specific antibody containing the mutant Fc region is excellent in terms of safety, and also discovered an improved method for producing Fc-containing molecules (particularly antibodies) using a one-pot method, thereby completing the present invention.

[0016] That is, the present invention relates to, but is not limited to, the following: [1] A compound represented by the following formula (II): (In the formula, m １ is in the range of 1 to 10, Ab represents an antibody or a functional fragment of the antibody, which may have a remodeled sugar chain, and the amino acid residues at positions 234, 235, and 265 (all EU numbering) of the Fc region of the antibody or functional fragment of the antibody are Ala, Ala, and Gly, respectively, wherein the antibody represents an anti-EGFR antibody or an anti-CDH6 antibody, L represents a linker connecting Ab and D, Ab may be bound to L directly from its amino acid residue, or may be bound to L from a sugar chain or a remodeled sugar chain of Ab, and D represents a linker represented by the following formula (I): (where L is L １ and binds to the hydroxy group contained in L １ is the following formula: (wherein the wavy line indicates the substitution position) groups, Q and Q ’ each independently represents a hydroxy group or a thiol group, R ２１ and R ２２ each independently represent a hydroxy group or a fluorine atom, and W represents -NH- or a sulfur atom. [2] An antibody-drug conjugate represented by the following formula: (Here, L １, Q, Q', and W are as defined above. [3] The antibody-drug conjugate according to [1], wherein D is represented by any one of the following two formulae: (wherein the asterisk indicates binding to L, and Q, Q', and W are as defined above). [4] The antibody-drug conjugate according to [1] or [2], wherein D is represented by any one of the following three formulae: (wherein the asterisk indicates binding to L, and W is as defined above). [5] 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 the asterisk indicates binding to L). [6] The antibody-drug conjugate according to any one of [1] to [4], wherein D is represented by any one of the following four formulae: (wherein the asterisk indicates binding to L). [7] The antibody-drug conjugate according to any one of [1] to [4], wherein D is represented by the following formula: (wherein the asterisk indicates binding to L). [8] The antibody-drug conjugate according to any one of [1] to [4] or [6], wherein the linker L is represented by -Lb-La-Lp-Lc-*, wherein the asterisk indicates binding to the drug D, Lp is a linker consisting of an amino acid sequence cleavable in target cells or is absent, and La is any one selected from the group consisting of: -C(=O)-(CH ２ CH ２ ) n ２ -C(=O)-, -C(=O)-(CH ２ CH ２ ) n ２ -CH ２ -C(=O)-, -C(=O)-(CH ２ CH ２ ) n ２ -C(=O)-NH-(CH ２ CH ２ ) n ３ -C(=O)-, -C(=O)-(CH ２ CH ２ ) n２ -C(=O)-NH-(CH ２ CH ２ ) n ３ -CH ２ -C(=O)-, -C(=O)-(CH ２ CH ２ ) n ２ -C(=O)-NH-(CH ２ CH ２ O)n ３ -CH ２ -C(=O)-, -(CH ２ ) n ４ -O-C(=O)-, and -(CH ２ ) n ９ -C(=O)- (where n ２ represents an integer of 1 to 3, and n ３ represents an integer of 1 to 5, and n ４ represents an integer of 0 to 2, and n ９ represents an integer of 2 to 7), Lb represents a spacer that connects the sugar chains or remodeled sugar chains of La and Ab, or a spacer that connects the cysteine ​​residues of La and Ab, and Lc represents -NH-CH ２ -, -NH-phenyl group -CH ２ -O(C=O)- or -NH-heteroaryl group -CH ２ [9] The antibody-drug conjugate according to any one of [1] to [7], wherein Lc represents -O(C=O)- or is absent. ２

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

[11] The antibody-drug conjugate according to

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

[12] La is any one of the following: -C(=O)-CH ２ CH ２ -C(=O)-, -C(=O)-CH ２ CH ２ -C(=O)-NH-(CH ２ CH ２ O)３ -CH ２ -C(=O)-, -C(=O)-CH ２ CH ２ -C(=O)-NH-(CH ２ CH ２ O) ４ -CH ２ -C(=O)-, and -(CH ２ ) ５

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

[11] , wherein Lb represents any one selected from the group consisting of -C(=O)-. or (In the structural formula of Lb shown above, the asterisk indicates binding to La, and the wavy line indicates binding to the sugar chain of Ab or a remodeled sugar chain.)

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

[12] , wherein Lb is -(succinimide-3-yl-N)-, wherein -(succinimide-3-yl-N)- is represented by the following structural formula:

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

[12] , wherein the linker L is represented by -Lb-La-Lp-Lc-*, wherein the asterisk indicates binding to the drug D, Lp is -GGGFG- or -GGPI-, and La is -C(=O)-CH ２ CH ２ Lb represents —C(═O)—; (In the structural formula of Lb shown above, the asterisk indicates binding to La, and the wavy line indicates binding to the sugar chain of Ab or the remodeled sugar chain), and Lc is -NH-CH ２

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

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

[17] The antibody-drug conjugate according to

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

[18] The antibody-drug conjugate according to

[17] , wherein the average number of drugs bound 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 [1] to

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

[20] The antibody-drug conjugate according to

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

[21] 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: In the formula, the wavy line indicates binding to Asn297 of the antibody, and L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ -NH-, which indicates that the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2nd position of the sialic acid at the non-reducing end of the 1-3 chain side of the branched chain of β-Man in the N297 sugar chain; an asterisk indicates that the linker L is bonded to the nitrogen atom at the 1st or 3rd position 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 binding to Asn297 of the antibody, and L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH ２ -CH ２-NH-, which indicates that the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2-position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain; an asterisk indicates that the linker L is bonded to the nitrogen atom at the 1-position or the 3-position on the 1,2,3-triazole ring of Lb; and n ５ is an integer from 2 to 5.

[22] The following formula: (In the formula, m ２ represents an integer of 1 or 2, L is a linker connecting the N297 sugar chain and D, and is as defined above, Ab represents an antibody or a functional fragment of the antibody, in which the amino acid residues at positions 234, 235, and 265 (all EU numbering) of the Fc region of the antibody or functional fragment of the antibody are Ala, Ala, and Gly, respectively, wherein the antibody represents an anti-EGFR antibody or an anti-CDH6 antibody, and the N297 sugar chain is N297-(Fuc)MSG1 or N297-(Fuc)SG having the structure represented by the following formula, In the formula, the wavy line indicates binding to Asn297 of the antibody, and L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ -NH-, which indicates that the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2nd position of the sialic acid at the non-reducing end of the 1-3 chain side of the branched chain of β-Man in the N297 sugar chain; an asterisk indicates that the linker L is bonded to the nitrogen atom at the 1st or 3rd position on the 1,2,3-triazole ring of Lb; and n ５ represents an integer of 2 to 5; In the formula, the wavy line indicates binding to Asn297 of the antibody, and L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH ２ -CH ２-NH-, which indicates that the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2-position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain; an asterisk indicates that the linker L is bonded to the nitrogen atom at the 1-position or the 3-position on the 1,2,3-triazole ring of Lb; and n ５ represents an integer of 2 to 5, and D is represented by one of the following four formulas:

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

[19] to

[21] , represented by the following formula: In each of the structural formulas shown above, m ２ represents an integer of 1 or 2; Ab represents an antibody or a functional fragment of the antibody, in which the amino acid residues at positions 234, 235, and 265 (all EU numbering) of the Fc region of the antibody or functional fragment of the antibody are Ala, Ala, and Gly, respectively, and the antibody represents an anti-EGFR antibody or an anti-CDH6 antibody; and the N297 sugar chain is either N297-(Fuc)MSG1 or N297-(Fuc)SG having the structure represented by the following formula: In the formula, the wavy line indicates binding to Asn297 of the antibody, and L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ -NH-, which indicates that the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2nd position of the sialic acid at the non-reducing end of the 1-3 chain side of the branched chain of β-Man in the N297 sugar chain; an asterisk indicates that the linker L is bonded to the nitrogen atom at the 1st or 3rd position on the 1,2,3-triazole ring of Lb; and n ５ represents an integer of 2 to 5; In the formula, the wavy line indicates binding to Asn297 of the antibody, and L(PEG) is -(CH ２ -CH ２ -O)n５ -CH ２ -CH ２ -NH-, which indicates that the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2-position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain; an asterisk indicates that the linker L is bonded to the nitrogen atom at the 1-position or the 3-position on the 1,2,3-triazole ring of Lb; and n ５ represents an integer of 2 to 5.

[24] The antibody-drug conjugate according to

[22] , wherein R is a group represented by the following formula: In each of the structural formulas shown above, m ２ represents an integer of 1 or 2; Ab represents an antibody or a functional fragment of the antibody, in which the amino acid residues at positions 234, 235, and 265 (all EU numbering) of the Fc region of the antibody or functional fragment of the antibody are Ala, Ala, and Gly, respectively, and the antibody represents an anti-EGFR antibody or an anti-CDH6 antibody; and the N297 sugar chain is either N297-(Fuc)MSG1 or N297-(Fuc)SG having the structure represented by the following formula: In the formula, the wavy line indicates binding to Asn297 of the antibody, and L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ -NH-, which indicates that the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2nd position of the sialic acid at the non-reducing end of the 1-3 chain side of the branched chain of β-Man in the N297 sugar chain; an asterisk indicates that the linker L is bonded to the nitrogen atom at the 1st or 3rd position on the 1,2,3-triazole ring of Lb; and n ５ represents an integer of 2 to 5; In the formula, the wavy line indicates binding to Asn297 of the antibody, and L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH ２ -CH ２-NH-, which indicates that the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2-position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain; an asterisk indicates that the linker L is bonded to the nitrogen atom at the 1-position or the 3-position on the 1,2,3-triazole ring of Lb; and n ５represents an integer of 2 to 5.

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

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

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

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

[27] The antibody-drug conjugate according to

[25] , wherein the antibody comprises 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, or 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, or any one of the above antibodies, wherein the heavy chain comprises one or several amino acid residues deleted at the carboxyl terminus.

[28] The antibody-drug conjugate according to

[26] , wherein the antibody comprises 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, or the antibody comprises a heavy chain comprising one or several amino acid residues deleted at the carboxyl terminus.

[29] The antibody-drug conjugate of

[25] , wherein the antibody comprises a light chain comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 29, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 31, wherein 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.

[30] The antibody-drug conjugate of

[26] , wherein the antibody comprises a light chain comprising a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 43, and a heavy chain comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 45, wherein 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.

[31] The antibody-drug conjugate according to

[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 (all EU numbering) of the Fc region of the antibody are Ala, Ala, and Gly, respectively.

[32] The antibody-drug conjugate according to

[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 (all EU numbering) of the Fc region of the antibody are Ala, Ala, and Gly, respectively.

[33] The antibody-drug conjugate according to

[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 Ab represents any one selected from the following group: 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 any one of the above antibodies, a heavy chain in which one or several amino acid residues at the carboxyl terminus are deleted; the N297 sugar chain is represented by the following formula: (wherein the wavy line indicates binding to Asn297 of the antibody, L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH ２ -CH２ -NH-, which indicates that the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2-position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain; an asterisk indicates that the linker L is bonded to the nitrogen atom at the 1-position or the 3-position on the 1,2,3-triazole ring of Lb; n ５ is 3, and m ２

[34] An antibody-drug conjugate represented by the following formula: wherein Ab represents any one selected from the following group: 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 (all EU numbering) of the Fc region of said antibody are Ala, Ala, and Gly, respectively, and the N297 sugar chain is represented by the following formula: (wherein the wavy line indicates binding to Asn297 of the antibody, L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ -NH-, which indicates that the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2-position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain; an asterisk indicates that the linker L is bonded to the nitrogen atom at the 1-position or the 3-position on the 1,2,3-triazole ring of Lb; n ５ is 3, and m ２

[35] An antibody-drug conjugate represented by the following formula: wherein Ab represents any one selected from the following group: 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; wherein the amino acid residues at positions 234, 235, and 265 (all EU numbering) of the Fc region of said antibody are Ala, Ala, and Gly, respectively; The N297 sugar chain has the following formula: (wherein the wavy line indicates binding to Asn297 of the antibody, L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ -NH-, which indicates that the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2-position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain; an asterisk indicates that the linker L is bonded to the nitrogen atom at the 1-position or the 3-position on the 1,2,3-triazole ring of Lb; n ５ is 3, and m ２

[36] An antibody-drug conjugate represented by the following formula: wherein Ab represents any one selected from the following group: 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 any one of the above antibodies, a heavy chain in which one or several amino acid residues at the carboxyl terminus are deleted; the N297 sugar chain is represented by the following formula: (wherein the wavy line indicates binding to Asn297 of the antibody, L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ -NH-, which indicates that the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2-position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain; an asterisk indicates that the linker L is bonded to the nitrogen atom at the 1-position or the 3-position on the 1,2,3-triazole ring of Lb; n ５ is 3, and m ２

[37] An antibody-drug conjugate represented by the following formula: wherein Ab represents any one selected from the following group: 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 (all EU numbering) of the Fc region of said antibody are Ala, Ala, and Gly, respectively, and the N297 sugar chain is represented by the following formula: (wherein the wavy line indicates binding to Asn297 of the antibody, L(PEG) is -(CH ２ -CH２ -O)n ５ -CH ２ -CH ２ -NH-, which indicates that the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2-position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain; an asterisk indicates that the linker L is bonded to the nitrogen atom at the 1-position or the 3-position on the 1,2,3-triazole ring of Lb; n ５ is 3, and m ２

[38] An antibody-drug conjugate represented by the following formula: wherein Ab represents any one selected from the following group: 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; wherein the amino acid residues at positions 234, 235, and 265 (all EU numbering) of the Fc region of said antibody are Ala, Ala, and Gly, respectively; The N297 sugar chain has the following formula: (wherein the wavy line indicates binding to Asn297 of the antibody, L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH ２ -CH ２-NH-, which indicates that the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2-position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain; an asterisk indicates that the linker L is bonded to the nitrogen atom at the 1-position or the 3-position on the 1,2,3-triazole ring of Lb; n ５ is 3, and m ２

[39] A STING agonist comprising the antibody-drug conjugate according to any one of [1] to

[38] .

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

[38] .

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

[38] .

[42] The antitumor agent according to

[41] , wherein the tumor 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, ear cancer, thymic cancer, small intestine cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, sarcoma, EGFR mutation-positive cancer, or cancer with a mutation in EGFR downstream signaling. [42-2] The antitumor agent according to

[41] , wherein the tumor is CDH6-positive cancer. [42-3] The antitumor agent according to

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

[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 [1] to

[38] , the STING agonist according to

[39] , the pharmaceutical composition according to

[40] , and the antitumor agent according to

[41] ,

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

[44] The method of

[43] , wherein the cancer is lung cancer, renal 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, ear cancer, thymic cancer, small intestine cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, sarcoma, EGFR mutation-positive cancer, or cancer with a mutation in EGFR downstream signaling. [44-2] The method of

[43] , wherein the cancer is CDH6-positive cancer.[44-3] The method according to

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

[45] (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) of the Fc region of the antibody are Ala, Ala, and Gly, respectively; or (B) 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, wherein 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.[45-2] (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; or (B) 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, 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] (A) An antibody comprising a light chain including a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 29 and a heavy chain including a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 31, wherein 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; or (B) an antibody comprising a light chain including a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 43 and a heavy chain including a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 45, wherein 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.[46-2] (A) An antibody comprising a light chain including a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 29 and a heavy chain including a heavy chain variable region consisting of the amino acid sequence of 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; or (B) an antibody comprising a light chain including a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 43 and a heavy chain including a heavy chain variable region consisting of the amino acid sequence of 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] (A) 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, or 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; (B) 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, or (C) an antibody comprising a heavy chain in which one or several amino acid residues at the carboxyl terminus are deleted in any one of the antibodies (A) to (B). [47-2] (A) 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, or 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, and having binding activity to EGFR; (B) 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 having binding activity to CDH6; or (C) An antibody comprising any one of the antibodies (A) to (B), a heavy chain in which one or several amino acid residues are deleted at the carboxyl terminus.

[48] The pharmaceutical composition set forth in

[40] or the antitumor agent set forth in

[41] ,

[42] , [42-2], or [42-3], which is administered in combination with another pharmaceutical agent.

[49] The pharmaceutical composition set forth in

[40] or the antitumor agent set forth in

[41] ,

[42] , [42-2], or [42-3], which contains another pharmaceutical agent.

[50] The method of

[43] ,

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

[38] , the STING agonist according to

[39] , the pharmaceutical composition according to

[40] and the antitumor agent according to

[41] ,

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

[51] A polynucleotide encoding the amino acid sequence of the antibody according to any one of

[45] , [45-2],

[46] , [46-2],

[47] and [47-2].

[52] An expression vector containing the polynucleotide according to

[51] .

[53] A host cell transformed with the expression vector according to

[52] .

[54] A culture method comprising culturing the host cell according to

[53] .

[55] A method for producing an antibody, comprising the steps of culturing the host cell according to

[53] and collecting the antibody of interest from the culture obtained in the culture step.

[56] A method for producing an Fc-containing molecule having an N297-linked glycan containing a glycan derived from a glycan donor molecule, comprising the following step 1: (Step 1) reacting an acceptor molecule that is an Fc-containing molecule having a core GlcNAc to which fucose may be attached as the N297-linked glycan with a glycan donor molecule containing a glycan containing GlcNAc whose reducing end is not activated, in the presence of: - endo-β-N-acetylglucosaminidase (enzyme-A) which uses the N297-linked glycan of the Fc-containing molecule as a substrate; - endo-β-N-acetylglucosaminidase (enzyme-B) which uses the glycan of the glycan donor molecule as a substrate; and - an additive selected from a monovalent salt, an organic solvent, a surfactant, sugars, amino acids, or any combination thereof, to obtain a reaction mixture.

[57] The method according to

[56] , further comprising the following step 2: (Step 2) contacting the reaction mixture with a cation exchange chromatography medium or a multimode chromatography medium under acidic conditions to recover an Fc-containing molecule having an N297-linked glycan that contains a glycan derived from a glycan donor molecule.

[58] The method of

[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 functional fragment of the antibody are Ala, Ala, and Gly, respectively.

[59] The method of any one of

[56] to

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

[60] The method of any one of

[56] to

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

[61] The method of any one of

[56] to

[60] , wherein the additive is sodium chloride.

[62] The method of any one of

[56] to

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

[63] The method of any one of

[56] to

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

[64] The method according to any one of

[56] to

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

[65] The method according to any one of

[56] to

[64] , wherein the Fc-containing molecule is an antibody, and the antibody comprises 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] The method according to any one of

[56] to

[64] , wherein the Fc-containing molecule is an antibody, and the antibody comprises 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.

[0017] The present invention provides a molecule that maintains antitumor activity while exhibiting excellent safety. The present invention also provides a novel antibody-drug conjugate that can be administered systemically and exhibits antitumor effects in tumors expressing antigens. Furthermore, the present invention provides an improved method for producing Fc-containing molecules (especially antibodies) using a one-pot method.

[0018] 1A ) and 1B) are schematic representations of antibody-drug conjugates (molecule (II)) of the present invention, obtained from an SG-type glycosylation remodeling antibody (molecule (II) in FIG. 1A ), and an antibody-drug conjugate (molecule (II) in FIG. 1B) obtained from an MSG-type glycosylation remodeling antibody. (a) represents drug D, (b) represents linker L, (c) represents PEG linker (L(PEG)), and (d) represents N297 glycosylation (wherein the white circle represents NeuAc(Sia), the white hexagon represents Man, the black hexagon represents GlcNAc, the white diamond represents Gal, and the white inverted triangle represents Fuc). The white pentagon represents a triazole ring generated by reaction of the alkyne derived from linker L with the azide group derived from the PEG linker. The Y-shape represents antibody Ab. The PEG linker is linked via an amide bond to the carboxyl group at the 2-position of the sialic acid at the non-reducing end. This notation method is applied throughout the specification unless otherwise specified. Figures 2A and 2B are schematic diagrams showing the structures of (Fucα1,6)GlcNAc-antibody (molecule (III) in Figure 2A), SG-type glycosylation remodeling antibody (molecule (IV) in Figure 2B), and MSG-type glycosylation remodeling antibody (molecule (IV) in Figure 2C), which are production intermediates of the antibody-drug conjugates of the present invention. In all figures, the Y-shape represents the antibody Ab, as in Figure 1. In Figure 2A, (e) represents the N297 glycan, which consists of a disaccharide α-glycosidically linked between position 1 of Fuc and position 6 of GlcNAc. In Figures 2B and 2C, (d) represents the same N297 glycan as in Figure 1, and (f) represents a PEG linker having an azide group, which is used for binding to the linker L at its terminal. The binding mode of the PEG linker having an azide group is the same as that of the PEG linker in Figure 1. This is a schematic diagram of the process for producing an SG-type glycosylation remodeling antibody and an MSG-type glycosylation remodeling antibody from an antibody produced in animal cells. Molecules (III) and (IV) in the diagram represent a (Fucα1,6)GlcNAc-antibody and an SG-type glycosylation remodeling antibody or an MSG-type glycosylation remodeling antibody, respectively, as in Figure 2. Molecule (V) is an antibody produced in animal cells, and is a mixture of molecules with heterogeneous N297 glycans.Figure 3A shows the process for preparing a uniform (Fucα1,6)GlcNAc-antibody (III) by treating the heterogeneous N297 sugar chain (V) with a hydrolase such as EndoS. Figure 3B shows the process for preparing an SG-type sugar chain remodeled antibody (IV) by transferring an SG-type sugar chain donor molecule to the GlcNAc of the N297 sugar chain of antibody (III) using a glycosyltransferase such as EndoS D233Q / Q303L mutant. Figure 3C shows the process for preparing an MSG-type sugar chain remodeled antibody (IV) by transferring an MSG-type sugar chain donor molecule to antibody (III), similar to Figure 3B. The SG-type glycan donor molecule and MSG-type glycan donor molecule used here have their non-reducing terminal sialic acids modified with a PEG linker having an azide group, and the SG-type N297 glycan remodeling antibody and MSG-type N297 glycan remodeling antibody produced also have similarly modified non-reducing terminal sialic acids, as shown in Figures 2B and 2C. Figure 2 shows the antitumor effect of intravenous administration of anti-EGFR antibody 1-CDN conjugate 1 and anti-EGFR antibody 3-CDN conjugate 1. The average number of drugs bound is approximately 4. In the figure, the black square line indicates the vehicle group, the black triangle line indicates the anti-EGFR antibody 3-CDN conjugate 1-administered group, and the black circle line indicates the anti-EGFR antibody 1-CDN conjugate 1-administered group. The vertical axis indicates tumor volume (mm). ３ The horizontal axis represents the number of days after tumor implantation. Figure 1 shows the antitumor effect of intravenous administration of anti-EGFR antibody 2-CDN conjugate 1. The average number of drug bindings is approximately 4. The black square line in the figure represents the vehicle group, and the black circle line represents the anti-EGFR antibody 2-CDN conjugate 1-administered group. The vertical axis represents tumor volume (mm ３ The horizontal axis represents the number of days after tumor implantation. Figure 1 shows the antitumor effect of intravenous administration of anti-EGFR antibody 1-CDN conjugate 2 and anti-EGFR antibody 3-CDN conjugate 2. The average number of drugs bound is approximately 2. In the figure, the black square line 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 tumor volume (mm ３The horizontal axis represents the number of days after tumor implantation. This figure shows the antitumor effect of intravenous administration of anti-EGFR antibody 2-CDN conjugate 2. The average number of drugs bound is approximately 2. In the figure, the black square line represents the vehicle group, and the black circle line represents the anti-EGFR antibody 2-CDN conjugate 2-administered group. The vertical axis represents tumor volume (mm ３ The horizontal axis represents the number of days after tumor implantation. Figure 1 shows the antitumor effect of intravenous administration of anti-EGFR antibody 2-CDN conjugate 1. The average number of drug bindings is approximately 4. The black square line in the figure represents the vehicle group, and the black circle line represents the anti-EGFR antibody 2-CDN conjugate 1-administered group. The vertical axis represents tumor volume (mm ３ The horizontal axis represents the number of days after tumor implantation. Figure 1 shows the antitumor effect of intravenous administration of anti-EGFR antibody 2-CDN conjugate 1. The average number of drug bindings is approximately 4. The black square line in the figure represents the vehicle group, and the black circle line represents the anti-EGFR antibody 2-CDN conjugate 1-administered group. The vertical axis represents tumor volume (mm ３ The horizontal axis represents the number of days after tumor implantation. This figure shows the antitumor effect of intravenous administration of anti-CDH6 antibody 1-CDN conjugate 1 and anti-CDH6 antibody 1-CDN conjugate 2. In the figure, the black square line represents the vehicle group, the white circle 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 tumor volume (mm ３), the horizontal axis indicates the number of days after tumor implantation. 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 against reporter cells. In the figure, the black triangle line indicates the anti-EGFR antibody 3-CDN conjugate addition group, the black circle line indicates the anti-EGFR antibody 1-CDN conjugate addition group, the dashed line indicates CDN conjugate 2 with an average drug binding number of approximately 2, and the solid line indicates CDN conjugate 1 with an average drug binding number of approximately 4. The vertical axis indicates the count value, and the horizontal axis indicates the concentration. The figure shows the activity of anti-CDH6 antibody 1-CDN conjugate 1 and anti-CDH6 antibody 1-CDN conjugate 2 against reporter cells. In the figure, the black triangle line indicates the anti-CDH6 antibody 1-CDN conjugate addition group. The dashed line indicates CDN conjugate 2, which has an average drug binding number of approximately 2, and the solid line indicates CDN conjugate 1, which has an average drug binding number of approximately 4. The vertical axis indicates count values, and the horizontal axis indicates concentration. This shows the antitumor effect of anti-EGFR antibody 1-CDN conjugate 1 in a tumor re-implantation test. The vertical axis indicates tumor volume (mm ３ ) are shown. The antitumor effect of anti-EGFR antibody 1-CDN conjugate 2 in a tumor re-implantation test is shown. The vertical axis represents tumor volume (mm ３) are shown. Cytokine production in a human whole blood test for each antibody-CDN conjugate is shown. The vertical axis represents the amount of cytokine, and the horizontal axis represents the Fc portion of each antibody-CDN conjugate. The cytokine amount was calculated using a standard line on a logarithmic scale. Cytokine production in a human whole blood test for each antibody-CDN conjugate is shown. The vertical axis represents the amount of cytokine, and the horizontal axis represents the Fc portion of each antibody-CDN conjugate. The cytokine amount was calculated using a standard line on a linear scale. Figure 16A shows graphs of the binding activity of an IgG2-type anti-EGFR antibody (IgG2) (anti-EGFR antibody B), an Fc-substituted antibody in which the Fc portion was replaced with an IgG1-type antibody (IgG1 WT) (anti-EGFR antibody A), a mutant in which the L234A / L235A (LALA) mutation was introduced into the Fc portion of IgG1 WT (modified anti-EGFR antibody 3), and a mutant in which the L234A / L235A / D265G (LALA-DG) mutation was introduced (modified anti-EGFR antibody 1) to human FcγRI, as assessed by the SPR method. The horizontal axis represents antibody concentration, and the vertical axis represents the RU (Response Unit) value obtained for that antibody concentration. Figure 16B shows graphs of the binding activity of the antibody group used in Figure 16A to human FcγRIIa, as assessed by the SPR method. The horizontal axis represents antibody concentration, and the vertical axis represents the RU value obtained for that antibody concentration. Figure 16C is a graph showing the SPR assay of the binding activity of the antibody group used in Figure 16A to human FcγRIIb / c. The horizontal axis represents antibody concentration, and the vertical axis represents the RU value obtained for that antibody concentration. Figure 16D is a graph showing the SPR assay of the binding activity of the antibody group used in Figure 16A to human FcγRIIIa. The horizontal axis represents antibody concentration, and the vertical axis represents the RU value obtained for that antibody concentration. Figure 16E is a graph showing the SPR assay of the binding activity of the antibody group used in Figure 16A to human FcγRIIIb. The horizontal axis represents antibody concentration, and the vertical axis represents the RU value obtained for that antibody concentration.17A is a graph showing the RU values ​​obtained at an antibody concentration of 4.7 μM when the binding activity to human FcγRI was evaluated by the SPR method for the antibody group used in FIG. 16A and antibody-drug conjugates in which STING AGONIST was conjugated to these antibody groups at 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). FIG. 17B is a graph showing the RU values ​​obtained at an antibody concentration of 4.7 μM when the binding activity to human FcγRIIa was evaluated by the SPR method for the antibody group used in FIG. 17A. Figure 17C is a graph showing RU values ​​obtained at an antibody concentration of 4.7 μM when the antibody group used in Figure 17A was evaluated for binding activity to human FcγRIIb / c by SPR. Figure 17D is a graph showing RU values ​​obtained at an antibody concentration of 4.7 μM when the antibody group used in Figure 17A was evaluated for binding activity to human FcγRIIIa by SPR. Figure 17E is a graph showing RU values ​​obtained at an antibody concentration of 4.7 μM when the antibody group used in Figure 17A was evaluated for binding activity to human FcγRIIIb by SPR. Figure 18 shows the T of anti-EGFR antibody A-CDN conjugate 1 (IgG1 WT), anti-EGFR antibody 3-CDN conjugate 1 (LALA), and modified anti-EGFR antibody 1-CDN conjugate 1 (LALA-DG). ｍ The table also shows the T values ​​of anti-EGFR antibody A-CDN conjugate 1 and various anti-EGFR antibody-CDN conjugates 1. ｍ The difference in values ​​(ΔT ｍ19 shows (a) the amino acid sequence of human STING wild-type (SEQ ID NO: 1), (b) the amino acid sequence of human STING REF mutant (R232H) (SEQ ID NO: 3), and (c) the amino acid sequence of human STING HAQ mutant (R71H, G230A, R293Q) (SEQ ID NO: 5). FIG. 20 shows (a) the amino acid sequence of CDRL1 (SEQ ID NO: 23) of the anti-EGFR antibody (modified anti-EGFR antibodies 1 to 3), (b) the amino acid sequence of CDRL2 (SEQ ID NO: 24) of the anti-EGFR antibody (modified anti-EGFR antibodies 1 to 3), (c) the amino acid sequence of CDRL3 (SEQ ID NO: 25) of the anti-EGFR antibody (modified anti-EGFR antibodies 1 to 3), (d) the amino acid sequence of CDRH1 (SEQ ID NO: 26) of the anti-EGFR antibody (modified anti-EGFR antibodies 1 to 3), (e) the amino acid sequence of CDRH2 (SEQ ID NO: 27) of the anti-EGFR antibody (modified anti-EGFR antibodies 1 to 3), and (f) the amino acid sequence of CDRH3 (SEQ ID NO: 28) of the anti-EGFR antibody (modified anti-EGFR antibodies 1 to 3). Figure 21-1 shows (a) the amino acid sequence (SEQ ID NO: 29) of the light chain variable region of the anti-EGFR antibody (modified anti-EGFR antibodies 1 to 3), (b) the amino acid sequence (SEQ ID NO: 30) of the light chain of the anti-EGFR antibody (modified anti-EGFR antibodies 1 to 3), and (c) the amino acid sequence (SEQ ID NO: 31) of the heavy chain variable region of the anti-EGFR antibody (modified anti-EGFR antibodies 1 to 3). Figure 21-2 shows (d) the amino acid sequence (SEQ ID NO: 32) of the heavy chain of the anti-EGFR antibody LALA-DG mutant (modified anti-EGFR antibody 1), (e) the amino acid sequence (SEQ ID NO: 33) of the heavy chain of the anti-EGFR antibody LALA-DG mutant (modified anti-EGFR antibody 2), and (f) the amino acid sequence (SEQ ID NO: 34) of the heavy chain of anti-EGFR antibody A. Figure 21-3 shows (g) the amino acid sequence of the heavy chain of anti-EGFR antibody B (Vectibix) (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).FIG. 22 shows (a) the amino acid sequence of CDRL1 of the anti-CDH6 antibody (modified anti-CDH6 antibody 1) (SEQ ID NO: 37), (b) the amino acid sequence of CDRL2 of the anti-CDH6 antibody (modified anti-CDH6 antibody 1) (SEQ ID NO: 38), (c) the amino acid sequence of CDRL3 of the anti-CDH6 antibody (modified anti-CDH6 antibody 1) (SEQ ID NO: 39), (d) the amino acid sequence of CDRH1 of the anti-CDH6 antibody (modified anti-CDH6 antibody 1) (SEQ ID NO: 40), (e) the amino acid sequence of CDRH2 of the anti-CDH6 antibody (modified anti-CDH6 antibody 1) (SEQ ID NO: 41), and (f) the amino acid sequence of CDRH3 of the anti-CDH6 antibody (modified anti-CDH6 antibody 1) (SEQ ID NO: 42). Figure 23 shows (a) the amino acid sequence (SEQ ID NO: 43) of the light chain variable region of the anti-CDH6 antibody (modified anti-CDH6 antibody 1), (b) the amino acid sequence (SEQ ID NO: 44) of the light chain of the anti-CDH6 antibody (modified anti-CDH6 antibody 1), (c) the amino acid sequence (SEQ ID NO: 45) of the heavy chain variable region of the anti-CDH6 antibody (modified anti-CDH6 antibody 1), and (d) the amino acid sequence (SEQ ID NO: 46) of the heavy chain of the anti-CDH6 antibody LALA-DG mutant (modified anti-CDH6 antibody 1). Figure 24 shows the anti-tumor effect when CT26.WT-hCDH6 tumors were re-implanted into the left axilla of mice whose tumors had completely regressed after intravenous administration of anti-CDH6 antibody 1-CDN conjugate 1 and anti-CDH6 antibody 1-CDN conjugate 2. The vertical axis represents tumor volume (mm). ３ ) Figure 25 shows the antitumor effects of 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 ), and the horizontal axis represents the number of days after tumor inoculation. Figure 26 shows the antitumor effect of intravenous administration of anti-CDH6 antibody 1-CDN conjugate 1. The vertical axis represents tumor volume (mm ３ ), and the horizontal axis represents the number of days after tumor inoculation. Figure 27 shows the antitumor effect of intravenous administration of anti-CDH6 antibody 1-CDN conjugate 2. The vertical axis represents tumor volume (mm ３ ), and the horizontal axis represents the number of days after tumor inoculation. Figure 28 shows the antitumor effect of intravenous administration of anti-CDH6 antibody 1-CDN conjugate 1. The vertical axis represents tumor volume (mm ３), and the horizontal axis represents the number of days after tumor inoculation. Figure 29 shows the antitumor effect of intravenous administration of anti-CDH6 antibody 1-CDN conjugate 2. The vertical axis represents tumor volume (mm ３), and the horizontal axis represents the number of days after tumor implantation. Figure 30 shows the amino acid sequence of wild-type Endo-S (SEQ ID NO: 47). Amino acid residues corresponding to the catalytically active center of the enzyme are underlined in bold, and amino acid residues whose mutations are expected to control the enzyme activity are shown in bold. Figure 31 shows the amino acid sequence of wild-type Endo-S2 (SEQ ID NO: 48). Amino acid residues corresponding to the catalytically active center of the enzyme are underlined in bold, and amino acid residues whose mutations are expected to control the enzyme activity are shown in bold. Figure 32 shows the amino acid sequence of wild-type Endo-Si (SEQ ID NO: 49). Amino acid residues corresponding to the catalytically active center of the enzyme are underlined in bold, and amino acid residues whose mutations are expected to control the enzyme activity are shown in bold. Figure 33 shows the amino acid sequence of wild-type Endo-M (SEQ ID NO: 50). Amino acid residues corresponding to the catalytically active center of the enzyme are underlined in bold, and amino acid residues whose mutations are expected to control the enzyme activity are shown in bold. Figure 34 shows the amino acid sequence of wild-type Endo-Rp (SEQ ID NO: 51). Amino acid residues corresponding to the catalytically active center of the enzyme are underlined in bold, and amino acid residues that are expected to be able to control the enzyme activity by mutation are shown in bold. Figure 35 shows the amino acid sequence of wild-type Endo-CC (SEQ ID NO: 52). Amino acid residues corresponding to the catalytically active center of the enzyme are underlined in bold, and amino acid residues that are expected to be able to control the enzyme activity by mutation are shown in bold. Figure 36 shows the amino acid sequence of wild-type Endo-Om (SEQ ID NO: 53). Amino acid residues corresponding to the catalytically active center of the enzyme are underlined in bold, and amino acid residues that are expected to be able to control the enzyme activity by mutation are shown in bold. Figure 37 shows the amino acid sequence of wild-type Endo-Sd (SEQ ID NO: 54). Amino acid residues corresponding to the catalytically active center of the enzyme are underlined in bold, and amino acid residues that are expected to be able to control the enzyme activity by mutation are shown in bold. Figure 38 shows the amino acid sequence of wild-type Endo-Sz (SEQ ID NO: 55). Amino acid residues corresponding to the catalytically active center of the enzyme are underlined in bold, and amino acid residues that are expected to be able to control enzyme activity by mutation are shown in bold. Figure 39 shows the amino acid sequence of wild-type Endo-Se (SEQ ID NO: 56).The amino acid residues corresponding to the catalytically active center of the enzyme are underlined in bold, and amino acid residues that are expected to be able to control the enzyme activity by mutation are shown in bold.

