Α ray emission antibody-drug conjugate

Abstract

To provide an α ray emission antibody-drug conjugate, a production method thereof, use thereof, and a pharmaceutical composition containing the α ray emission antibody-drug conjugate and inspection, diagnosis, and treatment methods.SOLUTION: Provided is an antibody-drug conjugate in which an introduction position of 211At is controlled by introducing 211At into an N-binding type sugar chain binding site (Asn297) in an antibody molecule via a linker using a sugar oxazoline derivative. The antibody-drug conjugate can be produced by a production method including: (1) a step of binding an antibody or an antigen-binding fragment B thereof and a first linker L1 in the presence of sugar hydrolase; (2) a step of binding an astatine-binding structure A and the first linker L1 via or without via a second linker L2; and (3) a step of binding 211At to an antibody conjugate containing the astatine-binding structure produced by the steps (1) and (2).SELECTED DRAWING: Figure 7

Description

[Technical field]

[0001] In this specification, the contents of International Application No. PCT / JP / 2021-012997 are incorporated by reference as part of this specification. The present disclosure relates to alpha-emitting antibody-drug conjugates, methods for their production, their uses, as well as pharmaceutical compositions and testing, diagnostic and therapeutic methods comprising said alpha-emitting antibody-drug conjugates. [Background technology]

[0002] Nuclear medicine therapy is radiation therapy using radioisotopes (RI) administered into the body (intravenously or orally) or drugs that incorporate these, and is also called RI internal therapy. Traditionally, nuclides that emit β-particles have been mainly used in nuclear medicine therapy. Compared to β-particles, α-particles have a shorter range and emit higher energy, making them highly toxic to cancer cells and causing fewer side effects on normal cells. Attempts have been made to use nuclides that emit α-particles, which have such characteristics, as antibody-drug conjugates (ADCs) by binding them to antibodies that specifically bind to antigens specifically expressed in target cells such as cancer cells.

[0003] The inventors have developed a method for non-specifically condensing decaborane-bearing polyethylene glycol (PEG) linkers with randomly occurring lysine residues in an antibody, which is capable of emitting alpha-particles. 211 We have successfully produced antibodies labeled with At (first generation 211 At-labeled antibody). 211 In an antitumor effect test using tumor-bearing mice, it was suggested that At-labeled antibodies have a strong tumor growth suppression effect. 211 Attempts have been made to prepare At-labeled antibodies and use them as antitumor drugs or pretreatment drugs for hematopoietic stem cell transplantation (Non-Patent Documents 1 to 5).

[0004] On the other hand, in order to use α-emitting drugs as medicines, they must be capable of binding to antibodies. 211It is essential to control the introduction position of α-particle emitting nuclides such as At. For example, first generation 211 In the general method of binding α-emitting nuclides to the side chain amino or thiol groups of IgG antibodies via a linker, which was also used for At-labeled antibodies, it is difficult to control the position of introduction of the α-emitting nuclides. In these general methods, the α-emitting nuclides are randomly introduced to the amino or thiol groups present at multiple locations on the antibody, resulting in a diverse group of ADC isomers with a wide distribution of the positions and numbers of introduction. Such ADC isomers raise clinical concerns, such as quality control and efficacy (reduced circulating half-life and efficacy).

[0005] As one method for solving this problem, a method has been reported in which drugs are introduced site-selectively using the N-linked glycan in the Fc region of an IgG antibody as a foothold (Non-Patent Documents 6 and 7). These methods can selectively introduce drugs only to the glycan portion of the antibody, and therefore have the advantages of higher site selectivity and the ability to control the number of drugs introduced compared to conventional methods of randomly introducing drugs.

[0006] In all of these methods, it is necessary to introduce a functional group for drug introduction to the N-acetylneuraminic acid at the non-reducing end of the glycan. However, it has been pointed out that N-acetylneuraminic acid may be released from the glycan in the blood, and there is a concern that the drug may be released from the ADC outside the target cells. In addition, the glycans used in these methods are prepared from chicken eggs, so egg-derived allergens may be a problem in drug production. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2019-156796 A [Patent Document 2] International Publication No. 2019 / 151384 [Patent Document 3] JP 2020-100865 A [Non-patent literature]

[0008]

Non-licensed literature 1

Non-licensed Document 2

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

[0009] In light of the above background, an objective of the present disclosure is to provide a novel α-ray emitting ADC in which the site of introduction of an α-ray emitting nuclide on an antibody molecule is controlled while fully maintaining the physiological effect brought about by α-ray emission. In another aspect, an objective of the present disclosure is to provide a method for producing the α-ray emitting ADC, a medicine containing the α-ray emitting ADC, and a treatment method using the α-ray emitting ADC. [Means for solving the problem]

[0010] As a result of intensive research, the inventors of the present disclosure have discovered that a sugar oxazoline derivative is linked to the N-linked glycan binding site (Asn297) of an antibody molecule via a linker. 211 By introducing At, 211 The present inventors have found that the above-mentioned problems can be solved by obtaining an antibody-drug conjugate in which the introduction site of At is controlled, and by discovering that the antibody-drug conjugate exerts an excellent physiological effect.

[0011] In one aspect, the present disclosure provides an α-emitting antibody-drug conjugate having formula (1): Equation (1)(N m -AL 2 -L 1 ) n -B [In the formula, B is an antibody or antigen-binding fragment thereof; L 1 is a linker having the formula (2): Formula (2) TIFF2024178476000002.tif37151[In the formula, X's each independently represent a single bond, an oxygen atom, -NH-, -COHN-, -COO-, or a group represented by formula (4) or (5): TIFF2024178476000003.tif43146[where, R 1 is a trivalent branched hydrocarbon group having 1 to 6 carbon atoms, and R 2 and R 3 each independently represents an alkylene group having 1 to 3 carbon atoms; at least one of Y1 to Y3 is present and each independently represents a PEG chain, a substituted PEG chain, or a PEG chain containing an oxygen atom, -NH-, -COHN-, or -COO- in the main chain, the PEG chain having a linear structure or a branched structure, and in the case of a branched structure, the number of branches is 2 to 10; Z is independently -O-, -CH 2 -, —N-, an optionally substituted tetrazine group, a norbornene group, a trans-cyclooctene group, a dibenzylcyclooctyl group, or a bicyclo[6.1.0]non-4-yn-9-ylmethyl group, or an optionally substituted cyanobenzothiazole group; R is L-fucose or a hydrogen atom; When any of Y1-3 is absent, Z is hydrogen and X is oxygen; When X is a group represented by the formula (4) or (5), Y1 to Y3 are each a branched R 2 and R 3 When the PEG chain has a branched structure, Z is bonded to each branch. #B represents the point of attachment to B, which is an asparagine residue present in the antibody molecule; #L 2-1 , #L 2-2 and #L 2-3 are each independently present when Y1 to Y3 are present, and each independently represents L 2 represents a hydrogen or a point of attachment to; Existing #L 2-1 , #L 2-2 and #L 2-3At least one of the following is L 2 Represents a connection point to . ]; L 2 is a bond or linker; A is the astatine bond structure; N is 211 At; m is an integer greater than or equal to 1; n is an integer from 1 to 10.

[0012] In one aspect, the present disclosure provides a pharmaceutical composition comprising the above-described α-emitting antibody-drug conjugate.

[0013] In one aspect, the present disclosure provides a testing, diagnostic, or therapeutic method using the above-mentioned α-ray emitting antibody-drug conjugate.

[0014] In one aspect, the present disclosure provides a method for producing the above-mentioned α-ray emitting antibody-drug conjugate.

[0015] In one aspect, the present disclosure provides compounds of the following formula (5) and formula (6), which are intermediates of the above-mentioned α-ray-emitting antibody-drug conjugate. Equation (5)(AL 2 -L 1 ) n -B Formula (6) AL 2 -L 1 In the formula, B, L 1 , L 2 and A has the same meaning as above.

[0016] More specifically, the present disclosure provides the following: [Item 1] A complex having the following formula (1): Equation (1)(N m -AL 2 -L 1 ) n -B [In the formula, B is an antibody or antigen-binding fragment thereof; L 1 is a first linker having the following formula (2): Formula (2) TIFF2024178476000004.tif34150[In the formula, X's each independently represent a single bond, an oxygen atom, -NH-, -COHN-, -COO-, or a group represented by formula (4) or (5): TIFF2024178476000005.tif44163[where, R 1 is a trivalent branched hydrocarbon group having 1 to 6 carbon atoms, and R 2 and R 3 each independently represents an alkylene group having 1 to 3 carbon atoms; at least one of Y1 to Y3 is present and each independently represents a PEG chain, a substituted PEG chain, or a PEG chain containing an oxygen atom, -NH-, -COHN-, or -COO- in the main chain, the PEG chain having a linear structure or a branched structure, and in the case of a branched structure, the number of branches is 2 to 10; Z is independently -O-, -CH 2 -, —N-, an optionally substituted tetrazine group, a norbornene group, a trans-cyclooctene group, a dibenzylcyclooctyl group, or a bicyclo[6.1.0]non-4-yn-9-ylmethyl group, or an optionally substituted cyanobenzothiazole group; R is L-fucose or a hydrogen atom; When any of Y1-3 is absent, Z is hydrogen and X is oxygen; When X is a group represented by the formula (4) or (5), Y1 to Y3 are each a branched R 2 and R 3 When the PEG chain has a branched structure, Z is bonded to each branch. #B represents the point of attachment to B, which is an asparagine residue present in the antibody molecule; #L 2-1 , #L 2-2 and #L 2-3 are each independently present when Y1 to Y3 are present, and each independently represents L 2 represents a hydrogen or a point of attachment to; Existing #L 2-1 , #L 2-2 and #L2-3 At least one of the following is L 2 Represents a connection point to . ]; L 2 is a bond or a second linker; A is the astatine bond structure; N is 211 At; m is an integer greater than or equal to 1; n is an integer from 1 to 10. [Item 2] #B in formula (2) is When the antibody class is IgG, this represents the attachment point to the asparagine residue at position 297; when the antibody class is IgA, it represents a binding site that binds to at least one of the group consisting of asparagine residues 144 and 340; when the antibody belongs to the IgA2 class, the binding site is at least one of the group consisting of asparagine residues 131, 205, and 327; when the antibody belongs to the IgE class, it exhibits a binding site that binds to at least one of the group consisting of asparagine residues 140, 168, 218, 265, 371, 383, and 394; when the antibody belongs to the IgM class, it represents a binding site that binds to at least one of the group consisting of asparagine residues 171, 332, 395, 402, and 563; 2. The conjugate according to item 1, wherein the position of the asparagine residue is according to the numbering according to the EU index of Kabat. [Item 3] 3. The complex according to any one of items 1 or 2, wherein the astatine-binding structure A is selected from the group consisting of decaborane and boron cluster lipid structures. [Item 4] A pharmaceutical composition comprising the conjugate according to any one of items 1 to 3. [Item 5] 5. The pharmaceutical composition according to item 4, which is used for cancer treatment. [Item 6] 6. The pharmaceutical composition according to item 5, wherein the cancer is selected from the group consisting of lymphoma, thyroid cancer, melanoma, prostate cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical cancer, lung cancer, head and neck cancer, and cancer stem cells. [Item 7] A method for producing an antibody-drug conjugate according to item 1, comprising: (1) In the presence of a glycosyl hydrolase, an antibody or an antigen-binding fragment thereof B and a first linker L 1 and combining the same; (2) Astatine-binding structure A and a first linker L 1 and a second linker L 2 binding with or without an intervening (3) subjecting the astatine-binding structure-containing antibody complex produced by steps (1) and (2) to 211 Step of binding At A manufacturing method comprising: [Item 8] A compound represented by the following formula (5): Equation (5)(AL 2 -L 1 ) n -B [In the formula, B is an antibody or antigen-binding fragment thereof; L 1 is the first linker; L 2 is a bond or a second linker; A is the astatine bond structure; n is an integer from 1 to 10; The first linker L 1 has the following formula (2). Formula (2) TIFF2024178476000006.tif36157[In the formula, #B is a point of attachment to an antibody molecule or antigen-binding fragment; X's each independently represent a single bond, an oxygen atom, -NH-, -COHN-, -COO-, or a group represented by formula (3) or (4): TIFF2024178476000007.tif45158[where, R 1 is a trivalent branched hydrocarbon group having 1 to 6 carbon atoms, and R2 and R 3 each independently represents an alkylene group having 1 to 3 carbon atoms; at least one of Y1 to Y3 is present and each independently represents a PEG chain, a substituted PEG chain, or a PEG chain containing an oxygen atom, -NH-, -COHN-, or -COO- in the main chain, the PEG chain having a linear structure or a branched structure, and in the case of a branched structure, the number of branches is 2 to 10; Z is independently -O-, -CH 2 -, —N-, an optionally substituted tetrazine group, a norbornene group, a trans-cyclooctene group, a dibenzylcyclooctyl group, or a bicyclo[6.1.0]non-4-yn-9-ylmethyl group, or an optionally substituted cyanobenzothiazole group; R is L-fucose or a hydrogen atom; When any of Y1-3 is absent, Z is hydrogen and X is oxygen; When X is a group represented by the formula (3) or (4), Y1 to Y3 are each a branched chain R 2 and R 3 When the PEG chain has a branched structure, Z is bonded to each branch. #B represents the point of attachment to B, which is an asparagine residue present in the antibody molecule; #L 2-1 , #L 2-2 and #L 2-3 are each independently present when Y1 to Y3 are present, and each independently represents L 2 represents a hydrogen or a point of attachment to; Existing #L 2-1 , #L 2-2 and #L 2-3 At least one of the following is L 2 represents the attachment point to .] [Item 9] A compound having the following formula (6): Formula (6) AL 2 -L 1 [In the formula, L 1 is the first linker; L2 is a bond or a second linker; A is the astatine bond structure; The first linker L 1 has the following formula (7). Formula (7) TIFF2024178476000008.tif35155[in the formula, X's each independently represent a single bond, an oxygen atom, -NH-, -COHN-, -COO-, or a group represented by formula (3) or (4): TIFF2024178476000009.tif47152[where, R 1 is a trivalent branched hydrocarbon group having 1 to 6 carbon atoms, and R 2 and R 3 each independently represents an alkylene group having 1 to 3 carbon atoms; at least one of Y1 to Y3 is present and each independently represents a PEG chain, a substituted PEG chain, or a PEG chain containing an oxygen atom, -NH-, -COHN-, or -COO- in the main chain, the PEG chain having a linear structure or a branched structure, and in the case of a branched structure, the number of branches is 2 to 10; Z is independently -O-, -CH 2 -, —N-, an optionally substituted tetrazine group, a norbornene group, a trans-cyclooctene group, a dibenzylcyclooctyl group, or a bicyclo[6.1.0]non-4-yn-9-ylmethyl group, or an optionally substituted cyanobenzothiazole group; R is L-fucose or a hydrogen atom; When any of Y1-3 is absent, Z is hydrogen and X is oxygen; When X is a group represented by the formula (3) or (4), Y1 to Y3 are each a branched chain R 2 and R 3 When the PEG chain has a branched structure, Z is bonded to each branch. #L 2-1 , #L 2-2 and #L 2-3 are each independently present when Y1 to Y3 are present, and each independently represents L 2 represents a hydrogen or a point of attachment to; Existing #L 2-1 , #L 2-2 and #L 2-3 At least one of the following is L 2 represents the attachment point to .] Effect of the Invention

[0017] The present disclosure provides a method for the preparation of a sugar oxazoline derivative-based linker for the preparation of a saccharide-based oxazoline derivative-based linker for the preparation of a sugar ... 211 This has the effect of providing an antibody-drug conjugate in which the introduction site of At is controlled. 211 By controlling the introduction site of At, it is possible to control the biological and physical properties of the antibody in more detail. In addition, the sugar oxazoline derivative and At-binding substances such as decaborane that bind the antibody and At in the ADC production of the present disclosure are fully chemically synthesized products, and therefore suitable for mass synthesis. Furthermore, since they do not have N-acetylneuraminic acid in the molecule, it is expected that the problem of stability in blood can be overcome. In addition, since the sugar oxazoline derivative can be introduced site-selectively into the sugar moiety of the antibody by using endo-β-N-acetylglucosaminidase (ENGase), which is one of the glycohydrolases, it is possible to synthesize a site-selectively modified antibody for drug introduction. By using the modified antibody obtained in this way as an ADC precursor, it is possible to introduce drugs site-selectively into the antibody and control the number of drugs introduced. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 is a schematic diagram showing the binding reaction between a rituximab antibody and an antibody-bound sugar linker. [Diagram 2] FIG. 2 shows a schematic diagram of the structure of a conjugate of a rituximab antibody and an antibody-binding sugar linker. [Diagram 3] FIG. 3 shows the results of SDS-PAGE experiments confirming the formation of carborate-containing structures. [Figure 4]FIG. 4 shows the results of flow cytometry confirming the binding ability of carborate-containing constructs to CD20-expressing cells. [Diagram 5] FIG. 5 shows the results of a study confirming the binding of 211At to carborate-containing structures. [Figure 6] FIG. 6 shows the results of a test confirming the ability of the antibody-drug conjugate RTX-[B]10-2nd of the present disclosure and the comparative antibody-drug conjugate RTX-[B]10-1st to produce double-stranded breaks. [Figure 7] FIG. 7 shows the results of a test confirming the antitumor effects of the antibody-drug conjugate RTX-[B]10-2nd of the present disclosure and the comparative antibody-drug conjugate RTX-[B]10-1st on transplanted tumor tissues. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] First, a PEG linker and decaborane were introduced into the cyclic alkyne DIBAC to synthesize decaborane-labeled DIBAC. Next, an antibody bearing an azide glycan at the non-reducing end of Asn297 was linked to decaborane-labeled DIBAC using a copper-free click reaction to successfully obtain a decaborane-labeled antibody, and it was confirmed that the resulting labeled antibody bound to the cell membrane. 211 When At was labeled, the labeling rate was similar to that of the first generation. 211 At-labeled antibody (second generation 211 Furthermore, we succeeded in obtaining a 3T-labeled antibody (a 3T-labeled antibody). 211 A comparative evaluation of the At-labeled antibodies suggested that they had comparable performance.

[0020] Hereinafter, embodiments of the present disclosure will be described. The scope of the present disclosure is not limited to these descriptions, and other than the following examples, the present disclosure can be appropriately modified and implemented without departing from the spirit of the present disclosure.

[0021] (definition) In the present disclosure, when multiple numerical ranges are indicated, the same applies to any combination of lower and upper limits of those multiple ranges.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0023] In this disclosure, the term "substantially" has the same meaning as commonly understood by a person skilled in the art to which this disclosure pertains, but is intended to encompass a desired state as well as a state that is unavoidably not achieved due to biological or chemical properties, taking into consideration, for example, that a biological or chemical phenomenon may not completely achieve a desired state.

[0024] In this disclosure, when the term "about" is used in reference to a numerical value x, it means that the value can vary within a range of, for example, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01%.

[0025] In this disclosure, the term "comprise" has the same meaning as commonly understood by a person skilled in the art to which this disclosure belongs, and includes, for example, "comprises" and "consists of." Specifically, a composition "comprising" A may contain another component, B, in addition to only A.

[0026] In the present disclosure, the terms "consisting of" or "composed of" used in reference to a composition have the same meaning as commonly understood by a person skilled in the art to which the present disclosure pertains, but are used to indicate a component that exclusively constitutes the composition. For example, a composition "consisting of" A contains exclusively A. However, in one embodiment, a composition "consisting of" A includes an embodiment in which a contaminant other than A that is unavoidable in the production process based on biological and chemical properties is included.

[0027] In the present disclosure, the term "antibody" has its general meaning in the art, and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, and antibody fragments that exhibit the intended antigen-binding activity. A full-length antibody includes a heavy chain and a light chain that are mainly composed of polypeptides. The heavy chain and the light chain each include a site called a variable region that recognizes an antigen, and the site is generally called a heavy chain variable region and a light chain variable region, respectively. The variable region has sites called CDR1 to 3, which are further specified as sites that recognize antigens, in order from the amino terminus. These CDR1 to 3 are also referred to in more detail as heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, light chain CDR3, etc. In addition, the regions other than CDR1 to 3 of the heavy chain and the light chain are referred to as heavy chain FR1 to 4 and light chain FR1 to 4, in order from the amino terminus, respectively. The antibody may be in a form consisting of two heavy chains and two light chains, or in a form consisting of one heavy chain and one light chain (also called a single-chain antibody).