[0019] In one aspect, 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 ability to produce inflammatory cytokines, and equivalent or enhanced in vivo anti-tumor activity compared to a corresponding molecule having a wild-type Fc region.

[0020] As used herein, the term "target molecule-binding moiety" refers to a moiety that binds to any target molecule, preferably a target molecule involved in a human disease. Examples of such moieties 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 protein scaffolds, aptamers, antisense nucleic acid molecules, synthetic organic small molecules, and naturally occurring small molecules. Preferred examples include antibodies, functional fragments of antibodies, and antigen-binding fragments of antibodies. More preferred examples include antibodies or functional fragments of antibodies. Antibodies are particularly preferred, but the term is not limited to these. In one aspect of the present invention, when the "target molecule-binding moiety" contains an Fc region (e.g., when the "target molecule-binding moiety" is an antibody), the Fc region contained in the target molecule-binding moiety may be a mutant Fc region. In other words, in one aspect of the present invention, the mutant Fc region may be contained in the target molecule-binding moiety.

[0021] "Antibodies," "functional fragments of antibodies," and "antigen-binding fragments of antibodies" will be described in detail below.

[0022] In the present invention, "immunostimulation" refers to inducing in some way the activation of immune cells involved in anti-tumor immunity, such as monocytes, macrophages, dendritic cells, T cells, B cells, NK cells, and neutrophils, and refers to causing any structural or functional changes in immune cells, such as the production of cytokines and chemokines, increased expression of immune activation markers, decreased expression of immunosuppressive markers, changes such as phosphorylation in intracellular signaling pathways, and changes in gene expression. It also includes causing changes in tumor cells that induce anti-tumor immunity, and refers to inducing, for example, the production of cytokines and chemokines that activate or induce migration of immune cells, and increased sensitivity to immune cells.

[0023] In the present invention, the term "immunostimulant" refers to any substance that brings about immunostimulation as defined above. In one aspect 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, an NLRC3 agonist, an NLRP1-14 agonist, a DEC205 agonist, an MMR agonist, a Dectin1 agonist, a Dectin2 agonist, or a Mincle agonist. agonist, DC-SIGN agonist, DNGR-1 agonist, MBL agonist, AIM2 agonist, MDA-5 agonist, RIG-I agonist, AhR agonist, more preferably TLR3 agonist, TLR7 agonist, TLR8 agonist, TLR9 agonist, RIG-I agonist, STING agonist, particularly preferably STING agonist.

[0024] As used herein, "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain, which region contains a CH2 domain and a CH3 domain. In one embodiment of the present invention, the "Fc region" comprises at least a portion of a hinge domain. In another embodiment of the present invention, the "Fc region" comprises at least a portion of a hinge domain, two CH2 domains, and two CH3 domains. The term "Fc region" generally refers to a homo- or heterodimer or multimer in which the C-terminal regions of two or more immunoglobulin heavy chains are linked via an S-S bond, but may also refer to a single-chain Fc region (scFc) in which these form a single chain.

[0025] In one aspect, the "variant Fc region" of the present invention comprises at least three amino acid substitutions compared to a wild-type Fc region. In one aspect of the present invention, the "wild-type Fc region" and "variant Fc region" may be derived from any biological species, but are preferably derived from humans, monkeys (including, but not limited to, cynomolgus monkeys, rhesus monkeys, common marmosets, and squirrel monkeys), mice, rats, or rabbits, more preferably from humans or monkeys, and particularly preferably from humans. In one aspect of the present invention, the "wild-type Fc region" and "variant Fc region" may be derived from any immunoglobulin molecule, including IgG, IgE, IgM, IgD, IgA, and IgY, but are preferably derived from IgG. Furthermore, the subclass may be any of IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, but IgG1, IgG2, or IgG4 is preferred, IgG1 or IgG4 is more preferred, and IgG1 is particularly preferred. Thus, in one aspect of the present invention, the Fc region in the "wild-type Fc region" and "mutant Fc region" is a human IgG1 Fc region or a human IgG4 Fc region, preferably a human IgG1 Fc region.

[0026] As described above, in one aspect of the present invention, the variant Fc region comprises at least three amino acid substitutions compared to a 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 variant Fc region, but are preferably present in the CH2 domain of the variant Fc region. In one aspect of the present invention, the at least three amino acid substitutions are present at positions 234, 235, and 265 (all according to EU numbering) of the variant Fc region. In a further aspect of the present invention, the at least three amino acid substitutions preferably comprise 234Ala, 235Ala, and 265Gly (all according to EU numbering). In the present invention, such a mutation in the Fc region consisting of 234Ala, 235Ala, and 265Gly (all according to EU numbering) can also be represented as "LALA-DG," "L234A / L235A / D265G," "-L234A, L235A, D265G," etc.

[0027] In one aspect of the present invention, a molecule comprising a target molecule-binding portion, a variant Fc region, and an immunostimulatory agent may comprise an additional Fc region, which may be a variant Fc region. In another aspect of the present invention, the "variant Fc region" comprises two CH2 domains, and one or both of the two CH2 domains contain the at least three amino acid substitutions.

[0028] In one aspect, a molecule comprising a target molecule-binding portion, a mutant Fc region, and an immunostimulatory agent of the present invention has reduced binding to human Fcγ receptors compared to a corresponding molecule having a wild-type Fc region. As used herein, the term "corresponding molecule having a wild-type Fc region" refers to a molecule that is identical or equivalent to a molecule comprising a target molecule-binding portion, a mutant Fc region, and an immunostimulatory agent of the present invention, except that it contains a wild-type Fc region rather than a mutant Fc region. As used herein, "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, the "human Fcγ receptor" refers to 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 isoforms listed above, and is preferably one or more receptors selected from the group consisting of human FcγRI, human FcγRIIa, human FcγRIIb / c, human FcγRIIIa, and human FcγRIIIb, more preferably human FcγRI. The binding ability of an analyte to a human Fcγ receptor can be determined using any known method, for example, by surface plasmon resonance (SPR).In one embodiment of the present invention, the binding affinity of an analyte to a human Fcγ receptor can be determined by capturing a His-tagged human Fcγ receptor as a ligand on a sensor chip to which an anti-His antibody has been immobilized, then adding the analyte to the sensor chip and measuring the interaction between the ligand and the analyte by surface SPR. Specifically, the binding affinity can be determined using the methods disclosed in Test Examples 15 to 20 of this specification.

[0029] In one aspect, when the target molecule-binding portion of the present invention is an antibody and a mutant Fc region is encapsulated in the antibody, the molecule comprising the target molecule-binding portion of the present invention, the mutant Fc region, and an immunostimulatory agent has reduced binding to human Fcγ receptors to the same extent as the parent antibody. In one aspect, reduced binding to human Fcγ receptors means reduced effector function.

[0030] In one aspect, a molecule comprising a target molecule-binding portion, a mutant Fc region, and an immunostimulant of the present invention 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 response or inflammatory symptoms. In one aspect of the present invention, "inflammatory cytokine" refers to a cytokine that causes or promotes an inflammatory response or inflammatory symptoms, and whose production is not expected or desired to be increased more than necessary by a test substance. For example, as in Test Example 14 of the present specification, a cytokine whose production is increased in the absence of cancer cells targeted by the target molecule-binding portion generally corresponds to a cytokine whose production is not expected or desired to be increased more than necessary, and therefore can be included in the "inflammatory cytokine" of the present invention. In one embodiment of the present invention, the "inflammatory cytokine" is an interleukin, TNF-α, TNF-β, interferon, or chemokine, preferably TNF-α, IL-1, IL-6, IL-10, CCL2, CCL4, or IFN-γ, and particularly preferably IL-6. The ability of a test substance (e.g., a molecule comprising a mutant Fc region of the present invention) to produce an inflammatory cytokine can be determined using any known method, for example, by immunoassay such as ELISA. In one embodiment of the present invention, the ability of a test substance to produce an inflammatory cytokine can be determined using a method such as that disclosed in Test Example 14 of this specification.

[0031] In one aspect, a molecule comprising a target molecule-binding portion of the present invention, a variant Fc region, and an immunostimulatory agent has equivalent or enhanced in vivo anti-tumor activity compared to a corresponding molecule having a wild-type Fc region.

[0032] In this specification, the terms "cancer," "carcinoma," and "tumor" are used interchangeably.

[0033] In the present invention, the term "antitumor effect" refers to the direct or indirect effect of a drug on tumor cells, thereby inducing a reduction or regression of tumors. For example, an antitumor effect can be caused by a drug directly damaging tumor cells, by tumor cells stimulating with the drug to activate antitumor immunity, or by a drug delivered to tumor cells being released extracellularly to activate antitumor immunity around the tumor cells, thereby reducing the number of tumor cells, damaging them, or causing tumor regression.

[0034] The in vivo antitumor activity of a test substance can be determined using any known method, for example, the methods disclosed in Test Examples 3 to 9 of this specification.

[0035] In one aspect, a molecule comprising a target molecule-binding portion, a mutant Fc region, and an immunostimulator of the present invention has the same or improved thermal stability, preferably improved thermal stability, as compared to a corresponding molecule having a wild-type Fc region. The thermal stability of such a molecule can be determined using any known method, for example, by measuring the heat capacity C of a solution containing the molecule when the temperature is increased. ｐ The thermal denaturation midpoint (T) of the domain into which the mutation has been introduced can be determined by examining the change in the thermal denaturation temperature (T), specifically, by using the method disclosed in Test Example 21 of the present specification. The peaks of the curve showing the change in heat capacity indicate the thermal denaturation of each domain of the antibody, and the thermal denaturation peaks are generally observed in the order of CH2, Fab, and CH3 (Biochem. Biophys. Res. Commun. 355, 751-757 (2007)). Therefore, the thermal denaturation midpoint (T ｍ The change in thermal stability can be determined by calculating the thermal capacity change (T) from the thermal denaturation peak and comparing the temperature at the peak of the mutant Fc region with that of the corresponding molecule having a wild-type Fc region. In this specification, the temperature at the peak top of the curve showing the change in heat capacity is referred to as T ｍ In this specification, T ｍ The change in value (ΔT ｍIf the difference in Tc between the peaks in the CH2 domain and the mutant Fc region is less than 1°C, the two molecules are considered to have equivalent thermal stability. If the difference is improved by 1°C or more, the molecule containing the mutant Fc region is considered to have improved thermal stability compared to the corresponding molecule having a wild-type Fc region. For example, if the mutation is in the CH2 domain, the Tc between the peaks in the CH2 domain and the mutant Fc region is considered to have improved thermal stability. ｍ The change in (ΔT ｍ If the difference in thermal stability between the mutant Fc region and the wild-type Fc region is less than 1°C, the two molecules are considered to have equivalent thermal stability, and if the difference is improved by 1°C or more, the molecule containing the mutant Fc region is considered to have improved thermal stability compared to the corresponding molecule having the wild-type Fc region.

[0036] In one aspect, when the mutant Fc region contains amino acid substitutions at 234Ala, 235Ala, and 265Gly (EU numbering), the molecule comprising the target molecule-binding portion of the present invention, the mutant Fc region, and the immunostimulant has excellent safety while retaining its efficacy, and its effector function is reduced to the same extent as that of the parent antibody. Also, in one aspect, when the mutant Fc region contains amino acid substitutions at 234Ala, 235Ala, and 265Gly (EU numbering), the molecule comprising the target molecule-binding portion of the present invention, the mutant Fc region, and the immunostimulant has excellent thermal stability.

[0037] In one aspect, the present invention relates to a pharmaceutical composition comprising a molecule comprising a target molecule-binding portion, a mutant Fc region, and an immunostimulatory agent as described above. In one aspect of the present invention, the pharmaceutical composition is an antitumor agent. The term "pharmaceutical composition" will be described in more detail below.

[0038] In one aspect, the present invention relates to an antibody-drug conjugate comprising a CDN derivative with STING agonist activity and uses thereof. The CDN derivative has STING agonist activity and activates immune cells to induce the production of interferons and cytokines. Furthermore, the CDN derivative exerts an antitumor effect by activating the immune cells. In one aspect, the antibody-drug conjugate of the present invention is prepared by linking the CDN derivative to an antibody capable of recognizing and binding to target cells (e.g., tumor cells or immune cells) via an optional linker, and can be administered systemically. Specific examples of systemic administration include intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. The antibody-drug conjugate can be considered as one embodiment of a molecule comprising a target molecule-binding portion, a mutant Fc region, and an immunostimulant, as described above.

[0039] STING (Stimulator of Interferon Genes) is a transmembrane adaptor protein localized in the endoplasmic reticulum. It is known that congenital polymorphisms of STING frequently exist (PLoS One, 2013 Oct, 21, 8(10), e77846). Examples of STING mutants include the R232H mutation in which the 232nd amino acid is mutated from arginine (R) to histidine (H), and the HAQ mutation in which the 71st arginine (R) is mutated to histidine (H), the 230th glycine (G) is mutated to alanine (A), and the 293rd arginine (R) is mutated to glutamine (Q). It is known that such STING polymorphisms result in differences in the strength 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 act stably in humans, it is desirable for it to have activity against each STING form.

[0040] In the present invention, "cytotoxic activity" refers to causing pathological changes in cells in some form, and refers to causing not only direct trauma but also any damage to the structure or function of cells, such as DNA breakage, formation of base dimers, chromosome breakage, damage to the cell division apparatus, and reduced activity of various enzymes.

[0041] In the present invention, the term "cells" includes cells in an animal body and cultured cells.

[0042] <1. CDN Derivative> The CDN derivative is a compound represented by the following formula (I):

[0043] It has the structure shown below.

[0044] L １ is a group represented by the following formula:

[0045] Q and Q ’ each independently represents a hydroxy group or a thiol group. Preferably, Q and Q' are both thiol groups.

[0046] R ２１ and R ２２ R each independently represents a hydroxy group or a fluorine atom. ２１ is preferably a hydroxy group. ２２ is preferably a fluorine atom.

[0047] W is —NH— or a sulfur atom, and preferably —NH—.

[0048] The method for producing the CDN derivative will be described later in <3. Production Method>.

[0049] 2. Antibody-Drug Conjugates In one aspect, the antibody-drug conjugate of the present invention can be systemically administered as an antibody-drug conjugate in which the above-described CDN derivative and an antibody capable of recognizing and binding to a target cell (e.g., a tumor cell or an immune cell) are linked via an optional linker. Hereinafter, when simply referring to the "antibody-drug conjugate of the present invention," in principle, it means an antibody-drug conjugate that includes the above-described CDN derivative as a drug moiety, but exceptionally, it may also mean the above-described "molecule that includes a target molecule-binding portion, a mutant Fc region, and an immunostimulant."

[0050] The antibody drug conjugate of the present invention has the following formula (II):

[0051] It is shown by m １ is in the range of 1 to 10 and indicates the number of drugs bound per antibody molecule in the antibody-drug conjugate. Ab represents an antibody or a functional fragment of the antibody, L represents a linker connecting Ab and D, and D represents the above-mentioned CDN derivative (herein, when a CDN derivative is used as a part of an antibody-drug conjugate, it is also simply referred to as a "drug").

[0052] Drug D is a compound having immune cell activation activity, specifically, STING agonist activity. When part or all of the linker is cleaved within a target cell (e.g., a tumor cell or an immune cell), Drug D is released in its original structure, thereby exerting its immunostimulatory effect. The desired function is exerted by enhancing the sensitivity of the target cell to immune cells or by activating the immune cell via the target cell. The desired function is not particularly limited as long as it is related to STING agonist activity, but is preferably antitumor activity. That is, Drug D linked to a tumor-targeting antibody (e.g., an anti-EGFR antibody, an anti-CDH6 antibody) via an optional linker is delivered to the target cell or tissue, where part or all of the linker is cleaved, thereby exerting an antitumor effect through enhancing the sensitivity of the target cell to immune cells or activating the immune cell via the target cell (e.g., production of interferon or cytokine).

[0053] The drug D conjugated to the antibody-drug conjugate of the present invention has the following formula (I):

[0054] (Here, L １ , Q, Q', R ２１ , R ２２ and W are as defined above in <1. CDN derivatives>. The drug D used in the antibody-drug conjugate of the present invention is preferably represented by the following two formulae:

[0055] (Here, L １ , Q, Q', and W are as defined above in <1. CDN Derivatives>).

[0056] Furthermore, the drug D used in the antibody-drug conjugate of the present invention is preferably represented by the following two formulas:

[0057] (wherein the asterisk indicates binding to L, and Q, Q', and W are as defined above in <1. CDN derivatives>).

[0058] Furthermore, the drug D used in the antibody-drug conjugate of the present invention is preferably represented by the following formula:

[0059] (wherein the asterisk indicates binding to L, and W is as defined in <1. CDN derivatives> above).

[0060] Furthermore, the drug D used in the antibody-drug conjugate of the present invention is preferably represented by the following formula:

[0061] (where the asterisk indicates the bond to L).

[0062] Furthermore, the drug D used in the antibody-drug conjugate of the present invention is preferably represented by the following formula 4:

[0063] (where the asterisk indicates the bond to L).

[0064] Furthermore, the drug D used in the antibody-drug conjugate of the present invention is more preferably a drug represented by the following formula:

[0065] It is shown as follows.

[0066] <2.1. Linker Structure> The linker structure that binds a drug to an antibody in the antibody-drug conjugate of the present invention will be 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 an antibody and a drug. Examples of linkers used in the antibody-drug conjugate of the present invention include, but are not limited to, linkers described in Protein Cell, 2018, 9(1):33-46, Pharm Res, 2015, 32:3526-3540, or Int. J. Mol. Sci., 2016, 17, 561. The linker may be a linker that is cleaved in vivo or a linker that is not cleaved in vivo, but is preferably a linker that is cleaved in vivo.

[0067] Examples of linkers used in the antibody-drug conjugates of the present invention include, but are not limited to, linkers that bind a drug to a sugar chain or a remodeled sugar chain of the Fc portion of an antibody (sometimes referred to herein as "sugar chain conjugation") (e.g., as described in WO2018 / 003983), or linkers that bind a drug to any amino acid residue of an antibody (e.g., a cysteine ​​residue or a lysine residue) (e.g., as described in WO2014 / 057687). Linkers that bind a drug to any amino acid residue of an antibody are preferably those that bind a drug via a thioether bond with a sulfhydryl group (SH group) of cysteine ​​of an Ab (sometimes referred to herein as "cysteine ​​conjugation") or an amino group (NH ２ Examples include cases where the conjugation is via an amide bond with a hydroxyl group (sometimes referred to as "lysine conjugation" in this specification), and preferred is cysteine ​​conjugation.

[0068] A preferred linker L for use in the antibody drug conjugates of the present invention is represented by the formula: -Lb-La-Lp-Lc-* (where the asterisk indicates attachment to drug D).

[0069] First, Lp will be described. Lp may be a linker consisting of an amino acid sequence that is cleavable in vivo or in target cells (hereinafter also referred to as a peptide linker in this specification), or may be absent.

[0070] Lp is cleaved by the action of an enzyme such as peptidase or esterase. Lp is a peptide composed of 2 to 7 (preferably 2 to 4) amino acids. Lp forms an amide bond at its N-terminus with the carbonyl group at the right end of La (described below), and forms an amide bond at its C-terminus with the amino group (—NH—) of Lc. The amide bond at the C-terminus of Lp is cleaved by the enzyme such as peptidase.

[0071] The amino acids constituting Lp are not particularly limited, but may be, for example, L- or D-amino acids, preferably L-amino acids. In addition to α-amino acids, amino acids having structures such as β-alanine, ε-aminocaproic acid, and γ-aminobutyric acid may also be used, and non-natural amino acids such as N-methylated amino acids may also be used. The amino acid sequence of Lp is not particularly limited, but examples of the amino acids constituting it include 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). Among these, 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 overlap, and the amino acid sequence may include any amino acid selected. Furthermore, the drug release pattern can be controlled by the type of amino acid.

[0072] Specific examples of Lp include -GGFG-, -GGPI-, -GGVA-, -GGFM-, -GGVCit-, -GGFCit-, -GGICit-, -GGPL-, -GGAQ-, and -GGPP-. The linker Lp is preferably -GGFG- or -GGPI-, more preferably -GGFG-.

[0073] Next, La will be described. La is the following: —C(═O)—(CH ２ CH ２ ) n ２ -C(=O)-, -C(=O)-(CH ２ CH ２ ) n ２ -CH ２ -C(=O)-, -C(=O)-(CH ２ CH ２ ) n ２ -C(=O)-NH-(CH ２ CH ２ ) n ３ -C(=O)-, -C(=O)-(CH ２ CH ２ ) n ２ -C(=O)-NH-(CH ２ CH ２ ) n ３ -CH ２ -C(=O)-, -C(=O)-(CH ２ CH ２ ) n ２ -C(=O)-NH-(CH ２ CH ２ O)n ３ -CH ２ -C(=O)-, -(CH ２ ) n ４ -O-C(=O)-, and -(CH ２ ) n ９ -C(=O)- (wherein, n ２ represents an integer of 1 to 3 (preferably 1 or 2), and n ３ represents an integer of 1 to 5 (preferably an integer of 2 to 5, more preferably 3 or 4), and n ４ represents an integer of 0 to 2 (preferably 0 or 1), n ９represents any one selected from the group consisting of integers of 2 to 7 (preferably an integer of 2 to 5, more preferably 2, 3, or 5).

[0074] La is preferably the following: —C(═O)—CH ２ CH ２ -C(=O)-, -C(=O)-CH ２ CH ２ -C(=O)-NH-(CH ２ CH ２ O) ３ -CH ２ -C(=O)-, -C(=O)-CH ２ CH ２ -C(=O)-NH-(CH ２ CH ２ O) ４ -CH ２ -C(=O)-, -C(=O)-(CH ２ CH ２ ) ２ -C(=O)-, -C(=O)-CH ２ CH ２ -C(=O)-NH-(CH ２ CH ２ ) ２ -C(=O)-, -C(=O)-CH ２ CH ２ -C(=O)-NH-(CH ２ CH ２ ) ２ -CH ２ -C(=O)-, -CH ２ -OC(=O)-, -OC(=O)-, and -(CH ２ ) ５ -C(=O)- represents any one selected from the group consisting of:

[0075] La is more preferably —C(═O)—CH ２ CH ２ -C(=O)-, -C(=O)-CH ２ CH ２ -C(=O)-NH-(CH ２ CH ２ O) ３ -CH ２ -C(=O)-, -C(=O)-CH ２ CH２ -C(=O)-NH-(CH ２ CH ２ O) ４ -CH ２ -C(=O)-, or -(CH ２ ) ５ -C(=O)-.

[0076] La is more preferably —C(═O)—CH ２ CH ２ -C(=O)-.

[0077] Next, Lb will be described. Lb represents a spacer used in a linker for sugar chain conjugation (also referred to herein as a "spacer for a linker for sugar chain conjugation") or a spacer used in a linker for cysteine ​​conjugation (also referred to herein as a "spacer for a linker for cysteine ​​conjugation").

[0078] <When Lb is a "spacer of a linker for sugar chain conjugation"> When Lb is a "spacer of a linker for sugar chain conjugation", Lb is not particularly limited, and examples thereof include spacers represented by the following formula:

[0079] or

[0080]

[0081] In each structural formula shown above, the asterisk (*) represents the -(C=O)-, -CH ２ The wavy line indicates binding to the sugar chain of Ab or a remodeled sugar chain.

[0082] When Lb-1 or Lb-3 is selected as Lb, the triazole ring moiety has a geometric isomeric structure, and one Lb contains either one of two types of structures or a mixture thereof. The antibody-drug conjugate of the present invention can bind multiple drugs to one antibody molecule. When multiple drugs are bound to one antibody molecule, multiple Lbs will also be present (see, for example, the schematic diagram (1e) of the antibody-drug conjugate shown in Method E in <3. Production Method> described below). When Lb is selected from Lb-1 or Lb-3 and multiple Lbs exist per antibody molecule (see, for example, m ２ is 1 or 2), in each Lb, the triazole ring moiety has a geometric isomeric structure, and one Lb contains either one of two types of structures or a mixture thereof.

[0083] <When Lb is a "spacer of a linker for cysteine ​​conjugation"> When Lb is a "spacer of a linker for cysteine ​​conjugation", Lb is not particularly limited, and examples thereof include -(succinimide-3-yl-N)-. In the present invention, "-(succinimide-3-yl-N)-" refers to a group represented by the following formula:

[0084] In the structural formula shown above, the asterisk indicates binding to La, and the wavy line indicates binding via a thioether with the side chain of a cysteine ​​residue in the antibody.

[0085] Next, Lc will be described. Lc is —NH—CH ２ -, -NH-phenyl group -CH ２ -O(C=O)- or -NH-heteroaryl group -CH ２ Lc represents —O(C═O)— or is absent. Here, the phenyl group is preferably a 1,4-phenyl group, and the heteroaryl group is preferably a 2,5-pyridyl group, a 3,6-pyridyl group, a 2,5-pyrimidyl group, or a 2,5-thienyl group. Lc is preferably —NH—CH ２ - or does not exist.

[0086] A more preferred linker L used in the antibody-drug conjugate of the present invention is -Z when the binding mode between the drug and the antibody is "sugar chain conjugation." Ｌ１ —C(═O)—CH ２ CH ２ -C(=O)-GGFG-, -Z Ｌ１ —C(═O)—CH ２ CH ２ -C(=O)-GGVA-, -Z Ｌ１ —C(═O)—CH ２ CH ２ -C(=O)-GGVCit-, -Z Ｌ１ —C(═O)—CH ２ CH ２ -C(=O)-GGFCit-, -Z Ｌ１ —C(═O)—CH ２ CH ２ -C(=O)-GGICit-, -Z Ｌ１ —C(═O)—CH ２ CH ２ -C(=O)-GGFM-, -Z Ｌ１ —C(═O)—CH ２ CH ２ -C(=O)-GGPI-, -Z Ｌ１ —C(═O)—CH ２ CH ２ -C(=O)-GGLM-, -Z Ｌ１ —C(═O)—CH ２ CH ２ -C(=O)-FG-, -Z Ｌ１ —C(═O)—CH ２ CH ２ -C(=O)-VA-, -Z Ｌ１ —C(═O)—CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ -, -Z Ｌ１ —C(═O)—CH ２ CH ２ -C(=O)-GGVA-NH-CH ２ -, -Z Ｌ１ —C(═O)—CH ２ CH ２ -C(=O)-GGVCit-NH-CH ２ -, -Z Ｌ１—C(═O)—CH ２ CH ２ -C(=O)-GGFCit-NH-CH ２ -, -Z Ｌ１ —C(═O)—CH ２ CH ２ -C(=O)-NH-(CH ２ CH ２ O) ３ -CH ２ -C(=O)-, or -Z Ｌ１ —C(═O)—CH ２ CH ２ -C(=O)-NH-(CH ２ CH ２ O) ４ -CH ２ -C(=O)- (where Z Ｌ１ is the structural formula of Lb shown below:

[0087] or when the binding mode between the drug and the antibody is "cysteine ​​conjugation", -Z Ｌ２ - (CH ２ ) ５ -C(=O)-GGFG-, -Z Ｌ２ - (CH ２ ) ５ -C(=O)-GGVA-, -Z Ｌ２ - (CH ２ ) ５ -C(=O)-GGVCit-, -Z Ｌ２ - (CH ２ ) ５ -C(=O)-GGFCit-, -Z Ｌ２ - (CH ２ ) ５ -C(=O)-GGICit-, -Z Ｌ２ - (CH ２ ) ５ -C(=O)-GGFM-, -Z Ｌ２ - (CH ２ ) ５ -C(=O)-GGPI-, -Z Ｌ２ - (CH ２ ) ５ -C(=O)-GGLM-, -Z Ｌ２ - (CH ２ ) ５-C(=O)-FG-, -Z Ｌ２ - (CH ２ ) ５ -C(=O)-VA-, -Z Ｌ２ - (CH ２ ) ５ -C(=O)-GGFG-NH-CH ２ -, -Z Ｌ２ - (CH ２ ) ５ -C(=O)-GGVA-NH-CH ２ -, -Z Ｌ２ - (CH ２ ) ５ -C(=O)-GGVCit-NH-CH ２ -, -Z Ｌ２ - (CH ２ ) ５ -C(=O)-GGFCit-NH-CH ２ -, -Z Ｌ２ - (CH ２ ) ５ -C(=O)-NH-(CH ２ CH ２ O) ３ -CH ２ -C(=O)-, or -Z Ｌ２ - (CH ２ ) ５ -C(=O)-NH-(CH ２ CH ２ O) ４ -CH ２ -C(=O)-, (where Z Ｌ２ is the structural formula of Lb shown below:

[0088] It represents -(succinimide-3-yl-N)-.

[0089] A more preferred linker L used in the antibody-drug conjugate of the present invention is one in which the binding mode between the drug and the antibody is "sugar chain conjugation" and is represented by the formula -Z Ｌ１ —C(═O)—CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ - or -Z Ｌ１ —C(═O)—CH ２ CH ２-C(=O)-GGPI-NH-CH ２ -, (where Z Ｌ１ is the structural formula of Lb shown below:

[0090] (Indicates

[0091] An even more preferred linker L used in the antibody-drug conjugate of the present invention is one in which the binding mode between the drug and the antibody is "sugar chain conjugation", and is represented by the formula -Z Ｌ１ —C(═O)—CH ２ CH ２ -C(=O)-GGFG-NH-CH ２ -, (where Z Ｌ１ is the structural formula of Lb shown below:

[0092] (Indicates

[0093] The right ends of the above-mentioned "preferred linker L", "more preferred linker L", "even more preferred linker L" and "even more preferred linker L" are bonded to the drug D.

[0094] The "linker L-drug D" used in the antibody drug conjugates of the present invention preferably has the following four formulas:

[0095] (where the wavy line indicates binding to the sugar chain of the Ab or the remodeled sugar chain).

[0096] The "linker L-drug D" used in the antibody drug conjugates of the present invention more preferably has one of the following four formulas:

[0097] (where the wavy line indicates binding to the sugar chain of the Ab or the remodeled sugar chain).

[0098] The "linker L-drug D" used in the antibody drug conjugates of the present invention is more preferably represented by the following formula:

[0099] (where the wavy line indicates binding to the sugar chain of the Ab or the remodeled sugar chain).

[0100] 2.2. Antibodies and Glycosylation Modifications Thereof 2.2.1 Antibodies As used herein, the term "gene" refers to a nucleotide or nucleotide sequence comprising a nucleotide sequence encoding the amino acids of a protein, or a complementary chain thereof. For example, the term "gene" includes polynucleotides, oligonucleotides, DNA, mRNA, cDNA, RNA, etc., which are nucleotide sequences comprising a nucleotide sequence encoding the amino acids of a protein, or complementary chains thereof.

[0101] As used herein, "nucleotide," "polynucleotide," or "nucleotide sequence" is synonymous with "nucleic acid," and for example, DNA, RNA, probes, oligonucleotides, polynucleotides, primers, etc. are also included in the meaning of "nucleotide" or "nucleotide sequence."

[0102] In this specification, the terms "polypeptide," "peptide," and "protein" are used interchangeably.

[0103] As used herein, the term "functional fragment of an antibody," also referred to as an "antigen-binding fragment of an antibody," refers to a partial fragment of an antibody that has antigen-binding activity, and includes linear antibodies and multispecific antibodies formed from antibody fragments. However, the term is not limited to these molecules as long as it has antigen-binding activity. Furthermore, these antigen-binding fragments include not only those obtained by treating full-length antibody protein molecules with appropriate enzymes, but also proteins produced in appropriate host cells using genetically engineered antibody genes.

[0104] The functional fragment of an antibody used in the antibody-drug conjugate of the present invention includes a functional fragment that retains an asparagine (Asn297) that is modified by an N-linked sugar chain, which is well conserved in the Fc region of an IgG heavy chain, and its surrounding amino acids, and that has antigen-binding ability.

[0105] The antibody used in the antibody-drug conjugate of the present invention refers to an immunoglobulin, and is a molecule containing an antigen-binding site that immunospecifically binds to an antigen. The antibody used in the antibody-drug conjugate of the present invention may be of any class, including IgG, IgE, IgM, IgD, IgA, and IgY, with IgG being preferred. The subclass may also be any of IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, with IgG1, IgG2, or IgG4 being preferred, IgG1 or IgG4 being more preferred, and IgG1 being particularly preferred (including antibodies with mutations in the Fc region of the IgG heavy chain that affect ADCC and ADCP activity). Thus, in one aspect 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 IgG1 or IgG4, preferably IgG1, antibody.

[0106] 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. Herein, the numbering of amino acid residues is in accordance with 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), unless otherwise specified.

[0107] In one aspect of the invention, when the antibody used in the antibody drug conjugate is an IgG1 or the functional fragment of the antibody used in the antibody drug conjugate is a functional fragment of an IgG1 antibody, the mutated Fc region comprises the IgG1 LALA-DG mutation (IgG1-L234A, L235A, D265G).

[0108] It has been known that effector functions can be adjusted by substituting a portion of the amino acid residues in the constant region (see WO88 / 07089, WO94 / 28027, WO94 / 29351). In particular, it has been known that reducing effector functions can avoid the possibility of activating immune cells outside the tumor site. However, it has also been reported that in conjugates using drugs that have the activity of activating immune cells, reducing the effector function of the antibody also reduces the efficacy (Non-Patent Documents 4 to 6).

[0109] In one aspect of the present invention, when an antibody or functional fragment of an antibody used in an antibody-drug conjugate contains the above-mentioned mutations in the Fc region, the effector function can be reduced while maintaining the pharmaceutical efficacy. Furthermore, in another aspect of the present invention, when an antibody or functional fragment of an antibody used in an antibody-drug conjugate contains the above-mentioned mutations in the Fc region, the antibody-drug conjugate has reduced human Fcγ receptor binding, reduced proinflammatory cytokine production, and / or equivalent or enhanced in vivo anti-tumor activity compared to a corresponding antibody-drug conjugate having a wild-type Fc region. Here, "a corresponding antibody-drug conjugate having a wild-type Fc region" refers to a molecule that is identical or equivalent to the antibody-drug conjugate of the present invention, except that it contains a wild-type Fc region instead of a mutant Fc region.

[0110] Furthermore, in one aspect of the present invention, when the antibody or functional fragment of the antibody used in the antibody-drug conjugate contains the above-mentioned mutations in the Fc region, the antibody-drug conjugate has reduced effector function to the same extent as the antibody containing the above-mentioned mutations in the Fc region (parent antibody; the antibody used to prepare the antibody-drug conjugate). In addition, in one aspect of the present invention, when the antibody or functional fragment of the antibody used in the antibody-drug conjugate contains the above-mentioned mutations in the Fc region, the antibody-drug conjugate has equivalent or improved thermal stability compared to a corresponding molecule having a wild-type Fc region, and in particular, when the antibody or functional fragment of the antibody used in the antibody-drug conjugate contains the LALA-DG mutation, the antibody-drug conjugate has improved thermal stability compared to a corresponding antibody-drug conjugate having a wild-type Fc region. In general, IgG2 antibodies are known to have lower effector functions than IgG1 antibodies (Front. Immunol. 2014, 5, 520).

[0111] In one aspect of the present invention, when the mutant Fc region contains the LALA-DG mutation, the antibody-drug conjugate of the present invention is excellent in terms of safety while retaining its efficacy, and has reduced effector function to the same extent as that of the parent antibody.Furthermore, in one aspect, when the mutant Fc region contains the LALA-DG mutation, the antibody-drug conjugate of the present invention is also excellent in thermal stability.

[0112] It is known that there are multiple allotypes of antibody constant regions. For example, for IgG1 heavy chains, examples include G1m17, G1m3, G1m1, and G1m2. The antibody constant region used in the present invention is not particularly limited, but it is preferable to use G1m17 or G1m3. The amino acid sequence of Fc without a Fab region is common to G1m17 and G1m3, and the LALA-DG mutant of Fc without a Fab region also exhibited attenuated FcγR binding activity and improved thermal stability compared to the LALA mutant (data not shown). Therefore, it is believed that the attenuated FcγR binding activity and improved thermal stability of the molecules of the present invention (particularly antibodies and antibody-drug conjugates) are independent of the G1m17 and G1m3 allotypes.

[0113] It is known that the heavy and light chains of an antibody molecule each contain three complementarity determining regions (CDRs). CDRs, also known as hypervariable regions, are located within the variable regions of the heavy and light chains of an antibody and are particularly highly variable in their primary structure. They are separated into three regions in the primary structure of the heavy and light chain polypeptide chains. In this specification, the CDRs of an antibody are represented as CDRH1, CDRH2, and CDRH3 from the amino-terminus of the heavy chain amino acid sequence, and CDRL1, CDRL2, and CDRL3 from the amino-terminus of the light chain amino acid sequence. These regions are close to each other in the three-dimensional structure and determine the specificity for the antigen to which they bind.

[0114] In this specification, the amino acid sequences of CDRs contained in the variable regions of antibodies are 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)).

[0115] The antibody may be derived from any species, preferably human, rat, mouse, or rabbit. When derived from a species other than human, it is preferably chimerized or humanized using well-known techniques. The antibody used in the present invention may 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, human antibodies, functional fragments thereof, or modified versions thereof.

[0116] The antibody is preferably, but not limited to, an antibody that targets tumor cells or immune cells, and more preferably, an antibody that targets tumor cells.

[0117] When an antibody that targets tumor cells is used, the antibody preferably has one or more of the following properties: the ability to recognize tumor cells, the ability to bind to tumor cells, the ability to be taken up and internalized into tumor cells, and the ability 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, when an antibody that targets tumor cells is used in the antibody-drug conjugate of the present invention, the antibody-drug conjugate is administered into the body, delivered to the tumor site, and taken up into tumor cells. After that, the linker moiety is cleaved by a peptidase or the like, releasing the drug moiety. The released drug moiety is thought to enhance the sensitivity of tumor cells to immune cells and activate anti-tumor immunity through its STING agonist activity, thereby exerting an anti-tumor effect. Alternatively, even if the antibody-drug conjugate accumulated in tumor cells is not internalized, the tumor cells and / or the antibody-drug conjugate may be taken up by immune cells by phagocytosis or the like, activating anti-tumor immunity through STING agonist activity and exerting an anti-tumor effect.

[0118] The binding of an antibody to tumor cells can be confirmed using flow cytometry. The uptake of an antibody into tumor cells can be confirmed using (1) an assay in which a secondary antibody (fluorescently labeled) that binds to a therapeutic antibody is used to visualize the antibody taken up into the cells using a fluorescence microscope (Cell Death and Differentiation (2008) 15, 751-761), (2) an assay in which a secondary antibody (fluorescently labeled) that binds to a therapeutic antibody is used to measure the amount of fluorescence taken up into the cells (Molecular Biology of the Cell Vol. 15, 5268-5282, December 2004), or (3) a Mab-ZAP assay in which an immunotoxin that binds to a therapeutic antibody is used, and upon uptake into the cells, the toxin is released, thereby suppressing cell proliferation (Bio Techniques 28:162-165, January 2000). As an immunotoxin, a recombinant conjugated protein of the catalytic domain of diphtheria toxin and protein G can also be used.

[0119] When an antibody that targets tumor cells is used in the antibody-drug conjugate of the present invention, it is preferable, but not essential, that the antibody itself have an anti-tumor effect.

[0120] The antitumor activity of a drug or antibody-drug conjugate refers to cytotoxic activity against tumor cells, anticellular effect, and tumor volume regression. Antitumor activity can be confirmed using known in vitro or in vivo evaluation systems.

[0121] The effects and immunostimulatory activity of drugs and antibody-drug conjugates refer to the enhancement of tumor cell sensitivity to immune cells or tumor cell-mediated activation of immune cells. The effects and immunostimulatory activity of drugs and antibody-drug conjugates can be confirmed using known in vitro or in vivo evaluation systems.

[0122] In vitro or in vivo evaluation systems that can be used in the present invention include a BALB / c mouse system in which CT26.WT-hEGFR cells, in which the human EGFR gene has been introduced into the mouse colon cancer cell line CT26.WT, have been subcutaneously transplanted, as described in Test Examples 3, 4, 5, and 6; a BALB / c-nu mouse system in which the human lung cancer cell line PC-9 cells have been subcutaneously transplanted, as described in Test Example 7; a BALB / c-nu mouse system in which the human lung cancer cell line NCI-H358 has been subcutaneously transplanted, as described in Test Example 8; and a CT26.WT mouse system in which the human CDH6 gene has been introduced into the CT26.WT, as described in Test Example 9. Examples of assay systems include, but are not limited to, a BALB / c mouse system into which WT-hCDH6 cells have been subcutaneously transplanted, a co-culture assay system of THP1 reporter cells and the human lung cancer cell line HCC827 described in Test Example 10, a co-culture assay system of THP1 reporter cells and the human ovarian cancer cell line OVCAR4 described in Test Example 11, a BALB / c mouse system into which CT26.WT cells have been subcutaneously transplanted again described in Test Examples 12 and 13, and an assay system using human whole blood described in Test Example 14.

[0123] Antibodies used in the present invention include, for example, anti-CDH6 antibodies and anti-EGFR antibodies.

[0124] The antibodies used in the present invention can be obtained by immunizing an animal with an antigenic polypeptide and collecting and purifying the antibodies produced in the body using methods commonly used in this field. The origin of the antigen is not limited to humans; animals can also be immunized with antigens derived from animals other than humans, such as mice or rats. In this case, antibodies that are applicable to human diseases can be selected by testing the cross-reactivity of the obtained antibodies that bind to heterologous antigens with human antigens.

[0125] Alternatively, a monoclonal antibody can be obtained by fusing antibody-producing cells that produce an antibody against an antigen with myeloma cells to establish a hybridoma according to known methods (e.g., Kohler and Milstein, Nature (1975) 256, pp. 495-497; Kennett, R. ed., Monoclonal Antibodies, pp. 365-367, Plenum Press, N.Y. (1980)).

[0126] The antigen can be obtained by genetically manipulating a gene encoding the antigen protein in a host cell to produce it.

[0127] The antibody used in the antibody-drug conjugate of the present invention can be obtained according to known methods (e.g., Proc. Natl. Acad. Sci. U.S.A., 81, 6851-6855, (1984), Nature (1986) 321, pp. 522-525, WO90 / 07861).

[0128] For example, anti-EGFR antibodies (WO1998 / 050433, WO2002 / 092771, etc.) and anti-CDH6 antibodies (WO2018 / 212136, etc.) can be obtained by known means.

[0129] The anti-EGFR antibody used in the present invention is not particularly limited, but preferably has the following properties: (1) An anti-EGFR antibody that specifically binds to EGFR. (2) The antibody according to (1) above that binds to the extracellular domain of human EGFR. (3) The antibody according to (1) or (2) above, which is a monoclonal antibody. (4) The antibody according to any one of (1) to (3) above, which is a mouse monoclonal antibody, a chimeric monoclonal antibody, a human monoclonal antibody, or a humanized monoclonal antibody. (5) The antibody according to (1) to (4) above, whose heavy chain constant region is that of human IgG1 and contains mutations that result in reduced ADCC and ADCP activity. (6) The antibody according to (4) above, whose heavy chain constant region is that of human IgG1 and whose Fc region has amino acid residues at positions 234, 235, and 265 (all EU numbering) that are Ala, Ala, and Gly, respectively. (7) The antibody according to (6) above, which is a humanized or human monoclonal antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 32 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 30. (8) The antibody according to (6) above, which is a humanized or human monoclonal antibody comprising a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 33 and a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 30. (9) The antibody according to (6) above, which is a humanized or human monoclonal antibody comprising a heavy chain variable region contained in a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 32 or 33 and a light chain variable region contained in a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 30. (10) The antibody according to (6) above, which is a humanized or human monoclonal antibody comprising a heavy chain comprising a CDRL1 consisting of the amino acid sequence set forth in SEQ ID NO: 23, a CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 24, and a CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 25, and a heavy chain comprising a CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 26, a CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 27, and a CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 28. (11) The antibody according to any one of (1) to (10) above, wherein one or two amino acids are deleted at the carboxyl terminus of the heavy chain.(12) An antibody obtained by a method for producing the antibody, comprising the steps 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 collecting the antibody of interest from the culture obtained in the step.

[0130] Examples of anti-EGFR antibodies include panitumumab, nimotuzumab, cetuximab, ametumumab (SY-101), SYN-004, SCT-200, tomuzotuximab, GC-1118, GR-1401, depatuxizumab (ABT-806), serculutamab, AMG595, and matuzumab, and preferred examples include panitumumab and ABT806.

[0131] The anti-CDH6 antibody used in the present invention is not particularly limited, but preferably has the following properties: (1) An anti-CDH6 antibody that specifically binds to CDH6. (2) The antibody according to (1) above that binds to the extracellular domain of human CDH6. (3) The antibody according to (1) or (2) above, which is a monoclonal antibody. (4) The antibody according to any one of (1) to (3) above, which is a mouse monoclonal antibody, a chimeric monoclonal antibody, a human monoclonal antibody, or a humanized monoclonal antibody. (5) The antibody according to (1) to (4) above, whose heavy chain constant region is that of human IgG1 and contains mutations that result in reduced ADCC and ADCP activity. (6) The antibody according to (5) above, whose heavy chain constant region is that of human IgG1 and whose Fc region contains amino acid residues at positions 234, 235, and 265 (all EU numbering) are Ala, Ala, and Gly, respectively. (7) The antibody according to (6) above, which 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. (8) The antibody according to (6) above, which is a humanized or human monoclonal antibody comprising a heavy chain variable region contained in a heavy chain consisting of the amino acid sequence set forth in SEQ ID NO: 46 and a light chain variable region contained in a light chain consisting of the amino acid sequence set forth in SEQ ID NO: 44. (9) The antibody according to (6) above, which is a humanized or human monoclonal 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. (10) The antibody according to any of (1) to (9) above, in which one or two amino acids are deleted at the carboxyl terminus of the heavy chain. (11) An antibody obtained by a method for producing the antibody, comprising the steps of 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 antibody of interest from the culture obtained in the step.

[0132] Examples of anti-CDH6 antibodies include antibodies (antibody X), NOV0712, and LTV977 having a light chain consisting of the amino acid sequence set forth in positions 21 to 233 of SEQ ID NO: 61 described in WO2018212136 and a heavy chain consisting of the amino acid sequence set forth in positions 20 to 471 of SEQ ID NO: 69, and preferably antibody X.

[0133] The antibody used in the present invention may have 80% to 99% amino acid identity with the heavy and / or light chains of the above-mentioned antibodies. Here, the term "identity" has the general definition used in the art. The percent identity refers to the percentage of identical amino acids per total number of amino acids (including gaps) when two amino acid sequences are aligned to maximize amino acid identity. Such identity is generally 80% or more, preferably 90, 91, 92, 93, or 94% or more, more preferably 95, 96, 97, or 98% or more, and even more preferably 99% or more. Furthermore, antibodies having various functions equivalent to those of the above-mentioned antibodies can be selected by combining amino acid sequences in which one to several amino acid residues are substituted, deleted, and / or added to the amino acid sequences of the heavy and / or light chains. The number of amino acid residues to be substituted, deleted and / or added is generally 10 amino acid residues or less, preferably 5 to 6 amino acid residues or less, more preferably 2 to 3 amino acid residues or less, and even more preferably 1 amino acid residue.

[0134] It is known that antibodies produced in cultured mammalian cells have deletions of lysine residues at the carboxyl terminus of their heavy chains (Journal of Chromatography A, 705: 129-134 (1995)). It is also known that two amino acid residues, glycine and lysine, are deleted from the carboxyl terminus of the heavy chain, and a proline residue at the carboxyl terminus is newly amidated (Analytical Biochemistry, 360: 75-83 (2007)). However, these deletions or modifications of the heavy chain sequence do not affect the antigen-binding ability or effector functions (e.g., complement activation and antibody-dependent cellular cytotoxicity) of the antibody. Therefore, the antibodies used in the present invention also include antibodies with such deletions or modifications and functional fragments of such antibodies. Therefore, in one aspect of the present invention, the antibody is an antibody having a heavy chain with one or several amino acid residues deleted from the carboxyl terminus. In this context, "one or several amino acid residues" preferably refers to 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 aspect of the present invention, the antibodies used in the present invention also include deletions in which one or two amino acids are deleted from the carboxyl terminus of the heavy chain, and amidated versions of such deletions (e.g., heavy chains in which a proline residue at the carboxyl terminus is amidated). However, as long as the deletion or modification (amidation) of the carboxyl terminus does not significantly affect the antigen-binding ability and effector function, the deletions in the carboxyl termini of the heavy chains of the antibodies used in the present invention are not limited to the above types. The two heavy chains constituting the antibodies used in the present invention may be any one of heavy chains selected from the group consisting of full-length heavy chains and the deletions described above, or a combination of two of these heavy chains. The quantitative ratio of each deletion may be affected by the type of mammalian cultured cells producing the antibody used in the present invention and the culture conditions, but the antibody used in the present invention preferably has one amino acid residue deleted at the carboxyl terminus of each of the two heavy chains.

[0135] 2.2.2 Polynucleotides, etc. In one aspect of the present invention, there are provided polynucleotides encoding the amino acid sequences of the antibodies described above. For example, in one aspect of the present invention, there are provided polynucleotides encoding the amino acid sequence of any one of the antibodies described in

[45] to

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

[0136] In one aspect of the present invention, an expression vector containing the polynucleotide is provided. The expression vector is not particularly limited, and any vector, such as a plasmid or virus, capable of expressing the polynucleotide can be used. Any known method can be used to construct such an expression vector. For example, an animal cell expression vector containing a wild-type Fc region gene can be used as a template, and the desired effector-less mutation can be introduced into the Fc region by PCR using a KOD-Plus-Mutagenesis Kit (TOYOBO Corporation). Alternatively, an Fc region gene containing the desired effector-less mutation can be synthesized and then incorporated into the animal cell expression vector by ligation.

[0137] In one embodiment of the present invention, a host cell transformed with the expression vector is provided. The host cell is not particularly limited, and any type of eukaryotic or prokaryotic cell capable of expressing the polynucleotide contained in the expression vector can be used. In one embodiment of the present invention, the host cell of the present invention is a eukaryotic cell, such as an animal cell, a plant cell, or a eukaryotic microorganism. In particular, animal cells include mammalian cells, such as monkey COS cells (Cell (1981) 23, pp. 175-182, ATCC CRL-1650), mouse fibroblast NIH3T3 (ATCC No. CRL-1658), a dihydrofolate reductase-deficient strain of Chinese hamster ovary cells (CHO cells, ATCC CCL-61) (Proc. Natl. Acad. Sci. USA (1980) 77, pp. 4126-4220), FreeStyle 293F cells (Invitrogen), lymphocytes, and myeloma cells. In another embodiment of the present invention, the host cell of the present invention is a prokaryotic cell, for example, Escherichia coli or Bacillus subtilis.

[0138] In one aspect of the present invention, there is provided a culture method comprising culturing the host cell. The culture method is not particularly limited as long as the host cell is capable of expressing the antibody of interest, and the culture conditions and the like can be appropriately determined depending on the type of host cell and expression vector used, etc.

[0139] In one aspect of the present invention, a method for producing an antibody is provided, comprising culturing the host cells and recovering a target antibody from the culture obtained in the culturing step. The "host cell culturing step" is not particularly limited as long as the host cells are capable of expressing the target antibody, and the culture conditions can be appropriately determined depending on the type of host cells and expression vector used. The "recovery of the target antibody from the culture obtained in the culturing step" step is also not particularly limited and can be appropriately determined depending on the type of host cells and expression vector used, the properties of the target antibody, and other factors. Examples of methods for producing the antibody include, but are not limited to, the production method described in the Vectibix Intravenous Infusion 100 mg Review Report (March 5, 2010, Pharmaceutical and Food Safety Bureau, Evaluation and Licensing Division) and the production method described in WO 2018 / 212136, as mentioned in Examples 13 and 14 of the present specification.

[0140] 2.2.3 Antibody Glycosylation Remodeling Recently, a method has been reported in which heterogeneous antibody glycosylation is remodeled by enzymatic reaction to uniformly introduce glycosylation with functional groups (ACS Chem. Biol. 2012, 7, 110-122, ACS Med. Chem. Lett. 2016, 7, 1005-1008). Attempts have also been made to use this glycosylation remodeling technique to site-specifically introduce drugs and synthesize homogeneous ADCs (Bioconjugate Chem. 2015, 26, 2233-2242, Angew. Chem. Int. Ed. 2016, 55, 2361-2367, US2016361436).

[0141] In glycan remodeling, first, a hydrolase is used to remove heterogeneous glycans attached to a protein (e.g., an antibody) leaving only the terminal GlcNAc, thereby preparing a homogeneous protein portion with GlcNAc attached (hereinafter referred to as "acceptor"). Next, a separately prepared glycan of your choice (hereinafter referred to as "donor") is prepared, and this acceptor and donor are linked using a glycosyltransferase. This allows the synthesis of a homogeneous glycoprotein with a desired glycan structure.

[0142] In the present invention, the term "sugar chain" refers to a structural unit in which two or more monosaccharides are linked by glycosidic bonds. Specific monosaccharides and sugar chains may be represented by abbreviations such as "GlcNAc-" or "SG-". When these abbreviations are used in structural formulae, the oxygen atom or nitrogen atom at the reducing end that forms a glycosidic bond with another structural unit is not included in the abbreviation representing the sugar chain, unless otherwise defined.

[0143] In the present invention, unless otherwise specified, monosaccharides, which are the basic units of sugar chains, are described in terms of convenience, with the carbon atom in the ring structure that is bonded to the oxygen atom constituting the ring and that is directly bonded to a hydroxy group (or an oxygen atom belonging to a glycosidic bond) being at position 1 (position 2 only in sialic acid). The names of the example compounds are given based on the entire chemical structure, and this rule does not necessarily apply.

[0144] In the present invention, when a sugar chain is described as a symbol (e.g., SG, MSG, GlcNAc, etc.), unless otherwise defined, the symbol includes the carbon atom at the reducing end, and does not include the N or O attributable to the N- or O-glycosidic bond.

[0145] The antibody drug conjugate of the present invention has the following formula:

[0146] The antibody Ab or a functional fragment thereof is bound to L either directly from the side chain of its amino acid residue (e.g., cysteine, lysine, etc.) or via a sugar chain or a remodeled sugar chain of the Ab.

[0147] The sugar chain of Ab in the present invention is an N-linked sugar chain or an O-linked sugar chain, preferably an N-linked sugar chain.

[0148] N-linked glycans are bound to amino acid side chains of antibodies via N-glycosidic bonds, and O-linked glycans are bound to amino acid side chains of antibodies via O-glycosidic bonds.

[0149] The Ab in the present invention is IgG, preferably IgG1, IgG2 or IgG4, more preferably IgG1 or IgG4, and particularly preferably IgG1.

[0150] IgG has a well-conserved N-linked glycan (hereinafter referred to as "Asn297 glycan or N297 glycan") at the 297th asparagine residue (hereinafter referred to as "Asn297 or N297") in the Fc region of its heavy chain, and is known to contribute to the activity, kinetics, etc. of antibody molecules (Eon-Duval, A. et al., Biotechnol. Prog. 2012, 28, 608-622; Sanglier-Cianferani, S., Anal. Chem. 2013, 85, 715-736).

[0151] The amino acid sequence in the constant region of IgG is well conserved, and in a report by Edelman et al. (Proc. Natl. Acad. Sci. U.S.A., 63, 78-85, (1969)), each amino acid is identified by an EU number (EU INDEX). For example, Asn297, to which an N-linked sugar chain is added in the Fc region, corresponds to position 297 in the EU numbering. Even if the actual amino acid position changes due to molecular fragmentation or region deletion, the amino acid can be uniquely identified by displaying it by EU numbering.