[0028] The antibody may be of any class, such as IgG, IgE, IgM, IgD, IgA or IgY, or subclass, such as IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2.

[0029] In the present disclosure, the "antigen-binding fragment" of an antibody has the general meaning in the art and refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been found that the ability of an antibody to specifically bind to an antigen can also be maintained by a fragment consisting of a part of the antibody. In one embodiment, the "antigen-binding fragment" of an antibody can be, but is not limited to, a Fab fragment consisting of a light chain variable region (VL), a heavy chain variable region (VH), a light chain constant region (CL) and a CH1 domain that is a part of the heavy chain constant region, a F(ab')2 fragment containing two Fab fragments linked by a disulfide bridge at the hinge region, an Fd fragment consisting of a VH and a CH1 domain, an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, a dAb fragment containing a single variable domain, and an isolated complementarity determining region (CDR).

[0030] In this disclosure, "pharmaceutical composition" or simply "composition" has its general meaning in the art and encompasses not only a product that contains active ingredients and inactive ingredients that constitute a carrier (a pharma- ceutically acceptable excipient), but also any product that results directly or indirectly as a result of the association, complexation or aggregation of any two or more components, or as a result of the dissociation of one or more components, or as a result of another type of reaction or interaction of one or more components.

[0031] 1. Antibody-drug conjugates and methods for producing same (Antibody-drug conjugates) In one aspect, the present disclosure provides an antibody-drug conjugate having formula (1): Equation (1)(N m -AL 2 -L 1 ) n -B [In the formula, B is an antibody or antigen-binding fragment thereof; L 1 is a first linker which is an antibody-binding saccharide linker; L 2 is a second linker which is a bond or an alpha-emitting nuclide-binding linker; A is an α-emitting nuclide binding structure; N is an alpha-emitting nuclide; m is an integer greater than or equal to 1; n is 1 to 10; Antibody-binding sugar linker L 1 has the following formula (2). Formula (2) TIFF2024178476000010.tif38149[In the formula, X's each independently represent a single bond, an oxygen atom, -NH-, -COHN-, -COO-, or a group represented by formula (3) or (4): TIFF2024178476000011.tif45152[where, R 1 is a trivalent branched hydrocarbon group having 1 to 6 carbon atoms, and R 2 and R 3each independently represents an alkylene group having 1 to 3 carbon atoms; at least one of Y1 to Y3 is present and each independently represents a PEG chain, a substituted PEG chain, or a PEG chain containing an oxygen atom, -NH-, -COHN-, or -COO- in the main chain, the PEG chain having a linear structure or a branched structure, and in the case of a branched structure, the number of branches is 2 to 10; Z is independently -O-, -CH 2 -, —N-, an optionally substituted tetrazine group, a norbornene group, a trans-cyclooctene group, a dibenzylcyclooctyl group, or a bicyclo[6.1.0]non-4-yn-9-ylmethyl group, or an optionally substituted cyanobenzothiazole group; R is L-fucose or a hydrogen atom; When any of Y1-3 is absent, Z is hydrogen and X is oxygen; When X is a group represented by the formula (3) or (4), Y1 to Y3 are each a branched chain R 2 and R 3 When the PEG chain has a branched structure, Z is bonded to each branch. #B represents the point of attachment to B, which is an asparagine residue present in the antibody molecule; #L 2-1 , #L 2-2 and #L 2-3 are each independently present when Y1 to Y3 are present, and each independently represents L 2 represents a hydrogen or a point of attachment to; Existing #L 2-1 , #L 2-2 and #L 2-3 At least one of the following is L 2 represents the attachment point to .]

[0032] In the antibody-drug conjugates of the present disclosure, m is an integer of 1 or greater, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30.

[0033] In the antibody-drug conjugates of the present disclosure, n is an integer from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0034] (Binding of antibody to antibody-binding sugar linker) The antibody of the present disclosure can be reacted with an antibody-bound sugar linker (sugar oxazoline derivative) in the presence of a glycosyl hydrolase such as ENGase to bind the antibody-bound sugar linker to a specific site on the antibody, particularly to a specific asparagine residue in the Fc region.

[0035] For example, an antibody-binding sugar linker can be attached to the 297th asparagine residue of the Fc region of IgG, the 144th and / or 340th asparagine residue of IgA1, the 131st, 205th, and / or 327th asparagine residue of IgA2, the 140th, 168th, 218th, 265th, 371st, 383rd, and / or 394th asparagine residue of IgE, the 354th, 445th, and / or 496th asparagine residue of IgD, and the 171st, 332nd, 395th, 402nd, and / or 563rd asparagine residue of IgM.

[0036] (Binding of antibody-binding sugar linker to α-particle-emitting compound) In the antibody-drug conjugate of the present disclosure, an α-ray emitting compound can be bound to the group at the Z position of the antibody-binding sugar linker, for example, with or without a second linker (a molecule that links the antibody and the α-ray emitting compound and controls the drug release rate, release mechanism, and release site). The group at the Z position may be an azide group, a derivative of a tetrazine group, a norbornene group, a trans-cyclooctene group, a dibenzylcyclooctyl group, a bicyclo[6.1.0]non-4-yn-9-ylmethyl group, or a derivative of a cyanobenzothiazole group, which allows the introduction of a drug under mild conditions. For example, the reaction can proceed rapidly at room temperature in the neutral pH range. Therefore, the antibody is not exposed to high temperatures and does not deteriorate.

[0037] (Antibody) In the antibody-drug conjugate of the present disclosure, the antibody is not particularly limited, and any antibody capable of binding to an antibody-binding sugar linker can be used. In one embodiment, it is desirable to use an antibody that binds to a biomolecule present on the cell surface of a tissue to which it is desired to deliver an α-ray emitting nuclide in vivo. For example, an antibody that specifically binds to a biomolecule specifically expressed in tumor cells (including cancer cells and cancer stem cells) can be used. For example, non-limiting examples of biomolecules that can be bound by the antibodies of the present disclosure include CD1a, CD2, CD3, CD4, CD5, CD6, CD7, CD9, CD10, CD13, CD19, CD20, CD21, CD22, CD25, CD28, CD30, CD32, CD33, CD38, CD40, CD40 ligand (CD40L), CD44, CD45, CD46, CD47, CD52, CD54, CD55, CD55, CD59, CD63, CD64, CD66b, CD69, CD70, CD74, CD80, CD89, C D95, CD98, CD105, CD134, CD137, CD138, CD147, CD158, CD160, CD162, CD164, CD200, CD227, adrenomedullin, angiopoietin related protein 4(ARP4), aurora, B7-H1, B7-DC, integlin, bone marrow stromal antigen 2(BST2), CA125, CA19.9, carbonic anhydrase 9(CA9), cadherin, cc-chemokine receptor(CCR)4, CCR7, carcinoembryonic antigen(CEA), cysteine-rich fibroblast growth factor receptor-1(CFR-1), c-Met, c-Myc, collagen, CTA, connective tissue growth factor(CTGF), CTLA-4, cytokeratin-18, DF3, E-catherin, epidermal growth factorer receptor(EGFR), EGFRvIII, EGFR2(HER2), EGFR3(HER3), EGFR4(HER4), heparin-binding EGF-like growthfactor(HB-EGF), endoglin, epithelial cell adhesion molecule(EpCAM), endothelial protein C receptor (EPCR), ephrin, ephrin receptor (Eph), EphA2, endotheliase-2 (ET2), FAM3D, fibroblast activating protein (FAP), Fc receptor homolog 1(FcRH1), ferritin, fibroblast growth factor-8(FGF-8), FGF8 receptor, basic FGF(bFGF), bFGF receptor, FGF receptor(FGFR)3, FGFR4, FLT1, FLT3, folate receptor, Frizzled homologue 10(FZD10), frizzled receptor 4(FZD-4), G250, G-CSF receptor, ganglioside(GD2, GD3, GM2, GM3, globe). H, gp75, gp88, GPR-9-6, heparanase I, hepatocyte growth factor(HGF), HGF receptor, HLA antigen(HLA-DR function), HM1.24, human milk fat globule (HMFG), hRS7, heat shock protein 90(hsp90), idiotype epitope, insulin-like growth factor (IGF), IGF receptor (IGFR), interleukin (including IL-6, IL-12, IL-15) interleukin receptor(from IL-2R, IL-3R, IL-6R, IL-10R, integrin, immune receptor translocation associated-4(IRTA-4), kallikrein 1, KDR, KIR2DL1, KIR2DL2 / 3, KS1 / 4, lamp-1, lamp-2, laminin-5, Lewis y, sialyl Lewis x, lymphotoxin-betareceptor(LTBR)、LUNX、melanoma-associated chondroitin sulfate proteoglycan(MCSP)、mesothelin、MICA、Mullerian inhibiting substance type II receptor(MISIIR)、mucin、neural cell adhesion molecule(NCAM)、Necl-5、Notch1、osteopontin、platelet-derived growth factor(PDGF)、PDGF receptor、platelet factor-4(PF-4)、phosphatidylserine、Prostate Specific Antigen(PSA)、prostate stem cell antigen(PSCA)、prostate specific membrane antigen(PSMA)、Parathyroid hormone related protein / peptide(PTHrP)、receptor activator of NF-kappaB ligand(RANKL)、receptor for hyaluronic acid mediated motility(RHAMM)、ROBO1、SART3、semaphorin 4B(SEMA4B)、secretory leukocyte protease inhibitor(SLPI)、SM5-1、sphingosine-1-phosphate、tumor-associated glycoprotein-72(TAG-72)、transferrin receptor(TfR)、TGF-beta、Thy-1、Tie-1、Tie2 receptor、T cell immunoglobulin domain and mucin domain 1(TIM-1)、human tissue factor(hTF)、Tn antigen、tumor necrosis factor(TNF)、Thomsen-Friedenreich antigen(TF antigen)、TNF receptor、tumor necrosis factor-related apoptosis-inducingLigand (TRAIL), TRAIL receptor (e.g., DR4 and DR5, etc.), system ASC amino acid transporter 2 (ASCT2), trkC, TROP-2, TWEAK receptor Fn14, urokinase plasminogen activator receptor (uPAR), type IV collagenase, urokinase receptor, vascular endothelial growth factor (VEGF), VEGF receptor (e.g., VEGFR1, VEGFR2, VEGFR3, etc.), vimentin, GPC1, xCT, CD24, CD34, CD133, ESA, HER1, LYVE-1, LAT1, CAT1, S1PR1, TfR, and VLA-4, etc., and their homologs. For example, Rituximab can be used as an antibody that specifically binds to CD20. In the antibody-drug conjugate of the present disclosure, an antigen-binding fragment that includes a binding site with the antibody-binding sugar linker can be used as the antibody.

[0038] (Antibody-binding sugar linker)

[0039] The antibody-binding sugar linker of the present disclosure has the structure of formula (2): TIFF2024178476000012.tif37162[In the formula, X's each independently represent a single bond, an oxygen atom, -NH-, -COHN-, -COO-, or a group represented by formula (4) or (5): TIFF2024178476000013.tif45157[where, R 1 is a trivalent branched hydrocarbon group having 1 to 6 carbon atoms, and R 2 and R 3 each independently represents an alkylene group having 1 to 3 carbon atoms; at least one of Y1 to Y3 is present and each independently represents a PEG chain, a substituted PEG chain, or a PEG chain containing an oxygen atom, -NH-, -COHN-, or -COO- in the main chain, the PEG chain having a linear structure or a branched structure, and in the case of a branched structure, the number of branches is 2 to 10; Z is independently -O-, -CH 2 -, —N-, an optionally substituted tetrazine group, a norbornene group, a trans-cyclooctene group, a dibenzylcyclooctyl group, or a bicyclo[6.1.0]non-4-yn-9-ylmethyl group, or an optionally substituted cyanobenzothiazole group; R is L-fucose or a hydrogen atom; When any of Y1-3 is absent, Z is hydrogen and X is oxygen; When X is a group represented by the formula (5) or (6), Y1 to Y3 are each a branched chain R 2 and R 3 When the PEG chain has a branched structure, Z is bonded to each branch. #B represents the point of attachment to B, which is an asparagine residue present in the antibody molecule; #L 2-1 , #L 2-2 and #L 2-3 are each independently present when Y1 to Y3 are present, and each independently represents L 2 represents a hydrogen or a point of attachment to; Existing #L 2-1 , #L 2-2 and #L 2-3 At least one of the following is L 2 represents the attachment point to .]

[0040] The antibody-binding sugar linker of the present disclosure is synthesized from a sugar oxazoline derivative represented by the following general formula (A1). [ka] In the formula, X is each independently a single bond, an oxygen atom, -NH-, -COHN-, -COO-, or a group represented by formula (A2) or (A3): [ka] [ka] (In the formula, R 1 is a trivalent branched hydrocarbon group having 1 to 6 carbon atoms, and R 2 and R 3 are each independently an alkylene group having 1 to 3 carbon atoms. and at least one of Y1 to Y3 is present and each independently represents a PEG chain, a substituted PEG chain, or a PEG chain containing an oxygen atom, -NH-, -COHN-, or -COO- in the main chain, the PEG chain has a linear structure or a branched structure, and in the case of a branched structure, the number of branches is 2 to 10; each Z is independently a hydroxy group, a methoxy group, an azide group, an optionally substituted tetrazine group, a norbornene group, a trans-cyclooctene group, a dibenzylcyclooctyl group, a bicyclo[6.1.0]non-4-yn-9-ylmethyl group, or an optionally substituted cyanobenzothiazole group, and at least one Z is not a hydroxy group or a methoxy group; When any of Y1-3 is absent, Z is hydrogen and X is oxygen; When X is a group represented by the formula (A2) or (A3), Y1 to Y3 are each a branched R 2 and R 3 are bonded to When the PEG chain has a branched structure, Z is bonded to each branched chain.

[0041] X is a bond between a sugar and a PEG chain or the like, and each X is independently a single bond, an oxygen atom, -NH-, -COHN-, -COO-, or a group represented by the formula (A2) or (A3): [ka] [ka] (In the formula, R 1is a trivalent branched hydrocarbon group having 1 to 6 carbon atoms, and R 2 and R 3 are each independently an alkylene group having 1 to 3 carbon atoms. When X is a single bond, an oxygen atom, -NH-, -COHN-, or -COO-, one PEG chain or the like is bonded to X. When X is a group represented by formula (A2) or (A3), a PEG chain or the like is bonded to each branch.

[0042] Y1 to Y3 are PEG chains, substituted PEG chains, or PEG chains containing oxygen atoms, -NH-, -COHN-, or -COO- in the main chain (hereinafter, these may be collectively referred to as PEG chains, etc.), and the structure of the PEG chains, etc. may be a linear structure or a branched structure. PEG chains, etc. are non-toxic and non-immunogenic polymers, and a protein therapeutic drug modified with a PEG chain, etc. can obtain the effect of increasing stability in the body by reducing immunogenicity, protecting it from degradation by various enzymes, and increasing its half-life. The repeat number of the PEG chain is not particularly limited, but is, for example, 2 to 1000, in one embodiment, 2 to 100, in one embodiment, 2 to 50, in one embodiment, 2 to 30, in one embodiment, 2 to 20, in one embodiment, 2 to 10, and in one embodiment, 2 to 5. The repeat number is the repeat number in one PEG chain, and in a branched PEG chain, it means the repeat number in each branched PEG chain. Well, the repeat number in a PEG chain containing an oxygen atom, -NH-, -COHN-, or -COO- in the main chain described below means the repeat number in one PEG chain containing these groups.

[0043] The substituted PEG chain refers to a PEG chain in which a hydrogen atom in a side chain of the PEG chain has been substituted. The substituent is not particularly limited as long as the effects of the present disclosure can be obtained, and examples of the substituent include an alkyl group, an aralkyl group, an alkenyl group, an alkynyl group, a hydroxyl group, an amino group, an aldehyde group, a ketone group, a carboxyl group, an amide group, a thiol group, and an ether group, with an alkyl group having 1 to 6 carbon atoms being particularly preferred.

[0044] The PEG chain containing the oxygen atom, -NH-, -COHN-, or -COO- in the main chain is a polyethylene glycol (CH 2 CH 2 The repeating units of the main chain of polyethylene glycol (CH 2 CH 2 In place of the oxygen atom (-O-), -NH-, -COHN-, or -COO- may be included.

[0045] The PEG chain etc. has a linear structure or a branched structure. In the case of a branched structure, the number of branches is not limited, but is preferably 2 to 10. The degree of polymerization of the PEG chain is also not particularly limited as described above, but is particularly preferably 2 to 10.

[0046] In the general formula (1), Z is a hydroxy group, a methoxy group, an azide group, a tetrazine group which may be substituted, a norbornene group, a trans-cyclooctene group, a dibenzylcyclooctyl group, or a bicyclo[6.1.0]nona-4-yn-9-ylmethyl group, or a cyanobenzothiazole group which may be substituted, where Y1 to Y3 are present. Where any of Y1 to Y3 is not present, X represents oxygen, and hydrogen. At least one or more Zs are not a hydroxy group or a methoxy group. That is, at least one Z is an azide group, a tetrazine group which may be substituted, a norbornene group, a trans-cyclooctene group, a dibenzylcyclooctyl group, or a bicyclo[6.1.0]nona-4-yn-9-ylmethyl group, or a cyanobenzothiazole group which may be substituted. Other compounds can be introduced into these groups. At least one Z is an azide group, an optionally substituted tetrazine group, a norbornene group, a trans-cyclooctene group, a dibenzylcyclooctyl group, a bicyclo[6.1.0]non-4-yn-9-ylmethyl group, or an optionally substituted cyanobenzothiazole group, so that other compounds can be efficiently introduced.

[0047] The azide group is -N 3 It is a group represented by the following formula:

[0048] The tetrazine group is a 1,2,4,5-tetrazine group. The substituent of the substituted tetrazine group is one in which the hydrogen atom at the 3-position and / or 6-position is substituted. The substituent at the 3-position is, but is not limited to, an alkyl group having 1 to 3 carbon atoms, a pyridinyl group, a bipyridinyl group, -CF 3 Group (fluoroalkyl group), -COOCH 3(ester group), or phenyl group. In addition, the substituent at the 6-position may be, but is not limited to, an amino group, an alkylene group having 1 to 3 carbon atoms (e.g., an ethylene group), a phenylene group, or a pyridinylene group. Furthermore, the amino group, the alkylene group having 1 to 3 carbon atoms (e.g., an ethylene group), a phenylene group, or a pyridinylene group, which is a substituent at the 6-position, may be bonded to Y1 to 3 via an amide bond or an amino group. In addition, the tetrazine group or the substituted tetrazine group may be directly bonded to the PEG chain or the like, or may be bonded via, for example, an ether bond, an ester bond, a carbonate bond, a carbamate bond, or an amide bond. Specific substituted tetrazine groups include, but are not limited to, groups represented by the following formula: [ka]

[0049] The norbornene group has the following formula (A4): [ka] The norbornene group may be directly bonded to the PEG chain or the like, or may be bonded via, for example, an ether bond, an ester bond, a carbonate bond, a carbamate bond, an amide bond, or the like.

[0050] The trans-cyclooctene group has the following formula (A5): [ka] The trans-cyclooctene group may be directly bonded to the PEG chain or the like, or may be bonded via, for example, an ether bond, an ester bond, a carbonate bond, a carbamate bond, or an amide bond.

[0051] The dibenzylcyclooctyl group has the following formula (A6): [ka] The dibenzylcyclooctyl group may be directly bonded to the PEG chain or the like, or may be bonded via, for example, an ether bond, an ester bond, a carbonate bond, a carbamate bond, an amide bond, or the like.

[0052] The bicyclo[6.1.0]non-4-yn-9-ylmethyl group has the following formula (A7): [ka] The bicyclo[6.1.0]nona-4-yn-9-ylmethyl group may be directly bonded to the PEG chain or the like, or may be bonded via, for example, an ether bond, an ester bond, a carbonate bond, a carbamate bond, or an amide bond.

[0053] The cyanobenzothiazole group has the following formula (A8): [ka] The substituted cyanobenzothiazole group is a group in which Y1 to Y3 are bonded via an amide bond, an ether bond, or an ester bond. The cyanobenzothiazole group and the substituted cyanobenzothiazole group may be bonded directly to the PEG chain or the like, or may be bonded via, for example, an ether bond, an ester bond, or an amide bond.