[0152] The diagram below shows the case where the antibody-drug conjugate of the present invention is bound to the N297 sugar chain of an antibody or a functional fragment thereof at L.

[0153] An antibody having such a remodeled sugar chain is called a sugar chain remodeling antibody.

[0154] SGP (α2,6-SGP) is an abbreviation for sialylglycopeptide and is a representative N-linked glycopeptide. SGP can be isolated and purified from chicken egg yolk, for example, according to the method described in WO 2011 / 027868. Purified SGP products are also commercially available from Tokyo Chemical Industry Co., Ltd. and Fushimi Pharmaceutical Co., Ltd. In this specification, the sugar chain portion of SGP is referred to as SG, and a sugar chain lacking one GlcNAc at the reducing end of SG is referred to as SG(10). SG(10) can be prepared by enzymatic hydrolysis of SGP, for example, with reference to the report by Umekawa et al. (Biochim. Biophys. Acta 2010, 1800, 1203-1209). SG(10) can also be purchased from Tokyo Chemical Industry Co., Ltd. and Fushimi Pharmaceutical Co., Ltd.

[0155] In this specification, the sugar chain structure in which sialic acid is deleted from the non-reducing end of only one of the branched chains of β-Man of SG(10) is designated as MSG(9), the sugar chain structure having sialic acid only in the 1-3 sugar chain of the branched chain is designated as MSG1, and the sugar chain structure having sialic acid only in the 1-6 sugar chain of the branched chain is designated as MSG2.

[0156] The remodeled sugar chains used in the antibody-drug conjugates of the present invention are 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, more preferably N297-(Fuc)SG or N297-(Fuc)MSG1.

[0157] N297-(Fuc)SG is represented by the following structural formula or sequence formula:

[0158]

[0159]

[0160] In the above formula, the wavy line indicates binding to Asn297 of the antibody, and L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH２ -CH ２ -NH-, which indicates that the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at the 2-position of the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man of the N297 sugar chain; an asterisk indicates that the linker L, particularly the linker L, is bonded to the nitrogen atom at the 1-position or 3-position on the 1,2,3-triazole ring of Lb; ５ is an integer of 2 to 10, preferably an integer of 2 to 5.

[0161] N297-(Fuc)MSG1 is represented by the following structural formula or sequence formula:

[0162]

[0163]

[0164] In the above formula, the wavy line indicates binding to Asn297 of the antibody, and L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ -NH-, which indicates that the amino group at the right end of L (PEG) is amide-bonded to the carboxyl group at the 2-position of the sialic acid at the non-reducing end of the 1-3 chain side of the branched chain of β-Man in the N297 sugar chain; an asterisk indicates that the linker L, particularly the linker L, is bonded to the nitrogen atom at the 1- or 3-position on the 1,2,3-triazole ring of Lb; ５ is an integer of 2 to 10, preferably an integer of 2 to 5.

[0165] N297-(Fuc)MSG2 is represented by the following structural formula or sequence formula:

[0166]

[0167]

[0168] In the above formula, the wavy line indicates binding to Asn297 of the antibody, and L(PEG) is -(CH ２ -CH ２ -O)n ５ -CH２ -CH ２ -NH-, which indicates that the amino group at the right end of L (PEG) is amide bonded to the carboxyl group at the 2nd position of the sialic acid at the non-reducing end of the 1-6 chain side of the branched chain of β-Man in the N297 sugar chain; an asterisk indicates that the linker L, particularly the linker L, is bonded to the nitrogen atom at the 1st or 3rd position on the 1,2,3-triazole ring of Lb; ５ is an integer of 2 to 10, preferably an integer of 2 to 5.

[0169] 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 therefore the antibody-drug conjugate is a molecule to which four linkers L and four drugs D are bound (the above m ２ = 2) (see Figure 1A). That is, the number of drugs D bound to each heavy chain in the antibody-drug conjugate is two.

[0170] 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 therefore the antibody-drug conjugate is a molecule to which two linkers L and two drugs D are bound (the above-mentioned m ２ = 1) (see Figure 1B). That is, the number of drugs D bound to each heavy chain in the antibody-drug conjugate is one.

[0171] The N297 sugar chain is preferably N297-(Fuc)SG or N297-(Fuc)MSG1 or N297-(Fuc)MSG2, more preferably N297-(Fuc)SG or N297-(Fuc)MSG1, and even more preferably N297-(Fuc)SG.

[0172] 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, a highly homogeneous antibody-drug conjugate can be obtained.

[0173] <3. Production Method> A representative method for producing an antibody-drug conjugate containing a CDN derivative of the present invention or a production intermediate thereof will be described. Note that, hereinafter, the compound numbers shown in each reaction scheme will be used to indicate the compounds. That is, they will be referred to as "compound of formula (1)," "compound (1)," etc. Compounds with other numbers will also be referred to in the same manner.

[0174] In the following Methods A to E, the substituent L １ has the same meaning as defined above. ２ is the following (i) or (ii): (i) when combined with L, L ２ represents —NHR′, a hydroxy C1-C6 alkyl group, or an amino C1-C6 alkyl group, where R′ represents a hydrogen atom, a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, or a C3-C6 cycloalkyl group, and the C1-C6 alkyl group, C2-C6 alkenyl group, or C2-C6 alkynyl group is optionally substituted with 1 to 6 halogen atoms; or (ii) when not bound to L, L ２ represents a hydrogen atom or a halogen atom, and represents a group selected from the following. １ represents —NH— or a sulfur atom. ２ represents -CH=. １ ~Z ３ together, -CH ２ -CH ２ -CH ２ - represents the substituent R １ ~R ３ are each independently a hydrogen atom, a halogen atom, —OR′, —OC(═O)R′, —N ３ , —NHR′, —NR′R″, or —NHC(═O)R′ (wherein 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). ４ represents a hydrogen atom. ５ Is W １ is a nitrogen atom, R ５ indicates a hydrogen atom, and W １ is an oxygen atom, R ５is not present. ａ , R ｃ , R ｅ and R ｇ represents the side chain of a natural α-amino acid, such as a methyl group, an isopropyl group, a sec-butyl group, an isobutyl group, or a benzyl group. １ represents a protecting group for a primary alcohol. Preferred is a 4,4'-dimethoxytrityl group, a 4-methoxytrityl group, etc. ２ , PRO ３ , PRO ７ , PRO ８ represents a protecting group for a secondary alcohol. Preferred examples include a tert-butyldimethylsilyl group, a triisopropylsilyloxymethyl group, a benzoyl group, a 2-nitrobenzyl group, and a 4-methoxytetrahydropyran-4-yl group. ６ represents a protecting group for carboxylic acid. Preferred is a tert-butyl group, a benzyl group, etc. ５ , PRO ９ indicates an amine protecting group. ５ is preferably a tert-butyloxycarbonyl group, a 9-fluorenylmethyloxycarbonyl group, an allyloxycarbonyl group, a 2,2,2-trichloroethoxycarbonyl group, a benzyloxycarbonyl group, or the like, and PRO ９ is preferably a 9-fluorenylmethyloxycarbonyl group or a 2-(trimethylsilyl)ethoxycarbonyl group. ４ represents a protecting group for an alcohol or an amine. In the case of an alcohol, preferred protecting groups include a tert-butyldimethylsilyl group, a benzoyl group, etc., and in the case of an amine, preferred protecting groups include a 2-(trimethylsilyl)ethoxycarbonyl group, an allyloxycarbonyl group, a tert-butyloxycarbonyl group, etc. ａ represents an oxygen atom or a sulfur atom, and Q ｂ represents a hydroxyl group or a thiol group. ａ’ and Q ｂ’ are each independently a negatively charged oxygen atom (O － ) or a sulfur atom (S － ) indicates. ｘ and R ｙare each independently a halogen atom or —O—PRO ２ n represents an integer of 1 to 3.

[0175] Method A The CDN derivative represented by (1) used in the antibody-drug conjugate of the present invention can be produced according to Method A described below.

[0176]

[0177] This production method is a method for producing a compound represented by general formula (1). Steps A-1 to A-5 of this production method can be performed as a one-pot synthesis, which can be carried out by referring to the report by Gaffney et al. (Org. Lett. 2010, 12, 3269-3271).

[0178]

[0179] (Step A-1) This step is a step of producing a compound of formula (2a) by successively carrying out hydrolysis and removal of the cyanoethyl group on a compound of formula (1a) using known organic chemistry techniques. The hydrolysis reaction was 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 a temperature ranging from −10°C to the boiling point of the solvent used in the reaction, preferably from 15°C to 35°C. Relative to 1 mole of compound (1a), 2 moles to an excess of molar amounts, preferably 2 to 10 moles, of water was used, and 1 mole to an excess of molar amounts, preferably 1 to 5 moles, of acid was used. The reaction time was 1 minute to 3 hours, preferably 5 to 30 minutes. Next, a base (tert-butylamine, etc.) was added to the reaction solution to remove the cyanoethyl group. The base was used in excess molar amount, preferably 30 to 50 moles, relative to 1 mole of compound (1a). The reaction time was 5 minutes to 6 hours, preferably 15 minutes to 1 hour. The reaction solution was concentrated under reduced pressure to obtain crude compound (2a). The crude compound (2a) can be used in the next step without purification.

[0180] (Step A-2) This step is a step of removing the protecting group of the hydroxy group from the compound of formula (2a) using a known organic chemistry method to produce a compound of formula (3a). Before starting the reaction of this step, the crude product of formula (2a) was dried by azeotropy with acetonitrile one to three times, if necessary. １ When is a 4,4'-dimethoxytrityl group, compound (2a) was treated with water and an acid (dichloroacetic acid, trifluoroacetic acid, etc.) in a solvent (dichloromethane, chloroform, dichloroethane, etc.) at a temperature ranging from -10°C to the boiling point of the solvent used in the reaction, preferably from 15°C to 35°C, to remove the 4,4'-dimethoxytrityl group. Water was used in excess, preferably 10 to 20 moles, per mole of compound (2a). The acid was diluted with the solvent used in the reaction to a concentration of 1% to 50% (v / v), preferably 5% to 10% (v / v), and the diluted solution was used in excess, preferably 5 to 15 moles. The reaction time was 1 minute to 3 hours, preferably 5 to 30 minutes. The reaction was quenched by adding pyridine to the reaction solution. Pyridine was used in an amount sufficient to sufficiently neutralize the acid used, preferably 2 to 10 moles per mole of acid. The reaction solution was concentrated under reduced pressure to obtain crude compound (3a). The crude compound (3a) was azeotroped three to five times with dehydrated acetonitrile. The acetonitrile was left over from the final azeotropic distillation to obtain a 0.01 M to 1 M acetonitrile solution of compound (3a). The resulting acetonitrile solution was used directly in the next step.

[0181] (Step A-3) This step is a step for producing a compound of formula (5a) by sequentially carrying out a coupling reaction of a compound of formula (3a) with a compound of formula (4a) using known organic chemistry techniques and a sulfurization reaction of the resulting coupling product. Prior to the start of the reaction in this step, compound (4a) was azeotropically distilled three to five times with dehydrated acetonitrile. Acetonitrile was left over from the final azeotropic distillation to prepare a 0.01 M to 1 M acetonitrile solution of compound (4a). A desiccant (powdered or pelleted molecular sieves 3A or molecular sieves 4A) was added to this solution, and the solution was stored under a nitrogen or argon atmosphere until use. The coupling reaction was carried out by adding an acetonitrile solution of compound (4a) dried by azeotropy to an acetonitrile solution of compound (3a) at 5°C to 35°C. The reaction time was 1 minute to 24 hours, preferably 5 minutes to 6 hours. Next, a sulfurizing agent (N,N-dimethyl-N'-(3-sulfanylidene-3H-1,2,4-dithiazol-5-yl)methanimidamide, 3H-1,2-benzodithiol-3-one, etc.) was added to this reaction solution to carry out a sulfurization reaction. The sulfurizing agent was used in an amount of 1 to 5 moles, preferably 1 to 2 moles, per mole of compound (3a). The reaction time was 5 minutes to 24 hours, preferably 30 minutes to 6 hours. The reaction solution was concentrated under reduced pressure to obtain a crude product of compound (5a). The obtained crude product of compound (5a) was directly used in the next step.

[0182] (Step A-4) This step is a step in which the protecting group of the hydroxy group is removed from the compound of formula (5a) using a known organic chemistry method to produce a compound of formula (6a). １When the 4,4'-dimethoxytrityl group is a 4,4'-dimethoxytrityl group, compound (5a) was treated with water and an acid (dichloroacetic acid, trifluoroacetic acid, etc.) in a solvent (dichloromethane, chloroform, dichloroethane, etc.) at a temperature ranging from -10°C to the boiling point of the solvent used in the reaction, preferably from 15°C to 35°C, to remove the 4,4'-dimethoxytrityl group. Water was used in excess, preferably 10 to 20 moles, per mole of compound (5a). The acid was diluted with the solvent used in the reaction to a concentration of 1% to 50% (v / v), preferably 5% to 10% (v / v), and the diluted solution was used in excess, preferably 5 to 15 moles. The reaction time was 1 minute to 3 hours, preferably 5 to 30 minutes. The reaction was terminated by adding pyridine to the reaction solution. Pyridine was used in an amount sufficient to sufficiently neutralize the acid used, preferably 10 to 200 moles per mole of acid. The reaction mixture was concentrated under reduced pressure to give crude compound (6a), which was used directly in the next step.

[0183] (Step A-5) This step is a step of producing a compound of formula (7a) by successively carrying out a cyclization reaction and a sulfurization reaction on a compound of formula (6a) using known organic chemistry techniques. Compound (6a) was dissolved in pyridine and then concentrated under reduced pressure to prepare a 0.01 M to 0.5 M pyridine solution. This pyridine solution was added with a dehydration condensation agent (2-chloro-5,5-dimethyl-1,3,2λ) at 5°C to 35°C. ５The cyclization reaction was carried out by adding a dehydration condensation agent (e.g., 3H-1,2-benzodithiol-3-one, N,N-dimethyl-N'-(3-sulfanylidene-3H-1,2,4-dithiazol-5-yl)methanimidamide, etc.) to the reaction mixture. The dehydration condensation agent was used in an amount of 1 mole to an excess of 1 mole, preferably 3 to 5 moles, relative to 1 mole of compound (6a). The reaction time was 1 minute to 6 hours, preferably 5 minutes to 1 hour. Next, water and a sulfurizing agent (e.g., 3H-1,2-benzodithiol-3-one, N,N-dimethyl-N'-(3-sulfanylidene-3H-1,2,4-dithiazol-5-yl)methanimidamide, etc.) were added to the reaction mixture to carry out a sulfurization reaction. Relative to 1 mole of compound (6a), water was used in an excess of 1 mole, preferably 30 to 50 moles, and the sulfurizing agent was used in an amount of 1 mole to 5 moles, preferably 1 to 2 moles. The reaction time was 5 minutes to 12 hours, preferably 30 minutes to 3 hours. The reaction mixture was added to an aqueous sodium bicarbonate solution (0.1 M to 1 M), and the mixture was stirred for 15 minutes to 24 hours to terminate the reaction. The reaction mixture was extracted one to five times with an organic solvent (ethyl acetate, diethyl ether, toluene, or a mixture thereof), and the combined extracts were dried over anhydrous sodium sulfate or anhydrous magnesium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting 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) as a mixture of two or more diastereomers or two or more pure diastereomers. In most cases, this step yields two diastereomers, but depending on the starting materials (1a) and (4a), one or two additional diastereomers may also be obtained. Even if the resulting compound (7a) is a mixture of multiple diastereomers, it can be carried on to the next step without further purification.

[0184] (Step A-6) In this step, the cyanoethyl group and all acyl protecting groups are simultaneously removed from the compound of formula (7a) using a known organic chemistry method to produce the compound of formula (8a). This step was carried out in an autoclave or a sealed tube, as necessary. ４When is a benzoyl group, compound (7a) was treated with 28% (v / v) aqueous ammonia in a solvent (methanol, ethanol, tetrahydrofuran, or a mixture thereof) at a temperature between 5°C and the boiling point of the solvent used in the reaction to remove the cyanoethyl and benzoyl groups. Ammonia was used in excess molar amount, preferably 300 to 3,000 moles, per mole of compound (7a). The reaction time was 30 minutes to 96 hours, preferably 2 to 48 hours. If necessary, the reaction solution was concentrated, and the residue was 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 (8a). Even if the resulting compound (8a) is a diastereomeric mixture, it can be advanced to the next step without further purification. Alternatively, it can be advanced to the next step without further purification in this step.

[0185] (Step A-7) This step is a step in which all silyl-based protecting groups are simultaneously removed from the compound of formula (8a) using a known organic chemistry method to produce a compound of formula (9a). ２ and PRO ３When is a tert-butyldimethylsilyl group, compound (8a) was directly treated with triethylamine trihydrofluoride at 5°C to 100°C, preferably 35°C to 60°C, to remove the tert-butyldimethylsilyl group. Triethylamine trihydrofluoride was used in excess molar amount, preferably 100 to 200 moles, per mole of compound (8a). The reaction time was 30 minutes to 24 hours, preferably 2 to 12 hours. After cooling the reaction solution to room temperature, an ice-cooled mixture of 1 M aqueous triethylammonium bicarbonate and triethylamine (3:1 to 10:1 (v / v)) was gradually poured into the reaction solution to terminate the reaction. If necessary, the reaction solution may be poured into an ice-cooled mixture of 1 M aqueous triethylammonium bicarbonate and triethylamine. In this case, the reaction vessel was washed with acetonitrile and water. Triethylamine is used in an amount sufficient to change the pH of the reaction solution to weak basicity, preferably about 2 moles of triethylamine per mole of triethylamine trihydrofluoride. After the organic solvent component of the reaction solution was distilled off under reduced pressure, the remaining aqueous solution was 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.

[0186] (Step A-8) This step involves ion-exchanging the compound of formula (9a) using a known organic chemistry technique to produce the compound of formula (1). A cation exchange resin (BT AG (registered trademark) 50W-X2 resin, 100-200 mesh, hydrogen type) was suspended in pure water and packed into an empty column cartridge. The amount of cation exchange resin used was 10 to 50 times the weight of the compound (9a). After allowing excess pure water to flow by gravity, 3 column volumes of 1 M aqueous sodium hydroxide were allowed to flow by gravity, followed by 6 column volumes of pure water. Compound (9a) was dissolved in approximately 3 column volumes of pure water and charged onto the column. If the compound is poorly soluble in pure water, a mixture with a small amount of organic solvent (acetonitrile, methanol, etc.) may be used. The solution that flowed by gravity was collected and then eluted with 6 column volumes of pure water, etc., to collect the fractions. The fractions containing the target product were combined and lyophilized to obtain compound (1) as a single diastereomer.

[0187] Method A' The CDN derivative represented by (1') used in the antibody-drug conjugate of the present invention can be produced according to Method A' described below.

[0188]

[0189] This production method is a method for producing a compound represented by general formula (1') by partially modifying Method A. Specifically, the compound of general formula (1') can be produced by modifying step A-5 of Method A to step A'-5 shown below. ｘ and R ｙ When both are halogen atoms, step A-7 can be omitted.

[0190]

[0191] (Step A'-5) This step is a step of producing a compound of formula (7a') by successively carrying out a cyclization reaction and an oxidation reaction on the compound of formula (6a') using known organic chemistry techniques. Compound (6a') was dissolved in pyridine and then concentrated under reduced pressure to prepare a 0.01M to 0.5M pyridine solution. This pyridine solution was added with a dehydration condensation agent (2-chloro-5,5-dimethyl-1,3,2λ) at 5°C to 35°C. ５A cyclization reaction was carried out by adding an oxidizing agent (e.g., dioxaphosphinan-2-one) to the reaction mixture. The dehydration condensation agent was used in an amount of 1 mole to an excess mole, preferably 3 to 5 moles, relative to 1 mole of compound (6a'). The reaction time was 1 minute to 6 hours, preferably 5 minutes to 1 hour. Next, water and an oxidizing agent (e.g., iodine) were added to the reaction mixture to carry out an oxidation reaction. Water was used in an amount of 0 mole to an excess mole, preferably 30 to 50 moles, relative to 1 mole of compound (6a'), and the oxidizing agent was used in an amount of 2 moles to 10 moles, preferably 3 to 5 moles. The reaction time was 5 minutes to 12 hours, preferably 30 minutes to 3 hours. The reaction mixture was added to an aqueous sodium bicarbonate solution (0.1 M to 1 M) and stirred for 15 minutes to 24 hours to terminate the reaction. The reaction mixture was extracted once to five times with an organic solvent (ethyl acetate, diethyl ether, toluene, or a mixture thereof), and the combined extracts were dried over anhydrous salts (anhydrous sodium sulfate or anhydrous magnesium sulfate). The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure. The resulting 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').

[0192] Method A″ The CDN derivative represented by (1″) used in the antibody-drug conjugate of the present invention can be produced according to Method A″ described below.

[0193]

[0194] This production method is a method for producing a compound represented by general formula (1″) by partially modifying Method A. Specifically, the compound of general formula (1″) can be produced by modifying step A-3 of Method A to step A″-3 shown below. In addition, when the substituent R ｘ and R ｙ When both are halogen atoms, step A-7 can be omitted.

[0195]

[0196] (Step A''-3) This step is a step for producing a compound of formula (5a'') by sequentially carrying out a coupling reaction of a compound of formula (3a'') with a compound of formula (4a'') using a known organic chemistry method and an oxidation reaction of the resulting coupling product. Before starting the reaction of this step, compound (4a'') was azeotropically distilled three to five times with dehydrated acetonitrile. Acetonitrile was left over from the final azeotropic distillation to prepare a 0.01 M to 1 M acetonitrile solution of compound (4a''). A desiccant (powdered or pelleted molecular sieves 3A or molecular sieves 4A) was added to this solution, and the solution was stored under a nitrogen or argon atmosphere until use. The coupling reaction was carried out by adding the acetonitrile solution of compound (4a'') dried by azeotropy to the acetonitrile solution of compound (3a'') at 5 to 35°C. The reaction time is 1 minute to 24 hours, preferably 5 minutes to 6 hours. Next, an oxidizing agent (such as tert-butyl hydroperoxide) was added to this reaction solution to carry out an oxidation reaction. The oxidizing agent was used in an amount of 1 to 5 moles, preferably 2 to 3 moles, per mole of compound (3a″). The reaction time was 5 minutes to 24 hours, preferably 30 minutes to 6 hours. A saturated aqueous solution of sodium thiosulfate was added to the reaction solution, and the mixture was stirred for 10 minutes to 12 hours to quench the reaction. The reaction solution was extracted once to five times with an organic solvent (such as a mixed solvent of dichloromethane and methanol), and the combined extracts were dried over anhydrous salt (anhydrous sodium sulfate or anhydrous magnesium sulfate). The drying agent was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain crude compound (5a″). The crude compound (5a″) obtained was directly used in the next step.

[0197] Method A''' The CDN derivative represented by (1''') used in the antibody-drug conjugate of the present invention can be produced according to Method A''' described below.

[0198]

[0199] This production method is a method for producing a compound represented by general formula (1''') by partially modifying Method A. Specifically, the compound of general formula (1''') can be produced by changing step A-3 of Method A to step A''-3 and step A-5 to step A'-5. In addition, ｘ and R ｙ When both are halogen atoms, step A-7 can be omitted.

[0200]

[0201] Method B: Conjugation Precursor (Sugar Chain Conjugation) The conjugation precursor represented by (2) used in the antibody-drug conjugate of the present invention can be produced according to Method B described below.

[0202]

[0203] This manufacturing method is L １ -NH at any position ２ is a method for preparing the conjugation precursor (2) where:

[0204]

[0205] (Step B-1) 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 chemistry method. ５When is a tert-butyloxycarbonyl group, the protecting group was removed by treating compound (1b) with trifluoroacetic acid in a solvent (dichloromethane, dioxane, acetonitrile, ethyl acetate, tetrahydrofuran, or a mixed solvent thereof) at a temperature ranging from −10°C to the boiling point of the solvent used in the reaction, preferably from 15°C to 35°C. Trifluoroacetic acid was used in excess molar amount, preferably 20 to 50 moles, per mole of compound (1b). The reaction time ranged from 5 minutes to 24 hours, preferably 30 minutes to 6 hours. The reaction solution was concentrated under reduced pressure, suspended in toluene, and then concentrated again under reduced pressure. This procedure was repeated two to five times. A solvent (diethyl ether, diisopropyl ether, hexane, dichloromethane, ethyl acetate, or a mixed solvent thereof) was added to form a slurry, and the solid was collected by filtration to obtain crude compound (2b). The crude compound (2b) was carried to the next step without further purification.

[0206] (Step B-2) 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 chemistry method. The amidation was 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. Relative to 1 mole of compound (2b), 1 mole to 5 moles of base and 0.5 moles to 1.5 moles of compound (3b) were used. The reaction time was 10 minutes to 72 hours, preferably 1 hour to 24 hours. The reaction solution was poured into a mixture of two layers: an organic solvent (dichloromethane, chloroform, ethyl acetate, methanol, or a mixture thereof) and water or an acidic aqueous solution (e.g., 0.1 to 1 M hydrochloric acid, citric acid, etc.), and extracted with the organic solvent one to five times. The combined extracts were washed with saturated saline and then dried over anhydrous salts (anhydrous sodium sulfate or anhydrous magnesium sulfate). The desiccant was filtered off, and the filtrate was concentrated under reduced pressure. Alternatively, the above-mentioned separation procedure can be omitted, and the reaction solution can be directly concentrated under reduced pressure and subjected to subsequent silica gel column purification. The resulting residue was purified by silica gel column chromatography [dichloromethane / methanol, ethyl acetate / methanol, etc.] to obtain compound (4b). If necessary, the purity can be increased by dissolving the obtained compound (4b) in a good solvent (e.g., ethyl acetate, acetonitrile, dichloromethane, methanol, or a mixture thereof) and then reprecipitating it by adding a poor solvent (e.g., diethyl ether, diisopropyl ether, hexane), and filtering the solid.

[0207] (Step B-3) This step is a step of producing a compound of formula (5b) by esterifying the compound of formula (4b) using a known organic chemistry method. Esterification was 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. N-hydroxysuccinimide and the condensing agent were used in amounts of 1 mol to 3 mol per mol of compound (4b). The reaction time was 30 minutes to 72 hours, preferably 2 hours to 24 hours. The reaction solution was diluted with an organic solvent (dichloromethane, chloroform, ethyl acetate, or a mixture thereof) and then washed with ice water three to five times. The organic layer was dried over anhydrous salts (anhydrous sodium sulfate or anhydrous magnesium sulfate). After filtering off the drying agent, the filtrate was concentrated under reduced pressure to obtain crude compound (5b). If necessary, the resulting compound (5b) may be purified by C18 silica gel column chromatography (acetonitrile only). Alternatively, the purity can be increased by dissolving the resulting compound (5b) in a good solvent (ethyl acetate, acetonitrile, dichloromethane, or a mixture thereof) and then adding a poor solvent (diethyl ether, diisopropyl ether, hexane, etc.) to reprecipitate the solid, and then filtering the solid.

[0208] (Step B-4) This step is a step of producing a compound of formula (2) by condensing a compound of formula (5b) with a compound of formula (6b) using a known organic chemistry method. The condensation reaction was 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 a temperature of −10°C to 100°C, preferably 15°C to 35°C. Relative to 1 mole of compound (6b), 2 to 5 moles of base and 1 to 2 moles of compound (5b) were used. The reaction time was 5 minutes to 24 hours, preferably 1 to 6 hours. The reaction was terminated by adding benzylamine to the reaction solution. Relative to 1 mole of compound (6b), 4 to 10 moles of benzylamine were used. If necessary, the reaction solution was partially concentrated under reduced pressure, and the remaining solution was purified by preparative HPLC [buffer solution / acetonitrile, buffer solution / methanol, etc.], C18 silica gel column chromatography [buffer solution / acetonitrile, buffer solution / methanol, etc.], or a combination thereof to obtain compound (2).

[0209] Method B': Conjugation Precursor (Cysteine ​​Conjugation) The conjugation precursor represented by (2') used in the antibody-drug conjugate of the present invention can be produced according to Method B' described below.

[0210]

[0211] This manufacturing method is L １ -NH at any position ２ is a method for producing a conjugation precursor (2') in which:

[0212]

[0213] (Step B-5) 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 chemistry method. Compound (8b) was obtained according to the method described in Step B-2 of Method B, except that a base was not used.

[0214] (Step B-6) 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 chemistry method. ６ When is a tert-butyl group, 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 purification.

[0215] (Step B-7) This step is a step of producing a compound of formula (10b) by esterifying the compound of formula (9b) using a known organic chemistry method. Compound (10b) was obtained according to the method described in Step B-3 of Method B.

[0216] (Step B-8) This step is a step of producing a compound of formula (2') by condensing a compound of formula (6b) with a compound of formula (10b) using a known organic chemistry method. Compound (2') was obtained according to the method described in Step B-4 of Method B.

[0217] Method C The conjugation precursor represented by (3) used in the antibody-drug conjugate of the present invention can be produced according to Method C described below.

[0218]

[0219] This manufacturing method is L １ The present invention relates to a method for producing a conjugation precursor (3) in which a hydroxy group is substituted at any position of the above.

[0220]

[0221] (Step C-1) This step is a step of producing a compound of formula (3c) by amidating a compound of formula (1c) with a compound of formula (2c) using a known organic chemistry method. Compound (3c) was obtained according to the method described in Step B-2 of Method B.

[0222] (Step C-2) This step is a step of producing a compound of formula (4c) by esterifying the compound of formula (3c) using a known organic chemistry method. Compound (4c) was obtained according to the method described in Step B-3 of Method B.

[0223] (Step C-3) This step is a step for producing a compound of formula (7c) by sequentially carrying out a coupling reaction (aminomethylenation) of a compound of formula (5c) with a compound of formula (6c) using a known organic chemistry technique and deprotecting the resulting coupling product. ９ When R is a 9-fluorenylmethyloxycarbonyl group, aminomethylenation was carried out by reacting compound (5c) with compound (6c) and an acid (e.g., p-toluenesulfonic acid) in tetrahydrofuran at 5°C to 35°C. Relative to 1 mole of compound (5c), 1 to 20 moles, preferably 2 to 10 moles, of compound (6c) were used, and 0.05 moles to an excess molar amount, preferably 0.1 to 3 moles, of acid were used. The reaction time was 30 minutes to 72 hours, preferably 2 to 24 hours. Next, a base (e.g., 1,8-diazabicyclo[5.4.0]-7-undecene) was added to the reaction mixture to carry out deprotection. If the reaction mixture was in suspension, a solvent (e.g., N,N-dimethylformamide) could be added as needed to dissolve the mixture before the reaction. The base was used in excess molar amount, preferably 5 to 20 moles, relative to 1 mole of compound (5c). The reaction time is 10 minutes to 24 hours, preferably 2 hours to 12 hours. Water is added to the reaction mixture, and the mixture is purified directly by C18 silica gel column chromatography [buffer / acetonitrile, etc.] to obtain compound (7c).