[0054] When Y1 to Y3 have a branched structure and a plurality of Z's are present in Y1 to Y3, the Z's may be the same or different.

[0055] In the general formula (1), the substituents Y1 to Y3 are not particularly limited, and examples thereof include organic groups and functional groups. The organic group is an assembly of atoms that essentially contains a carbon atom, and examples thereof include an alkyl group, an aralkyl group, an alkenyl group, and an alkynyl group, and in particular, an alkyl group and an alkenyl group are preferred. The structure of the alkyl group and the alkenyl group is not particularly limited, and may be a saturated or unsaturated chain hydrocarbon group or an alicyclic hydrocarbon group. The number of carbon atoms in the alkyl group and the alkenyl group is not particularly limited, but is preferably 1 to 30, and more preferably 1 to 20. The functional group is an assembly of atoms that determines the properties of an organic compound, and examples thereof include a hydroxyl group, an amino group, an aldehyde group, a ketone group, a carboxyl group, an amide group, a thiol group, and an ether group, and in particular, a hydroxyl group, an amino group, a carboxyl group, an amide group, and an ether group are preferred.

[0056] The sugar oxazoline derivative is preferably an oxazoline derivative represented by the following formula (A9). [ka] In the general formula (A9), the degree of polymerization of the polyethylene glycol chain is not particularly limited, but a degree of polymerization of 2 to 10 is preferred.

[0057] More specifically, the sugar oxazoline derivative may be an oxazoline derivative represented by the following formula: [ka] [ka] [ka] [ka] [ka] [ka] [ka] The antibody-bound sugar linker of the present disclosure can be site-selectively introduced into the sugar moiety of an antibody using a glycosyl hydrolase such as endo-β-N-acetylglucosaminidase (ENGase), allowing the synthesis of site-selectively modified antibodies for drug introduction. In this way, it becomes possible to site-selectively introduce drugs into the antibody and to control the number of drugs introduced.

[0058] (Alpha-emitting nuclide conjugate linker) In the antibody-drug conjugate of the present disclosure, the antibody-binding sugar linker and the α-ray-emitting nuclide conjugate can be directly bound, or can be bound using an α-ray-emitting nuclide conjugate-binding linker. The structure of the α-ray emitting nuclide conjugate-binding linker is not particularly limited, and various structures can be adopted depending on the structure of the antibody-bound sugar linker and / or the α-ray emitting nuclide conjugate, the release control mechanism to be adopted, etc. As the α-ray emitting nuclide conjugate-binding linker, linkers known to those skilled in the art can be adopted, and in addition, a structure having desired properties can be newly synthesized and used. In one embodiment, non-limiting examples of linkers that may be used as alpha-emitting nuclide conjugate attachment linkers of the present disclosure include the following: Val-Cit linker, MCC linker, Gly-Gly-Phe-Gly linker, linkers having the structure TIFF2024178476000033.tif159158 The linker having the above 11,12-didehydrodibenzo[b,f]azocin ring structure is typically bonded to, for example, an azo group of the antibody-bound sugar linker via the azocin structure and bonded to an α-particle-emitting nuclide conjugate via the amino group.

[0059] (α-particle-emitting nuclide conjugate) In the antibody-drug conjugates of the present disclosure, the α-emitter is conjugated to the antibody via an α-emitter conjugate. Typically, the α-emitter is 211 At, and the alpha-emitting nuclide conjugate is 211 This is the At bond structure. The structure of the α-ray emitting nuclide conjugate is not particularly limited, and various structures can be adopted depending on the state of the α-ray emitting nuclide, the structure of other parts of the antibody-drug conjugate, such as the antibody, the antibody-bound sugar linker, the structure of the α-ray emitting nuclide conjugate, and / or the release control mechanism employed, etc. As the α-ray emitting nuclide conjugate, α-ray emitting nuclide conjugates known to those skilled in the art can be adopted, and in addition, structures having desired properties can be newly synthesized and used. In one embodiment, non-limiting examples that can be used as the α-ray emitting nuclide conjugate of the present disclosure include a carborane structure, which is a cluster consisting of a boron atom, a carbon atom, and a hydrogen atom, a boron cluster lipid structure, which is a cluster consisting of a boron atom and a hydrogen atom (Patent Document 1, JP 2019-156796 A), a stannylbenzoic acid structure (Non-Patent Document 11, E. Aurlien, British Journal of Cancer (2000) 83(10), 1375-1379), a neopentyl halide structure (Patent Document 2, WO 2019 / 151384 A), an arylboronic acid structure (Non-Patent Document 12, Shirakami et al., Scientific Reports, 11, Article number: 12982 (2021), and a benzene ring (Patent Document 3, JP 2020-100865 A):

[0060] (alpha-particle emitting nuclide) The α-ray emitting nuclide used in the antibody-drug conjugate of the present disclosure is not particularly limited, but typically 211 Use At. 211 At is a radioisotope of astatine (At), an element in the halogen family. 211 At decays to stable lead (207Pb) emitting high-energy alpha rays that are cell-killing. 211 The half-life of At is 7.2 hours. 211 At has a short life span and high cell killing activity, and therefore can efficiently destroy cancer tissues.

[0061] Alpha-emitting nuclides can be produced using known methods, for example: 211At is produced by the (α,2n) nuclear reaction using an accelerator such as a cyclotron with Bi-209 as the target material, and is then purified by a dry method (distillation) and dissolved in water. 211 It is supplied as an aqueous solution of At.

[0062] (Method of producing antibody-drug conjugate) In one aspect, the disclosure provides a method for producing an antibody-drug conjugate. The method for producing an antibody-drug conjugate includes the following steps. (1) In the presence of a glycosyl hydrolase, an antibody or an antigen-binding fragment thereof B and an antibody-bound sugar linker L 1 and combining the same; (2) α-particle-emitting nuclide conjugate A and antibody-bound sugar linker L 1 and an α-particle-emitting nuclide conjugate linker L 2 binding with or without an intervening (3) The α-ray emitting nuclide conjugate of the α-ray emitting nuclide conjugate-containing antibody conjugate produced by steps (1) and (2), 211 Step of binding At

[0063] In the method for producing an antibody-drug conjugate of the present disclosure, the order of the above steps (1) and (2) can be appropriately selected. An α-ray emitting nuclide conjugate may be conjugated to the conjugate of the antibody and the antibody-binding sugar linker obtained in step (1) in step (2), or an α-ray emitting nuclide conjugate-containing sugar linker obtained by conjugating the α-ray emitting nuclide conjugate and the antibody-binding sugar linker in step (2) may be conjugated to the antibody in step (1).

[0064] The glycohydrolase used in step (1) is not particularly limited as long as it is an enzyme capable of site-specifically binding the antibody-bound saccharide linker of the present disclosure to an antibody. In one embodiment, the glycohydrolase can specifically bind the antibody-bound saccharide linker to a single or multiple amino acid residues on an antibody. For example, the glycohydrolase can bind the antibody-bound saccharide linker to specific sites 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 on an antibody. For example, by using endo-β-N-acetylglucosaminidase (ENGase) as the glycohydrolase, the antibody-bound saccharide linker can be specifically bound to the 297th asparagine residue of an IgG antibody. Those skilled in the art can use known information to select a hydrolase that binds the antibody-bound saccharide linker to a specific site on a specific antibody.

[0065] The antibody or antibody-binding fragment used in step (1) may be in the form of an acceptor having a glycan bound thereto (Non-Patent Document 9, Aoyama, M., et al., Mabs, 11:826-836 (2019)).

[0066] In step (2), the α-ray emitting nuclide conjugate is bound to the antibody-bound sugar linker of the antibody-saccharide linker conjugate with or without an α-ray emitting nuclide conjugate binding linker. As the α-ray emitting nuclide conjugate and the α-ray emitting nuclide conjugate binding linker, those described above in the sections (α-ray emitting nuclide conjugate) and (α-ray emitting nuclide conjugate binding linker) can be used. Those skilled in the art can select an appropriate reaction system by using known information regarding the method of binding between the α-ray emitting nuclide conjugate and the antibody-bound sugar linker, or between the α-ray emitting nuclide conjugate, the α-ray emitting nuclide conjugate binding linker and the antibody-bound sugar linker. The group at the Z position of the antibody-binding sugar linker may be an azide group, a derivative of a tetrazine group, a norbornene group, a trans-cyclooctene group, a dibenzylcyclooctyl group, a bicyclo[6.1.0]non-4-yn-9-ylmethyl group, or a derivative of a cyanobenzothiazole group, which can introduce drugs under mild conditions. For example, the reaction can proceed rapidly at room temperature in the neutral pH range. For example, an antibody-bound sugar linker having an azide group at the Z position of the antibody-bound sugar linker and an α-ray emitting nuclide conjugate consisting of decaborane bound to an α-ray emitting nuclide conjugate binding linker consisting of methyl 5-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-5-oxopentanoate (DIABAC) can be reacted in PBS buffer to bind the antibody-bound sugar linker and the α-ray emitting nuclide conjugate consisting of decaborane via the α-ray emitting nuclide conjugate binding linker consisting of DIABAC.

[0067] (Antibody-drug conjugate intermediate) In one aspect, the disclosure provides an intermediate product produced in a method for producing an antibody drug conjugate.

[0068] In one aspect, the disclosure provides the following compound: In one aspect, the present disclosure provides an antibody-drug conjugate having formula (5): Equation (5)(AL 2 -L 1 ) n -B [In the formula, B is an antibody or antigen-binding fragment thereof; L 1 is the first linker; L 2 is a bond or a second linker; A is an α-emitting nuclide binding structure; n is 1 to 10; The first linker L 1 has the following formula (2). Formula (2) TIFF2024178476000034.tif39157[in the formula, #B is the point of attachment to the antibody molecule; X's each independently represent a single bond, an oxygen atom, -NH-, -COHN-, -COO-, or a group represented by formula (3) or (4): TIFF2024178476000035.tif43156[where, R 1 is a trivalent branched hydrocarbon group having 1 to 6 carbon atoms, and R 2 and R 3 each independently represents an alkylene group having 1 to 3 carbon atoms; at least one of Y1 to Y3 is present and each independently represents a PEG chain, a substituted PEG chain, or a PEG chain containing an oxygen atom, -NH-, -COHN-, or -COO- in the main chain, the PEG chain having a linear structure or a branched structure, and in the case of a branched structure, the number of branches is 2 to 10; Z is independently -O-, -CH 2 -, —N-, an optionally substituted tetrazine group, a norbornene group, a trans-cyclooctene group, a dibenzylcyclooctyl group, a bicyclo[6.1.0]non-4-yn-9-ylmethyl group, or an optionally substituted cyanobenzothiazole group, and at least one Z is not a hydroxy group or a methoxy group; R is L-fucose or a hydrogen atom; When any of Y1-3 is absent, Z is hydrogen and X is oxygen; When X is a group represented by the formula (3) or (4), Y1 to Y3 are each a branched chain R 2 and R 3When the PEG chain has a branched structure, Z is bonded to each branch. #B represents the point of attachment to B, which is an asparagine residue present in the antibody molecule; #L 2-1 , #L 2-2 and #L 2-3 are each independently present when Y1 to Y3 are present, and each independently represents L 2 represents a hydrogen or a point of attachment to; Existing #L 2-1 , #L 2-2 and #L 2-3 At least one of the following is L 2 represents the attachment point to .]

[0069] In one aspect, the present disclosure provides a compound having formula (6): Formula (6) AL 2 -L 1 [In the formula, L 1 is the first linker; L 2 is a bond or a second linker; A is the astatine bond structure; The first linker L 1 has the following formula (7). Formula (7) TIFF2024178476000036.tif30153[In the formula, X's each independently represent a single bond, an oxygen atom, -NH-, -COHN-, -COO-, or a group represented by formula (3) or (4): TIFF2024178476000037.tif43154 [In the formula, R 1 is a trivalent branched hydrocarbon group having 1 to 6 carbon atoms, and R 2 and R 3 each independently represents an alkylene group having 1 to 3 carbon atoms; at least one of Y1 to Y3 is present and each independently represents a PEG chain, a substituted PEG chain, or a PEG chain containing an oxygen atom, -NH-, -COHN-, or -COO- in the main chain, the PEG chain having a linear structure or a branched structure, and in the case of a branched structure, the number of branches is 2 to 10; Z is independently -O-, -CH 2 -, —N-, an optionally substituted tetrazine group, a norbornene group, a trans-cyclooctene group, a dibenzylcyclooctyl group, a bicyclo[6.1.0]non-4-yn-9-ylmethyl group, or an optionally substituted cyanobenzothiazole group, and at least one Z is not a hydroxy group or a methoxy group; R is L-fucose or a hydrogen atom; When any of Y1-3 is absent, Z is hydrogen and X is oxygen; When X is a group represented by the formula (3) or (4), Y1 to Y3 are each a branched chain R 2 and R 3 When the PEG chain has a branched structure, Z is bonded to each branch. #B represents the point of attachment to B, which is an asparagine residue present in the antibody molecule; #L 2-1 , #L 2-2 and #L 2-3 are each independently present when Y1 to Y3 are present, and each independently represents L 2 represents a hydrogen or a point of attachment to; Existing #L 2-1 , #L 2-2 and #L 2-3 At least one of the following is L 2 represents the attachment point to .]

[0070] 2. (Pharmaceutical Compositions) In one embodiment, the antibody-drug conjugate of the present disclosure is provided in the form of a pharmaceutical composition comprising the same, which is used to obtain a physiological or physical effect obtained by administering an α-emitting nuclide to a living body, for example, but not limited to, for the purpose of testing, diagnosis, treatment, or a combination thereof.

[0071] As used herein, "treatment" has its general meaning in the art and refers to the administration of a composition of the present disclosure to a subject for the purposes of ameliorating, eradicating, or curing any disorder or disease state, preventing or delaying the onset of such disorder or disease state (prophylaxis), or preventing or reducing the likelihood of recurrence of such disorder or disease state (recurrence prevention).

[0072] In one embodiment, the pharmaceutical composition comprising the antibody-drug conjugate of the present disclosure is a pharmaceutical composition for use in tumor treatment. In one embodiment, the pharmaceutical composition of the present disclosure is used for inhibiting progression, regression, elimination, inhibiting metastasis, and preventing recurrence of tumors. The pharmaceutical composition comprising the antibody-drug conjugate of the present disclosure can be applied to the treatment of various tumors by adjusting its composition, for example, the antibody used, according to the tumor to be targeted. The tumors to be treated or otherwise treated by the pharmaceutical composition of the present disclosure are not particularly limited and include any benign or malignant tumor, including cancers in the digestive system, urological system, nervous system, connective tissue, skin cancer, and the like. Non-limiting examples of tumors to be treated or otherwise treated by the pharmaceutical composition of the present disclosure include lymphoma, thyroid cancer, melanoma, prostate cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical cancer, lung cancer, head and neck cancer, and cancer stem cells.

[0073] In one aspect, the pharmaceutical composition of the present disclosure can be used for testing or diagnosis, including detection of the presence or absence of cancer cells in a living body, visualization of cancer tissue, and quantification of the amount of cancer tissue, either in conjunction with or independently of the treatment of the above-mentioned tumor.

[0074] The pharmaceutical compositions of the present disclosure can be administered orally or parenterally to humans or other mammals, such as mice, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.

[0075] Parenteral administration is not particularly limited, but may be intravascular administration, subcutaneous administration, intramuscular administration, rectal administration, transdermal administration, nasal administration, or the like.

[0076] The dosage form of the pharmaceutical composition of the present disclosure is not particularly limited, and it can be prepared as a pharmaceutical composition for oral administration in the form of granules, fine granules, powders, hard capsules, soft capsules, syrups, emulsions, suspensions, or liquids, etc., or as a pharmaceutical composition for parenteral administration in the form of injections for intravenous administration, intramuscular administration, or subcutaneous administration, drips, transdermal absorption agents, transmucosal absorption agents, nasal drops, inhalants, suppositories, etc. These preparations can be prepared according to conventional methods. Preferably, they are liquid preparations for oral administration or injection.

[0077] When the pharmaceutical composition of the present disclosure is provided as an injection, it can be prepared by dissolving, suspending or emulsifying it in a sterile aqueous or oily liquid. As an aqueous liquid for injection, for example, physiological saline, an isotonic liquid containing glucose or other auxiliary drugs, etc., can be used, and a suitable solubilizing agent, for example, alcohol such as ethanol, polyalcohol such as propylene glycol, polyethylene glycol, nonionic surfactant such as polysorbate 80, etc. can be used in combination. As an oily liquid, for example, sesame oil, soybean oil, etc. can be used in combination with benzyl benzoate, benzyl alcohol, etc. as a solubilizing agent. When providing as an injection, drip, etc., it can also be provided in the form of a freeze-dried preparation that is dissolved when used.

[0078] When the pharmaceutical composition of the present disclosure is administered by injection, it can be administered systemically or by direct injection into the tumor site or its surrounding area.

[0079] The dosage and / or schedule of the pharmaceutical composition of the present disclosure can be appropriately determined by a doctor or medical professional, taking into consideration the size of the cancer, the age, weight, condition, etc. of the patient.

[0080] In one embodiment, the pharmaceutical composition of the present disclosure can contain a combination of multiple types of antibody-drug conjugates of the present disclosure to the extent that their functions are not impaired. Also, the pharmaceutical composition of the present disclosure can be used in combination with other therapeutic agents, such as other anti-cancer therapeutic agents, to the extent that their functions are not impaired.

[0081] In one embodiment, the pharmaceutical composition of the present disclosure may further contain a probe compound capable of detecting a target tissue to the extent that the function is not impaired. Preferably, such a probe compound is a probe compound for positron emission tomography (PET). As the PET probe compound (PET agent), any known compound in the art may be used. By using the probe compound in combination, the delivery of the drug to the affected area or the condition of the affected area after administration can be monitored simultaneously with treatment.

[0082] When the antibody-drug conjugates of the present disclosure are used in combination, or when they are used in combination with other therapeutic agents or probe compounds, these agents may be included in a single formulation or may be included in separate formulations. When these are provided in separate formulations, they may be provided in the form of a kit containing the combination of formulations, or each may be provided as a single agent to be combined with other formulations. These formulations may be administered simultaneously or sequentially.

[0083] 3. (Testing, diagnostic or treatment methods) In one aspect, the disclosure includes a method of testing, diagnosing, preventing or treating using the antibody drug conjugates of the disclosure. The examination, diagnosis, or treatment method according to the present disclosure includes administering to a subject the composition described above in the "pharmaceutical composition." The specific administration method and dosage may be the same as those described above in the "pharmaceutical composition according to the present disclosure." EXAMPLES

[0084] The present disclosure will be described in more detail below using examples. However, these examples are illustrative of the present disclosure and do not limit the present disclosure in any way.

[0085] Example 1 Preparation of antibody-drug conjugates (1) Preparation of antibody-binding sugar linker

[0086] (1-1) Preparation of antibody-binding sugar linker compound 12 An antibody-binding sugar linker compound 12 having an azide-terminated PEG chain was prepared as follows. (Preparation of Compound 2) Compound 1 (2.03 g, 8.52 mmol) was dissolved in toluene (40 mL), and p-methoxybenzyl chloride (1.27 mL, 9.37 mmol), potassium iodide (283 mg), and silver (I) oxide (2.96 g, 12.8 mmol) were added in that order, and the mixture was stirred at 60°C overnight. The solid matter was filtered off and washed with chloroform, and the filtrate and washings were combined and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (developing solvent chloroform:methanol = 50:1) to obtain compound 2 (1.66 g, 58% (yield)) as a yellow oil. [ka] compound 2 1 H NMR (600MHz, CDCl 3 ):δ=2.66(brs,1H),3.57-3.62(m,4H),3.63-3.69(m,14H),3.69-3.74(m,2 H),3.80(s,3H),4.50(s,2H),6.87(d,J=8.9Hz,2H),7.27(d,J=7.1Hz,2H).