[0224] (Step C-4) In this step, the protecting group is removed from the compound of formula (7c) using a known organic chemistry method to produce a compound of formula (8c).７ and PRO ８ When is a tert-butyldimethylsilyl group, compound (8c) was obtained according to the method described in Method A, Step A-7.

[0225] (Step C-5) This step is a step of producing a compound of formula (3) by condensing a compound of formula (8c) with a compound of formula (4c) using a known organic chemistry method. Compound (3) was obtained according to the method described in Step B-4 of Method B.

[0226] Method C' The conjugation precursor represented by (3') used in the antibody-drug conjugate of the present invention can be prepared according to Method C' described below.

[0227]

[0228] This manufacturing method is L １ The present invention relates to a method for producing a conjugation precursor (3') in which a hydroxy group is substituted at any position of the above.

[0229]

[0230] (Step C'-1) This step is a step of producing a compound of formula (2c') by successively carrying out hydrolysis and removal of the cyanoethyl group on a compound of formula (1c') using known organic chemistry techniques. Compound (2c') was obtained according to the method described in Step A-1 of Method A.

[0231] (Step C'-2) This step is a step of removing the protecting group of the hydroxy group from the compound of formula (2c') using a known organic chemistry method to produce a compound of formula (3c'). Compound (3c') was obtained according to the method described in Step A-2 of Method A.

[0232] (Step C'-3) This step is a step for producing a compound of formula (5c') by sequentially carrying out a coupling reaction of a compound of formula (3c') with a compound of formula (4c') using known organic chemistry techniques and a sulfurization reaction or oxidation reaction of the resulting coupling product. Compound (5c') was obtained according to the method described in Step A-3 of Method A or Step A''-3 of Method A''.

[0233] (Step C'-4) This step is a step of removing the protecting group of the hydroxy group from the compound of formula (5c') using a known organic chemistry method to produce a compound of formula (6c'). Compound (6c') was obtained according to the method described in Step A-4 of Method A.

[0234] (Step C'-5) This step is a step in which a compound of formula (7c') is produced by successively carrying out a cyclization reaction and a sulfurization reaction or an oxidation reaction on a compound of formula (6c') using known organic chemistry techniques. Compound (7c') was obtained according to the method described in Step A-5 of Method A or Step A'-5 of Method A'.

[0235] (Step C'-6) This step is a step in which the cyanoethyl group and all acyl protecting groups are simultaneously removed from the compound of formula (7c') using a known organic chemistry method to produce the compound of formula (8c'). Compound (8c') was obtained according to the method described in Step A-6 of Method A.

[0236] (Step C'-7) This step is a step in which all silyl protecting groups are simultaneously removed from the compound of formula (8c') using a known organic chemistry method to produce a compound of formula (9c'). ９ When R is a 2-(trimethylsilyl)ethoxycarbonyl group, compound (8c') was treated with a tetrahydrofuran solution of tetrabutylammonium fluoride at 5°C to 100°C, preferably 35°C to 60°C, to remove the 2-(trimethylsilyl)ethoxycarbonyl group. Tetrabutylammonium fluoride was used in excess molar amount, preferably 10 to 30 moles, per mole of compound (8c'). The reaction time was 1 to 48 hours, preferably 4 to 24 hours. After diluting the reaction solution with a buffer solution, the organic solvent component was distilled off under reduced pressure as needed. The residue was purified by preparative HPLC [buffer solution / acetonitrile, buffer solution / methanol, etc.], C18 silica gel column chromatography [buffer solution / acetonitrile, buffer solution / methanol, etc.], or a combination thereof to obtain compound (9c').

[0237] (Step C'-8) This step is a step of producing a compound of formula (3') by condensing a compound of formula (9c') with a compound of formula (4c) using a known organic chemistry method. Compound (3') was obtained according to the method described in Step B-4 of Method B.

[0238] Method D: Production of Glycosylation Remodeling Antibodies Glycosylation remodeling antibodies can be produced by the method shown in the following scheme, for example, in accordance with the methods described in WO2018 / 003983, WO2020 / 050406, WO2021 / 177438, WO2022 / 050300, PLos ONE 2018, 13, e0193534, etc.

[0239]

[0240] (Step D-1) This step involves hydrolyzing and cleaving the glycosidic bond between GlcNAcβ1-4GlcNAc in the reducing-end chitobiose structure of the N-linked glycan (N297-linked glycan) attached to asparagine at position 297 in the antibody heavy chain using a known enzymatic reaction to produce a glycosylated antibody. The target antibody (1d) (10 mg / mL) was placed in a buffer solution (e.g., phosphate buffer) at 0°C to 40°C, and the glycosidic bond between GlcNAcβ1 and 4GlcNAc in the reducing-end chitobiose structure was hydrolyzed using a hydrolase such as wild-type EndoS enzyme. The reaction time was 10 minutes to 72 hours, preferably 1 hour to 6 hours. The wild-type EndoS enzyme was used in an amount of 0.1 mg to 10 mg, preferably 0.1 mg to 3 mg, per 100 mg of antibody (1d). After completion of the reaction, the reaction mixture was purified by affinity chromatography (HiTrap rProtein A FF (5 ml) (GE Healthcare)) and / or a hydroxyapatite column (Bio-Scale Mini CHT Type I cartridge (5 ml) (BIO-RAD)) to obtain (Fucα1,6)GlcNAc antibody (2d).

[0241] (Step D-2) This step is a step of producing a glycan remodeling antibody (3d) by binding an SG-type or MSG (MSG1, MSG2)-type glycan oxazoline derivative having an azide group-containing PEG linker (hereinafter referred to as "azidoglycan oxazoline derivative") to the (Fucα1,6)GlcNAc antibody (2d) obtained in Step D-1 using a known enzymatic reaction.

[0242] Antibody (2d) was reacted with an azido-oligosaccharide-linked oxazoline derivative in the presence of a glycosyltransferase such as EndoS (D233Q / Q303L) in a buffer solution (e.g., phosphate buffer) at 0°C to 40°C to carry out the glycosyltransferase reaction. The reaction time was 10 minutes to 72 hours, preferably 1 hour to 6 hours. EndoS enzyme (D233Q / Q303L) was used in an amount of 1 mg to 10 mg, preferably 1 mg to 3 mg, per 100 mg of antibody, and the azido-oligosaccharide-linked oxazoline derivative was used in an amount of 2 equivalents to an excess equivalent, preferably 4 equivalents to 20 equivalents. After completion of the reaction, the product was purified by affinity chromatography (HiTrap rProtein A FF (5 ml) (GE Healthcare)) and a hydroxyapatite column (Bio-Scale Mini CHT Type I cartridge (5 ml) (BIO-RAD)) to obtain a sugar chain remodeling antibody (3d).

[0243] In the preparation of the above-mentioned sugar chain remodeling antibody, concentration of the aqueous antibody solution, concentration measurement, and buffer exchange can be carried out according to the common procedures A to C described below.

[0244] The SG-type azide sugar chain oxazoline compound was synthesized according to the method described in WO2018 / 003983. ３ -PEG(3)] ２ The synthesis method of -SG(10)-Ox (compound 1-10 described in WO2018 / 003983) is shown in the following scheme.

[0245]

[0246] The MSG-type azidoglycan oxazoline derivative was also synthesized according to the method described in WO2018 / 003983. ３-PEG(3)]-MSG1(9)-Ox (compound 1-11 described in WO2018 / 003983) is synthesized as follows.

[0247]

[0248] (Step D-3) This step is a step of producing a glycosylated remodeling antibody (3d) by subjecting the (Fucα1,6)GlcNAc antibody (2d) obtained in Step D-1 to a glycosylation reaction using two types of Endo enzymes. By using two types of enzymes simultaneously, glycosylation can be directly performed on the N297 glycosylation of the antibody using a glycosylation donor such as SGP or (SG)Asn, whose reducing end is not activated.

[0249] Regarding the two types of Endo enzymes used, Enzyme-B (EndoM-like enzyme) and Enzyme-A (EndoS-like enzyme) can be suitably combined.

[0250] Examples of enzyme-B include EndoM, EndoOm, EndoCC, and EndoM mutants, EndoOm mutants, and EndoCC mutants with reduced hydrolytic activity. Preferred enzyme-B are EndoM N175Q, EndoCC N180H, and EndoOm N194Q.

[0251] Examples of enzyme-A include EndoS, EndoS2 (EndoS49), and EndoS mutants with reduced hydrolytic activity, EndoS2 (EndoS49) mutants, etc. Preferred enzyme-A include EndoS D233Q, EndoS D233Q / Q303L, EndoS D233Q / E350A, EndoS D233Q / E350Q, EndoS D233Q / E350D, EndoS D233Q / E350N, EndoS D233Q / D405A, EndoS2 D184M, and EndoS2 T138Q.

[0252] The sugar chain donor is ([N ３ -PEG(3)] ２ -SG)-Asn-PEG(3)-N ３ , [N ３ -PEG(3)]-MSG1-Asn-PEG(3)-N ３、[N ３ -PEG(3)]-MSG2-Asn-PEG(3)-N ３ etc. can be used.

[0253] The antibody (2d) is dissolved in a buffer solution (such as Tris buffer) in the presence of glycosyltransferases Enzyme-B (EndoM-like enzyme) and Enzyme-A (EndoS-like enzyme) ([N ３ -PEG(3)] ２ -SG)-Asn-PEG(3)-N ３ A glycosyltransfer reaction is carried out by reacting the glycan with the glycan remodeling antibody (3d). The reaction temperature can be selected appropriately depending on the optimum temperature of the enzyme used, but is usually 15 to 50°C, preferably 25 to 40°C. The reaction time can be selected appropriately between 2 and 48 hours. After completion of the reaction, a purification method (affinity chromatography, hydroxyapatite column, etc.) or ultrafiltration method (ultrafiltration membrane) appropriate for the reaction scale can be selected to obtain the glycan remodeling antibody (3d).

[0254] In the preparation of the above-mentioned sugar chain remodeling antibody, concentration of the aqueous antibody solution, concentration measurement, and buffer exchange can be carried out according to the common procedures A to C described below.

[0255] In addition, ([N ３ -PEG(3)] ２ -SG)-Asn-PEG(3)-N ３ is synthesized according to the method described in WO2018 / 003983. In step 1-2A described in WO2018 / 003983, a free form of Fmoc-(SG-)Asn prepared from Fmoc-(SG-)Asn (1S2S-11NC-Asn-Fmoc, manufactured by Glycotechnology Institute) is reacted with 11-azido-3,6,9-trioxaundecan-1-amine to obtain [N ３ -PEG(3)] ２ -SG)-Asn-PEG(3)-N ３ (Compounds 1-13 described in WO2018 / 003983) can be obtained.

[0256] MSG-type sugar chain donor [N ３ -PEG(3)]-MSG1-Asn-PEG(3)-N ３、 [N ３-PEG(3)]-MSG2-Asn-PEG(3)-N ３ can also be synthesized in accordance with the method described in steps 1 to 3 of Example 154 of WO2019065964.

[0257] Step D-3 can also be carried out in accordance with the description in <5. Method for producing Fc-containing molecules> below.

[0258] Method E: Antibody-drug conjugation (glycoconjugation 1)

[0259] (Here, the two asterisks (*) on the left side of antibody-drug conjugate (1e) represent the drug linker moiety indicated by the asterisk on the right side.) This production method is a method for producing antibody-drug conjugate (1e) by binding the glycosylation remodeling antibody (3d) obtained in step D-2 of Method D with the conjugation precursor (2) obtained in step B-4 of Method B via the SPAAC (strain-promoted azide-alkyne cycloaddition: J. Am. Chem. Soc. 2004, 126, 15046-15047) reaction.

[0260] (Step E-1) The SPAAC reaction was carried out by mixing a buffer solution of the sugar chain remodeling antibody (3d) (phosphate buffer, acetate buffer, borate buffer, etc.) with a solution of the conjugation precursor (2) dissolved in an appropriate solvent (dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, propylene glycol, or a mixture thereof). The conjugation precursor (2) is present in an amount of 2 moles to a molar excess, preferably 4 to 30 moles, per mole of the sugar chain remodeling antibody (3d). The ratio of the organic solvent to the antibody buffer solution is preferably 1% to 200% (v / v). 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 was purified according to the method described in Common Procedure D below to obtain the antibody-drug conjugate (1e).

[0261] Method E': Conjugation of Antibody and Drug (Cysteine ​​Conjugation) The antibody-drug conjugate of the present invention having a cysteine ​​conjugation can be produced in accordance with the method described in WO2014 / 057687, etc., using the antibody of interest prepared according to Reference Example 3, etc., and the conjugation precursor (2') having a maleimide group obtained in Step B-8 of Method B'.

[0262] Method E″: Conjugation of antibody and drug (sugar chain conjugation 2) In Method E, the conjugation precursor (2) was changed to the conjugation precursor (3′) obtained in Step C′-8 of Method C′, thereby obtaining the antibody-drug conjugate (1e″) shown in the following formula.

[0263] (where the two asterisks (*) on the left side of the antibody drug conjugate (1e'') １ ) indicates the drug linker moiety indicated by an asterisk on the right.

[0264] The antibody-drug conjugate can be identified by buffer exchange, purification, measurement of antibody concentration, and measurement of the average number of drugs bound per antibody molecule according to the common procedures D to G described below.

[0265] Method F: Production of antibodies containing mutant Fc regions Antibodies containing mutant Fc regions can be produced by known methods using an Fc effector-less mutant animal cell expression vector. Methods for constructing an Fc effector-less mutant animal cell expression vector include, for example, the following methods.

[0266] Construction of Fc effectorless mutant animal cell expression vectors Using an animal cell expression vector containing a wild-type Fc region gene as a template, the desired effectorless mutation is introduced into the Fc region by PCR using the KOD-Plus-Mutagenesis Kit (TOYOBO Co., Ltd.). Alternatively, the Fc region gene containing the desired effectorless mutation may be synthesized and then incorporated into the animal cell expression vector by ligation.

[0267] The antibody-drug conjugate containing the CDN derivative of the present invention or a production intermediate thereof can also be produced with reference to WO2022163846. Furthermore, molecules of the present invention that contain a target molecule-binding portion, a mutant Fc region, and an immunostimulant, and that contain an immunostimulant other than the CDN derivative, can be produced by appropriately modifying the production method described above with reference to known methods, or by referring to known methods, for example.

[0268] Common Procedure A: Concentration of Aqueous Antibody Solution The antibody or antibody-drug conjugate solution was placed in an Amicon (registered trademark) Ultra centrifugal filter device (50,000 NMWL, Merck Millipore Ltd.), and the antibody or 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.).

[0269] Common Procedure B: Measurement of Antibody Concentration Antibody concentrations were measured using a UV meter (Nanodrop 1000, Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. －１ cm －１ to 1.8 mL mg －１ cm －１ ) was used.

[0270] Common Procedure C: Buffer Exchange of Antibody A buffer solution (phosphate buffered saline (pH 6.0), phosphate buffer (pH 6.0), etc.) was added to an aqueous antibody solution, and the solution was concentrated according to the method described in Common Procedure A. After repeating this procedure several times, the antibody concentration was measured according to the method described in Common Procedure B. An appropriate buffer solution (phosphate buffered saline (pH 6.0), phosphate buffer (pH 6.0), etc.) was added to this antibody buffer solution to prepare an antibody buffer solution of the desired concentration (for example, about 10 mg / mL).

[0271] Common Procedure D: Purification of Antibody-Drug Conjugate (Gel Filtration Chromatography) A NAP column (NAP-5, NAP-10, NAP-25 (manufactured by GE Healthcare)) was equilibrated with acetate buffer (10 mM acetate buffer, 5% sorbitol, pH 5.5; referred to herein as ABS) or another appropriate buffer. The antibody-drug conjugate reaction solution was charged onto this NAP column, and a manufacturer-specified amount of buffer was allowed to flow down by gravity, and the antibody fraction was collected. This fraction was again charged onto the NAP column, and a manufacturer-specified amount of buffer was allowed to flow down by gravity, and the antibody fraction was collected. This procedure was repeated two to three times in total to obtain an antibody-drug conjugate from which unbound drug linker, dimethyl sulfoxide, and propylene glycol had been removed. If necessary, the concentration of the antibody-drug conjugate solution was adjusted by common procedures A and C.

[0272] Common Procedure E: Measurement of Antibody Concentration in Antibody-Drug Conjugate and Average Number of Drugs Bound per Antibody Molecule (UV Method) The concentration of bound drug in an antibody-drug conjugate can be calculated by measuring the absorbance of an aqueous antibody-drug conjugate solution at two wavelengths, 280 nm and 250 nm, using an absorptiometer (UV / VIS Spectrometer Lambda 25, PerkinElmer, Inc.), and then performing the following calculation. Since 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), assuming that there is no change in the molar extinction coefficients of the antibody and drug before and after conjugation of the antibody and drug, the antibody concentration and drug concentration in the antibody-drug conjugate are expressed by the following relationship: A ２８０ = A Ｄ , ２８０ +A Ａ , ２８０ = ε Ｄ , ２８０ C Ｄ +ε Ａ , ２８０ C Ａ Formula (I) A ２５０ = A Ｄ , ２５０ +A Ａ , ２５０ = ε Ｄ, ２５０ C Ｄ +ε Ａ , ２５０ C Ａ Formula (II) where A ２８０ indicates the absorbance of an aqueous solution of an antibody-drug conjugate at 280 nm, and A ２５０ indicates the absorbance of an aqueous solution of an antibody-drug conjugate at 250 nm, and A Ａ , ２８０ indicates the absorbance of the antibody at 280 nm, and A Ａ , ２５０ indicates the absorbance of the antibody at 250 nm, and A Ｄ , ２８０ denotes the absorbance of the conjugate precursor at 280 nm, and A Ｄ , ２５０ denotes the absorbance of the conjugate precursor at 250 nm, and ε Ａ , ２８０ denotes the molar extinction coefficient of the antibody at 280 nm, and ε Ａ , ２５０ denotes the molar extinction coefficient of the antibody at 250 nm, and ε Ｄ , 280 denotes the molar extinction coefficient of the conjugate precursor at 280 nm, and ε Ｄ , ２５０ denotes the molar extinction coefficient of the conjugate precursor at 250 nm, and C Ａ indicates the antibody concentration in the antibody drug conjugate, and C Ｄ denotes the drug concentration in the antibody-drug conjugate, where ε Ａ , ２８０ , ε Ａ , ２５０ , ε Ｄ , ２８０ , ε Ｄ , ２５０ A value prepared in advance (calculated estimated value or actual measured value) is used for ε. Ａ , ２８０ can be estimated from the amino acid sequence of the antibody by a known calculation method (Protein Science, 1995, vol. 4, 2411-2423). Ａ , ２５０ is the difference between the measured value obtained from UV measurement of the antibody and ε Ａ , ２８０In the examples, the molar extinction coefficient of anti-EGFR antibody 1 was calculated as ε Ａ , ２８０ = 203460 and ε Ａ , ２５０ The molar extinction coefficient of anti-EGFR antibody 2 was ε = 70151 or 63837. Ａ , ２８０ = 203460 and ε Ａ , ２５０ = 63051 or 63370 was used. The molar extinction coefficient of anti-EGFR antibody 3 was ε Ａ , ２８０ = 203460 and ε Ａ , ２５０ The molar extinction coefficient of anti-EGFR antibody A was ε = 62692 or 63853. Ａ , ２８０ = 203460 and ε Ａ , ２５０ The molar extinction coefficient of anti-EGFR antibody B was ε Ａ , ２８０ = 197750 and ε Ａ , ２５０ The molar extinction coefficient of anti-CDH6 antibody 1 was ε Ａ , ２８０ = 223400 and ε Ａ , ２５０ = 74671 or 71530 was used. Ｄ , ２８０ and ε Ｄ , ２５０ The A of the antibody-drug conjugate solution was measured using the Beer-Lambert law (absorbance = molar concentration × molar extinction coefficient × cell path length) by measuring the absorbance of a solution in which the conjugate precursor used was dissolved at a certain molar concentration. The molar extinction coefficient of the conjugate precursor in the examples was obtained by UV measurement each time. ２８０ and A ２５０ These values ​​are substituted into the equations (I) and (II) to solve the simultaneous equations, thereby obtaining C Ａ and C Ｄ Furthermore, C Ｄ C Ａ By dividing by this, the average number of drugs bound per antibody molecule can be calculated.

[0273] Common Procedure F: Measurement of antibody concentration in antibody-drug conjugates and average number of drugs bound per antibody molecule (reverse-phase high-performance liquid chromatography: RP-HPLC) The antibody concentration in antibody-drug conjugates and average number of drugs 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.

[0274] [F-1. Preparation of sample for HPLC analysis (reduction of antibody-drug conjugate)] The antibody-drug conjugate solution (approximately 1 mg / mL, 60 μL) was mixed with an aqueous dithiothreitol (DTT) solution (100 mM, 15 μL). The mixture was incubated at 37°C for 30 minutes to cleave the disulfide bond between the L chain and H chain of the antibody-drug conjugate. This reaction solution was used directly for HPLC analysis.

[0275] [F-2. HPLC analysis] Typical analytical conditions are as follows: HPLC system: Agilent 1290 HPLC system (Agilent Technologies) Detector: UV spectrophotometer (measurement wavelength: 280 nm) Column: Acquity BEH Phenyl (2.1 x 50 mm, 1.7 μm, manufactured by Waters) Column temperature: 75°C Flow rate: 0.8 mL / min Sample injection volume: 10 μL Mobile phase A: 0.1% trifluoroacetic acid (TFA), 15% isopropyl alcohol aqueous solution Mobile phase B: 0.075% TFA, 15% isopropyl alcohol acetonitrile solution 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)

[0276] [F-3. Data Analysis] [F-3-1] In the case of sugar chain conjugation in the SPAAC reaction, the H chains to which drugs are bound (H chains to which one drug is bound: H1, H chains to which two drugs are bound: H2) become more hydrophobic and have longer retention times in proportion to the number of drugs bound, compared to the L chain (L0) and H chain (H0) of an antibody to which no drug is bound. Therefore, they are eluted in the order of L0, H0, H1, and H2 in principle. By comparing the retention times of L0 and H0, the detected peak can be assigned to either L0, H0, H1, or H2. Similarly, in the case of cysteine ​​conjugation, the hydrophobicity of drug-bound L chains (L chains with one drug bound: L1) and drug-bound H chains (H chains with one drug bound: H1, H chains with two drugs bound: H2, H chains with three drugs bound: H3) increases in proportion to the number of drugs bound, and the retention time increases, so they are eluted in the order of L0, L1, H0, H1, H2, and H3 in principle. By comparing the retention times of L0 and H0, the detected peak can be assigned to either L0, L1, H0, H1, H2, or H3.

[0277] [F-3-2] Because the drug linker has UV absorption, in the case of sugar chain conjugation in the SPAAC reaction, the peak area was corrected according to the number of drug linkers bound using the molar extinction coefficients of the H chain and drug linker according to the following formula: In the case of cysteine ​​conjugation in which the drug is also bound to the L chain, the peak area was similarly corrected for the L chain.

[0278]

[0279] Here, the molar absorption coefficients (280 nm) of the L chain and H chain of each antibody were estimated values ​​calculated by the known calculation method described in Common Procedure E. For 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. For 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. For 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. As the molar extinction coefficient (280 nm) of the drug linker, in the case of sugar chain conjugation by SPAAC reaction, the actual measured value of the conjugation precursor was used, and in the case of cysteine ​​conjugation, the actual measured value of the compound in which the conjugation precursor was reacted with mercaptoethanol or N-acetylcysteine ​​to convert the maleimide group to a succinimide thioether was used.

[0280] [F-3-3] The ratio (%) of each chain peak area to the total corrected peak area was calculated according to the following formula.

[0281]

[0282] [F-3-4] The average number of drugs bound per antibody molecule (DAR) in the antibody-drug conjugate was calculated according to the following formula.

[0283]

[0284] [F-3-5] The antibody concentration in the antibody-drug conjugate was calculated according to the following formula.

[0285]

[0286] Here, the absorbance (280 nm) of the antibody-drug conjugate was determined using the actual measured value of the antibody-drug conjugate aqueous solution. The dilution factor indicates how many times the antibody-drug conjugate aqueous solution was diluted when measuring absorbance, and is typically 4-fold. The molar extinction coefficient (280 nm) of the antibody was an estimated value calculated using the known calculation method described in Common Procedure E. The average number of drugs bound was determined using the value obtained in [F-3-4]. For the molar extinction coefficient (280 nm) of the drug linker, the actual measured value of the conjugation precursor was used in the case of sugar chain conjugation via SPAAC reaction, and the actual measured value of the compound obtained by reacting the conjugation precursor with mercaptoethanol or N-acetylcysteine ​​to convert the maleimide group to succinimide thioether was used in the case of cysteine ​​conjugation.

[0287] Common Procedure G: Measurement of antibody concentration in antibody-drug conjugates and average number of drugs bound per antibody molecule (hydrophobic interaction-high performance liquid chromatography: HI-HPLC) The antibody concentration in antibody-drug conjugates and average number of drugs 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.

[0288] [G-1. Preparation of sample for HPLC analysis] The antibody-drug conjugate solution (approximately 1 mg / mL, 60 μL) was used directly for HPLC analysis.

[0289] [G-2. HPLC Analysis] The following two typical analytical conditions are used. HPLC system: SHIMADZU CBM-20A (Shimadzu Corporation) Detector: UV spectrophotometer (measurement wavelength: 280 nm) Column: TSK-gel Butyl-NPR (4.6 x 100 mm, 2.5 μm, manufactured by TOSOH) Column temperature: constant temperature around 25°C Mobile phase A: 25 mM phosphate buffer (pH = 7.0) containing 1.5 M ammonium sulfate Mobile phase B: 25 mM phosphate buffer (pH = 7.0) / isopropyl alcohol mixture (3:1) Flow rate: 0.8 mL / min Sample injection volume: 15 μL Gradient program (mobile phase B): 10% - 15% (0 min - 5 min), 15% - 65% (5 min - 20 min) or HPLC system: SHIMADZU CBM-20A (Shimadzu Corporation) Detector: UV spectrophotometer (measurement wavelength: 280 nm) Column: PolyPROPYL A (4.6 x 100 mm, 3 μm, 1500 Å, manufactured by PolyLC) Column temperature: constant temperature around 40°C Mobile phase A: 20 mM phosphate buffer (pH = 7.4) containing 1.5 M ammonium sulfate Mobile phase B: 20 ​​mM phosphate buffer (pH = 7.4) Flow rate: 0.8 mL / min Sample injection volume: 15 μL Gradient program (mobile phase B): 40% - 80% (0 min - 20 min)

[0290] [G-3. Data Analysis] [G-3-1] Since hydrophobicity increases in proportion to the number of drugs bound to the antibody and retention time increases, in the case of sugar chain conjugation in the SPAAC reaction, elution generally occurs in the order of DAR=0, DAR=2, and DAR=4. By comparing the retention time with DAR=0, the detected peak can be assigned to either DAR=2 or DAR=4. Depending on the type of antibody and drug linker, peaks with DAR=1 and DAR=3 may also be detected. The DAR of the detected peak may also be estimated by measuring the mass spectrum after fractionating the peak by HI-HPLC.

[0291] [G-3-2] Because the drug linker has UV absorption, the peak area value was corrected according to the number of drug linkers bound using the molar absorption coefficients of the antibody and drug linker according to the following formula.

[0292]

[0293] Here, the molar extinction coefficient (280 nm) of the antibody was an estimated value calculated by the known calculation method described in Common Procedure E. The molar extinction coefficient (280 nm) of the drug linker was the actually measured value of the conjugation precursor.

[0294] [G-3-3] The antibody peak area ratio (%) to the total corrected peak area was calculated according to the following formula.

[0295]

[0296] [G-3-4] The average number of drugs bound per antibody molecule in the antibody-drug conjugate was calculated according to the following formula.

[0297]

[0298] [G-3-5] The antibody concentration in the antibody-drug conjugate was calculated according to the formula described in [F-3-5], and the average number of bound drugs was calculated using the value obtained in [G-3-4].

[0299] The antibody-drug conjugate of the present invention or a production intermediate thereof may exist as a stereoisomer, an optical isomer derived from an asymmetric carbon atom, a geometric isomer, a tautomer, or an optical isomer such as a d-isomer, an l-isomer, or an atropisomer, and all of these isomers, optical isomers, and mixtures thereof are included in the present invention.

[0300] In the antibody-drug conjugates of the present invention, the number of drugs bound to one antibody molecule is an important factor affecting their efficacy and safety. Antibody-drug conjugates are produced by specifying reaction conditions, such as the amounts of raw materials and reagents used, so that a certain number of drugs are bound. However, unlike chemical reactions of small molecules, a mixture of different numbers of drugs is usually obtained. The number of drugs bound to one antibody molecule can be specified as an average value, i.e., the average drug binding rate (DAR). The number of cyclic dinucleotide derivatives bound to an antibody molecule can be controlled, and the average number of drugs bound to one antibody can range from 1 to 10, preferably 1 to 8, more preferably 1 to 5, and even more preferably 2 to 4. In the present invention, the terms "average drug binding rate" and "average drug binding rate" have the same meaning and can be used interchangeably.

[0301] In the antibody-drug conjugate of the present invention, when the antibody Ab is bound to L via a remodeled sugar chain of the antibody Ab, the number of drugs bound per antibody heavy chain in the antibody-drug conjugate, m ２ is an integer of 1 or 2. When the sugar chain is an N297 sugar chain and the sugar chain is N297-(Fuc)SG, m ２ is 2, and DAR is in the range of 3 to 5 (preferably in the range of 3.2 to 4.8, 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 is 1, and DAR is in the range of 1 to 3 (preferably in the range of 1.0 to 2.5, more preferably in the range of 1.2 to 2.2).

[0302] Furthermore, a person skilled in the art would be able to design a reaction for binding the required number of drugs to an antibody based on the description of the examples of the present application, and obtain an antibody in which the number of cyclic dinucleotide derivatives bound is controlled.

[0303] The antibody-drug conjugate of the present invention or a production intermediate thereof may absorb moisture, become adsorbed with water, or become a hydrate when left in the air or when recrystallized, and such water-containing compounds and salts are also encompassed by the present invention.

[0304] When the antibody-drug conjugate of the present invention or a production intermediate thereof has a basic group such as an amino group, it can be converted into a pharmaceutically acceptable salt, if desired. Examples of such salts include hydrohalide salts such as hydrochloride and hydroiodide; inorganic acid salts such as nitrate, perchlorate, sulfate, and phosphate; lower alkanesulfonate salts such as methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate; arylsulfonate salts 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.