[0087] (Preparation of Compound 3) Compound 2 (773 mg, 2.28 mmol) was dissolved in dichloromethane (15 mL), triethylamine (1.57 mL, 11.4 mmol) and tosyl chloride (610 mg, 3.20 mmol) were added, and the mixture was stirred at room temperature overnight. Water was added to the reaction solution, and the mixture was extracted twice with chloroform. The organic phase was washed with a saturated aqueous solution of sodium bicarbonate and saturated saline in this order, and then sodium sulfate was added to dry the mixture. The drying agent was filtered off, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent toluene:ethyl acetate=2:3) to obtain pale yellow compound 3 (1.08 g, 92% (yield)). [ka] compound 3 1 H NMR (600MHz, CDCl 3):δ=2.44(s,3H),3.55-3.71(m,18H),3.80(m,3H),4.15(t,J=4.8Hz,2H),4.49(s,2H),6 .87(d,J=8.2Hz,2H),7.26(d,J=8.9Hz,2H),7.34(d,J=8.2Hz,2H),7.80(d,J=8.2Hz,2H).

[0088] (Preparation of Compound 5) Compound 4 (435 mg, 522 μmol) and compound 3 (1.07 g, 2.09 mmol) prepared by the method described in Non-Patent Document 3 were dissolved in DMF (2.1 mL), sodium hydride (50.1 mg, 1.15 mmol) was added, and the mixture was stirred at 0° C. for 10 minutes and then at room temperature overnight. After adding 5% citric acid aqueous solution (2 mL) to the reaction liquid to stop the reaction, water was added and the mixture was extracted twice with ethyl acetate. The organic phase was washed with saturated sodium bicarbonate aqueous solution and saturated saline in that order, and then magnesium sulfate was added and dried. After filtering off the desiccant, the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent: ethyl acetate) to obtain colorless oily compound 5 (624 mg, 79% (yield)). [ka] compound 5 1 H NMR (600MHz, CDCl 3 ):δ=1.66(s,3H),3.23-3.30(m,2H),3.42-3.47(m,2H),3.47-3.87(m,52H),3. 90(t,J=6.2Hz,1H),4.05(t,J=6.2Hz,1H),4.45(d,J=11.7Hz,1H),4.46-4.50( m,5H),4.52-4.60(m,3H),4.64(d,J=11.7Hz,1H),4.75-4.91(m,6H),6.11(d,J =8.2Hz,1H),6.86(d,J=8.9Hz,4H),7.19-7.34(m,27H),7.40(d,J=7.6Hz,2H).

[0089] (Preparation of Compound 6) Compound 5 (627 mg, 414 μmol) was dissolved in dichloromethane (6 mL), water (0.6 mL) and DDQ (263 mg, 1.16 mmol) were added, and the mixture was stirred at 0°C for 15 minutes and then at room temperature for 2.5 hours. Saturated aqueous sodium bicarbonate solution (18 mL) and chloroform (12 mL) were added to the reaction solution, and the mixture was stirred at room temperature for 10 minutes, after which water was added and the mixture was extracted twice with chloroform. The organic phase was washed with saturated aqueous sodium bicarbonate solution and saturated saline in that order, and then sodium sulfate was added to dry it. After filtering off the desiccant, the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent ethyl acetate:methanol=8:1) to obtain colorless oily compound 6 (286 mg, 54% (yield)). [ka] compound 6 1 H NMR (600MHz, CDCl 3 ):δ=1.68(s,3H),2.83(brs,1H),2.95(brs,1H),3.26-3.31(m,2H),3.42- 3.84(m,48H),3.91(t,J=6.9Hz,1H),4.07(t,J=6.9Hz,1H),4.46(d,J=11.7 Hz,1H),4.50(s,1H),4.53-4.61(m,3H),4.64(d,J=11.7Hz,1H),4.75-4.9 1(m,6H),6.26(d,J=8.2Hz,1H),7.18-7.36(m,23H),7.40(d,J=7.6Hz,2H).

[0090] (Preparation of Compound 7) Compound 6 (663 mg, 520 μmol) was dissolved in dichloromethane (6 mL), triethylamine (582 μL, 4.16 mmol) and tosyl chloride (397 mg, 2.08 mmol) were added in that order, and the mixture was stirred at room temperature overnight. Water was added to the reaction solution, and the mixture was extracted twice with ethyl acetate. The organic phase was washed with a saturated aqueous solution of sodium bicarbonate and saturated saline in that order, and then magnesium sulfate was added to dry the mixture. The drying agent was filtered off, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent toluene:ethyl acetate=1:20) to obtain colorless oily compound 7 (658 mg, 80% (yield)). [ka] compound 7 1 H NMR (600MHz, CDCl 3 ):δ=1.66(s,3H),2.43(s,6H),3.24-3.30(m,2H),3.43-3.85(m,44H),3.91(t,J =6.2Hz,1H),4.05(t,J=6.2Hz,1H),4.11-4.14(m,4H),4.44(d,J=11.7Hz,1H),4. 49(s,1H),4.52-4.60(m,3H),4.63(d,J=11.7Hz,1H),4.75-4.91(m,6H),6.08(d ,J=8.2Hz,1H),7.17-7.38(m,27H),7.40(d,J=6.9Hz,2H),7.78(d,J=8.2Hz,4H).

[0091] (Preparation of Compound 8) Compound 7 (422 mg, 279 μmol) was dissolved in 1,4-dioxane (16 mL) and added to a suspension of Pd / C (260 mg) in 1,4-dioxane (4 mL), then water (5 mL) was added and stirred overnight at room temperature under a hydrogen atmosphere. The solid matter was filtered off and washed with a 50% aqueous solution of 1,4-dioxane, and the filtrate and washings were combined and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (developing solvent chloroform:methanol:water=85:15:0.8) to obtain colorless oily compound 8 (245 mg, 78% (yield)). [ka] compound 8 13 C NMR (150MHz, CD 3OD):δ=21.65,22.70,23.00,55.65,58.27,58.36,62.10,67.58,69.14, 69.17,69.79,69.90,70.63,71.03,71.37,71.47,71.52,71.55,71.61, 71.82,71.86,71.88,73.91,76.49,76.89,81.62,82.11,83.78,83.80, 92.25,97.32,102.07,102.15,129.12,131.13,146.52,173.46,174.03

[0092] (Preparation of Compound 9) Compound 8 (222 mg, 196 μmol) was dissolved in pyridine (2 mL), acetic anhydride (1 mL) was added, and the mixture was stirred at room temperature overnight. Methanol (2 mL) was added to the reaction solution to decompose the excess reagent, and the reaction solution was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent chloroform:methanol:water=20:1) to obtain colorless oily compound 9 (131 mg, 50% (yield), α / β=7 / 3). [ka] compound 9 1 H NMR (600MHz, CDCl 3):δ=1.93(s,3H),2.07(s,3H),2.09(s,0.9H),2.10-2.13(m,9H),2.18(s,2.1H),2.45(s,6H),3.44-3.77(m,40H),3.77-3.83 (m,0.3H),3.86-3.95(m,1.7H),4·13-4.19(m,4H),4.22-4.40(m,3H),4.55(s,0.7H),4.58(s,0.3H),4.91-5.01(m,1H),5.12 (t,J=8.9Hz,0.3H),5.20(dd,J=8.9Hz,11.0Hz,0.7H),5.45(d,J=3.4Hz,0.3H),5.48(d,J=3.4Hz,0.7H),5.61(d,J=8.9Hz,0. 3H),5.62(d,J=8.9Hz,0.7H),5.88(d,J=9.6Hz,0.3H),6.10(d,J=4.1Hz,0.7H),7.35(d,J=8.2Hz,4H),7.80(d,J=8.2Hz,4H).

[0093] (Preparation of Compound 10) Compound 9 (76.2 mg, 56.8 μmol) was dissolved in DMF (1.5 mL), sodium azide (36.9 mg, 568 μmol) was added, and the mixture was stirred at 50° C. overnight. Water was added to the reaction solution, and ethyl acetate was added for two extractions. The organic phase was washed with a saturated aqueous solution of sodium bicarbonate and saturated saline, and then magnesium sulfate was added for drying. The drying agent was filtered off, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent chloroform:methanol:water=30:1) to obtain colorless oily compound 10 (48.9 mg, 80%, α / β=75 / 25). [ka] compound 10 1 H NMR (600MHz, CDCl 3):δ=1.93(s,3H),2.08(s,3H),2.10(s,0.75H),2.11-2.14(m,9H),2.18(s,2.25H),3.39(t,J=4.8Hz,4H),3.44-3.7 8(m,40H),3.78-3.83(m,0.25H),3.87-3.95(m,1.75H),4.20-4.40(m,3H),4.53(s,0.75H),4.56(s,0.25H),4.92-5 .00(m,1H),5.11(t,J=9.6Hz,0.25H),5.19(dd,J=8.9Hz,11.0Hz,0.75H),5.45(d,J=3.4Hz,0.25H),5.48(d,J=3.4H z,0.75H),5.56(d,J=8.9Hz,0.75H),5.61(d,J=8.2Hz,0.25H),5.81(d,J=9.6Hz,0.25H),6.11(d,J=4.1Hz,0.75H).

[0094] (Preparation of Compound 11) Compound 10 (41.3 mg, 38.1 μmol) was dissolved in methanol (10 mL), a catalytic amount of 28% sodium methoxide in methanol was added dropwise, and the mixture was stirred at room temperature overnight. + After neutralization with the addition of chloroform, the resin was filtered off. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: chloroform:methanol:water=85:15:0.1) to obtain compound 11 (14.6 mg, 44% (yield)) as a colorless oil. [ka] compound 11 1 H NMR (600MHz, CD 3 OD):δ=1.93(s,3H),3.37-3.89(m,51H),4.18-4.23(m,1H),4.62-4.67(m,1.4H),5.12(d,J=2.7Hz,0.6H).

[0095] (Preparation of Compound 12) Compound 11 (6.6 mg, 7.55 μmol) was added with 0.780 M 2-chloro-1,3-dimethylimidazolinium chloride aqueous solution (300 μL, 234 μmol) and triethylamine (82.1 μL, 589 μmol) and stirred overnight at 0° C. The reaction solution was purified by gel filtration chromatography (developing solvent: 0.05% triethylamine aqueous solution) to quantitatively obtain compound 12 (6.57 mg). [ka] compound 12 1 H NMR (600MHz, D 2 O):δ=1.93(s,3H),3.23-3.28(m,1H),3.30(dd,J=2.7Hz,9.6Hz,1H),3.32-3.40(m,6H), 3.46-3.78(m,41H),4.01-4.07(m,2H),4.22(s,1H),4.55(s,1H),5.93(d,J=7.5Hz,1H).

[0096] (1-2) Preparation of antibody-binding sugar linker compound 34 An antibody-binding sugar linker compound 34 having a PEG chain with a tetrazine group at its end was prepared as follows. (Preparation of Compound 18) The commercially available compound 17 (5.01 g, 11.2 mmol) was dissolved in dichloromethane (6 mL), 25% hydrogen bromide-acetic acid solution (15 mL) was added, and the mixture was stirred at room temperature for 2.5 hours. Ice water was added to the reaction solution, and the mixture was extracted with chloroform. The organic phase was washed with water (twice), saturated aqueous sodium bicarbonate solution, and saturated saline solution in this order, and then dried over sodium sulfate. The drying agent was filtered off, and the solvent was distilled off under reduced pressure. The resulting residue (4.96 g) was dissolved in benzyl alcohol (14 mL), and the mixture was stirred overnight at 90°C. Acetone (40 mL), water (15 mL), and hydrochloric acid (10 mL) were added to the reaction solution, and the mixture was stirred at 75°C for an additional 5 hours. Triethylamine (20 mL) was added to the reaction solution to stop the reaction, and the mixture was concentrated under reduced pressure. Water was added to the residue, and the mixture was extracted with ethyl acetate (twice). The organic phase was washed with saturated aqueous sodium bicarbonate solution and saturated saline solution in this order, and then dried over sodium sulfate. After filtering off the drying agent, the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (eluent: CHCl 3 :MeOH=10:1) to obtain compound 18 (3.05 g, 68% (yield)). [ka] compound 18 1 H NMR (600MHz, CDCl 3 ):δ=7.82-7.75(m,2H),7.73-7.69(m,2H),7.14-7.03(m,5H),5.25(d,J=8 .2Hz,1H),4.80(d,J=12.4Hz,1H),4.53(d,J=12.4Hz,1H),4.36-4.30(m,1 H),4.16(dd,J=8.2Hz,11.0Hz,1H),3.97-3.86(m,2H),3.70(dtJ=2.7Hz,8 .9Hz,1H),3.52-3.48(m,2H),3.17(d,J=4.8Hz,1H),2.59(t,J=6.2Hz,1H).

[0097] (Preparation of Compound 19) Compound 18 (3.03 g, 7.59 mmol) was dissolved in acetonitrile (125 mL), and benzaldehyde dimethyl acetal (2.27 mL, 15.2 mmol) and CSA (250 mg) were added in that order, followed by stirring at room temperature overnight. Triethylamine (0.5 mL) was added to the reaction solution, which was then concentrated under reduced pressure. Water was added to the residue, which was then extracted with ethyl acetate (twice). The organic phase was washed with saturated aqueous sodium bicarbonate and saturated saline, and then dried over sodium sulfate. The drying agent was filtered off, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (developing solvent Hexane: EtOAc = 2: 1) to obtain compound 19 (3.36 g, 91% (yield)). [ka] compound 19 1 H NMR (600MHz, CDCl 3 ):δ=7.86-7.70(m,4H),7.53-7.48(m,2H),7.41-7.35(m,3H),7.11-7.02(m,5 H),5.58(s,1H),5.28(d,J=8.2Hz,1H),4.85(d,J=12.4Hz,1H),4.69-4.60(m, 1H),4.52(d,J=12.4Hz,1H),4.43(dd,J=3.4Hz,9.6Hz,1H),4.30(dd,J=8.9Hz ,10.3Hz,1H),3.87(t,J=9.6Hz,1H),3.69-3.61(m,2H),2.46(d,J=3.4Hz,1H).

[0098] (Preparation of Compound 20) Compound 19 (4.36 g, 8.96 mmol) was dissolved in DMF (40 mL), and benzyl bromide (5.32 mL, 44.8 mmol) and sodium hydride (589 mg, 13.4 mmol) were added in that order. The mixture was stirred at 0°C for 30 minutes and at room temperature overnight. After 5% citric acid solution (5 mL) was added to the reaction solution to stop the reaction, water was added and the mixture was extracted with ethyl acetate (twice). The organic phase was washed with saturated sodium bicarbonate solution and saturated saline, and dried over sodium sulfate. After filtering off the drying agent, the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (developing solvent Toluene: EtOAc = 20: 1) to obtain compound 20 (4.14 g, 80% (yield)). [ka] compound 20 1 HNMR (600MHz, CDCl 3 ):δ=7.88-7.64(m,3H),7.54-7.48(m,3H),7.43-7.36(m,3H),7.07-6.94(m,7H ),6.92-6.83(m,3H),5.63(s,1H),5.19(d,J=8.2Hz,1H),4.80(d,J=12.4Hz,1H) ,4.77(d,J=12.4Hz,1H),4.48(d,J=12.4Hz,2H),4.45-4.36(m,2H),4.26(dd,J= 8.9Hz,10.3Hz,1H),3.88(t,J=10.3Hz,1H),3.83(t,J=8.9Hz,1H),3.67(m,1H).

[0099] (Preparation of Compound 21) Compound 20 (4.05 g, 7.01 mmol) was dissolved in dichloromethane (120 mL), and triethylsilane (3.34 mL, 21.0 mmol) and boron trifluoride diethyl ether complex (2.91 mL, 23.1 mmol) were added in sequence under an argon atmosphere, and the mixture was stirred at -78 ° C for 10 minutes and at 0 ° C overnight. A mixture of methanol (13 mL), triethylamine (6.5 mL) and chloroform (19.5 mL) was added to the reaction solution to terminate the reaction, and then a saturated aqueous sodium bicarbonate solution was added and extracted with chloroform (twice). The organic phase was washed with saturated saline and dried over sodium sulfate. After filtering off the desiccant, the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (developing solvent Hexane: EtOAc = 2: 1) to obtain compound 21 (2.98 g, 73% (yield)). [ka] compound 21 1 H NMR (600MHz, CDCl 3 ):δ=7.85-7.51(m,4H),7.41-7.35(m,4H),7.35-7.30(m,1H),7.11-7.01(m,7H),6.99 -6.90(m,3H),5.15(d,J=7.6Hz,1H),4.78(d,J=12.4Hz,1H),4.72(d,J=12.4Hz,1H),4. 67(d,J=12.4Hz,1H),4.60(d,J=12.4Hz,1H),4.51(d,J=12.4Hz,1H),4.47(d,J=12.4H z,1H),4.26-4.19(m,2H),3.89-3.78(m,3H),3.68-3.61(m,1H),2.89(d,J=2.1Hz,1H).

[0100] (Preparation of Compound 23) The commercially available compound 22 (5.65 g, 14.5 mmol) was dissolved in dichloromethane (56 mL), and 5-tert-butyl-2-methylbenzenethiol (5.27 mL, 28.9 mmol) and boron trifluoride diethyl ether complex (3.37 mL, 28.9 mmol) were added in sequence, followed by stirring at room temperature overnight. Saturated saline was added to the reaction solution, and the mixture was extracted with chloroform (twice). The organic phase was washed with a saturated aqueous solution of sodium bicarbonate, and then dried over sodium sulfate. The drying agent was filtered off, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (developing solvent Hexane: EtOAc = 2: 1) to obtain compound 23 (6.75 g, 91% (yield)). [ka] compound 23 1 H NMR (600MHz, CDCl 3 ):δ=7.53(d,J=2.1Hz,1H),7.23(dd,J=2.1Hz,8.2Hz,1H),7.14(d,J=7.6Hz,1H),5.53(s,1H),5.43(s,1H),5.41-5.34(m,2H),4.58-4.49(m,1H) ,4.35(dd,J=4.8Hz,12.4Hz,1H),4.08(dd,J=2.7Hz,12.4Hz,1H),2.41(s,3H),2.17(s,3H),2.08(s,3H),2.06(s,3H),2.03(s,3H),1.30(s,9H).

[0101] (Preparation of Compound 24) Compound 23 (8.07 g, 15.8 mmol) was dissolved in methanol (80 mL), and a catalytic amount of 28% sodium methoxide in methanol was added and stirred at room temperature overnight. + After neutralization with the addition of 1,000g ... 3 The mixture was purified using an aqueous hexane-MeOH mixture (10:1) to obtain compound 24 (5.29 g, 98% yield). [ka] compound 24 1 H NMR (600MHz, CD 3 OD):δ=7.60(d,J=2.7Hz,1H),7.23(dd,J=2.1Hz,8.2Hz,1H),7.14(d,J=8.2Hz,1H),5.33(s, 1H),4.11(t,J=2.1Hz,1H),4.06-3.97(m,1H),3.79-3.73(m,4H),2.39(s,3H),1.29(s,9H).

[0102] (Preparation of Compound 25) Compound 24 (5.01 g, 14.6 mmol) was dissolved in acetonitrile (250 mL), and benzaldehyde dimethyl acetal (3.29 mL, 21.9 mmol) and CSA (507 mg) were added in that order, followed by stirring at 0°C for 1 hour. Triethylamine (1.5 mL) was added to the reaction solution to terminate the reaction, and the solution was concentrated under reduced pressure. The residue was purified by silica gel chromatography (developing solvent Toluene: EtOAc = 3: 1) to obtain compound 25 (4.12 g, 67% (yield)). [ka] compound 25 1 H NMR (600MHz, CDCl 3 ):δ=7.58-7.48(m,3H),7.42-7.35(m,3H),7.25-7.20(m,1H),7.16(d,J=7.6Hz,1H),5.59(s,1H),5.52(s,1H),4.41-4.33(m,2H) ),4.24-4.17(m,2H),4.02(t,J=9.6Hz,1H),3.85(t,J=9.6Hz,1H),2.85(s,1H),2.77(d,J=3.4Hz,1H),2.41(s,3H),1.30(s,9H).