[0305] The antibody-drug conjugates of the present invention contain a phosphate group and / or a thiophosphate group in their structure and are therefore generally capable of forming base addition salts. Furthermore, when their production intermediates contain an acidic group such as a carboxy group, they are also generally capable of forming base addition salts. Examples of pharmaceutically acceptable salts include alkali metal salts such as sodium salt, potassium salt, and lithium salt; alkaline earth metal salts such as calcium salt and magnesium salt; inorganic salts such as ammonium salt; and organic amine salts such as dibenzylamine salt, morpholine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, diethylamine salt, triethylamine salt, cyclohexylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, diethanolamine salt, N-benzyl-N-(2-phenylethoxy)amine salt, piperazine salt, tetramethylammonium salt, and tris(hydroxymethyl)aminomethane salt.

[0306] The antibody-drug conjugates of the present invention and their production intermediates may exist as hydrates due to, for example, absorption of moisture from the air. The solvates of the present invention are not particularly limited as long as they are pharmaceutically acceptable, but specifically preferred are hydrates, ethanol solvates, 2-propanol solvates, and the like. Furthermore, when nitrogen atoms are present in the antibody-drug conjugates of the present invention and their production intermediates, they may be in the form of N-oxides, and these solvates and N-oxides are also included within the scope of the present invention. Furthermore, when sulfur atoms are present in the antibody-drug conjugates of the present invention and their production intermediates, they may be in the form of sulfoxides, and these solvates and sulfoxides are also included within the scope of the present invention.

[0307] The present invention also encompasses compounds labeled with various radioactive or non-radioactive isotopes. One or more atoms constituting the antibody-drug conjugates of the present invention and their production intermediates may contain unnatural proportions of atomic isotopes. Examples of atomic isotopes include deuterium (2H), tritium (3H), iodine-125 (125I), and carbon-14 (14C). The compounds of the present invention may also be radiolabeled with radioactive isotopes such as tritium (3H), iodine-125 (125I), or carbon-14 (14C). Radiolabeled compounds are useful as therapeutic or prophylactic agents, research reagents, e.g., assay reagents, and diagnostic agents, e.g., in vivo diagnostic imaging agents. All isotopic variants of the antibody-drug conjugates of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

[0308] <4. Pharmaceuticals> The antibody-drug conjugate of the present invention exhibits antitumor immune activity or cytotoxic activity against cancer cells and is also excellent in terms of safety, and therefore can be used as a pharmaceutical, particularly as a therapeutic and / or preventive agent for cancer, or an antitumor agent. Furthermore, the antibody-drug conjugate of the present invention can be used as a pharmaceutical, particularly as a therapeutic and / or preventive agent for infectious diseases. In one aspect of the present invention, the antibody-drug conjugate of the present invention can be used as a pharmaceutical having high heat stability.

[0309] Examples of cancer types to which the antibody-drug conjugates of the present invention can be applied include lung cancer (non-small cell lung cancer, small cell lung cancer, etc.), renal cancer, urothelial cancer, colon cancer, prostate cancer, glioblastoma multiforme, ovarian cancer (surface epithelial tumor, stromal tumor, germ cell tumor, etc.), pancreatic cancer, breast cancer, melanoma, liver cancer, hepatocellular carcinoma, bladder cancer, gastric cancer, esophageal cancer, uterine cancer, testicular cancer (seminoma, nonseminoma), cervical cancer, placental choriocarcinoma, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, gastrointestinal stromal tumor (GIST), gallbladder cancer, bile duct cancer, adrenal cancer, pharyngeal cancer, tongue cancer, ear cancer, thymus cancer, small intestine cancer, squamous cell carcinoma, leukemia, malignant lymphoma, plasmacytoma, myeloma, sarcoma, etc. Furthermore, the antibody-drug conjugate of the present invention can be applied to EGFR-positive cancer or CDH6-positive cancer. EGFR-positive cancer is cancer that expresses EGFR, and examples thereof include lung cancer (non-small cell lung cancer, small cell lung cancer, etc.), renal cancer, urothelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer (surface epithelial tumor, stromal tumor, germ cell tumor, etc.), pancreatic cancer, breast cancer, melanoma, liver cancer, hepatocellular carcinoma, bladder cancer, gastric cancer, esophageal cancer, uterine cancer, cervical cancer, brain tumor, head and neck cancer, thyroid cancer, mesothelioma, gastrointestinal stromal tumor (GIST), gallbladder cancer, bile duct cancer, squamous cell carcinoma, pharyngeal cancer, tongue cancer, thymic cancer, and small intestine cancer. CDH6-positive cancers are cancers that express 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 tumor, stromal tumor, germ cell tumor, etc.), pancreatic cancer, uterine cancer, cervical cancer, thyroid cancer, mesothelioma, gallbladder cancer, bile duct cancer, and sarcoma. Furthermore, the antibody-drug conjugate of the present invention can be applied to EGFR signaling mutation-positive cancers. EGFR signaling mutation-positive cancers include cancers with a mutation in EGFR (EGFR mutation-positive cancers) and cancers with a mutation in an EGFR signaling molecule (cancers with a mutation in an EGFR downstream signal). Examples of cancers with an EGFR mutation include lung cancer and head and neck cancer containing at least one mutation in exons 18 to 21, and preferably lung cancer with an exon 19 mutation.Examples of cancers with a mutation in an EGFR signal molecule include cancers with a mutation in at least one of RAS, RAF, PI3K, etc. Preferred examples include RAS-mutated cancers such as lung cancer, colon cancer, and pancreatic cancer, which have a mutation in at least one of RAS (KRAS, NRAS, HRAS), more preferably lung cancer or colon cancer with a KRAS mutation, and even more preferably lung cancer with a KRAS mutation. Cancers to which the antibody-drug conjugate of the present invention can be applied are not limited to these, as long as the cancer to be treated expresses a protein that can be recognized by the antibody in the antibody-drug conjugate.

[0310] The antibody-drug conjugate of the present invention can be suitably administered to mammals, and more preferably, the mammal is a human.

[0311] Substances used in pharmaceutical compositions containing the antibody-drug conjugates of the present invention can be appropriately selected from pharmaceutical additives and other substances commonly used in this field in terms of dosage amount and administration concentration.

[0312] The antibody-drug conjugates of the present invention can be administered as pharmaceutical compositions containing one or more pharmaceutically compatible ingredients. For example, the pharmaceutical compositions typically contain one or more pharmaceutical carriers, such as sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.). Water is a more typical carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients are known in the art. The composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. The formulation will correspond to the mode of administration.

[0313] Various delivery systems are known and can be used to administer the antibody-drug conjugate of the present invention.Introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes.Administration can be, for example, by infusion or bolus injection.In certain preferred embodiments, the antibody-drug conjugate is administered by infusion.Parenteral administration is the preferred administration route.

[0314] In a representative embodiment, a pharmaceutical composition comprising the antibody-drug conjugate is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If necessary, the pharmaceutical composition may also include a solubilizing agent and a local anesthetic (e.g., lignocaine) to ease pain at the injection site. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. If the pharmaceutical composition is to be administered by infusion, it can be dispensed, for example, in an infusion bottle containing sterile pharmaceutical-grade water or saline. If the pharmaceutical composition is to be administered by injection, an ampoule of sterile water for injection or saline can be provided, for example, so that the ingredients can be mixed prior to administration. The pharmaceutical composition may also be provided as a solution.

[0315] The pharmaceutical composition of the present invention may be a pharmaceutical composition containing only the antibody-drug conjugate of the present invention, or may be a pharmaceutical composition containing the antibody-drug conjugate of the present invention and other drugs (including cancer therapeutic agents). The antibody-drug conjugate of the present invention can also be administered together (simultaneously, separately, or one after the other) or in combination with one or more other drugs (including, but not limited to, cancer therapeutic agents), thereby enhancing the anti-tumor effect and improving safety. The other drugs used for such purposes may be administered to an individual simultaneously, separately, or consecutively with the antibody-drug conjugate, or the respective drugs may be administered at different administration intervals. Examples of such other cancer therapeutic agents include hormone regulators (LH-RH analogs such as leuprorelin, goserelin, estramustine, and estrogen antagonists such as tamoxifen and raloxifene), aromatase inhibitors (anastrozole, letrozole, exemestane, etc.), kinase inhibitors, PARP inhibitors, bone destruction inhibitors, bone formation promoters, metastasis inhibitors, anti-regulatory T cell agents (anti-GITR antibodies, anti-GARP antibodies, anti-TIGIT antibodies, anti-CCR8 antibodies, etc.), immune activators (anti-4-1BB antibodies, anti-OX40 antibodies, anti-CD40 antibodies, anti-CD3 antibodies, anti-CD28 antibodies, IL-2 analogs, cytokines, TLR agonists, etc.), immunomodulators (anti-CD47 antibodies, anti-SIRPα antibodies, inhibitory myeloid modulators, etc.), and ADCC (Antibody Dependent Cellular Examples of such antibodies include antibody drugs having anti-tumor activity, antibody-dependent cellular phagocytosis (ADCP) activity, or complement activity, BiTE (bi-specific T-cell engagers), ADC combined with photodynamic therapy, 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 are not limited thereto as long as they have anti-tumor activity. Furthermore, the antibody-drug conjugate of the present invention can be administered together with other antibody-drug conjugates of the present invention having anti-tumor activity, thereby enhancing the anti-tumor effect.Furthermore, the antibody-drug conjugate of the present invention can enhance the antitumor effect not only by using a drug but also by using in combination with a treatment that brings about an antitumor effect, such as radiation, quantum beams, surgery, bone marrow transplantation, etc., but is not limited thereto as long as it is a treatment that has an antitumor effect.

[0316] Such pharmaceutical compositions may be formulated as lyophilized or liquid preparations with the selected composition and required purity. When formulated as a lyophilized preparation, it may be a preparation containing appropriate formulation additives used in this field. Similarly, liquid preparations may be formulated as liquid preparations containing various formulation additives used in this field.

[0317] Although the composition and concentration of the pharmaceutical composition vary depending on the administration method, the antibody-drug conjugate contained in the pharmaceutical composition of the present invention can exert its medicinal effect even at a smaller dose, in terms of the affinity of the antibody-drug conjugate for the antigen, i.e., the dissociation constant (Kd value) for the antigen, the higher the affinity (the lower the Kd value). Therefore, when determining the dose of the antibody-drug conjugate, the dose can be set based on the affinity between the antibody-drug conjugate and 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 may be administered once or multiple times at intervals of once every 1 to 180 days.

[0318] 5. Method for Producing Fc-Containing Molecules In one aspect of the present invention, a method for producing an Fc-containing molecule having an N297-linked sugar chain containing a sugar chain derived from a sugar chain donor molecule is provided.

[0319] In one aspect of the present invention, there is provided a method for producing an Fc-containing molecule having an N297-linked glycan that includes a glycan derived from a glycan donor molecule, comprising the following step 1: (Step 1) reacting an acceptor molecule that is an Fc-containing molecule having a core GlcNAc to which fucose may be attached as the N297-linked glycan with a glycan donor molecule that includes a glycan that has a GlcNAc in which the reducing end is not activated, in the presence of: endo-β-N-acetylglucosaminidase (enzyme-A) that uses the N297-linked glycan of the Fc-containing molecule as a substrate; endo-β-N-acetylglucosaminidase (enzyme-B) that uses the glycan of the glycan donor molecule as a substrate; and additives selected from a monovalent salt, an organic solvent, a surfactant, sugars, amino acids, or any combination thereof, to obtain a reaction mixture.

[0320] <N297-linked glycan> In one aspect of the present invention, the term "N297-linked glycan" refers to an N-linked glycan bound to the side chain of Asn at position 297 of an IgG heavy chain. Even if the attachment position of an N-linked glycan on an IgG heavy chain is changed by any method, the N-linked glycan is still included in the N297-linked glycan as long as it is subjected to the action of enzyme-A. For example, in one aspect of the present invention, when an IgG heavy chain is fragmented, even a glycan bound to the corresponding Asn in a peptide fragment containing the Asn is included in the N297-linked glycan. Typically, an N297-linked glycan in IgG produced in animals or the like has a basic structure represented by the following formula (I) or (II), and its non-reducing end may be further chemically modified, for example, by the addition of Gal or sialic acid.

[0321]

[0322]

[0323] Many of the N297-linked glycans of IgG produced by cells have diverse glycan structures, including those with additional glycans attached to the reducing terminal GlcNAc (core GlcNAc), non-reducing terminal, branched sugar, etc. of this basic structure. The N297-linked glycan may have a structure having a core fucose (Fuc) linked to position 6 of the core GlcNAc via an α1,6 bond ((Fucα1,6)GlcNAc). In the case of the branched sugar Man, a tri-antennary glycan may be formed in which a glycan containing GlcNAc is further attached to position 5. The non-reducing terminal GlcNAc may be further attached to a glycan containing galactose (Gal) or sialic acid (Sia).

[0324] <Sugar Chain Donor Molecule> 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.

[0325] When used for glycan remodeling for the purpose of drug discovery, it is preferable to employ glycan donor molecules having human-type or human-compatible glycans, which pose few problems when applied to humans. Such glycans are known to be non-antigenic in the human body, and N-linked glycans include high-mannose, hybrid, and complex types. These three types share a common basic structure. The high-mannose type is a glycan with a mannose-rich structure in which multiple mannoses are consecutively attached to two branched chains (1-3 chain and 1-6 chain) branched from a mannose (β-mannose) located near the reducing end. The hybrid type is a structure in which one of the two branched chains (1-3 chain and 1-6 chain) branched from a mannose (β-mannose) located near the reducing end has a GlcNAc residue. The complex type has a structure in which GlcNAc is attached to two branched chains (1-3 chain and 1-6 chain) branched from a mannose (β-mannose) located near the reducing end, and has a variety of structures including the presence or absence of galactose, the presence or absence of sialic acid, and the bond and position isomerism of these. Known complex type sugar chains include bi-, tri-, and tetra-antennary types.

[0326] Examples of the high-mannose, hybrid, and complex structures are shown below. The common basic structure is indicated by the dotted box (the dotted box contains both structures with and without GlcNAc introduced at the 4-position of β-mannose (bisecting GlcNAc), but in this specification, all of these structures refer to the common basic structure).

[0327]

[0328] In one aspect of the present invention, the glycan portion of the glycan donor molecule may be any of a high-mannose type glycan, a hybrid type glycan, and a complex type glycan, but is preferably a high-mannose type glycan or a complex type glycan, and particularly preferably a complex type glycan. Therefore, in one aspect of the present invention, the glycan donor molecule comprises a complex type glycan. Furthermore, in the present invention, a complex type glycan may be any of a biantennary type, a triantennary type, or a tetraantennary type, but preferably, the glycan donor molecule of the present invention comprises a biantennary type complex type glycan.

[0329] In one embodiment of the present invention, the glycan donor molecule is a glycan-containing molecule having an inactivated GlcNAc at the reducing end of the glycan, preferably SGP, (SG-)Asn, (MSG1-)Asn, (MSG2-)Asn, or a mixture of (MSG1-)Asn and (MSG2-)Asn. These glycan moieties are representative of N-linked glycans and contain sialyl glycans (hereinafter referred to as "SG") having the structures shown in the structural and sequence formulas below. The linkage mode of the reducing end glycan may be changed from N-linked to O-linked. In this specification, unless otherwise specified, partial structures in which the glycan is linked to the side chain of an amino acid will be represented by the side chain portion in parentheses, as shown above, for example, "(SG-)Asn."

[0330]

[0331] (In the formula, "-(N / O)" indicates a glycosidic bond with an N atom or an O atom.)

[0332] Specific examples of the sugar chain portion in the sugar chain donor molecule that can be used in the present invention include AG(9) (AG(9) of the structural formula and sequence formula below) obtained by treating SG with neuraminidase and deleting two NeuAcs at the non-reducing ends, and AG(7) (AG(7) of the structural formula and sequence formula below) obtained by treating AG(9) with galactosidase and deleting two Gals at the non-reducing ends.

[0333]

[0334] (In the formula, "-(N / O)" indicates a glycosidic bond with an N atom or an O atom.)

[0335]

[0336] (In the formula, "-(N / O)" indicates a glycosidic bond with an N atom or an O atom.)

[0337] In the present invention, the sugar chain donor molecule may be chemically modified, for example, the non-reducing end may be chemically modified.

[0338] Examples of the sugar chain moiety in the sugar chain donor molecule in which the non-reducing end may be chemically modified and can be used in the present invention include derivatives in which a site on the wild-type sialic acid is chemically modified with an arbitrary substituent (SG type).

[0339]

[0340] (In the formula, X represents a substituent introduced onto the sialic acid at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man, and is a substituent that can be synthesized by applying various methods known in the field of organic synthetic chemistry. With reference to the substituents used in the conjugation method applied to the synthesis of antibody-drug conjugates (Bioconjugate Chem. 2015, 26, 2198-2215), for example, an acetylene group, an azide group (N ３-), dibenzylcyclooctene (DBCO) group, bicyclo[6.1.0]non-4-ene (BCN) group, a structure containing a 1,2,4,5-tetrazine ring, an aldehyde group, an SH group, a methylsulfonyltriazole group, etc., are preferred, and an acetylene group, an azide group (N ３ -), and more preferably an azido group (N ３ Examples of substituents include, but are not limited to, those having an "azide group (N -)". ３ Examples of the "substituent having N-" include ３ a polyethylene glycol linker having an ethylene glycol unit (—CH ２ -CH ２ The upper limit of the number of "azide groups (N-O-)" is about 20 or less, preferably about 11 or less, and more preferably about 7 or less. ３ Examples of the "substituent having N-" include ３a polyethylene glycol linker having, for example, 35-azido-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontane-1-amino group, 32-azido-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontane-1-amino group, 29-azido-3,6,9,12,15,18,21,24,27-nonaoxanonacosane -1-amino group, 26-azido-3,6,9,12,15,18,21,24-octaoxahexacosane-1-amino group, 23-azido-3,6,9,12,15,18,21-heptaoxatricosane-1-amino group, 20-azido-3,6,9,12,15,18-hexaoxaeicosane-1-amino group, 17-azido-3,6,9,12,15-pentaoxaheptadecane-1-amino group, 14 -azido-3,6,9,12-tetraoxatetradecane-1-amino group, 11-azido-3,6,9-trioxaundecane-1-amino group, 2-[2-(2-azidoethoxy)ethoxy]ethylamino group, 2-(azidoethoxy)ethylamino group, 2-azidoethylamino group, 2-[2-[2-(2-azidoethoxy)ethoxy]-N-[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy ]ethyl]ethanamine group, or branched substituents such as a 1,3-bis[2-[2-(2-azidoethoxy)ethoxy]ethoxy]propan-2-amino group or a 3-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]-2-[2-[2-(2-azidoethoxy)ethoxy]ethoxymethyl]propan-1-amino group. In the formula, "-(N / O)" indicates a glycosidic bond with an N atom or an O atom.

[0341] Among the X-SG type structures shown here, X-MSG1 type, which is a structure lacking chemically modified sialic acid on the 1-6 chain side of the branched chain of β-Man, and X-MSG2 type, which is a structure lacking chemically modified sialic acid on the 1-3 chain side, are also included as examples of the sugar chain moiety in a sugar chain donor molecule whose non-reducing end may be chemically modified.

[0342] Furthermore, sites that may be chemically modified may be other than sialic acid, and examples thereof include AG(9) type derivatives and AG(7) type derivatives.

[0343]

[0344] (In the formula, X represents a substituent introduced onto the galactose at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man, and specifically represents the same meaning as defined above. In the formula, "-(N / O)" represents a glycosidic bond to an N atom or an O atom.)

[0345]

[0346] (In the formula, X represents a substituent introduced onto the GlcNAc at the non-reducing end of both the 1-3 chain side and the 1-6 chain side of the branched chain of β-Man, and specifically represents the same meaning as defined above. In the formula, "-(N / O)" represents a glycosidic bond to an N atom or an O atom.)

[0347] Among the X-AG(9) structures shown here, structures lacking chemically modified galactose on the 1-6 chain side of the branched chain of β-Man, and structures lacking chemically modified galactose on the 1-3 chain side, as well as among the X-AG(7) structures, structures lacking chemically modified GlcNAc on the 1-6 chain side of the branched chain of β-Man, and structures lacking chemically modified GlcNAc on the 1-3 chain side, are also examples of the sugar chain moiety in the donor molecule.

[0348] The sugar chain donor molecule of the present invention preferably includes 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, each of which may have a chemically modified non-reducing end, or a sugar chain donor molecule having the structure shown below.

[0349]

[0350] In the above structural formula, "P" represents a peptide moiety derived from SGP, and "X" has the same meaning as defined above. More preferably, the sugar chain donor molecule of the present invention contains an N-linked sugar chain, and the sugar chain donor molecule of the present invention is ([N ３ -PEG(3)] ２ -SG)-P-PEG(3)-N ３ , ([N ３ -PEG(3)] ２ -SG)-Asn-PEG(3)-N ３ , ([N ３ -PEG(3)]-MSG1)-Asn-PEG(3)-N ３ , ([N ３ -PEG(3)]-MSG2)-Asn-PEG(3)-N ３ , or ([N ３ -PEG(3)]-MSG1)-Asn-PEG(3)-N ３ and ([N ３ -PEG(3)]-MSG2)-Asn-PEG(3)-N ３ Examples of mixtures containing O-linked glycans include ([N ３ -PEG(3)] ２ -SG)-OR, ([N ３ -PEG(3)]-MSG1)-OR, ([N ３ -PEG(3)]-MSG2)-OR, or ([N ３ -PEG(3)]-MSG1)-OR and ([N ３ -PEG(3)]-MSG2)-OR.

[0351] In the above chemical formula, "R" represents an arbitrary substituent linked to the oxygen atom via at least one carbon atom, preferably a C1-C6 alkyl group or an optionally substituted aryl group. A "C1-C6 alkyl group" refers to a linear or branched alkyl group having 1 to 6 carbon atoms, including, but not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, and n-hexyl. An "aryl group" includes, but is not limited to, phenyl, benzyl, indenyl, naphthyl, fluorenyl, anthranyl, and phenanthrenyl. Substituents for the aryl group include, but are not limited to, C1-C6 alkoxy groups (e.g., methoxy, ethoxy, and isopropoxy), C1-C6 alkyl groups (e.g., methyl and ethyl), and halogen groups. Substituted aryl groups include, but are not limited to, para-methoxyphenyl, para-methylphenyl, etc. In one embodiment of the present invention, "R" is selected from PMP, Me, A-Mor, iPr, nPr, PrOMe, and A-PEG-N. ３ (A represents the acetyl group in the glycolic acid unit, i.e., the moiety sandwiched between the anomeric hydroxyl group and the amino group in Mor or PEG).

[0352] Furthermore, in one embodiment of the present invention, preferred examples of the sugar chain donor molecule include the following sugar chain donor molecules.

[0353] The sugar chain donor molecule of the present invention is preferably selected appropriately depending on various conditions such as the combination of enzyme-A and enzyme-B to be used.

[0354] In one embodiment of the present invention, the glycan donor molecule comprises a high mannose type glycan. Such a glycan donor molecule includes Man9-GlcNAc having the following structure: ２-Asn ("M9-Asn"), and Man8-GlcNAc having a structure in which one, two, or three mannose residues are deleted from M9-Asn. ２ -Asn (“M8-Asn”), Man7-GlcNAc ２ -Asn (“M7-Asn”), Man6-GlcNAc ２ Examples of suitable amino acids include, but are not limited to, M6-Asn ("M6-Asn").

[0355]

[0356] The sugar chain donor molecule can be purchased as a commercially available product or can be prepared by utilizing or applying a known method.

[0357] <Fc-containing molecule> In one aspect of the present invention, an "Fc-containing molecule" (sometimes referred to as an "Fc region-containing molecule") refers to a molecule containing an Fc region, and examples thereof include antibodies (immunoglobulins), Fc-fusion proteins, and Fc fragments (or Fc-containing fragments). Examples of Fc-containing fragments other than antibodies include CLCH, such as an Fc fragment of IgG (particularly, an IgG monoclonal antibody) or a CLCH consisting only of a constant region (Patent Document 1), and a bispecific antibody containing an Fc fragment (Expert Opin Ther Pat. 2018 28, 251-276). In one aspect of the present invention, the Fc-containing molecule is an antibody or a functional fragment of the antibody, and examples of the antibody or functional fragment include all of those mentioned above. Thus, for example, in one embodiment of the present invention, the Fc-containing molecule is an antibody or a functional fragment of such an antibody in which 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; and 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.

[0358] <Acceptor Molecule> In one aspect of the present invention, an "acceptor molecule" refers to a molecule that receives a sugar chain from a sugar chain donor molecule. A typical acceptor molecule is an IgG or Fc fragment thereof derived from a monoclonal antibody, which has an N297-linked sugar chain consisting only of core GlcNAc to which core Fuc may be bound. In this specification, "core Fuc" (or "core fucose") refers to fucose bound to the core GlnNAc. Core GlcNAc may or may not be bound to core Fuc, depending on the antibody from which it is derived or the method for producing it. Therefore, in one embodiment of the present invention, the acceptor molecule is an Fc-containing molecule (e.g., an antibody or an Fc-containing fragment thereof) having a core GlcNAc to which fucose may be attached as the N297-linked glycan, preferably an Fc-containing molecule (e.g., an antibody or an Fc-containing fragment thereof) having an N297-linked glycan consisting of GlcNAc or (Fucα1,6)GlcNAc. The acceptor molecule can be derived from various monoclonal antibodies, glycan-containing molecules, or Fc region-containing molecules (e.g., Fc, CLCH, which combines a CH consisting only of the constant region obtained by deleting the variable region from the heavy chain with a CL consisting only of the light chain constant region, etc.). Preferred examples of the acceptor molecule of the present invention include (Fucα1,6)-GlcNAc-IgG (for example, (Fucα1,6)GlcNAc-mAb1), (Fucα1,6)-GlcNAc-Fc, (Fucα1,6)-GlcNAc-CLCH, and GlcNAc-mAb4.

[0359] In one aspect of the invention, the Fc-containing molecule having an N297-linked carbohydrate chain can be derived from an antibody.

[0360] Acceptor molecules can be purchased commercially or prepared by utilizing or adapting known methods, as appropriate. Acceptor molecules can typically be prepared by treating the N297-linked glycan of an Fc-containing molecule (typically an IgG monoclonal antibody or an Fc fragment of the antibody or an Fc-containing molecule such as CLCH consisting only of the constant region) to be subjected to glycosylation (also referred to as glycosylation remodeling) with ENGase that retains the activity to specifically hydrolyze the 1,4-glycosidic bond (GlcNAcβ1-4GlcNAc) between GlcNAcs in the core chitobiose structure of the N297-linked glycan. Various ENGases can be used in this case, including Endo-Si WT, Endo-A, Endo-D, Endo-E, Endo-F3, Endo-H, Endo-S, Endo-S2, and Endo-Si. Therefore, in one aspect of the present invention, the acceptor molecule can be prepared in a step independent of the glycosylation reaction in step 1 by, for example, reacting an enzyme identical to or different from the enzyme-A used in step 1 (an enzyme having the activity of transferring a glycosylation chain in a glycosylation donor molecule to an acceptor molecule) with a starting Fc-containing molecule (an Fc-containing molecule to be subjected to glycosylation modification, typically an Fc-containing molecule having a host cell-derived N297-linked glycosylation chain, a heterogeneous N297-linked glycosylation chain, or a host cell-derived heterogeneous N297-linked glycosylation chain). Furthermore, one feature of the enzyme-A that can be used in step 1 is that it "may have residual hydrolytic activity." Therefore, instead of pre-treating the acceptor molecule with these ENGases, the enzyme-A may act on an Fc-containing molecule having a host cell-derived heterogeneous glycosylation chain, thereby preparing the acceptor molecule in situ. Therefore, in one aspect of the present invention, the acceptor molecule is prepared in situ and / or in one pot by allowing enzyme-A to act on a starting Fc-containing molecule (an Fc-containing molecule to be subjected to glycosylation, typically an Fc-containing molecule having a host cell-derived N297-linked glycan, a heterogeneous N297-linked glycan, or a host cell-derived heterogeneous N297-linked glycan).Furthermore, the IgG or Fc-containing molecule used for glycosylation (glycan remodeling) is preferably derived from an IgG heavy chain consisting of the same amino acid sequence and produced in a form having an N297-linked glycan. The production method is not limited, and IgG or IgG CLCH produced by commonly known monoclonal antibody production methods, or Fc fragments obtained by enzymatic treatment thereof, can be used. Furthermore, such IgG or Fc fragments may be a mixture of samples obtained by different production methods or different lots.

[0361] <Endo-β-N-acetylglucosaminidase> "Endo-β-N-acetylglucosaminidase" is a glycosidase also known as ENGase (Endo-β-N-acetylglucosaminidase), which is responsible for cleaving chitobiose structures in glycan structures and forming bonds. It is also called differently depending on its origin. In this specification, endo-β-N-acetylglucosaminidase that uses the N297-linked glycan of an Fc-containing molecule as a substrate is referred to as "Enzyme-A," and endo-β-N-acetylglucosaminidase that uses the glycan of a glycan donor molecule, for example, the glycan of a glycan raw material (e.g., SGP) that does not contain fucose, as a substrate is referred to as "Enzyme-B." Enzyme-A and Enzyme-B in the present invention are described below.

[0362] <Enzyme-A> "Enzyme-A" refers to an endo-β-N-acetylglucosaminidase that utilizes the N297-linked glycan of an Fc-containing molecule (e.g., an antibody) as a substrate. Enzyme-A generally comprises a domain with high affinity for Fc-containing molecules, a domain with high affinity for glycans, and a glycosyltransferase catalytic domain. Typically, Enzyme-A possesses both hydrolytic activity and glycosyltransferase activity using the N297-linked glycan of an Fc-containing molecule (e.g., an antibody) as a substrate. However, what is important in the present invention is the activity of transferring a glycan on a glycan donor molecule (e.g., a glycosyltransferase activated by a chemical or enzymatic method) to an acceptor molecule. Therefore, the hydrolytic activity may be weakened or eliminated. Rather, since an enzyme that retains strong hydrolytic activity may hydrolyze a glycan transferred to the core GlcNAc of an acceptor molecule by glycosyltransferase activity, the hydrolytic activity of Enzyme-A in the present invention is preferably relatively reduced. As described above, when Enzyme-A has residual hydrolytic activity, it can be advantageously prepared in situ in the same reaction vessel as Step 1, without the need for separate preparation of the acceptor molecule in Step 1. Therefore, in one aspect of the present invention, Enzyme-A has an activity that can act on the sugar chain of an Fc-containing molecule (particularly, a glycosylation activity using the N297-linked sugar chain of an Fc-containing molecule as a substrate), and in another aspect of the present invention, Enzyme-A has an activity that can act on the sugar chain of an Fc-containing molecule and either retains hydrolytic activity or has lost its hydrolytic activity.

[0363] The hydrolytic activity of Enzyme-A means the activity of specifically hydrolyzing the β1,4 glycosidic bond contained in core-chitobiose in the common basic structure of glycans, and in particular, means the activity of specifically hydrolyzing the β1,4 glycosidic bond contained in core-chitobiose in the common basic structure of N297-linked glycans of Fc-containing molecules.