[0103] (Preparation of Compound 26) Compound 25 (4.08 g, 9.48 mmol) was dissolved in DMF (40 mL), and sodium hydride (996 mg, 22.7 mmol) and benzyl bromide (2.70 mL, 22.7 mmol) were added in that order. The mixture was stirred at 0°C for 15 minutes and at room temperature overnight. Methanol (3 mL) was added to the reaction solution to decompose the excess reagent, and then water was added and the mixture was extracted with ethyl acetate (twice). The organic phase was washed with a saturated aqueous solution of sodium bicarbonate and saturated saline, and then dried over sodium sulfate. The desiccant was filtered off, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (developing solvent: Toluene) to obtain compound 26 (4.46 g, 77% (yield)). [ka] compound 26 1 H NMR (600MHz, CDCl 3 ):δ=7.52(d,J=6.9Hz,2H),7.48(d,J=2.1Hz1H),7.43-7.27(m,13H),7.22(dd,J=2.1Hz ,8.2Hz,1H),7.13(d,J=8.2Hz,1H),5.67(s,1H),5.42(s,1H),4.84(d,J=12.4Hz,1H),4. 74(s,2H),4.67(d,J=12.4Hz,1H),4.36-4.30(m,2H),4.22-4.19(m,1H),4.08(d,J=3.4 Hz,1H),4.02(dd,J=3.4Hz,9.6Hz,1H),3.91(t,J=9.6Hz,1H),2.31(s,3H),1.29(s,9H).

[0104] (Preparation of Compound 27) Compound 26 (2.17 g, 3.55 mmol), BSP (818 mg, 3.91 mmol) and TTBP (1.77 g, 7.11 mmol) were dissolved in dichloromethane (36 mL), and molecular sieves 3A (4.11 g) was added under an argon atmosphere and stirred at -60°C for 15 minutes. 2After adding 2H2O (717 mL) and stirring for 5 minutes, a solution of compound 21 (1.65 g, 2.84 mmol) in dichloromethane (36 mL) was added. The reaction solution was stirred at -60°C for 1 hour, and then warmed to room temperature over 1.5 hours. Triethylamine (1.2 mL) was added to the reaction solution to stop the reaction, and the solid matter was filtered off by silica gel filtration and washed with ethyl acetate. The filtrate was combined with the washings and concentrated under reduced pressure. The residue was purified by silica gel chromatography (developing solvent Toluene: EtOAc = 10: 1) to obtain compound 27 (1.83 g, 64% (yield)). [ka] compound 27 1 H NMR (600MHz, CDCl 3 ):δ=7.83-7.49(m,4H),7.49-7.42(m,4H),7.39-7.21(m,16H),7.11-7. 03(m,5H),6.93-6.89(m,2H),6.83-6.80(m,3H),5.51(s,1H),5.11(d,J= 8.2Hz,1H),4.89-4.82(m,3H),4.80(d,J=12.4Hz,1H),4.74(d,J=12.4H z,1H),4.68(d,J=12.4Hz,1H),4.58(d,J=12.4Hz,1H),4.54(s,1H),4.49 (d,J=12.4Hz,1H),4.41(d,J=12.4Hz,1H),4.40(d,J=11.0Hz,1H),4.26-4.15(m,3H),4.07(t,J=9.6Hz,1H),4.03(t,J=9.6Hz,1H),3.74(d,J=2. 7Hz,1H),3.67(dd,J=1.4Hz,11.0Hz,1H),3.59-3.55(m,2H),3.49-3.46(m,1H),3.41(dd,J=3.4Hz,10.3Hz,1H),3.14(dt,J=4.8Hz,9.6Hz,1H),.

[0105] (Preparation of Compound 28) Compound 27 (1.72 g, 1.70 mmol) was mixed with 1.0 M H 3 A solution of 1.0Mn-Bu in THF (12.8 mL, 12.8 mmol) 2A solution of BOTf in dichloromethane (2.56 mL, 2.56 mmol) was added in sequence and stirred at 0°C for 1.5 hours. Triethylamine (1.5 mL) was added to the reaction solution to terminate the reaction, and the solution was concentrated under reduced pressure. The residue was purified by silica gel chromatography (developing solvent Toluene: EtOAc = 5: 1) to obtain compound 28 (1.53 g, 89% (yield)). [ka] Compound 28δ=7.83-7.52(m,4H),7.44-7.40(m,2H),7.35-7.23(m,18H),7.11-7.02(m,5H),6.90-6.81(m,5H),5.13(d,J =8.2Hz,1H),4.90-4.82(m,4H),4.80(d,J=12.4Hz,1H),4.67(d,J=11.7Hz,1H),4.56(d,J=11.0Hz,1H),4.53-4.45 (m,5H),4.40(d,J=12.4Hz,1H),4.27-4.19(m,2H),4.00(t,J=9.6Hz,1H),3.79-3.70(m,4H),3.66(dd,J=3.4Hz,11 .0Hz,1H),3.58-3.54(m,1H),3.47-3.41(m,1H),3.36(dd,J=3.4Hz,9.6Hz,1H),3.22-3.18(m,1H),2.10(brs,1H).

[0106] (Preparation of Compound 29) Compound 28 (790 mg, 782 μmol) was dissolved in 1,2-dichloroethane (8 mL), triethylamine (437 μL, 3.13 mmol) and mesyl chloride (121 μL, 1.56 mmol) were added in that order, and the mixture was stirred at 60° C. overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate (twice). The organic phase was washed with a saturated aqueous solution of sodium bicarbonate and saturated saline, and then dried over sodium sulfate. The drying agent was filtered off, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (developing solvent Toluene: EtOAc = 10: 1) to obtain compound 29 (720 mg, 85% (yield)). [ka] compound 29 1 H NMR (600MHz, CDCl 3 ):δ=7.83-7.52(m,4H),7.41-7.21(m,20H),7.13-7.08(m,1H),7.06(d,J=4.1Hz,4H),6.84-6.75(m,5H),5.14(d,J=8.2Hz,1H),4 .91(d,J=11.0Hz,1H),4.90(d,J=12.4Hz,1H),4,83(s,2H),4.79(d,J=12.4Hz,1H),4.71(d,J=11.7Hz,1H),4.58(d,J=11.0Hz,1H ),4.55(s,1H),4.50(d,J=12.4Hz,1H),4.49-4.43(m,3H),4.37(d,J=13.1Hz,2H),4.25-4.18(m,2H),4.14(dd,J=8.2Hz,10.3Hz, 1H),4.02(t,J=9.6Hz,1H),3.79-3.72(m,3H),3.69(dd,J=3.4Hz,11.0Hz,1H),3.58-3.54(m,1H),3.37-3.32(m,2H),2.86(s,3H).

[0107] (Preparation of Compound 30) Compound 29 (956 mg, 877 μmol) was dissolved in DMF (9.5 mL), sodium azide (285 mg, 4.38 mmol) was added, and the mixture was stirred at 80° C. overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate (twice). The organic phase was washed with saturated aqueous sodium bicarbonate and saturated saline in that order, and then dried over sodium sulfate. The drying agent was filtered off, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (developing solvent Toluene: EtOAc = 10: 1) to obtain compound 30 (862 mg, 95% (yield)). [ka] compound 30 1 H NMR (600MHz, CDCl 3):δ=7.81-7.60(m,3H),7.46-7.43(m,3H),7.35-7.23(m,18H),7.10-7. 01(m,5H),6.91-6.88(m,2H),6.77-6.74(m,3H),5.11(d,J=8.2Hz,1H),4 .91(d,J=11.7Hz,3H),4.86(d,J=12.4Hz,1H),4.78(d,J=12.4Hz,1H),4. 67(d,J=12.4Hz,1H),4.56(d,J=11.0Hz,1H),4.56(s,1H),4.51-4.41(m, 5H),4.26-4.19(m,2H),4.05(dd,J=8.2Hz,9.6Hz,1H),3.82(t,J=9.6Hz,1H),3.77(d,J=2.7Hz,1H),3.72(dd,J=2.1Hz,11.0Hz,1H),3.65(dd,J= 3.4Hz,11.0Hz,1H),3.55-3.51(m,1H),3.45(d,J=12.4Hz,1H),3.32(dd,J=3.4Hz,9.6Hz,1H),3.27-3.24(m,1H),3.22(dd,J=5.5Hz,12.4Hz,1H).

[0108] (Preparation of Compound 31) Compound 30 (156 mg, 150 μmol) was dissolved in a mixture of methanol (4 mL) and THF (1 mL), ethylenediamine (1 mL) was added, and the mixture was heated to reflux overnight. The reaction solution was concentrated under reduced pressure, and pyridine (1 mL) and acetic anhydride (2 mL) were added to the residue and stirred at room temperature overnight. Methanol (2 mL) was added to the reaction solution to stop the reaction, and the mixture was concentrated under reduced pressure. Water was added to the residue and extracted with ethyl acetate. The organic phase was washed with 1M hydrochloric acid, saturated aqueous sodium bicarbonate solution, and saturated saline in that order, and dried over sodium sulfate. The drying agent was filtered off, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (developing solvent Toluene: EtOAc = 5: 1) to obtain compound 31 (137 mg, 96% (yield)). [ka] compound 31 1 H NMR (600MHz, CDCl 3):δ=7.39(d,J=6,9Hz,2H),7.35-7.22(m,28H),5.92(d,J=8.2Hz,1H),4.94-4.86(m,3H), 4.85-4.78(m,3H),4.65(d,J=11.7Hz,1H),4.60-4.50(m,5H),4.47(d,J=11.7Hz,1H),4.4 2(d,J=11.7Hz,1H),4.04(t,J=6.2Hz,1H),3.96(t,J=6.2Hz,1H),3.85-3.69(m,6H),3.38 (dd,J=2.7Hz,9.6Hz,1H),3.30(dd,J=1.4Hz,12.4Hz,1H),3.27-3.18(m,2H),1.65(s,3H).

[0109] Preparation of Compound 32 Compound 31 (101 mg, 103 μmol) was dissolved in 1,4-dioxane (10.0 mL) and added to a suspension of palladium hydroxide (99.5 mg) in 1,4-dioxane (2.0 mL). Water (3.0 mL) and 1N hydrochloric acid (155 μL, 155 μmol) were added, and the mixture was stirred at room temperature under a hydrogen atmosphere for 4 hours. The solid matter was filtered off and washed with water, and the filtrate and the washings were combined and freeze-dried to obtain compound 32 (42.9 mg). [ka] compound 32 1 H NMR (600MHz, CDCl 3 ):δ=7.39(d,J=6,9Hz,2H),7.33-7.21(m,28H),5.80(d,J=7.6Hz,1H),4.92-4.86(m,4H),4.8 4-4.79(m,2H),4.63-4.55(m,4H),4.53-4.44(m,4H),4.03(t,J=7.6Hz,1H),3.89(t,J=6.9Hz ,1H),3.79-3.75(m,2H),3.70-3.61(m,4H),3.38(dd,J=2.7Hz,8.9Hz,1H),3.09-3.05(m,1H) ,2.92(dd,J=2.7Hz,13.1Hz,1H),2.61(dd,J=7.5Hz,13.1Hz,1H),1.69(s,3H),1.48(brs,2H).

[0110] Preparation of Compound 34 Compound 32 (42.9 mg) was dissolved in 50% acetonitrile aqueous solution (0.80 mL), and triethylamine (6.96 μL, 50.0 μmol) and commercially available compound 33 (15.1 mg, 25.0 μmol) were added in that order, followed by stirring at room temperature for 2 hours. Water (2.0 mL) was added to the reaction solution, which was then freeze-dried. The residue was purified by silica gel column chromatography (developing solvent 7.5:2.5:0.3CHCl 3 -MeOH-H 2 O) to obtain compound 34 (22.2 mg, 99% yield for two steps). [ka] compound 34 1 H-NMR (600MHz, D 2 O):δ=10.35(s,1H),8.55(d,J=8.2Hz,2H),7.58(d,J=8.2Hz,2H),5.10(d,J=2.1Hz,0.8H),4.64(s,1H),4.58(d,J=8.2Hz,0.2H), 4.53(s,2H),3.93-3.56(m,28H),3.49-3.43(m,3H),3.31-3.27(m,1H),2.55(t,J=6.2Hz,2H),2.48(t,J=6.2Hz,2H),1.98(s,3H) The obtained compound 34 can be converted to an oxazoline by using a conventional oxazolination method.

[0111] (1-3) Preparation of antibody-binding sugar linker compound 12, part 2 Antibody-binding sugar linker compound 12 was prepared by a process different from that of (1-1) above. Preparation of Compound 36 Compound 35 (4.07 g, 4.35 mmol) (Tokyo Chemical Industry Co., Ltd., Japan) was dissolved in 1.0 M H 3 A solution of 1.0Mn-Bu in THF (32.6 mL, 32.6 mmol) 2A dichloromethane solution of BOTf (6.52 mL, 6.52 mmol) was added in sequence and stirred at 0°C for 1 hour under an argon atmosphere. Triethylamine (12 mL) was added to the reaction solution to terminate the reaction, and the reaction solution was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent n-hexane:ethyl acetate = 3:2 → 1:1) to obtain compound 36 (4.08 g, 88% (yield)) as a white solid. [ka]

[0112] Preparation of Compound 37 Compound 36 (3.57 g, 3.81 mmol) was dissolved in methanol (48 mL), ethylenediamine (12 mL) was added, and the mixture was heated to reflux for 17 hours. The reaction solution was concentrated and azeotroped three times with DMF. Pyridine (30 mL) and acetic anhydride (15 mL) were added to the residue in that order, and the mixture was stirred at room temperature for 18 hours. Methanol (10 mL) was added to the reaction solution to stop the reaction, and the mixture was concentrated under reduced pressure. Water was added to the residue, and the mixture was extracted twice with ethyl acetate. The organic phase was washed with 1M hydrochloric acid, saturated aqueous sodium bicarbonate solution, and saturated saline in that order, and then dried over anhydrous magnesium sulfate. The drying agent was filtered off, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent n-hexane:ethyl acetate = 1:1) to obtain compound 37 (3.45 g, 97% (yield) for two steps) as a white solid. [ka]

[0113] Preparation of Compound 38 Compound 37 (3.44 g, 3.68 mmol) was dissolved in methanol (60 mL), and a catalytic amount of 28% sodium methoxide solution was added and stirred at room temperature for 24 hours. + After neutralization with 1,000 ml of 1,000 ml of ethyl acetate, the resin was filtered off and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: n-hexane:ethyl acetate=2:3) to obtain compound 38 (3.02 g, 96% yield) as a white solid. [ka]

[0114] (Preparation of Compound 39) Compound 38 (3.00 g, 3.52 mmol) and compound 3 (7.41 g, 14.5 mmol) were dissolved in DMF (15 mL), and sodium hydride (323 mg, 1.84 mmol) was added under an argon atmosphere, followed by stirring at 0°C for 1 hour and at room temperature for 20 hours. After adding 5% citric acid aqueous solution (9.0 mL) to the reaction solution to terminate the reaction, water was added and the mixture was extracted twice with ethyl acetate. The organic phase was washed with saturated sodium bicarbonate aqueous solution and saturated saline in this order, and then dried over anhydrous magnesium sulfate. After filtering off the desiccant, the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate) to obtain colorless oily compound 39 (3.98 g, 74% (yield)). [ka]

[0115] (Preparation of Compound 40) Compound 39 (3.98 g, 2.60 mmol) was dissolved in dichloromethane (60 mL), and a mixed solvent of dichloromethane (54 mL), TFA (5.7 mL), and water (0.3 mL) was added and stirred at 0° C. for 2.5 hours. A saturated aqueous solution of sodium bicarbonate (240 mL) was added to the reaction solution and stirred for 1 hour to terminate the reaction, and then water was added and extracted twice with chloroform. The organic phase was washed with a saturated aqueous solution of sodium bicarbonate and saturated saline in this order, and then dried over anhydrous sodium sulfate. The desiccant was filtered off, and the solvent was distilled off under reduced pressure. The resulting residue (5.27 g) was dissolved in methanol (40 mL), and a catalytic amount of 28% sodium methoxide solution was added and stirred at room temperature for 2 hours. Amberlite IR-120 (H + After neutralization with ethyl acetate, the resin was filtered off, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate → chloroform:methanol = 20:1) to obtain compound 40 (3.26 g, 97% (yield) for two steps) as a pale yellow oil. [ka]

[0116] (Preparation of Compound 41) Compound 40 (3.26 g, 2.53 mmol) was dissolved in dichloromethane (30 mL), and triethylamine (4.24 mL, 30.3 mmol) and tosyl chloride (2.89 g, 15.2 mmol) were added in sequence, followed by stirring at room temperature for 16 hours. The reaction solution was diluted with chloroform, and extracted with saturated sodium bicarbonate solution. The organic phase was washed with saturated saline and then dried over anhydrous sodium sulfate. The drying agent was filtered off, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent chloroform:methanol=50:1) to obtain compound 41 (3.48 g, 86% (yield)) as a yellow oil. [ka]

[0117] Preparation of Compound 42 Compound 41 (3.45 g, 2.16 mmol) was dissolved in 1,4-dioxane (50 mL) and added to a suspension (10 mL) of Pd / C (2.86 g) in 1,4-dioxane. Water (10 mL) was then added and the mixture was stirred at room temperature under a hydrogen atmosphere for 22 hours. The solid matter was filtered off and washed with a 50% aqueous solution of 1,4-dioxane. The filtrate and the washings were combined and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (developing solvent chloroform:methanol:water=9:1:0.06) to obtain colorless oily compound 42 (2.51 g, 94% (yield)). [ka]

[0118] Preparation of Compound 43 Compound 42 (2.41 g, 1.95 mmol) was dissolved in DMF (25 mL), sodium azide (1.27 g, 19.5 mmol) was added, and the mixture was stirred for 20 hours at 50° C. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (developing solvent chloroform:methanol:water=9:1:0.06) to obtain colorless oily compound 43 (1.76 g, 92% (yield)). [ka]

[0119] Preparation of Compound 44 Compound 43 (1.75 g, 1.79 mmol) was dissolved in pyridine (8.0 mL), acetic anhydride (4.0 mL) was added, and the mixture was stirred at room temperature for 26 hours. Methanol (8 mL) was added to the reaction solution to decompose the excess reagent, and the reaction solution was then concentrated under reduced pressure. Water was added to the residue, and the mixture was extracted twice with ethyl acetate. The organic phase was washed with 1 M hydrochloric acid, saturated aqueous sodium bicarbonate, and saturated saline in that order, and then dried over anhydrous magnesium sulfate. The drying agent was filtered off, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent chloroform:methanol:water=20:1) to obtain colorless oily compound 44 (2.07, quant.). [ka]

[0120] Preparation of Compound 45 Compound 44 (1.50 g, 1.31 mmol) was dissolved in 25% acetonitrile aqueous solution (28 mL), and CAN (2.15 g, 3.92 mmol) was added and stirred at 0°C for 2.5 hours. The reaction solution was diluted with chloroform and water was added to separate the liquids twice. The organic phase was washed with saturated sodium bicarbonate aqueous solution and saturated saline in that order, and then dried over anhydrous magnesium sulfate. After filtering off the drying agent, the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent chloroform:methanol = 40:1 → 20:1) to obtain compound 45 (900 mg, 66% (yield)) as an orange oil. [ka]

[0121] Preparation of Compound 46 Compound 45 (900 mg, 864 μmol) was dissolved in dichloromethane (18 mL), and triethylamine (724 μL, 5.18 mmol) and DMC (438 mg, 2.59 mmol) were added in that order, followed by stirring at room temperature for 1.5 hours. The reaction solution was diluted with chloroform, and saturated aqueous sodium bicarbonate was added and separated five times. The organic phase was washed with saturated saline and then dried over anhydrous magnesium sulfate. The drying agent was filtered off, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent chloroform:methanol=60:1 (containing 1% triethylamine)) to obtain orange oily compound 46 (660 mg, 75% (yield)). [ka]

[0122] (Preparation of Compound 12) Compound 46 (16.1 mg, 27.3 μmol) was dissolved in methanol (3.0 mL), and a catalytic amount of 28% sodium methoxide solution was added and stirred at room temperature for 18 hours. The reaction solution was concentrated under reduced pressure to obtain compound 12 as a pale yellow oil. [ka]

[0123] (1-4) Preparation of antibody-binding sugar linker compound 66 An antibody-binding sugar linker compound 66 having a PEG chain with a terminally substituted tetrazine group was prepared as follows. Preparation of Compound 53 Compound 52 (2.46 g, 2.36 mmol) (compound 2 in K. Goto, et al, Tetrahedron Lett., 61, 151475 (2020)) was added with 1.0 M H 3 A solution of 1.0M n-Bu in THF (17.7 mL, 17.7 mmol) 2A dichloromethane solution of BOTf (3.35 mL, 3.35 mmol) was added in sequence and stirred at 0°C for 1 hour under an argon atmosphere. Triethylamine (5.0 mL) was added to the reaction solution to terminate the reaction, and the reaction solution was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent toluene:ethyl acetate=4:1) to obtain compound 53 (2.32 g, 99% (yield)) as a white powder. [ka]

[0124] Preparation of Compound 54 Compound 53 (2.22 g, 2.13 mmol) was dissolved in 1,2-dichloroethane (20 mL), and triethylamine (1.78 mL, 12.8 mmol) and mesyl chloride (495 μL, 6.39 mmol) were added in that order, followed by stirring at 60 ° C for 24 hours. Water was added to the reaction solution, and the mixture was extracted twice with chloroform. The organic phase was washed with saturated sodium bicarbonate solution and saturated saline, and then dried over anhydrous magnesium sulfate. The desiccant was filtered off, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent toluene: ethyl acetate = 7: 1) to obtain compound 54 (1.27 g, 62% (yield)) as a white powder. [ka]

[0125] Preparation of Compound 55 Compound 54 (1.27 g, 1.13 mmol) was dissolved in DMF (13 mL), sodium azide (368 mg, 5.67 mmol) was added, and the mixture was stirred at 80° C. for 15 hours. Water was added to the reaction solution, and the mixture was extracted twice with ethyl acetate. The organic phase was washed with saturated aqueous sodium bicarbonate and saturated saline in this order, and then dried over anhydrous magnesium sulfate. The drying agent was filtered off, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent toluene:ethyl acetate=8:1) to obtain compound 55 (1.11 g, quant.) as a white powder. [ka]

[0126] Preparation of Compound 56 Compound 55 (1.16 g, 1.09 mmol) was dissolved in a mixed solvent of methanol (12 mL) and THF (3.0 mL), and ethylenediamine (3.0 mL) was added and the mixture was heated to reflux for 18 hours. The reaction solution was concentrated under reduced pressure and azeotroped three times with DMF. Pyridine (4.5 mL) and acetic anhydride (3.0 mL) were added to the residue in order and stirred at room temperature for 18 hours. Methanol (4.5 mL) was added to the reaction solution to stop the reaction, and the mixture was concentrated under reduced pressure. Water was added to the residue and extracted twice with ethyl acetate. The organic phase was washed with 1M hydrochloric acid, saturated aqueous sodium bicarbonate solution, and saturated saline in that order, and then dried over anhydrous magnesium sulfate. The desiccant was filtered off, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent toluene:ethyl acetate=3:1) to obtain compound 56 (1.08 g, 98% (yield) for two steps) as a white solid. [ka]

[0127] Preparation of Compound 57 Compound 56 (195 mg, 199 μmol) was dissolved in 1,4-dioxane (10 mL) and added to a suspension of Pd / C (128 mg) in 1,4-dioxane (2.0 mL), followed by the addition of water (10 mL) and 1 M hydrochloric acid in that order, and stirred at room temperature for 21 hours under a hydrogen atmosphere. The solid matter was filtered off and washed with a 50% aqueous solution of 1,4-dioxane. The filtrate and washings were combined and concentrated under reduced pressure to give compound 57 (79.0 mg). [ka]

[0128] Preparation of Compound 59 12-Hydroxy-4,7,10-trioxadodecanoic acid tert-butyl (compound 58) (204 mg, 0.73 mmol) was dissolved in dichloromethane (7 mL), and triethylamine (305 μL, 2.19 mmol) and tosyl chloride (419 mg, 2.19 mmol) were added in sequence, and the mixture was stirred at room temperature for 18 hours. The reaction solution was diluted with chloroform, washed with 5% citric acid aqueous solution and saturated saline, and dried over anhydrous magnesium sulfate. The drying agent was filtered off, and the solvent was distilled off under reduced pressure. The resulting residue was dissolved in DMF (7 mL), sodium azide (233 mg, 3.60 mmol) was added, and the mixture was stirred at 65 ° C. for 18 hours. The reaction solution was diluted with chloroform, washed with water and saturated saline in that order, and dried over anhydrous magnesium. The drying agent was filtered off, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (eluent: n-hexane:ethyl acetate=3:1) to obtain compound 59 (209 mg, 96% yield) as a colorless oil. [ka]

[0129] (Preparation of Compound 60) Compound 59 (185 mg, 0.61 mmol) was dissolved in ethyl acetate (8 mL), palladium-activated carbon ethylenediamine complex (167 mg) was added, and the mixture was stirred at room temperature under a hydrogen atmosphere for 1 hour. The reaction solution was filtered through Celite, and the solvent was distilled off under reduced pressure to quantitatively obtain compound 60. [ka]

[0130] Preparation of Compound 62 Compound 61 (84.0 mg, 0.39 mmol) was dissolved in DMF (4 mL), DIEA (82 μL, 0.47 mmol) and PyBOP (243 mg, 0.47 mmol) were added in sequence, and the mixture was stirred at room temperature for 10 minutes. Compound 60 (107 mg, 0.39 mmol) was then added and the mixture was stirred at room temperature for 18 hours. The reaction solution was diluted with ethyl acetate, washed with saturated aqueous sodium carbonate, water, and saturated saline, and dried over anhydrous magnesium sulfate. The drying agent was filtered off, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (developing solvent n-hexane:ethyl acetate=1:5) to obtain compound 62 (132 mg, 71% (yield)) as a red solid. [ka]

[0131] Preparation of Compound 63 Compound 62 (62.2 mg, 137 μmol) was dissolved in dichloromethane (2.0 mL), trifluoroacetic acid (0.2 mL) was added, and the mixture was stirred at room temperature for 4 hours. The reaction solution was concentrated under reduced pressure, and then toluene was added and azeotroped three times. The residue was purified by silica gel column chromatography (developing solvent chloroform:methanol:water=9:1:0.06) to obtain compound 63 (54.1 mg, 94% (yield)) as a pink solid. [ka]

[0132] Preparation of Compound 64 Compound 63 (52.6 mg, 125 μmol) was dissolved in dichloromethane (3.8 mL), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (52.9 mg, 276 μmol) and HOSu (19.5 mg, 169 μmol) were added in that order, followed by stirring at room temperature for 16 hours. Water was added to the reaction solution, which was then extracted twice with ethyl acetate. The organic phase was washed with a 5% aqueous citric acid solution, a saturated aqueous sodium bicarbonate solution, and saturated saline, and then dried over magnesium sulfate. The drying agent was filtered off, and the solvent was removed under reduced pressure to obtain compound 64 (63.3 mg, 86% (NMR yield)) as a pink solid. [ka]

[0133] Preparation of Compound 65 Compound 57 (79.0 mg, 189 μmol) was dissolved in water (0.85 mL), and triethylamine (13.3 μL, 94.5 μmol) and a solution of compound 64 (24.4 mg, 47.2 μmol) in acetonitrile (0.8 mL) were added in sequence, and the mixture was stirred at room temperature for 2 hours. Water (3 mL) was added to the reaction solution, which was then frozen and then freeze-dried. The residue was purified by silica gel column chromatography (developing solvent chloroform; methanol:water = 8:2:0.15 → 7:3:0.3) to obtain compound 65 (28.7 mg, 78% (yield)) as a pink solid. [ka]

[0134] Preparation of Compound 66 Compound 65 (3.1 mg, 3.96 μmol) was dissolved in a mixed solvent of deuterated acetonitrile (0.20 mL) and deuterium oxide (0.10 mL), and 2-chloro-1,3-dimethylimidazolinium chloride (20.0 mg, 119 μmol) and triethylamine (41.5 μL, 297 μmol) were added in sequence and stirred overnight at 0° C. The reaction solution was analyzed by MALDI-TOFMS to confirm the formation of compound 66. MALDI-TOFMS.:CalcdforC 33 H 47 N 7 O 14 Nam / z[M+Na] + :788.3,Found788.7. [ka]

[0135] (1-5) Preparation of antibody-binding sugar linker compound 72 A branched antibody-binding sugar linker compound 72 having a PEG chain with an azide group and a substituted tetrazine group at its terminus was prepared as follows. Preparation of Compound 67 Compound 59 (557 mg, 1.83 mmol) was dissolved in dichloromethane (25 mL), trifluoroacetic acid (2.6 mL, 34 mmol) was added under ice cooling, and the mixture was stirred at room temperature for 3 hours. Toluene was added to the reaction solution to concentrate it, and the residue was purified by silica gel column chromatography (developing solvent hexane:ethyl acetate=1:2) to obtain compound 67 (375 mg, 83% (yield)). [ka]

[0136] Preparation of Compound 68 H-Lys(Boc)-OtBu hydrochloride (122 mg, 0.36 mmol) and compound 67 (89 mg, 0.36 mmol) were dissolved in DMF (4 mL), and DIEA (138 μL, 0.79 mmol) and PyBOP (255 mg, 0.43 mmol) were added in sequence, and the mixture was stirred at room temperature for 18 hours. The reaction solution was diluted with ethyl acetate, washed with saturated aqueous sodium carbonate, water, and saturated saline, and dried over anhydrous magnesium sulfate. After filtering off the drying agent, the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (developing solvent n-hexane:ethyl acetate = 1:3) to obtain compound 68 (172 mg, 90% (yield)) as a white solid. [ka]

[0137] Preparation of Compound 69 Compound 68 (170 mg, 0.32 mmol) was dissolved in 4 M hydrogen chloride-1,4-dioxane solution (4 mL) and stirred at room temperature for 4 hours. The reaction solution was concentrated under reduced pressure and then freeze-dried to obtain compound 69 (128 mg, 98% (yield)) as a colorless oil. [ka]

[0138] Preparation of Compound 70 Compound 69 (270 mg, 0.43 mmol) and compound 64 (182 mg, 0.44 mmol) were dissolved in DMF (5 mL), triethylamine (178 μL, 1.28 mmol) was added, and the mixture was stirred at room temperature for 3 hours. The reaction solution was distilled under reduced pressure, and the residue was purified by silica gel chromatography (developing solvent chloroform:methanol:water=10:1:0.06) to obtain compound 70 (347 mg, 96% (yield)) as a pink solid. [ka]

[0139] Preparation of Compound 71 Compound 70 (10.4 mg, 13.3 μmol) was dissolved in acetonitrile (0.5 mL), and HOSu (2.2 mg, 19.1 μmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.8 mg, 19.8 μmol) were added in sequence, and the mixture was stirred at room temperature overnight. HOSu (1.1 mg, 9.6 μmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.9 mg, 9.9 μmol) were further added in sequence to the reaction solution, and the mixture was stirred at room temperature for 5 hours. An aqueous solution (150 μL) of compound 57 (5.0 mg, 13.3 μmol) and triethylamine (3.7 μL, 26.6 μmol) were added in sequence to the reaction solution, and the mixture was stirred at room temperature overnight. The reaction solution was freeze-dried, and the residue was purified by silica gel chromatography (eluent: chloroform:methanol=80:20→70:30) to obtain Compound 71 (3.1 mg, 20% yield) as a pink solid. [ka]

[0140] Preparation of Compound 72 Compound 71 (3.1 mg, 2.72 μmol) was dissolved in a mixed solvent of deuterated acetonitrile (0.10 mL) and deuterium oxide (0.10 mL), and 2-chloro-1,3-dimethylimidazolinium chloride (13.8 mg, 81.5 μmol) and triethylamine (28.5 μL, 204 μmol) were added in sequence and stirred overnight at 0° C. The reaction solution was analyzed by MALDI-TOFMS to confirm the formation of compound 72. MALDI-TOFMS.:CalcdforC 48 H 74 N 12 O 19 Nam / z[M+Na] + :1145.5,Found1146.3. [ka] (1-6) Preparation of antibody-binding sugar linker compound 78 Antibody-binding sugar linker compound 78 having a PEG chain with an azide group at its end was prepared as follows. Preparation of Compound 74 Compound 73 (55.9 mg, 192 μmol) was dissolved in dichloromethane (2 mL), and N-hydroxysuccinimide (39.0 mg, 339 μmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (64.7 mg, 338 μmol) were added in that order, followed by stirring at room temperature overnight. Water was added to the reaction solution, and the solution was extracted twice with chloroform. The organic phase was washed with water and saturated saline in that order, and then dried by adding anhydrous magnesium sulfate. After filtering off the drying agent, the solvent was distilled off under reduced pressure to obtain compound 74 (72.4 mg, 97% (yield)). [ka]

[0141] Preparation of Compound 75 Compound 43 (45.2 mg, 46.1 μmol) was dissolved in dioxane (1 mL), and a 1,4-dioxane suspension (3 mL) of 10% palladium activated carbon (29.47 mg) was added, followed by water (1 mL). The mixture was stirred overnight at room temperature under a hydrogen atmosphere. The solid matter was filtered off and washed with a 50% aqueous 1,4-dioxane solution, and the filtrate and washings were combined and concentrated under reduced pressure. Compound 74 (72.4 mg, 186 μmol) in a 50% aqueous acetonitrile solution (1.6 mL) and triethylamine (54 μL, 390 μmol) were added to the resulting residue. After stirring overnight at room temperature, water (2.0 mL) was added to the reaction solution and freeze-dried. The residue was purified by silica gel column chromatography (eluent: CHCl 3 -MeOH-H 2 The mixture was purified by a 3:1 ratio (O=9:1:0.06). Pyridine (3 mL) and acetic anhydride (1.5 mL) were added to the purified product and stirred at room temperature overnight. Methanol (4 mL) was added to the reaction mixture to terminate the reaction, and the mixture was concentrated under reduced pressure. Azeotropy with toluene (4 mL) was repeated five times, and the residue was purified by silica gel chromatography (eluent CHCl 3 -MeOH-H 2 O=15:1:0.04) to obtain compound 75 (30.4 mg, 40% yield for three steps). [ka]

[0142] Preparation of Compound 76 Compound 75 (8.7 mg, 5.3 μmol) was dissolved in acetonitrile (2 mL), and water (0.5 mL) and ammonium cerium (IV) nitrate (11.2 mg, 21 μmmol) were added at 0°C, and the mixture was stirred for 1.5 hours and then at room temperature for 2 hours. Furthermore, ammonium cerium (IV) nitrate (10.5 mg, 19 μmmol) was added to the reaction solution, and the mixture was stirred at 0°C for 1.5 hours. A saturated aqueous solution of sodium bicarbonate was added to the reaction solution, and the mixture was extracted twice with chloroform. The organic phase was washed with saturated saline, and then dried by adding anhydrous magnesium sulfate. The desiccant was filtered off, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent CHCl 3 -MeOH-H 2O=9:1:0.06) to obtain compound 76 (4.6 mg, 57% yield). [ka]

[0143] Preparation of Compound 77 Compound 76 (4.6 mg, 3.0 μmol) was dissolved in dichloromethane (1 mL), and triethylamine (20 μL, 140 μmmol) and a dichloromethane solution of 2-chloro-1,3-dimethylimidazolinium chloride (0.2 M, 180 μL, 36 μmol) were added, followed by stirring at room temperature for 2 hours. Water was added to the reaction solution, and the mixture was extracted twice with chloroform. The organic phase was washed with a saturated aqueous solution of sodium bicarbonate and then with saturated saline, and then dried by adding magnesium sulfate. The drying agent was filtered off, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent: CHCl 3 -MeOH-H 2 O=20:1:0.2) to obtain compound 77 (4.0 mg, 89% yield). [ka]

[0144] Preparation of Compound 78 Compound 77 (4.0 mg, 2.6 μmol) was dissolved in methanol (1 mL), and sodium methoxide (0.5 mol / L methanol solution, 2 μL, 1 μmol) was added at 0°C. Water (5 mL) was then added, and the mixture was stirred for 1 hour and then at room temperature for 1 hour. After the solvent was distilled off under reduced pressure, MALDI-TOFMS was measured, and the presence of compound 78 was confirmed. [ka] Compound 78 MALDI-TOFMS:[M+Na]+m / z1372.4(calcd1372.7). (1-7) Preparation of antibody-binding sugar linker compound 83 A branched antibody-binding sugar linker compound 83 having a PEG chain with an azide group at its end was prepared as follows. Preparation of Compound 79 Compound 67 (305 mg, 1.23 mmol) was added with L-lysine methyl ester dihydrochloride (108 mg, 462 μmol) and dichloromethane (1.6 mL). N,N-diisopropylethylamine (0.45 mL, 3.2 mmol) and 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide tetrafluoroborate (390 mg, 1.21 mmol) were added to this solution under ice cooling, and the mixture was stirred at room temperature overnight. After concentrating the reaction solution, the residue was purified by silica gel column chromatography (developing solvent chloroform:methanol=60:1). The purified product was dissolved in chloroform, and the organic phase was washed with a saturated aqueous solution of sodium bicarbonate, and then anhydrous magnesium sulfate was added to dry it. After filtering off the desiccant, the solvent was distilled off under reduced pressure to obtain compound 79 (283 mg, 98% (yield)). [ka]

[0145] Preparation of Compound 80 Compound 79 (103 mg, 167 μmol) was dissolved in methanol (3.0 mL), and 1 M aqueous sodium hydroxide solution (194 μL) was added under ice cooling, followed by stirring at room temperature for 5 hours. The reaction solution was extracted with water and washed with diethyl ether. 1 M aqueous hydrochloric acid was added to the aqueous phase to adjust the pH to 1, followed by extraction with ethyl acetate and chloroform, and anhydrous magnesium sulfate was added to the organic phase to dry it. After filtering off the drying agent, the solvent was distilled off under reduced pressure to obtain compound 80 (71.7 mg, 72% (yield)). [ka]

[0146] (Preparation of Compound 81) Compound 80 (52 mg, 86 μmol) was dissolved in dichloromethane (0.86 mL), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (36.5 mg, 190 μmol) and N-hydroxysuccinimide (13.4 mg, 116 μmol) were added, and the mixture was stirred at room temperature overnight. Water was added to the reaction solution, and the mixture was extracted twice with chloroform. The organic phase was washed with a saturated aqueous ammonium chloride solution and saturated saline in this order, and then dried by adding anhydrous magnesium sulfate. After filtering off the drying agent, the solvent was distilled off under reduced pressure to obtain compound 81 (52.3 mg, 87% (yield)). [ka]

[0147] Preparation of Compound 82 Compound 56 (88.0 mg, 91.2 μmol) was dissolved in 1,4-dioxane (10.8 mL), palladium hydroxide (89 mg), water (2.64 mL), and 1M hydrochloric acid (136 μL) were added in that order, and the mixture was stirred overnight at room temperature under a hydrogen atmosphere. The solid matter was filtered off and washed with 50% 1,4-dioxane aqueous solution, and the filtrate and washings were combined and concentrated under reduced pressure. The resulting residue and compound 81 (52.3 mg, 74.5 μmol) were dissolved in 50% acetonitrile aqueous solution (0.8 mL), triethylamine (20 μL, 144 μmol) was added, and the mixture was stirred at room temperature for 5 hours. Water was added to the reaction solution, which was then freeze-dried. The residue was purified by silica gel column chromatography (developing solvent chloroform:methanol:water=7.5:2.5:0.3) to obtain compound 82 (22.7 mg, 38% (yield)). [ka]

[0148] Preparation of Compound 83 Compound 82 (11.4 mg, 11.8 μmol) was dissolved in water (0.482 mL), and 0.757 M 2-chloro-1,3-dimethylimidazolinium chloride aqueous solution (0.482 mL, 365 μmol) and triethylamine (235 μL, 1.70 mmol) were added under ice cooling, and the mixture was stirred at room temperature for 18 hours. Water was added to the reaction solution, and the mixture was freeze-dried. The residue was purified by gel filtration chromatography (developing solvent: 0.05% triethylamine aqueous solution), and 0.1 M sodium hydroxide aqueous solution (129 μL, 12.9 μmol) was added to the eluate containing compound 83. This solution was freeze-dried, and 22.0 mg of a mixture of compound 83, sodium hydroxide, and triethylamine was obtained. The structure of oxazoline was confirmed from the 1H-NMR spectrum, and the amount of compound 83 was calculated from the integral value, and compound 83 (9.2 mg, 83% (yield)) was obtained. [ka] compound 83 1 H NMR (600MHz, D 2 O): δ = 6.14 (d, J = 6.14 Hz, 1H).