[0364] The transglycosylation activity of Enzyme-A means the activity of glycosidically linking the reducing end of the sugar chain donor molecule to an acceptor molecule containing an Fc moiety having only core GlcNAc (which may or may not have core fucose attached) at N297.

[0365] 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 (Cambridge)). 2018, 54, 6161-6164), Endo-F3 (an enzyme derived from Flavobacterium meningosepticum) (see Huang W, et al., Chembiochem. 2011, 12, 932-941, and Giddens JP, et al., J Biol Chem. 2016, 291, 9356-9370), or mutant enzymes thereof. Examples of the enzyme-A of the present invention include endo-β-N-acetylglucosaminidase (hereinafter referred to as "Endo-Si", the amino acid sequence of which (SEQ ID NO: 49) is shown in Figure 32) derived from Streptococcus iniae (see Pier GB and Madin SH., Int J Syst Bacteriol. 1976 26, 545-553), or a mutant enzyme thereof. Examples of the enzyme-A of the present invention include an enzyme whose sequence is identified from the genome information of the genus Streptococcus (see Vincent P. Richard, et al., Genome Biol. Evol. 2014, 6, 741-753), or a mutant enzyme thereof.

[0366] The enzyme-A of the present invention is not limited to the specific enzymes used in the examples, as long as it has the above-mentioned properties, and may 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. When isolated from nature, the organism species from which it is isolated is not particularly limited, but bacteria are preferred.

[0367] The active domain and Carbohydrate-binding module (CBM) of Endo-Si are presumed to be regions of amino acids 106 to 447 and 762 to 897 of SEQ ID NO: 49, respectively, based on sequence comparison with EndoS, whose crystal structure has been analyzed (B. Trastoy et al., PNAS (2014) vol. 111, No. 18, pp. 6714-6719). These two regions are considered to be important sites for hydrolysis activity and / or transfer activity, and for interaction with antibodies. Therefore, examples of the enzyme-A of the present invention include polypeptides that contain the amino acid sequence of amino acid numbers 106 to 447 and / or 762 to 897 of SEQ ID NO: 49, preferably the amino acid sequence of amino acid numbers 106 to 897 of SEQ ID NO: 49, more preferably the amino acid sequence of amino acid numbers 106 to 928 of SEQ ID NO: 49, and even more preferably the amino acid sequence of amino acid numbers 34 to 928 of SEQ ID NO: 49, and that exhibit transglycosylation activity.

[0368] As described above, endo-β-N-acetylglucosaminidases usually possess both hydrolytic and transglycosylation activities, but enzymes with strong hydrolytic activity may hydrolyze, as a substrate, the sugar chain transferred to the core GlcNAc of an acceptor molecule (an antibody having a core GlcNAc as the N297-linked sugar chain or an Fc domain-containing molecule thereof) by the transglycosylation activity, and may fail to properly obtain the desired transglycosylated product. Therefore, in the synthesis of glycosylation-remodeling antibodies or glycosylated compounds, mutant enzymes with reduced hydrolytic activity relative to their transglycosylation activity are useful. Therefore, in one embodiment of the present invention, Enzyme-A is a mutant enzyme of Endo-S, Endo-S-2, Endo-Si, Endo-Sd, Endo-Se, or Endo-Sz, and is a mutant enzyme having a lower hydrolytic activity than the corresponding wild-type enzyme, and is preferably a mutant enzyme in which the amino acid residues shown in bold in Figures 30 to 32 and Figures 37 to 39 have been appropriately substituted, and particularly preferably Endo-S D233Q, Endo-S D233Q / Q303L, Endo-S D233Q / E350A, Endo-S D233Q / E350Q, Endo-S D233Q / E350D, Endo-S D233Q / E350N, Endo-S D233Q / D405A, Endo-Si D241Q, Endo-Si D241Q / Q311L, Endo-Si D241Q / 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 D241X １ (X １ represents any amino acid residue other than K, R, and D, specifically A, N, C, Q, E, G, H, I, L, M, F, P, S, T, W, Y, or V, preferably A, Q, E, H, I, L, M, F, P, S, T, W, Y, or V), Endo-Si T190X ２ (X ２ represents an amino acid residue of F, H, K, M, Q, R, W, or Y), Endo-Si Q311X ３ (X ３indicates the amino acid residue F, N, or Y), Endo-S2 E360K, Endo-S2 D184M, Endo-S2 T138Q, Endo-S2 D184Q, Endo-S2 D184Q / Q250L, Endo-S2 D184Q / E289Q, Endo-S2 D184M / Q250L, Endo-S2 D184M / E289Q, Endo-S2 D182Q, Endo-S2 D226Q, Endo-S2 T227Q, Endo-S2 T228Q, Endo-Sd D232Q, Endo-Sd D232M, Endo-Sd Examples of the endotoxin-containing peptide include, but are not limited to, Endo-Sz D232Q / Q302L, Endo-Sd D232M / Q302L, Endo-Se D233Q, Endo-Se D233M, Endo-Se D233Q / Q303L, Endo-Se D233M / Q303L, Endo-Sz D234Q, Endo-Sz D234M, Endo-Sz D234Q / Q304L, and Endo-Sz D234M / Q304L. In addition to the specific mutations mentioned above, enzyme-A may also have further mutations added, for example, with reference to patent literature (WO 2022 / 050300), etc., to the extent that it does not affect the enzyme activity, 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 residues may be substituted, deleted, inserted, and / or added. Further, as the amino acid sequence of enzyme-A, with reference to patent literature (WO 2022 / 050300), etc., specific mutated amino acid residues other than the amino acid residues, at least 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95%, 96%, 97%, 98% or 99% or more homology or identity can be mentioned.

[0369] <Enzyme-B> "Enzyme-B" refers to an endo-β-N-acetylglucosaminidase that uses the sugar chain of a sugar chain donor molecule as a substrate, particularly an endo-β-N-acetylglucosaminidase that uses the sugar chain of a sugar chain donor molecule as a substrate but not an N297-linked sugar chain (in other words, that has low reactivity, preferably extremely low reactivity, to N297-linked sugar chains). The sugar chain moiety on the sugar chain donor molecule that Enzyme-B uses as a substrate may be any of a high-mannose-type sugar chain, a hybrid-type sugar chain, and a complex-type sugar chain, but is preferably a high-mannose-type sugar chain or a complex-type sugar chain, and is particularly preferably a complex-type sugar chain. Therefore, in one aspect of the present invention, the sugar chain moiety on the sugar chain donor molecule that Enzyme-B uses as a substrate is a high-mannose-type sugar chain or a complex-type sugar chain, and is preferably a complex-type sugar chain. In the present invention, the complex glycan may be any of biantennary, triantennary, or tetraantennary, but preferably the glycan moiety on the glycan donor molecule used as a substrate by Enzyme-B is a biantennary complex glycan. In one aspect of the present invention, Enzyme-B is an endo-β-N-acetylglucosaminidase that uses as a substrate a glycan source material (glycopeptides or glycoproteins such as SGP) that does not contain fucose, and preferably an endo-β-N-acetylglucosaminidase that uses as a substrate a glycan source material containing a biantennary complex glycan that does not contain fucose.

[0370] The enzyme-B of the present invention is preferably an enzyme that has the property of acting on a glycan donor molecule containing a glycan containing GlcNAc whose reducing end is not activated in the presence of enzyme-A to generate an activated intermediate in which the reducing end is available for a glycosyltransfer reaction, thereby promoting the glycosyltransfer reaction of a glycan derived from the glycan donor molecule to an acceptor molecule by enzyme-A. Here, since the transglycosylation activity of Enzyme-B itself is thought to correlate with its ability to activate a glycan donor molecule, in one embodiment of the present invention, Enzyme-B can be selected from endo-β-N-acetylglucosaminidases that use as substrates the glycans of glycan donor molecules (e.g., SGP) containing GlcNAc whose reducing terminus is not activated, but do not use N297-linked glycans, using the transglycosylation activity to an acceptor having GlcNAc (e.g., a GlnNAc derivative such as 1-methylethyl-2-(acetylamino)-2-deoxy-β-D-glucopyranoside, 2-(acetylamino)-2-deoxy-β-D-glucopyranoside) as an indicator. Therefore, in one embodiment of the present invention, Enzyme-B is an enzyme that has the activity of transglycosylating SGP to a GlcNAc derivative.

[0371] The activation of glycan donor molecules by Enzyme-B acts on glycan donor molecules containing GlcNAc whose reducing end is not activated, generating an activated intermediate in which the reducing end is available for the glycosyltransferase reaction. When the hydrolysis activity of Enzyme-B is high, the activated intermediate does not react with the acceptor molecule via Enzyme-A, but with the H present in the reaction system. ２ Therefore, it is preferable that the enzyme B in the present invention has a relatively reduced hydrolysis activity compared to its sugar chain activation activity.

[0372] 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), and Endo-Om (an enzyme derived from Ogataea minuta-derived enzyme) (see Murakami S, et al., Glycobiology. 2013, 23, 736-744), Endo-Rp (Rhizomucor pusillus-derived enzyme) (WO2018101454), or mutant enzymes thereof (preferably mutant enzymes in which hydrolysis activity is reduced 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, which has a lower hydrolytic activity than the corresponding wild-type enzyme, and is preferably a mutant enzyme in which the amino acid residues shown in bold in Figures 33 to 36 have been appropriately substituted, and particularly preferably 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, ... N172P, Endo-Rp N172S, Endo-Rp N172T, Endo-Rp N172V, Endo-Rp W278F / S216V, Endo-Rp W278F / N246D, Endo-Rp W278F / D276N, 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 Examples of the enzymes include, but are not limited to, enzymes selected from the group consisting of Endo-M N175Q, Endo-M N175Q / Y217F, Endo-CC N180H, and Endo-Om N194Q. In addition to the specific mutations mentioned above, Enzyme-B may also have further mutations added. For example, with reference to patent literature (WO 2022 / 050300), 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 may be substituted, deleted, inserted, and / or added within a range that does not affect the enzyme activity. Furthermore, the amino acid sequence of Enzyme-B may be, with reference to patent documents (WO 2022 / 050300) and the like, an amino acid sequence having at least 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95%, 96%, 97%, 98%, or 99% or more homology or identity to amino acid residues other than specific mutant amino acid residues.

[0373] In a typical embodiment of the production method of the present invention, Enzyme-A and Enzyme-B are added as separate molecules in Step 1. However, Enzyme-A and Enzyme-B may be added to the reaction system in a directly or indirectly linked state, as long as they each perform their desired functions. Accordingly, in one embodiment of the present invention, Enzyme-A and Enzyme-B may be added to the reaction system in a directly or indirectly chemically linked state. For example, the two molecules may be added to the reaction system in the form of an immobilized enzyme immobilized on a solid support, or in the form of a fusion protein linked by any amino acid, polymer such as PEG, oligomer linker, or the like. In other words, in one embodiment of the present invention, Enzyme-A and Enzyme-B are directly or indirectly chemically linked (i.e., Enzyme-A and Enzyme-B are fused), and may be added to the reaction system in the form of an immobilized enzyme immobilized on a solid support, or in the form of a fusion protein linked by any amino acid, polymer such as PEG, oligomer linker, or the like, preferably an amino acid linker. In the present invention, even if Enzyme-A and Enzyme-B are not full-length molecules without deletions but are functional fragments, as long as the functional fragments have the desired function, the fragments are also considered to be one embodiment of Enzyme-A and Enzyme-B. Therefore, Enzyme-A and Enzyme-B in the present invention also include, for example, states in which "Enzyme-A" and "Enzyme-B," "an enzyme fragment having the function of Enzyme-A" and "an enzyme fragment having the function of Enzyme-B," "Enzyme-A" and "an enzyme fragment having the function of Enzyme-B," and "an enzyme fragment having the function of Enzyme-A" and "Enzyme-B" are chemically bonded directly or indirectly.Examples of the "enzyme fragment having the function of Enzyme-A" include the above-mentioned Enzyme-A, or Endo-BI1 (GenBank Accession number: ACJ53522.1), Endo-BI2 (GenBank Accession number: BAJ71450.1), Endo-BT-3987 (GenBank Accession number: AAO79092.1), Endo-E (GenBank Accession number: AAR20477.1), Endo-F (GenBank Accession number: AAA24922.1), and Endo-F2 (GenBank Accession number: Examples of such fragments include amino acid sequences obtained by fragmenting the full-length sequences of Endo-F3 (GenBank Accession number: AAA24923.1), Endo-F3 (GenBank Accession number: AAA24924.1), Endo-CoM (GenBank Accession number: XP006673222.1), and Endo-SB (GenBank Accession number: BBB35949.1) into specific functional sequences, and fragments having amino acid sequences that may contain specific mutations.Furthermore, examples of the "enzyme fragment having the function of Enzyme-B" include the catalytically active domain of the above-mentioned Enzyme-B, or Endo-A (GenBank Accession number: AAD10851.1), Endo-CE (GenBank Accession number: BAB84821.1), Endo-Tsp1006 (GenBank Accession number: CCY37287.1), Endo-Tsp1263 (GenBank Accession number: CDD88945.1), Endo-Tsp1457 (GenBank Accession number: CDD89351.1), Endo-BB ... Accession number: AAN25135.1), Endo-BH (GenBank Accession number: BAB04504.1), Endo-BN (GenBank Accession number: WP_007484749.1), Endo-CC1 (GenBank Accession number: XP_001839402.1), Endo-CC2 (GenBank Accession number: XP_002911817.1), Endo-D (GenBank Accession number: Examples of such fragments include amino acid sequences obtained by fragmenting the full-length sequences of Endo-Pm (GenBank Accession number: ADK97032.1) into specific functional sequences, which may also contain specific mutations.For example, with respect to the functional sequences of enzyme-A and enzyme-B, with reference to patent documents (WO 2022 / 050300) and the like, 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 residues may be substituted, deleted, inserted, and / or added, and each amino acid sequence, with reference to patent documents (WO 2022 / 050300) and the like, includes amino acid sequences having at least 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95%, 96%, 97%, 98% or 99% or more homology or identity to amino acid residues other than specific amino acid residues that affect activity.

[0374] Any combination of enzyme A and enzyme B can be used as long as the enzymes A and B fulfill their respective roles. For example, any combination of enzyme A and enzyme B listed in the following table can be used.

[0375]

[0376] Furthermore, as described above, when Enzyme-A and Enzyme-B are fused, any combination of Enzyme-A and Enzyme-B can be used as long as they perform their respective roles. More specifically, for example, Enzyme-A is an Endo-Si mutant and Enzyme-B is an Endo-Rp mutant, e.g., Enzyme-A is Endo-Si D241M / Q311L or Endo-Si D241Q / Q311L, and Enzyme-B is Endo-Rp N172H or Endo-Rp N172V. In yet 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 a fusion protein of Endo-Si D241Q / Q311L and Endo-Rp N172V, and examples thereof include the [Enzyme-A]-[Enzyme-B] fusion protein enzyme and the [Enzyme-B]-[Enzyme-A] fusion protein enzyme shown in the table below.

[0377]

[0378] Fusion proteins of enzyme-A and enzyme-B can be produced according to standard methods, for example, by binding enzyme-A and enzyme-B to any solid support via physical or chemical interaction, with reference to non-patent literature (Joaquim, et al., Processes. 2022, 10, 494.). Alternatively, fusion proteins can be produced in host cells by genetic engineering, 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). That is, after linking the genes encoding Enzyme-A and Enzyme-B, a single enzyme having functions derived from Enzyme-A and Enzyme-B can be produced in a host cell by genetic engineering. Producing a fusion enzyme in a host cell by genetic engineering in this way eliminates the need to prepare Enzyme-A and Enzyme-B separately, reducing the number of enzymes to be prepared, potentially reducing the cost and labor required for enzyme production. When Enzyme-A and Enzyme-B are linked via a linker composed of amino acids, the linker, in one embodiment, is composed of one or more amino acids. Linkers having typical properties known in the art can be used, with reference to non-patent literature (Chen, et al., Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357-1369.), for example. Flexible or rigid linkers, cleavable or non-cleavable linkers, and long or short linkers can be used. In one aspect, the linker can comprise, as an amino acid sequence fragment, (SGGS)n, (GGGS)n, (GGGGS)n, (G)n, (EAAAK)n, (EF)n, or (GGS)n, or a combination thereof (where n is an integer of 1 to 30, preferably an integer of 1 to 20, more preferably an integer of 1 to 10, and even more preferably an integer of 1 to 6). Furthermore, the linker can comprise an amino acid sequence commonly used as a purification tag, such as a polyhistidine tag, HA tag, FLAG tag, CBD tag, Fc tag, or GST tag.In one aspect, the purification tag is a polyhistidine tag, an HA tag, a FLAG tag, an Fc tag, or a GST tag, preferably a polyhistidine tag, an HA tag, a FLAG tag, or a CBD tag, more preferably a polyhistidine tag or a FLAG tag, and more preferably a polyhistidine tag.

[0379] <Additives> In one aspect of the present invention, step 1 involves reacting an acceptor molecule with a glycan donor molecule in the presence of enzyme-A, enzyme-B, and a specific additive to obtain a reaction mixture. Additives can positively affect the transglycosylation rate in a one-pot reaction through electrostatic interactions, hydrophobic interactions, solvent effects, and the like. In particular, mutations in the Fc region of an Fc-containing molecule (typically an antibody) can alter the conformation of the Fc region, resulting in a change in the transglycosylation rate. For example, when an antibody used as an acceptor molecule has a mutation in its Fc region, the transglycosylation rate can be reduced compared to when the antibody does not have the mutation. Additives are useful for regulating the transglycosylation rate of an Fc-containing molecule (typically an antibody) used as an acceptor molecule, and are particularly useful when the Fc region of the Fc-containing molecule (typically an antibody) used as an acceptor molecule has a mutation, which can reduce the transglycosylation rate.

[0380] In one embodiment of the present invention, the additive used in step 1 is a monovalent salt. As used herein, the term "monovalent salt" refers to a salt containing a monovalent anion or a monovalent cation. Examples of monovalent salts include, but are not limited to, inorganic salts (such as alkali metal salts of sodium chloride, lithium chloride, potassium chloride, potassium iodide, sodium iodide, lithium iodide, potassium bromide, sodium fluoride, and potassium fluoride; and ammonium salts of ammonium fluoride, ammonium chloride, and ammonium sulfate), and organic salts (such as ammonium acetate, sodium acetate, and potassium acetate). In one embodiment of the present invention, the additive used in step 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 step 1 is a monovalent alkali metal salt, preferably sodium chloride, potassium acetate, lithium chloride, or sodium acetate, and more preferably sodium chloride.

[0381] In one embodiment of the present invention, the additive used in step 1 is an organic solvent. Examples of organic solvents that can be used in step 1 include, but are not limited to, 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. In one embodiment of the present invention, the organic solvent used in step 1 is ethanol or dimethyl sulfoxide.

[0382] In one embodiment of the present invention, the additive used in step 1 is a surfactant. Examples of surfactants that can be used in step 1 include, but are not limited to, anionic surfactants, cationic surfactants, nonionic surfactants, and amphoteric surfactants.Examples of anionic surfactants include carboxylic acid surfactants such as sodium octanoate and sodium decanoate, sulfonic acid surfactants such as sodium 1-hexanesulfonate and sodium 1-octanesulfonate, sulfate ester surfactants such as sodium lauryl sulfate and sodium myristyl sulfate, and phosphate ester surfactants such as lauryl phosphate and sodium lauryl phosphate. Examples of cationic surfactants include quaternary ammonium salt surfactants such as tetramethylammonium chloride and hexadecyltrimethylammonium bromide, alkylamine salt surfactants such as monomethylamine hydrochloride and dimethylamine hydrochloride, and pyridine ring-containing surfactants such as butylpyridinium chloride and cetylpyridinium chloride. Examples of nonionic surfactants include ester surfactants such as glycerin laurate and sorbitan fatty acid esters, polyethylene glycols (polyethylene glycol 2000, polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 11000, polyethylene glycol Examples of the surfactants include surfactants of ether type such as polyoxyethylene alkylphenyl ether, ester ether type such as polyoxyethylene glycerin fatty acid ester and polyoxyethylene 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 such as cetanol and stearyl alcohol, and examples of the amphoteric surfactants include surfactants of alkyl betaine type such as lauryl dimethylaminoacetic acid betaine, fatty acid amidopropyl betaine type such as cocamidopropyl betaine and cocamidopropyl hydroxysultaine, amino acid type such as sodium lauroyl glutamate and potassium lauroyl glutamate, and amine oxide type such as lauryl dimethylamine N-oxide and oleyl 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 a polyoxyethylene sorbitan fatty acid ester, preferably polyethylene glycol 6000 or polysorbate 20.

[0383] In one embodiment of the present invention, the additive used in step 1 is a sugar. Examples of sugars that can be used in step 1 include, but are not limited to, monosaccharides, disaccharides, oligosaccharides (trisaccharides or higher are referred to as oligosaccharides herein), deoxysugars, uronic acids, amino sugars, sugar alcohols, lactones, and polysaccharides. Examples of monosaccharides that can be used as additives in step 1 include aldotrioses such as glyceraldehyde, aldotetroses such as erythrose and threose, aldopentoses such as ribose and xylose, aldohexoses such as glucose, mannose, and galactose, ketotrioses such as dihydroxyacetone, ketotetroses such as erythrulose, ketopentoses such as xylulose and ribulose, and ketohexoses such as fructose and sorbose. When an Endo-Rp enzyme (including mutants) is used as Enzyme-B in step 1, glucose may act as an acceptor for the sugar chain activated by the Endo-Rp enzyme, thereby reducing the rate of glycosylation. Therefore, in one embodiment of the present invention, the sugars used as additives in step 1 do not contain glucose. Examples of disaccharides used as additives in step 1 include trehalose, isotrehalose, kojibiose, sophorose, nigerose, laminaribiose, maltose, cellobiose, isomaltose, gentiobiose, lactose, sucrose, etc.; oligosaccharides include fructooligosaccharides and galactooligosaccharides, etc.; deoxysugars include deoxyribose and fucose, uronic acids include glucuronic acid and galacturonic acid, amino sugars include glucosamine and galactosamine sugar, sugar alcohols include glycerin, xylitol, sorbitol, etc.; lactones include ascorbic acid and glucuronolactone, etc.; and polysaccharides include starch, amylose, amylopectin, glycogen, cellulose, pectin, glucomannan, etc. 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.

[0384] In one embodiment of the present invention, the additive used in step 1 is an amino acid. The amino acids that can be used as additives in step 1 are not particularly limited as long as they are organic compounds having both amino and carboxy functional groups. 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; and neutral nonpolar amino acids such as isoleucine, leucine, valine, methionine, phenylalanine, tryptophan, alanine, glycine, and proline. In one embodiment of the present invention, the amino acids used as additives in step 1 are basic amino acids, preferably arginine, histidine, or lysine, and more preferably arginine or histidine. Furthermore, the amino acids that can be used as additives in step 1 may be added in the form of a salt; for example, the basic amino acids arginine, histidine, and lysine can be added as hydrochlorides. Therefore, in one embodiment of the present invention, the amino acid used as an additive in step 1 is arginine hydrochloride, histidine hydrochloride or lysine hydrochloride, more preferably arginine hydrochloride or histidine hydrochloride.

[0385] The concentration of the additive used in step 1 can be set arbitrarily as long as the addition of the additive does not precipitate the Fc-containing molecule or the enzyme, i.e., as long as the reactivity of the one-pot reaction remains, and can be set, for example, by using or applying the method described in Example 15-3 of the present 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, 40 mM or more, 50 mM or more, In another embodiment of the present invention, when the additive used in step 1 is a monovalent salt (e.g., sodium chloride), its concentration can be set within any concentration range between any of the upper and lower limits exemplified above, for example, 1 to 700 mM, 10 to 600 mM, 100 to 550 mM, or 50 to 1000 mM. In one embodiment of the present invention, the additive may be contained in advance or may be added in step 1.

[0386] In one embodiment of the present invention, when the additive used in step 1 is an organic solvent (e.g., ethanol or 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, even more preferably 0.4 to 3% v / v, particularly preferably 0.5 to 2% v / v, and most preferably about 1% v / v.

[0387] In one embodiment of the present invention, when the additive used in step 1 is a surfactant (e.g., polyethylene glycol 6000 or 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, even more preferably 0.4 to 3% v / v, particularly preferably 0.5 to 2% v / v, and most preferably about 1% v / v.

[0388] In one embodiment of the present invention, when the additive used in step 1 is a sugar (e.g., trehalose or sorbitol), its concentration is 1 mM to 10 M, preferably 5 to 1000 mM, more preferably 10 to 500 mM, even more preferably 15 to 200 mM, particularly preferably 20 to 100 mM, and most preferably about 40 mM.

[0389] In one embodiment of the present invention, when the additive used in step 1 is an amino acid (e.g., arginine hydrochloride or histidine hydrochloride), the concentration thereof is 1 mM to 10 M, preferably 5 to 1000 mM, more preferably 10 to 500 mM, even more preferably 15 to 200 mM, particularly preferably 20 to 100 mM, and most preferably about 40 mM.

[0390] <Step 1> In one aspect of the present invention, Step 1 is a step in which an acceptor molecule and a glycan donor molecule are reacted in the presence of enzyme A, enzyme B, and a specific additive to obtain a reaction mixture. The reaction conditions for Step 1 can be appropriately selected according to the reaction conditions of a known transglycosylation reaction using GlcNAc whose reducing end is not activated as the glycan donor molecule, or by utilizing or applying the reaction conditions of a known transglycosylation reaction.

[0391] The reaction in step 1 is preferably carried out in a buffer solution, desirably under conditions that do not promote the decomposition of glycan donor molecules containing GlcNAc at an unactivated reducing end. From this perspective, in one embodiment of the present invention, the pH of the reaction system in step 1 can be appropriately selected between 5.8 and 9.5, preferably between 6.2 and 8.5, more preferably between 6.5 and 8.0, and even more preferably between 6.5 and 7.5. The buffer solution can be appropriately selected from phosphate buffer (pH 6.0-7.5), MOPS-NaOH buffer (pH 6.5-8.0), Tris-HCl buffer (pH 7.0-9.0), etc., and is preferably Tris-HCl buffer (pH 7.0-9.0). For the purpose of stabilizing the enzyme, an additive that does not inhibit the enzymatic reaction may be added to the reaction solution.

[0392] The reaction temperature can be appropriately selected between 4°C and 50°C, but is preferably 15°C to 45°C, more preferably 20°C to 40°C, and even more preferably 25°C to 40°C.

[0393] The reaction time can be appropriately selected between 10 minutes and 96 hours, preferably between 0.5 hours and 80 hours, more preferably between 2 hours and 70 hours, even more preferably between 4 hours and 60 hours, particularly preferably between 6 and 48 hours or between 8 and 30 hours, and most preferably between 16 and 24 hours. The completion of the reaction can be determined by sampling a small amount of the reaction solution over time and checking the progress of the transglycosylation reaction. Generally, the progress of the transglycosylation reaction can be monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis, microchip electrophoresis, a fully automated electrophoresis system, or liquid chromatography mass spectrometry (LC-MS). For example, after fragmenting a glycosylation remodeling antibody into heavy and light chains, the reaction can be monitored by confirming a change in retention time of only the heavy chain to which the N297-linked glycan is attached using a fully automated electrophoresis system.

[0394] Step 1 can be performed as a one-pot method in which the sugar chain of a sugar chain donor molecule is directly transferred to an acceptor molecule, an antibody or an Fc region-containing molecule thereof, having a core GlcNAc optionally having fucose attached thereto as an N297-linked sugar chain.

[0395] In glycan remodeling using a glycan donor molecule containing an oxazolinated glycan at the reducing end, glycation, a non-Endo enzyme-mediated chemical reaction, is more likely to occur with Lys in the acceptor molecule, so it is common to set the antibody concentration in the reaction solution low. On the other hand, when a glycan donor molecule containing GlnNAc at an inactivated reducing end is used, a chemically synthesized oxazoline solution, which is the cause of glycation, is not added, so there is no need to worry about glycation, and therefore a relatively high acceptor molecule concentration can be used. Rather, adding a high concentration of acceptor molecule is effective from the perspective of increasing the frequency of intermolecular contact. Therefore, the acceptor molecule concentration in step 1 can be appropriately set in accordance with known methods. For example, the upper limit of the final acceptor molecule concentration in the reaction system in step 1 can be 200 mg / mL, preferably 160 mg / mL, more preferably 120 mg / mL, even more preferably 100 mg / mL, and particularly preferably 80 mg / mL. The lower limit can be 10 mg / mL, preferably 14 mg / mL, more preferably 18 mg / mL, and even more preferably 20 mg / mL. The concentration range can 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, even more preferably 20 mg / mL to 100 mg / mL, and particularly preferably 20 mg / mL to 80 mg / mL, but is not limited thereto.

[0396] The amount of sugar chain donor molecule added in step 1 can be appropriately determined by utilizing or applying a known method. For example, in one embodiment of the present invention, the lower limit of the amount of glycan donor molecule added (used) in step 1 can be 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 glycan donor molecule can be 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 to these. Furthermore, the range of the amount of the sugar chain donor molecule to be added (used) in step 1 can be any combination of the upper and lower limits mentioned above, 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, but is not limited to these.

[0397] In one embodiment of the present invention, the transglycosylation rate in step 1 can be set to any value, for example, 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. Alternatively, 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 transglycosylation rate in step 1 exceeds 80%, preferably exceeds 85%, and particularly preferably exceeds 90%. The "transglycosylation rate" can be determined according to standard methods, for example, using the fully automated electrophoresis system disclosed in Example 15-2B. The timing of measurement of the transglycosylation rate varies depending on the enzyme used, but is typically between 1 minute and 100 hours, preferably between 1 hour and 80 hours, and more preferably between 2 hours and 72 hours.

[0398] The acceptor molecule used in step 1 can be prepared by a separate preparation method independent of step 1. Therefore, in one embodiment of the present invention, the method of the present invention further comprises a step of separately preparing an acceptor molecule prior to step 1. In this embodiment, the acceptor molecule can be prepared in a step independent of the glycosylation reaction in step 1, for example, by reacting an enzyme that is the same as or different from Enzyme-A (an enzyme having the activity of transferring a glycosylation chain in a glycosylation donor molecule to an acceptor molecule) used in step 1 with a starting Fc-containing molecule (an Fc-containing molecule to be subjected to glycosylation modification, typically an Fc-containing molecule having a host cell-derived N297-linked glycosylation chain, a heterogeneous N297-linked glycosylation chain, or a host cell-derived heterogeneous N297-linked glycosylation chain). In one embodiment of the present invention, the acceptor molecule can be prepared, for example, by treating the N297-linked glycan of an Fc-containing molecule to be subjected to glycosylation with ENGase that retains the activity of specifically hydrolyzing the 1,4-glycosidic bond between GlcNAcs (GlcNAcβ1-4GlcNAc) in the core chitobiose structure of the N297-linked glycan.