[0149] (1-8) Preparation of antibody-binding sugar linker compound 94 Antibody-binding sugar linker compound 94 having three azide groups at its termini was prepared as follows. Preparation of Compound 84 Compound 35 (797.4 mg, 852 μmol) was dissolved in methanol (6.80 mL) and ethylenediamine (1.70 mL) was added at room temperature. The reaction solution was stirred under reflux for 17 hours, diluted with dimethylformamide, and the solvent was distilled off under reduced pressure. The crude product was obtained by azeotroping with dimethylformamide three times and drying the residue under vacuum. This crude product was dissolved in methanol (6.82 mL) and purified water (1.70 mL), and sodium bicarbonate (218.3 mg, 2.60 mmol), copper sulfate pentahydrate (45.2 mg, 181 μmol), imidazole-1-sulfonyl azide hydrochloride (271.3 mg, 1.29 mmol), and dichloromethane (8.52 mL) were added in that order at room temperature. The reaction solution was stirred at room temperature for 1.5 hours and copper sulfate pentahydrate (44.4 mg, 178 μmol) was added. The reaction solution was stirred at room temperature for 18 hours, and sodium bicarbonate (152.3 mg, 1.81 mmol) and imidazole-1-sulfonyl azide hydrochloride (359.3 mg, 1.71 mmol) were added. The reaction solution was stirred at room temperature for 3 hours, diluted with chloroform, added with 1M aqueous hydrochloric acid, and extracted three times with chloroform. The organic layer was washed with saturated aqueous sodium bicarbonate and extracted three times with chloroform. The organic layer was dried over anhydrous magnesium sulfate and filtered, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent toluene:ethyl acetate=7:1) to obtain colorless solid compound 84 (641.8 mg, 91% (yield)). [ka] compound 84 1 H NMR (600MHz, CDCl 3)δ:7.46(dd,J=7.2,1.7Hz,2H),7.42-7.27(m,18H),7.06-7.02(m,2H),6.85-6.81(m,2H),5.45(s,1H),5.08(d,J=11.0 Hz,1H),4.93(d,J=11.0Hz,1H),4.72-4.67(m,3H),4.66(d,J=11.7Hz,1H),4.61(s,1H),4.47(d,J=11.7Hz,1H),4.09(dd ,J=10.3,4.8Hz,1H),4.06(t,J=9.3Hz,1H),3.78(s,3H),3.76-3.71(m,2H),3.70-3.65(m,3H),3.57(td,J=9.3,3.2Hz,1 H),3.49(t,J=10.3Hz,1H),3.47-3.44(m,1H),3.41(t,J=9.6Hz,1H),3.10(td,J=9.6,4.8Hz,1H),2.33(d,J=8.2Hz,1H). MALDI-TOFMS (AXIMA-TOF 2 )m / z[M+Na] + calcdforC 47 H 49 N 3 O 11 Na,854.3,found854.2.

[0150] Preparation of Compound 85 Compound 84 (460.1 mg, 553.4 μmol) was dissolved in dichloromethane (4.61 mL), purified water (461 μL) was added at room temperature, and trifluoroacetic acid (461 μL) was added under ice cooling. The reaction solution was stirred at room temperature for 20 hours, diluted with chloroform, saturated aqueous sodium carbonate was added, and extracted three times with chloroform. The organic layer was dried over anhydrous sodium sulfate and filtered, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel chromatography (toluene: ethyl acetate = 1: 2) to obtain colorless solid compound 85 (379.8 mg, 92% (yield)). [ka] compound 85 1 H NMR (600MHz, CDCl 3)δ:7.38-7.27(m,15H),7.06-7.01(m,2H),6.85-6.80(m,2H),5.06(d,J=11.0Hz,1H),4.97(d,J=11.0Hz,1H),4.71 (d,J=8.9Hz,2H),4.69(d,J=12.4Hz,1H),4.57(d,J=11.0Hz,1H),4.52(s,1H),4.49(d,J=11.7Hz,1H),4.00(t,J=9. 6Hz,1H),3.78(s,3H),3.76(dd,J=11.0,2.1Hz,1H),3.71-3.63(m,3H),3.61(d,J=3.4Hz,1H),3.55-3.49(m,2H),3 .43-3.36(m,2H),3.22(td,J=8.9,2.7Hz,1H),3.05-3.02(m,1H),2.37(s,1H),2.35(s,1H),2.25(d,J=10.3Hz,1H). MALDI-TOFMS (AXIMA-TOF 2 )m / z[M+Na] + calcdforC 40 H 45 N 3 O 11 Na,766.3,found765.5.

[0151] Preparation of Compound 86 Compound 85 (379.8 mg, 511 μmol) and compound 3 (1.576 g, 3.07 mmol) were azeotroped with toluene three times, then vacuum dried, dissolved in dimethylformamide (5.10 mL), and sodium hydride (370.1 mg, 8.49 mmol, 45% oil complex) was added. The reaction solution was stirred at room temperature for 20 and a half hours, diluted with ethyl acetate, added with 1M aqueous hydrochloric acid, and extracted three times with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and filtered, and the solvent was distilled off under reduced pressure. The residue was crudely purified by silica gel column chromatography (developing solvent chloroform:methanol = 50:1). The resulting mixture was purified by gel filtration chromatography (developing solvent methanol) to obtain colorless oily compound 86 (775.8 mg, 86% (yield)). [ka] Compound 86 1 H NMR (600 MHz, CDCl 3 )δ:7.40-7.20(m,21H),7.04-7.01(m,2H),6.88-6.84(m,6H),6.82-6.79(m,2H),5.15(d,J=11.0Hz,1H),4.84(d,J=11.7H z,1H),4.76(d,J=12.4Hz,1H),4.67(d,J=8.2Hz,1H),4.67(d,J=11.0Hz,1H),4.60(d,J=11.7Hz,1H),4.50-4.47(m,7H),4 .45(d,J=11.0Hz,1H),3.97(t,J=9.3Hz,1H),3.94-3.90(m,1H),3.81-3.76(m,10H),3.73-3.57(m,56H),3.56-3.52(m,4H ),3.52-3.49(m,4H),3.48-3.42(m,3H),3.42-3.39(m,3H),3.22(ddd,J=9.6,6.2,1.4Hz,1H),3.13(dd,J=9.6,2.7Hz,1H). MALDI-TOFMS (AXIMA-TOF 2 )m / z[M+Na] + calcdforC 94 H 129 N 3 O 29 Na,1786.9,found1786.3.

[0152] (Preparation of compound 88) Zinc powder was suspended in water, ultrasonically washed for 30 minutes, and then 2% copper sulfate aqueous solution was added and stirred. The precipitate was collected by filtration and then air-dried to prepare Zn-Cucomplex. Compound 86 (635.0 mg, 359.8 μmol) was dissolved in tetrahydrofuran (18.0 mL) and acetic acid (18.0 mL), and Zn-Cucomplex (1.19 g) was added. The reaction solution was stirred at room temperature for 2 hours, diluted with toluene, and the solid was filtered off. The solvent was distilled off under reduced pressure and dried in vacuum to obtain a residue. This residue was dissolved in acetic anhydride (4.0 mL) and pyridine (4.0 mL). The reaction solution was stirred at room temperature for 18 hours, diluted with toluene, and the solvent was distilled off under reduced pressure. The residue was azeotroped with toluene three times and dissolved in chloroform. A saturated aqueous solution of sodium bicarbonate was added and extracted three times with chloroform. The organic phase was dried over anhydrous sodium sulfate and filtered, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (eluent: chloroform:methanol=50:1) to obtain crude product 87. [ka] The crude product 87 was dissolved in dichloromethane (5.5 mL), and 1,3-dimethoxybenzene (107.5 μL, 832 μmol) was added at room temperature. Trifluoromethanesulfonic acid (36.7 μL, 416 μmol) was added under ice cooling. The reaction solution was stirred at room temperature for 1 hour, then diluted with chloroform, and saturated aqueous sodium bicarbonate was added, followed by extraction with chloroform three times. The organic phase was dried over anhydrous sodium sulfate and filtered, after which the solvent was distilled off under reduced pressure. The residue was dried in vacuum to obtain the crude product. This crude product was dissolved in dichloromethane (2.77 mL) and methanol (2.77 mL), and 28% sodium methoxide-methanol solution (50 μL) was added at room temperature. The reaction solution was stirred at room temperature for 6 hours, then diluted with chloroform, and Amberlite IR-120 (H + The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: chloroform:methanol=20:1) to obtain compound 88 (322.6 mg, 63% (yield) for four steps) as a colorless oil. [ka] Compound 88 1 H NMR (600 MHz, CDCl 3 )δ:7.38(d,J=6.9Hz,2H),7.33-7.21(m,13H),6.97(d,J=8.9Hz,2H),6.80(d,J=8.9Hz,2H),6.50(d,J=8.2Hz,1H),5.34(d,J =5.5Hz,1H),4.86(d,J=11.7Hz,1H),4.78(s,1H),4.76(s,1H),4.71(d,J=11.7Hz,1H),4.50(s,1H),4.46(d,J=11.7Hz,1H),4 .41(d,J=11.7Hz,1H),4.17(t,J=5.8Hz,1H),4.07(dd,J=14.1,5.8Hz,1H),3.98-3.93(m,2H),3.91-3.84(m,2H),3.83(d,J= 2.7Hz,1H),3.76(s,4H),3.74-3.47(m,59H),3.30-3.24(m,2H),2.88(t,J=6.2Hz,2H),2.80(t,J=6.2Hz,1H),1.78(brs,6H). MALDI-TOFMS (AXIMA-TOF 2 )m / z[M+Na] + calcdforC 72 H 109 NO 27 Na,1442.7,found1443.3.

[0153] (Preparation of compound 89) Compound 88 (322.6 mg, 227 μmol) was dissolved in dichloromethane (4.54 mL), and triethylamine (286 μL, 2.05 mmol) and paratoluenesulfonyl chloride (358.4 mg, 2.10 mmol) were added successively at room temperature. The reaction solution was stirred at room temperature for 1 hour, and 4-dimethylaminopyridine (8.4 mg, 68.8 μmol) was added. The reaction solution was stirred at room temperature for 18 hours, and then diluted with chloroform, and a saturated aqueous solution of sodium bicarbonate was added, followed by extraction with chloroform three times. The organic layer was dried over anhydrous sodium sulfate and filtered, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent chloroform:methanol = 50:1) to obtain colorless oily compound 89 (403.4 mg, 94% (yield)). [ka] compound 89 1 H NMR (600MHz, CDCl 3 )δ:7.82-7.76(m,6H),7.40-7.19(m,21H),6.97(d,J=8.9Hz,2H),6.79(d,J=9.6Hz,2H),6.39(d,J=8.9Hz,1H), 5.33(d,J=5.5Hz,1H),4.86(d,J=11.7Hz,1H),4.79-4.75(m,2H),4.71(d,J=11.7Hz,1H),4.49(s,1H),4.46(d,J =11.7Hz,1H),4.40(d,J=12.4Hz,1H),4.17-4.11(m,8H),4.07(dd,J=13.4,5.8Hz,1H),3.97-3.92(m,2H),3.90 -3.83(m,2H),3.82(d,J=2.7Hz,1H),3.76(s,3H),3.74-3.46(m,65H),3.29-3.22(m,2H),1.80(d,J=6.2Hz,3H). MALDI-TOFMS (AXIMA-TOF 2 )m / z[M+Na] + calcdforC 93 H 127 NO 33 S 3 Na, 1904.7, found 1904.9.

[0154] Preparation of Compound 90 Compound 89 (403.4 mg, 214 μmol) was dissolved in 1,4-dioxane (18.4 mL) and purified water (3.05 mL), and 10% Pd / C (482.2 mg, 204 μmol) was added at room temperature. The reaction solution was stirred at room temperature for 21.5 hours under a hydrogen atmosphere, and then diluted with a mixed solution (chloroform:methanol = 1:1). The solid matter was filtered through Celite and washed with a mixed solution (chloroform:methanol = 1:1). The filtrate and washings were combined and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent chloroform:methanol = 10:1) to obtain colorless solid compound 90 (265.9 mg, 77% (yield)). [ka] compound 90 1 H NMR (600MHz, CDCl 3 )δ:7.82-7.75(m,6H),7.37-7.32(m,6H),6.94-6.90(m,2H),6.80-6.76(m,2H),6.27( d,J=8.2Hz,1H),5.19(d,J=8.2Hz,1H),4.80(s,1H),4.58(s,1H),4.18-4.10(m,7H),4. 06(t,J=9.3Hz,1H),3.94(dt,J=11.0,4.5Hz,1H),3.91-3.86(m,1H),3.82-3.43(m,63 H),3.39(dd,J=8.2,2.7Hz,1H),2.45(s,3H),2.44(s,6H),2.05(brs,3H),1.99(s,3H). MALDI-TOFMS (AXIMA-TOF 2 )m / z[M+Na] + calcdforC 72 H 109 NO 33 S 3 Na,1634.6,found1634.4.

[0155] (Preparation of Compound 91) Compound 90 (265.9 mg, 165 μmol) was dissolved in dimethylformamide (3.30 mL) and sodium azide (171.4 mg, 2.64 mmol) was added at room temperature. The reaction solution was stirred at 50°C for 20 hours, then diluted with toluene and methanol, and the solvent was distilled off under reduced pressure. The residue was azeotroped twice with toluene and dried in vacuum. The residue was purified by silica gel column chromatography (developing solvent chloroform:methanol=10:1) to obtain colorless solid compound 91 (176.6 mg, 87% (yield)). [ka] compound 91 1 H NMR (600MHz, CDCl 3 )δ:6.95-6.91(m,2H),6.82-6.78(m,2H),6.23(d,J=7.6Hz,1H),5.24(d,J =8.9Hz,1H),4.79(brs,1H),4.58(s,1H),4.15-4.08(m,2H),3.94(dt,J=11 .0,4.8Hz,1H),3.91-3.87(m,1H),3.83-3.55(m,60H),3.50-3.46(m,2H),3 .41-3.34(m,7H),3.29(s,1H),2.29(dd,J=8.6,5.2Hz,1H),2.01(brs,5H). MALDI-TOFMS (AXIMA-TOF 2 )m / z[M+Na] + calcdforC 51 H 88 N 10 O 24 Na,1247.6,found1247.3.

[0156] Preparation of Compound 92 Compound 91 (176.6 mg, 144 μmol) was dissolved in acetic anhydride (1.50 mL) and pyridine (1.50 mL). The reaction solution was stirred at room temperature for 18 hours, then diluted with chloroform, and the solvent was distilled off under reduced pressure. The residue was azeotroped three times with toluene and dried in vacuum. The residue was purified by silica gel column chromatography (chloroform:methanol=20:1) to obtain colorless oily compound 92 (191.9 mg, 99% (yield)). [ka] compound 92 1 H-NMR (CDCl 3 )δ:6.95-6.89(m,2H),6.82-6.76(m,2H),5.84(d,J=9.6Hz,1H),5.40(d,J=2.7Hz,1H),5. 21(t,J=9.3Hz,1H),4.95(d,J=7.6Hz,1H),4.48(s,1H),4.36(dd,J=12.4,2.7Hz,1H),4.28 (dd,J=12.0,5.2Hz,1H),4.21(dd,J=17.5,9.3Hz,1H),3.97(dt,J=11.0,4.5Hz,1H),3.91 (t,J=8.9Hz,1H),3.82-3.32(m,68H),2.13(s,3H),2.11(s,3H),2.08(s,3H),1.97(s,3H). MALDI-TOFMS (AXIMA-TOF 2 )m / z[M+Na] + calcdforC 57 H 94 N 10 O 27 Na,1373.6,found1373.4.

[0157] Preparation of Compound 93 Compound 92 (1.2 mg, 0.89 μmol) was dissolved in acetonitrile (142 μL) and purified water (35.5 μL), and ammonium hexanitratocerate (IV) (2.3 mg, 4.20 μmol) was added under ice cooling. The reaction solution was stirred at 0°C for 1.5 hours, then diluted with chloroform, saturated aqueous sodium bicarbonate was added, and extracted three times with chloroform. The organic layer was dried over anhydrous sodium sulfate and filtered, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent chloroform:methanol = 20:1) to obtain colorless oily compound 93 (0.6 mg, 54% (yield)). [ka] compound 93 1 H NMR (600MHz, CDCl3 )δ:6.10(s,1H),5.90(d,J=8.9Hz,1H),5.40-5.37(m,2H),5.34(d,J=2.7Hz,1H),5.17(t,J=2.7Hz,1H),4.55(s,1 H),4.37-4.14(m,8H),4.00-3.84(m,5H),3.83-3.34(m,56H),2.11(s,3H),2.10(s,3H),2.09(s,3H),1.96(s,3H). MALDI-TOFMS (AXIMA-TOF 2 )m / z[M+Na] + calcdforC 50 H 88 N 10 O 26 Na,1267.6,found1267.7.

[0158] Preparation of Compound 94 Compound 93 (0.6 mg, 0.482 μmol) was dissolved in dichloromethane (231 μL), and triethylamine (0.4 μL, 2.89 μmol) and 2-chloro-1,3-dimethylimidazolinium chloride (0.24 mg, 1.45 μmol) dissolved in dichloromethane (10 μL) were added in sequence at room temperature. The reaction solution was stirred at room temperature for 1.5 hours, then diluted with chloroform, saturated aqueous sodium bicarbonate solution was added, and extracted three times with chloroform. The organic layer was dried over anhydrous sodium sulfate and filtered, and the solvent was distilled off under reduced pressure. The residue was purified by gel filtration chromatography (developing solvent chloroform:methanol = 1:1, +1% triethylamine) to obtain a crude product. The crude product was dissolved in methanol (241 μL), and 28% sodium methoxide-methanol solution (5.0 μL) was added at room temperature. The reaction solution was stirred at room temperature for 24 hours, then diluted with methanol, and the reaction solution was subjected to mass spectrometry to confirm the production of compound 94. [ka] Compound 94 MALDI-TOFMS (AXIMA-TOF 2 )m / z[M+Na] + calcdforC 44 H 80 N10 O 22 Na,1123.5,found1122.9.

[0159] (2) Example of antibody-antibody-linker conjugate (Manufacturing example B-1) The antibody-bound sugar linker compound 12 obtained in (1) above was bound to rituximab (antibody). The reaction scheme is shown in Figure 1. Rituximab acceptor (13) was prepared according to Non-Patent Document 9 (Aoyama, M., et al., Mabs, 11: 826-836 (2019)) using rituximab manufactured by Zenyaku Kogyo Co., Ltd. The transfer reaction of compound 12 to the sugar chain binding site of rituximab acceptor 13 was carried out by adding the prepared rituximab acceptor 13 (2 mg), compound 12 (1.384 μmol), and wild-type enzyme Endo-F3 WT (200 μg) expressed and purified in E. coli to 50 mM Tris-HCl buffer (pH 7.0) and leaving it at 37 ° C for 1 hour in a total volume of 0.5 mL. A portion of the reaction solution was taken and examined by SDS-PAGE (Coomassie Brilliant Blue staining) using a 9.0% polyacrylamide gel, and a heavy chain band shifted to the high molecular weight side due to the transfer of compound 12 was confirmed (Figure 1). 80 μL of Ab-Capcher ExTra (Protenova) equilibrated with the same buffer was added to the reaction solution in wet volume and rotated at room temperature for 1 hour to adsorb rituximab to the gel carrier. The gel carrier was washed three times at room temperature with 1.8 mL of NETN buffer (50 mM Tris-HCl buffer (pH 8.0), 150 mM sodium chloride, 1 mM EDTA and 0.1% (w / v) NP-40) by inversion mixing, washed once at room temperature with 1.8 mL of NETN buffer by rotation shaking for 5 minutes, and rinsed four times with 1 mL of PBS. Rituximab was eluted by adding 200 μL of 0.1 M glycine hydrochloride buffer (pH 2.7) to the washed carrier, and 6.67 μL of 1 M Tris-HCl buffer (pH 9.0) was added to the eluate to neutralize it. This elution procedure was performed four times in total, and the eluate was concentrated with Amicon Ultra-0.5 (molecular weight cutoff 30 kDa, Merck Millipore) and the buffer was replaced with PBS. As a result, 1.46 mg of a mixture of fully azide PEGylated rituximab (14), hemi azide PEGylated rituximab (15), and unreacted rituximab acceptor (13) was obtained as the transfer product of compound 12 to the glycan binding site of rituximab (Figure 1). As a result of mass spectrometry analysis, the transfer yield of antibody-bound sugar linker compound 12 to the glycan site of the heavy chain was 46.7%.

[0160] (Manufacturing example B-2) A conjugate was prepared by binding an antibody-binding sugar linker having the following structure to N297 of the rituximab antibody through a reaction similar to that in Production Example B-1. TIFF2024178476000114.tif45153The obtained reaction products included fully PEGylated RTX, in which a complete antibody-binding sugar linker was bound to both of the two heavy chains; hemi-PEGylated RTX, in which the PEG moiety was deleted using an antibody-binding sugar linker bound to one of the heavy chains; and non-PEGylated RTX, in which the PEG moiety was deleted from the antibody-binding sugar linker bound to both heavy chains (Figure 2). The structures of glycopeptide fragments obtained by trypsin digestion of the antibody were confirmed by mass spectrometry (VelosPro / Ultimate3000 (Thermo Fisher Scientific)). The content of light chain-heavy chain pairs binding antibody-bound saccharide linkers with fucose was 46.49%, the content of light chain-heavy chain pairs binding antibody-bound saccharide linkers without fucose was 0.25%, the content of light chain-heavy chain pairs binding antibody-bound saccharide linkers with fucose and lacking PEG chains was 46.91%, and the content of light chain-heavy chain pairs binding antibody-bound saccharide linkers without fucose and lacking PEG chains was 6.35% (Figure 2).

[0161] (Manufacturing example B-3) In this example, compound 12 obtained in (1) above was introduced into a trastuzumab antibody. (Transfer reaction of compound 12 to the glycan binding site of trastuzumab (one-pot reaction. No prior preparation of antibody acceptor is required; alternative method to Production Example B-1)) Trastuzumab (80 μg) manufactured by Chugai Pharmaceutical Co., Ltd., compound 12 (54 nmol), and the enzyme Halo-Endo-S wild type (8 μg) expressed and purified using E. coli as a host were added to 50 mM Tris-HCl buffer (pH 7.0) in a total volume of 20 μL, and the reaction was carried out by leaving it at 37 ° C for up to 3 hours. A portion of the reaction solution was taken at 0.5, 1, 2, and 3 hours and confirmed by SDS-PAGE (Coomassie Brilliant Blue staining) using a 10% polyacrylamide gel, and a heavy chain band shifted to the high molecular weight side due to the transfer of compound 12 was confirmed from 0.5 hours. After 3 hours of reaction, a 76:24 mixture of fully azido-PEGylated trastuzumab and hemi azido-PEGylated trastuzumab was obtained as the transfer product of compound 12 to the glycan binding site of trastuzumab. Mass spectrometry analysis revealed that the transfer yield of compound 12 to the glycan site of the heavy chain was 87.4%.

[0162] (Manufacturing example C-1) (3) Preparation of a conjugate of an α-emitter conjugate and an α-emitter conjugate linker A scheme for the synthesis of these conjugates (compound C4) from an alpha-emitter conjugate linker (compound C1) and an alpha-emitter conjugate (compound C2) is shown below as scheme C1. (Scheme C1) TIFF2024178476000115.tif77158 Compound C1 was synthesized according to (Non-Patent Document 10: Kuzmin, A.; Poloukhtine, A.; Wolfert, MA; Popik, VV Bioconjugate Chem. 2010, 21, 2076-2085). Compound C2 (142 mg, 0.439 mmol) was dissolved in anhydrous CH 3 CN (4.4 mL) was added and stirred. Then, (COCl) 2 (25 μL, 0.202 mmol) was added at room temperature and stirred for 2 hours. The reaction solvent was removed under reduced pressure to give compound C3. Compound C3 was dissolved in anhydrous CH 3 CN (2.0 mL) was added and stirred. Then, compound C1 (38.3 mg, 0.0731 mmol) was added to CH 3The mixture was dissolved in CN (1.7 mL) and added at room temperature. Triethylamine (30.5 μL, 0.219 mmol) was then added at room temperature. The reaction mixture was stirred overnight and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (CHCl 3 / MeOH 10:1) to give compound C4 (15.1 mg, 0.017 mmol, 24%).

[0163] (4) Decaborate-containing conjugates (RTX-[B] 10 -2 nd ) A decaborate-containing conjugate RTX-[B] containing an antibody, an antibody-bound sugar linker, an α-ray emitting nuclear conjugate linker, and decaborate as an α-ray emitting nuclear conjugate is obtained from the α-ray emitting nuclear conjugate bond linker and α-ray emitting nuclear conjugate compound C4 produced in Production Example C-1, and the conjugate compound B2 of rituximab and an antibody-bound sugar linker produced in Production Example B-2. 10 -2 nd The synthesis scheme is shown below as Scheme D. (Scheme D) TIFF2024178476000116.tif83152 A solution of compound C4 (decaborane-labeled DIBAC) in PBS-buffer, pH 7.4 (50 mM, 20 μL) was added to a solution of compound B2 (azido-PEG-RTX) in PBS-buffer, pH 7.4 (100 μg, 80 μL), and the mixture was left at room temperature overnight. The labeled and unlabeled antibodies were separated from unreacted reagents and phosphates by ultrafiltration using centrifugal ultrafiltration filter units (10 kDa MWCO; Merck Millipore) and stocked in 50% glycerol. SDS-PAGE revealed that the decaborate-containing conjugates (RTX-[B] 10 -2 nd ) was confirmed (Figure 3). Specifically, the difference in molecular weight between the presence and absence of glycosylation was detected by silver staining using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the band obtained was determined by comparing the position of the band with that of compound B2, and the formation of a decaborate-containing conjugate (RTX-[B]10-2nd) was confirmed (Figure 3). The position of the band was measured from the top of the gel and the molecular weight was calculated by comparing it with a molecular weight marker. It was calculated that up to 3.8 molecules of compound C4 (decaborane-labeled DIBAC) were bound to one molecule of compound B2 (azido-PEG-RTX).

[0164] (5) Synthesis of Comparative Decaborate-Containing Conjugates Using the decaborane-containing structure (D1) reported in Non-Patent Document 1 (Wilbur, D. et al. Bioconjug. Chem. 2009, 20, 1983-1991), the decaborane-containing structure (D1) was nonspecifically bound to the amide group on rituximab (RTX) to obtain the decaborate-containing conjugate RTX-[B]. 10 - 1st was prepared. TIFF2024178476000117.tif34150

[0165] (6)RTX-[B] 10 -2 nd and RTX-[B] 10 - 1st Binding characteristics of to CD20 expressing cells Flow cytometry revealed that RTX-[B] binds to CD20-expressing Raji cells. 10 -2 nd and RTX-[B] 10 - 1st The affinity of the IgG was confirmed (Figure 4). Raji cells were harvested from the culture vessel and incubated in 1×PBS. 2nd (0.34 fluorescent groups per antibody) and RTX-[B]10- 1st(0.11 fluorescent groups per antibody) were added at the same concentration according to the number of fluorescent groups labeled, and the mixture was allowed to bind at 4°C (on ice) for 1 hour. After five washing steps (the cell suspension was adjusted to 500 μL with ice-cold 1×PBS, and centrifuged at 4°C and 1600 rpm for 6 minutes using a centrifuge (HITACHI, himac CF15R) and rotor (HITACHI, T15A33), 400 μL of the supernatant was removed), the fluorescence intensity was measured using a flow cytometer (Invitrogen, Attune NxT). As a result, RTX-[B] 10 -2 nd and RTX-[B] 10 - 1st It was confirmed that all of these antibodies maintained their binding ability to CD20-expressing cells.

[0166] (7) 211 At binding The decaborate-containing conjugate RTX-[B] prepared in (4) above 10 -2 nd and the comparative decaborate-containing conjugate RTX-[B] prepared in (5). 10 - 1st To 211 Specifically, 100 μg / mL of RTX-[B] 10 -2 nd or RTX-[B] 10 - 1st In contrast, 211 At aqueous solution (1MBq), Chloramine-T·3H 2 O (20 μg / mL) and 1× PBS (pH 7.5) were added and incubated for 10 min, followed by Na 2 S 2 O 5 (20 μg / mL) was added and subjected to gel filtration (PDMidi Trap G-25, gravity flow ×5). 211 At binding complex, RTX-[B] 10 -2 nd - 211 At and RTX-[B] 10 -1 st - 211The same procedure was carried out for the complexes lacking the decaborane structure, and the results were analyzed by thin layer chromatography (TLC). 211 It was confirmed that At specifically binds to the decaborane structure in the complex (Figure 5).

[0167] Example 2 Antitumor activity of antibody-drug conjugates

[0168] (1) Double-stranded DNA break test The antibody-drug conjugate RTX-[B] of the present disclosure prepared in Example 1 was administered to Raji cells derived from B lymphocytes of a Burkitt lymphoma patient. 10 -2 nd - 211 At and comparative antibody-drug conjugate RTX-[B] 10 -1 st - 211 At was applied and assessed for its ability to generate double-stranded DNA breaks (DSBs). Specifically, a suspension of Raji cells (5x10 5 0, 5, 50, 500 kBq of RTX-[B]10- 2nd - 211 At and RTX-[B]10- 1st - 211After adding At, the cells were left to stand for 5 minutes. After washing once with PBS and resuspending in 20 μL of PBS, 1.5 μL of the mixture was taken and mixed with 1 μL of Smear Gel (Nihon Genetics). The above mixture was spread on a slide glass and washed three times with PBS. After that, 4% paraformaldehyde in PBS was added and left to stand at room temperature for 15 minutes to fix the cells. After washing twice with PBS-Glycine, 0.1% (v / v) saponin in PBS was added and left to stand at room temperature for 5 minutes to permeabilize the cell membrane. After washing twice with PBS, Anti-gamma H2A.X (phospho S139) antibody [EP854(2)Y] (Alexa Fluor 594) was added at a 100-fold dilution and left to stand overnight at 4°C for staining. The next day, the supernatant was aspirated, DAPI in SlowFade (anti-fading mounting medium, Thermo Fisher) was added, and the sections were covered with cover glasses and photographed under a microscope (all-in-one fluorescence microscope (KEYENCE, BZ-X810)). As a result, RTX-[B] 10 -2 nd - 211 At and RTX-[B] 10 -1 st - 211 Both RTX-[B] and At were observed to generate DSBs in a dose-dependent manner. 10 -2 nd - 211 At is RTX-[B] 10 -1 st - 211 In relation to this result, the antibody-drug conjugate of the present disclosure has a tendency to have a higher DSB generating ability than At. 211 By introducing At, it is considered that the influence on functional sites such as the binding site of neonatal Fc receptor (FcRn) that may affect intracellular retention may be minimized. Note that this discussion does not limit the scope of the present disclosure. This experiment demonstrated that the antibody-drug conjugate of the present disclosure: 211 It was confirmed that this antibody exhibits superior antitumor effects while providing the advantage of introducing At into a specific site on the antibody.

[0169] (2) In vivo antitumor test The antibody-drug conjugate RTX-[B] of the present disclosure was administered to mice transplanted with Raji cells. 10 -2 nd - 211 At and comparative antibody-drug conjugate RTX-[B] 10 -1 st - 211 At was administered at 100 kBq / individual, and tumor size was measured for 29 days after administration. The tumors were then removed and the tissues were observed (n=4 each). Specifically, BALB / c nu / nu mice were purchased from Japan SLC Co., Ltd. (Hamamatsu City), given food (CRF-1: Oriental Yeast, Tokyo, Japan) and water ad libitum, and housed at 25 ± 1°C under a 12-h dark-light cycle (light: 08:00-20:00). Cultured Raji cells were washed with PBS and diluted to 1 × 10 in 0.2 mL of serum-free medium and Matrigel (1:1; BD Bioscience, Franklin Lakes, NJ, USA). 7 Tumor xenograft models were established by subcutaneously injecting 1000 cells into female BALB / c-nu / nu mice. Raji xenograft mice were used when the tumor size reached an average of 50 mm3. For dose-dependent experiments, mice were divided into five groups according to the injection dose: control (n=4), RTX-[B]10- 1st (n=4), RTX-[B]10- 2nd (n=4), RTX-[B]10- 1st - 211 At(n=4), RTX-[B]10- 2nd - 211 At (n=4)]. Controls received only the solvent. Similarly, 211 Unlabeled decaborate conjugate RTX-[B] that does not bind At 10 -2 nd and RTX-[B] 10 -1 st Observations were also performed on mice administered with the buffer as a negative control (n=4 for each group). As a result, the antibody-drug conjugate RTX-[B] 10 -2 nd - 211 At and comparative antibody-drug conjugate RTX-[B] 10 -1 st - 211 It was observed that all At exhibited excellent antitumor effects. This experiment demonstrated that the antibody-drug conjugate of the present disclosure: 211 It was confirmed that this antibody exhibits superior in vivo antitumor activity while providing the advantage of introducing At into a specific site on the antibody.

Claims

1. A complex having the following formula (1): Formula (1) (N ｍ -A-L ２ -L １ ) ｎ -B [In the formula, B is an antibody or antigen-binding fragment thereof; L １ is a first linker having the following formula (2): Formula (2) [In the formula, X's each independently represent a single bond, an oxygen atom, -NH-, -COHN-, -COO-, or a group represented by formula (4) or (5): [In the formula, R １ is a trivalent branched hydrocarbon group having 1 to 6 carbon atoms, R ２ and R ３ each independently represents an alkylene group having 1 to 3 carbon atoms; any one or more of Y1 to Y3 are present and each independently represents a PEG chain, a substituted PEG chain, or a PEG chain containing an oxygen atom, -NH-, -COHN-, or -COO- in the main chain, the PEG chain having a linear structure or a branched structure, and in the case of a branched structure, the number of branches is 2 to 10; Each Z is independently -O-, -CH ２ -, -N-, an optionally substituted tetrazine group, a norbornene group, a trans-cyclooctene group, a dibenzylcyclooctyl group, or a bicyclo[6.1.0]non-4-yn-9-ylmethyl group, or an optionally substituted cyanobenzothiazole group; R is L-fucose or a hydrogen atom; When any of Y1-3 is absent, Z is hydrogen and X is oxygen; When X is a group represented by the formula (5) or (6), Y1 to Y3 are each a branched R ２ and R ３ When the PEG chain has a branched structure, Z is bonded to each branched chain. #B represents the point of attachment to B, which is an asparagine residue present in the antibody molecule; #L ２－１ , #L ２－２ and #L ２－３ each independently represents a group when Y1 to Y3 are present, and each independently represents L ２ represents a hydrogen or a point of attachment to Existing #L ２－１ , #L ２－２ and #L ２－３ At least one of ２ represents the point of attachment to L ２ is a bond or a second linker; A is an astatine-binding structure; N is ２１１ At; m is an integer of 1 or greater; n is an integer from 1 to 10.

2. #B in formula (2) is When the antibody class is IgG, this represents the attachment point at asparagine residue 297; When the antibody class is IgA, it represents an attachment point that binds to at least one of the group consisting of asparagine residues 144 and 340; when the antibody is of the IgA2 class, it exhibits a binding site that binds to at least one of the group consisting of asparagine residues 131, 205, and 327; when the antibody belongs to the IgE class, it exhibits a binding site that binds to at least one of the group consisting of asparagine residues 140, 168, 218, 265, 371, 383, and 394; when the antibody belongs to the IgM class, it represents an attachment point binding to at least one of the group consisting of asparagine residues 171, 332, 395, 402, and 563; 2. The conjugate of claim 1, wherein the position of the asparagine residue is according to the numbering of the EU index of Kabat.

3. 3. The complex of claim 1, wherein the astatine-binding structure A is selected from the group consisting of decaborane and boron cluster lipid structures.

4. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 3.

5. The pharmaceutical composition according to claim 4, which is used for the treatment of cancer.

6. The pharmaceutical composition according to claim 5, wherein the cancer is selected from the group consisting of lymphoma, thyroid cancer, melanoma, prostate cancer, colon cancer, pancreatic cancer, liver cancer, breast cancer, cervical cancer, lung cancer, head and neck cancer, and cancer stem cells.

7. A method for producing an antibody-drug conjugate according to claim 1, comprising the steps of: (1) In the presence of a glycosyl hydrolase, an antibody or an antigen-binding fragment thereof B and a first linker L 1 and combining the (2) Astatine binding structure A and a first linker L 1 and a second linker L 2 binding, with or without an intervening (3) subjecting the astatine-binding structure-containing antibody complex produced by steps (1) and (2) to ２１１ Binding At A manufacturing method comprising:

8. A compound represented by the following formula (5): Formula (5) (A-L ２ -L １ ) ｎ -B [In the formula, B is an antibody or antigen-binding fragment thereof; L １ is the first linker; L ２ is a bond or a second linker; A is an astatine-binding structure; n is an integer from 1 to 10; First linker L １ has the following formula (2). Formula (2) [In the formula, #B is a point of attachment to an antibody molecule or antigen-binding fragment; X's each independently represent a single bond, an oxygen atom, -NH-, -COHN-, -COO-, or a group represented by formula (3) or (4): [In the formula, R １ is a trivalent branched hydrocarbon group having 1 to 6 carbon atoms, R ２ and R ３ are each independently an alkylene group having 1 to 3 carbon atoms; any one or more of Y1 to Y3 are present and each independently represents a PEG chain, a substituted PEG chain, or a PEG chain containing an oxygen atom, -NH-, -COHN-, or -COO- in the main chain, the PEG chain having a linear structure or a branched structure, and in the case of a branched structure, the number of branches is 2 to 10; Each Z is independently -O-, -CH ２ -, -N-, an optionally substituted tetrazine group, a norbornene group, a trans-cyclooctene group, a dibenzylcyclooctyl group, or a bicyclo[6.1.0]non-4-yn-9-ylmethyl group, or an optionally substituted cyanobenzothiazole group; R is L-fucose or a hydrogen atom; When any of Y1-3 is absent, Z is hydrogen and X is oxygen; When X is a group represented by the formula (3) or (4), Y1 to Y3 are each a branched R ２ and R ３ When the PEG chain has a branched structure, Z is bonded to each branched chain. #B represents the point of attachment to B, which is an asparagine residue present in the antibody molecule; #L ２－１ , #L ２－２ and #L ２－３ each independently represents a group when Y1 to Y3 are present, and each independently represents L ２ represents a hydrogen or a point of attachment to Existing #L ２－１ , #L ２－２ and #L ２－３ At least one of ２ represents the point of attachment to

9. A compound having the following formula (6): Formula (6) A-L ２ -L １ [In the formula, L １ is the first linker; L ２ is a bond or a second linker; A is an astatine-binding structure; First linker L １ has the following formula (7). Formula (7) [In the formula, X's each independently represent a single bond, an oxygen atom, -NH-, -COHN-, -COO-, or a group represented by formula (3) or (4): [In the formula, R １ is a trivalent branched hydrocarbon group having 1 to 6 carbon atoms, R ２ and R ３ each independently represents an alkylene group having 1 to 3 carbon atoms; any one or more of Y1 to Y3 are present and each independently represents a PEG chain, a substituted PEG chain, or a PEG chain containing an oxygen atom, -NH-, -COHN-, or -COO- in the main chain, the PEG chain having a linear structure or a branched structure, and in the case of a branched structure, the number of branches is 2 to 10; Each Z is independently -O-, -CH ２ -, -N-, an optionally substituted tetrazine group, a norbornene group, a trans-cyclooctene group, a dibenzylcyclooctyl group, or a bicyclo[6.1.0]non-4-yn-9-ylmethyl group, or an optionally substituted cyanobenzothiazole group; R is L-fucose or a hydrogen atom; When any of Y1-3 is absent, Z is hydrogen and X is oxygen; When X is a group represented by the formula (3) or (4), Y1 to Y3 are each a branched R ２ and R ３ When the PEG chain has a branched structure, Z is bonded to each branched chain. #L ２－１ , #L ２－２ and #L ２－３ each independently represents a group when Y1 to Y3 are present, and each independently represents L ２ represents a hydrogen or a point of attachment to Existing #L ２－１ , #L ２－２ and #L ２－３ At least one of ２ represents the point of attachment to

Images