[0399] Alternatively, if the enzyme-A used in step 1 retains hydrolytic activity, the acceptor molecule in step 1 can be prepared in situ in the same vessel as step 1. Thus, step 1 of the present invention encompasses an embodiment in which the acceptor molecule is prepared in situ. In this embodiment, the step of separately preparing the acceptor molecule prior to step 1 can be omitted, thereby simplifying the production process. This simplification of the production process is highly beneficial because it obviates the need for a step to remove the ENGase used in that other step, and also obviates the need for procedures such as separating and purifying the relatively unstable acceptor molecule produced in that other step. Furthermore, because the same enzyme-A is used as both the enzyme that hydrolyzes the N297-linked glycan of the starting Fc-containing molecule and the enzyme that transfers the glycan on the glycan donor molecule, the production process can benefit from the reduction in the number and types of enzymes used (this advantage is even more pronounced when a fusion protein of enzyme-A and enzyme-B is used). In this embodiment, an Fc-containing molecule to be subjected to glycosylation (typically, an IgG monoclonal antibody or an Fc fragment of the antibody or an Fc-containing molecule such as CLCH consisting only of the constant region) is introduced into the reaction system in step 1, rather than an acceptor molecule itself, which is a molecule that directly receives a glycosylation chain from a glycosylation donor molecule. The N297-linked glycosylation chain of the Fc-containing molecule to be introduced as such a raw material is not particularly limited as long as it is different from the glycosylation chain derived from the glycosylation donor molecule of interest, but typical examples include an N297-linked glycosylation chain derived from the host cells used in producing the Fc-containing molecule, a heterogeneous N297-linked glycosylation chain, or a heterogeneous N297-linked glycosylation chain derived from the host cells. When such an Fc-containing molecule is introduced into the reaction system of step 1, the hydrolytic activity of enzyme-A specifically hydrolyzes the 1,4-glycosidic bond (GlcNAcβ1-4GlcNAc) between GlcNAcs in the core chitobiose structure of the N297-linked sugar chain of the Fc-containing molecule, generating an acceptor molecule in situ.Subsequently, the sugar chain of the sugar chain donor molecule is transferred to the in situ generated acceptor molecule by the transglycosylation activity of Enzyme-A, thereby producing the target Fc-containing molecule having an N297-linked sugar chain containing a sugar chain derived from the sugar chain donor molecule in one pot. In this embodiment, although the acceptor molecule is generated in situ, it is not isolated or purified, so it appears as if the N297-linked sugar chain of the Fc-containing molecule input as a raw material has been directly exchanged with the sugar chain derived from the sugar chain donor molecule in one step. Therefore, in this specification, this embodiment is appropriately referred to as the "direct exchange reaction of N297-linked sugar chains."

[0400] In the direct exchange reaction of N297-linked glycans, any kind of glycan donor molecule as described above can be used without any particular limitation. Note that, although the reaction conditions such as reaction temperature in the direct exchange reaction of N297-linked glycans are as described above, it is preferable to set the reaction time longer than in the embodiment including the step of separately preparing the acceptor molecule.

[0401] In one aspect of the present invention, the method for producing an Fc-containing molecule further comprises a step of recovering an Fc-containing molecule having an N297-linked glycan that includes a glycan derived from a glycan donor molecule from the reaction mixture obtained in step 1. The recovery can be carried out appropriately by a method well known in the art or a method based on such a method, but is particularly preferably carried out by step 2 described below. Thus, in one aspect of the present invention, the method for producing an Fc-containing molecule further comprises step 2 described below.

[0402] <Step 2> In one aspect of the present invention, Step 2 is a step of contacting the reaction mixture obtained in Step 1 with a cation exchange chromatography medium or a multimode chromatography medium under acidic conditions to recover an Fc-containing molecule having an N297-linked glycan that contains a glycan derived from a glycan donor molecule, and preferably a step of contacting the reaction mixture obtained in Step 1 with a cation exchange chromatography medium or a multimode chromatography medium under acidic conditions to isolate and recover an Fc-containing molecule having an N297-linked glycan that contains a glycan derived from a glycan donor molecule contained in the reaction mixture from partly or entirely unreacted Fc-containing molecules (acceptor molecules). In one aspect of the present invention, step 2 is a step in which the reaction mixture obtained in step 1 is used as a load solution as is, or a load solution is prepared by appropriately adding a buffer to the reaction mixture, and the load solution is brought into contact with a cation exchange chromatography medium or a multimode chromatography medium under acidic conditions to selectively adsorb unreacted acceptor molecules onto the medium, while the flow-through liquid containing Fc-containing molecules having an N297-linked glycan that includes a glycan derived from a glycan donor molecule is collected, thereby selectively recovering the Fc-containing molecules.

[0403] In step 2, the target glycosylated Fc-containing molecule can be separated from the unreacted Fc-containing molecule (acceptor molecule). This allows the production of highly purified Fc-containing molecules with uniform glycosylation structures even when only a small amount of glycosylation donor molecule is used in step 1. According to this concept, in order to produce an Fc-containing molecule with a glycosylation structure with a high glycosylation rate while suppressing the amount of glycosylation donor molecule used in step 1, step 2 must have high selectivity and recovery rate. If the selectivity is low, the target glycosylated Fc-containing molecule cannot be isolated from the unreacted Fc-containing molecule (acceptor molecule), preventing an increase in the glycosylation rate. Furthermore, if the recovery rate is low, the amount of acceptor molecule added in step 1 must be increased to increase the yield of the Fc-containing molecule, resulting in an increased amount of glycosylation donor molecule used. Furthermore, when the present invention is applied to an industrial-scale method for producing therapeutic antibodies, it is necessary to efficiently isolate the target antibody in step 2 while maintaining the desired biological activity.

[0404] As a conventional isolation technique, capillary gel electrophoresis (CE-SDS) is known as an analytical method for detecting antibodies without a glycosylated chain (i.e., without a core GlcNAc) contained in antibodies with an N297-linked glycosylated chain (Richard R. Rustandi et al., Electrophoresis. 2008 Sep; 29(17): 3612-20). While this method can isolate antibody heavy chains fragmented under reducing conditions according to the hydrodynamic size of the molecules, it denatures the antibodies during the pretreatment process, causing them to lose their biological activity, and the processing capacity is limited to approximately several hundred μg because the antibodies are isolated using a medium filled in a fine capillary. Furthermore, a method using hydrophilic interaction chromatography (HILIC) is known as an analytical technique for separating antibodies having a core GlcNAc group prepared using ENGase from antibodies having an N297-linked glycan before the treatment with ENGase (Matthew A et al., Measuring the Glycan Occupancy of Intact mAbs using HILIC and Detection by Intrinsic Fluorescence). While this method can separate antibodies having an N297-linked glycan from antibodies having a core GlcNAc group without requiring antibody fragmentation, heating and exposure to organic solvents can denature the antibodies, causing them to lose their biological activity. In addition, isolation requires an analytical column with a high theoretical plate count and an ultra-high performance liquid chromatography (UHPLC) system, limiting the throughput to a few μg. Furthermore, a method using cation exchange chromatography is known as an analytical technique for isolating an antibody having a core GlcNAc group prepared using ENGase from an antibody having an N297-linked glycan before the treatment with ENGase (Masaki Kurogochi et al., PLOS ONE | DOI: 10.1371 / journal.pone.0132848 July 22, 2015, and Shiyi Wang et al., Journal of Chromatography A, 1217 (2010) 6496-6502).While this method can separate antibodies with N297-linked glycans from those with core GlcNAc without denaturing the antibodies, it has the drawbacks of requiring an analytical column with a high theoretical plate number and a gradient elution method in which two solutions are mixed using a complex program to achieve high-resolution separation, limiting the processing capacity to several hundred μg, and not achieving a satisfactory yield. Therefore, prior to the present invention, no method for isolating antibodies based on the presence or absence of glycosylation that could be applied for industrial-scale isolation and purification had been reported.

[0405] Under these circumstances, the present inventors have discovered that the target glycosylated Fc-containing molecule can be selectively isolated and purified in high yield without denaturation by a very simple method of contacting the reaction mixture with a cation exchange chromatography medium or a multimode chromatography medium under acidic conditions.

[0406] Step 2 can be carried out according to conventional methods for cation exchange chromatography or multimode chromatography. For example, the reaction mixture obtained in step 1 is mixed with an acidic buffer to obtain a loading solution, and the loading solution is passed through a column packed with a cation exchange chromatography medium or a multimode chromatography medium, thereby separating molecules adsorbed to the column from molecules that pass through. In step 2, an equilibration solution (or a washing solution) is subsequently passed through the column to wash away molecules nonspecifically adsorbed to the column, and then an elution solution (or a regeneration solution) is passed through the column to elute molecules specifically adsorbed to the column. After step 2, a cleaning solution can be passed through as needed for the purpose of cleaning in place.

[0407] In step 2, cation exchange chromatography or multimodal chromatography is performed under acidic conditions. In one embodiment of the present invention, the loading solution (or the loading solution and equilibration solution) in step 2 has an acidic pH. In step 2, the lower the pH of the loading solution (o...

Claims

1. The following formula (II): 【Chemistry 1】 (In the formula, m １ The range is from 1 to 10. Ab represents an antibody or a functional fragment of the antibody, the antibody or functional fragment of the antibody may have a remodeled glycan, and the amino acid residues at positions 234, 235, and 265 (all EU numbered) of the Fc region of the antibody or functional fragment of the antibody are Ala, Ala, and Gly, respectively, where the antibody represents an anti-EGFR antibody. L represents the linker connecting Ab and D. Ab may be bound to L directly from its amino acid residue, or it may be bound to L from Ab's sugar chain or a remodeled sugar chain. D is given by the following equation (I): 【Chemistry 2】 (Here, L is L １ It binds with the hydroxyl group contained in, L １ The formula is as follows: 【Transformation 3】 (Here, the wavy line indicates the substitution position.) This shows the basis of Q and Q ’ Each of these independently represents either a hydroxyl group or a thiol group. R ２１ and R ２２ Each of these independently represents either a hydroxyl group or a fluorine atom. (W represents -NH- or a sulfur atom) (This indicates a compound represented by the formula.) An antibody-drug conjugate represented by [the specified format].

2. D is expressed in the following two equations: 【Chemistry 4】 (Here, L １ (Q, Q', and W are as previously defined.) Indicated by one of the following: In some cases, D is expressed in the following two equations: 【Transformation 5】 (Here, the asterisk indicates that it is coupled with L, and Q, Q', and W are as previously defined.) Indicated by one of the following: In some cases, D is expressed by the following three equations: 【Transformation 6】 (Here, the asterisk indicates that it is coupled with L, and W is as defined previously.) The antibody-drug conjugate according to claim 1, represented by any one of the following.

3. D is represented by the following three equations: 【Transformation 7】 (Here, the asterisk indicates that it is bound to L.) The antibody-drug conjugate according to claim 1, represented by any one of the following.

4. D is represented by the following four equations: 【Transformation 8】 (Here, the asterisk indicates that it is bound to L.) The antibody-drug conjugate according to claim 1, represented by any one of the following.

5. D is given by the following equation: 【Chemistry 9】 (Here, the asterisk indicates that it is bound to L.) The antibody-drug conjugate according to claim 1, as shown in [the provided symbol].

6. The linker L is denoted as -Lb-La-Lp-Lc-*, In the formula, the asterisk indicates that it is bound to drug D. Lp either exhibits or does not exhibit a linker consisting of an amino acid sequence that can be cleaved in the target cell. La indicates one of the following groups: -C(=O)-(CH ２ CH ２ )n ２ -C(=O)-、 -C(=O)-(CH ２ CH ２ )n ２ -CH ２ -C(=O)-、 -C(=O)-(CH ２ CH ２ )n ２ -C(=O)-NH-(CH ２ CH ２ )n ３ -C(=O)-、 -C(=O)-(CH ２ CH ２ )n ２ -C(=O)-NH-(CH ２ CH ２ )n ３ -CH ２ -C(=O)-、 -C(=O)-(CH ２ CH ２ )n ２ -C(=O)-NH-(CH ２ CH ２ O)n ３ -CH ２ -C(=O)-、 - (CH ２ )n ４ -O-C(=O)-, and, -(CH ２ )n ９ -C(=O)- (Here, n ２ represents an integer between 1 and 3, and n ３ represents an integer from 1 to 5, and n ４ represents an integer between 0 and 2, and n ９ (where represents an integer between 2 and 7) Lb indicates a spacer that connects the glycans of La and Ab or a remodeled glycan, or a spacer that connects the cysteine ​​residues of La and Ab, and, Lc is -NH-CH ２ -, -NH-phenyl group-CH ２ -O(C=O)- or -NH- heteroaryl group -CH ２ It either shows -O(C=O)- or does not exist. In some cases, Lc is -NH-CH ２ - The antibody-drug conjugate according to claim 1.

7. Lp is one of the following: -GGGF-, -GGPI-, -GGVA-, -GGFM-, -GGVCit-, -GGFCit-, -GGICit-, -GGPL-, -GGAQ-, or -GGPP-. In some cases, The antibody-drug conjugate according to claim 6, wherein Lp is -GGGF- or -GGPI-.

8. La is as follows: -C(=O)-CH ２ CH ２ -C(=O)-、 -C(=0)-CH ２ CH ２ -C(=O)-NH-(CH ２ CH ２ O) ３ -CH ２ -C(=O)-、 -C(=O)-CH ２ CH ２ -C(=O)-NH-(CH ２ CH ２ O) ４ -CH ２ -C(=O)-, and -(CH ２ ) ５ -C(=O)- The antibody-drug conjugate according to claim 6, which represents one selected from the group consisting of the following.

9. Lb is given by the following equation: 【Chemistry 10】 or 【Chemistry 11】 The antibody-drug conjugate according to claim 6, represented by either (in the structural formula of Lb shown above, an asterisk indicates binding to La, and a wavy line indicates binding to the sugar chain or remodeled sugar chain of Ab).

10. Lb is -(succinimido-3-yl-N)-, Here, -(succinimido-3-yl-N)- has the following structural formula: 【Chemistry 12】 Show, Herein, the asterisk indicates binding to La, and the wavy line indicates binding by forming a thioether with the side chain of a cysteine ​​residue of the antibody, as described in claim 6.

11. The linker L is denoted as -Lb-La-Lp-Lc-*, In the formula, the asterisk indicates that it is bound to drug D. Lp is -GGGFG- or -GGPI-, La is -C(=O)-CH ２ CH ２ Show -C (=O)- Lb is given by the following equation: 【Chemistry 13】 (In the structural formula of Lb shown above, the asterisk indicates that it is bonded to La, and the wavy line indicates that it is bonded to the sugar chain or remodeled sugar chain of Ab.) Lc is -NH-CH ２ The antibody-drug conjugate according to claim 6, which indicates -.

12. The average number of drug bindings per antibody molecule in antibody-drug conjugates is in the range of 1 to 10. In some cases, The average number of drug-bound antibodies per antibody molecule in antibody-drug conjugates is in the range of 1 to 5. In some cases, The antibody-drug conjugate according to claim 1, wherein the average number of drug-bound antibodies per molecule in the antibody-drug conjugate is in the range of 1 to 3 or 3 to 5.

13. The antibody is linked to L via a sugar chain (N297 sugar chain) that binds to Asn297 of the antibody. In some cases, The antibody-drug conjugate according to claim 1, wherein the N297 glycan is a remodeled glycan.

14. The antibody-drug conjugate according to claim 13, wherein the N297 glycan is N297-(Fuc)MSG1 or N297-(Fuc)SG having the structure shown in the following formula: 【Chemistry 14】 In the formula, the wavy line indicates that the antibody is bound to Asn297. L (PEG) is - (CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ It shows -NH-, indicating that the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at position 2 of the sialic acid at the non-reducing end of the β-Man branched chain on the 1-3 chain side of the N297 sugar chain. The asterisk indicates that Lb in the linker L is bonded to the nitrogen atom at position 1 or 3 on the 1,2,3-triazole ring, and n ５ is an integer between 2 and 5; 【Chemistry 15】 In the formula, the wavy line indicates that the antibody is bound to Asn297. L (PEG) is - (CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ It indicates -NH-, and that the amino group at the right end of L(PEG) is amide-bonded to 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 β-Man branched chain of the N297 sugar chain. The asterisk indicates that Lb in the linker L is bonded to the nitrogen atom at position 1 or 3 on the 1,2,3-triazole ring, and n ５ The integer is between 2 and 5.

15. The following formula: 【Chemistry 16】 (In the formula, m ２ represents an integer of 1 or 2, L is a linker that connects the N297 glycan and D, as defined previously. Ab represents an antibody or a functional fragment of the antibody, where the amino acid residues at positions 234, 235, and 265 (all EU numbered) of the Fc region of the antibody or functional fragment of the antibody are Ala, Ala, and Gly, respectively, where the antibody represents an anti-EGFR antibody. The N297 glycan is N297-(Fuc)MSG1 or N297-(Fuc)SG having the structure shown in the following formula. 【Chemistry 17】 In the formula, the wavy line indicates that the antibody is bound to Asn297. L (PEG) is - (CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ It shows -NH-, indicating that the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at position 2 of the sialic acid at the non-reducing end of the β-Man branched chain on the 1-3 chain side of the N297 sugar chain. The asterisk indicates that Lb in the linker L is bonded to the nitrogen atom at position 1 or 3 on the 1,2,3-triazole ring, and n ５ This represents an integer between 2 and 5; [Chemistry 18] In the formula, the wavy line indicates that the antibody is bound to Asn297. L (PEG) is - (CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ It indicates -NH-, and that the amino group at the right end of L(PEG) is amide-bonded to 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 β-Man branched chain of the N297 sugar chain. The asterisk indicates that Lb in the linker L is bonded to the nitrogen atom at position 1 or 3 on the 1,2,3-triazole ring, and n ５ represents an integer of 2 to 5, D is represented by one of the following four equations: 【Chemistry 19】 The antibody-drug conjugate according to claim 6, represented by (wherein the formula, the asterisk indicates that it is bound to L).

16. The following formula: 【Chemistry 20】 【change】 Selected from, In each of the structural formulas shown above, m ２ This indicates that it is an integer of 1 or 2. Ab represents an antibody or a functional fragment of the antibody, where the amino acid residues at positions 234, 235, and 265 (all EU numbered) of the Fc region of the antibody or functional fragment of the antibody are Ala, Ala, and Gly, respectively, where the antibody represents an anti-EGFR antibody. The N297 glycan is either N297-(Fuc)MSG1 or N297-(Fuc)SG, which have the structure shown in the following formula. 【Chemistry 21】 In the formula, the wavy line indicates that the antibody is bound to Asn297. L (PEG) is - (CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ It shows -NH-, indicating that the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at position 2 of the sialic acid at the non-reducing end of the β-Man branched chain on the 1-3 chain side of the N297 sugar chain. The asterisk indicates that it is bonded to the nitrogen atom at position 1 or 3 on the 1,2,3-triazole ring, and n ５ represents an integer of 2 to 5; 【Chemistry 22】 In the formula, the wavy line indicates that the antibody is bound to Asn297. L (PEG) is - (CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ It indicates -NH-, and that the amino group at the right end of L(PEG) is amide-bonded to 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 β-Man branched chain of the N297 sugar chain. The asterisk indicates that it is bonded to the nitrogen atom at position 1 or 3 on the 1,2,3-triazole ring, and n ５ This represents an integer between 2 and 5. In some cases, The following formula: 【Chemistry 23】 【change】 An antibody-drug conjugate according to claim 15, selected from the above.

17. The antibody is an antibody comprising a light chain consisting of the amino acid sequence described in SEQ ID NO: 30 and a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 32, or an antibody comprising a light chain consisting of the amino acid sequence described in SEQ ID NO: 30 and a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 33, or an antibody comprising a heavy chain in which one or more amino acid residues at the carboxyl terminus of any one of the above antibodies are deleted; or The antibody comprises a light chain containing a light chain variable region consisting of the amino acid sequence described in SEQ ID NO: 29 and a heavy chain containing a heavy chain variable region consisting of the amino acid sequence described in SEQ ID NO: 31, wherein the amino acid residues at positions 234, 235, and 265 (all EU numbered) of the Fc region of the antibody are Ala, Ala, and Gly, respectively; or The antibody comprises a light chain containing CDRL1 consisting of the amino acid sequence described in SEQ ID NO: 23, CDRL2 consisting of the amino acid sequence described in SEQ ID NO: 24, and CDRL3 consisting of the amino acid sequence described in SEQ ID NO: 25, and a heavy chain containing CDRH1 consisting of the amino acid sequence described in SEQ ID NO: 26, CDRH2 consisting of the amino acid sequence described in SEQ ID NO: 27, and CDRH3 consisting of the amino acid sequence described in SEQ ID NO: 28, wherein the amino acid residues at positions 234, 235, and 265 (all EU numbered) of the Fc region of the antibody are Ala, Ala, and Gly, respectively. The antibody-drug conjugate according to claim 1.

18. The following formula: 【Chemistry 24】 It is expressed as follows, and in the formula Ab is An antibody comprising a light chain consisting of the amino acid sequence described in SEQ ID NO: 30 and a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 33; or An antibody comprising a light chain consisting of the amino acid sequence described in SEQ ID NO: 30 and a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 33, wherein the heavy chain comprises a heavy chain in which one or more amino acid residues at the carboxyl terminus of the heavy chain are deleted. Show, The N297 glycan is given by the following formula: 【Chemistry 25】 (In the formula, the wavy line indicates that the antibody is bound to Asn297.) L (PEG) is - (CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ It indicates -NH-, and that the amino group at the right end of L(PEG) is amide-bonded to 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 β-Man branched chain of the N297 sugar chain. The asterisk indicates that it is bonded to the nitrogen atom at position 1 or 3 on the 1,2,3-triazole ring. n ５ is 3, and I understand ２ An antibody-drug conjugate represented as (where is 2).

19. The following formula: 【Chemistry 26】 It is expressed as follows, and in the formula Ab is The present invention relates to an antibody comprising a light chain containing a light chain variable region consisting of the amino acid sequence described in SEQ ID NO: 29, and a heavy chain containing a heavy chain variable region consisting of the amino acid sequence described in SEQ ID NO:

31. The amino acid residues at positions 234, 235, and 265 (all EU numbered) of the Fc region of the antibody are Ala, Ala, and Gly, respectively. The N297 glycan is given by the following formula: 【Chemistry 27】 (In the formula, the wavy line indicates that the antibody is bound to Asn297.) L (PEG) is - (CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ It indicates -NH-, and that the amino group at the right end of L(PEG) is amide-bonded to 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 β-Man branched chain of the N297 sugar chain. The asterisk indicates that it is bonded to the nitrogen atom at position 1 or 3 on the 1,2,3-triazole ring. n ５ is 3, and I understand ２ An antibody-drug conjugate represented as (where is 2).

20. The following formula: 【Chemistry 28】 It is expressed as follows, and in the formula Ab is An antibody comprising a light chain containing CDRL1 consisting of the amino acid sequence described in SEQ ID NO: 23, CDRL2 consisting of the amino acid sequence described in SEQ ID NO: 24, and CDRL3 consisting of the amino acid sequence described in SEQ ID NO: 25, and a heavy chain containing CDRH1 consisting of the amino acid sequence described in SEQ ID NO: 26, CDRH2 consisting of the amino acid sequence described in SEQ ID NO: 27, and CDRH3 consisting of the amino acid sequence described in SEQ ID NO:

28. Show, The amino acid residues at positions 234, 235, and 265 (all EU numbered) of the Fc region of the antibody are Ala, Ala, and Gly, respectively. The N297 glycan is given by the following formula: 【Chemistry 29】 (In the formula, the wavy line indicates that the antibody is bound to Asn297.) L (PEG) is - (CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ It indicates -NH-, and that the amino group at the right end of L(PEG) is amide-bonded to 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 β-Man branched chain of the N297 sugar chain. The asterisk indicates that it is bonded to the nitrogen atom at position 1 or 3 on the 1,2,3-triazole ring. n ５ is 3, and I understand ２ An antibody-drug conjugate represented as (where is 2).

21. The following formula: 【Transformation 30】 It is expressed as follows, and in the formula Ab is An antibody comprising a light chain consisting of the amino acid sequence described in SEQ ID NO: 30 and a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 33; or An antibody comprising a light chain consisting of the amino acid sequence described in SEQ ID NO: 30 and a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 33, wherein the heavy chain comprises a heavy chain in which one or more amino acid residues at the carboxyl terminus of the heavy chain are deleted. Show, The N297 glycan is given by the following formula: 【Chemistry 31】 (In the formula, the wavy line indicates that the antibody is bound to Asn297.) L (PEG) is - (CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ It shows -NH-, indicating that the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at position 2 of the sialic acid at the non-reducing end of the β-Man branched chain on the 1-3 chain side of the N297 sugar chain. The asterisk indicates that it is bonded to the nitrogen atom at position 1 or 3 on the 1,2,3-triazole ring. n ５ is 3, and I understand ２ An antibody-drug conjugate represented as (where is 1).

22. The following formula: 【Chemistry 32】 It is expressed as follows, and in the formula Ab is An antibody comprising a light chain containing a light chain variable region consisting of the amino acid sequence described in SEQ ID NO: 29, and a heavy chain containing a heavy chain variable region consisting of the amino acid sequence described in SEQ ID NO:

31. Show, The amino acid residues at positions 234, 235, and 265 (all EU numbered) of the Fc region of the antibody are Ala, Ala, and Gly, respectively. The N297 glycan is given by the following formula: 【Transformation 33】 (In the formula, the wavy line indicates that the antibody is bound to Asn297.) L (PEG) is - (CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ It shows -NH-, indicating that the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at position 2 of the sialic acid at the non-reducing end of the β-Man branched chain on the 1-3 chain side of the N297 sugar chain. The asterisk indicates that it is bonded to the nitrogen atom at position 1 or 3 on the 1,2,3-triazole ring. n ５ is 3, and I understand ２ An antibody-drug conjugate represented as (where is 1).

23. The following formula: 【Transformation 34】 It is expressed as follows, and in the formula Ab is An antibody comprising a light chain containing CDRL1 consisting of the amino acid sequence described in SEQ ID NO: 23, CDRL2 consisting of the amino acid sequence described in SEQ ID NO: 24, and CDRL3 consisting of the amino acid sequence described in SEQ ID NO: 25, and a heavy chain containing CDRH1 consisting of the amino acid sequence described in SEQ ID NO: 26, CDRH2 consisting of the amino acid sequence described in SEQ ID NO: 27, and CDRH3 consisting of the amino acid sequence described in SEQ ID NO:

28. Show, The amino acid residues at positions 234, 235, and 265 (all EU numbered) of the Fc region of the antibody are Ala, Ala, and Gly, respectively. The N297 glycan is given by the following formula: 【Chemistry 35】 (In the formula, the wavy line indicates that the antibody is bound to Asn297.) L (PEG) is - (CH ２ -CH ２ -O)n ５ -CH ２ -CH ２ It shows -NH-, indicating that the amino group at the right end of L(PEG) is amide-bonded to the carboxyl group at position 2 of the sialic acid at the non-reducing end of the β-Man branched chain on the 1-3 chain side of the N297 sugar chain. The asterisk indicates that it is bonded to the nitrogen atom at position 1 or 3 on the 1,2,3-triazole ring. n ５ is 3, and I understand ２ An antibody-drug conjugate represented as (where is 1).

24. A STING agonist containing an antibody-drug conjugate according to any one of claims 1 to 23.

25. A pharmaceutical composition containing an antibody-drug conjugate according to any one of claims 1 to 23.

26. An antitumor agent containing an antibody-drug conjugate according to any one of claims 1 to 23, In some cases, An antitumor agent for tumors such as 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, stomach 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 tract cancer, thymic cancer, small intestine cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, sarcoma, EGFR mutation-positive cancer, or cancer with mutations in the EGFR signaling molecule.

27. ​​A cancer treatment agent comprising an antibody-drug conjugate according to any one of claims 1 to 23.

28. A cancer treatment agent comprising the STING agonist described in Claim 24.

29. A cancer treatment agent comprising the pharmaceutical composition described in Claim 25.

30. A cancer treatment agent comprising the antitumor agent described in Claim 26.

31. The cancer treatment agent according to claim 27, 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, stomach cancer, esophageal cancer, endometrial 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 having a mutation in the EGFR signaling molecule.

32. The cancer treatment agent according to claim 28, 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, stomach cancer, esophageal cancer, endometrial 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 tract cancer, thymic cancer, small intestine cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, sarcoma, EGFR mutation-positive cancer, or cancer having a mutation in the EGFR signaling molecule.

33. The cancer treatment agent according to claim 29, 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, stomach 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 tract cancer, thymic cancer, small intestine cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, sarcoma, EGFR mutation-positive cancer, or cancer having a mutation in the EGFR signaling molecule.

34. (A) An antibody comprising a light chain containing CDRL1 consisting of the amino acid sequence described in SEQ ID NO: 23, CDRL2 consisting of the amino acid sequence described in SEQ ID NO: 24, and CDRL3 consisting of the amino acid sequence described in SEQ ID NO: 25, and a heavy chain containing CDRH1 consisting of the amino acid sequence described in SEQ ID NO: 26, CDRH2 consisting of the amino acid sequence described in SEQ ID NO: 27, and CDRH3 consisting of the amino acid sequence described in SEQ ID NO: 28, wherein the amino acid residues at positions 234, 235, and 265 (all EU numbered) of the Fc region of the antibody are Ala, Ala, and Gly, respectively; (B) An antibody comprising a light chain containing a light chain variable region consisting of the amino acid sequence described in SEQ ID NO: 29, and a heavy chain containing a heavy chain variable region consisting of the amino acid sequence described in SEQ ID NO: 31, wherein the amino acid residues at positions 234, 235, and 265 (all EU numbered) of the Fc region of the antibody are Ala, Ala, and Gly, respectively; (C) An antibody comprising a light chain consisting of the amino acid sequence described in SEQ ID NO: 30 and a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 32; (D) An antibody comprising a light chain consisting of the amino acid sequence described in SEQ ID NO: 30 and a heavy chain consisting of the amino acid sequence described in SEQ ID NO: 33; or An antibody comprising a heavy chain in which one or more amino acid residues at the carboxyl terminus of the heavy chain are deleted, in any one of the antibodies (E), (C), to (D).

35. The pharmaceutical composition according to claim 25, which is administered in combination with other pharmaceuticals.

36. The antitumor agent according to claim 26, which is administered in combination with other pharmaceuticals.

37. The pharmaceutical composition according to claim 25, comprising other pharmaceuticals.

38. The antitumor agent according to claim 26, comprising other pharmaceuticals.

39. The cancer treatment agent according to claim 27, wherein the antibody-drug conjugate according to any one of claims 1 to 23 is administered in combination with other pharmaceuticals.

40. The cancer treatment agent according to claim 28, wherein the STING agonist according to claim 24 is administered in combination with other pharmaceuticals.

41. The cancer treatment agent according to claim 29, wherein the pharmaceutical composition according to claim 25 is administered in combination with other pharmaceuticals.

42. The cancer treatment agent according to claim 30, wherein the antitumor agent according to claim 26 is administered in combination with other pharmaceuticals.

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